Deepak Juyal, Rajat Prakash, Shamanth A. Shanakarnarayan, Munesh Sharma, Vikrant Negi, Neelam Sharma

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1 [Downloaded free from on Thursday, May 15, 2014, IP: ] Click here to download free Android application for this jou Original Article Prevalence of non fermenting gram negative bacilli and their in vitro susceptibility pattern in a tertiary care hospital of Uttarakhand: A study from foothills of Himalayas Deepak Juyal, Rajat Prakash, Shamanth A. Shanakarnarayan, Munesh Sharma, Vikrant Negi, Neelam Sharma Department of Microbiology and Immunology, Veer Chandra Singh Garhwali Government Medical Sciences and Research Institute, Srinagar Garhwal, Uttarakhand, India Address for correspondence: Mr. Deepak Juyal, Department of Microbiology and Immunology, Veer Chandra Singh Garhwali Government Medical Sciences and Research Institute, Srinagar Garhwal, Uttarakhand, India. E mail: deepakk787@gmail.com Abstract Introduction: Non fermenting gram negative bacilli (NFGNB) are taxonomically diverse group of pathogens that has emerged as a major cause of health care associated infections especially in immunocompromised hosts. Identification of NFGNB and monitoring their susceptibility pattern are important for proper management of infections caused by them. Prevalence and antibiogram of NFGNB has not yet been reported from this part of India. Aim of the present study was to characterize the prevalence of NFGNB along with their antimicrobial sensitivity pattern among the patients coming to our hospital a tertiary care center. Materials and Methods: A total of 2585 various clinical specimens were received in laboratory and were processed. Non fermenters were identified using a standard protocol. Antimicrobial susceptibility testing was performed by Kirby Bauer disc diffusion method. Results: Among 2585 clinical samples 241 yielded NFGNB accounting for an isolation rate of 9.32% and a total of 246 non fermenters were grown as five samples yielded two types of NFGNB. Pseudomonas species (49.59%) and Acinetobacter species (43.09%) were the most commonly isolated NFGNB. A high level of antibiotic resistance was recorded for most of the first and second line drugs. Imipenem and amikacin were the drugs with maximum activity. Overall imipenem resistance was found to be 30.54%. Conclusion: Identification of NFGNB and monitoring their susceptibility patterns will help in proper management of infections caused by them. Improved antibiotic stewardship and infection control measures will be needed to prevent or slow the emergence and spread of multidrug resistant NFGNB in the healthcare setting. Key words: Acinetobacter, antibiotics, nonfermenters, nosocomial infection, Pseudomonas INTRODUCTION Aerobic non fermenting gram negative bacilli (NFGNB) are a taxonomically diverse group of organisms that either do not utilize glucose as a source of energy or utilize it oxidatively. [1] They are saprophytic in nature but can cause a significant number of infections particularly in hospitalized patients, immunocompromised hosts and patients with hematological malignancies. [2] Inherent resistance of these bacterial agents to commonly used disinfectants and there tendency Website: DOI: / Access this article online Quick Response Code to colonize various surfaces have been pivotal in their emergence as important nosocomial pathogens [3] NFGNB are known to account for 15% of all bacterial isolates from clinical microbiological laboratory. [4] This heterogeneous group includes organisms like Pseudomonas.spp, Acinetobacter.spp, Alkaligenes.spp, Stenotrophomonas maltophilia, Burkholderia cepacia complex (BCC). Currently Pseudomonas aeruginosa and Acinetobacter baumannii are the most commonly isolated nonfermenters pathogenic for humans. Infections caused by other species are relatively infrequent. [5] In recent years due to the indiscriminate use of antimicrobials, NFGNB have emerged as important health care associated pathogens. They have been incriminated in infections such as bacteremia, meningitis, pneumonia, urinary tract infections, surgical site infections, wound infections, osteomyelitis etc. [6] Risk factors include immunosuppression (oncology patients on cytotoxic therapy/radiotherapy, organ transplant patients and even patients with AIDS), neutropenia, mechanical ventilation, cystic fibrosis, indwelling catheters, invasive diagnostic 108 Saudi Journal for Health Sciences - Vol 2, Issue 2, May-Aug 2013

