Prevalence and in vitro antibiogram of non-fermenting gram negative bacilli in a tertiary care hospital of Karnataka

Introduction

Non fermenting gram negative bacilli (NFGNB) are taxonomically diverse heterogenous group of aerobic, nonsporing, bacilli that either do not utilize carbohydrates as source of energy or degrade them by various metabolic pathways other than fermentation. NFGNB are known to account for about 15% of all bacterial isolates from a clinical microbiology laboratory.^[1], ^[2] They are saprophytes which may be found inhabiting the soil and water or as commensals in humans and animals. Their emergence as important nosocomial pathogens can be attributed to the liberal use of antibiotics, their inherent resistance to common disinfecting agents and their ability to colonize a variety of surfaces.^[3]

These bacteria can be frequently isolated from samples of patients suffering from septicemia, meningitis, pneumonia, urinary tract infection and surgical wound infection. Some of the risk factors that can contribute to NFGNB infections 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. Currently Pseudomonas aeruginosa and Acinetobacter baumannii are the most commonly isolated nonfermenters pathogenic for humans. Other species that can be isolated include opportunist pathogens like P. fluorescence, P.stutzeri, Stenotrophomonas maltophilia, P.putida and P.cepacia.^[4], ^[5]

A difficulty has been encountered recently in treating NFGNB infections as they exihibit a multidrug resistance (MDR). MDR is common and increasing among these bacteria. A number of strains have been identified to be resistance towards various groups of drugs including Beta-lactam, Aminoglycosides and fluroquinolones. This in turn is posing a barrier to treatment and hospital infection control.^[6], ^[7]

There have been very few studies to report on the prevalence of NFGNB and their antibiogram from this part of the country. The present study was hence taken up to help bridge this gap of knowledge. This study was aimed to isolate, identify and carry out in vitro susceptibility antibiogram of NFGNB from various clinical specimens and also to highlight their clinical significance among the in-patients admitted at Basaveshwar Teaching and General Hospital, Gulbarga, Karnataka.

Materials and Methods

This study was carried out on 120 samples of clinical specimens collected from in- patients admitted to various departments of Basaveshwar teaching and general hospital, Gulbarga during a period of 1 year study from January 2012 to December 2012. A detailed history of the patients was recorded including the current underlying disease like Diabetes or malignancy.

The samples for the study were collected from patients with NFGNB infection, admitted during the period of study. NFGNB was isolated from blood, CSF and other body fluids. Patients with colonisation of NFGNB with no apparent clinical infection and isolates from improperly collected samples were excluded from the study.

The samples were collected from respiratory tract (RT), pus, wound, urine and blood. A preliminary (gram stain) examination was carried out for RT, pus, wound and urine samples, following which they were incubated in appropriate media. The RT samples were inoculated in 5% sheep blood agar (BA), Macconkey agar (MA), and chocolate agar (CA) which were incubated overnight at 37⁰c and were observed for growth for 48 hrs.

The pus and wound swab sample were plated on 5% sheep BA, MA, Thioglycollate which were incubated overnight at 37⁰c and were observed for growth for 48 hrs. For the urine samples, gram stain smear were made by placing a loopful urine sample on a clean slide and allowed to air dry. These samples were then plated by a 4mm loop onto 5% sheep BA and MA for semi-quantitative analysis. Isolates which were significant in semi quantitative culture of urine were included in the study.

A Brain Heart Infusion broth was used for blood culture. The bottle was examined duly for turbidity and subculture was made at regular intervals on to BA, MA and any growth was processed further for identification. Cultures that showed growth in the first three days were included in the study.

For identification of species, biochemical tests were performed which include OF medium(Hugh and Leifson), Nitrate reducing broth, Citrate utilization test, Growth at room temperature 25⁰c-30⁰c, 37⁰c, 44⁰c, Hemolysis on a 5% sheep BA, Gelatin liquefaction and Hanging drop preparation for motility testing. Antibiogram was done by KIRBY-BAUER disc diffusion method. The various antibiotics with their concentration that were used are shown in [Table 1].

Only those NFGNB which grew either in pure culture or as predominant growth were identified in the study. All the tests were performed with positive and negative control.

