Krutarth Ppt 9 April

7/31/2019 Krutarth Ppt 9 April

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Presented by: Krutarth C. GautamDepartment of

MicrobiologyD.B. Science College,

Gondia.


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BACKGROUND:

Nosocomial infections are important cause of

morbidity and mortality all over the world. It hasbeen shown that appropriate environmental,hygienic and disinfection practices can be veryhelpful to hospital infection control. Large quantities of disinfectants are used inhospitals, externally on human skin or toeliminate microorganisms from inanimate objects.After use, residual quantities of these productsreach the hospital waste and wastewater,

exposing the bacteria that survive in hospitalwaste. Increasing attention has been directedrecently to the resistance of bacteria todisinfectants.

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Large quantities of disinfectants are used in hospitals for a

range of purposes. Usually disinfectant compounds are usedexternally on human skin to prevent or limit microbialinfection or for preoperative skin disinfection. The micro-floraof hospital wastewaters is composed by saprophytic bacteriafrom the atmosphere, soil, medical devices and water employed in the hospital practice; the pathogens are mainlyreleased with the patient excreta. They are also used toeliminate microorganisms from inanimate objects (surfaces,instruments). After use residual quantities of disinfectants

reach the wastewater. The hospital effluents are discharged,usually, in the urban sewer system were they mix with other effluents and finally reach the sewage treatment plant. The laststep of this process is the release of purified wastewaters to ariver, a lake, or to seawaters. Some of these water bodies aresources of drinking water .

INTRODUCTION


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Disinfectant-resistant bacterial strains have arisen as a resultof the lack in standardization of some factors, such as criteriafor use of chemicals agents, specifications in the labels of available products and scarcity of well-trained personnel. Theselection, use and control of the effectiveness of thedisinfectants have been emphasized, since environmental

surfaces and medical and surgical instruments can serve asvehicles to infectious agents in susceptible hosts associatedwith the hospital setting. The bacteria that survive in hospitalwastewaters may be exposed to a wide range of biocides that

could act as a selective pressure for the development of resistance. Acquired resistance to biocides may arise bycellular mutation or by the acquisition of genetic elements inthe form of plasmids or transposons.

INTRODUCTION cont


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There has been an interest in improving the sterilization anddisinfection procedures to reduce the infection risk for hospitalized

patients and healthcare workers . Disinfectant exposed bacteriahad also developed resistance to a commonly used antibioticknown as ciprofloxacin, without ever being exposed to it - causingalarm that the use of such disinfectants in a hospital setting couldbe promoting the growth of 'Superbugs'. The team demonstratedthat the bacteria had adapted to more efficiently pump outantimicrobial agents from the cell - both disinfectant, andantibiotic. It was also discovered that the adapted bacteriacontained a mutation in their DNA that yielded resistance tociprofloxacin-type antibiotics specifically. The selection, use andcontrol of the effectiveness of the disinfectants have beenemphasized, since environmental surfaces and medical andsurgical instruments can serve as vehicles to infectious agents insusceptible hosts associated with the hospital setting .Considering the importance of the disinfection for the prevention of nosocomial infections, the aim of this study was to evaluate the

bactericidal action of five disinfectants commonly used in hospitalsagainst antibiotic-susceptible and antibiotic-resistant clinical

INTRODUCTION cont


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A variety of substances such as pharmaceuticals, radionucleides andsolvents are used in hospitals for medical diagnostics, disinfection and

research. After application many non-metabolized drugs are excreted bythe patient and enter into wastewater. The microflora of hospitalwastewaters is composed by saprophytic bacteria from the atmosphere,soil, medical devices and water employed in the hospital practice; thepathogens are mainly released with the patient excreta. Large quantitiesof disinfectants are used in hospitals for a range of purposes. Usually

disinfectant compounds are used externally on human skin to prevent or limit microbial infection or for preoperative skin disinfection. They are alsoused to eliminate microorganisms from inanimate objects (surfaces,instruments). After use residual quantities of disinfectants reach thewastewater . The bacteria that survive in hospital wastewaters may beexposed to a wide range of biocides that could act as a selective pressurefor the development of resistance. Acquired resistance to biocides mayarise by cellular mutation or by the acquisition of genetic elements in theform of plasmids or transposons (10). Disinfectants and antiseptics areextensively used in hospital practice around the world, nevertheless theprofile of resistance to biocides in the hospital effluents has been barelystudied.

