Multi-drug Resistant Bacteria Isolated from Pus and Phenotypic Detection of Metallo β-lactamase Activity of Pseudomonas Aeruginosa

(MRSA), 14(8%) were Methicillin sensitive Staphylococcus aureus (MSSA), 21(13.0%) were Klebsiella spp, 19(11.7%) were Escherichia coli, 14 (8.6%) were Pseudomonas aeruginosa, 10(6.2%) were Acinetobacter spp, 5(3.1%) were Streptococcus spp, 4(2.5%) were Enterococci ,3(1.9%) were Enterobacter spp, 2(1.2%) were Coagulase negative S. aureus, 1(0.6%) were Proteus vulgaris, and1(0.6%) were Citrobacter spp. Antibiogram of Gram positive cocci revealed that they showed susceptibility towards vancomycin, clindamycin, gentamycin, amikacin. Similarly, Gram negative bacilli showed good response towards gentamycin, amikacin, nitrofurantoin, colistin. Out of 162 isolates, 102(62.96%) were Multi-Drug Resistant (MDR) and 60 (37.04%) were Non-MDR. Out of 14 (8.6%) Pseudomonas aeruginosa isolates, 8(57.1%) were Imepenem resistant and among imepenem resistant, only 3(37.5%) of them showed MβL production activity.

Klebsiella spp., Pseudomonas spp., Escherichia coli, Enterobacter, Providencia, Acinetobacter, in which Staphylococcus. aureus is the most frequent bacteria. 3 Antibiotics sensitivity test (AST) is the susceptibility of bacteria to antibiotics which is usually carried out to determine suitable antibiotics in treating a bacterial infection in-vivo. 4 Multidrug resistance in bacteria may occur by one of two mechanisms. First, these bacteria may create multiple genes, each coding for resistance to a single drug, within a single cell on resistance (R) plasmids. Second, it may also occur by the increased gene expression coding different multidrug efflux pumps, expelling variety of drugs. 5,6 The present study was designed to evaluate the profile of aerobic pyogenic bacteria along with their susceptibility to antimicrobial agents including phenotypic detection of Metallo Beta-Lactamase (MβL) activity of Pseudomonas aeruginosa in pus isolates.
It was a hospital based cross-sectional descriptive study from 16th October 2016 AD to 15th December 2016 AD, Nepal. Pus sample were collected from various hospitals of Pokhara valley. All the age groups were included in the study. Samples were collected from the limited hospitals of the Pokhara Valley. The sample size was calculated based on the approximate number of patients visiting 4 different hospitals where they were recommended for pus culture. Keeping 95% confidence level and 5% confidence interval, we estimated that there were at least 500 patients. So, by using the above parameters, we determined our sample size to be 217. Total 179 samples of pus was collected from any site of the body either by using swab or aspirated by using syringe and cultured in respective hospitals. Thus, obtained isolates were preserved in the vials containing glycerol (16%) and Brain Heart Infusion Broth (84%) and were transported to the Laboratory of School of Health and Allied Sciences, Pokhara University for further processing. Isolates were next asceptically inoculated onto Nutrient Agar, Blood Agar and MacConkey Agar plate with standard inoculation loop (4 mm) using Quadrant Streaking Technique and incubated for 24 hours at 37°C. Colony morphology was studied for shape, size, elevation, margin, surface, edge, colour, transparency and consistency. MacConkey plate was observed for the utilization pattern of lactose as lactose fermenter (LF) and Non-Lactose fermenter (NLF) and type of hemolysis was observed in Blood Agar plate (BA) namely alpha (greenish color around the colony), beta (clear zone around the colony) and gamma hemolysis (no detectable hemolysis). Out of 179 samples, there was no growth of 25 samples in culture media in which 154 samples showed growth. Among 154 samples, 162 isolates were obtained and 9 samples showed polymicrobial growth. Gram staining was performed for the differentiation of gram positive and gram negative bacteria, gram positive cocci (GPC) were further subjected to one step enzymatic test like catalase and coagulase (free and bound coagulase). Gram negative bacilli (GNB) were only enrolled for biochemical testing and identified by the different biochemical reaction patterns exhibited in different media like: Oxidative and Fermentative (OF) media, Sulphur Indole Motility (SIM), Urease media, Citrate media, and Triple sugar iron (TSI) media, Oxidative test was carried out for confirmation of aerobic gram negative bacteria.
For the Phenotypic detection of MβL activity, test organism was inoculated onto plates of Mueller-Hinton agar plate (opacity adjusted to 0.5 McFarland opacity standards). 7 A 0.5-m EDTA solution was prepared by dissolving 186.1 g of disodium EDTA 2H 2O in 1000 ml of distilled water and adjusting it to pH 8.0 by using 1% NaOH. The mixture was sterilized by autoclaving. Two 10-µg imipenem discs were placed on the plate; 5 µl of EDTA solution was added to one of the disc each. The inhibition zones of the imipenem and imipenem-EDTA discs were compared after 16-18 h of incubation at 35°C. An increase in the zone size of at least 7 mm around the imipenem-EDTA disc was recorded as an MβL-positive strain. 7,8 Data was entered and analysis was done by Microsoft excel, SPSS V20.0 software. Table 1, highest occurrence was seen in male (54.3%) as compared to female (45.7%). Table 2 all bacteria were resistant to ceftazidine whereas 57 isolates were sensitive to amikacin followed by vancomycin (53 isolates). Likewise, in case of 14 isolates of MSSA, all the isolates were resistant to ceftazidime. In case of 5

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With MRSA AND MSSA p value <0.05 Hint: S= Sensitive, I= Intermediate, R= Resistant As shown in Table 3 Gram negative bacteria were most susceptible to amikacin followed by gentamicin.
Staphylococcus aureus (50%) was the most common gram positive isolate. These findings are similar to the results obtained by Zafar Grace BN et.al. 12 In our study, P.aeruginosa was isolated from 4(2.5%) females and 10(6.2%) males which is statistically significant (p=0.028). P. aeruginosa were sensitive to amikacin, gentamycin and colistin which corresponds with the results obtained by Thapa P et.al. 18  On the basis of our study the following recommendation are suggested, • MRSA is the predominant cause of pyogenic infection • Antibiotics that are commonly used for the management of pyogenic infection are being less effective. • For instance cefoxitin, and penicillin are being highly resistant to pus isolates. • Universal and rational recommendations might not reflect the local scenario so survelliance programs should be strengthened for the establishment of local antibiogram and should be constantly renewed over time. • MβL detection showed the presence of MβL production in imepenem resistant P.aeruginosa that might harbor resistant genes.