Diversity and biomass of bacteria
Totally, 109 bacterial isolates were retrieved and tentatively identified in different culture media. Among the diverse bacterial isolates, 80.7% (n = 88) comprised Gram-positive cocci and the remaining 19.2% (n = 21) were Gram-negative bacilli (Table III). Based on the colony morphology, culture, biochemical characteristics and subsequent comparison with previous reports, the isolates were identified and sorted into nine species including Gram-negative bacilli such as Acinetobacter sp., E. coli, Klebsiella sp., Enterobacter sp., Citrobacter sp., Serratia sp., Salmonella sp. and Gram-positive cocci such as S. aureus and CNS. Among the Gram-positive cocci identified, notably, CNS 46 (52.2%) was the predominant bacteria followed by S. aureus 42 (47.7%). In the case of Gram-negative bacilli, the most common isolate was Acinetobacter sp. representing 28.5%, followed by Klebsiella sp., 23.8%.
It was found that bacteria isolated from each ward showed variation in species diversity and biomass (Table III). The Gram-positive cocci, CNS and S. aureus were isolated from all the wards. However, the Gram-negative bacteria, Acinetobacter sp. and Klebsiella sp. were exclusively retrieved from the surgical and paediatric wards. Isolates of Enterobacter sp. and Salmonella sp. were recovered from the surgical ward and NICU. Meanwhile, E. coli and Citrobacter sp. were predominately isolated from the paediatric ward. Finally, the Serratia sp. was recovered from NICU. Bacteria retrieved and identified from the diverse patient-care equipments and inanimate objects in each ward showed variations in species diversity and biomass. Among the different inanimate objects analyzed in three wards, beds and tables showed the highest contamination. Twenty-nine Gram-positive and seven Gram-negative bacteria were recovered from the beds and tables. In the case of different patient-care equipments, stethoscopes showed the highest contamination, from which five Gram-positive and one Gram-negative bacteria were retrieved.
Results of the microbiological analysis revealed that among the three wards, the most contaminated one was the surgical ward. It demonstrated the highest number of isolates both from the inanimate objects and patient-care equipments (n = 41) which corresponds to 33(37.7%) Gram-positive and 8 (38.0%) Gram-negative bacteria. Notably, isolates of S. aureus (n = 17) and Acinetobacter sp. (n = 5) were found to be the predominant Gram-positive and Gram-negative bacteria respectively. The heavily contaminated inanimate objects in the surgical ward were tables and beds. Totally, twenty-five Gram-positive and 5 Gram-negative bacteria were isolated. Gram-negative species comprising Klebsiella sp., Salmonella sp. Enterobacter sp. and Acinetobacter sp. were identified. However, the patient-care equipments such as stethoscopes and sphygmomanometers showed minimum contamination with 5 Gram-positive and 1 Gram-negative bacterium (Acinetobacter sp.) (Table III). The second most contaminated was the paediatric ward. From the paediatric ward, 38 isolates comprising 29 (32.9%) Gram-positive and 9 (42.8%) Gram-negative bacteria were isolated. Particularly, the isolates of CNS (15) and Klebsiella sp. (4) respectively were found to be the major Gram-positive and Gram-negative bacteria that were retrieved. In the paediatric ward, the highest contamination was associated with inanimate objects. Twenty-seven Gram-positive and nine Gram-negative bacteria were isolated. Gram-negative species comprising Klebsiella sp., Citrobacter sp. Enterobacter sp., E. coli, and Acinetobacter sp. were identified. Among the inanimate objects, the bed was found to be the most contaminated one. Thirteen Gram-positive and three Gram-negative bacteria were isolated from beds. The number of bacteria isolated from patient-care equipments was minimal. NICU showed the least species diversity and biomass of bacterial isolates. Of the total 30 isolates, 26 (29.5%) corresponds to Gram-positive and the rest 4 (19.04%) were Gram-negative bacteria. Notably, the isolates of CNS (n = 15) and Enterobacter sp. (n = 2) were the predominant Gram-positive and Gram-negative bacteria. Like in the case of other wards, inanimate objects were found to be heavily contaminated compared to patient-care equipments. Totally 19 bacteria were isolated from inanimate objects. The heavily contaminated inanimate object in NICU was beds. Contamination in patient-care equipments was moderate in NICU. Among the different patient-care equipments, baby incubators were observed to be heavily contaminated (Table III). Notably, in all the three wards, fomites such as tables, bed- frames, medical charts and patient-care equipments like stethoscopes, ambu bags, suction machines, sphygmomanometers and baby incubators were contaminated with Gram-positive bacteria particularly the species of genus Staphylococcus (Table IV).