Results

In the present study, 120 samples were collected from clinical specimens with local infections, Septicemia, Respiratory tract infections, Urinary tract infections, Ear infections, meningitis and cervicitis samples from patients admitted to Basaveshwar Teaching and General Hospital, Gulbarga.

The various samples from which NFGNB were isolated are shown in [Table 2]. Pus samples constituted majority of specimens accounting for 50.83%. Urine and Sputum samples accounted for 14.17% & 15% of specimens respectively. Stool, Blood, Pleural fluid, Ascitic fluid and CSF samples accounted for remaining 20%.

Various bacterial species isolated from each clinical diagnosis have been shown in [Table 3]. Out of all the cases, Pseudomonas aeruginosa was isolated in 48 cases which included 18 cases of local infection, 7 cases of respiratory tract infection (RTI), 4 cases each of Gastro intestinal tract (GIT) and post traumatic, 3 cases each of urinary tract infection (UTI) and septicaemia and 9 cases of post-operative (post OP) infection.

Pseudomonas fluorescens was isolated from 5 cases including 2 cases of RTI and 3 of post OP infection. Acinetobacter baumanii was isolated from 15 cases which included 5 cases of local infection, 3 cases of RTI, 2 cases each of GIT and post OP and 1 case each of UTI, post traumatic and septicaemia.

Mixed growth (Proteus mirabilis, Proteus vulgaris, Citrobacter, MRSA, E coli, Klebsiella, Enterococci, Enterobacter species, Group A beta haemolytic streptococci, Salmonella spp, Shigella spp) were seen a total of 52 cases which included 16 cases of local infection, 13 cases of UTI, 9 cases of RTI, 5 cases of GIT, 4 case of septicaemia, 3 cases of post OP infection and 2 cases of post traumatic infection.

The antibiotic susceptibility pattern for various NFGNBs for penicillin group of drugs is shown in [Table 4]. In the decreasing order of frequency, the NFGNB showed 54.41% sensitivity to Carbenicillin, 45.58% to Piperacillin and Tazobactam, 29.42% to Piperacillin, 26.48% sensitivity to Ticarcillin, 23.53% sensitivity to Amoxyclav, 16.18% sensitivity to Netilmicin and 7.36% sensitivity to Penicillin. Maximum resistance was exhibited against penicillin (92.64% cases) and minimum against carbenicillin (45.58% cases).

Ps.aeruginosa showed a sensitivity of 68.75% to Carbenicillin followed by 41.66% sensitivity to Piperacillin+Tazobactam. Ps.fluroscens showed a uniform sensitivity of 60% to Carbenicillin and Piperacillin+Tazobactam. Acinetobacter baumanii showed a sensitivity of 40% to Piperacillin+Tazobactam, 26.66% sensitivity to Carbenicillin and Amoxyclav.

The antibiotic susceptibility pattern of NFGNB for cephalosporin group of drugs is shown in [Table 5]. NFGNB showed a sensitivity of 52.95% to Ceftazidime, 48.53% to Cefaperazonea and 42.65% to Ceftriaxone. Maximum resistance was observed for Cefuroxime and minimum for Ceftazidime.

Ps.aeruginosa showed a sensitivity of 54.16% for Cefoperazone, 60.42% sensitivity for Ceftazidime, 49.84% sensitivity for Cefepime. Ps. Fluorescens showed uniform sensitivity of 60% to Ceftriaxone, Ceftazidime, and Cefaperazone. Ac. baumanii showed a susceptibility of 46.66% to Ceftazidime, 53.34% to Ceftriaxone and 33.33% to Cefepime and Cefaperazone.

The antibiotic susceptibility pattern of NFGNB for aminoglycosides and quinolone group of drugs is shown in [Table 6]. NFGNB showed a sensitivity of 69.11% to Amikacin, 55.18% to Gentamicin, 61.76% to Ciprofloxacin and 55.88% to Ofloxacin.

Ps.aeruginosa showed a sensitivity of 75% to Amikacin, 60.42% sensitivity to Gentamicin, 72.92 % sensitivity to Ciprofloxacin and 64.58 % sensitivity to Ofloxacin. Ps.fluorescens showed a sensitivity of 40% to Amikacin and Ofloxacin and 20% sensitivity to Gentamicin and Ciprofloxacin. Ac.baumanii showed a sensitivity of 60% to Amikacin and Gentamicin, 40% sensitivity to Ciprofloxacin and 33.33% sensitivity to Ofloxacin.

