MOLECULAR TAXONOMY OF E.C(?/.i HARBOURING R-PLASMIDS

ISOLATED FROM GANGA WATER

DISSERTATION SUBMITTED

IN PARTIAL FULFILMENT FOR THE DEGREE OF

MASTER OF PHILOSOPHY

IN

BIOCHEMISTRY

BY

PIR SHAFAT A. QADRI

DEPARTMENT OF BIOCHEMISTRY FACULTY OF LIFE SCIENCES

ALIGARH MUSLIM UNIVERSITY ALIGARH

1990

DEPARTMEMT OF BIOCHEMISTRY F a c u l t y O f L l f® Se lonc©? ALIGARH MUSLIM UNIVERSITY. ALI6ARH-202002 U. P. INDIA

TELEX ! 564230 CEA IN PHONE OFF.-5741

CERTJflCATE

Thii i6 to ce.itl^y that thz iZ'tt^aich Moik Qjnbodlzd in thl6 dl66&.fLtation entitled "Mol^culai Taxonomy o^ E.coli kaibouiing R-PZaimld6 i6alat&d j om Ganga watzn." 74 an oiiglnaZ woik, unlz66 othe,iwi&z 6tate.d, catiizd oat by M/i. Sha^at A. Qadii undzt my 6apzivl6lon and i6 6uitablz ^ot 6ubmi66ion fiOn. tha avoaid of^ M.Phlt dzg\ze. in Bioakzmi6t'iy.

P a t t d . - ^ ^ " ^ 2>'''' > '•''''' ( MASOOV AHMAV

DEDICATED

TO

MY

PARENTS

ACKNOWLEVGEMENTS

I u)l6h to e.Kpfie.66 my mo6t i,lnce.fiz and pio^ound giatitude. to my 6upeivi6o^ V>i. Ma6ood Ahmad ^on. hl6 Indi6pe.n6abtt guidance., con6tn.uc.tlve. ciitici6m, ancea6ing encouiagement, con&tant 6uppo>it and tiu6t duilng the. couue. o^ thl6 inve.itlgatlon.

J am giatefiUt to Pio^. M. Sale^ddln, Chairman, Vepait-me.nt o^ Blochzmi6tiy, faculty o^ Llie. Sciznce.6 {^on. he.lp^ul Augge6tlon6 and providing mce66aiy ^acilltlz6 to cafifiy out thl& VOOKk.

I am highly thankf^ul to ?Kol. A.M. Slddiqi f^oK ^lult^ul advlcz and e.ncouiagzme.nt throughout thl& 6tudy.

My Alnceie. thanki ane. due. to Pio^.S.M. Hadl, PiU) A.U.K. yu6u^l, MfL6. 8. Bano, Mu. W. Banu, ¥ai,lh Ahmad, Rlaz Mahmood, Qayyum Huiain, Mi66. J. Naitem and An.6had Rahman /ioi thelt helpful 6ugge6tlon6.

J am al6o thankful to Vfil6) Saad Tayyab, M.M. Mil, K. fazlli and Mi. Javzd H. itlanl ^oi the.lfi valuable, ituggt&tlom and moial zncouiagement.

A token o{ deep appieciatlon to Vi. Javed Muiaiiat ioK hi6 con6tiuctivz 6ugge6tion6 and kind help throughout thi6 6tudy.

My gratitude, too, to Shabana ^or genuine help and con6-tant encourage.me.nt.

My iinczrz thanki are alio due to my ^rie.nd6 and lab. colleaguzi, Shahid, Ra6hid, Said, Malik, A&ghar, Adit, Vahim, Qa6im, Gopal, M 44(j) Z. Rehana, A. Naheed, R. Zaidi, M. Mirza, R.V. Gupta, T. Zehra, Ai6ha, Veena, Mtifi) A. laldi, Fabzha and Farah.

7 have no u)ord6 to expreii thz infinite gratitude to my parents ior their benedictiou6 and con&tant in&piration throughout my itudieh. I warmly acknowledge my brothzr6 who contributed 6igni^icantty with their a^^ection and love.

La6t but not lea-^t M . Khan M. Akmal and Mr. Sabir Ali de6erve6 word o^ praiie ^or expert word proce66ing o^ thi& di66ertation.

Ministry o^ Environment and Fore6t6, Govt, o^ India i& acknowledged {,or financial a66i6tance.

{ SHAFAT A. QAPRI )

Contents

Page Certificate

Acknovj ledgemen b

List of abbreviations (i>

Lisst of tables (ii)

List of illustrations (iii)

Preface (iv-v)

Introduct i on 1 - 40

Material and Methods 41-58

Results 59-80

Discussion 81-88

Bibliography 89-101

l.it;!/ o f ahbrt . 'V i <i I-i o i i s :

A a m p i o i l l i n

Ak a m i k a c i n

B b a c i t r a c i n

C c h 1 (jr'arfii>hen i c o 1

CFU c o l o n y f o r m i n g u n i t

C t cFi] o r o l , e l . r a c y c ; ] i n c

Cx c l o x i c i l l i n

E c r y t-hrom?/!'; i n

E t B r e t h i d i ) . u n b r o m i d e

F r f u r a w o 1 i d o n t ;

G {^cn t amy c i n

h h o u r

K k a n a m y c i n

min tfiJnuLciJ

Mg m i c r o g r a m s

M1 mi c i o 1 i l ; c r «

Wa n a l i d a x i c a c i d

N n e o m y c i n

0/1-i O v e r n i g h t

P I ' c n i c i ] 1 i n

Pb p o l y m y x i n B

H r i f amf> i <: ill

S s t r c p l . o m y c - i n

stta wer;or)df;

T i c t - r a c y c 1 i in;

(11)

List, o C t;.aVj!f'!;

Table Paf?e

I. Genera of gi-arn-nef ati ve and f^ram-pool t i vo 8

bacteria in which plasifiid« have Vjeen detected.

II. Properties determined by bacterial plasrnids. 11-12

III. g.coli strains used in this syudy. 49 IV. Antibiotic re- i. -tance pattern of 19 E. coli GW 60

strains isolated from Ganfja water.

V. Percent resistance af^ainst individual anbibio - S2 bic.

VI. Transformation frequency of E.coli AB1157 with 66 plasrnids Rpl, Rp2, Rp3 an<i Rp4 isolated from four E.coli GW strains.

VII. Plasmid transfer by conjugation from E. c.olj, GV 74 strain to recipient E.colJt.

VIII. Effect of pll on conjugation in E.coli GV 3 strain 75 and E. coli AB11.57 recipient strain.

( I l l )

l.irrh of i 11 MR tr fit ions

Fit'ure Pa/io

1. Agarose tifel t;l e<.;Li'<)ph<.)rey3 a pal,torn of plasmid DNA 63 isolated from nine Bi.ooli GW strains.

2. Agarose gel elecLrophoresi s of i»J Hsmid DMA isol- 64 ated from four E.ooli GW strains and purified on sucrose density gradient.

3. Plasmid curing and survival of plasmid harbouring 67 E.coJLi GW strains by treatment with ethidium bromide.

A. Comparative pal/Lerns of curing and survival of 69 E.coli GW3 strain with ethidium bromide and caffi ene.

.). Effect of pll on i>\ tx::iu\<\ ciuing of I'l.coij CiWi strain 70 with ethidium bromide, at pH 7.0 and 7.4.

Ci. Effect of 1/rcatiri<;ri t {. mg/ml for various 1,ime inter- 71 vals) witli r;thidiurfi biotiiidc on f>la:;mi(] <;uring in ¥••(19.11 GV 3 strain.

7. Agarose ge;l electroi'h<;;rcsis of the cell lysatf; 73 mini preps) derived frtjm E-colA GW3 prior and after treatment with EtBr.

8. Agarose gel ol cctror'hort;si .s of R pla.'jifiids i so] atf'd 77 from four iL c£il i. GW strains.

9. Mol . si we vs mf)>>ility plot- of l,he test and standard 78 DMA.

10. .Agarose gel electrophoresis of the multiple plasmid 79 species isolated from E.ooli GW5 strain.

11. Mol. siae vs mobility plot of the test (multiple 80 species of the R-plasmids isolated from E.coli GW.5) and standard DMA.

(iv)

i'KEFACK

The Ganga in t.he lartjj'-nt and the rnost important river of

India. It iy also suppoyod to bo the holiest river of the

country and thu,s not <3nly houses the most important pilgrim

centres of India, but the soil of the basin also provides the

home of more than one-third of Indian population. The river

basin covers one fourth of India's geographical area. The

problem of pollution of the Ganga water due to discharge of

industrial effluents and sewage is getting more and more serious

(Gupta., 1984).

Usually the oucui-iuricu of pollution indicator bacteria

like total an<l fecal coliform is used as a sanitary parameter

for evaluation of the water quality. It is also known that these

indi<.-at(jrs are asso<;iatcd with discatjo causing genera of

importance to public health (Armstrong et al., 1981). The holy

water of this riv<ir is <;rjnaunicd afl^cr a long storage in addition

to its common use for drinking, washing, irrigation etc. by the

inhabitants of the Gangetic V:>elt.

As a consequence of v/idc si>read use of antibiotics,

R-factors v/hich transfer antibiotic resistance among

enterobacteiia, have be<.;omc c;omrnon in the non i<athog(;nic E - S Q U

of the alimentary trarjt ol' th<.- man and domestic animals. Many R

(v)

organisms eai>ecially those of huraati oritUn are exi>eebed i.o enter

oowa<?(3 and from thin, obvious ly to rivorf! (ornith, 1970). l»].(,-oli,

with Iransmiasible leoifjtance to aome broad speetrum ant.ibiol.ics

is considered to be potentially very danr?erourj because its

resiatance might be tancmitted to other i>athogenic bacterial

strains, thus rendering ttie treatment of infectious diseases

more difficult.. Hence, river is not only a source of infection,

but can also promote the spread of antibiotic resistance.

First step tov/ards prevention of environmental pollution

with R bacteria is to study their incidence of occurrence

eapeein] ly in rivt r iii«; r;y;;l,fm.

Antibiol-ic; rer; i stnrKJO in R >iMf;toriM in gf;iierMl]y j>]asifiid

mediated, and their characterisation on the basis of

antibiograms and plasmid profiles is feister and cost effective

than other methods. Thus an attempt v/as made to study the

incidence of R-plasmid harbouring E.coli in the Ganga river

within the short stretch from Warora to Kannauj. These criteria

would provide an appropriate inde;x for their classification.

INTRODUCTION

Escherichia coli and its Qh&racterjgtjc featurg

Escherichia coli for the first time was described by Buchner

(1885) and the genus was named after Theoder Escherich {1888)

who made a detailed study of this microorganism. E.coli is the

predominant facultative anaerobic species in the large intestine

and thus the species name represents this characteristic

feature. According to the Bergey's Manual the genus Escherichia

has been grouped under the family of EnterobacteriaoeQe

(Bergey's Manual, 1984). It is a gram negative straight rod,

measuring (1-3) x (0.4-0.7) micron arranged singly or in pairs

(Alcamo, 1987).

Cultural characteristic and colony morphology

When grown in liquid media, E.coli produces a well o o

dispersed turbidity in the temperature range of 10-46 C, but 37

C is the optimum temperature. Most E.coli strains have flagella

and are motile. It forms a variety of colonies on solid media

e.g; large, thick, moist, smooth, greyish, white or colorless,

opaque or partially transluscent. Smooth (S) type strains form

shining convex colonies but when repeatedly subcultured they

become rough (R) type and form lustureless, granular colonies.

The S > R variation is associated with the loss of surface

antigen and, usually, of virulence. Encapsulated varients

produce mucoid colonies, particularly when incubated at low

temperature and when grown in media containing limited amount of

nitrogen and phosphorous and a high concentration of

carbohydrate. Typical E.coli colonies are easily recognized by

their characteristic appearance on certain differential media.

This bacterium is largely a lactose fermenter and forms bright

pink colonies on MacConkey's medium. The colonies on the

eosin-methylenc-blue and endo agar display a metallic sheen

character, p~hemolysin is also produced on blood agar in certain

cases (Davis ^ aJl-* 1980).

Serotypjng and biotypifAg

E.coli can be characterised on the basis of antigenic

properties in various serotypes and based on some physiological

properties to biotypes. It has three antigenic types: the

somatic antigen O, the capsular antigen K and flagellor antigen

H. The K antigen at the cell surface often masks the deeper 0

antigen (Kaufman, 1975; Pelcsar e± &!., 1986).

Biochemical reactions

E.jS2£Lli usually ferments carbohydrates with the production

of acid and gas. A few strains are, however, anaerogenic

producing acid without- gas. Other substances with which

different biotypes of E.coli react differently include

raffinose, rharnnose, sucrose, xylose, adonitol, arginine,

glutamic acid and ornithine. Gram's staining and IMViC reaction

are commonly used for the identification of enteric bacilli, and

their identification is of prime importance in controlling

intestinal infections by preventing contaminations of food and

water supplies. E. coli displays indol pc^jitive, methyl-red

positive, voges-proskaur negative and citrate utilisation

negative reaction i.e. IMViC -f+—. E.coli is also distinguished

from other coliforms by its ability to form gas from lactose in o

toot syotom inr:!ubatod at 44 C (C/Jii»uc»)ii:io and tJhorman, 1907).

E.coli is usually similar to other Enterobacteria in its

susceptibility to hasardous agents and conditions, though it is

slightly more resistant to heat, to some chemicals and drying

than are Salmonella and Shigella. It is killed by moist heat at o

60 C usually within 30 min. It can survive for several days v;hen

dried on clothing or in dust. Pathogenic serotypes have been

found to be viable in floor-dust, in air and on clothing,

napkins and ward equipment in hospitals containing infants with

gastroenteri tis.

HasJi Pftragit>9 cglat^jQar^-hip

E-COli is a commensal "that thrives best in the human

intestine. However, some microorganisms have the ability to

change genetically and become virulent. E.coli which was long

oonsidered an avirulent commensal to humans, sometimes Vjecome

opportunistic and attains pathogenic character because certain

toxin-producing strains have been isolated in the outbreaks of

human diarrhoea and urinary tract infections (Middlebrook and

Dorland, 1984).

Pathogenicity

Ej. coli causes four main types of syndromes which are

extra-intestinal and Intestinal dis-oasori (Sack, 1975).

gxtra-intestinal diSfiaRQS: E-GQH most comrnonly causes disease-

in the urinary tract. Organisms conceivably travel from the

intestinal tract to the urinary passages and kidneys via

hematogenous and lymphatic routes. The normal urinary tract is

relatively free of bacteria, but asymptomatic bacilluria is

common, particularly in women and patients with obstructive

lesions.

E.poll is often found, alongwibh other enteric bacteria,

in sepsis adjacent to the gut: peritonitis, appendicitis and

infections of the gallbladder and biliary tract. E.coli raay also

cause pyogenic infections such as wound infections as it occurs

on the skin of the perineum and genitalia and abscesses or deep

infections such as cholecystitis and meningitis. E.coli is

common cause of meningitis in newborn, but is much less so in

older patients.

Intestinal diseases: E.coli is the dominant member of the

aerobic bacterial flora of the human (Gordon, 1971; Sack at al..

1971) and animal {Sack, 1975; Smith and Gyles, 1970) intestine.

It is usually nonpathogenic in this location. But there are four

groups of E.fiGl-i. which can cause human diarrhooal disea.ye

particularly in infants and adults in developing countries

(Gordon, 1971). These strains are called enteropathogenic,

enteroinvasive and enterohaemorragic E-fiolj.. (Sack, 1975).

In 1968, studies were made in patients in Calcutta (India)

who had cholera liko illness, but from whom no VibrJo cholerae

could be isolated. Inhibition studies during the acute phase of 7 9

diarrhoea demonstrated a largo population of E.op1j (10 - 10

viable counts/ml) in the proximal bowl of these patients

(Gorbaoh et al. , 1971).

Plasmid harbouring bacteria

Many bact-eria of diverse type and habitat are known to harbour

plasraid DNA. This observation lends credence to the view that

these extrachroraosornal genetic elements are ubiquitous among

prokaryotes. The bacteria screened for the extrachromosomal DNA

have often been chosen because of some distinctive property tliat

they display. This may be a pathogenic property, an unusual

characteristics or the ability to survive in an extreme

environment (Cohen, 1976; Stanisich, 1984). A single bacterial

cell can harbour one or more than one type of plasmids or

multiple plasmids. These multiple plasmids have genetic markers

for different phenotypic traits (Elwell and Shipley, 1980;

Shinji and Yokiko, 1988). But the bacterial cell may contain one

or more than one copy of the same plasmid. Depending upon its

cellular needs this bacterial cc;ll can accordingly control the

copy number of this plasmid, ranging from 1 to 100 replicons

(Ohman, 1988).

Several r«]fir;mi<l li/irb<niri riK l>nc;t<»rin linvri boon idou t J f i (;cl

without any additional phenotypic characteristics. These

extrachromosomal genetic elements without any visible phenotypic

trait are called the "cryptic" plasmids. In most instances such

cryptic DNAs are typical pla.ymids but in others they may

represent bacteriophage DNAs comparable to the coliphages of the

Pi type that exist intracel lularly as extrachrorfiosomally

replicating molecules (Falkow, 1975).

The first Vjacterial plasmids were identified amonr^ the

members of the family Enterobacteriaceae {Meynell, 1972), bub

subsequently the plasmids have been found in almost every

bacterial group (Cohen, 1976). Naturally occuring plasmids vary

in size from the 2,250- nucleotide-long minicircular "cryptic"

plasmid of Escherichia coll strain 15 (Cosaarelli fit . , 1968)

to the large and complex F plasmids (Low, 1972) which may

contain more than 400,000 nucleotide pairs and carry upto 600

genes (Cohen, 1976). Different genera of gram-negative and

gram-possitive bacteria in which plasmids have been detected are

given in table I.