INTRODUCTION cont


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The aim of this paper was to determine thedisinfectant bacterial resistance pattern of themicroflora released to the urban sewer system bythe hospital effluent. In a first stage, thecharacterization of the waste water microflora wasperformed by determination of the number of colony forming units( CFU) of heterotrophicbacteria, fecal indicator bacteria, Pseudomonas sp.and Staphylococcus sp ., in hospital effluent

samples. In the next step, the bacterial resistanceto disinfectants was studied. The disinfectantsmore frequently used locally in the hospitalpractice, glutaraldehyde, chlorhexidine phenol,Lysol and dettol were selected for this study. Theresistant strains were isolated and typified for the 7

OBJECTIVES


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Pseudomonas sp. and Staphylococcus sp .: Tenfold dilutionof each sample was plated on Cetrimide agar and Baird Parker

agar, used for enumeration of Pseudomonas and S taphylococci, respectively.

Isolation of disinfectant resistant bacteriaThe phenol resistant subpopulation of effluent was selected fromthe bacterial population by treating wastewater samples 4minutes with phenol 0.1%. The lyzol resistant subpopulation of effluent was selected from the bacterial population by treatingwastewater samples 4 minutes with lyzol 0.1% . The dettol

resistant subpopulation of effluent was selected from the bacterial population by treating wastewater samples 4 minuteswith dettol 0.1% . The chlorhexidine resistant subpopulation of effluent was selected from the bacterial population by treatingwastewater samples 4 minutes with chlorhexidine 0.1% .

METHODOLOGY cont.


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Disinfectant Tolerance testing:Isolates were tested for their resistance patterns determined by on the basisof MIC values for different disinfectants. MIC values were determined by brothdilution method.Bacterial identification and test of sensitivity to the biocides Bacteria tolerant to each disinfectant were identified by different biochemicaltest, according to the Manual of Clinical Microbiology . The susceptibility of the isolated strains to each disinfectant was analyzed: MIC values of chlorhexidine were determined on tryptic soy agar (TSA) plates containing a

range of chlorhexidine concentrations (500-250-125-100-50-25 ppm),inoculated with 0.1 mL of bacterial suspension. All plates were incubated for 48 h at 35C. The MIC was the lowest concentration that prevented bacterialgrowth . Iodination was performed on suspension ca 10 7 CFU/mL. Povidoneiodine was added to a final concentration of 1%. Samples were removed atintervals, sodium thiosulphate added (final concentration 0.1%) and bacterial

dilutions were placed on Tryptic soy agar (TSA).For the determination of tolerance level to glutaraldehyde 0.1 mL of thesuspension was added to 9.9 mL of glutaraldehyde 2%. Samples wereremoved at intervals (5-20-60 minutes) and mixed with neutralization medium(14). Bactericide activity was expressed as reduction factors, that is,logarithmic reductions in viable organisms:

Reduction factor = log 10 CFU/mL (control) - log 10 CFU/mL (treated)

METHODOLOGY cont.


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RESULT AND DISCUSSION:

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1.Occurrence of bacterial flora in hospital waste:

Sr No Sample Sample origin Bacterial count S. aureus

count Heterotrophic

count E.coli count

Pseudomonas

count 1 Blood KTS, Gondia 5 X 10 2 40 X 10 5 20 X 10 2 25 X 10 3

2 Syringe KTS, Gondia 80 X 10 2 30 X 10 5 10 X 10 2 15 X 10 3

3 Sewer line KTS, Gondia 40 X 10 3 200 X 10 5 25 X 10 3 30 X 10 3

4 Dressing KTS, Gondia 60 X 10 2 20 X 10 5 5 X 10 2 50 X 10 2


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1.Occurrence of disinfectant resistant bacteria in hospital waste and waste water:

SrNo

Sample Sample origin Bacterial count S. aureus

count Heterotrop

hic

count

E.coli count

Pseudomonas

count 1 Blood KTS, Gondia 25 2 X 10 2 60 25 X 10 2

2 Syringe KTS, Gondia 8 X 10 2 3 X 10 2 70 15 X 10 2

3 Sewer line KTS, Gondia 40X 10 3 20 X 10 2 200 30 X 10 2

4 Dressing KTS, Gondia 6 X 10 2 2 X 10 2 25 5 X 10 2


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count

Heterotrop

hiccount

E.coli

count

Pseudomon

ascount

1 Blood KTS, Gondia 25 2 X 10 2 60 25 X 10 2


3 Sewer line KTS, Gondia 40X 10 3 20 X 10 2 2 X 10 2 30 X 10 2


TABLE 5.3.Occuerence of Lysol Resistant Bacteria


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count

Heterotr

ophiccount

E.coli

count

Pseudom

onascount





TABLE 5.4.Occuerence of Chlorhexidine Resistant Bacteria


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count Heterotrop

hiccount

E.coli count

Pseudomonas

count





TABLE 5.5.Occuerence of Detol Resistant Bacteria


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1.Disinfectant tolerance and susceptibility testing:

Sr. No Disinfectant Conc. Timemin

cfu/ml Log cfu/ml Reduction factor C T C T

1 Dettol 2% 10 10 6 90 x10 4 6 5.9542 0.04582% 20 10 6 85x10 4 6 5.9294 0.07062% 30 10 6 80x10 4 6 5.9030 0.09702% 40 10 6 70x10 4 6 5.8450 0.15502% 50 10 6 60x10 4 6 5.7781 0.22192% 60 10 6 50x10 4 6 5.6989 0.3011

2 Phenol 2% 10 10 6 75x10 4 6 5.8750 0.12502% 20 10 6 70x10 4 6 5.8450 0.15502% 30 10 6 60x10 4 6 5.7781 0.22192% 40 10 6 40x10 4 6 5.7781 0.22192% 50 10 6 20x10 4 6 5.3010 0.69902% 60 10 6 0000 0 0 0.0000

3 Lyzol 2% 10 10 6 95x10 4 6 5.9777 0.02232% 20 10 6 85x10 4 6 5.9294 0.07062% 30 10 6 75x10 4 6 5.8750 0.12502% 40 10 6 00 6 00 0.00002% 50 10 6 50x10 4 6 5.6989 0.30112% 60 10 6 30x10 4 6 5.4771 0.5229

4 Glutaraldehyde 2% 10 10 6 60x10 4 6 5.7781 0.22192% 20 10 6 40x10 4 6 5.6020 0.39802% 30 10 6 20x10 4 6 5.3010 0.6990

2% 40 10 6 10x10 4 6 5.0000 1.00002% 50 10 6 000 6 00 6.0000


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1 Dettol 2% 10 10 6 95x10 5 6 5.9777 0.0223

2% 20 10 6 93x10 4 6 5.9684 0.0316

2% 30 10 6 90x10 4 6 5.9546 0.04542% 40 10 6 85x10 4 6 5.9294 0.07062% 50 10 6 78x10 4 6 5.8920 0.10802% 60 10 6 60x10 4 6 5.7781 0.2219

2 Phenol 2% 10 10 6 80x10 4 6 5.9030 0.09702% 20 10 6 70x10 4 6 5.8450 0.15502% 30 10 6 55x10 4 6 5.7403 0.25972% 40 10 6 40x10 4 6 5.6020 0.39802% 50 10 6 20x10 4 6 5.3010 0.69902% 60 10 6 0000 6 00 6.0000

3 Lyzol 2% 10 10 6 90x10 4 6 5.9542 0.04582% 20 10 6 85x10 4 6 5.9294 0.07062% 30 10 6 80x10 4 6 5.9030 0.09702% 40 10 6 70x10 4 6 5.8450 0.15502% 50 10 6 55x10 4 6 5.7403 0.2597

2% 60 106

40x104

6 5.6020 0.39804 Glutaraldehyde 2% 10 10 6 60x10 4 6 5.7781 0.22192% 20 10 6 50x10 4 6 5.6989 0.30112% 30 10 6 25x10 4 6 5.3979 0.60212% 40 10 6 00 0 00 0.00002% 50 10 6 00 0 00 0.00002% 60 10 6 00 000 00 0.0000

5 Chlorhexidine 2% 10 10 6 90x10 5 6 5.9549 0.04510

2% 20 10 6 70x10 4 6 5.8450 0.15502% 30 10 6 70x10 4 6 5.8450 0.1550


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cfu/ml Log cfu/ml Reduction factor