2 and therapeutic procedures. Prolonged hospital stay broad spectrum antibiotic use and underlying host factors are the best predictors of outcome. [7] Multidrug resistances is common and increasing among NFGNB and they are known to produce extended spectrum beta lactamases (ESBL s) and metalo beta lactamases (MBL s). [6] As Pseudomonas and Acinetobacter are the most predominantly isolated NFGNB so carbapenem resistance among them is of major concern. Carbapenemase activity in A.baumannii is mainly due to carbapenem hydrolyzing class D b lactamases (CHDLs) that is mostly specific for this species. These enzymes belong to 3 unrelated groups of clavulanic acid resistant b lactamases represented by OXA 23, OXA 24, and OXA 58 that can be either plasmid or chromosomally encoded. [8] In case of P.aeruginosa the dominant mechanism of carbapenem resistance is loss of carbapenem specific porin OprD2. [9] Number of strains have now been identified that exhibit resistance to essentially all commonly used antibiotics. There are few studies from India that provide identification and antimicrobial susceptibility pattern of NFGNB especially. [4,10 18] Prevalence of NFGNB and their antibiogram has not yet been reported from this part of India and the study was therefore taken up to close this gap in our knowledge. To the best of our knowledge this is the first report on prevalence and antibiogram of NFGNB from Garhwal hills of Uttarakhand State. Aim of the present study was to isolate, identify and characterize the prevalence of NFGNB along with their antimicrobial sensitivity pattern among the patients coming to our hospital a tertiary care center. MATERIALS AND METHODS This study was conducted for a period of 6 months (January 2012 to July 2012) in a tertiary care hospital of Uttarakhand state, India with a total capacity of 550 beds including 30 Intensive care unit (ICU) beds. A total of 2585 clinical specimens were received in laboratory which included urine (791), blood (657), pus (463), sputum and other respiratory samples (295), ear swab (238) and other body fluids (141). Samples were plated on blood agar (BA) and Mac Conkey s agar (MA) and incubated at 37 C for 48 hours before being reported as sterile. The isolates that showed non lactose fermenting (NLF) colonies on MA and failed to acidify the butts of triple sugar iron (TSI) agar were provisionally considered as NFGNB and they were further identified by using a standard protocol for identification. [1] The characters assessed were gram staining morphology, motility (by hanging drop), catalase test, oxidase test, citrate utilization, urea hydrolysis, hemolysis on 5% sheep blood agar, growth on 6.5% NaCl, nitrate reduction, pigment production, indole production, lysine and ornithine decarboxylation, arginine dihydrolase test, growth at 40 C and 42 C, oxidation of 1% glucose, lactose, sucrose, maltose, mannitol, xylose (Hugh and Leifson s medium), growth on 10% lactose agar and gelatin liquefaction test. Antimicrobial sensitivity was determined by Kirby Bauer disc diffusion method on Muller Hinton agar (MHA). Briefly a suspension of each isolate was made so that the turbidity was equal to 0.5 McFarland standards and then plated as a lawn culture onto MHA. Antibiotic discs were placed and plates were incubated at 37 C for hrs. Results were interpreted in accordance with central laboratory standards institute (CLSI) guidelines. [19] Escherichia coli ATCC and Pseudomonas aeruginosa ATCC were used as control strains. All dehydrated media, reagents and antibiotic discs were procured from Hi Media Laboratories Pvt. Ltd, Mumbai, India. RESULTS Among 2585 clinical samples, NFGNB were isolated from 241 samples accounting for an isolation rate of 9.32%. A total of 246 NFGNB were isolated from 241 clinical samples as five samples yielded two types of NFGNB. Monomicrobial growth was seen in 138 (57.26%) specimens whereas 103 (42.74%) specimens showed polymicrobial growth where nonfermenters were isolated with other organisms, of which S.aureus, E. coli, K.pneumoniae and Citrobacter species were common. Nonfermenters were isolated from variety of clinical specimens. Majority were isolated from pus, ear swab and sputum. Table 1 depicts the distribution and percentage of NFGNB isolated from various clinical samples. P.aeruginosa was the most common isolate, accounting for 94 (38.21%) followed by A.baumannii 72 (29.27%), A.lwoffii 34 (13.82%) and P.fluorescens 28 (11.38%). S.maltophilia and Al.fecalis were rarely isolated together accounting for 11 (4.47%) of the isolates. Three isolates of Table 1: Non fermenting gram negative bacilli isolated from various clinical samples Organism Pus Ear swab Sputum Urine Blood Other body fluids* Total no (%) P.aeruginosa (38.21) A.baumannii (29.27) P.fluorescens (11.38) A.lwoffii (13.82) S.maltophilia (2.44) Al.fecalis (2.03) Others (2.85) Total no. of isolates (%) 85 (34.55) 61 (24.80) 49 (19.92) 32 (13.01) 13 (5.28) 6 (2.44) 246 *Pleural fluid, ascitic fluid, knee aspirate, Cryseomonas species, Sphingomonas species, P.stutzeri Saudi Journal for Health Sciences - Vol 2, Issue 2, May-Aug

