Relationship of ESBL production with fluoroquinolones resistance in

Escherichai coli and Klebsiella pneumonia isolates: The remaining antibiotics

as drug of choice for treatment the infections caused by these strains

Nihad Yusuf *, Shaker Faris**, Ibrahim Al Subal***

*Dept. of Botany, Faculty of Sciences. University of Aleppo

**Dept. of Laboratory Medicine. Faculty of Medicine. University of Aleppo

***Postgraduate Student (M.Sc.) Dept. of Botany, Faculty of Sciences,

University of Aleppo

Abstract: Background. The importance of infections due to extended-spectrum β-

lactamase–producing Escherichia coli and Klebsiella pneumoniae has been

increasingly recognized in recent years. Infections caused by ESBL producers are

of clinical concern, because few antimicrobials are available as therapeutic

options. Although fluoroquinolones offer effective therapy for ESBL-E.coli and

K.pneumoniae infections, their usefulness is threatened by increasing

fluoroquinolones resistant strains. This lead up to Increase reliance on

carbapenems subsequently increase carbapenem resistance.

Aim. In this study we aimed to investigate the prevalence of ESBL-producing

E.coli. and K.pneumoniaee as well as flouroquinolones resistance in ESBL-

producing isolates, and determination the best antibiotics as drug of choice for

treatment the infections caused by these species in three university hospitals in

Aleppo.

Results. Of 197 episodes of infection, 39 (19.80%) were caused by K.pneumoniae

and 158 (80.20%) were caused by E.coli. Of 158 E.coli, 100 (63.29%) were

ESBL producing, of 39 K.pneumoniaee, 27 (69.23%) were ESBL producing. Of

65 ciprofloxacin-resistant E.coli isolates, 57 (87.69%) were ESBL producers. Of

34 ofloxacin-resistant E.coli isolates, 30 (88.24%) were ESBL producers. All 10

ciprofloxacin-resistant, and 6 ofloxacin-resistant K.pneumoniae (100%) were

ESBL producers.The percentages of sensitivity of ESBL producing E.coli and

K.pneumoniae were found as, 100% and 96.3% for imepenem, 100% and 96.3%

for meropenem, 100% and 92.59% for amikacin, 89% and 85.19% for

chloramphenicol, 70% and 77.78% for ofloxacin, 43% and 62.96% for

ciprofloxacin respectively. In conclusion, meropenem , imipenem and amikacin

seem to be better choice of antibiotics should be used for ESBL positive

lifethreatening infections, because of their remaining highest activity.

Key words : ESBL, fluoroquinolones, E.coli., K.pneumoniae.

Introduction: Escherichia coli and Klebsiella pneumoniae strains are opportunistic pathogens

and have been associated with various ailments such as urinary tract infection,

septicemia, respiratory tract infection, wound infection and diarrhea [1,2].

Extended-spectrum beta-lactamase (ESBL)-producing organisms have spread

worldwide since their first description in 1983 [3,4,5, 6]. Over 300 Ambler class

A ESBL genotypes have now been described, and they have been reported in

numerous species of the Enterobacteriaceae family, including Escherichia coli,

Klebsiella pneumoniae and Salmonella species, as well as in non-

Enterobacteriaceae [4,5,6]. Furthermore, ESBLs have continued to increase in

variety and prevalence and are now a global health concern [7,8,9]. Extended

spectrum β-lactamases (ESBLs) hydrolyze most β-lactam antibiotics, such as

penicillins, cephalosporins and monobactams, but not carbapenems and

cephamycins, and are inhibited by β-lactamase inhibitors. [3,4,10]. E.coli and

K.pneumoniae isolates with resistance to fluoroquinolones, sulfonamides,

aminoglycosides and tetracyclines, in addition to their ESBL phenotype, are

common and severely limit therapeutic options [3,6,7]. Flouroquinolones are

potential therapeutic options for treatment of infections caused by ESBL-

producing enterobacteriaceae [11,12]. The introduction of flouroqiunolones in

the med-1980s considered medical evolution not only because of their potency

and broad spectrum of activity, but also because of the potential for development

of resistance to these agents was predicted to be very low, particularly among

enterobacteriaceae [13,14]. In the five years after their initial introduction, there

were few reports of resistance against to these agents [15,16]. However, in the

last 10 years , cases of infections with flouroquinolones-resistant E.coli and

K.pneumoniaee have increased in number, together with upward trends in the use

of fouroquinolones in the community and hospitals [13,17,]. In this study we

aimed to investigate the relationship between flouroquinolones-resistant E.coli.