Plasmids and their characteristic features

Plasmids are covalently closed circular extrachromosomal DMAs

capable of autonomous replication. These are ubiquitous among

the bacterial genera and are al.TO found in some oukaryoti*;

organisms (Davis and Rov/nd, 1972; Meynell, 1973; Lew in, 1977;

Broda, 1979),

Plasmids were discovered soon after the discovery of

Fnblf? 1 . InfJtiFT ri o t qt r i i i t -neaa t 1 r- nnrl n t r u n - f i o B i t i v e b n r t ' r 1 ."i i n i i l i i ! (i I) 1 ri'Md 1 (t' h,i>.f l i ! ' ( ' i i df^'p.- t r-cl .

Gr am—neqa t i vc? nf?rr)l>ir ror ls tintl ro t r i Ps(?udomonfifSi .V,.u) thamnn,} n Rh I zabitim Aqrohac t ni .i t im Hal J abac tc'r.iiim Alcaligenes Bot cif?tel la 1hermus

Or a m - n e q a t i vi? ( at u 11 . I i v c l y a iu i« 'r<ih ir r CRI* .

Escherichia Salmonella Stiine?l la K1 fpt.><3ie I 1 n Entcrobac t rv Serratia Proteus Vprr^i nia Er^s) I ni a Vibrio Provi dene .1 a ^eromonais Haemophi JuB Ci trobacter

Gr am- i ieqa t i vr? . i i iacr iit) i i h a c l J T i .i Bacteroides

Gram—neqa t i vf? r (x ( i ,»iiil t rx <nh.u i I I i Nei •s'ieria ^cmetobac ter

Gr am-pnr i i I Ivr^ ( o t t i Sta/>hy I oc oc cw Strep tococc LID

E n d o s p o r p - f or m i n(| r r»il'. Bac J 1 Jus CI OS tridiuiv

Gram—pasi t i vn ar,|»(ircun'iui-'. r (u l nl»apc(t lia( t c r i fi Lactabacj 1lus

A d a p t e d f r o m " M " l t i o d ' - i n M i ( l o h i 11 1 (u iy " ( 1 9 0 ' 1 ) . D e i u i e L . P . t i and R r i n s t - . o d , >I ( ' • d ' ^ . ; . TK .idr-.aii r I'r r.-.r-.

bacterial conjugation in early 1950s by J. Lederberg (Lederberg,

1952) in family Enberobacteriaceae when it became clear that

there were two genetically determined "ma4.Ij-ng" types and that

genetic information v/as physically transferred from a "male" or

donor type to a "female" or recipient presumably involving the

transfer of F (fertility) factor. Lederberg demonstrated that

this character for "maleness" was present in an extrachromosomal

genetic element and in 1952 he coined the term iilasmid to refer

to all such extrachromosomal genetic system.

Plasmids encode a number of specialised function that are

generally nonessential for their bacterial host. However, these

plasmid encoded functions provide their host cells with

versatility and adaptability for growth and survival under a

variety of conditions. Plasmids and transposable elements that

they often contain may also have played an integral role in the

evolution of bacterial species by promoting the distribution and

exchange of genetic information {Arber gt ai., 1985).

Plasmid modiaLod functions include genetic transfer, bact -

eriocin. Production and their resistance, antiobiotic resistance

and their production, resistance to heavy metals and DWA

damaging agents, metabolism of carbohydrates and hydrocarbons,

toxin and hemolsin production, virulence and colonisation

factors, tumorigenicity and nitrogen fixation in plants.

10

Recently, plasrnids have become the indispensable tools of modern

molecular biological research {Womble and Rownd, 1988). A

detailed list of properties determined by bacterial plasrnids is

given in table II.

CLasaiXicatiorA P I pj^siaids

Identification and classification of plasrnids are especially

important in medicine, because genes for clinical traits such as

drug resistance and virulence factors are frequently present on

plasrnids (Couturier, jei. &1.-> 1988). Three major classes of

plasrnids have beeti oharacborised most extonsivoiy, which include

conjugative plasrnids, bacteriocinogenic plasrnids and drug

roaistanco (R) piaomid.'j. The K piawmid io tlie claoaio fortiilLy

factor that is capable of its own transfer, the transfer of

other plasrnids and transfer of the host bacterial chromosome

during bacterial conjugation. Col-plasmids and R-plasmida in

contrast mediate the production of colicins and confer drug

resistance to their bacterial host cells respectively (Womble

and Rownd, 1988).

E.coli plasrnids

fi.Sfiii contain all types of plasmids. It was Joshua Lederberg in

1952 who di Bcovered F—plasmids in E.col_i. Clinical isolates of

n

Table - 11: i'roi'orLloo doLermiiiod by bucLoriui piaumidH

1. Resistance prQPQrtJQS

(a) Resis'bance to antibiotics - Aminoglycosides ( e.g. streptomycin, gentamicin,

amikacin-as a result of enzymic modification by N-acetylation. 0-nucleotidylation or 0-phosphorylation)

- Chloramphenicol {as a result of enaymic modification by 0-acetylation)

- Fusidic acid - Furans - B-Lactam antibiotics (e.g. benayl pencillin, ampicil-

lin, carbenicillin (as a result of enzymic cleavage of the lactam ring)

- Sulphenamides, trimethoprim (as a result of alternative drug-resistant target enzymes).

- Tetracyclines (as a resv^lt of altered transport sysbem) - Erythromycin (as a result of altered ribosome binding

site.

(b) Resistance to heavy-metal cations - Mercury and organomercurials (as a result of reductases

and hydro1ases) - Nickel, cobalt, lead, cadmimum, bismuth, antimony,

sine, silver, thallium

(c) Resistance to anions - Arsenate, arsenite, tellurite, borate, chromate

(d) Other resistances - Radiation (e.g. u.v.. X-rays) - Phage and bacteriocin resistance (e.g. Inc P2 plasmids) - Plasmid- specified restriction, modification systems

(e.g. Inc N piasmids)

2. Metabolic properties

- Antibiotic and bnct'Ori ocin T>roduction ( o.fT. by Streptomyces. lilsch^richia. Pseudomonas. Bacillus. Str^BtCCPccus)

- Metabolism of simj<le carbohydrates ( e.g. lactose, sucrose, raffinose)

- Metabolism of complex carbon compounds (e.g. octane and other n-alkanes, p or m-xylenes, p or m-toluenes, camphor, nicotine)

- Metabolism of i>roteins ( e.g. cfisein, gelatin ) - Metabolism of opines (by Ti+ Agrobacterium ) - Nitrogen fixation ( by Rhiaobium ) - S -Endotoxin by sporulating B- thurin^iensis - Other properties ( e.g. citrate utilization, H S

production, leucine biosynthesis) (Con td. )

12

Table II.

3. Properties fiOEitriiaitiBE L9. IlfvUhofieriiMiiy. iiC siafliiia&is

- Antibiotic refsistance and bacteriocin production - Toxin production (e.g. enterotoxins of B],gcherichia coli and Staphylococcus aureus. exfoliative toxin by S. aureus. haernolytsins by Escherichia. Staphylococcus and Streptococcus)

- Colonisation antigens of E.coli (e.g. K88, K89, CFAI, CFAII)

- TuHiorigenicity ( by Ti Agrobacterium ) - Host specificity (of Agrobacterium and Rhiaobiurn) - Modulation ( by Rhi^shium )

4. CQO.JMgftl propertyies.

- Sex-pili and associatt;d sensitivity to pilus-specific phages

- Surface exclusion - Response to and inhibition of pheroxnones (in Streptococcus)

- F e r t i l i t y i n h i b i t i o n

5. Replication-rnainbonance p r o p e r t i e s

- S e n s i t i v i t y t o curing agents - Incompatibility - Host range - Copy nutfiber - Temperature-sensitive replication

6. Other properties

- Gas vacuole formation in Halobacteriufn - Interference in sporulation of Bacillus pumilis - Sensitivity to bacteriocins ( in Agrobacterium and

SireDLfcococQus ) - Translucent-opaque colony change in Mycobacterium - Regulation of melanin production in Streptomyces

Adopted from "Methods in Microbiology" (1984), Bennet, P.M. and Grinsted, J (eds.). Academic Press.

13

E.coli both from humans and animals were found to carry

R-factors (Huber fit ai, 1971; Grabow and Proaesky, 1973).

Largely, as a consequence of widespread use of antibiotics,

R-factors have become common in tho non-pathogenic E.coli of the

alimentary canal of man and domestic animals. Smith has

determined the E.coli with transmissible resistance bo

chloramphenicol from British rivers (Smith, 1970). Furthermore,

the R-factors harboured by the non-pathogenic E.coli c^n be

transferred to drug susceptible pathogens such as SSullBaDella, and

Shigella (Kasuya, 1964; Watanabe, 1971).

R-plasmids present in E.coli isolated from various sources

have also shown resistance to a number of heavy metal salts

(Ogunseitan ei al. , 1987).

Much earlier before the discovery of bacterial plasmids.

Gratia in 1925 in Belgium discovered that a protein released by

a strain of E-c.Qli inhibited the growth of a limited number of

other bacterial strains and this protein in E.coli was known as

oolicin (Koneskey, 1978). The oolicin was found to be coded by

Col-plasmid (Novick, 1969).

R-plasmid bearing E.coli strains may occasionally exhibit

colicin production or oolicin resistance (Siccardi, 1966;

-V-Novakova st al., 1969). Colicin production by R strains may

14

result from the coexistance of a col-plasmid or probably by

association of a col determinant with the R-plaGmids.

In case of iiivasivc' E-OOli l/hat causo extra-intestinal

infeotions in man and domestic animals, some specific phenotypes

namely hemolysin production, colicin biosynthesis and specific

adherence properties have all boon shown to be plasmid-mediated

characteristics (Novick, 1969; Helinsiki, 1973).

The entoropathof?enic E.coli. strains rovatinely harbour-

five or more distinctive plasmid species. One or more of theses

plasmids is frequently an antibiotic resistence plasmid (Elwell

and Shiply, 1980).

R-plasmid and their salient features

The best-known plasmids from the standpoint of human medicine

are those that specify entibiotic resistance (Elwell and Shiply,

1980). R-plasmids in bacteria have resistance markers, either

for one antibiotic (Thomson and Dilgeri, 1982; Elirsh fit al.,

1989) or for more than one antibiotics (Hardy and Haeflei, 1982;

Hirsh fiik al.> 1989; Amyes, 1989). R-plasmid mediated antibiotics

resistance in enteric bacteria also referred to as "multiple

antibiotic resistance" or infectious antibiotic resistance is

known eversince its discovery in Japan in 1959 (Akiba, 1959;

15

Feary fit &!• , 1972; Womble and Rowrid, 1988). The R-plasmids are

moFJb common in bacteria frrjm clinicai and veterinary Houreeu

where exposure to antibiotics is likely to be high (Linton,

1977; Smith, 1977; 0,Brien., et ftl-. 1986). Many R organisms

especially those from human origin, v/ould be expected ultimately

to enter sewage and from thL-i', po.ssibly to rivers (Smith, 1970).

Many authors have reported their occurence at low frequency in

bacteria from water and other sources (Sisemore and Colwell,

1977; Talboot et al., 1980). E.£oii harbouring R-plasmids have

also been isolated from meat (Jayaratne et QX- > 1987) and from

cosmonauts (I1'in, 1989).

The R-plasmid WRI, which is also referred to as R100 or

222 is 94.5 kilobase (kb), self transmissible, multiple

antibiotic resistance plasmid, HRI is the original incB'II

bacterial resistance factor, the so-called R-factor isolated by

Rintaro Nakaya (Wakaya et ai., 1960), in Japan in late 1950s and

is archetype of a new large collection of similar R-plasmids

that have been discovered worldwide (Watanabe, 1963; Hashimoto

and Mitsuhashi, 1971; Davios and Rownd, 1972; Datta, 1975).

Besides antibiotic resistance, R-plasmids can often confer

certain additional properties on their host cell and can be

accordingly charactf,Tis<;d. Some- of the jt>i:oi)crtie,y int.-Judc:

resistance to heavy metals and their salts like arsenate,

16

arsenite, nickel and cobalt ions (Smit-h, 1967; Dabbes and Sole,

1900). R-plaairiid, NRI \-t(\;i f<juti<l Lu coni^tn r«j!;iM Lunce Lo morcurio

ions {Korflura et ai. , 1971; Kornura and Isaki, 1971). Plasmids

also confer resistance to UV radiations in: Streptococcus,

Pseudomonas and Enterobacteriaceae {Jacob et aJL., 1977; Jacoby

and Shapiro, 1977; Ciowcli, i9fJi). in addition Lo Ihuiu,

resistance properties some of the other properties of R-plasmids

include: fertility inhibition to a male bacterium, propagation

of specific phages, phage restriction, . colicin production,

colioin rosistnnoe, ijlasinid transfor, plasmid iticompatibility,

plasrnid instability and plasmid curing (Siccardi, 1966; Meynell

et fiJL., 1968; Bannister and Glover, 1968; Wovakova et al-, 1969;

Khatoon gt al., 1972; Stanisich, 1984; Couturier et al.. 1988;

Womble and Rownd, 1988; Hirsh et al., 1989; Jewell and

Collins-Thompson, 1989).

Some authors have classified the R-plasmids on the basis

of (a) their transfer by conjugation, (b) incompatibility and

(c) curing or elimination (Hovick, 1969; Couturier fit al_. ,

1988).

The R plasmi<hj cjapahlf; of inhibiting F associated

fertility have been referred to as fi and those lacking this

property as fi R-i>lasmida (Meynell et al. , 1968). Most of fi

R-plasmids, when present in E.coli F cells were found to

17

doLojiHiitiu [Alt) iiynLli'-'ii i!! of MII !•' tyj-c pilu.'; and Llmti inado Lht; i r-

host cella f3erif3itivc' bo the male apocifio pha^e MS2 (Meynoli BI.

fil., 1968). On the other hand, most of the fi R-plasmids made

their hosts sensitive to the phage IF! or to the phage IKe

(Meynell et &l., 1968; Khatoon et al., 1972).

Restriction of phages is a property associated with

some R-plasmido find IR indtspondonl- of thfrir fi l-yi'O (Sicoiirdi,

1966; Bannister and Glover, 1968). Plasmids other than

R-plasmido nro al;;o found l.o r,\^(;<:if\<Mi\]y r<;Rl,rict f:(;iLMiii

phages. Examples are restriction of phage T.3, T7 and it 11 or

phyll by E.coli F hosts (Schell ^i, ^1.., 1963; Make la et al.*

1964; Linial and Malamy, 1970).

Plasrnid transfer

The ability of a property to be; transferred from one

bacterium to another, provides a good presumptive evidence of

plasmid involvement, particularly if the frequency of transfer

is high. Transfer oxporimenbfj bhat are (;(>ndv,ujbod by dimply

mixing cultures of the test bacterium vith a .suitable recipient

strain can giv<j rise bo progeny by con,jugabion, branuducbion or

transformation (Brooks-Low and Porter, 1978).

Con .jugation: Conjugation is the highly specific process whereby

18

DNA is transferred from donor to recipient bacteria by a

mechanism invoivitig cell Lo coii conLaob (Lederberg, 19b2).

Conjugation has been detected in many members of

Enterobacteriaceae (Jacob et al., 1977) and in other

grarn-negative and gram-variable bacteria. It also occurs in some

gram-positive bacteria (Clewell, 1981; Stanisioh^ 1984).

The conjugation process is usually encoded by conjugal,ive

plasmids which have been isolated from a diverse range of

gram-negative bacteria and include the members of more than 20

incompatibility groups (Bukhari et aj,., 1977; Datta, 1979;

6 Bradely, 1980). Almost all types of R-plasmids bigger than 5x10

daltons are conjugative in nature. R-plasmids are composed of

tv/o genetically and t>hysically distinguishable components: a

resistance transfer factor (RTF) that harbour.s the genes for

self transmissibility (tra), autonomous replication (rep) and a

resistance determinant (R-determinant) markers. The

r-determinant harbours the majority of resistance genes (Clones,

1972; Davies and Rownd, 1972).

The plasmid transfer by conjugation is possible bo h

within and outside the boundaries of the genus (Marmur ftt ai.,

1963; Datta and Hedges, 1972; Datta, 1975). E.coli is also

reported to transfer its R-plasmids to various pathogenic as

well as non-pathogenic bacteria (Baron and Falkow, 1961; Jones

19

and Sneath, 1970; Oleson and Gonzales, 1974; Jean Claude et al.,

1988).

Transforma-bion: This is the process by which DNA from one cell

(the donor) is taken up by another (the recipient) directly from

the surrounding medium {Cohen et a1., 1972). There are

many genera of bacteria in which transformation has been

successfuly demonstrated e.g.: all genera in Entgrobacteriaceae,

Bacillus. Heamophilus (Smith et al., 1981). Stephy1ococcus

(Lncoy, iy7i») Mini {,'>l,r*JplrO(;o<;r<,;uf! (('J «;v/<! I I , I9MJ). fiWiiiio g';ii<Tn ol"

bacteria are able to undergo physiological transformation, bub

E.iiQjJL is not c<>mr>ot,C!nt to undergo pFiyfrJ olcjjri t;nl transf orrriMt-ion

(Saunders ei aj, .,1984). However, the E.coli can be made

susceptible to take up foreign DNA under artifipial conditions

like treatment with transition metals (Ca , Rb ) , with

intermittent heat shock (Lederberg and Cohen, 1974; Kushner,

1978).

In addition to other plasmid transfer processes in

bacteria, transformation by plasmid DWA can bo exploited a:; a

support that the phenotypic traits are expressed by the genes

present on plasmids. Elewell and Coworkers (1975) have shown

tViat B-lactamase resistance marker is present on the plasmid in

ampicillin resistant nR-7 and HR-885 strains of Haemophilus

influeng ae. Ampicillin resistant H. j.nfluenzae strains G32 were

20

obtained with the piasinids isolated from HR-7 and HR-885 strains

(Elewoll si, al. . 19Vb).

