Isolation and Molecular Characterization of Multi Drug Resistant Gram-Negative Bacteria from Clinical Samples in Tertiary Care Hospital, South India

Introduction

The spread of Multi drug resistant (MDR) Gram negative bacteria (GNB) has become a serious threat to public health and remains a major cause of mortality and morbidity worldwide.^1-3 The use of higher rate of prolonged antimicrobial treatment is believed to be associated with the spread of antibiotic resistance in clinical settings. A survey from Van Boeckel et al. reported that highest consumption of antibiotics was observed in India, followed by China and United States.⁴ This is alarming. In India, Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii are the most frequently encountered pathogens in clinical settings and increasing rate of extended spectrum β-lactamase (ESBL) production and carbapenem resistance among these infections is a cause for concern.⁵

The ESBL’s represents the most important group of bacterial beta-lactamases that belong to BushJacoby functional subgroup 2be and Ambler class A, that can hydrolyze penicillins and higher generation cephalosporins and cause resistance to third-generation cephalosporins.⁶ Escherichia coli and Klebsiella pneumoniae are the most common ESBL producing organisms followed by A. baumannii, Enterobacter sp and P. aeruginosa.^7,8 Evidences have suggested that the excessive usage of carbapenem against ESBL producing GNB mediates the occurrence and spread of carbapenemases producing bacteria.⁹ The GNB harboring the ESBL genes can also carry resistance to many other antibiotics. The bacteria which shows resistance to multiple classes of antibiotics are called as multi-drug resistant bacteria.^1-3 The carbapenem antibiotics are considered as a last resort for treating infections caused by MDR-GNB; however, the increasing rate of carbapenem resistant bacteria poses greater challenge in the clinical settings. In 2017, WHO published that carbapenem resistant P. aeruginosa, carbapenem resistant A. baumannii and carbapenem resistant Enterobacteriaceae are designated as critically prioritized organisms of crucial importance for the development of new antibiotics.^10,11 The occurrence of carbapenem resistance in GNB is mainly due to production of carbapenemases enzymes, mutations in genes encoding porins and over expression of efflux pump.¹¹ Carbapenemases are beta-lactamases that belong to Ambler molecular classes A (KPC and GES enzymes), B metallo-beta-lactamases (NDM, VIM and IMP) and D (OXA type), which can hydrolyze carbapenem group of antibiotics and involved in acquired resistance.¹²

Hence, screening and reporting of the antibiotic resistance patterns in GNB plays a vital role in successful infection control, antimicrobial stewardship and public health interventions aimed at managing the emergence of such life-threatening organisms. In this context, we aimed (i) to examine the antimicrobial resistance patterns and

(ii) to characterize the ESBLs- and carbapenemasesproduction among GNB strains recovered from clinical samples.

Materials and Methods

Isolate collection, identification and antibiotic susceptibility test: A total of 934 non-duplicate gram negative bacteria (GNB) strains were isolated from different clinical samples including urine, pus, sputum, wound swabs, tissue, blood, endotracheal aspirate, surgical site swabs, diabetic foot and cerebrospinal fluid (CSF) from patients admitted in different wards and intensive care units from January 2017 to September 2017 in a tertiary care hospital, Karnataka, South India. The bacterial identification was carried out using standard microbiological techniques including microscopy, colony morphology and biochemical characteristics. Antimicrobial susceptibility testing was performed against Imipenem (10 μg), Meropenem (10 μg), Ceftazidime (30 μg), Ciprofloxacin (5 μg), Piperacillin/Tazobactam (100/10 μg), Amikacin (30 μg) and Gentamycin (10 μg) using CLSI described Kirby Bauer disk diffusion method.

