International Journal of Pharma and Bio Sciences CLINICAL DISTRIBUTION AND ANTIBIOTIC RESISTANCE PATTERN OF NON- FERMENTING GRAM NEGATIVE BACILLI.

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1 International Journal of Pharma and Bio Sciences RESEARCH ARTICLE MICROBIOLOGY CLINICAL DISTRIBUTION AND ANTIBIOTIC RESISTANCE PATTERN OF NON- FERMENTING GRAM NEGATIVE BACILLI. S.JAYANTHI AND M.JEYA* Department of Microbiology, Chettinad Hospital and Research Institute, Chettinad University, Chennai, Tamilnadu, India. M.JEYA Department of Microbiology, Chettinad Hospital and Research Institute, Chettinad University, Chennai, Tamilnadu, India. ABSTRACT Nonfermenting Gram negative bacilli (NFGNB) emerged as important nosocomial pathogen causing opportunistic infections. Aim of the study is to detect the clinical distribution and antibiotic resistant pattern of nonfermenting Gram negative bacilli isolated from the clinical samples and to detect Mettalobeta lactamase production by multidrug resistant isolates. A total of 6284 clinical samples were processed for the period of six months. The species of the NFGNB isolates were identified by API ID 32 GN system (bio merieux).screening for Metallo beta lactamase (MBL) production by combined disc test method was done on the multidrug resistant isolates. Out of 6284 clinical samples 327 (5.2%) nonfermenting Gram negative bacilli were isolated. The split up of NFGNB were Pseudomonas aeruginosa 135(41.2%),Pseudomonas species other than P.aeruginosa were 91(27.8%), Acinetobacter species 88(26.9%) and others 13(3.9%). Multidrug resistant strains among these isolates were 129 (39.4%) and the metallo beta lactamase enzyme producers were 40 (31%). B - 487

2 KEY WORDS Nonfermenting gram negative bacilli, drug resistance, metallo betalactamase INTRODUCTION Nonfermenting Gram negative bacilli (NFGNB) emerged as important nosocomial pathogen causing opportunistic infections. 1,2 Pseudomonas aeruginosa and Acinetobacter baumanii are most common nonfermenters which are pathogenic to humans. They are generally saprophytic in nature. 3,4 These isolates have also been responsible for serious infections such as septicaemia and pneumonia. 5 Pseudomonas aeruginosa showing resistance to carbapenem which is currently the most effective treatment option and the number of reported 6,7 incidences are gradually increasing. Resistance to carbapenems is often mediated by production of metallobetalactamase (MBL) a class of B type of beta- lactamase that require bivalent metal ions, usually zinc for their activity. 6,7 The emergence of multi drug resistant strains of Acinetobacter species, that suddenly causes an outbreak of infection involving several patients in clinical units are reported. 8,9 Within the genus, Acinetobacter baumanii appears to be the species of greatest clinical importance. The Acinetobacter baumanii and Acinetobacter baumanii complex contains isolates that are multiresistant to antibiotics and that have been responsible for many outbreaks of infection throughout the world. 10 The process influenced by various risk factors like surgery, endotracheal tube, intravascular, ventricular and urinary catheters can result in colonization by this opportunistic bacilli. It gets disseminated via the hands of staff often remains undetected. This study gives the clinical distribution and antibiotic resistance pattern of the clinical isolates of nonfermenters in a tertiary care hospital. MATERIALS AND METHODS Source of bacterial strains: Nonfermenters were isolated and identified from various clinical samples received during the period of six months from June to December 2010 in a tertiary care hospital. Total samples of 6284 were processed includes urine, blood, sputum, swabs from ear, nose, throat, wound, pus, CVP tip, pleural effusion and tracheal aspirates. From 6284 samples, 328 NFGNB were isolated. They were identified by routine colony morphology and biochemical reactions as described in Bailey and Scotts diagnostic microbiology. 11 The strains for which the species could not be identified by routine; tests were identified by using API ID 32 GN automated system (Biomeriux).All the nonfermentors isolated were subjected for antimicrobial susceptibility testing according to the CLSI guidelines and the results were interpreted. 12 The isolates showing multidrug resistance (resistance to more than three classes of drugs) were again tested for MBL production. The list of antibiotics tested were ampicillin (10µg) of the synthetic penicillin, amikacin (30µg), gentamicin (10µg) of the aminoglycosides, ciprofloxacin (5µg) ofloxacillin (5µg),norfloxacillin (10µg), of quinolones, cefotaxime (30µg), ceftazidime (30µg), cefepime (30µg), of cephalosporins, piperacillin tazobactum (100/10µg)of the betalactum inhibitor antibiotics, imepenem (10µg), meropenem (10µg) of carbapenems, aztreonam (30µg) of monobactum, and cotrimoxazole (23.75/1.25µg). Eight categories of antibiotics were tested. 4. Bacterial strains that demonstrated B - 488