Transduction: Transduction is the transfer of bacterial DNA

from one cell (the donor) to another (the recipient) mediated by

a bacteriophage. Transduction has been used to demonstrate

whether or not the genes in question are plasmid associated and

form a single linkage group (Watanabe at. ixl-> 1968).

Phages containing double stranded DNA ranging in siae

from 12x10 to 480x10 daltons have been detected in a wide

varioty of baoLrjria (Roannoy nt\<\ Ar/kr rm/iriti, 108 1). Thoffo phngr'i;

are capable of transduction in a number of bacterial genera of

family En terobac tor iacoae as w«:'il a:j soveral other /jotirjra

including Acinetobacter. Bacillus. Haemophilus. Staphylococcus.

Streptococcus. Corvnebacterium and Streptomycps (Stanisich,

1984). Gene transfer by transduction occurs at a frequency of 10 -7

- 10 , depending on the phage. Transduction has been used to

separate individual plasmids from celLs harbouring several

different plasmidsj and the range of properties associated v/ith a

particular plasmid. More usually, transduction is confined to

"the same species but can be extended to a larger number of

strains if the effects of restriction can be overcome. The sise

of DNA to be transduced is also a matter of interest. This is

limited by the DNA capacity of the phage. Plasmids of the

21

similar or smaller siae than the phage can be transduced intact

and (?ive rise to }!l,al)Jr; l,rMn!!du(;Laijl,!t. In coriLrasL, iixriW,

plasmids are transduced as fragments and will nc t form stable

transductants unless appropriate recipie.'nts are used, that allov/

recombinational "rescue" of properties from the DNA {Stanisich

fit ai., 1976). Phage PI of E.coli is known to transduce an

R-plasmid as a whole. The Salmonella phage, P22, on the other

hand, can transduce only segments of an R plasmid (Watanabe and

Fukasawa, 1961; Watanabe et al., 1968).

Transduction has been used to differentiate a plasmid

associated phenotype from a chromosomally associaLed phenoLypo

in a bacteria (Stanisich, 1984).

Plasmid instability and curing

One of the ways to ascertain v/hether or not a particular

character is associated with plasmid, the elimination (curing)

of plasmids provides an approj^riato experimonbal basis. The

criterion of curing can be used for both, c<bnjugativo and

non-conjugative plasmids (Khatoon and Ali-Mohammad. 1986). Th irr

expected, therefore, that in any growing population of plasmid

harbouring bacteria, plasmid-less segregants will occasionally

be produced as the error in the process of plasmid replication

or partitioning to daughter bacteria. Thus this Ipss of plasmid,

22

or curing can occur either spontaneously (Wovick, 1969) or under

tho influonco of the £>biy«ic<iJ (Sl.adlor atid AdoJbort^ 197i!) arid

chemical agents (Novick, 1969; Stanirich, 1984). Some of these

curing agont,f< CMII miil-/il-'! 1>NA (VJi 1 I <jl,L<rt:, 1907). 'l'li'»tio ii(i<j[i\,\t nrc

individually effective only against some plasmids, and their

effect on newly diacoveirod iilasinid cannot be predicted

(Mitsuhashi et al. , 1961; Stani&ijh, 1984).

The generally used curing agents are acridine dyes

(Hirota, 1956; Hirota and lijiman, 19.57), ethidium bromide

(Bounchaud ejt A 1 . , 1969; Jones et aJL. , 1982; Hardman eJt al-i

1986; Khatoon and Ali~Mohammad, 1986; El-Syed et _al. , 1988),

many mutagens (Willebtos, 1967; Chakrabtirty, 1972), some

antibiotics (Ikeda ejb al., 1967; Yoshikawa and Gevag, 1967; Hahn

and Caik, 1971; Lacy, 1975; McHugh and Swartz, 1977; Molnar et

al-, 1978; Fu et a,l. , 1988; Selan et al. > 1988), chemicals like

sodium dodecyl sulphate {Tomoeda et al., 1968; Inusaka et al..

1969; Tomoeda gt SLI-, 1970), metal ions like Ni , Co (Hirota,

1956) and also some physical agents like heat (May gt a_l. , 1964;

Stadler and Adolhorrt, 1972; .JnynraLne n\, al- . 1900).

BlQchiafd<,;w] jn't?h«nif'mf:J of drug resistance

Various biochemical mechanisms have been proposed for

resistance to different antibiotics (Davis and Maas, 1952; Davis

23

and Smith, 1978). These resistance mechanisffis include (a)

alteration of torf^et site in the bacterial cell, (\>)

interference with drug bransporb, (c) enaymatic detoxificabion

of antibiotics and <d) by-pass mechanism. In addition to these

mechanisms there are some unknown drug resistance mechanisms

also (Davie and Smith, 1978).

(a) Alteration of target site: In the bacterial cell there is

reduction or elimination of binding of the drug to the target

Bite. Several examples of mutational alterfition of antimicrobial

target sites are known e.g.: resistance to streptomycin {Flakes

sit fll-> 1962), spoctinomycin (Bollon et fll-> 1969), erythromycin

(Tanaka et al., 1968), chloramphenicol (Osawa et al., 1973),

rifamyoin {Robussay and Zillig, 1969) and to many other

antibiotics some of which are of clinical significance. Weisblum

and his coworkers have provided one well established case of

plasraid-determined resistance, that is due to the modification

of the target site with macrolide (erythromycin)- lincosmide

(lincomycin) resistance in gram-positive Vjacteria (Lai ond

Weisblum, 1971; Yogi ot al., 1975; Ail(Mi. 1977). In thii; <-nr,<<

RNA of large ribosome subunit (50S) is dimethylated by a planmid

determined enzyme at two adenine residues. The methylation of

RNA prevents the binding of a variety of erythromycin,

lincomycin type antibiotics to the ribosome and the cells become

resistant to high levels of drug.

24

(b) Interference with drug transport: For those antimicrobial

agents with active tratisport syotem, inutationo or pias-rnid

determined modifications can lead potentially to a block in

transport system that prevetits entry of drug into the cell. One

of the most intriguing and best studied examples of antibiotic

transport and its apparent organisms by piasmid-coded functions

is the case of tetracycline resistance. Resistance to this drug

has been suggested to be due to inhibition of the normal active

transport system (Franklin, 1967).

The most common form of resistance to aminoglycoside

antibiotics depends on the presence of plasmid-coded modifying

enaymes. The aminoglycoside modifying ensymes are classified

according to the mechanism of their modification (e.g.

N-acetylation, o -phosphorylation or o-nucleotidylation) and

their site of jfiodification on the aminoglycoside (Davis and

Smith, 1978).

(c) Epzymatic detoxification .Q,f antibiotics: Certain antibiotics

are detoxified by the modification of these antibiotics as in

case of chloramphenicol. Chloramphenicol resistance in

gram-positive and gram-negative bacteria is predominantly the

result of acetylation of chloramphenico] by chloramphenicol

acetyl transfera.se. Another mechanism of chloramphenicol

detoxification known, is by the reduction of p-nitro group to

25

give the inao-tive amino derivative (0,Brien and Morris, 1971).

Apart from the chloramphenicol, resistance to several

other antimicrobial agents have been demonstrated to accomplish

through detoxification. The most important f roup is the B-lactam

antibiotics, the most widely used for all therapeutic agents.

B-lactamases, catalyse hydrolysis of the B-lactam ring with

concomitant detoxification of the drug. B-lactamases are both

chromosomal and plasmid determined (Davis and Smith, 1978).

(d) By-pass mechanism: Two of the most v^noxpected findings of

recent years came with the demonstration of the mechanism of

resistance to sulfonamides and to trimethoprim. In these two

examples the plasmid provides the cell with an entirely new

metabolic enzyme that being insensitive to the inhibitor,

substitutes for the inhibited chromosomal ensyme and allow

continued functioning of blocked pathway and growth in the

presence of drug (Davis and Smith, 1978).

Sulphonamidos exert their bacteriostatic effect by compe­

titive inhibition of the enayme dihydropteroate synthetase.

Plasmid determined sulfonamide resistance was one of the first

R-factor characters to have been discovered in Japan in

1950s.Many workers have provided a convincing evidence that

sulfonamide resistance was due to R-plasmid inhibition of

26

transport of the drug (Akibo and Yakota, 1962; Wise and

Abou-Donia, 1975).

A high level of resistance to trimethoprim was observed

in E-flflii involving the by-pas^3 mechanism. In E-OQli cells

carrying plasmid R388, Amyes and Smith (1974) found a new

dihydrofolate reductase in addition to the normal trimethoprim

sensitive chromosomally coded enayme.

(d) Unknown resistance mechanism: Inspito of extensive studios

on the mechanism of plasmid-determined drug resistance, a number

of resistance phenotypes still lack accurate biochemical

definition (Davis and Smith, 1978).

Resistance to fusadic acid is well known in Staphy1ococcus

AUCSVtEl> but still unclear irj fTram nogaLivo bacteria. Similarly

the plasmid determined mechanism of novobiocin rosistatmc in

Stgphy 1 ococci is unknown (Davis utjd Smith, 1978).

Although it is obvious that our knowledge of plasmid-

deterrained resistance mechanisms have increased greatly in the

past few years, but many plasmid genes presently have no

recognizable phenotype and thus may be the determinants of

resistance for antibiotics yet to be discovered and introduced

(Davis and Smith, 1978).

27

illAasifiCf»1'Jc»ri of bficteria aricl their tfi>;priornic . 'tyUlf

The classification of organisrnfs or their taxonomy has tv/o

purposes: one is to group the organisms of same kind, the

descriptive classification and the second purpose is to provide

a "natural" phylogenetic classification. In the descriptive

classification, organisms have been grouped on the basis of

information col J trf.-t-r-d abouL t\ jri v<;ii kind, l.hfiL can bo i>oo.l<Kl and

compared. This type of classification is based on the characters

of the organism, which a Laxonomi MI- can f.'XpJoJL (Mandcl, I9G9).

Accordingly, in bacteria, where a species include a

spectrum of organisms with a wide range of properties, a species

represent a cluster of biotypes (Mandel, 1969).

A classification system was given by Kaufman and his

collaborators in 1940s. In this system E.coll was subdivided on

the basis of antigens like O (lipopolysaccharide), K (capsuler)

and H (flagellar) (Vahlne, 1945; Sjostedt, 1946; Kaufman, 197.5).

In 1976, an analysis of 0:11 ooroLypcu and biotypes was used for

classifying E-Ciaii. causing diarrhoeal diseases (Orskov e_b al. ,

1976). A mixed collection of E.coli isolated from Boston USA,

was analysed by serotyping and biotyping (Myerowits ej; al.,

1977). However, v/ith certain exceptions these classical methods

did not allow a generally applicable taxonomic analysis of

28

E.coli isolates, nor vas it clear that which criteria should be

used for successful analysis. Numerous taxonomists continue bo

use serotyping and biotyping for classifying bacteria, not

solely because of tradition, bub the results also allow ready

pigeonholing of E-coli isolates and often correlate'well v/ibh

disease specificiby. [lowever, those rnebhods do not on their ov/n

result to definitive analysis of taxonornic structure. A

successful taxonornic analysis of E.coli population structure

would include quantitative estimates of the re]atednesr; betv;oeri

bacterial groups. This might yield valuable insights into

microbial ecology and pathogenicity (Achtrnan and Fluschke,

1986).

Many classification methods have been used in E.coli

taxonornic studies. Among the.se, starch gel electrophoretic

analysis of cytoplasmic isoenzyme, a classical technique in the

taxonomy of the eukaryotic organism, has been extensively used.

Other methods based on resistance to colicins and other toxic

chemicals, DNA-DNA hybridisation or aoquence analysis of rliNA or

proteins have also been used in taxonornic studies (Achtrnan and

Pluschke, 1986).

SDS-PAGE OMP (outer membrane protein) patterns and 0:K

serotypes were used to classify E.coli from epidemiological

sources into various groups. The major OMPs exhibited many

29

different elect-rophoret-ic migration patterns when random samples

of E. coli are analysed {Paahkanen e.t al. , 1979; Jann and Jann,

1980; Overbeeko and Luin^Unibfjrit, 1900). In the last few yoars

the usefulness of this property for identification of bacterial

clones has been demonstrated.

Isoenzyme analysis (Ochman and Salander, 1984), OMP

analysis and serotyping collectively defined 15 clones of E.coli

(Achtman and Pluschke, 1986). More than 160 E-cP.li O groups have

been described. The 0 serotyping in based on the reactivity of

the somatic O sugar chains of LPS (lipopolysaccharide) with

hyperimmune rabbit antigens (Oskov and Oskov, 1984). Recently,

electrophoretic separation of cellular proteins have been

employed to differentiate methicillin-resistant Staphylococcus

aureus (Gaston et al., 1988). Sutherland and Kennedy, (1986)

compared lipid-free polysaccharides from gram-negative bacteria.

This comparison was accomplished by high performance liquid

chromatography.

Bacterial strains have also been groui>ed on the basis of

phagrj Lyi'ing. •ialuiuiiuiin tyi'liiiuuriyin i;/iu;itjd outl.>ruak;i ^^^!

gastroentritis in a number of cities throughout India since

1977. The Salmonella strains v/ere divided into various groups on

the basis of phage typing (Fz-ost et ai. , 1982). Phage typing v/as

also employed by Burnie and his coworkers fnr the identification

30

and comparison of epidemic strains of methecillin resis"bant

Staphy1OCQCCUS aureus (Burnie et al., 1989).

Restriction of phages is a property associated with some

plasmids including the R-plasmids (Siccardi, 1966; Bannister and

Glover, 1968). Plasmids also interfere v/ith phage propagation

(Duckworth fit al-> 1981). These two properties of phages with

their bacterial hosts have been cxi»loited for the grouj;>irjg of

bacteria like E.coli (Meyenell et ai., 1968) and Pseudomonas

aeruginosa (Jaooby, 1970).

Phage typing of certain bacteria is sometimes difficult

and confusing. Staphy1ococcus aureus strains are not typable by

bacteriophages of international typing set. Supplementary

bacteriophages therefore, have been isolated to distinguish

these non-typable strains of S.aureus {Vickery et al-. 1983;

Richardson ei al-, 1988).

The approaches like serotyping, biotyping, phagetyping and

numerical taxonumy dijuuribfjd fur l[\i; ci/iij!jifioation have ordorc-d

bacteria rather tentatively, in terms of phenotypic traitf.;. The

development of molecular genetics, however, has now revolution­

ized taxonomy. This taxonomy is based on macromolecular sequence

homology (Brenner and Falkow, 1971).

31

The simplest comparison is the base composition of DNA.

This can be estimated easily from melting temperature or buoyant

density. The base composition is essentially the same in all

vertebrates (about 40 moles percent gaunine, cytosine [G+C] and

60 percent adenine, thymine [A+T]. But the base Qomposition

among bacteria vary remarkably from about 30-70 moles percent

(Marmur fit M . , 1963.

DNA sequence homoloogy (DNA-DNA homology) can be measured

quantitatively in terms of the ability of the DHA strand from

two different sources to form molecular hybrids ixi vitro. Among

bacteria DNA-DNA hybridisation is useful only within closoly

related groups, because it quickly vanishes in the broad range

of variation between more distant organisms. Ribosomal i?.NA

hybridization to DNA is useful for estimating more distant

kinship among bacteria. These processes have been employed in

descriptive classification of bacterial strains (Brenner and

Falkow, 1971; Schiffer and Stackebrandt, 1983).

DNA-DNA hybridisation of R i>lasmid harbouring arai>ic;i J 1 in

resistant gram-negative Vjacterial strains was used for tkiolr

characterisation and classification. All of the ampicillin

resistant gram-negative strains demonstrated sequence homology

(Fock-Ruediger and Rainer, 1982).

32

Antibiotic sensitivity patterns or antibiograrns have been

used for the classification and grouping of bacterial strains by

a number of workers. This typing method is faster and more-cost

effective than biochemical analysis and bacteriophage tyj'ing

(Gillespie st aJL-. 1990). E.coli isolated from porcine fecal

origin, patients of urinary tract infection and patients from

Egypt have been divided into various groups on the basis of

their multiple antibiotic resiE;tance pattern or antibiograrns

(Ohmae gi si., 1980; Elkhouly, 1981; Cees ei al., 1982).

Gillespie and his collaborators have used antibiograrns for

the identification and comparison of Gtaphviococcus aureus

(Gillespie fit si-> 1990). The antibiotic resistance in bacteria

is usually conferred by plasmids. Thus plasmid profiles have

been used for identification and comparison of bacterial strains

in addition to antibiograrns and phage typing. Electrophoretic

patterns of plasmids were used for bacterial identification. 72

E.coli strains conferring resistance to rhultiple antibiotics

have been shown to contain single type of plftsmid by thcji-

electrophoretic patterns (Ohmae et al., 1980). Similarly 5-6

plasmids ranging from 30-70 Mdal, have been isolated from 30

strains of bacteria (Vakulenko et al., 1980). Plasmid profiles

of Salmonella typhirnurium isolates in an outbreak of

gastroenteritis in India was compared by Frost and his coworkers

(Frost St al., 1982). They found that this group of strains

33

belongs to single clone which has become widespread in India.

Plasrnid typinff hfiw bt;<?ii ufiod by many oliicr workois l,o \,rn<.:f.:

bacterial strains prevalent in USA (Koaarsky et ai., 1986) and

in Ireland (Col(;man ^,1. exX. > lOBli). Lyon and his coworkers havt;

used plasmid profiles to meniter the spread of epidemic strains

of inethicillin resistant S.-Uyireus in Australia (Lyon et ai. ,

1983, 1984a).

Antibiograms, plasmid profiles and phage typing have been

jointly used for the classification of E.coli. Salmonella

typhi murium and S:baphy 1 OCOQCUS ay^eus. strains from clinical

origin (Frost gt ai., 1982; Martinea et al., 1987; Gillespie ej.

si., 1990).