The antibiotic susceptibility pattern of NFGNB for carbapenems group of drugs is shown in [Table 7]. NFGNB’s showed a sensitivity of 82.35% to Imipenem and 58.82% sensitivity to Meropenem.

Ps.aeruginosa showed a sensitivity of 85.42% to Imipenem and 62.5% sensitivity were seen with Meropenem. Ps.fluorescens showed a sensitivity of 80% to Imipenem and 60% sensitivity were seen with Meropenem. Ac.baumanii showed a sensitivity of 73.34% to Imipenem and 40% sensitivity were seen with Meropenem.

The antibiotic susceptibility pattern of NFGNB for other group of drugs is shown in [Table 8]. The isolated organisms showed a sensitivity of 30.88% to Tetracycline, 25% to Co-trimoxazole, 19.12% to Chloramphenicol and a susceptibility of 35.29% to Polymyxin B.

Ps. aeruginosa showed a sensitivity of 39.58% to Polymyxin B, 31.25% to Tetracycline, 22.92% to Cotrimoxazole and 12.5% to Chloramphenicol. Ps. fluorescens showed a uniform sensitivity of 40% to Tetracycline, Cotrimoxazole and Chloramphenicol. Ac. baumanii showed a uniform sensitivity of 26.66% to Tetracycline, Cotrimoxazole and Chloramphenicol.

The individual sensitivity and resistance of P. aeruginosa, P. fluorescens and A. baumannii to various antibiotics has been shown in [false].

Discussion

The non-fermenters are present everywhere in the environment. Usually they are considered as commensals or Contaminants but their pathogenic potential has been established owing to their frequent isolation from clinical materials.^[4], ^[8] Their association with nosocomial infections has also been observed frequently. The facts of an epidemiological complexity, efficacy to cause outbreaks of infection and antimicrobial resistance has brought the focus of attention on these NFGNBs.^[9], ^[10], ^[11], ^[12] Resistance to antimicrobials has increased over the years as a result of which many strains exhibit resistance to all commonly used antibiotics. This multi-drug resistance in turn increases the difficulty and cost of treatment for these infections.^[13], ^[14], ^[15], ^[16]

During the study period from January 2012 to December 2012 at Basaveshwar Teaching and General Hospital, Gulbarga, 120 samples from various clinical conditions like septicaemia, local infection, post-operative infection, post traumatic infection, RTI, UTI and GIT were collected and subjected for further processing. NFGNB were isolated from 68 samples.

The most common NFGNB isolated in our study was P. aeruginosa in 48/120 isolates (40%) followed by A. baumannii in 15/120 cases (12.5%) which is similar to the results obtained by Malini et al. who reported P. aeruginosa as the most common isolate accounting for 104/189 (53.8%) isolates, followed by A. baumannii (43/189, 22.2%).^[3]

In the present study, the highest number of isolates were isolated from pus swabs (50.83%), which is in accordance with the observations made by Rit et al.^[17] and Gokale and Metgud^[18] who also reported pus swabs as the source of maximum percentage of the isolates i.e., 27.86% and 58.4%, respectively. P. aeruginosa and A. baumannii were mostly associated with local infections (37.5% and 33.33% respectively) while P. fluorescens was mostly associated with RTI (40%) and post OP infections (60%) as is evident from [Table 3].