C T C T

1 Dettol 2% 10 10 6 95x10 4 6 5.9777 0.0223

2% 20 10 6 90x10 4 6 5.9542 0.04582% 30 10 6 80x10 4 6 5.9030 0.0970

2% 40 10 6 60x10 4 6 5.7781 0.2219

2% 50 10 6 50x10 4 6 5.6989 0.3011

2% 60 10 6 40x10 4 6 5.6020 0.3980

2 Phenol 2% 10 10 6 80x10 4 6 5.9030 0.0970

2% 20 10 6 70x10 4 6 5.8450 0.1550

2% 30 10 6 55x10 4 6 5.7403 0.2597

2% 40 10 6 40x10 4 6 5.6020 0.3980

2% 50 10 6 20x10 4 6 5.3010 0.6990

2% 60 10 6 0000 6 00 6.0000

3 Lyzol 2% 10 10 6 95x10 4 6 5.9777 0.0223

2% 20 10 6 85x10 4 6 5.9294 0.0706

2% 30 10 6 75x10 4 6 5.8750 0.1250

2% 40 10 6 60x10 4 6 5.7781 0.2219

2% 50 10 6 50x10 4 6 5.6989 0.3011

2% 60 10 6 30x10 4 6 5.4771 0.5229

4 Glutaraldehyde 2% 10 10 6 65x10 4 6 5.8129 0.1871

2% 20 10 6 55x10 4 6 5.7403 0.2597

2% 30 10 6 40x10 4 6 5.6020 0.3980

2% 40 10 6 20x10 4 6 5.3010 0.6990

2% 50 10 6 0x10 4 6 00 6.0000

2% 60 10 6 0x10 4 6 00 6.0000

5 Chlorhexidine 2% 10 10 6 85x10 4 6 5.9294 0.0706

2% 20 10 6 75x10 4 6 5.8750 0.125

2% 30 10 6 65x10 4 6 5.8129 0.1871

2% 40 10 6 50x10 4 6 5.6989 0.3011

2% 50 10 6 40x10 4 6 5.6020 0.398

2% 60 10 6 20x10 4 6 5.3010 0.699


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Sr. No Disinfeactant Conc. Timemin


1 Dettol 2% 10 10 6 90x10 4 6 5.9030 0.0970

2% 20 106

85x104

6 5.9294 0.07062% 30 10 6 80x10 4 6 5.9.30 0.09702% 40 10 6 70x10 4 6 5.8450 0.15502% 50 10 6 60x10 4 6 5.7781 0.22192% 60 10 6 50x10 4 6 5.6989 0.3011

2 Phenol 2% 10 10 6 75x10 4 6 5.8750 0.12502% 20 10 6 70x10 4 6 5.8450 0.15502% 30 10 6 60x10 4 6 5.7781 0.22192% 40 10 6 45x10 4 6 5.6532 0.34682% 50 10 6 25x10 4 6 5.3979 0.60212% 60 10 6 0000 6 00 6.0000

3 Lyzol 2% 10 10 6 85x10 4 6 5.9294 0.07062% 20 10 6 80x10 4 6 5.9030 0.09702% 30 10 6 75x10 4 6 5.8750 0.12502% 40 10 6 65x10 4 6 5.8129 0.18712% 50 10 6 50x10 4 6 5.6982 0.30182% 60 10 6 30x10 4 6 5.4771 0.5229

4 Glutaraldehyde 2% 10 10 6 65x10 4 6 5.8129 0.18712% 20 10 6 55x10 4 6 5.7403 0.25972% 30 10 6 40x10 4 6 5.6020 0.39802% 40 10 6 20x10 4 6 5.3010 0.69902% 50 10 6 00 6 00 6.00002% 60 10 6 00 6 00 6.0000

5 Chlorhexidine 2% 10 10 6 95x10 4 6 5.9777 0.02232% 20 10 6 90x10 4 6 5.9549 0.0451


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1.Bacterial identification of disinfectant resistant bacteria:

Sr. No. Isolates Identification

1 Sa1, Sa2, Sa3, Sa4, Sa5, Sa6, Sa7,

Sa8, Sa9, Sa10.

Staphylococcus aureus

2 Ps1, Ps2, Ps3, Ps4 Ps5, Ps6, Ps7, Ps8,Ps9, Ps10.

Pseudomonas areuginosa

3 E1,E2,E3,E4,E5,E6,E7,E8,E9,E10 Enterococcus spp.4 Ec1,Ec 2,Ec 3,Ec4,Ec5,Ec6,Ec7,Ec8,Ec9,Ec1

0

Escherichia coli


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1.Test of sensitivity to some biocides:

Chlorhexidine is also effective at higher concentration.Study of Thomas et al carry out some experiments whichgiving an idea that reflects, expose of biocides to a sub-inhibitory concentration increases MIC of biocides tohospital bacteria. It is reasonable to assume that in aclinical environment and in the hospital effluent bacteriawill be exposed to sub inhibitory amount of biocidesremaining in environment .Several investigators have analyzed MICs to assess the

susceptibility of antibiotic-resistant pathogens todisinfectants.