Restriction endonuclease; fragmental,ion of DNA or plasudds

and molecular siae differences have also been used for taxonomic

studies of various bacterial species (Achtman and Pluschke,

1986). Hardman et al (1986) have used restriction fragmentation

and molecular sise difference of plasmids for the characterisa­

tion and claf3si C Lcal> ion of f(.it.ir ri;';uduin','tia:j and bv/o alcal 1 "H^<HICS

species. Shinji and Yokiko (1988) have classified E.fi.Qli strains

by comparing their plasmid profiles and the restriction

fragments of these plasmids. Burnie and his coworkers have used

EcoRI restriction ensyme fragmentation patterns and immunoblott-

ing to differentiate and classify methicillin resistant isolates

34

of Staphylococcujs aureus (Burnie et aj. , 1989).

Bacterial pollution in riverine system

Sewage polluted water is often a common source of disease

in man and animals (Craun, 1972). Various bacterial spoaie-s

pollute different water sources through the city and hospital

sewage. The bacteria polluting water sources are various genera

of Ent^erobftcteriaceae and some other gram-variable organisms

like Streptococci. Xecsinla, LQ££miQxm&.. SttmAxylusAOHQ^AS, Vibrio

and Clostridium. Among the known bacborial species, coli form

group of bacteria is the principal indicator of suitability of a

water for domestic, dietetic or other uses. The coliform group

density is a criterion of the degree of pollution and thus of

sanitary quality. Fecal Streptococci also are indicators of

fecal pollution (McCarthy et a_l. , 1961; APHA, 1985).

Presence of any coliform Vjacteria renders the water

potentially unsatisfactory and unsafe. Among Lhe coliform group,

E-££2li is predominant in all types of water sources including

river water (Smith, 1970; Dell et al., 1980; Wiemi fii al. , J983;

Al-Ghazali et al., 1988).

Although E.coll is usually found in fresh pollution

derived from warm blooded animals, other coliform organisms may

35

be found in fresh pollution in the absence of E.cpli. The f^enera

Enterobacter. Klebsiella. Citrobacber and Escherichia usualiy

represent the majority of isolations made from raw and treated

municipal supplies (APHA, 1985). Coliform organisms some of

which are free-living saprobes, can multiply on leather' v/asliers,

plant material, wood, swimming pool ropes or jute packings and

may produce slimes inside pipes {Eller and Edwords, 1968; Clark

and Pagel, 1977).

Industrial wastes containing high concentration of

baoborial nutrients are capable of promotitjg significant after­

growth of coliform types in effluents and recreiving waters.

Aftergrowth also may appear in treated water distribution

systems. Aeromonas and other oxidase- positive, gram-negative

bacteria as well as Erwinia may be expected in raw and treated

waters (Gorden and Fliermans, 1978; APIIA, 198D).

Fecal Streptococc i is also found in different water

sources. This also provides the extent of fecal pollution in

these water sources (Kenner et al., 1961). The normal habitat of

fecal SliUQP-Lyifyuui if! tlw' int'-Ntinr- of liutiiMrif; and Miiimalfi. thui;

these organisms are the indicators of fecal pollution.

S«£fiiecsl.i£ subspocicf! J ic|V4ef(u.;i r?nf: i r; not r(:Kl,rJ rjttrd to [,hc;

intestine of humans and animals. It has been found associated

with vegetation, insects and certain types of soils. Because of

36

limited survival in the environment, ib is not recommended trj

use only fecal Streptococci when determining Lhe water qualily.

In combination with fecal coliform, data on fecal strexdiococci

may provide more information about pollution sources (Sureau,

1958; Hartley and Slanets, 1969; Geldrich and Kenner, 1969;

Geldrich, 197,6).

A wide variety of enteric pathogenic microorganJ fimu

occurs in waste water. With increasing demands on water sources,

•the potential of cjoiitamination of surface and ground waLcr by

enteric microorganisms could be expected to increase (Seligman

and Reitler, 1965). The most common and important bacterial

pathogens include Salmorie.lJ./Ji, Shigillla. Campylobacter,

enteropathogenic E.coli. Leptospirella and Yersinia (Galton et

ai. , 1958; Ewing, 1962; Greenberg nn<10ngorhh, 19B6; Sack. 1975;

Knill 8±, Q1., 1978; Mentaig, 1961). Other organisms such as

Vibrio cholerae. also may be found in water becavase of exbenrjivo

and rapid world travel. Leg i one 11 acea. ttppears v/idely

distributed in the aquatic environmetit, pneumonia outbreaks

associated with tap water and aerosol transmission route have

been reported (Wadowsky and Yoe, 1981; EdeleHtein, 1982). The

occurence of Salmonella in water is highly variable; more than

1700 Salmonella serotypes have been isolated and currently

recognised. Besides, Shigella sp. have also been found in

various polluted waters. Outbreaks of waterborne shigellosis

37

frequently result from accidental interruption of v/ater

treatment and waste water seepage into water supply linos.

Enteropathogenic E.coli has been isolated from tap waters,

drinking water sources and mountain streams {Seigneurin et al.,

1951; Peterson and Boring, 1962; Ewing, 1962; Greenberg emd

Ongerth, 1966; Edwards and Ewing, 1972). Campylobacter je..jjjni

has been isolated from inadequately treated or untreated river

water and mountain streams (Knill et al., 1978) and both endemic

and epidemic water-borne diseases have been reported (Mentsing,

1981). While water-borne outbreaks of cholera often is a sr-rious

problem in near East and Orient, the potentifil risk of similar

epidemics in other parts of tlie world oiiinot bo ignored. Varioufj

Qoncholerae vibrio strains are found in both fresh and c;stuarlnc

.environment (APHA, 1985).

A large number of isolates of bacterial population

contaminating river and other water sources have been found to

confer drug resistance, v/hen tested for their sensitivity. The

distribution of antibiotic resistant bacteria in the aquatic

environment, mainly in surface v/aters, has been investigated by

a number of workorrs (Koditschok and Guyrc, 1974; Goyol et fyl.,

1979; Ogunseitan et al., 1987), v/ith particular attention to

members of the Enterobacteriacea {Feary et al., 1972; Grahovf and

Prozeskey, 1973; Niemi et al., 1983; Al-Ghaaali et al-. 1988).

Coliform group of bacteria showing drug resistance has been

38

generally isolated from almost all types of water sources

world-wide. Besides coliforms few pathogenic drug resistant

bacteria like Shigella dysenteriae (Ganr^arosa et fxl- , 1972) and

Sftlmonella typhi (Baine et al., 1977) have also been isolated

from river waters. The drug resistance in these bacterial

S'brains was due to the R-plasmids (Gmitki, 1970; Baine eL; al. ,

1977; Bell gt al., 1980). The emergence of drug resistance In

bacteria is common due to widespread utilisation and misuse (jf

antibiotics (Smith, 1970; Farid et al. , 1975; Levy, 1983;

Al-Ghazali Qi al. , 1988).

4-

R bacterial strains found in the water sources particu­

larly from river.s wore of focal (jrigin (Smith, 1970; Jaai'awi and

Ishaq,1983; Niemi et al., 1983). Smith (1970) isolated E.coli of

alimentary canal of man and dome.stic animals from British ITJIOM.

Al-Doori and his coworkers (1986), liave found a significant rise

in multiplicity of resistance among clinical isolates of E. coli.

A high percentage of multiple drug resistant pollution indicator

bacteria was found in river waters in different studies (Goyel

fit fil-, 1979; Belletal. , 1980; Al-Ghaaali ejt al. , 1988).

Majority of the.se antibicjtic re^jistan*- U bnr;terial strain.f!

were able to transfer their drug resistant markers to other

bacterial species (Smith, 1970; Bell et al., 1980, Stotzky and

Krasovsky, 1981). E.coli. with transmissible lesistance to

ampicillin, tetracycline, chloramphenicol and riome other broad

39

BpecLrum ariLibioLics is cronfjjdorod t-a bo- poLenbialiy more-

dangerous because its resistance might be transferred to

pathogenic bficl-o-iia] riLrMiri!; likr,- t>alrnor»cl I'l hyjlhk> <ihii',<:\^'\

dysenterai etc. Thus rendering the treatment of some diseases

like typhoid fever and dysentry more difficult (Smith, 1970;

Bell fit Ski.-, 1980). E-Gfili as a focal pollution indicabor

organism is used to indicate the transfer of antibiotic

resistance in aquatic environment where polluted surface waters

can be considoi-ed tis a j'olnt sourcf.- for SF>roadirifT the antibiotic

resistance via many possible routes which ultimately reach

humans (Al-Ghasali ejt al. , 1988).

During recent years, the distribution of antibiotic-

resistant strains of Enterobacte;riacea in the aquatic environ­

ment has been studied in the different parts of the world. For

example sewage polluted British Isel (Smith, 1970), water-borne

enteric pathogens in Guetamala (Gangarosa .e_t al. , 1972), river

and sewage waters in South Africa (Grabow and Prosesky, 1973)

surface waters, sea waters and shellfish in New Zealand (Cooke,

1976a, 1976b), Mexican outbreaks of S. typhi (Baine eit al. ,

1977), surface and sea waters in USA (Koditschek and Guyrc,

1974; Kelch and Lee, 1976) and Al-Khair river, Baghdad Iraq

(Al-Ghazali et ^ i . , 1988).

40

In view of the present lit-erat-ure GVArvey it seemed worl-h

while to carry out the systematic study on the R-plasmid

harbouring g.poli strains isolate;d from Ganga Water. Such type

of studies are need of the day and have also been conducted all

over the worJd, ov/jrii [,<> the aJarmJn(7 JovoJ of Lhc d J vcriili" i t-d

"type of pollution in the riverine system.

MATERIAL AND METHODS

MATERIALS

M£QIA

41

m& fifiat (pH 7.2)

Peptone

Lactose

Sucrose

Potassium phosphate

Agar

Eos in Y

Methylene Blue

10.0 g/1

5.0 g/1

5.0 g/1

2.0 g/1

13.5 g/1

0.4 g/1

0.065g/l

MacConkev^s agar (pH 7.1)

Pancreatic digesL cif gelatin

Pancreatic digest of casein

Tissue

Bile salts

Sodiurn ch lo r ide

Agar

Neutral red

Crystal v i o l e t

17.0 g/1

1.5 g/1

1.5 g/1

1.5 g/1

5.0 g/1

15.0 g /1

30.0fng/l

1.0 rrig/1

42

MacConkey^s broth (pH 7.4)

Peptone : 20.0 g/1

Lactose : 10.0 g/1

Bile salts : 5.0 g/1

Sodium chloride •• b.0 g/1

Neutral red •- 0.07. Jg/l

HMtrient &Sj&r (pH 7.4)

Peptone : 5.0 g/1

Sodium chlorldr.' : f>. 0 ii/\

Beef extract •- 1.5 g/1

Yeast extract 1.5 g/1

Agar •• 1.5 g/1

Nutrient broth <pH 7.4)

Peptone '• 5.0 g/1

Sodium chloride : 5.0 g/1

Beef extract : 1.5 g/1

Yeast extract 1.5 g/1

M M figSE (pH 7.3)

Peptone : 30.0 g/1

Beef extract : 3.0 g/1

Ferrous ammonium sulfate : 0.2 g/1

Sodium thiosulfate : 0.2 g/1

Agar : 3.0 g/1

43

Simniona cifcr^i,© ML^V. (I'H 6.9)

Ammonium hydrogen phosphate 1.0 g/1

Dipotassium phosphate 1.0 g/1

Sodium chloride : 5.0 g/1

Sodium citrate : 2.0 g/1

MagneQsium fsulfate : 0.2 g/1

Agar : 15.0 g/1

Bromo thymol blue : 0.08 g/1

Soft fir IfiK Sger

Nutrient broth : 13.0 g/1

Agar powder •- 7.0 g/1

TryptJgftSg £?Qy broth (pH 7.2)

Trypticase : 15.0 g/1

Phytene : 50.0 g/1

Sodium chloride : 5.0 g/1

REAGENTS AND BUFFERS :

SDS : 1.0 %

NaOH : 0.2 N

Ammonium acetate Solnution.

Ammonium acetate : 0.1 M

44

Soln. A: Alpha-naphbhol

Ethanol (absolute)

Soln. B: Potassium hydroxide

Calcium chloride solution

Calcium chloride

Cryafcel yiolat

Crystal violet (85 % dye content)

Distilled water

Ethanol (95 %)

Acetate

Dilution buf;fer

Megnesium sulphate

Creatine

Distilled Water

EcoRI assay buffer

Sodium chloride

Tris-Cl (pH 7.5)

Megnesium chloride

Dithiothreitol

Electrophoresis buffer (Trio-aceLuLe,

Tris

EDTA

Acetic acid (glacial)

Distilled water

: 5.0gm

: 5.0ml

: 40 gm

: 0. 1 M

: 1.0gm

: 100ml

: 250ml

: 250tni

: 0.01M

: 0.3gm

: 100ml

: 100mM

: 50 mM

: 10 mM

: 1.0mM

pll 8 . 0 )

: 9 . 7 gm

: 0 . 7 gm

: 2 .28ra l

: 197 ml

45

Gram^ s Iodine

Iodine crystalG 1.0 Hm

Potassium Iodide •• 2.0 gm

Distilled water : 300 ml

Kovac's reafienb

p-Dimethyl amino benwaldehyde 5.0 Km

Amyl alcohol : 75.0ml

HCl (concentrated) : 25.0ml

Trio-cl (pH 8.0) : 25.0ffiM

EDTA : 10 mM

Glucose : 50 mM

Lysoayme : 2mg/ml

Marker dye

Glycerol : 50 %

Bromophenol blue "- 0.05 %

EDTA : 40 mM

MethV1 red solution

Methyl red : 0.1 gm

Ethyl alcohol (9.5%) : 300 ml

Rga^ent h

TE buffer containing

5 % dow corning antifoam RD emulsion.

Rgafieot fi o

1 M NaOH saturated at 20 C

with SDS.DisbiliGd water : 200 ml

46

Safranin 0

(25 % solution in 95 % ethyl alcohol)

Distilled water

Sodium flggtftte sfijl_ution

Sodium a c e t a t e

10 ml

100 ml

3 . 0 M

The fo l lowing chemicals were used

Ch^nijgftlp Source

Acetic acid

Agarose

Agar powder

Ampicillin

Ammonium acetate

Antifoam RD emulsion

Butanol

Caffeine

Calcium chloride

Ch1oramphen i co1

Dipotassium phosphate

BDII, India

Sigma, USA

Hi-Media^ India

Ranbaxy, India

Hi-Media, India

tlopkins and W i l l i ams,

Eng1and

S i s c o L a b o r a t o r i e s , l i i ' l ia

A l d r i c h , USA

S i s c o I .aboraboricfi , I n d i a

Ranbaxy, I n d i a

BDH, I n d i a

47

Dithiothreitol

EcoRI endonuclease

Ethylene diaminetetra acetate

Eosin methylene blue agar

Glucose

Glycerol

Kanamycin

Lambda DNA

Lysoayme

MacConkey's agar

MacConkey's broth

Magnesium sulphate

Nutrient agar

Nutrient broth

Potassium acetate

Potassium dihydrogen

ortho phosphate

Sodium acetate

Sodium citrate

Sodium chloride

Sodium hydroxide

Sodium thiaaulphate

Simmons citrate agar

SIM agar

Hi Mf.'dia, India

CSIR Centre for

Biochf;micals, India

sd Fine Chemicals, India

lli-Media, India

[li-Media, India

lli-Media, India

Ran b axy, Ind i a

Sigma, USA

Sisco Laboratories, India

M i-Med j a, Ind i a

Hi-Media, India

E. McTf ;k, Indi a

Hi-Media, India

Hi-Modi fi, India

City Chemicals Corpn. , fJSA

BDH, India

BDH, India

Loba Chemicals, India

BDH, India

Sisco Labora tor ies , India

sd I'iiie Chemicals, Iri'lia

Hi-Media, India

Difco, USA

48

Sucrose

Sbropbotnyo Iti

Tetracycline

Tris HCl

Tryp-bicase soy broth

Qua I LfZonu f ine Chern i c/il fi,

I n d i a

I DIM.. luiWn

P f i z e r L t d . , I n d i a

Sifltna, USA

Difco , USA.

Note : The chernicalis which have n o t been i n c l u d e d in t h i s l i s t

v/ere of a n a l y t i c a l g r a d e .

49

l ah ln 111 . follei-'ing E . co l > Strairxs have been used in t h ' S Si*^dy.

S t r a i n de-- Re l evan t g e n e t i c markers Source Signait ion

AB1157 t H i - l , a r g E S j t f i r - 1 , i e u 8 6 . P r o n 2 , S r i -yas t^»Va j fl-S.

C60W t h r . l e u , t h i , l a c , >^ • Thotnas, R-

E"cRp4 Pt*" J 1C , r ' ' . S r i vr ts - tava , 9-S

GVn A ' ' , B^ , C'", C t ^ , E ' ^ . F r ' ' . K ' ' , ft^, J s o l a t r r i f , rwr,

S' , Sz^, T''. lac. B.Aiujn l ^ i v f r .

e W 2 A ' ' , A k ' ^ , B*", C*^, r t * ' . F*", F r ' ' , K ' ' I s n l a b i M l ( r (KC

R ' ' , S * ' . S z * ' , T ' ' , l a c . Barun,! i n v o . .

BW3 A ' ' , Ak '^ . B ' ' , C t * " . E T , F r*^ , k*", R* I n o l a t p d t rcvn

Sr' ' , T^ . lac. (5,uuir> m v t . ' i .

6W4 A ' ' , B ' ' . C t ' ' . E ' ' , F r * ^ , K^, 9.^ ,Sz^. I s o l n t p r ) ( m m

T , 1 a c . (I c_i n (h I f n / ' •' .

