EXTENDED SPECTRUM BETA LACTAMASE PRODUCING MULTI DRUG RESISTANT (MDR) GRAM NEGATIVE BACTERIA FROM CLINICAL SAMPLES

Phenotypic detection of ESBL producing Gram negative bacilli in clinical samples such as urine and exudates Study of the antibiotic susceptibility pattern of the ESBL producers

Morphological Identification
`

Gram staining Biochemical test

Anti-biotic Susceptability test is done by Kirby Bauer Disc Diffusion method , mueller Hinton Agar is used as media , where it has good porosity and diffusion of antibiotics

PREPARATION OF INOCULAM: From the culture ,small volume is transferred into peptone water and
for 2hours at 370C incubated

INOCULATION:
A sterile cotton-wool swab is dipped into the suspension and surplus removed by rotation of the swab against the side of the tube above the fluid level Swabbing done all around petriplate and finally Inoculam is dried with closed lid

APPLICATION OF ANTIBIOTICS: A susceptibility disc containing Amoxicillin- Clavulanic acid disc (20/10 g) was placed in the centre of the plate and a disc of Cefotaxime (30 g) was placed 20 mm apart, centre to centre, from Amoxicillin-Clavulanic acid disc. The Aztreonam (30 g) disc was placed at 25 mm and the Ceftazidime (30 g) disc was placed at 30 mm and Piperacillin (110 g) Tazobactam (10 g), Piperacillin/Tazobactam (110 g) disc was placed 25 mm from Cefepime . single discs are applied with forceps and pressed gently to ensure even contact with the medium. The discs should be about 15mm from the edge of the plate and no closer than about 25mm from disc to disc.

INCUBATION: Plates are incubated for 16-18 hours (overnight) at 35 to 370C aerobically or in CO2 atmosphere

READING OF ZONES OF INHIBITION: The diameters of zones are measured to the nearest millimeter with a thin transparent millimeter scale on the under-surface of the plate without opening the lid. inhibition is judged by the naked eye INTERPRETATION OF THE ZONE SIZES: Zone size interpretation is done in susceptible, resistant, or intermediate- pattern susceptible - when the zone edge is outside the black circle, resistantwhen there is no zone, or when it lies within the white circle; and intermediate- when the edge of the zone of inhibition lies on the black circle. The results should be interpreted according to the critical diameters given in the Kirby-Bauers Standard Chart.

Petriplate shows zone of inhibition

TOTAL NUMBER OF SAMPLES PROCESS ED 150

TOTAL NUMBER GRAM NEGATIVE BACTERIA

ESBL producing organisms

Non ESBL producing organisms

Number

Percentage (%)

Number

Percentage (%)

39

15

38.47

24

61.53

The different organisms which cause ESBL production are E.coli, species of citrobacter, Klebsiella, Pseudomonas , proteus , morgella are detected from the samples like urine, exudates and their frequency of occurrence is interpret in the following table.
SNO Organisms Sample Percentage% Exudate 5.128 2.564 2.564 2.564 Exudate 1 2 3 4 E.coli Klebsella Citrobacter Psedomona s spp 5 Proteus spp. 6 Morgella TOTAL 5 1 34 12.814 2.568 87.186 3 7.692 2 1 1 1 Urine 27 2 1 Urine 69.230 5.128 2.564 -

120

Percentage Urine

Percentage Exudate

100 Organisms 80 Organisms

60

40

20

0 E.coli Klebsella Citrobacter Psedomonas sps Proteus sps. Morgella TOTAL

AGE 3months - 20 20-40 40-60 60-80 TOTAL

120

FEMALE 2.5% 15.3% 12.8% 23.07% 53.8%

MALE 7.6% 15.3% 15.3% 7.6% 46.1%

100

80

60

MALE FEMALE

40

20

0 3months - 20 20-40 40-60 60-80 TOTAL

Total ESBL 15

organism E.coli Klebsiella Citrobacter species

Percentage 13(86.6%) 1(6.6%) 1(6.6%)

Among the 15 ESBLs , E.coli is found to be 86.6 % ,klebsiella 6.6% and citrobacter species is 6.6 %.

ESBL producing organisms pose a major problem for clinical therapeutics. The incidence of ESBL producing strains among clinical isolates has been steadily increasing over the past few years . Indiscrimination use of antibiotics should be avoided A knowledge of resistance pattern of bacterial strains in a geographical area will help to guide the appropriate and judicious antibiotic use and formulate antibiotic policy.