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Extended spectrum [beta]-lactamase producing Enterobacteriaceae & antibiotic co-resistance.

Sir,

Extended spectrum [beta]-lactamases (ESBLs) are plasmid mediated enzymes inactivating [beta]-lactam antibiotics containing oxyimino group such as oxyiminocephalosporins and oxyimino-monobactam, except cephamycins and carbapenems (1). They are derived from the point mutation of plasmid determined TEM or SHV [beta]-lactamases (1,2). ESBLs are inhibited by clavulanic acid and placed under Bush's functional class 2be (3). Till date more than 200 different types of ESBLs have been described. In recent years, there is a dramatic increase in the prevalence of CTX-M type of ESBLs among clinical isolates of Enterobacteriaceae in Europe and Asia (4).

ESBLs are the most evolving mechanism of antibiotic resistance among the family Enterobacteriaceae due to the selective pressure imposed by inappropriate use of third generation cephalosporins, most often encountered in ICU settings (2). Plasmids coding for ESBL enzymes may carry co-resistance genes for other non-[beta]-lactam antibiotics, thus limiting the number of useful drugs against these bacteria (5-7). Reliable detection of ESBL production by clinical microbiology laboratory is essential to guide the clinicians to provide appropriate therapy. Hence this study was designed to know the presence of ESBLs among members of the family Enterobacteriaceae isolated at Victoria hospital, Bangalore and to know the antibiotic susceptibility pattern among ESBL producers and non-ESBL-producers.

A total of 239 consecutive, non-repetitive, clinical isolates of Enterobacteriaceae isolated from various clinical samples such as exudates (95), urine (71), sputum (54), blood (15) and vaginal swab (4) obtained between July 2009 and November 2009 were included in the study. Samples were processed and isolates were identified by standard laboratory methods (8).

Antibiotic susceptibility was determined by Kirby Bauer disc diffusion method according to Clinical Laboratory Standard Institute (CLSI) guidelines (9). Antibiotics were chosen depending on the organism and the sample and results were interpreted as sensitive or resistant as per CLSI recommendations (10).

ESBL was confirmed by CLSI described phenotypic confirmation method along with routine antibiotic susceptibility testing (9,10). A stock solution of clavulanic acid (2000 [micro]g/ml) was prepared, aliquoted into small vials and stored at -20[degrees]C. One vial was removed just before antibiotic susceptibility testing and 5 [micro]l of clavulunate solution was added to the cefotaxime (30 [micro]g) disc (Hi-Media, Mumbai). A lawn of test organism was made on Mueller-Hinton agar (MHA) after adjusting the inoculum to 0.5 McFarland and cefotaxime and cefotaxime/clavulunate discs were placed along with CLSI described antibiotic discs, incubated at 37[degrees]C for 18-24 h. A zone difference of >5 mm between cefotaxime and cefotaxime/ clavulunate was considered as confirmative for ESBL production.

Though CLSI described phenotypic confirmatory test is applicable for Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis, an attempt was made to look for ESBL production among other members of Enterobacteriaceae also. Throughout the study K. pneumoniae ATCC 700603 and E. coli ATCC 25922 (HiMedia Laboratories, Mumbai) were used as positive and negative controls respectively, for ESBL production.

Among the 239 Enterobacteriaceae isolates, 96 (40.2%) were E. coli, 79 (33.1%) K. pneumoniae, 26 (10.9%) Enterobacter spp, 21 (8.8%) Proteus spp, 15 (6.3%) Citrobacter spp and 2 (0.8%) Salmonella Typhi. Prevalence of ESBL is known to vary according to geographical regions (11,12). In our study, ESBL production was seen in 149 (62.3%) isolates of Enterobacteriaceae. Fifty four (22.6%) isolates were non-ESBL producers and 36 (15.1%) showed no zone difference in phenotypic confirmatory test. Such isolates showed uniform resistance to cefoxitin and all were susceptible to cefepime. On further testing, all the 36 isolates were found to be AmpC producers by modified three dimensional test (13).

ESBL production was more common among isolates obtained from exudates 67/95 (70%) followed by blood 10/15 (66.7%) and urine 42/71 (59.1%). Among the 239 isolates, 136 (56.9%) were obtained from inpatients and 103 (43.1%) from out patients. ESBL production was more among the isolates from inpatients 97/136 (71.3%) when compared to outpatients 52/103 (50.5%). The AmpC producing isolates among the in- and out-patients were 22/136 (16.2%) and 14/103 (13.6%) respectively.