6W5 ft'', B'" . C ' ' . C\^. E ' " , F r ' " . K ' ' , R^ . i ' ( i i , . t . " i ' M I ­

C V= r e s i s t - ^ v x t , s = s e n s i t i v e )

50

METHODS

Maintenance Mid gCQWJfch ol hacierlfl

Each strain of E.cpli. was streaked over nutrient agar

plates. A single colony was picked up and repurified by

streaking over agar plates. The culture was tested on the basis

of associated genetic markers raising it from a single colony

from the master |>late. Then the culture was raised and streaked

over nutrient agar slanbeo. It was then allowed to grov/ nl 37

o deg. C and stored at 4 C. Every month culltures were transfered

over fresh slanbes. Media used in slant preparation for F.r-'Up'i

was supplemented with 20 ug/ml ampicillin and 10 ug/ml

kanamycin.

Overnight cultures of E.coli strains v/ere raised in

O nutrient broth at 37 C. The culture was diluted fifty times in

o fresh nutrient broth follov/ed by shaking at 37 C till the cell

density reached to about 2 x 10 viable r.-ounts/rnl. Such f.-xfonen

tial cultures were used in all the experiments.

Collection QZ water samples

Water samples were collected routinel2f' from the i-oint

sources at four selected stations (Narora, Katchla, Fateligarh

51

and Kannauj) along the river Ganga. These four sites were

oovorely affected by human activities and were near the hirihly

populous area. Sampling v/as done seasonally starting frorti surmner

1988 to spring 1990. 4 to 6 samples per season were collected in

these two years. Samples v/ere collected from the bank and

midstream of the river in 2b0 ml sterile glass bottles and

stored in an ice box. The elapsed time betv/een the collection of

sample and initial processing did not exceed 8 h.

Isolation and idenialjQatjon Q£ E.00.11 tvsim Sanga VM^SLL

The isolation of E.coli was done according to the standard

methods des cribed in APHA, 198b. 10 ml aliquots of water sami'les

were inoculated into 10 ml aliquots of double strength

MacConkey's b roth and incubated at 37 C for 24 h. Furthermore

the sample indicating acid and gas production was spread on

eosin-methylene-blue (EMB) agar plates and incubated at 37 C for

24 h. The colonies which were showing metallic green sheen

character were picked and grown individually in nutrient broth

O at 37 C for 12 to 16 h.

grwn staining

The gram staining of all E.coli isolates was done accor­

ding to the standard procedure of Cappuccino and Sherman (1907).

52

heroical rfi&ctions

The isolated E-co_li. strains were finally identified on

the basis of their biochemical properties and enzymatic

reactions in the presence of specific substrates. The IMViC

series of tessts were performed according bo the method of

Cappuccino and Sherman (1987).

Antibiotic afinsltiyity t_ej5t

All the E.coli isolates were tested for sensitivity to

anti^" !Imicrobial agents by meanrj of disc diffusion method

employing multi discs (Bauer gt aJL. , 1966; Coleman et al. ,

1985). This test was carried out by mixing 0.3 ml of fresh

exponential culture of the test E.coli strain with 3.0 ml of

o molten top agar (held at 4b C ) . The mixture was layered over a

freshly prepared nutrient agar plate and allowed to set for aout

half an hour. The antibiotic discs were applied using sterilised

O forceps and the plates were incubated overnight at 37 C. The

zone of growth inhibition around the antibiotic discs v/ere

meaaurod and tho rr-fMiltt! rt •(:<>{<]<•<]. Tlir- ftillov/ing nri t i h i r •!. i r-

discs (all from Hi-media) were used; concentration of the

antibiotics used was* in ^gramS i>er disc;". Amikacin(Ak 30),

Ampicillin(A 10), Bacitracin(B 10units), Chloramphenicol(C 30),

Chlorotetracycline(Ct 30), Cloxacillin(Cx 5 ) , Erythromycin(E

53

15), Furazolidone(Fr 15), Geritamycin(G 10), Kanamycin(K 30),

Nalidixic acid (Wa 30), Neomycin{N 30), Periicillin-G{P 10units),

Polymyxin-B(pb 300^, Rifampicin(R 5), Str-eptomycin(S 10) ,(Sz zo\

Tet-racycline(T 30). These antibiotics were selected on the basis

of> their common use by fche humans and domestic animals in the

Gangetic belt.

Isolation Q 1 plasmids

The isolation of plasmid fiom satisfactory cleared lysatc

from any bacterial species is frequently a combination of skill,

luck and patience, especially if large plasmids are to be

isolated (Ohman, 1968). Plasmids were routinely isolated from

jE.coli isolates of river Ganga. Two methods were followed for

the isolation of plasmids:

1. The miniprei* method wat; follov/(,-d csijcntially as described by

Birnboim and Doly (1979).

2. Plasmid DNA was also extracted from E.coli cells isolated

from river water by the method of Wheatcroft and Williams

(1981). Freshly harvested cells fiom 50ml volumes of bacterial

o u l L u r e s w o r e utM.inl I y rr;r;i.i;;f>(:ii<ler<l i ti '/.. 0 m l R<.-'\(/ciil.. A . 1 . () m i

aliquots of this suspension wtis transferred to centrifuge tube.

0.4 ml Reagent B \iffxr, then added with Kentl*.- rjhaking. After that

the tube was stirred vigorously on a vortex mixer for 5 min. The

suspension was boiled for 40 sec before electrophore.sis in order

54

t o p r e c i p i t a t e more c-hrornosomal DNA find c e n t r i fvjf ed a t 12000 T\m

for h rnin. Tho tn,ir"jriiMl 'ml. wnu <.i>l I ()r.-l..r;(J MIKI .'JWJ.I I fraf.-L i on;; W<M '•

e lec t rophoresed .

1,0 rnl f r ac t i ons of t he above suspensions were laye;red

over a 3.5 ml uucruso g rad ien t in 4.5 mi poiyaliomer u lLraoent-

r i fuge tubes . These were centrifufied in a 6.0x4.5 ml av/inc out o r o t o r a t 31000 rpm for 1 h a t 20 C. 0 .5 ml f r ac t ions v/ere

c o l l e c t e d in eppendorf tubes v/ith the he lp of a syr inge .

P repara t ion of &ufirQ&£ gJCS<lieo_t

A so lu t i on of 20% (v//v) sucrose in s t e r i l e d i s t i l l e d water

was slowly frosen s o l i d and then thawed in cen t r i fuge tubes t o

form sucrose g rad ien t s (Baxter-Gabbard,1972). Those were afUtin O

frosen and s to red a t -20 C and f i n a l l y thawed slowly j u s t before

use.

Petermination of jriolecular weifjh.t

The plasmid siae estimates of th<j; isolated plasmid were

obtained by comparing their relative mobilities on agarose gel

with standard known molecular weight DNA fragments such as

lambda DNA, its EcoRI fragments and plasmid Rp4 isolated from

E-£iili EcRp4. The standard curve was dravm by plotting relative

55

mobilit.y vs log of the mol.wt. of standard markers. The rnol, v/t.

of the unknown plasmids was deterrained by plotting the relative

mobility on the standard curve (Ohinan, 1988) .

Agarose tfel filactCOPhoreRiR

The isolated plasmids were characteriBed by agaros-e f e]

electrophoresis according to the standard procedure of Maniatis

fit jal. . (1982). Abovit 30 to 50pl fractions along with glycerol

dye mixtures (50% glycerolj 0.25% bromophenol blue) were applied

on the slots. Horizontal slab ge;l electrophore.sis was carried

out using 0.8% agarose. The gel was pre-electrophoresed at 40mA

for 20 min and the normal run was performed at 20mA in

electrophoresis buffer (0.004M Tris-acetabe, 0.002M EDTA, pH

8.0) for 3 to 5 h. The DNA iMtridfi wore HLaintsd with el,hidium

bromide and fluorescent profile was photographed by UV

illumination through photodyne UV300 transillvaminator.

Plasmid tre^osfer

Transformation: The tremsfer of U }:>lasmids to recipient

E.fiaii AB1157 cells was studied through transformation following

the method of Lederberg and Cohen (1974). The overnight grov/n

AB1157 cells were made competent by suspending in 0.IM MgCl for

30 min on ice and then in 0.IM CaClg under similar conditions.

56

To 1.0 ml competent cells, 2.0ug of plasmid DMA (Rpl, Rp2, Rp3

and Rp4) was added separately and left on ice for 30 min. Heat

O shook was given to these cells at 42 C for 3 min. These cells

o were then allowed to grow in nutrient broth for 2 h at 37 C, The

cells were spread on the antibiotic supplemented plates after o

suitable dilution. The plates were incubattsd at 37 C for 16 h.

Con .luxation: Standard method of conjugation was followed

employing the donor, tost K.coli GW strains and l-he rcfripicnt

AB11&7 and C(300 strains {Ohman, i9}3B) . An overnight culture of

recipient and donor E.coli cells were added to fresh nutrient

O broth and incubated at 37 C for 30 min, 45 min and 60 min. For

control the parental culture alone v/as added to fresh nutrient

broth and incubated at 37 C. These cultures were serially

diluted and sprefid on plain and antibiotic supi»lemented i>lates.

The selection v/as done on the basis of appearance of

plasraidial markers alongwith the original markers of the parent

recipient strain. The C600 and ABllf'>7 are ia^ mutants and aiiio

lacking antibiotic re;sistance markei's, except the streptomycin

resistance marker which is cnrru.-d by l;h<,' A1J1157 strain. Ttir-

selection of transconjugants was therefore Vjased on appeare-nce

of the donor's antibiotic resistance plus non-pink colonies on

the MacConkey's agar plates.

57

The curing of R-plasmids v/as performed by the rnet-hod

doocribod by UirnLn (JUf)0) 'uid il/itdrri»Hi oL ui (IWMG). Ov.-rn i f hL

cultures of E-Coli were £»rowri in the nutrient broth in ihe

presence of different curing agents (ethidium bromide and

caffeine) at concentration« rariging from 0. 5 to 100 jag/ml . A

control tube lacking the curing agent was also included. All the

o tubes were incubated overnight (it 37 C. Contfjnt.i; of tPic l-t.ibc

were then plated, after making required dilutions, on antibiotic

supplemented and plain nutrient agar plates. The plate.'; v/ore

O incubated at 37 C overnight. The number of colonies appeared on

the plates were then recorded. The number of colonies on

antibiotic supplemented plate was subtracted from those on the

plain plate, i.e.

The number of cured cells =: No. of colonies on the plain

plate at a specific concentration - No. of colonies on the

antibiotic supplemented plate tit the same concentration.

To study the effect of time on the curing of plasmid

harbouring E.coli strains, 5jjg/ml ethidium bromide treated

cultures were incubated at different time intervals starting

from sero h to 10 h of incubation. A control without curing

agent was also run simultaneausly. Samples v/ere withdrawn at

various time intervals, suitably diluted and plated on plain

58

and antibiotic supplemented plates to assay the colony forming

ability. The colonies on the plates v?ere counted and percent

curing was calculated at each time interval.

Effect of P H on Eiasinid curing

To study the effect of lAl on curinf?, the pU of the

nutrient broth before autoclaviiifj was adjusted t<j pll 7.0 and

7.2. The media were selected for the curing of plasinids with

ethidium bromide. The procedure was same as described ah<jve.

The plasmidless or cured E.coli cell clones were selected

by the patch pattern method as described by Ohman (1988). These

cells were selected for their plasmid isolation, to confirm the

physical loss of the R-plasmids after treatment with ethidium

bromide.

RESULTS

59

Iir«fl Eesisilence rntt,<!rri

All i he 245 K. colj Qinxi^n v/al-'-r if;olnt,(vr; wcrr(; fouml [.<> he

multiply resistant to antibiobics. Out of 18 commonly used

antibiotics/drug tested for sensitivity, 19 different multiple

antibiotio/dru(? t-osi!;;l/anoe paLI.(,Tns wcro ubsorved. The numb<.T of

antibiotics or drugs against which resistance v/as found ranged

from 3 to 13. Thus out of 245 E-Cali isolates, 19 different

E-Cilli. strains were identified on the basis of antibiograms as

shown in Table IV. These iji.colj, isolates were grouped inbo 9

categories on the basis of number of antibiotics against v/hich

resistance was conferred. E.co1i GW13 among the 19 E.co1i GW

strains showed r<?f;iMl-?irico to 3 ant-ibi ot u:r! v/hilf; K.oolJ HWIG v/a;;

resistant against 13 different atjbibiobics (Table IV).

It is a wt ll oatftbliehGd fact that a single antibiotic

class contains many antibiotics somehow related in their

mechanism of action, e.g. terramycin, tetracycline,

ohlorotetracycline and oxytetracycline belong to same antibiotic

class. Thus another classification could be based upon the

nature of antibiobic/drug classes vis. Penicillins, sulpha,

tetracyclines, aminoglycosides, macrolides etc.,19 groups of

K. coli, GW strains were is<jlabod on this basis of resisbance

against different antibiotic classes.

60

Tab^e IV. Antibiotic resistance pattern of 19 E.coli GW strains isolated from Ganga Water.

S.No. E.coli strains

Resistance against antibiotics/ drugs

No. of an­tibiotics against which re­sistance was obser­ved

3

5

5

5

5

6

6

7

7

7

7

7

a

8

10

10

11

No. of strains resist­ant to difft. antibio­tic cla­sses

T

11

III

IV

V

VI

VII

VIII

IX

X

XI

XII

XTTT

XIV

XV

XVI

XVII

Total no. of antibi otics used

IQ

1.0

IH

18

18

18

18

18

18

18

18

18

18

18

IH

18

1.8

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

CWI3

GW4

GW5

GW7

GW15

GW6

GW12

GWl

GW2

GW8

GW9

GWll

GW3

GWIO

GWl 7

GW19

GWl 8

18.

19.

G'\^'l4

GWl 6

A, n, P

A, D, P, Pb, R,

A, n, N, P, I'b.

A, B, P, Pb, Sz.

A, Ct, P, S, T.

A, B, Fr, P, Pb, R.

A, D, Ct, P, R, T.

A, B, Cx, P, Pb, R, T.

A, B, Cx, Na, P, Pb, R.

A, B, Ct, P, R, SZ, T.

A, B, Fr, T, R, Sz, T.

A, B, C, Ct, E, R, T.

A, n, Cx, N, Na, P, Pb, R.

A, B, E, Fr, Na, P, R, Sz.

A, B, Ct, K, l''r, K, C, U, Sz, T.

A, B, C, Ct, E, Fr, K, P, R, T.

A, Ak, B, Ct, E, Fr, K, P, K, Sz, T.

A, Ak, C, Ct, E, Fr, K, P, R, S, Sz, T

A, Ak, B, C, Ct, E, Fr, K, P, R, S, Sz, T,

12

13

XVTTI

XIX

18

18

for abbreviations : See list of abbreviations

61

Table V shows the percent resistance against the indivi­

dual antibioti Of;. Out of the 18 antibacl-er i al ngentfj Lf;;i\,<:d,

gentamycin sensitivity was invariably observed in all the 245

isolates (i.e. percent resistance is 0) whereas, resistance to

ampioillin and penicillin was exhibited by all these bacterial

strains. 89 % isolates were resistant to bacitracin and 79 %

against rifaropicin.

Agarose ftei filfictrQEhoreais pf KlasmidfJ

All the 19 aforementioned E.coli GW isolates were found

"bo harbour plasrnids (data not shown). Fig. 1 shows the agarose

gel electrophoretic profiles of plasrnids isolated from the 9

distinct E.coli GW strains.Only a slight difference was observed

in their relative mobility.

Fig. 2 shows the agarose gol olecUrophoretic |<rofiles

of 4 plasrnids isolated by the method of Binrboim and Doly

(1979) followed by their jjurification on sucrose density

gradient. Three E.coJ-i GW strains seemed lo contain two plafifitids

each (lanes b, c and d) while one sLrtiin coifitained sin^de

plasmid (lane e). Lane a shows the linear and supercoiled forms

of plasmid pBR322 used as molecular sixc; marker. This

photograph, however, could not rule out the possibility of the

same plasmid in one lane but having different si>ecies i.e. CCC

and relaxed forms.

62

Table - V : P'ercent resistance; against individual antibiotics.

Total No. of E.ijoii N '- <^i' isolates resistant Percent rcjsistanc GW isolates to antibiotics/drugs

245 Ampicillin 245 100

VG

89

23

47

16

42

0

26

11

16

100

37

7y

16

42

Ampicillin

Aifiikucin

Bacitracin :

Chloramphenicol :

Chlorotetracycline :

Cloxicillin

Erythromicin

Furazolidone :

Gentamycin :

Kanamycin

Neomycin

Nalidaxic acid :

Penicillin :

Polymixin-B

Rifampicin

Streptomycin :

Sulfadiaaine :

Tetracycline

245

'jy

219

56

116

39

77

103

0

64

26

39

245

90

193

39

103

142

63

Fig. 1. Agarose gel elect-rophoreGis patterns of plas-

mid DHA isolated from nine E-co2.i GW strains

(lanes a to i).

G b c d e f g h i

i _, - - 1

\

m^

^

1

6/t

J**

Fig. 2. Agarose gel electropVioresis of plasraid

DNA ifjolated from four E. c^olj GW s"tr-ains

p u r i f i e d on sucrose dens i ty g rad ien t .

lane a : marker plasmid i>\Ml322.

lanes h,Q.,d & e • t e s t plasmids.

a b c d e

65

Trans forrnfltiou

To show l,hab the druf^ rosistonce in E-.QQli GW strfiins v/ar;

plasmid mediated character, the transformation of E.qoli AB1157

with R-plasmids isolated from four GW J?. fictLi test strains was

performed. Thrs transformation frequency of ABli57 with R--piasmid

Rpl, Rp2, Rp3 and Rp4 is shown in Table VI. The transformation

frequency of recipient cells with R-plasmid Rpl and Rp2 was

5.5x10 and 8.5x10 .On the other hand,the plasmids Rp3 and RF"1 —<r -s-

displayed the transformation frequency of 1.4x10 and i..'Sxl0

respectively.