3 resistance to three or more categories of antibiotics were defined as multidrug resistant isolates. MBL Screening Procedure (combined disc test) Screening for MBL production was done in multidrug resistant strains by combined disc test using imepenem (10µg) and imepenem with EDTA (10µg/750µg) discs. 13,14 The imepenem EDTA combined disk test was performed as described by Young et al. 17 Test organisms were inoculated on to Muller Hinton agar plates. Two 10µg imepenem disks were placed with about 30mm distance apart on the plate and 10µL of EDTA solution was added to one of disks to obtain the desired concentration of EDTA (750µg).The inhibition zones of the imepenem and imepenem EDTA disks were compared after hrs of incubation at 35 0 C. In the combined disc test, if the zone of inhibition with the imepenem and EDTA disc was7mm greater than plain imepenem disc, it was considered as positive MBL production. 13,14,15 (Fig -1) Figure 1 Enhanced Zone of inhibition in imepenem with EDTA than imepenem alone RESULTS From 6284 clinical samples cultured, 327 (5.2%) nonfermenting Gram negative bacilli were isolated. The split up was Pseudomonas aeruginosa 135(41.2%), Pseudomonas species 91(27.8%), Acinetobacter species 88 (26.9%) and others 13 (3.9%). Among the NFGNB, Pseudomonas aeruginosa were more prevalent than Acinetobacter baummanni (Table1). The clinical samples from the inpatients yielded more number of the nonfermenting gram negative isolates than out patients (Table 2). B - 489

4 Clinical samples Total no. of samples ( 6284 ) Table 1 Clinical distribution of nonfermentors. Total no. of nonfermenters 327 (5.2 %) Pseudomonas aeruginosa 135( 41.2% ) Pseudomonas species 91(27.8 % ) Acinetobacter species 88 (26.9% ) Blood Respiratory Urine Exudates Pus/ bodyfluids Others 13 (3.9% ) Clinical distribution of nonfermentors The prevalence of NFGNB were higher from the exudate samples (43.4%), followed by the respiratory samples (35.4 %), urine (11.6%) and blood samples (9.4%).The overall drug resistance to different classes of drugs were higher among Acinetobacter baumanii than Pseudomonas species. The split up of Pseudomonas species (27.8%) isolated other than P.aeruginosa were 10% P. stutzeri, 6.1% P. orizihabitans,10.7% P.putida, 0.6% P.fluorescence. Other species isolated were 0.3% Ralstonia picketti, 1.2% Stenotrophomonas maltophillia, and 2.4% Burkholderia cepacia. All the Acinetobacter isolated from the clinical samples were Acinetobacter baumanii.. Table 2 Nonfermenting Gram negative bacilli isolated from OP and IP Clinical isolates Pseudomonas aeruginosa Pseudomonas species Acinetobacter species Out Patient isolates S R Inpatient isolates S R S-sensitive strains, R-resistant strains Drug resistance pattern of P.aeruginosa The resistant pattern of Pseudomonas isolates to specific anti - Pseudomonal drugs such as carbenicillin was 22%, followed by aztreonam 24.4% and tobramycin 27.4%. Among the aminoglycosides, gentamycin (30.3%) demonstrated higher resistance than amikacin (15.5%), in quinolones, ofloxacillin ( %) than ciprofloxacillin (24-31%), of the third generation cephalosporins the ceftrioxazone (25.1%) showed less resistance than ceftazidime (34%) and cefotaxime (37.7%).The betalactum inhibitors such as cefeperazone sulbactum demonstrated 26.6% resistance and piperacillin tazobactum 22.9%. The resistance towards carbapenem drugs such as meropenem (14.8%) was higher than imepenem (6.6%). In-vitro resistance pattern of polymyxin (4.4%) was lesser compared to colistin (6.6%)(Fig 2). B - 490