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Sr.No

Bacteria MIC of disinfectants (M)

Phenol Lysol Chlorhexidine Dettol Glutaraldehyde

1 E.coli 60 65 80 60 552 Enterococci 80 70 90 80 65

3 S. aureus 100 80 110 100 85

4 Ps. areuginosa 100 110 130 120 95

TABLE 5.19 Bacterial Tolerances to Disinfectant: MIC value


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Sr.No Bacteria CFU/100MLPhenol Lysol Chlorhexidine Dettol Glutaralde

hyde

1 E.coli 20x10 6 30x10 6 20x10 6 50x10 6 10x10 6

2 Enterococci 25x10 6 30x10 6 20x10 6 40x10 6 20x10 6

3 S. aureus 25x10 6 30x10 6 30x10 6 50x10 6 20x10 6

4 Ps. areuginosa 20x10 6 40x10 6 40x10 6 60x10 6 25x10 6


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1.Antibiotic Susceptibility Testing: E.coli

Sr.No.

Antibiotics No. of isolates %

Tested Resistant Sensitive Intermediate R S I

1 Amoxicillin 10 10 00 00 100 00 00

2 Streptomycin 10 04 05 01 40 50 10

3 Tobramycin 10 04 06 00 40 60 00

4 Erythromycin 10 08 00 02 80 20 00

5 Piperacillin 10 10 00 00 100 00 00

6 Imipenem 10 08 02 00 80 20 00

7 Gentamycin 10 05 05 00 50 50 008 Ciprofloxacin 10 01 08 01 10 80 10

9 Co-trimaxazole 10 01 04 05 10 40 50


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Sr.No. Antibiotics No. of isolates %Tested Resistant Sensitive Intermediate R S I

1 Ofloxacin 10 02 08 00 20 80 00

2 Nalidixic acid 10 10 00 00 100 00 00

3 Norfloxacin 10 02 08 00 20 80 00

4 Amoxicillin 10 10 00 00 100 00 00

5 Carbenicillin 10 10 00 00 100 00 00

6 Piperacillin 10 10 00 00 100 00 00

7 Ampicillin 10 10 00 00 100 00 008 Amikacin 10 00 10 00 00 100 00

9 Ciprofloxacin 10 00 10 00 00 100 00

1.Antibiotic Susceptibility Testing: Enterococcus spp.


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1.Antibiotic Susceptibility Testing: S. aureus .

Sr.No.



1 Amoxicillin 10 08 02 00 80 20 00

2 Ceftriaxone 10 10 00 00 100 00 00

3 Tobramycin 10 10 00 00 100 00 00

4 Lomefloxacin 10 10 00 00 100 00 00

5 Norfloxacin 10 00 10 00 00 100 00

6 Kanamycin 10 10 00 00 100 00 00

7 Vancomycin 10 09 01 00 90 10 00

8 Erythromycin 10 07 03 00 70 30 00

9 Amikacin 10 10 00 00 100 00 00

10 Gentamycin 10 08 02 00 80 20 00


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1.Antibiotic Susceptibility Testing: Pseudomonas spp.

Sr.

No.



1 Penicillin 10 09 01 00 90 10 00

2 Kanamycin 10 06 04 00 70 30 00

3 Tetracycline 10 10 00 01 00 90 10

4 Chloramphenicol 10 08 02 00 100 00 00

5 Amoxicillin 10 10 00 00 100 00 00

6 Ciprofloxacin 10 10 00 00 30 70 00

7 Gentamycin 10 09 01 05 40 10 50

8 Ceftriaxone 10 10 00 01 90 00 10

9 Cotrimaxazole 10 10 00 00 10 90 00

10 Carbenicillin 10 10 00 00 100 00 00


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This study giving information regarding in between antibioticresistance and disinfectant resistance among hospital bacteria.

SIGNIFICANCE:


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CONCLUSION

Disinfectants present in the hospital

waste and wastewater may act asselective pressure for the retention of the plasmids that often contain thegenes for resistance to numerous

antibiotics. It is generally agreed that the selection and dissemination of resistant bacteria in nature should beavoided in order to ensure effectivetreatment against infectious diseases inhumans and maintain an ecologicalbalance that favors the predominance of a susceptible bacterial flora in nature.The indiscriminate use of antimicrobial 39


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Krutarth Ppt 9 April