and K.pneumoniaee and ESBLs production, and determination the best

antibiotics as drug of choice for treatment the infections caused by these species.

Materials and methods: Study location. The study was performed at three University Hospitals in

Aleppo city centre between October 2010 and June 2011. These are the major

teaching hospitals in the northern part of Syria, offers secondary as well as

tertiary medical care to patients from city centre and the surrounding regions. It is

also the referral centre for the surrounding area, and patients with severe,

complicated cases are transferred to these hospitals from other hospitals.

Bacterial isolates. Total 158 E. coli (100 ESBL-producing, 58 non-ESBL

producing) and 39 K. pneumoniae strains (27 ESBLproducing, 12 non-ESBL

producing) isolated from all clinical specimens obtained at these hospitals were

included in this study. The isolates were non-consecutive (one per patient).

Identification of species were done by qualitative bacteriological methods

and API ID 32E system kits (bioMe´rieux, France) [18].

Antimicrobial susceptibility testing. Disk-diffusion tests were carried out with

antibiotic-containing disks (Abtek, UK) on Mueller-Hinton agar plate

(bioMerieux, France). The results were expressed as susceptible or resistant

according to the criteria recommended by the Clinical and Laboratory Standards

Institute (CLSI) [19]. The table (1) contains antimicrobial agents which were

used. Table (1) antibiotics used in the study.

Tetracycline (TET 30µg) Cefotaxime (CTX 30 µg)

Cefepime (CFP 30 µg) Ceftazidime (CAZ 30 µg)

Imipenem (IMP 10 µg) Cefteriaxone (CTR 30 µg)

Meropenem (MER 10 µg) Chloramphenicol (CHL 30 µg)

Nalidixic acid (NAL 30µg) Amoxycillin-clavulanic acid (AUG 03/10µg)

Ofloxacin (OFL 25 µg) Piperacillin-tazobactam (TZP 85 µg)

Ciprofloxacin (CPR 5 µg) Trimethoprim-sulfamethoxazol (COT 25 µg)

Amikacin (AMK 30 µg) Gentamycin (GEN 10 µg)

Detection of ESBL. E. coli and K. pneumoniae strains were screened for ESBL

production using double disk synergy test. Bacteria were suspended equivalent to

McFarland 0.5 turbidity standard in sterile serum physiologic. After inoculating a

Mueller-Hinton Agar plate, 30 µg ceftazidime (CAZ), cefotaxime (CTX),

ceftriaxone (CTR) and cefepime (CFP) disks were placed 22mm (center to

center) from a centered amoxicillin plus clavulanic acid (30/10 µg) disk. After

overnight incubation at 36 Cº , an enhanced inhibition area between the

amoxicillin-clavulanic acid disk and any one of other disks indicated the presence

of ESBL [19]. The ESBL production was confirmed by the E-test method (AB

Biodisk, Solna, Sweden). The E-test ESBL strips that are ceftazidime (TZ)

ceftazidime plus clavulanic acid (TZL) and cefotaxime (CT) cefotaxime plus

clavulanic acid (CTL) were both used for each strain, according to

recommendation of the manufacturer E. coli ATCC 25922 and K.pneumoniae

ATCC 700603 were used as a control strains in susceptibility test [19].

Statistical methods. We used the statistical program SPSS version /19/.

Differences between proportions were analyzed using Chi-square test (χ2). All

differences in which the probability of the null hypothesis was p ≤ 0.05 were

considered significant.