Disc diffusion test for ESBLs: The study isolates were inoculated on Mueller Hinton agar (MHA) with standard inoculum (0.5 McFarland) and ceftazidime (30 μg) & ceftazidime - clavulanic acid (30 μg/10 μg) disks were placed. The Extended spectrum β-lactamases (ESBLs) producers were interpreted by zone diameter of ≥5 mm in the presence of clavulanic acid than ceftazidime alone.¹³

DNA extraction: A total of 168 carbapenem resistant GNB isolates were subjected to DNA extraction using boiling lysis method.¹⁴ Briefly, a loop full of overnight grown bacteria sub cultured on MacConkey agar was emulsified in 100μL of nuclease free water and centrifuged at 10000g for 10 min. The supernatant was eliminated and the pellet was re-suspended in 100μL of nuclease free water and subjected to boiling at 100°C in a water bath for 5 min. After boiling, the total reaction mixture was immediately cooled on ice and centrifuged at 10000g for 10 min. The supernatant was carefully removed and stored at -20°C. Aliquots of 5 μL of template DNA was used for Multiplex PCR assays.

Multiplex PCR assay for Carbapenemases detection: Multiplex PCR assay was optimized to detect NDM1, OXA-48, VIM, IMP and KPC genes in carbapenem resistant GNB.14 The multiplex PCR assay was performed using the following parameters: initial denaturation step of 15 min at 95°C; followed by 30 cycles of 30 seconds at 94°C (denaturation), 1.5 min at 59°C (Annealing Temperature) and 72°C for 1.5 min (extension); and final extension at 72°C for 10 min. A negative control was run concurrently for each multiplex PCR experiment. The primers used in this study are listed in table 1.

The drug resistant patterns and detection of carbapenemases genes were expressed as percentages. The MS Excel was used for data analysis.

Results

Of the 934 Gram negative bacteria (GNB) strains obtained, E. coli (n=464, 49.7%) was most common, followed by K. pneumoniae (n=137, 14.7%), Acinetobacter Sp (n=131, 14%), P. aeruginosa (n=12, 12%), Enterobacter Sp (n=31, 3.3%), Klebsiella Sp (n=12, 1.5%), Proteus Sp (n=11, 1.2%), Citrobacter freundii (n=10, 1.1%), Salmonella Typhi (n=8, 0.9%), Providencia Sp (n=7, 0.7%), Serratia Sp (n=4, 0.4%), Morganella Sp (n=3, 0.3%) and 0.2% (n=2) of Aeromonas Sp. and S. Paratyphi A. These isolates were from 475 (50.9%) males and 459 (49.1%) females with ages between 2 days and 90 years. The highest proportion of strains were isolated from urine specimens (n=470, 50.3%), followed by pus (n=166, 17.8%), sputum (n=85, 9.1%), wound swabs (n=48, 5.1), tissue (n=45, 4.8%), blood (n=38, 4.1%), endotracheal aspirate (n=29, 3.1%), surgical site swabs (n=26,2.8%), diabetic foot (n=24, 2.6%) and CSF (n=3, 0.3%).

Based on the disc diffusion test, ceftazidime resistance was more frequently observed (n=635, 68.0%), followed by ciprofloxacin (n=587, 62.8%), piperacillin/ tazobactam (n=353, 37.8%), gentamicin (n=251, 26.9%), amikacin (n=201, 21.5%), meropenem (n=164,17.6%) and imipenem (121, 13.0%) (Table 2). In addition, 117 GNB isolates were found to be resistant to both imipenem and meropenem followed by meropenem alone (n=47) and imipenem alone (n=4) (Table 3). On the other hand, 8.6% (n=80) of isolates were found to be resistant to all tested antibiotics including carbapenems, fluoroquinolones, and aminoglycosides with high rate of resistance among Acinetobacter sp (n=45) followed by E. coli (n=13) and K. pneumoniae (n=10). The remaining consists of P. aeruginosa (n=3), Enterobacter Sp (n=3), Citrobacter Sp (n=3), Klebsiella Sp (n=1), Proteus Sp (n=1) and Morganella Sp (n=1).