ESBL production was higher in E. coli when compared to K. pneumoniae and Enterobacter spp. AmpC [beta]-lactamases were seen more among Citrobacter spp. followed by Proteus spp. and E. coli (Table I).

The presence of multi-drug resistance was higher among ESBL producers and carbapenems remained the most effective drug against such isolates (Table II). Non-betalactam antibiotic susceptibility among ESBL producing organisms showed least sensitive to co-trimoxazole (23.4%) followed by ciprofloxacin (29.5%) and gentamycin (46.9%).

In our study freshly prepared cefotaxime/ clavulunate discs were used for phenotypic confirmatory test.

The study showed ESBL producers were highly resistant to cefepime (97.3%) at standard inoculum, which is in contrast to the study of Thomson et al (14) who showed inoculum effect was more for cefepime among the ESBL producing Enterobacteriaceae.

The present study showed 15.1 per cent of isolates were pure AmpC producers. Among the 149 ESBL producers, 50 isolates though showed zone difference of >5mm in phenotypic confirmatory test, the combination with clavulanic acid did not enhance the zone to completely susceptible levels. Such phenotype may suggest production of both ESBL and AmpC or production of multiple [beta]-lactamases. Cefoxitin resistance in such isolates cannot be considered as indicator of AmpC production, as other mechanism of resistance such as porin channel mutation is also more often seen among ESBL producing organisms (15). Further studies are needed for appropriate detection of combined ESBL and AmpC enzyme production among such isolates.

Occurrence of ESBL producing Enterobacteriaceae at our centre was higher when compared to reports from other hospitals in India (11,12). The study indicated routine detection of ESBL production using phenotypic confirmatory test as simple, cost-effective and time saving method. Instead of screening and confirming ESBL production, direct phenotypic confirmatory test along with routine antibiotic susceptibility testing helped to report ESBL production within 48 h.

High degree of antibiotic co-resistance among ESBL producers emphasizes the judicious use of antimicrobials. Imipenem still remains most effective drug against ESBL producing organisms followed by piperacillin-tazobactam. The study showed phenotypic confirmatory test can reliably detect ESBL production among all the members of Enterobacteriaceae.

Acknowledgment

Authors thank Medrich Pharmaceuticals Pvt. Ltd., Bangalore, for providing pure form of potassium clavulunate for conducting the study.

Conflict of interest: None.

S.M. Rudresh & T. Nagarathnamma *

Department of Microbiology

Victoria Hospital

Bangalore 560 002

Karnataka, India

* For correspondence:

Dr Nagarathnamma T.

Prof. & Head of the Department of Microbiology

Victoria hospital campus

Fort, BMC & RI, Bangalore 560 002

Karnataka, India

rudreshsm@gmail.com

nagarathnammat@yahoo.co.in

References

(1.) Paterson DL, Bonomo RA. Extended-spectrum [beta]-lactamases: a clinical update. Clin Microbiol rev 2005; 18 : 657-86.

(2.) Bradford PA. Extended-spectrum [beta]-lactamases in the 21st century: Characterization, epidemiology and detection of this important resistance threat. Clin Microbiol rev 2001; 14 : 933-51.

(3.) Bush K, Jacoby GA, Medeiros AA. A functional classification scheme for [beta]-lactamases and its correlation with molecular structure. antimicrobagents Chemother 1995; 39 : 1211-33.

(4.) Bonnet R. Growing group of extended-spectrum [beta]-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother 2004; 48 : 1-14.

(5.) Schwaber MJ, Navon-Venezia S, Schwartz D, Carmeli Y. High levels of antimicrobial coresistance among extendedspectrum-[beta]-lactamase producing Enterobacteriaceae. Antimicrob Agents Chemother 2005; 49 : 2137-9.

(6.) Morosini MI, Garcia-Castillo M, Coque TM, Valverde A, Novais A, Loza E, et al. Antibiotic coresistance in extendedspectrum-[beta]-lactamase producing-Enterobacteriaceae and in vitro activity of tigecycline. Antimicrob Agents Chemother 2006; 50 : 2695-9.