Results:

During the study period, a total of 197 patient-specific isolates were included,

158 (80.20%) of the isolates were E.coli, 39 (19.80%) were K.pneumoniae. Of

197 isolates, 127 (64.47%) were ESBL producers. The proportions of ESBL-

producing isolates was 63.29% for E. coli (100 of 158 isolates), 69.23% for

K.pneumoniae

(27 of 39). Of 197 patients, 94 were inpatiens and 103 were outpatients, the

median age of patients was 43 years (range, 2–81 years) and male: female ratio

was 1 : 3.2, the distribution of patients were illustrated in table (2) and figure (1).

Table (2): factors affecting the distribution of isolates according to ESBL production or not.

characters ESBL (127) nonESBL (70)

Total P

value N % N %

Gender male 38 80.85 9 19.15 47

0.008 female 89 59.33 61 40.67 150

Acquisition inpatient 82 87.23 12 12.77 94

0.001 oupatient 45 43.69 58 56.31 103

Age ≥ 43 years 76 77.55 22 22.45 98

0.001 ≤ 43 years 51 51.52 48 48.48 99

Figur (1): factors affecting the distribution of isolates according to ESBL production or not.

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

mal

e

fem

ale

inp

atie

nt

ou

pat

ien

t

≥ 4

3 y

ears

≤ 4

3 y

ears

Gender Acquisition Age

ESBL

nonESBL

The isolates with intermediate resistance to ciprofloxacin (5 isolates) and

ofloxacin (12 isolates) were excluded. Of 192 isolates were tested against

ciprofloxacin, 75 (39.1% ) were due to ciprofloxacin- resistant E.coli and

K.pneumoniae, and 117 (60.9% ) were due to ciprofloxacin-susceptible E.coli

and K.pneumoniae. Of 185 isolates were tested against ofloxacin, 40 (21.62% )

were due to ofloxacin-resistant E.coli and K.pneumoniae, and 145 (78.38% )

were due to ofloxacin-susceptible E.coli and K.pneumoniae, figure(2).

S = sensitive, R = resistance

Figure (2): sensitivity and resistance ratios for all isolates to fluoroquinolones agents.

Fluoroquinolones resistance wase found with surprisingly high frequency in

extended-spectrum β-lactamase (ESBL)-producing E.coli and K.pneumoniae

isolates. Fifty four percent and 31% of the ESBL-producing isolates were

resistant to ciprofloxacin and ofloxacin respectively, in comparison with 11.76%

and 5.8% ciprofloxacin and ofloxacin resistance respectively in non ESBL-

producing isolates. Table (3) and figure (3).

Table (3): the relationship between ESBL enzymes production, and resistance to

fluoroquinolones.

Ciprofloxacin (192) Ofloxacin (185) ESBL

positive (124)

ESBL negative

(68) P

value

ESBL positive

(116)

ESBL negative

(69) P

value N % N % N % N % 67 54.03 8 11.76 0.00 36 31.04 4 5.8 0.00

0

10

20

30

40

50

60

70

80

ciprofloxacin ofloxacin

60.9

78.38

39.1

21.62

S

R

Figure (3): the relationship between ESBL enzymes production, and resistance to

fluoroquinolones.

ESBL production was significantly higher among fluoroquinolones-resistant E.

coli and K.pneumoniae strains than among fluoroquinilones-susceptible strains.

ESBL production was detected in 57 (87.69%) of the 65 ciprofloxacin- resistant

E. coli isolates. In comparison with 42 (46.67%) of the 90 ciprofloxacin-

susceptible E.coli isolates were ESBL producers (P = .000). Of 34 cases of

infection due to ofloxacin resistant E.coli, 30 (88.24%) were ESBL producers,

whereas 59 (52.68%) of the 112 ofloxacin susceptible E.coli were ESBL

porducers, (p=.000). Table (4), and figure (4).

