|Year : 2013 | Volume
| Issue : 2 | Page : 108-112
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
|Date of Web Publication||10-Sep-2013|
Department of Microbiology and Immunology, Veer Chandra Singh Garhwali Government Medical Sciences and Research Institute, Srinagar Garhwal, Uttarakhand
Source of Support: None, Conflict of Interest: None
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.
Keywords: Acinetobacter , antibiotics, nonfermenters, nosocomial infection, Pseudomonas
|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;2:108-12
|How to cite this URL:|
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 [serial online] 2013 [cited 2020 Jan 24];2:108-12. Available from: http://www.saudijhealthsci.org/text.asp?2013/2/2/108/117915
| 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.  They are saprophytic in nature but can cause a significant number of infections particularly in hospitalized patients, immunocompromised hosts and patients with hematological malignancies.  Inherent resistance of these bacterial agents to commonly used disinfectants and there tendency to colonize various surfaces have been pivotal in their emergence as important nosocomial pathogens  NFGNB are known to account for 15% of all bacterial isolates from clinical microbiological laboratory.  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.  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.  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 and therapeutic procedures. Prolonged hospital stay broad spectrum antibiotic use and underlying host factors are the best predictors of outcome.  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).  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 β-lactamases (CHDLs) that is mostly specific for this species. These enzymes belong to 3 unrelated groups of clavulanic acid resistant β-lactamases represented by OXA-23, OXA-24, and OXA-58 that can be either plasmid or chromosomally encoded.  In case of P.aeruginosa the dominant mechanism of carbapenem resistance is loss of carbapenem specific porin OprD2.  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. ,,,,,,,,,
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.  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 18-24 hrs. Results were interpreted in accordance with central laboratory standards institute (CLSI) guidelines.  Escherichia More Details coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 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 Pseudomonas stutzeri and two isolates each of Cryseomonas species and Sphingomonas species were found to be contaminants.
|Table 1: Non fermenting gram negative bacilli isolated from various clinical samples|
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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.
|Table 2: Antibiotic sensitivity pattern of the isolated non - fermenting gram negative bacilli|
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| 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. , The available data suggests that NFGNB are remarkable microorganisms because of their epidemiological complexity, propensity to cause outbreaks of infection and antimicrobial resistance. ,,, 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. 
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  where NFGNB were isolated in 10% of clinical samples. A study from Amritsar  reported very high isolation rate of 45.9% while another study from Bangalore  reported it to be 21.80%. In a study from Saudi Arabia  NFGNB accounted for 16% of all the gram negative bacilli isolated. In contrast a study from Kolar, Karnataka  reported NFGNB to be isolated only in 4.5% of clinical samples. A study from Brazil  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 isolation rate of Pseudomonas species and Acinetobacter species in various studies and is compared to our findings.
|Table 3: Isolation rate of Pseudomonas species and Acinetobacter species in various studies|
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The NFGNB are known to be responsible for wide range of nosocomial infections. , Resistance pattern among nosocomial bacterial pathogens may vary widely from country to country at any given time and within the same country over time.  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.  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. , High degree of resistance to almost all the antibiotics was seen and this finding is in line with the study from Chandigarh.  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. ,, 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 , 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  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. Continued awareness of the need to maintain good housekeeping, equipment decontamination, strict attention to hand washing and isolation procedures and control of antibiotic usage especially in high risk areas were implemented. Combination of all these measures is necessary to control the previously unabated spread of these organisms.
