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Isolation and Antibiogram of Nonfermentative Gram Negative Bacilli in various Clinical Specimens in a Tertiary Care Hospital, Jaipur, India

Nonfermenters are group of bacteria that are aerobic, non sporing, gram negative rod shaped bacilli and do not utilize carbohydrates as source of energy or degrade them by various metabolic pathways other than fermentation. These organisms although frequently considered to be commensals or contaminants, but the pathogenic potential of these organisms has been established beyond doubt because of their frequent isolation from clinical specimens and their association with the disease (Koneman et al., 2006). International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 12 (2017) pp. 1369-1380 Journal homepage: http://www.ijcmas.com


Introduction
Nonfermenters are group of bacteria that are aerobic, non sporing, gram negative rod shaped bacilli and do not utilize carbohydrates as source of energy or degrade them by various metabolic pathways other than fermentation. These organisms although frequently considered to be commensals or contaminants, but the pathogenic potential of these organisms has been established beyond doubt because of their frequent isolation from clinical specimens and their association with the disease (Koneman et al., 2006).

ISSN: 2319-7706 Volume 6 Number 12 (2017) pp. 1369-1380
Journal homepage: http://www.ijcmas.com Non Fermentative Gram Negative Bacilli were regarded to be Nonpathogenic and Contaminants in the past. Recently these bacilli became impending pathogenic with the ability to show resistance for commonly used antibiotics. These organisms isolated from clinical specimens with increasing frequency. So this study planned to isolate and antibiogram of Non-fermentative gram negative bacilli in various clinical specimens at tertiary care hospital at Jaipur. The present study was carried out on 49 of strains of Nonfermenters isolated from 397 non repetitive clinical samples like Pus, Wound discharge, Blood culture, Urine, Sputum, Ear discharge, Body fluid, ET Secretions and CSF etc. from all age's group. Conventional bacteriological methods were used for identification non fermenting gram negative bacilli and susceptibility testing was performed with the help of the Modified Kirby-Bauer disc diffusion method. This study comprises of 49 isolates of Non Fermentative Gram Negative Bacilli from total of 397 clinical specimens, collected from in-patients admitted to various departments during a period of 6 months. Pseudomonas aeruginosa was the most common isolate (57.14%) while Acinetobacter baumannii (24.48%) was the second most common isolated followed by Pseudomonas fluorescens (14.28%) and Acinetobacter lwoffii (4.08%). Among all clinical samples Pus yield maximum isolates of NFGNB i.e. 15(30.61%) followed by same account of Urine 8(16.32%) and Sputum 8(16.32%). Most sensitive drug against NFGNB were Polymyxin-B (100%) and Colistin (100%) followed by Imipenem (81.63%), Amikacin (71.42%). Pseudomonas aeruginosa and Acinetobacter baumannii have emerged as the most common Nonfermentative gram negative bacilli causing human infections. Alarming resistance rate was found towards commonly used antibiotics. Polymyxin B and Colistin showed excellent susceptibility against such bacteria followed by Imipenem and amikacin.
We encounter many problems with these organisms because they are invariable resistance to common disinfectants like as chlorhexidine and quaternary ammonium compounds and now a day they account approximate 15% of all bacterial isolates of any clinical laboratory (Malini et al., 2009).
Pseudomonas aeruginosa is a common inhabitant of soil and moist environments (Minhas Akbar et al., 2014;Ronald et al., 1969). In hospitals, Pseudomonas Aeruginosa can be isolated from a variety of sources, including respiratory therapy equipments, antiseptics, soap, sinks, mops, medicines, physiotherapy equipment and hydrotherapy pools (Pollack et al., 1995).
Data from more recent studies continue to show Pseudomonas aeruginosa as the second most common cause of nosocomical pneumonia, health care associated pneumonia and ventilator associated pneumonia (Gaynes et al., 2005;Kollef et al., 2005).
Acinetobacter species are ubiquitous in nature and have been found on or in soil, water, animals and humans. Acinetobacter baumannii is known to be recoverable from the skin, throat and rectum of humans and has been reported to be a healthcare-acquired colonizer of the respiratory tract. According to the Centres for Disease Control and Prevention (CDC), the species A. baumannii accounts for nearly 80% of reported Acinetobacter infections. This prolonged survival in the healthcare environment along with multidrug resistance, colonization potential, and contact transmission (hands, instruments, equipment) are some of the challenging factors in Acinetobacter prevention and control (An APIC guide, 2010).
Members of the genus Acinetobacter usually invade compromised hosts and are seen in patients who have undergone endotracheal intubation, peritoneal dialysis, or central venous catheterization (Kiran Chawla et al., 2013).
Pseudomonas aeruginosa and Acinetobacter species in particular are most often associated with carbapenem resistance. Carbapenem resistance appears to be due to metallo-β-Lactamase.
Excessive and indiscriminate used of broad spectrum antibiotics also responsible for antibiotic resistance (Mehta et al., 2007). Patients become susceptible to non fermentive bacterial infections after abusive use of wide spectrum antimicrobial agents, prolonged treatment with immunosuppressive drugs, prolonged hospital stay, inadequate mechanical instruments like tracheostomy, catheterization, in case of burns, low birth weight babies or radiation (Gerard et al., 1991).
The prevalence and rates of resistance in these organisms to available antimicrobials are increasing of particular concern is their resistance to β-lactams antibiotics, which is usually mediated by β-lactamase production. Widespread use of extended spectrum βlactams antibiotics has resulted in the formation of potent enzymes that are able to inactivate many of the new generation of antimicrobial agents.
Due to increasing frequency as isolation and increasing resistance against various antibiotics, it is important to know prevalence and antibiogram of Nonfermentive gram negative bacilli in hospital settings.