The antibiotic sensitivity profile of all isolates was inspected using fourteen antibiotics. The antibiogram of Gram-positive and Gram-negative bacteria are given in table IV and V. The isolated bacteria showed a broad spectrum of variations in the susceptibility/resistance patterns to all the antibiotics tested. It was observed that Gram-positive cocci were highly sensitive to most of the antibiotics tested. The highest degree of susceptibility was shown by S. aureus against eight antibiotics and the range was 61.9–95.2%. The drug sensitivity manifested by S. aureus to clindamycin was 95.2% followed by 92.9% against chloramphenicol, 90.5% (ciprofloxacin), 80.9% (erythromycin), 76.1% (amikacin), 69% (ceftriaxone), 64.3% gentamicin and 61.9% (tetracycline). In the case of CNS, the corresponding values were 82.6% against clindamycin followed by 78.2% (amikacin), 73.9% (ciprofloxacin) and 63.4% (chloramphenicol). Pertaining to the resistant profiles, 100% of S. aureus was resistant to penicillin, but for the trimethoprim-sulfamethoxazole, the percentage was just 30.9%. Among the 42 S. aureus, 8 isolates with a zone of inhibition ≤ 21 mm in cefoxitin disc diffusion assay were extrapolated as Methicillin-Resistant S. aureus (MRSA). The percentage of MRSA among S. aureus was 19.1%. Isolates of CNS showed a higher degree of resistance, 78.3% to penicillin and a moderate degree, 43.4% to trimethoprim-sulfamethoxazole, 32.6% to tetracycline, 28.1% to both chloramphenicol and erythromycin. In this study, the multi-drug resistance was inferred as the resistance to three or more groups of antibiotics tested. Altogether, 47.7% of Gram-positive cocci were multi-drug resistant. Among them, 42.8% and 52.17% of S. aureus and CNS respectively were resistant to three (and above) antibiotics.
The susceptibility pattern of Gram-negative bacilli revealed that the predominant isolate, Acinetobacter sp. showed a lower range of sensitivity to the five antibiotics tested (16.7–33.3%). Only 16.7% of Acinetobacter isolates showed sensitivity to tetracycline, norfloxacin, and trimethoprim-sulfamethoxazole. Besides, 33.3% of the isolates were sensitive to gentamicin and ciprofloxacin. Invariably, all the isolates were extremely susceptible (100%) to amikacin. The antibiogram of Klebsiella sp. revealed that 100% of the isolates were susceptible to amikacin, whereas susceptibility to chloramphenicol was 80% only. This phenomenon was also observed in all the isolates of Citrobacter sp., which showed sensitivity to amikacin. However, only 33.3% of isolates were sensitive to chloramphenicol, ciprofloxacin, and norfloxacin. Isolates of Enterobacter sp. exhibited a lower degree of susceptibility in the range of 33.3 to 66.7% against five antibiotics tested. It was observed that 66.7% of the isolates were sensitive to amikacin and ceftriaxone. Regarding the Salmonella sp., 50% of the isolates were susceptible to both amikacin and ceftriaxone. The antibiotic sensitivity patterns of E. coli and Serratia sp. indicated that both the isolates were extremely susceptible (100%) to tetracycline, amikacin, ceftriaxone, chloramphenicol, ciprofloxacin, and norfloxacin.
Concerning the antibiogram of Gram-negative bacilli, it was found that all the isolates of Acinetobacter showed 100% resistance against ceftriaxone and ampicillin. Besides, 83.3% of the isolates were resistant to amoxicillin followed by tetracycline, chloramphenicol, ciprofloxacin, and trimethoprim-sulfamethoxazole (66.7%). Isolates of Klebsiella sp. showed notable resistance in the range of 80 to 100% against amoxicillin, ampicillin, and trimethoprim-sulfamethoxazole. Invariably, all the isolates of Citrobacter sp. showed maximum resistance (100%) to amoxicillin, ampicillin, and trimethoprim-sulfamethoxazole. This phenomenon also occurred in the case of Enterobacter sp. Salmonella sp., and E. coli. The resistance of Salmonella sp. to gentamicin, amikacin, ceftriaxone, chloramphenicol, and ciprofloxacin correspond to 50%. Species wise, the least diverse bacterial isolate, Serratia sp. were resistant to gentamicin, amoxicillin, ampicillin, and trimethoprim-sulfamethoxazole (100%). It can be noted that 100% of Gram-negative bacteria were found to be multi-drug resistant. Therefore, the overall prevalence of multi-drug resistant isolates in this study was found to be 57.7% (Table VI).