NFGNB displays a wide and variable spectrum of antibiotic sensitivity. There is no antibiotic to which all strains are susceptible. NFGNB are uniformly resistant to Penicillin group of drugs. NFGNB showed sensitivity of 29.42% to Piperacillin in our study and the sensitivity ranged from 40% to 85% in other studies by Rajan R et al. and Prakash K S et al. Piperacillin+Tazobactam is a preferred drug for treating NFGNB infections and showed a sensitivity of 45.58% in our study.^[19], ^[20], ^[21], ^[22], ^[23]

NFGNB’s showed an overall 17.16 % resistance to Imipenem in our study. In a study by Taneja et al. it showed 36%.^[24] NFGNB’s showed a resistance of 41.18% to Meropenem which was higher, compared to Imipenem in our study. It is known that Meropenem develops resistance earlier than Imipenem. In study by Gupta E et al resistance to Meropenem was 22.16%.^[25]

Maximum sensitivity by Ps.aeruginosa was shown to Imipenem (85.42%) followed by carbenicillin (68.75%) and ofloxacin (64.58%) which are similar to results of studies conducted by Malini A. et al. and Rit et al. where the susceptibility to imipenem shown was 94.2% and 91.08%, respectively.^[3], ^[17] In agreement with the studies done by Benachinmardi et al. and Naqvi et al. that showed higher susceptibility to quinolones, P. aeruginosa isolates in the present study showed susceptibility of 75.92% and 64.58% to the quinolones such as ciprofloxacin and ofloxacin, respectively.^[11], ^[12]

In our study, P. aeruginosa showed lesser susceptibility to amoxicillin + clavulanic acid (18.75%) and least to chloramphenicol (12.5%). Ps.aeruginosa showed a resistance of 54.42 % to Piperacillin+Tazobactam in our study. The low sensitivity in our study could be due to excessive use of Piperacillin+Tazobactam combination in our hospital.

Ac.baumanii showed maximum sensitivity towards imipenem (73.34%) followed by amikacin and gentamicin (each 60%). The results obtained in our study are similar to the studies conducted by Rit et al. and Tunyapanit et al. where the susceptibility to imipenem was 90% and 100% respectively. ^{[13, 25]} Ac.baumanii showed a sensitivity of 30% to Piperacillin in our study. In studies conducted by Wong fu et al. it showed a sensitivity of 30% and in a study by Taneja et al it showed 40% sensitivity. Ac. baumanii showed a resistance of 60% to Piperacillin+Tazobactam in our study, similar to other studies.^[24], ^[9] Ac .baumanii showed a sensitivity of 26.66 % to Amoxyclav in our study. In studies conducted by Jawad et al. it showed a sensitivity of 57% and in study by Wong fu et al., it was 25%.^[9], ^[10]

NFGNBs showed resistance of 47.05 % to Ceftazidime, 51.47 % to Cefaperazone, 57.35% to Cefepime which are commonly used by the clinicians in our hospital. Ps.aeruginosa showed 27.08% resistance to Ciprofloxacin in our study. In various other studies by Taneja et al., Algun U et al., Prakash KS et al., Wong fu et al. and Smitha S et al., it ranged from 12.5% to 83%.^[23], ^[24], ^[9], ^[26], ^[27]

NFGNBs showed a good sensitivity to Amikacin 69.11% in our study which is similar to other studies by Prakash K S et al., Wong Fu et al. and Taneja et al. Gentamicin showed a sensitivity of 55.18 % in our study. However Ps.fluorescens showed least sensitivity of 20% to Gentamicin which was comparable to study by Yashodhara et al. where it was 25%.^[24], ^[9], ^[27], ^[28]

Conclusion

NFGNB though regarded as contaminants are important bacteria causing wide range of nosocomial infections. A variability in their sensitivity pattern emphasizes the need for identification and isolation of NFGNB and to perform antibiogram to help in decide proper line of treatment and antibiotic protocol.

P. aeruginosa and A. baumannii were the most common NFGNB isolated in our study. P. aeruginosa had shown good sensitivity to imipenem, carbenicillin and ofloxacin. A. baumannii showed good sensitivity to imipenem, amikacin and gentamicin. The different species of NFGNB have shown a varied sensitivity pattern in our study.

The multidrug resistance of these organisms is thought to be enhanced and maintained through repeated exposure of organisms to antibiotics. Presence of a sub lethal concentration of antibiotics creates a suitable environment for development of resistance. Also, the sensitivity pattern varies from hospital to hospital and population to population depending upon the frequently prescribed antibiotics to the infected patients.

Thus, it is essential to establish the clinical relevance of the isolated NFGNB, before they are considered as pathogens. This would avoid unnecessary usage of antibiotics and emergence of drug-resistant strains.

Source of Funding

None.

Conflict of Interest

None.