Among 168 (18%) carbapenem resistant GNB, NDM1 was most predominant in E. coli, (n=23, 13.7%) followed by K. pneumoniae (n=16, 9.5%), Acinetobacter Sp (n=15, 8.9%) and P. aeruginosa (n=8, 4.8%) (Table 4 and Figure 1). OXA-48 was most common in Acinetobacter Sp (n=41, 24.4%) followed by E. coli (n=7, 4.2%) and K. pneumoniae, (n=6, 3.6%). The cooccurrence of NDM-1 and VIM was observed among E. coli (n=3), Acinetobacter Sp (n=2), K. pneumoniae (n=2) and Citrobacter Sp (n=1) (Table 4). The coexistence of NDM-1 and OXA-48 was observed among Acinetobacter Sp (n=3), K. pneumoniae (n=3) and Citrobacter Sp (n=1). In addition, an isolate of K. pneumoniae was found to be harboring with both OXA48 and VIM (Figure 1).

Of the total of 934 GNB isolates, 221 (23.7%) were found to be producing ESBL. The E. coli (n=167, 17.9%) was most common followed by K. pneumoniae (n=30, 3.2%), Acinetobacter Sp (n=9, 1%), Enterobacter Sp (n=7, 0.7%), C. freundii (n=2, 0.2%), Proteus Sp (n=2, 0.2%) and Serratia Sp (n=1, 0.1%). All the ESBLs producing organisms were sensitive to both imipenem and meropenem.

Discussion

The emergence and spread of multi drug resistant (MDR) gram negative bacteria (GNB) in hospital settings has become a serious public threat and mortality rate due to these infections has been increasing worldwide.1-3 In the present study, we evaluated the distribution of antimicrobial resistance patterns among the GNB strains isolated from clinical samples. In our study, E. coli (49.7%) was most frequently isolated, followed by K. pneumoniae (14.7%), Acinetobacter Sp (14%), P. aeruginosa (12%), Enterobacter Sp (3.3%), Klebsiella Sp (1.5%), Proteus Sp (1.2%), Citrobacter freundii (1.1%), S. typhi (0.9%), Providencia Sp (0.7%), Serratia Sp (0.4%), Morganella Sp (0.3%), Aeromonas Sp. (0.2%) and S. paratyphi A (0.2%). Most of the strains were collected from urine specimens (50.3%), followed by pus (17.8%), sputum (9.1%), wound swabs (5.1%), tissue (4.8%), blood (4.1%), endotracheal aspirate (3.1%), surgical site swabs (2.8%), diabetic foot (2.6%) and CSF (0.3%). The frequency of GNB strains isolated from 50.9% of males and 49.1% of females, showed no appreciable difference. Several studies from south India suggested that E. coli and Klebsiella sp are the most common organisms isolated from urinary tract infections with high resistance to broad spectrum antibiotics.^5,15 Abhilash et al observed that 53% of drug resistant GNB strains isolated from patients with bacteraemia were E. coli and Klebsiella sp.¹⁵ Veeraraghavan et al isolated E. coli (44%) most frequently followed by K. pneumoniae (25%) and P. aeruginosa (11%).5 In Ghana, Agyepong et al isolated E. coli (24.5%) most frequently in clinical samples followed by P. aeruginosa (19.5%), K. pneumoniae (19%), Enterobacter spp. (6.0%), Serratia spp. (4.0%), Sphingimonas spp. (5.0%) and Acinetobacter spp. (4.0%).² They observed that most of these organisms were isolated from urine specimens followed by wound swabs and sputum. A recent study from Egypt by El-Kholy et al reported that, E. coli (54.8%) and K. pneumoniae (43%) were the most frequently isolated organisms predominantly in urinary tract infection.¹⁶ In this present study, we demonstrated that 18% (n=168) and 8.6% (n=80) of GNB were resistant to carbapenem and were multi drug resistant (resistant to ceftazidime, piperacillin/tazobactam, ceftacarbapenems, fluoroquinolones, and aminoglycosides), respectively. Among eighty MDR-GNB isolates, Acinetobacter Sp (n=45) was predominant, followed by E. coli (n=13), K. pneumoniae (n=10), P. aeruginosa (n=3), Enterobacter Sp. (n=3), Citrobacter Sp. (n=3) Klebsiella Sp. (n=1), Proteus Sp. (n=1) and Morganella Sp. (n=1) (Table 2). Similarly, a study from Nepal isolated most frequently MDR Acinetobacter sp (41%, 52/128) followed by MDR K. pneumoniae (28%, 36/128) and MDR Pseudomonas sp (21%, 27/128).³