| References|| |
|1.||In: Winn W Jr, Allen S, Janda W, Koneman E, Procop G, Schreckenberger P, et al., editors. Nonfermenting Gram negative bacilli. In: Koneman's color Atlas and textbook of Diagnostic Microbiology. 6 th ed. USA: Lippincott Williams and Wilkins Company; 2006. p. 305-91. |
|2.||Rampal R. Infections due to the Pseudomonas species and related organisms, Chapter 145. Harrison's Principles of Internal Medicine. 17 th ed. In: Fauci AS, Braunwald E, Kasper DL, Hauser SL, Longo DL, Jameson JL, et al., editors. USA: McGraw-Hill Medical; 2008. p. 949-56. |
|3.||Perez F, Hujer AM, Hujer KM, Decker BK, Rather PN, Bonomo RA. Global challenge of multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother 2007;51:3471-84. |
|4.||Meherwal SK, Taneja N, Sharma SK, Sharma M. Complicated nosocomial UTI caused by nonfermenters. Indian J Urol 2002;18:123-8. |
|5.||Fass RJ, Barnishan J, Solomon MC, Ayers LW. In vitro activities of quinolones, beta-lactams, tobramycin, and trimethoprim-sulfamethoxazole against nonfermentative gram-negative bacilli. Antimicrob Agents Chemother 1996;40:1412-8. |
|6.||Gales AC, Jones RN, Forward KR, Linares J, Sader HS, Verhoef J. Emerging importance of multidrug-resistant Acinetobacter species and Stenotrophomonas maltophila as pathogen in seriously ill patients: Geographical patterns, Epidemiological features, and trends in the SENTRY antimicrobial surveillance program (1997-1999). Clin Infect Dis 2001;32:104-13. |
|7.||Quinn JP. Clinical problems posed by multiresistant nonfermenting gram negative pathogens. Clinl Infect Dis 1998;27(Suppl 1):S117-24. |
|8.||Poirel L, Nordmann P. Carbapenem resistance in Acinetobacter baumannii: Mechanisms and epidemiology. Clin Microbiol Infect 2006;12:826-36. |
|9.||Quinn JP, Studemeister AE, DiVincenzo CA, Lerner SA. Resistance to imipenem in Pseudomonas aeruginosa: Clinical experience and biochemical mechanisms. Rev Infect Dis 1988;10:892-8. |
|10.||Samanta P, Gautam V, Thapar R, Ray P. Emerging resistance of non-fermenting gram negative bacilli in a tertiary care centre. Indian J Pathol Microbiol 2011;54:666-7. |
|11.||Gautam V, Ray P, Vandamme P, Chatterjee SS, Das A, Sharma K, et al. Identification of lysine positive non-fermenting gram negative bacilli (Stenotrophomonas maltophilia and Burkholderia cepacia complex). Indian J Med Microbiol 2009;27:128-33. |
|12.||Malini A, Deepa EK, Gokul BN, Prasad SR. Nonfermenting gram-negative bacilli infections in a tertiary care hospital in Kolar, Karnataka. J Lab Physicians 2009;1:62-6. |
|13.||Taneja N, Maharwal S, Sharma M. Imipenem resistance in nonfermenters causing nosocomial urinary tract infections. Indian J Med Sci 2003;57:294-9. |
|14.||Upgade A, Prabhu N, Gopi V, Soundararajan N. Current status of antibiotic resistant nonfermentative gram negative bacilli among nosocomial infections. Adv Appl Sci Res 2012;3:738-42. |
|15.||Vijaya D, Kamala, Bavani S, Veena M. Prevalence of nonfermenters in clinical specimens. Indian J Med Sci 2000;54:87-91. |
|16.||Gladstone P, Rajendran P, Brahmadathan KN. Incidence of carbapenem resistant nonfermenting gram negative bacilli from patients with respiratory infections in the intensive care units. Indian J Med Microbiol 2005;23:189-91. |
|17.||Sidhu S, Arora U, Devi P. Prevalence of nonfermentative gram negative bacilli in seriously ill patients with bacteraemia. JK Science 2010;12:168-71. |
|18.||Rajendra D, Ramana BV, Chaudhury A. Spectrum of Non fermenting gram negative bacilli infection (excluding Pseudomonads) in tertiary care hospital. Int J Biol Med Res 2012;3:1902-4. |