Table (4): the relationship between fluoroquinolones resistance, and ESBL enzymes production

in E.coli isolates.

characters

Ciprofloxacin (155) Ofloxacin (146)

S (90) R (65) S (112) R (34)

N % N % N % N %

ESBL 42 46.67 57 87.69 59 52.68 30 88.24

nonESBL 48 53.33 8 12.31 53 47.32 4 11.76

P value 0.00 0.00 S = sensitive, R = resistance , N = number of isolates

0

10

20

30

40

50

60

ciprofloxacin ofloxacin

54.03

31.04

11.76

5.8

ESBL positive

ESBL negative

S = sensitive, R = resistance , numbers above the columns = number of isolates

Figure (4): the relationship between fluoroquinolones resistance, and ESBL enzymes

production in E.coli isolates.

Ten (100%) of 10 ciprofloxacin-resistant K.pneumoniae isolates caused

infections were ESBL producers, in coparison with 15 (55.56%) of 27

ciprofloxacin-susceptible K.pneumoniae isolates were ESBL producers, (p=.015).

However there was not found statistically significant for the results of ofloxacicn

susceptibility study in K.pneumoniae and its relationship to ESBL producers

isolates. nevertheless 6 (100%) of 6 ofloxacin-resistant K.pneumoniae isolates

were ESBL producers, in coparison with 21 (63.64%) of 33 susceptible

K.pneumoniae isolates were ESBL producers (p=.151). Table (5), and figure (5).

Table (5): the relationship between fluoroquinolones resistance, and ESBL enzymes production

in K.pneumoniae isolates.

characters

Ciprofloxacin (37) Ofloxacin (39)

S (27) R (10) S (33) R (6)

N % N % N % N %

ESBL 15 55.56 10 100 21 63.64 6 100

nonESBL 12 44.44 0 0 12 36.36 0 0

P value 0.015 0.151 S = sensitive, R = resistance , N = number of isolates

0

10

20

30

40

50

60

S R S R

Ciprofloxacin Ofloxacin

42

57 59

30

48

8

53

4

E.coli ESBL

E.coli nonESBL

S = sensitive, R = resistance , numbers above the columns = number of isolates

Figure (5): the relationship between fluoroquinolones resistance, and ESBL enzymes

production in K.pneumoniae isolates.

In vitro susceptibility of ESBL-producing and non-producing E.coli strains

against others antibiotics were found for amikacin as (100%) and (100%), for

meropenem as (100%) and (100%), for imipenem as (100%) and (100%) and for

chloramphenicol as (89%) and (87.93%) respectively. There was not found

statistically significant between two groups of strains, Table (6).

Table (6): the sensitivity ratios of ESBL-producing and nonESBL-producing E.coli isolates

against some antibiotics as drugs of choice for treatment.

characters

ESBL

E.coli (100)

nonESBL

E.coli (58) P value

N % N %

Amikacin 100 100 58 100 NS

Meropenem 100 100 58 100 NS Imipenem 100 100 58 100 NS

Chloramphenicol 89 89 51 87.93 NS N = number of isolates, NS = no significance

Similar results appeared for k.pneumoniae strains, the susceptibility of ESBL-

producing isolates versus non-producing were, (92.59%) and (100%) for

0

5

10

15

20

25

S R S R

Ciprofloxacin Ofloxacin

15

10

21

6

12

0

12

0

K. pneu ESBL

K.pneu nonESBL

amikacin, (96.3%) and (100%) for imipenem, (96.3%) and (91.67%) for

meropenem and (85.19%) and (100%) for chloramphenicol respectively. There

was not found statistically significant between two groups of strains, table (7).

