In the present study, a total of 314 non-repetitive gram negative isolates were obtained. E.coli with the occurrence of 38% was highly prevalent pathogen predominantly isolated from urine specimens. It is identical to study by Fatima S et al (38%). Several studies reported E.coli to be the most isolated GNB. The incidence of Acinetobacter spp (20%) and Pseudomonas spp (12%) in our study coincides with the findings of Sundaram M et al (21%) and Mshana S et al (12.2%) respectively. Moreover, the occurrence of Proteus and Citrobacter species was similar with the study report of Kaur N. et al (2017).
On account of antibiotic resistance pattern for E.coli, maximum number of resistance was seen with ceftazidime and ofloxacin (62%) followed by piperacillin (56.2%), cefepime (52.1%) and levofloxacin (51.2%). The susceptibility of E.coli to amikacin, piperacillin—tazobactum and imipenem (table no.2) are in concordance with the study of Shanmugam et al (2017). Furthermore, colistin was observed effective against all strains with nil resistance except for intrinsically resistant Proteus and Providencia species. Conversely, almost all organisms were highly resistant to ceftazidime. The possible reason might be due to haphazard therapeutic use of third generation cephalosporins despite of knowing the severity of infections.
In our attempt for ESBL production, 84/314 (26.8%) were detected as ESBL producers. This finding coincides with Mshana S et al (29.8%). From various studies in Nepal, the proportion of ESBL production ranges from (18%) to (62.7%). Among the maximum isolated strains, the occurrence was high for E.coli (38%), comparable to the result of Sundaram M et al (35.3%) and the prevalence was in accordance with the study of Kaur N et al and Bhandari R et al. Slightly lower rates were detected by both Neupane S et al (2016) and Nepal K et al (2017) i.e. 33.2% of E.coli as ESBL producers due to the limitation of their study in specific organism. 30.6% of Klebsiella spp were ESBL producers. The incidence of ESBL production by Klebsiella spp in Nepal was found as high as 90.9% (Pathak J et al) and as low as 4.1% (Raut et al). Laudy A et al had the result of 15% of ESBL producing P.aeruginosa by phenotypic assay equal to our finding.
On the account of MBL producers, 16.2% of isolated strains were detected positive. It was lower than the record of N. Kaur et al (25.3%) but concomitant with the report of Kamble et al (19.9%).(19,29) 26.5% of Klebsiella species as the highest MBL producers followed by Pseudomonas spp (26.3%) and Acinetobacter spp (20.6%) were reported in our study. These are in accordance with N. Kaur et al (34.8%), Anuradha et al (28.6%) and Baniya et al (22%) for respective organisms.(19,30,31) Adding up another fact, most of MBL producers had the inhibited zones of ceftazidime and ceftazidime- clavulanic acid. The probable reason might be the inhibitory action of carbapenemases over beta lactam inhibitors.
Overall 62.7% of isolates were detected as biofilm formers in our study. This was in total agreement with the data of study by Allam NG (2017) i.e. 64.28%.(32) Rather, Fatima S et al (2015) and Singhai et al (2013) reported discording result of (41%) and (80%) biofilm positive GNB respectively.(18,33) In addition, similar study conducted in our center, BPKIHS, by Shrestha L.B et al, had the findings of 71.8% biofilm producers. Moreover, TAM (54.8%) was found better biofilm detector than CRA method (30.2%) in consistence with other studies.(13,34) The notable prevalence of biofilm producers from Klebsiella species (77.6%) in our study is in accordance with the study of Singhai et al (2012) in device related infections (61.1%) and Allam N G (2017) in UTIs (72.72%).(32,35) In the same manner, biofilm production by Pseudomonas species (73.7%), E.coli (60.3%) and Acinetobacter spp (54%) were comparable with the findings of Pittaya M et al (79.4%), Allam N G et al (55.8%) and Gurung et al (50%) respectively. Another study in Nepal demonstrated 51.92% of E.coli isolates to be biofilm producers.
On the comparative study of biofilm formers and ESBL producers, in spite of majority of ESBL producers being biofilm positive, there was no significant association between them (P> 0.05) which is concordant with the findings of Emami S et al (2015) but in contrast to the study by Shanmugam K et al (P=0.023) and Neupane S et al (P<0.05). On the other hand, the correlation between MBL production and biofilm formation was observed to be statistically significant (p<0.05). The reports of Singhai et al (2013) and Heydari S et al (2015) supported the result for non fermenters (Acinetobacter spp and Pseudomonas spp) but Baniya et al (2017) had the opposite outcome.
The antibiotic resistant pattern of ESBL producing strains was statistically insignificant for most of the antibiotics except for except for fluoroquinolones, cephalosporins and piperacillin. The study by Debora C et al also presented significant relation for ceftazidime and ciprofloxacin. Raut et al (2015) had documented p value significant for all groups except beta lactamase inhibitors. Amikacin and imipenem were observed as effective antibiotic against ESBL producers. Likewise, statistical significance was obtained (p<0.05) for all antibiotics except polymyxin B between the association of MBL production and antibiotic resistance (table no.7) Ceftazidime was 100% resistant to MBL positive strain which was compatible with the study of Lyra P A et al (2016). Besides, various studies have demonstrated MBL producers as pan drug resistant strains signifying an alarming threat.
In the matter of correlation between biofilm and antibiotic resistance, it was found statistically significant in our study for aminoglycosides, fluoroquinolones, cephalosporins, imipenem and piperacillin (table no.8). Hussain et al (2017) showed no correlation between antibiotic susceptibility pattern and biofilm formation while on contrary, it was found significant in the other studies. Several studies have documented that there is higher proportion of antibiotic resistance in biofilm producers than non producers. Colistin was sensitive for all strains as the last viable option for all multidrug resistant strain in spite of biofilm production or not.
From the present study, the knowledge of prevalence, relevance and emergence of biofilm forming and multidrug resistant organisms especially harbored in hospitalized and critically ill patients can be extracted. Thereby, weighing up the possible outcomes, it can be concluded that the efforts from the global level must be carried out for raising awareness against irrational use of antibiotics and for strict implementation of infection control and prevention activities as well as to seek newer therapies in order to counterattack this upcoming threat.
As simple methods of biofilm detection (TAM and CRA) and combined disc assay for ESBL and MBL detection can be used for screening purpose, the routine monitoring of biofilm and beta lactamases production can be recommended in clinical laboratories devising clinicians for rational decision making during the course of treatment.
As molecular study was not feasible in our set up, the lack of confirmation by molecular methods for biofilm, ESBL and MBL are the drawbacks of this study.
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