5 Figure 2 Percentage of the resistance exhibited by Pseudomonas aeruginosa. Drug resistance pattern of Acinetobacter The resistant pattern of Acinetobacter for ampicillin was 15%, cotrimoxazole was 7.8% only. Among aminoglycosides, gentamycin demonstrated higher resistance (39.3%) than amikacin (15.5%), in quinolones, ciprofloxacillin (3.4%) showed significantly lesser resistance than ofloxacillin (39.3%),of the cephalosporins, third generation showed higher resistance than other generations. The resistant pattern of betalactum inhibitors such as cefeperazone sulbactum (34.8%) and piperacillin tazobactum (33.7%) were similar. The resistance towards carbapenem drugs were 21.3% for meropenem and 22.4% for imepenem. In vitro resistance pattern of polymixin was 2.2% and to colistin 1.1%.(Fig 3) Among the NFGNB 129 multidrug resistant isolates were tested for the metallo beta lactamase (MBL) production by combined disc diffusion method. Out of which 40(31%) (Table 3) isolates exhibited MBL production. Table 3 Multi drug resistant Nonfermenting Gram negative bacilli and MBL producers. Clinical isolates MDR NFGNB MBL producers Pseudomonas aeruginosa ( 135) 48 (35.5%) 12 (25%) Pseudomonas species ( 91) 32 (35.1% ) 9 (28.1% ) Acinetobacter species (88 ) 49 (55.6% ) 19 ( 38.7%) Total (314 ) 129 (41.1%) 40 ( 31%) B - 491

6 Figure 3 Resistant pattern of the isolates of Acinetobacter baumannii from the clinical samples DISCUSSION Nonfermentors are ubiquitous in the environment. Of the total clinical samples tested 5.2% yielded nonfermenting gram negative bacilli. Among the NFGNB isolated Pseudomonas aeruginosa (41.2%) were higher than the Acinetobacter (26.9%). The prevalence of the multidrug resistant strains were 41.1 % which is in concordance with Behera et al, The prevalence of the metallo beta lactamase producers among the multidrug resistant strains were 31%. Resistance of nonfermentors to amikacin (15%) was remarkably lower than gentamycin (29-39%) which is similar to the studies of Taneja N et al, Resistance to the mono ring betalactam aztreonam for Pseudomonas (20-25 %) was less when compared to other studies (38%) Srinivasa rao et al, The resistance of Pseudomonas species to the fluorinated quinolones such as norfloxacillin was nil ( for urinary isolates) and 23 to 35% for ciprofloxacin and 35 to 40% to ofloxacin. This is lesser when compared with other studies 13,16,20. The first generation (cephalexin 13%), second generation cefalosporins (Cefuroxime 15-16%) demonstrated less resistance than the third generation cephalosporins (Cefotaxime 37-41%, Ceftazidime %, Ceftriaxone %).Since third generation cephalosporins are most commonly prescribed by clinicians they show higher resistance than first and second generation cephalosporins. The resistance pattern towards the fourth generation Cefipime and Cefperome were 20-40% similar to Shobha et al, The overall resistance pattern for Acinetobacter isolates (55.6 %) was higher than, Pseudomonas isolates (35.5 %) which is concordance with other studies 21. Carbapenem has always been regarded as a dependable drug for treating Pseudomonas aeruginosa infection. Due to the increasing clinical use, the problem of resistance to B - 492

7 carbapenem is gradually getting worse. 16,18 In our study carbapenem resistant strains exhibited significantly higher frequencies of resistance to other group of beta- lactam antibiotics (ceftazidime, cefepime, cefperazone sulbactum, Piperazine tazobactum, gentamycin, ofloxacillin) than non-carbapenem resistant strains. 14,15 The other nonfermentors Stenotrophomonas maltophilia, Burkholderia cepacia, Ralstonia picketti were sensitive strains in our study. Even though the Burkholderia species was reported 11 to have high intrinsic resistance to antimicrobial compounds, it did not correlate to our study. Over fifty percent of strains of Acinetobacter species isolated from the clinical samples, showed resistance to the various antibiotics similar to other studies 15. Acinetobacter baumanii is provoking more concentration because of potential ability to form bioflim might also explain its outstanding antibiotic resistance, survival properties, and increased virulence. The multi drug resistant strains were of 48/135(35.5%) Pseudomonas aeruginosa, 32/91(35.1%) Pseudomonas species other than P.aeruginosa and 49/88 (55.6%) Acinetobacter species similar to other studies 20. Combined disk method of detection of metallobetalactamase producers were cost effective and comfortable method in concordance with Lee et al, Great importance should be given to resistance surveillance and proper drug administration in accordance with the sensitivity test results, and following proper antibiotic policy is essential to minimize drug resistance. 15 REFERENCES 1. Shahid.M, Malik.A,Plasmid mediated amikacin resistance in clinical isolates of Pseudomonas aeruginosa.indian J Med Microbiol,22(3): (2004). 2. Taneja.N, Maharwal.S, Meera Sharma, Imepenam resistance in nonfermenters causing nosocomial urinary tract infections.indian J Med science,57:294(2003). 3. Shahid.M, Abida Malik, Sheeba,Multidrug resistant Pseudomonas aeruginosa strains harbouring R plasmids & Amp C beta lactamases isolated from hospitalized burn patients in tertiary care hospital.federation of European Microbiol Societies, (2003). 4. Mackie and McCartney.A text book of Practical Medical Microbiology,14 edi (Churchill Livingstone) New york, (1996). 5. Manoharan.A, Chatterjee.S.Mathai.D,Detection & Characterization of Metallobetalactamases producing Pseudomonas aeruginosa SARI study group.indian J Med Microbiol,28(3):241-4(2010). 6. Pitout J.D, Gregson, D.B,Poirel.L, Mc.llure,JA Le P, Church DL,Detection of Pseudomonas aeruginosa producing metallo beta lactamases in a large centralized laboratory.j.clinic. Microbiol, 43: (2005). 7. Cirioni O,Ghiselli R,Silverstri C, Kamysz W, Orlando F, Mocchegiani F,et al, Efficacy of tachyplesin III, colistin & Imipenem against a multiresistant Pseudomonas aeruginosa strain.antimicrobial agents chemotherapy, 51: (2007). 8. Bush.K,Beta lactamases of increasing clinical importance.current Pharm.des, 5: (1999). 9. Joly Guillou,Clinical impact & pathogenicity of Acinetobacter. European society Microbiol and Infec Dis, 01227: (2005). B - 493