Plasmid Curing

All the four E.fioli strains i.e. E.coli GWl, GW2, GW3 and

GW4 lost their R-plasmids on treatment with ethidium bromide.

Fig. 3 shows a plot of percent curinfc vs ethiduim bromide

concentration. Curing was observed at the minimum concentration

of 0.5 |jfj/ml and intrreafjod v/j tli the i nerfjnjt i n^ c;ono( ritf M I. i on of"

ethidium bromide. All the test E.coli GW strains displayed 50 %

curing on treatment with 0..'" ;,i(T (•tliidjum bromide per inl oC

culture on 16 h of treatment. A maximum curing of 70 % was

observed with the maximum dose (100 jjg/ml) of ethidium bromide

(Fig.3).

66

Table - VI Trann forrrini/ion frr^nunnny of E . r rol i ABllJi? wi th [•''J'if" i ' l-Hpl, Hp2, Ki>3 and ttp4 i soJaLed rroin four i'i. coJ i (.W s t r a i n s .

S.No. Plaomid Species

No. of Total No. TransformaLion Resistance marker i

1.

2 .

3 .

4 .

Rpl

Rp2

Rp3

Rp4

Cells/ ml Seeded

2x10

2x10 •7

2x10

2x10

of anti­biotic-resistant transfor-mants per plate (0.1 ml)

1.10x10

1.70x10

86x li

•frequency

.5.5x10

8..5x10

1.4x10

p r e s e n t in t r a n s -forrnants i . e . a f t e r trarnsforma-t i o n

3.03x10

A, T, Ct, C, K

A, Ak, CI, C, K, T

A, Ak, Ct, K, T

A, Ct, K, T

67

F i g . 3 . Plasmicl eu r ing" and s u r v i v a l of p lasmid

h a r b o u r i n g E. c o l i <3W s t r a i n s by t reabmet i t w i th

e th id iurn bromide. .

Symbols : c u r i n g { s o l i d l i n e ) ; s u r v i n a i {broken l i n e ) ;

E .co . l i GW1-{A ) - , .E.fioli GW2-{A ) - . E.GoLi GW3

- ( • ) - , K . c o l i GW4-(0 ) -.

100

o m

cc <

LU <

50

25

10 40 60 80

ETHIDIUM BROMIDE ( j j g / m l )

100

68

Curing phenomenon wafj also observed vfith caffeino. 'A0 %

curing was observed in E.ooli GW3 at the concentration of 0.5

jjg/rnl on 16 h of incubation while a high percentage of curing

(52%) was observed with ethidiurn bromide treatment of E. coli GV 3

strain {Fig. 4).

Lethal effects of cjjKing a^^entu

The curing agents ethidiurn bromide and caffeine were also

found to be lethal at the concentrations used {Eigs. 3 and 4).

The survival was decreased by 10% in all the four E-iioli GW

strains on 0. 5 jAg/xal ethidiurn bromide treatment and v/as only 40%

at the concentration of 100 jw^/ml (Fig. 3). ('affoJrif.- aliio

reduced the survival of E.coli GW3 strain upto 45% at the cone.

of 100 jjg/ml of culture after 16 li of i n<;ubatiori.

Fig. 5 shows the effect of two pH values (7.0 and 7.4)

on the plasmid curing of E.coli GW3 strain with ethidiurn

bromide. A protective effect on plasmid curing was ob.served with

the acidification of the medium vis. at pEl 7.4, the curing was

52% while at 7.0 it was reduf;erl to 2.3%.

Incubation time with ethidiurn bromide also affected the

curing efficiency. Plasmidless clones of E.coli GW3 started

appearing after 2 h and 60 % curing was observed after 8 Fi of

treatment with 5 y\g/xal ethidium bromide at pH 7.4 (Fig. 6).

f)9

Fig. 4. Compar-at.ive pat'berhs of curing and survival of

E. coli GW3 strain with etkiidiurn bromide and

caffeine.

Symbols : curing (solid line); survival (broken line),

Ethidiurn bromide -(# )- and caffeine -( O )""•

20 40 60 80 100

CURING AGENT {ug/ml)

/()

Fig. 5. Effect of pH on plasmid curing of E.coli GW3

straiti with ebhidium bromide at pH 7.0 - ( • ) - ; e

pH 7.4 - (O )-.

100-

o z

o

UJ <^

UJ

20 40 ETHDIUM BROMIDE (jjg)

60

71

Fig. 6. Effect of treatment (5 ug/rnl for varous time

intervals) with ethidium bromide on plasmid

curing in E.coll GV 3 strain. Treated - ( O ) - .

control -{ # ) -

o 2

s o

LU O Q: LU Q.

60-

30

•

' «

o /

A V

/ o

m w J

^ •• V —

/~i • v *

A —w •

2 6 TIME (h)

10

72

To dernonstrat'C Lhc; ftiyrrji ca l JOUH of iilnyrnidy from cfrJlL;

during the course of EtBr t rea tment the Birnboim and Doly (1979)

method of plasmid i s o l a t i o n v/ao ca r r i ed out before and aft'.;r

treatraent with t he cur ing agent . Fig.7 shows the agarose gel

electrophoresis of bht) i s o l a t e d pltismids. I.aries a and b contnirjcl

"the plasrnid i s o l a t e d from GW3 c e l l s before the t rea tment and

lanes c and d did not show any plasrnid band v/hich containd the

S2Lmples t r e a t e d with ethidium bromide.

Con .juration

Table VII shows the percent conjugation of four E.coli

GW strains. E. coli GWl demonstrated 64 percent conjugation Vi'hen

mated for 45 min v/ith recipient E. coli AB1157 strain. E. coli

QW3 and GW4 showed 47 and 48 percent conjugation respectively,

but in case of E.iS.oli GV 2 strain, the conjugation was found to

be only 40 percent.

Table VIII shown tht; <;ff<;<.rt of pH on th<; tron.iugntiori of

E-iSflli GW strains. The higher percentage of transconjugants

were found from pll 7.4 to i>II Q.[\, but the conjugation decreased

below pH 7.0 and above pH 9.0 of the medium.

73

Fig . 7. Agfiro.se gfj] f;lectroph()rf;.s i f; of l.he co l J ly«a--

t e s (mini p r e p s ) d e r i v e d from E . c o l i GW3 p r i o r

arid ari,f;r treatrnenl. v/il,h Kl.lVr.

l a n e s a Sc b : b e f o r e t r e a t m e n t of t h e c e l l

wi th EtBr.

l a n e s c & d : a f t e r t r e a t m e n t .

74

Table- VII Plasmid transfer by conjugation from test E._coli. GW strains to recipient .K.<;?pJi.

S.No.

1.

2.

3.

4.

Donor strains E.coli isola­ted from water

GWl

GW2

GW3

GW4

Recii'i oiit. E.coli strains

C600

C600

AB1157

AB1157

Fiicvil M\[- \ o n

time of mating in (min)

30

45

30

45

30

45

30

45

Col on lot; on Plain nutrient agar pla-U;r: (CbV)

431

482

200

241

121

154

150

197

(Joloniof! on selec— tivC/v supp­lemented pj aLcfi (Transof.Mi jugants )

91

307

57

97

37

72

48

94

P<M'<:(:lil conjug­ation

21

64

29

40

31

47

32

48

75

Table - VIII: Effo<::l. of fU cm IIK- corj juf al-i on in l>\. ci,)l'i C\'V.) Hn<\ E-.Qoii ABli57 recipient strain.

S.Mo. pll Colonies on Plain Coloniers on antibifr Ffjr(M'nl-Platea tic Supp. Plates (CFU) Conjugation

Transconjugants

1.

2.

3.

4.

5.

6.

7.

e.. 9.

6.0

6.5

7.0

7.4

8.0

8.5

9.0

9.5

10.0

142

150

163

167

170

161

142

132

10.^j

29

63

73

82

80

77

59

44

21

42

45

49

47

48

42

33

19

76

Molecular weight of Rijy.Lasif>iiis

Fig.8 shovs the relative mobility of R-plasmids on atfarose

gel, isolated from E-Qoii GWl, GW2, GW3 and GW4 strains in lanes

d, e, f and H r ofir>ecl,i vol.v. hnn*; a carrifd EcoR i di (((•!; I

fragments of lambda DNA, lanes h and c carried lambda DNA and

lane f containy plasmid Ri»4 isoJated from E..Qfjili EcRp4, us<;d ar;

molecular siae markers.

Fig.9 gives tlie molecular sise of six different plasmid

species isolated from four E. coli GW strains that ranged from

23 to 40 Mdals. The standard curve was plotted using log mole­

cular weight vs relative mobility of standard markers (lambda

DNA, its EcoRI fragment and plasmid EcRp4).

Fig.10 shows the relative mobility of multiple plasmids

isolated froiri E. coJ i GWii sLraiii. The jiLrain harbouj-f.jd four

plasmids (lane c), having molecular sise less than that of

lambda DNA. liinwn a, h <»nd <-• '.rontaln .s-tandnrd mo,l(;cular si wo UNA.

The molecular sise of the four plasmids ranged from 3.2 to

9.2 Mdals. Their molecular siae was determined by plotting

the relative mobilities on the standard curve (Fig.11).

77

Fig. 8. Agarose tiol elecbrophoreHiH of R-plasrnids

isolat-ed from four E. coli GW strains,

lane n : KcoUl dJ^fited larnbda DNA.

lanes b & c : intact lambda DWA.

lan« d : E.coli, tiWl mini fref.

lane e : E.coli GW2 mini prep,

lane f : K.coli GW3 mini prep,

lane g : E.coli GW4 mini prep,

lane h : R-plasmid isolated from marker

Eottp4.

* arrow indicates the position of the welLs

{starting point).

a b c

m^ 7AD f/fc

^.•fe\

78

F i g . 9 . Mol. s i s e v s m o b i l i t y p l o t o f t h e t e s t and

s t a n d a r d DWA.

Syrnbolt^ : K f l n i a n i d c ir,alni,<:<} f r ou iCWl . mr/., CWa nnd

GW4 E. c o l l s t r e y f n s - ( © ) - a p p r o x s i a e 40 Mdal .

P l a s r n i d i s o l | R , e d f rom E - c o l i GW2-( O ) - a p p r o x

l i s e 2 ' •7 ^ 1

Plasmid isolated fro E.colj GWl-{©)- approx

si;i!fe 23 Mdal.

standard DNA markers - { • )-.

Ui N Co

< - J

O LU

10 20 30 RELATIVE MOBILITY (mm)

79

F'ig. 10 . Affnc<)(Ui f^r;}. o l e c L r o p h o r c ; ; Iff o f t h o rnul b li:»i<;

p l a s r n i d s p e c i e s i s o l a t e d from E. c o l i GVJ5

nJ.rn i n .

l a n e a : p l a s m i d pSK 2 7 5 { 3 k b ) .

l a n e b : p l a s m i d s pSK 275 and pSK 279( 3kb

and 6 k b ) .

l a n e c : EcoRI d i g e s t of lamVjda DWA.

l a n e d : p - l asmids i.s-r»labed from }']..QQXi GWJ

s t r a i n .

* a r r o w inr l ica l .e . s \,]\n j<of;il-lori fjf t;he W O I I K

{ s t a r t i n g p o i n t ) .

a

80

l ' ' i g . l l . Mf>l. '.iiy.i.: vs- m o b i l i t y i ' loL uf. Llie Lo^t (muiL l

p i e s p e c i e s of t h e R - p l a s m i d s i s o l a t d from

K-C'olJ GV Jj) and s t a n d a r d DWAs.

syrnboir;: GH5[>1 ( Q ) iippKyy.. ijiwo 9 . 1 2 MdaL.

GW5p2 - ( O ) - a p p r o x . s i z e {j.20 Mdal .

<lW5p3 ( ® ) nf'pr<i>:. r; i :-;o ••J. K«0 M<l/tl .

GW5p4 - ( ® ) - a p p r o x . s i s e 3 . 2 0 Mdal .

f i t r indard DMA rnarkorfJ •{ • ) .

10 20 30

RELATIVE MOBILITY (mm)

DISCUSSION

81

AJriiotiL ail J'!. <;oJ i i !:<»] nl,f;;i irom Gatit a river v/ere i\>und

to be multiply resistant to antimicrobial agents. The organisms

were mostly resistant to ampicillin, amikacin, bacitracin,

chloramphenicol, chlorotetracycline, erythromycin, furaaolidone,

kanarnycin, rjoomycin, penicillin, volymyyiin h, rifampicin,

streptomycin, sulfadiasine and tetracycline. Conflicting data

have been rei»urtod from various paits of the world as far us the

percentage of the resistance character is concerned (Feary et

jal.,]972; Miemj etal.,U>Oy; Al flhaxali etol.,i988). All l.hr-

Ganga water E-col.i isolates were found to be resistant to

ampicillin (TabJr- V). AJfno:.il- siiriiijir r/.-wulLs v/«.;re obtained by

Al-Ghaahli and his coworkers in the E.co.li. isolated from

Al-khair river (Ai Gha'/;a]i ct al.,J980), but the incidtmce of

ampicillin resistant fecal coliforms isolated from Red river in

Canada was significantly lov/ (Bell et al;1980). Niemi and his

coworkers found a significant level of ampicillin resistant

bacteria in almost all samples of untreated sewage{Niemi et

fil.,1983). A high level of resistance against bacitracin and

rifampicin was ai.':!o observed in our K.t-oti isolates {Table V).

The information regarding these resistance markers in the

pollution indicating E./..-oli is meagre in the literature.

Contrary to the ampicillin, bacitracin and rifampicin the

incjjdence <:)f (uni n(<({i .v<<->s ide i <"!'.i :;t,avi<;f r V;M;; r<rlatively lev/. Th<;:;e

results were in t;une v/ii:.h the aminoglycoside resistance patterri

disj>layed hy Lha eoiifirm bael.eria fr<>m hoiipital and the '.;i ty

82

aG\fni^r; (Grabov; nnd I'r o:;r.-:!kf;y, iU73) . M:; woil /.is Cruifi th'; ii<:<i

river in Canada {Bell et al.,1980). Moreover, our results on the

100 % gentainyoin nr-nii i bi vity alno fjupi>i>rt bhoHe of Boll _et al

(1980).

The high level of resistance as conferred by these E.colji

isolabes against arnpicillin, botracycline arid some other

antibiotics (Table V) se;em to reflect their widespread

chemobherapeubic uso in bhc viciniby of the tesb Gangcbio

stretch. The indiscriminate use is one of the major factors that

resulted in the emergence of anbibiobic resistant bacteria

(Smith, 1970; Farid et al.,1975; Levy et a.l.,l983; Al-Doori et

&l.,1986).

Amyes and his cowoikers demonstrated that arnpicillin,

which is widely used in many clinical settings may select

bacteria harboviring plasmids encoding resistance to arnpicillin

and other related antibiotics (Amyes et a_l.,198l; Amyes, 1983).

The drainage of chemically polluted water into the river Ganga

may also contribute in providing a favourable selection pressure

that is responsible for the selection of the antibiotic

resistant bacteria. One of the possible sourceife of chemicals,

contaminating the rivf.-r, is drainage <>.{' effluents from vixti oua

chemical factories oj.g. the effluerjt from Glindia India Ltd. (a

uhemjt;al ra»;toi y niariu I/H; tui: i ii/{ (|ii.i(:; MIKI many t)l,li'.;r ch<!m J c;a J j;) ,

83

which in iti t[\<: vif;lnll;y of l,h<; fj. f!;l, tiattipi iiif j Latioti of Lho

river Ganga arjd scumr, bo exerl: l,he Kelocl/ion pleasure for the

observed resistatice iti tlie test E.coli strains.

Ttie E.PA'li ir;olMt«!cl from Cinnfc a rivisr- hMv<; Vx.-on <.;! aiii; i i" i <;d

into 19 groups on the basis of their antibiograms obtained

against different Miil-Ibiot j t; cJauije;; {TnbJe iV). Among the

various methods of taxonornio studies, the bacteria have been

oominonly eJ auu j i'i <(l r>ii I,he \>i\',;\;; of an t i t> ii i/Oamu aJ<.)ngwJth th<;ij-

plasrnid profiles (Gillespie Qt f%l,1990). Since different workers

have used different antibiotic classes for sensitivity testing

of the bacterial strains isolated from river sources, the

comparison of the results given in table IV with i>revious work

is not possible. The E.colj, isolated from water samples were

multiply resistant to antibacterial agents, ranging from three

to thirteen antibiotics. These results Vifere almost similar to

the results of Bell and his eoworker.y. These workers have

isolated fecal coliforms from Red river in Canada that conferred

resistance to tv/f;lvt; antibitjtif;;; (iW.;!! et ai. , 1980) . In oux-

study, resistance against seven antibiotics (heptable

resistance) was found l-o be dominant amorjg multiply resistant

isolates. Similar heptable resistance patterns v/ere also shov/n

by the clinical isolates of bacteria like Pseudoirionas aeru/lilipjsa

and E.coli (Al-Doori ^t al.,1986).

8A

The 19 rtiulLipiy ariLlbiol.ic ftjsi fjLant E.coXi Btrainy v/cre

divided into 9 classes on the basis of their resistance against

different number of antimicrobial agents {Table IV). All the 9

groups of E.coli isolated from Ganga river were found to harbour

R-plasraids (Fia ! ) • These results v/ere almost similar.to that of

the Vakulenko and his coworkers, who found that, 30 out of 32

antibiotic resisl,ant E..i;(:!, strains v/ere carryirif]J pladrnids

(Vakulenko gt a l. , 1980).