In India, ESBLs rates are alarmingly increasing in E. coli and Klebsiella sp. ranging from 16 to 94%.^8,17,18,19 In our study, ESBLs producers were documented predominantly in E. coli, 167 (17.9%) followed by K. pneumoniae, 30 (3.2%) and Acinetobacter Sp,9 (1.0%), Enterobacter Sp, 7 (0.7%), C. freundii, 2 (0.2%), Proteus Sp, 2 (0.2%) and Serratia Sp, 1 (0.1%). A multi centric study from India at four major tertiary care hospitals observed that ESBLs producing E. coli and K. pneumoniae was 55% and 60%, respectively, in PGIMER, Chandigarh; 37% and 53%, respectively, in CMC, Vellore; 38% and 33%, respectively, in AIIMS, New Delhi and 16% and 30%, respectively, in JIPMER, Puducherry.¹⁹ A neonatal study from North India by Roy et al observed that 70% of E. coli and 60% of K. pneumoniae were found to be ESBLs producers.²⁰ Another study from western India by Patel et al documented 94.8% and 92% of ESBLs producing K. pneumoniae and E. coli strains, respectively.²¹ Shaik et al from north India demonstrated 55.69% of the E. coli and 44.31% of the K. pneumoniae isolates were ESBL producers.¹⁸

Carbapenems are often considered as the most effective and potent therapeutic options for treating infections caused by MDR-GNB. However, the occurrence of carbapenem resistance significantly compromises their efficiency.⁹ Previous studies in India have reported about 35–38% of carbapenem resistant Acinetobacter Sp in clinical settings.^22,23 In this present study, Acinetobacter Sp was predominantly resistant to imipenem (n=66) and meropenem (n=66) followed by E. coli (imipenem, n=21 and meropenem, n=37) and K. pneumoniae (imipenem, n=16 and meropenem, n=32). A study from Chaudhary et al observed that 86.5% of Acinetobacter isolates were confirmed to be carbapenemase positive through PCR.²⁴ Several studies have reported the wide spread of NDM1, OXA-48 and VIM producing carbapenem resistant GNB in clinical settings.^25-28 In our hospital, NDM-1 was most predominant in E. coli (13.7%) followed by K. pneumoniae (9.5%), Acinetobacter Sp, (8.9%) and P. aeruginosa, (4.8%). The OXA-48 was most common in Acinetobacter Sp (24.4%) followed by E. coli (4.2%) and K. pneumoniae (3.6%). The co-occurrence of NDM-1 and VIM was observed among E. coli (n=3), Acinetobacter Sp (n=2), K. pneumoniae (n=2) and Citrobacter Sp (n=1). The co-existence of NDM-1 and OXA-48 was observed among Acinetobacter Sp (n=3), K. pneumoniae (n=3) and Citrobacter Sp (n=1). The co-occurrence of OXA48 and VIM was detected in one Klebsiella Sp. A study from south India by Nagaraj et al reported that majority of the carbapenem resistant K. pneumoniae (75%) and carbapenem resistant E. coli (66.6%) were found to be harboring with NDM.²⁹ Nachimuthu et al showed that among sixteen NDM harboring GNB, nine were E. coli and four K. pneumoniae were found to be harboring with OXA-like producer.³⁰

Conclusion

The increasing rate of multi drug resistance in clinical strains of Acinetobacter Sp, E. coli and K. pneumoniae at tertiary care hospital is worrisome. Our results highlighted that the NDM-1 producing E. coli and K. pneumoniae and OXA-48 in Acinetobacter Sp are spreading rapidly and warrants further studies in hospital environment. This sheds light on the importance of treatment options to be considered instantly as infection control measures.