|19.||Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 17 th informational supplement, CLSI document M100-S17. Wayne PA: Clinical and Laboratory Standards Institute; 2007. |
|20.||Prashanth K, Badrinath S. In vitro susceptibility pattern of acinetobacter species to commonly used cephalosporins, quinolones, and aminoglycosides. Indian J Med Microbiol 2004;22:97-103. |
|21.||Rahbar M, Hajia M. Detection and quantitation of the etiologic agents of ventilator-associated pneumonia in endotracheal tube aspirates from patients in Iran. Infect Control Hosp Epidemiol 2006;27:884-5. |
|22.||Boroumand MA, Esfahanifard P, Saadat S, Sheihkvatan M, Hekmatyazdi S, Saremi M, et al. A report of Pseudomonas aeruginosa antibiotic resistance from a multicenter study in Iran. Indian J Med Microbiol 2007;25:435-6. |
|23.||Japoni A, Alborzi A, Kalani M, Nasiri J, Hayati M, Farshad S. Susceptibility patterns and cross-resistance of antibiotics against Pseudomonas aeruginosa isolated from burn patients in the South of Iran. Burns 2006;32:343-7. |
|24.||Taherikalani M, Etemadi G, Geliani KN, Fatollahzadeh B, Soroush S, Feizabadi MM. Emergence of multi and pan-drug resistance Acinetobacter baumannii carrying blaOXA-type -carbapenemase genes among burn patients in Tehran, Iran. Saudi Med J 2008;29:623-4. |
|25.||Kharangate NV, Pinto MJ, Rodrigues S, Verenkar MP. Characterization of nonfermenters from clinical samples. J Assoc Physicians India 2001;49:324-6. |
|26.||Eltahawy AT, Khalaf RM. Antibiotic resistance among gram-negative non fermentative bacteria at a teaching hospital in Saudi Arabia. J Chemother 2001;13:260-4. |
|27.||Bruno D, Nishino MK, Priore WN, Remus PR, do Carmo AA, Stefanello VB, et al. Prevalence of Gram-negative non-fermenters patients in Porto Alegre-RS. Jornal Brasileiro de Patologia e Medicina Laboratorial2011;47:529-34. |
|28.||Wang H, Chen MJ. China Nosocomial Pathogens Resistance Surveillance Study Group. Changes of antimicrobial resistance among nonfermenting gram-negative bacilli isolated from intensive care units from 1994 to 2001 in China. Zhonghua Yi Xue Za Zhi 2003;83:385-90. |
|29.||Rahbar1 M, Mehragan H, Akbari AN. Prevalence of drug resistance in nonfermenter gram-negative bacilli. Iran J Pathol 2010;5:90-6. |
|30.||Zhang C, Liang J, Liu P. Monitoring to drug resistance of non-fermenting gram-negative bacilli isolated from clinics in county hospital. Chin J Nosocomiol 2011;7:1432-3. |
|31.||Memish ZA, Shibl AM, Kambal AM, Ohaly YA, Ishaq A, Livermore DM. Antimicrobial resistance among non-fermenting Gram-negative bacteria in Saudi Arabia. J Antimicrob Chemother 2012;67:1701-5. |
|32.||Larson EL. Persistent carriage of Gram negative bacteria on hands. Am J Infect Control 1981;9:112-9. |
|33.||Bergogne-Berezin E. Guidelines on antimicrobial chemotherapy for prevention and treatment of infections in the intensive care unit. J Chemother 2001;1:134-49. |
|34.||Takeyama K, Kunishima Y, Matsukawa M, Takahashi S, Hirose T, Kobayashi N, et al. Multidrug-resistant Pseudomonas aeruginosa isolated from the urine of patients with urinary tract infection. J Infect Chemother 2002;8:59-63. |
|35.||Jombo AG, Jonah P, Ayeni AJ. Multidrug resistant Pseudomonas aeruginosa in a contemporary Medical Practice: Findings from urinary isolates at a Nigerian University Teaching Hospital. Niger J Physiol Sci 2008;23:105-9. |
|36.||Gupta V, Yadav A, Joshi RM. Antibiotic resistance pattern in uropathogens. Indian J Med Microbiol 2002;20:96-8. |
|37.||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;54:556-60. |
[Table 1], [Table 2], [Table 3]