3 Pseudomonas stutzeri and two isolates each of Cryseomonas species and Sphingomonas species were found to be contaminants. The antibiotic susceptibility test results are given in Table 2 which shows the percentage of susceptible isolates. High level of resistance was recorded among most of the isolates. Except for S.maltophilia, amikacin and imipenem were found to be the most effective antibiotics. DISCUSSION Nonfermenters are ubiquitous in environment. Although frequently they are considered as commensals or contaminants, the pathogenic potential of NFGNB has been established beyond doubt by their frequent isolation from clinical materials and their association with disease. [1,20] The available data suggests that NFGNB are remarkable microorganisms because of their epidemiological complexity, propensity to cause outbreaks of infection and antimicrobial resistance. [21 24] They have emerged as important nosocomial pathogens especially in immunocompromised hosts and are responsible for causing variety of infections. Resistance to antimicrobials is common and has increased over the years among NFGNB and number of strains are now resistant nearly to all commonly used antibiotics. Multi drug resistance among these organisms makes the treatment of infections caused by them difficult and expensive. [25] Studies carried out by different researchers have reported varied isolation rates. In the present study NFGNB were isolated in 9.32% of clinical samples and this was parallel to the results of a study from Chandigarh [10] where NFGNB were isolated in 10% of clinical samples. A study from Amritsar [17] reported very high isolation rate of 45.9% while another study from Bangalore [15] reported it to be 21.80%. In a study from Saudi Arabia [26] NFGNB accounted for 16% of all the gram negative bacilli isolated. In contrast a study from Kolar, Karnataka [12] reported NFGNB to be isolated only in 4.5% of clinical samples. A study from Brazil [27] also reported a very low isolation rate of about 2.18%. Pseudomonas was found to be commonest non fermenter in all of these studies followed by Acinetobacter and this is in concordance to our finding. Table 3 depicts the Table 2: Antibiotic sensitivity pattern of the isolated non-fermenting gram negative bacilli Antibiotics Organisms P.aeruginosa n=94 A.baumannii n=72 P.fluorescens n=28 A.lwoffii n=34 S.maltophilia n=6 Al.fecalis n=5 Amikacin Imipenem Ticarcillin clavulanic acid Piperacillin tazobactam Gentamicin Cefoperazone sulbactam Cefipime Piperacillin Ceftazidime Cefoperazone Ciprofloxacin Cotrimoxazole Aztreonam Sensitivity pattern shown in the table is the percentage of isolates sensitive to the antibiotic, Intermediately sensitive isolates were considered as resistant, Table 3: Isolation rate of Pseudomonas species and Acinetobacter species in various studies Authors Year Place Pseudomonas % Acinetobacter % Vijya et al., [15] 2000 Banglore Eltahawy and Khalaf [26] 2001 Jeddah, Saudi Arabia Wang H and Chen [28] 2003 China Malini et al., [12] 2009 Karnataka Sidhu et al., [17] 2010 Amritsar Mohamad rahbar et al., [29] 2010 Iran Samanta et al., [10] 2011 Chandigarh Zhang et al., [30] 2011 China Deliberali Bruno et al., [27] 2011 Brazil Upgade et al., [14] 2012 Tamil nadu Memish et al., [31] 2012 Riyadh, Saudi Arabia Our study 2012 Uttarakhand Saudi Journal for Health Sciences - Vol 2, Issue 2, May-Aug 2013

4 isolation rate of Pseudomonas species and Acinetobacter species in various studies and is compared to our findings. The NFGNB are known to be responsible for wide range of nosocomial infections. [32,33] Resistance pattern among nosocomial bacterial pathogens may vary widely from country to country at any given time and within the same country over time. [20] Because of these variations a surveillance of the nosocomial pathogens for resistograms in a given set up is needed in order to guide appropriate selection of empiric therapy. Various international authorities emphasize that every hospital should have its individual antibiotic sensitivity pattern since the standard antibiotic sensitivity pattern may not hold true for every area. [15] Most of our patients were from surgical wards and not from ICU