Materials and Methods
This Descriptive cross sectional study was carried out in the Bacteriology Department of Microbiology of National Institute of Medical Sciences (NIMS) University, Jaipur, District-Jaipur, Rajasthan, India for a period of six months (January 2017 to June 2017).
A total of 397 clinical specimens were found culture positive in the laboratory during the study period.
The organisms isolated and identified provisionally as NFGNB (n=49) were identified further by using a standard protocol for identification. (Koneman et al., 2006

Antimicrobial susceptibility
Antibiotic susceptibility testing was done by Modified Kirby Bauer's disc diffusion method as per CLSI guidelines 2017 using commercially available discs. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as control strains.

Results and Discussion
A total 49 strains of nonfermenters were isolated from 397 various non-repetitive various clinical samples. Incidence of nonfermentative gram negative bacilli isolates from clinical samples was 12.34%.
Out of total 397 samples, maximum from age group between 21-30 years i.e. 23.17 % while minimum from <10 years of age i.e. 7.30 %.

Antibiotic sensitivity testing results
Out of 49 isolates of NFGNB were processed for Antibiotic susceptibility testing was done by Modified Kirby Bauer's disc diffusion method as per CLSI guidelines 2017 using commercially available discs.

Demographic profile of patients included in study
In our study higher samples were received in the age group of 21-30 years (23.17%) with over all preponderance of males (56.92%).This observations were compatible with study done by Jayapriya Sukumaran et al., (2014).
Non fermenting Gram negative bacilli considered to be contaminants in the past have now emerged as important major pathogenic organisms. Previous studies reported varied isolation rates of NFGNB. In our study, isolation rate of NFGNB was 12.34%, which is in agreement with the study conducted by Hariom Sharan et al., (2016) where the isolation rate was 13.82%. Our study is not in agreement with the studies conducted by Sidhu et al., (2010) (45.9%) and Vijaya et al., (2000) 21.80% where very high isolation rate was reported. However, studies conducted by Malini et al., (2009) and Bruno et al., (2011) showed very low isolation rate of 4.5% and 2.18% respectively. Pseudomonas aeruginosa was found to be commonest nonfermenter in previous studies followed by Acinetobacter baumannii and this is in concordance to our results.
In the present study the most common NFGNB isolate was Pseudomonas aeruginosa 57.14%. This result has correlated well with the study conducted by Malini et al., (2009) reported 64.4% while Arora et al., (2010) reported 72.83%, Vijaya et al., (2000) reported 78.94% while Juyal et al., (2013) reported a low isolation rate of 38.21% Pseudomonas aeruginosa. The variation is likely due to number and type of clinical samples and geographical area of the study.
In the present study the second commonest isolate was Acinetobacter baumannii 24.48%. The result correlated well with the study conducted by Malini et al., (2009) reported 25.3%. However, Juyal et al., (2013 reported 29.27% in their study while Arora et al., (2010) reported 8.4%. The variation is likely due to number and type of clinical samples and geographical area of the study.
Majority of Pseudomonas aeruginosa (32.14%) were isolated from pus specimens. This is comparable with the study conducted by Hariom Sharan et al., (2016) who reported 26.32% isolation rate of Pseudomonas aeruginosa from Pus samples as dominant pathogen. However Dipak Bhargava et al., (2015) reported 43.9% Pseudomonas aeruginosa from Pus samples.
In our study, majority of Acinetobacter baumannii were from Pus and Urine specimen (25% each). This is similar to study done by Kamalraj et al., (2015) However Sundaram Mohan et al., (2016) reported 56.52%. Acinetobacter baumannii from Respiratory Specimens such as Endotracheal Tube and Sputum.
Different types of pigments are produced by nonfermenters, which are helpful in making their species identification e.g. Pseudomonas, Roseomonas, Stenotrophomonas maltophila etc. Pseudomonas aeruginosa produces Pyocyanin pigment in 90-96% of strains.