As far as the number of plasmids in the plasmid harbourinf*

strains is con<;<;rn(-d, orif? ;t aLrl thr.- OWb wa:; carrying multiple

species of plasmids (Fig 10) and most of the E.coli strains

seemed to carry only one R-plasmid. These results are slightly

at variance with the observations made by Tantulivanchi and liis

coworkers who reported a very high inci<lanoe of multiple

plasmids in the clinical E-CCJI i s-l,rajns (Tantul i vanchi /.;t

ai->1981)- This diffrence is v/ell justified owing to the

different origins of isolates. Among the 19 E.coli strain;;

(Table IV), five were selected on the basis of their plasmid

profiles (Fig 8 &•, 10).

To provide evidence if the resistance markers vMtre

actually on thr- f>la;;tnid DNA, v/c carried oi.\t the tratis format ion

experiments employing plasmids isolated from test E-Qclj

strains. The expression of resistance markers in all

85

•trnrujforrrinriLiJ •,:nifi*nrA-';<\ l-hni l.li'r mil. ihi nl, ic t <•'. :]•,;[,i\r\<:n V/M!I

plasmid mediated. The transforrnat-ion frequency obtained v ith

both the recipient- strtiiny i.e. K.coii C600 and AB115V (Tablt-

VI > was cornparaVjle to that obtained by Bopp et al (1983) in case

of E.coli recipients transformed with P. putida derived Rp4

plasmid. All the test E.coli strains transferred four or more

resistance markers out of 7 to 13 markers (Table VI).

Tantulivanchi gt a.X (1981) had shov^n thai: 9 out of 15 resistance

markers were transferable in JE.(;.<;?li.

The final proof in favour of Lhc presence of R-plasmids

in the test E. coli strains was provided through the curing

experiments. A significant fraction of test strains was found to

lose the resistance markers in presence of curing agents

ethidium bromide and caffeine (Fig. 3 ^. 4). Our results of

curing with ethidium Vjromide v/ere even better than 1-hose

obtained by El-Sycd and Coworkers (1908) in case of clinical

isolates of E-<?oli-

The loss of a :;f'(?(ri f i (; trait i:; not an evidoncre that tVif.;

plasmid under consideration has been <:;ured since certain

"treatments may lead to selection for deletion derivatives. In

general, physical loss of a plasmid is best demonstrated by its

absence (Caro gt al., 1984) involving the analysis of partially

purified plasmid DHA «jti agarose gels (Birnboim and Doly, 1979 ;

86

Klein eic Jijl. , 1900). \'h; hnvf.- cotif i rrnocl I-IK; Jof;fJ of plaarriid nft,er

"the curing experiment through agarose gel electrophoresis (fig-

7) from E.fiQlJ, ^iWS bacterial strains, after treatrrient v/i th

ethidium bromide and caffeine.

The concentration of curing agents and pH of the ifiediv rn

have been reported to influence the rate of plasrnid loss

(Hirota, 1960). A prot-ective effect, on piasmid curing v/as

observed with the acidification of the inediurn. Higher curing

rate was observed al^ 7.4 pH tlian at 7.0 pH (Fig. 5) as well as

during the log phase of growth of the E.coli strain (Fig. 6 ) .

These results support the findings of Hirota (1960).

The test E.GSili. strains were able to transfer some of

their resistance markers to standard K-'-oli recsii'lcnt strains by

conjugation (Table VII). Moreover, the percent conjugation v/as

much higher than that observed by Bell and Cov/orkers (1980) in

fecal c o n f o r m s isolated from Red river in Canada, but coincide

with the results obtained by Genthner and his Coworkers in

aquatic gram negative bacteria (Genthner et al.. , 1988). This

transfer of muitii-le rc;sistanc'; markers strongly suggest that

this property of antibiotic resistance is plasmid mediated. We

had performed our f.:on juf: fit j on experiments in liquid v/hich might

not be a better environment as <;omparetl to solid media. Since

Genthner ot al (1988) demonstrated that the conjugation is much

87

efficient on solid moflia rather than in ii'iuid in cane of

aquatic grarn negative organisms.

Regarding the possibility of the transfer of resistance

markers through conjugation in the natural aquatic •habitat,

Stotzky and Krasovsky (1981) provided emperical evidence in

favour of this phenomenon. The higher percentage of resistance

•transfer in liquid under Ifiboratory conditions is of gre;at

concern, because aquatic environment of river could also provide

almost similar , conditions for the resistance transfer by

conjugation in l .-CQll- Morc(>vr,T, a high frequency of* O

transconjugants were observed around pH 8.5 at 37 C under

laboratory conditions (Table VIII). Thus the temperature of

river water (20-30 C) and its pH (8.1 - 8.7) is favourable for

the growth of E.coli 'ind would also fac-Llitate the geti'?tif;

transfer by means of conjugation in riverine system.

The R-plasmids isolated from four tefj). K. Qoli si,rains

(GWl, GW2, GW3 and GW4) ranged from 23 to 40 Mdal. (Fig. 8). The

R-plasmids of this s Lwe were als(.' Isolated by Martinez et al

(1987). The plasmid profiles have been used in this study for

typing E.coli isolated from river Ganga. The strains harbouring

plasmids coding for different i>henotypic traits can be easily

typed. Such typing would also be easy, rapid and inexpensive

(Gillespie :et :al. , 1990).

88

Our studies provide suppor-bive evidence in favour of

feasibility of the ciaHsirioabioti based on anbibiograms and

plasrnid profile in riverine system. Present study also has a

clinioal dimension, iti view of bho incroasLtu^ incLdenoo <j\]

multiple drug resistance in pathogens which is conceivable due

to the observed transmissible nature of the R-plasmids. These

findings call for particular concern towards the spread of

multiple drug resistance in the system to v/hioh the man in

general and an Indian in particular, is bound to essentially

expose in his daily life.

BIBLIOGRAPHY

89

Achtman, M- and Pluschke, G- (1986). Annu. Rev. Mi.crobiol. 40.1 185.

Akiba, T .(19^9). Vr<.><:. JJ.Lh Gr.-n. Mcol .lni-?in Mod. AftKoo. , b:?.d9.

Akiba, T and Yakota, T. (1962). Med. Biol. 63:155.

Alcaifio, E. (1987). In FundarnenLaiy of Microbiology, 2nd. Ed. (Eds.) Alcarno. E PP 75.

Al-Doori, Z., E-vaddouri, M. , Clor, A and Naorn, I (1986). J.Biol. Sci. Res. 17:195.

Al--Ghazali, M.R., Jasiarawi, S.F and AJ doori, Z.A. (1988). WaL. Res. a2-641.

Allen, N.E. (1977). Antimicrob. Af^ents Chemother. 11:661.

American Pi.il)lif; il<'/»llh Anrior-i al. MMI . (IVHS). ',>l,nri<lar(l utr<\Au.«\r. I"<«r the cxarninaLJ on of Wal^-j- and Wa;rtf.-waL*.T, 16Lh. V.d. American Public Health Association, Washington D.C.

Amyes, S.G.B. and Smith, J.T. (1974). Biochem. Biophys. Res. Commun. M:412. .

Aroyes, S.G.B., McMillan, C.J and Drysdale, C.J. (1981). Transferable trimethoprim resistance amongst hospitr^l isolates. In Grassi, G.G and SaliatVi, 1.. 1>. (Eds.) Hew Trends* in Antibiotics: Research and Therapy. Amesterd-im Elsevier/North Holland Biomedica] Press pp325.

Amyes, S.G.B. (1983). Health Bull. 4J.: 100.

Arftyes, S.G.B. (1989). J.Med. Microbiol. 28:73.

Arber, W., Sengestan, C., Caspers, P and Dalrymple, R. (1985). Evelutionarv relevance of genetic rearrangements involving plasmids. p21-31. In Helinsiki, D.R ., Cohen, S.N., Clemell, D.B., Jakson, D.A and Hollander, A (Eds.) plasmids in bacteria . Plenum publishing Corp. , Nevi' York.

Armstrong, J.I... ^ l ig<-nif>, D. r>. , C/i 1 f iTficr i;;. .1. .1 fmrl r>cidlfr. (V,..l (1981). Appl. Knviron. Microbiol. •}4::H)M.

Baine, W.B., Farmer, J.J., Gangarosa, E.J., Herman, G.T., thornberry, C and Rice. P. A. (1977). J. Infect. Di;,-. 1M:649.

Bannister, D and Glover, S.W. (1968). Biochem. Biophys. Res. Commun. 3i3:73.':-.

90

Baron, L. S ami FaJkov^ S. ( 1 9 6 1 ) . Reo. Genert. Soc . Am. 3 0 : 5 9 .

H a r t l e y , C.U nnd S l^metw. I..W. ( 1 9 6 9 ) . Am. .J. Pub. flr^nll.h. 4 9 : 1 3 7 9 .

Baur, A.W., Kirby, W.M.M., Sherris, J.C and turk, M. (1966). Am. J. Clinical Pathology. 45:493.

Baxter-Gabbard, K.L. (1972). FEES Lett. 20:117.

Bell, J.B., Macrae, W.P, and lilliobb, G.E. ( 19i30) . Appl. Environ. Microbiol. 40:486.

Birrnboirn, H.C and Doly, J. (1979). Nuclr-ic AoidK ROG . .7:1513.

Bollen, A., Davics, J., Oaaki, M and Miaushima, M. (1969). Science. 165:85.

Bopp, H.L ., Chakrabarty, A. M and Ehrlich.El.L. (1983). Appl. Environ. M icrob io1. 155:1105.

Bouanchaud, D.El., Scavipisii , M. R and Chabbert, Y.A. (1969). J, Gen. Microb in I. 54:417.

Bradley, D.E. (1980). Plasmid. 4:155.

Brenner, R.J and Ealkow, S. (1971). adv. Gonnb. 16:81.

Broda, P. (1979). Plasmids, W.H. Freeman, San Francisco.

Brooks-Low, K and Porter, D.D (1978). Annu. Rev. Geneb. 12:249.

Bukhari, A.I,. Shapiro, J.A and Adhya, S.L. (1977). DNA Insertion Element.s, Plasmids and Epitomes. Cold Sprin/f Harbour, N.Y.

Burnie, J. P., Ruth, CM., VJoei . 1. and Murdoch, D. (1989). J. Med. Microbiol. 29:255.

Capuccino, J.G and Sherman, H. (1987). In Microbiology a Labortary Manual. pp31.

Caro, L. , Chiurfliv/nrd, G /in<l rbmid Ir-r, M (1904). In MrdJiod;; in Microbiology (Eds.) Bennet, P.M and Grinsted, J. Academic Press, pp. 118.

Cees, W. , liorjch, V., .lohanncf;, 1'"., Ma(rlai(,-n, 1). M and Joh tniK-f , G.O. (1982). EEMS Microbiol. Lcbt 14:171.

Chakrabarty, A.M (1972). J. Bac ber i o1. 112:815.

91

Clark, J. A and pM({eJ , J.E. (1977). Can. J. Microbiol. .4:465.

C1 owo 11, D.I) (1 y !J 1 ) . M i <j t<A> i. o I. R. .• v . 4b : 405:).

Clowes, R. (1972) . Bacteriol. Rev. M-361.

Cohen, S.N., Chan^?, A.C.YandHsu, L. (1972). Proc. Watl. Acad. Sol. (USA). 69:210.

Cohen, S.N. (1976). Nature (London). 263:731.

Colnian, D.C., Pcincroy, 11., l<;;rl/rid(*(r, J.K., Kcanc, C. T. , Cafferkey, M.T., Hono, R and Foster, T.J. (1985). J. Med. Microbiol. 20:157.

Cooke, M.D. (1970). Antimicrob. Aticnlr Chemother. 9:879.

Cooke, M.D. (1976). W.Z.J. Mar. KreshwaLcr Rc;j. 10:391.

Couturier, M., Bex, F. , Bergquist, R.L and Maas, W.K. (1968). Microbiol. Revs.

Co7.zeralli, N.R., Kelly, R.B and Kornberg, A. (1968). Proc. Natl. Acad. Sci. (USA). 69:2110.

Craun, G.F. (1972).. J. V^att. Pollut. Control. Fed. 44:1175.

Dabbes,E,R and Sole, G.I. (1988). Mol. Gen. Genet. 211:148.

Datta, N. (1975). Ei'idetnlolof y and Claf;f;i :f icaUion of iilasmidr; ,p. 9-15. In D. Schlessinger (Ed.) Microbiology-1974. American Society for Microbiology, Washington, D.C.

Datta, N. (1979). Plasmid Classification: Incompatibility grouping, i>. 3-12. In , K. W. Timmiis and A. Puhler (Elds.), Plasmids of medical, environmental and commercial I irriportance. Elsevier/ North Holland Biomedical Press, Amsterdam.

Datta, N and Hedges, R.W. (1972). Ann. Inst. Pasteur (paris). 123:849.

Davis, B.D., Dulbecco, R., Eisen, H.N and Ginsberg, H.S. (1980). In Microbiology, 3rd Ed. Davie et al . , (Eds. ) pp 656.

Davis, B.D and Maai!, W.K. (1952). I'roc. Na(,l. Acad. ;k;i. (llliA). 2fi:775.

Daviee,J.E and Rownd, R. (1972). Sciencf •. 1 76 : 758 .

92

Davis, J and Smith, D.I. (1978). Annu. Rev. Microbiol. 32:469.

DuokwurLh. IKII.. ('.I<iin, J nnd Mc-Cnrc,. tr ,r|M hs D.-' (li'lt]), Microbiol. Rev. 45:52.

Ecielstein, P.H. (1982). J. Clin. Microbiol. 1.6:697.

Edwards, P.R and Ewing, W.H. (1972). Identification of Enterobacteriaceae. 3rd Ed. Burgess Publishng Co. , Men n e ap o1i f5, Minn.

Eller, C and Edwards, F.F (J968). Af-pl. Microbiol. 16:896

Elkhouly, A. E an<l Wicdernatin, B. (1981). J. Pharrn. Pbarriia<;t,<l.

aa:36 El-Syed, M.A., Ashour, M.S.E and Sassyoni, M.I. (1988). Mansoura

J. Pharrn. Gci. 4-57.

Elwell, L.P., CraaiT, J.l)., :.>cibcrL, 1) and Kalkow, S. (W/b). Infection and Iniinunity. 12.: 404.

Elwell, L.P and Shiply, P.l,. (1980). Annu. Rev. Microbiol. M:465

Ewing, W.H. (1962). J.Infect. Dis. 110:114.

Falkow, S. (1975). Infectious multiple drug resistance. Pion. Ltd., London.

Farid, I., Miner, W.F., Hassan, A and Tarobolsi, B. (1975). New. Engl. J. Med. 292:216.

Feary, T.W. , St-urtcvanb, Jr. A. B and I.ankfor, J. (1972). Arch. Bnv. Health. 25:215

Flakes, J.G., Cox, E.C., Witting, M.L and White, J.R. (1962). Biochem. Buji'liy;!. Res. CfMnmun. 7.: 390.

Fock-Reudiger, R.E and Rainer, L. (1982). Curr. Chemother. Immunother. In Proc. Int. Congr. Chemother. 12th. 1981. 1:215

Franklin, T.J. (1967). Biochem. J. 105:371.

Frost, J. A., Rowe. B. , Ward, L. R and Tlierlfall, E.J. (1982). J. Hyg. M:193.

93

Qaltori, M.M., MerifV!;, R.W. nrifl f)t.f;fl<-. .l.il. (lOf.n) Ann N Y Acad. Sci. 70:427.

Gangarosa, E.J., Benrif-I,, J.V., V^yaLt, C. , Pic-rco, P.B;., OJartr;, J., Hardnese, P.M., Vaaques, V and Bessudo, P. (1972). J. Infect. Dis. i26:21.! i.

Gaston, M.A., Duff, P.S., Naidoo, J., Ellis, K. , Roberts, J.I.S. and Richardson, J.F. (1988). J. Med. Microbiol. 26:189.

Geldriech, E.E. (1976). CMC Cril,i<u,l Rev. Environ. Cont/rol. £-.349.

Geldreich, E.E. and Kenner, B.A. (1969). J. Water. Pollut. Control. F«'ed. 41:R33G.

Genthner, F.J., Chetteirjee, P., Barkey, T. and Borquin, A.W. (1988). Appl. Environ. Microbiol. . 4:11- .

Gillespie, M.T., l.yon, B.R. and Skurray, R. A. (1990). J. Med. Microbiol. 31:57.

Gorbach, S.L., Banv/f.-ll, J. G. , Chalterjee, B.D., Jacobs, B. and Sacks, R.B. (1971). J. Clin. Invest. 50-881.

Gordon. J.E. (1071). Arm. My. Ae/irl. T.e i . 17C>-. 9.

Gorden, R.W. and Flierrnans, C.B. (1978). Wat. Res. 1_2:343.

Goyal, S.M., Gerba, C.P. and Melriir;k. J. (1979). Wat. Res. ia-349.

Grabow, W.O.K. and Prosesky, O.W. (1973). Antimicrob. Agents. Chemother. 3:175.

Greenberg, A. E. and Ongerth, H.J. (1966). J. Arner. Water Works Ass. M-1145.

Gupta, S.P.D. (1984). The GanjVi basin Part, 11 prei»arecl by I;}K; Centre for Study of man and environment, Central Board for the Prevention and Control of v/ater Pollution.

Hahn, F.E. and Ciak. .J. (1971). Ann. H.Y. Aead. Soi. J82-29»..

Hardman, D.J., Gov/land, P.C. and Slater, H.J. (1986). Appl. Environ. Microbiol. 51:44.

Hardy, K. and Haefeli, C. (1982). J. Bacteriol. 152:524.

94

Haohirnoto, 11. aii«A Mi buuhaslii, S. (1971). Genebic Propertioc oL' R-factors, P. 39-98. In S. Mitsuhashi (ed. ) Transferal-le drug res i r!l-an<-') i'fM:i:<)r R. IJn i vr;r!; i by of T<,ikyo Pr ffif;, Tokyo.