(7.) Agrawal P, Ghosh AN, Kumar S, Basu B, Kapila K. Prevalence of extended-spectrum [beta]-lactamases among Escherichia coli and Klebsiella pneumoniae isolates in a tertiary care hospital. Indian J Pathol Microbiol 2008; 51 : 139-42.

(8.) Enterobacteriaceae. In: Win WC, Allen SD, Janda WM, Koneman EW, Procop GW, Schreckenberger PC, Woods G, editors. Colour atlas & text book of diagnostic microbiology, 6th ed. Philadelphia: Lippincott Williams and Wilkins; 2006. p. 211-302.

(9.) Clinical and Laboratory Standards Institute (CLSI). M2-A9. Performance standards for antimicrobial disk susceptibility tests; approved standard, 9th ed. Wayne, PA: CLSI; 2006.

(10.) Clinical and Laboratory Standards Institute (CLSI). M100-S17. Performance standards for antimicrobial susceptibility testing; 17th informational supplement: Wayne, PA: CLSI; 2007.

(11.) Sridher Rao PN, Basavarajappa KG, Krishna GL. Detection of extended spectrum beta-lactamase from clinical isolates in Davangere. indian J Pathol Microbiol 2008; 51 : 497-9.

(12.) Kumar MS, Lakshmi V, Rajagopalan R. Occurrence of extended-spectrum P-lactamases among Enterobacteriaceae spp. isolated at a tertiary care institute. indian J Med Microbiol 2006; 24 : 208-11.

(13.) Singhal S, Mathur T, Khan S, Upadhyay DJ, Chugh S, Gaind R, et al. Evaluation of methods for Amp C [beta]-lactamase in gram negative clinical isolates from tertiary care hospitals. Indian J Med Microbiol 2005; 23 : 120-4.

(14.) Thomson KS, Moland ES. Cefepime, piperacillin-tazobactam, and the inoculum effect in tests with extended-spectrum [beta]-lactamase-producing Enterobacteriaceae. antimicrob agents Chemother 2001; 45 : 3548-54.

(15.) Ananthan S, Subha A. Cefoxitin resistance mediated by loss of a porin in clinical strains of Klebsiella pneumoniae and Escherichia coli. indian J Med Microbiol 2005; 23 : 20-3.
Table I. Comparison of ESBL production among clinical isolates

Organism ESBL+ (%) ESBL- (%) Amp C (%) Total

E. coli 65 (67.7) 15 (15.6) 16 (16.7) 96
K. pneumoniae 50 (63.3) 22 (27.8) 7 (8.9) 79
Enterobacter spp. 15 (57.7) 9 (34.6) 2 (7.7) 26
Proteus spp. 12 (57.1) 3 (14.3) 6 (28.6) 21
Citrobacter spp. 7 (46.7) 3 (20) 5 (33.3) 15
Salmonella Typhi 0 (00) 2 (100) 0 (00) 2

Total 149 (62.3) 54 (22.6) 36 (15.1) 239

Table II Comparison of antibiotic susceptibility pattern of ESBL,
non-ESBL and AmpC producing Enterobacteriaceae

Drug I PT NT C AK G

ESBL+ (%) 100 95.3 76.5 69.1 67.7 46.9
ESBL- (%) 100 100 98.1 88.8 94.4 88.8
AmpC (%) 100 69.4 41.6 55.5 41.6 38.8

Drug CF CO CPM NF NX CB

ESBL+ (%) 29.5 23.4 2.6 92.8 14.2 7.1
ESBL- (%) 79.6 70.3 100 93.3 53.3 60
AmpC (%) 22.2 22.2 100 85.7 28.5 00

I, Imipenem (10 [micro]g); PT, piperacillin/tazobactam
(100/10 [micro]g); NT, netilmicin (30 [micro]g); C, chloramphinicol
(30 [micro]g); AK, amikacin (30 [micro]g); G, gentamicin
(10 [micro]g); CF, ciprofloxacin (30 [micro]g); CO, co-trimoxazole
(1.25/23.75 [micro]g); CPM, cefepime (30 [micro]g); NF, nitrofurantoin
(300 [micro]g); NX, norfloxacin (10 [micro]g); CB, carbenicillin
(100 [micro]g)
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Title Annotation:Correspondence
Publication:Indian Journal of Medical Research
Article Type:Report
Geographic Code:9INDI
Date:Jan 1, 2011
Words:1767
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