settings. Furthermore our patients came from rural areas without much exposure to antibiotics. In the present study, from the antibiotic sensitivity pattern it is clear that most of the isolates showed high degree of resistance suggesting that majority of the first and second line drugs were ineffective and this further confirms the multi drug resistant (MDR) attribute of NFGNB. Among the Pseudomonas species high levels of resistance were recorded for cotrimoxazole (89.34%), ciprofloxacin (73.77%), ceftazidime (68.85%), cefipime (61.48%), gentamicin (51.64%) and cefoperazone sulbactam (59.02%). Imipenem, amikacin and ticarcillin clavulanic acid showed maximum activity with an overall susceptibility of 72.95%, 72.13% and 68.85% respectively. Pseudomonas aeruginosa was found to be more sensitive to most of the antibiotics tested than P.fluorescens. Among the Acinetobacter species also amikacin and imipenem were found to be the most effective drug with 75.47% and 68.87% susceptible isolates respectively. A.baumannii showed a higher rate of resistance than A.lwoffii. S.maltophilia showed 100% susceptibility for ciprofloxacin and cotrimoxazole but was completely resistant to imipenem, amikacin and most of the other drugs. Low level of resistance was seen among the Al.fecalis isolates in comparison to other NFGNB. Imipenem resistance among Pseudomonas species, Acinetobacter species, S.maltophilia and Al.fecalis was 27.05%, 31.13%, 100% and 20% respectively. Our study is in concordance with reports of other authors where MDR in P.aeruginosa has been reported. [34,35] High degree of resistance to almost all the antibiotics was seen and this finding is in line with the study from Chandigarh. [36] Though imipenem showed good activity to all the NFGNB except for S.maltophilia, but emerging resistance to this group of drug is of major concern. Previous studies by other authors also have reported carbapenem resistance among NFGNB. [13,16,37] In the present study 31.13% of Acinetobacter species and 27.05% of Pseudomonas species were imipenem resistant and this was in contrast to the findings of Gladstone P et al., from Tamil Nadu and Joseph NM et al. from Pondicherry [16,37] who have reported the same to be 12.2% and 50% respectively. In our study a overall imipenem resistance among NFGNB was 30.54% and this corroborates well with the study by Taneja N et al., from Chandigarh [14] who reported the same to be 36.4%. We believe that documenting resistance among NFGNB is very important especially the carbapenem resistance, as these strains may often cause outbreaks in the ICU setting and can limit therapeutic option due to the high degree of multi drug resistance. These organisms can also spread resistance to other susceptible bacteria by horizontal gene transfer. To conclude, NFGNB though regarded as contaminants are important bacteria causing wide range of nosocomial infections. Variability in sensitivity pattern emphasizes the need for identification of NFGNB and to monitor their susceptibility patterns as it will help in proper management of the infection caused by them. Prevalence of pathogens often varies dramatically between communities, hospitals in the same community and among different patient populations in the same hospital. Thus it is important for clinicians to remain updated with prevalence and antimicrobial susceptibility pattern of the circulating pathogens in their practice setting and the antimicrobials to be used for empiric therapy should be selected accordingly. More importantly these organisms have great potential to survive in hospital environment so improved antibiotic stewardship and infection control measures will be needed to prevent or slow the emergence and spread of multidrug resistant NFGNB in the healthcare setting. A combination of control measures were implemented to contain these organisms in our set up. 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Indian J Med Microbiol 2002;20: Joseph NM, Sistla S, Dutta TK, Badhe AS, Rasitha D, Parija SC. Reliability of Kirby Bauer disk diffusion method for detecting meropenem resistance among non fermenting gram negative bacilli. Indian J Pathol Microbiol 2011;54: How to cite this article: Juyal D, Prakash R, Shanakarnarayan SA, Sharma M, Negi V, Sharma N. Prevalence of non-fermenting gram negative bacilli and their in vitro susceptibility pattern in a tertiary care hospital of Uttarakhand: A study from foothills of Himalayas. Saudi J Health Sci 2013;2: Source of Support: Nil, Conflict of Interest: None declared. 112 Saudi Journal for Health Sciences - Vol 2, Issue 2, May-Aug 2013