8 10. Tower KJ,Clinical importance & Antibiotic resistance of Acinetobacter species.j.med.microbiol, 46: (1997). 11. Gautam.V, Ray.P, Chatterjee.SS, Das.A, Sharma.K,Rana.S et al,identification of lysine positive non fermenting Gram negative bacilli (Stenotrophomonas maltophilia and Burkholderia cepacia complex).indian J Med Microbiol,27(2):128-33(2009). 12. Agarwal KC, Antibiotic sensitivity test by disc diffusion method. Indian J Pathol Microbiol,17:149-59(1994). 13. Yu lingling,yang Jin Hong,Yang HaiWei,Distribution and drug resistance of nonfermenters in intensive care units.j Disease surveillance,24(1):50-53(2009). 14. Dongeun Yong,Yeoung seon choi, Kyoung HoRoh,Chang KiKim,Youn Hee, Park,Increasing prevalence and Diversity of Metallo-betalactamases in Pseudomonas spp,acinetobacter spp and Enterobacteriaceae from Korea.Antimicrobial Agents and Chemotherapy, p (2006). 15. Cheng-Hong Yang, Shaoyung Lee, Pai-Wei Su, Cheng-San Yang, Li- Yeh Chuang, Genotype and antibiotic susceptibitlity patterns of drug resistant pseudomonas aeruginosa and Acinetobacter baumanii isolates in Taiwan.Microbial drug resistance, Dec14(4): (2008). 16. Behera.B,Mathur.P, Das.A, Kapil A, Sharma.V,An Evaluation of four different phenotypic techniques for detection of Metallo beta lactamase producing Pseudomonas aeruginosa.indian J Med Microbiol, 26(3): (2008). 17. Young D, Lee K,Yum JH, Shin HB, Rossolini GM,Chong Y,Imepenem- EDTA disk method for differentiation of metallo beta lactamases producing clinical isolates of Pseudomonas spp and Acinetobacter spp.j Clin Microbiol,40: (2002). 18. Lee k, Lim YS, Young D, Yum JH, Chong Y,Evaluation of the Hodge test and the Imepenem EDTA double disk synergy test for differentiation of metallo β lactamase producing isolates of Pseudomonas spp and Acinetobacter spp.j Clin Microbiol,41:4623-9(2003). 19. Srinivasa Rao.R,Uma Karthika.R, Singh.SP, Shashikala. P.Kanungo,R,Jayachandran.S, Prashanth.K,Correlation between bioflim production and multiple drug resistance in imipenem resistant clinical isolates of Acinetobacter baumannii.indian.j.med Microbiol, 26(4):333-7,(2008) 20..Mohanasoundram.K.M,Antimicrobial resistance in Pseudomonas aeruginosa.j clinical and Diagnos Research, 5(3): ,(2008). 21. Irfan.S, Zafar.A, Guhar.D,Ahsan.T,Hasan.R, Metallo beta lactamase producing clinical isolates of Acinetobacter species and Pseudomonas aeruginosa from intensive care unit of a tertiary care hospital. Indian J Med Microbiol,26(3): ,(2008) 22. Shobha.KL, Mr.Gowrish, Rao.G, Mr.Anand, M Kukkumalla, Prevalance of non fermenters in urinary tract infections in a tertiary care hospital,webmed Central Microbiol,2(1):WM COO1464,(2008). B - 494

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