Table (7): the sensitivity ratios of ESBL-producing and nonESBL-producing K.pneumoniae

isolates against some antibiotics as drugs of choice for treatment.

characters

ESBL

K.pneumoniae (27)

nonESBL

K.pneumoniae (12) P value

N % N %

Amikacin 25 92.59 12 100 NS

Meropenem 26 96.3 11 91.67 NS Imipenem 26 96.3 12 100 NS

Chloramphenicol 23 85.19 12 87.93 NS N = number of isolates, NS = no significance

DISCUSSION:

The prevalence of ESBL-producing E.coli and K.pneumoniae strains vary greatly

from contry to country, and are increasing rapidly every year [10]. For example,

3% of enterobacteriaceae isolates from the USA, and 0.1% of E.coli and 0.3% of

K.pneumoniae isolates in Japan were reported to be ESBL-producing strains [2],

while 94.1% of E.coli and 52.4% of K.pneumoniae isolates from China [20],

And 67.3% of E.coli and K.pneumoniae isolates from india were ESBLs

producers [21]. The infections caused by ESBL-producing E.coli and

K.pneumoniae are considered an increasing risk associated with the treatment

failure [10]. Although several studies have addressed the issue of the emergence

of ESBL-producing strains of E.coli and K.pneumoniae worldwide. We don’t

find any study has addressed this issue in Syria. The results of our study showed

that, the prevalence of ESBL-producing E.coli and K.pneumoniae was (64.47% ).

This result agrees with many reports in previous studies, [22] , [21] . But our

result is higher than other reports, [23] and [24] . Our results showed that, ESBL

production was more prevalent among K.pneumoniae isolates (69.23% )

comparing with E.coli isolates (63.29% ). Similar results have been reported from

other studies, [20], [4], [10], [25], [26] and [2]. The high prevalence of ESBL

production among E.coli and K.pneumoniae may be attributed to extensive use of

extended spectrum betalactam agents especially third generation cephalosporins,

this lead up to pressure selection for resistant strains. Although betalactam and

fluoroquinolones antimicrobial agents are frequently used for the treatment of

E.coli and K.pneumoniae infections. ESBL-producing isolates have increased in

prevalence and are resistant to other antibiotic classes and thus increased

limitation of effective treatments [2]. Fluoroquinolones resistance varied from

country to country. For example, fluoroquinolones resistance rates for Canada

and United states were 22% and 24% respectively, whereas fluoroquinolones

resistance rates were 75% and 70% in India and Panama respectively [15]. In this

study, the ciprofloxacin and ofloxacin resistance rates were (39.1%) and

(21.62%) respectively. This can be compared to the results of previous studies,

[15], [9] and [27] . . The possible explanation for these results is the overuse of

fluoroquinolone agents for treatment in community and hospital settings.

Statistical analysis showed that, the incidence of ciprofloxacin and ofloxacin

resistance wase significantly higer among ESBL-producing strains, 54% and 31%

respectively. Also the incidence of ESBL production was significantly higher

among ciprofloxacin and ofloxacin resistant E.coli and K.pneumoniae strains,

(87.69%) and (100%) for ciprofloxacin respectively, and (88.24%) and (100%)

for ofloxacin respectively. This association of ESBL production with

fluoroquinolones resistance has been noted in several studies such as [28], [29],

[21], [12], [30], [31], [16], [6], [7], [17], [9] and [32] . The possible explanation

for the association between ESBL production and fluoroquinolones resistance is

the presence of genes of the two resistance mechanisms on the same plasmid.

There are other potential explanations for the association between resistance to

third-generation cephalosporins and fluoroquinolones, including active efflux and

outer membrane protein alterations. There may be other possible explanations for

the coexistence of resistance to β-lactams and fluoroquinolones in gram negative

bacteria. For example, bacteria that are able to acquire the ability to produce

ESBL can be selected by intensive fluoroquinolones use. In this study,

carbapenems, both imipenem and meropenem, also amikacin had the best activity

against K. pneumonia and E. coli, and there was no differences in the

susceptibility patterns between the ESBL producers and ESBL non-producers

strains.

In conclusion, ESBL-producing E. coli and K. pneumoniae are common in north

syria. Most of them are resistant to many classes of antibiotic, resulting in

limited treatment options. All ESBL producer were susceptible to imipenem and

meropenem, these finding suggest that carbapenems are the best choice for

treatment of ESBl related infections. The management of infections due to these

organisms is difficult and more complex, particularly in severe infections.

Therefore, it is important to control such strains closely in order to prevent and

reduce their spread.

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