Helinsiki, D.R. (1973). Annu. Rev. Microbiol. 27:437.

Hirota, Y. (1956). Nature. (London). 178:92.

Hirota, Y. (1960). Proc. Natl. Acad. Sci. (USA). 46/. 57.

Hirota, Y. and lijitna, T. (1957). Nature (London). 180:655.

Hirsh, D.C., Ilonricn, L. M. Drorrnan, L.C., Sniir>er, K.P., Carpenter, T.E., Hird, 1). and McCapes-, R.U. (1989). Antirnicrob. AcenUs Chemother. 3.3.: 670.

Huber, W.G., Korica. D. . Neal. T.P.. Schnurrenherr-fer, P.R., and Martin, R. J . ( 1971). Arch . Envi ron . [lea] th. 22: 561.

Ikeda, Y. , lijirna, T. and Tajirna, K. (1967). J. Gen. Appl. Microbiol. 13:247.

II'in, V.K. (1989). Kotirn. Bioi. Avia. Kcsm. Med. £3:90.

Inuzaka, N., Nakamura, S., Inuaaka, M and Tomoeda, M. (1969). J. Bacteriol. .100:827.

Jaari, B. and Jaan, K. (1980). FEMS. Microbiol. Lett. 7:19.

Jacob, A.E., Shapiro, J. A., Yamarfiol;o, L. , Smith, D.I., Cohen, S.N. and Berg, D. (1977). In "DNA Insertion Elements, Plasmids and Episomes" (A.I. Bukhari, J.A. Shapiro and S. Adhya, eda), PP. 607-638. Cr.ld SyrinfT Sarbor Laboraix»ry, New York.

Jacoby, G.A. (1979). In 'T-S.endom.orias aeruf tinos' : Clinical ManifesLah i on of In ffM.-l, i on:: of Curr«>nl, Therapy" (R..(i. Dogget ed. ) I'P 271 309. Academic Prer;f!, New York and London.

Jacoby, G.A. and Sl-iapiro, .J. A. (1977). In "DNA TriBcr t,al. i on Elements, Piasmi<l;,- and Episumr;;;" (A. I. Bukhari, J. A. Shapiro and S. Adhya. eds). PP 639-656. Cold Spring Harbor LaVjoratory, Nev/ York.

Jayaratne, A.H.G.P., Col 1 in?;-Thomp.s-on, D.L. and Trevors, d.T. (1987). Syst. Appl. Microbiol. 10:78.

95

Jazrawi, S.F. imd Js-haci, M.V^ (1983). J. Envjr. Sci. llJt.Pi. A.lfi:673.

Jeanclaude, P., Ar-hilio, A. nrid Joachim, F. {1988). Mol . Go;n. Genet. 212:215.

Jewell, B. and Collins-Thomprion, D.L. (1989). Curr. Microbiol. IS-343.

Jones, D. and Sneath, P.H.A. (1970). Bacterial. Rev. 34:40.

Kaufman, F. (197.^). Clasrii f i' at i f.-n of Baci.. A realisjt>ic .scFifmc v/ith special reference to the <.;ltiHaif icatiovi of Salmonella and Esoheri_cbi„a - species, Copenhaj^en: Munksgaard. PP 169.

Kasuya, M. (1964). J. Bacterid. 88-322.

Kelch, W.J. and I.ce, J.S. (1978). Ai>pl. Environ. Microhif.j. ^••450.

Kenner, B.A., Clark, H.F. and Kabler, P.W. (1961). Appl. Microbiol. 9_-lr>.

Klriatoon, H. and Ali-Mohammad (1986). Indian Exptl. Biol. 24:1.

Khatoon, [|. . lyr-r-. U..Wim<l \y>-r, V.N. (I!i72). Wi r<>\.<^^Vy • 48: It'..

Klein, R.D., Seising, E. and Wells, R.D. (1980). piasmid. 3:.388.

Knill, M. , Sueklinr^, W.G. and Pearson. A.D. (1978). Lan<-ot. 2:1002.

Koditschek, L.K. and Guyre, P. (1974). Bight. Mar. Pollut. Bull. S:71.

Komura, I. and laaki. K. (1971). J. Biochem. (Tokyo) 70:88r..

Komura, I., Funaba, T atjd Isaki, K. (1971). J. Biochein. (Tokyo). 7g:895.

Konisky, J. (1978). Tlio bactor i or; i nr;. Jn Orniiton I,.H., Bokalcli, J.R. (eds): The Bacteria Vol. VI. New York, Academic Press, P 71.

Kozarsky, P.E., Rimland, 1)., Terry, P.M. and Wachsrnuth, K. (1986). Infection Control. 7:577.

Krieg, N.R. (eds): (1984) Bergey's Manual of Systematic Bacteriology, Vol. I. Williams and Wilkins, Baltimore.

96

Kushtitjr, K. !:> {19V(J). An j rrifuoved rfn.'Mjod ij.tr T ra i i s ro r i f i a l io i i of E. c o l i w i t h Col EI d e r i v e d p las rn ids" . In G e n e t i c E n g i n e e r i n g P . 1 7 - 2 3 , H.W. Boye r and S. N i c o s i a ( e d s . ) . E l s i v e r / N o r b h H o l l a n d B i o m e d i c a l i>rGsri.

Lacey, R. W. ( 3 9 7 5 ) . B a c t e r i d . Rev. 3 9 : 1 .

L a i , C..J. and WeisVjlmn, B. ( 1 9 7 1 ) . P r o c . N a t l . Acad. S c i (USA). 6 a - 8 5 6 .

Lederberg , E. and Cohen, S.N. ( 1 9 7 4 ) . J. B a c t e r i d . 1 1 9 : 1 0 7 2 .

Lederberg , J . ( 1 9 5 2 ) . P h y s i o l . Rev. 3 2 : 4 0 2 .

Lederberg , J . , Caval^l Ivforxa , [.. f.. and h r - d c r h c r r ^ E.M. (19.'i'.'.)-G e n e t i c s . .3: / :720.

Levy, S .B . ( 1 9 8 3 ) . I n f e c t . C o n t r o l . 4 : 1 9 5 .

Lewin, B. ( 1 9 7 7 ) . iJene E x p r e s s i o n 3 , P l a s c a i d s and P h a g e s . .Jrjhn Wiley and S o n s , I n c . , New York.

L i n i a l , M. and Malarny, M.ll. ( 1 9 7 0 ) . J . Vi ro lo^?y . . ^ : 7 2 .

L i n t o n , A.H. ( 1 9 7 7 ) . I n " A n t i b i o t i c s and A n t i b i o s i s i n A g r i c u l t u r e " (M. Woodbine , e d . ) , PP 3 1 5 - 3 4 3 , B u t t e r w o r t h s , London.

Low, K . B . , ( 1 9 7 2 ) . B a c t . Rev. 3 6 : 5 8 7 .

Lyon, B.R., Luorio, J.L. and okuarry, R.A. (1984a). J. Med. Microbiol. L7:79.

Lyon, B.R., May, J.W and Skurray, R.A (1983). Antirnicrob. Agents Chemother. 23:817.

Wakela, 0., Makela, P.H. and Soikkelli, S. (1964). Annu. Med. Exptl. Biol. Fennie (flelsi nk i ) - 42:188.

Mandel, M. (19G9). Annu. Rev. Microbiol. 23.: 239.

Maniatis, T. , Fritsch, E.F. an<l Sambrook, .1. (1982). Molecular Cl<ininfJ[: ii 1 MK>oral,or.v m/mual. Cold r>i'riri< Ilathfu-Laboratory, Cold Spring Harbor, N.Y.

Marmur, J., Falkow, S. and Mandel. M. (1963). Annu. Rov. Microbiol. 17-329.

Martinez, L.Y., Arenas, M.M.P., Montes, M.Y.R., Martins, L.T and Baca, B.E. (1987). Can. J. Microbiol. 33:816.

97

May, J.W., lloufUil-on, li.ll. and PerrcL, C.J. (1964). J. Gen. Microbiol. 37:157.

McCarthy, J. A., Delanoy, J.E. ami GrriKso, H.J. {1961). V^abor Sewage Works. 1_08:238.

McHugh, G.L. and Swarts, M.N. (1977). Antirfiicrob. Agentr;. Chemother. L2:423.

Mentsirig, L.O. (1981). Lancet. (Vol. IT for 1981) : 3r>?..

Meynell, E. , Meynoli, (i.G. and Dabla, N. (1968). Bacterioi. Rov. 32:55.

Meynell, G.G. (1972). "Bactorial Plnrimids" Macrnillan, London and New York.

Meynell, G. (1973). Bacterial Plasmids. M.I.T. Press Cambridge, Mass.

Middlebrooke, J.L. and Dorland, R.B. (1984). "Bacterial Toxin: Cellular Mechanism of action". Microbiol; Rev. 48:199.

Mitsuhashi, S. , Ilarada, K. and Kameda, M. (1961). Nature (London). 189-947.

Molnar, J., Beladi, 1. and llollanb, I.B. (1978). Genet. Res. 31:197.

Myerowits, R.L., Albers, A.C.. Yec, R.B. and Orskov, F. (1977). J. Clin. Mi..TobioL. 6:124.

Nakaya, R., Nakamura, A. and Muiata, Y (1960). Biochem. Biophys. Res. Commun. 3,: 6.0 4.

Niemi, M., Sibakov, M. and Niemela, S. (1983). Appl. Envir. Microbiol. i4^:79.

Novakova, Z., Soykova, S., Krcmery, V. /md Parakova, E. (1909). Zeitschrift fur Allg Microbiol. 9:399.

Novick, R.P. (1969). Bacterioi. Revs. 33:210.

O'Brien, R.W. and Morris, J.G (1971). J. Gen. Microbiol. 6_7:265.

O'Brien, T.F., Mayer, K.H. and Hopkins, J.D. (1986). Global surveillance of the deployment of antibiotic resistance genes and plasmids, i>. 3. In Levy, S.B. and Novick, R.P. (ed.), Banbury report, no. 24. Antibiotic resistance genes: ecrolci/ y, transfer, and exrirrjssion. Cold si'rinf

Harbor Labora tory , Cold SPrirvg Harbor-^ N-Y-

98

Ochman, H. and Salander, R.K. (1984). Proc. Natl. Acad. Sol. (USA). 81 -tflB.

Ogunseitan, O.A., TedCord, Bl.T., Facia, D. , Sirotkin, K.M. and Sayler, G.S (1987). J. Indust. Microbiol. 1:311.

Ohmae, K. , Yoniv-HV/a, S. and ']'<:'.rf\kf\d<.>, M. (1980). Ani-irfticr<ib. Agents. Chemother. 19 •• 86.

Qhman, D.E. (1988). In Experiments in Gene Manipulation, by Prentice Hall, Inc., Englewood Cliffs, New Jersey.

Olesen, R.EL and GonMnle?:, C. (1974). Bioc:Viem. RiophyK. R<r;;. Commun. }i.9:377.

Osawa, S. , Takata, R., Tanaka, K. and Tamaki, M. (1973). Mol. Gen. Genet. .127:163.

Orekov, F. , Orskov, 1., Evan;!, D..!., Suck, R.B., Sack, D.A, , and Wadstorm, T. (1976). Med. Microbiol. Immunol. 162:73.

Oskov, F. and Oiikav ^ I. (1984). Methods. Microbiol. 14:43.

Overbeeke, N. and Lugtenberg, B. (1980). J. Gen. Microbiol. 121:373.

Paahkanen, J., Gotschlich, E.G. and Makela, P.H. (1979). J. Bacteriol. 131:835.

Pelcsar, M.J. (Jr.), Chan, E.C.S. and Krieg, N.R., (1986). In Microbiology (eds.) Pelcaar et ai (1986). PP 767.

Peterson, N. and Boring, J.R. (1960). Amor. J. Hyg. Zl.: 134.

Reanney, D.C. and Ackermann, H.W. (1981). Intervirology. 15:190.

Richardson, J.F., Chi ItasobVion, N. and Marpies, R.R. (1988). J. Med. Microbiol. 25:67.

Roubussay, D. and Zilling, W. (1969). FF.RS Lett. .5:104.

Sack, R.B. (1975). Annu. Rov. MifMohinl. 29:333.

Sack, R.B., Gorbach, S.L., Banwell, J.G., Jacobs, B., Chatterjee, B.D. and Mi bra, R.C. (1971). J. Infect. Dis. 123:378.

Saunders, J.R., Docherty, A. and Humphreys, G.O. (1984). In Methods in Microbiology (ejds. ) . Bennet P.M and Grinested, J. Academic Press. PP. 61-96.

99

Sohnll, .1.. ("11..v..I, ll.W.. :;i/..-.'y. K.A.. Ut..<l(i. I'.M.A. MM.I Syrrionds, N. ( H)0;O . <!f.Tiol,. \h::^. Camh. A : MVA.

Schiffer, K.El. and Sbackebrandb, E. (1983). Anrm.Rev. Microbiol. 31:143.

Seigneuriri, R. , MaKnin, K. and Ac-hard, M.L. (1951). Annu. Jn;;l.. Pasteur. (39:473.

Selan, L. , Scassocchio, F. , Oliva, B. , Schippa, S. , Allocal^i, N and Rensinia, G (1988). Chemioterapia. 7,:292.

Seligmann, R. and Rentier, R. (1965). d. Arner. VJater. Works Ass-. ^.1572.

Shinj, T. and Yukiko, W. (1988). J. Clin. Microbiol. 26:244.

Siccardi, A.G. (19GG). GonoI. Res. 8:2 L9.

Sissemore, R.K. and Colv/el], R.R. (1977). Antimicrol). AMc;nts Chemother. 12:373.

Sjostedt, S. (1946). Acta. Pathol. Microbiol. Scan. Suppl. 63:1.

Smith, D.H. (1967). Science, 156-1114.

Smith, El.O., Danner, D.B. and Deich, R. A. (1981). Annu. Rev. Biochem. 50:41.

Smith, H.W. (1970). Nature (London). 228:1286.

Smith, H.W. (1977). In "Antibiotics and Antibiosi,-^ in Agriculture" (M. Woodbine, cd.), PP 344. BuLterworths, London.

Smith, H.W. and Gyles, C.L. (1970). J. Med. Microbiol. .3:387.

Stadler, J. and AdollM^rri, li:. A. (1972). .1. Dacteriol. 10y: •1'''''•

Stanisich, V.A. (1984). In Methods in Micrcbiology (eds). Bennet, P.M and Grinsted, J (1984). Academic Press. PP 5.

Stanisich, V.A., Bennett, P.M. andOrtis, J.M. (1976). Mol. Gen. Genet. 143:333.

Sureau, P. (1958). Annu. Inst. Pastr-ur. 95:6.

Stotaky, G. and Krasovsky, V.W. (1981). In Molecular Biolor;y, Pathogeni ci t/y and Floojor y of Bact,eria] plasinids. (f;ds,).

100

Levy, 13.B., (MMV/I-!.'. R.C. >\f\<\ Kor;iii(T(. K. f.. l'l<;rii,un \^r<r.:r,, Now York. IM' :<! .

Sutherland, I.W. ond Kennedy, A.F.I). (1986). Appl. Env. Microbiology. 52:948.

Talboot, n.W., YMinMUK.I.M. D.K., r>inilh. II.VJ. and ;1<r id 1 «ri. , R..I. (1980). Api-I.. lilnviroti. Microbiol. 3.9:97.

Tanaka, K. , Teraoka, H. , Tainaki, M. , Ohaka, E. and Osav/a, S. (1968). Science. 162:576.

Tantulavanchi, S., Olexy, V.M., Prasad, T.R., Bird, T.J. and Talenda, F.C. (1981). J. Gen. Microbiol. 14:371.

Thomson, J.A. and Bilgeri, Y.R. (1982). Antimicrob. Agents Chemother. 22:689.

Tomeoda, M. , Inuf.aka, M. , Kubr>, N. and Nakarnura, 3. (1968). J. Bacteriol. 95:1078.

Tomoeda, M. , Kokubu, M. , Nabfvta, Fl and Minamikawa, S. (1970). J. Bacteriol. 104:864.

Vahlne, G. (1945). Acta. Pathol. Microbiol. Scan. Buyvpl . 62:1.

Vakulenko, S.B., Bodunkova, L.E., Garaev, M.M., Fomina, I.P. and Navashin, S.M. (1980). Antibiotic Resistance Transpo-fsition Ot,hcr Mncb. \r^t. Symp. . 'lib (M J IviMibaiib i . S. , Rosivai, L and Kracernery, V. eds.). PP 125.

Vickery, A.M., Beard-Pegler, M.A. and Rountree, P.M. (1983). Pathology. 15:235.

Wadowsky, R.M. andYee, R.B. (1981). Appl. Environ. Microbiol. 42:768.

Watanabe, T. (1963). Bacteriol. Rev. 27:87.

Watanabe, T. (1971). Curr. Top. Microbiol. Imriiunol. 56:4:1.

Watanabe, T. and Fukasav/a, T. (1961). J. Bacteit-iol. 82:202.

Watanabe, T. , Furuse, C. and Sakaiaurni, S. (1968). J. Bacteriol. aS:1791.

Wheatcroft, R. and Williams, P.A. (1981). J. Gen. Microbiol. iM:433.

Willetts, N.S. (1967). Biochein. Biophys. Res. Comrnun. 27: 112.

101

Wise, E.M. and AbouDonla, M.M. {1975). Proc. Natl. Acad. Sci. (USA). 1_?.:?.0,2\ .

Womble, D.D. andHov/nd, R.H. (1988). Microbiol. Revs. 52:433.

Yagi, Y., Franke, A.E. and Clewell, D.B. (1975). Antimicrob. Agents Cherno ther. 7:871.

Yoshikawa, M. and Sevag, M.G. (19G7). J. Bacterid. 93:245.