In this cross-sectional study, a total of 151 non-duplicate, Gram-negative bacteria belonging to 11 genera were studied which include Escherichia coli (n=57, 38%), Klebsiella pneumoniae (n=40, 26%), Klebsiella oxytoca (n=5, 3%), Pseudomonas aeruginosa (n=10, 7%), Salmonella typhi (n=8, 5%), Enterobacter cloacae (n=8, 5%), Acinetobacter baumannii (n=7, 5%), Serratia marcescens (n=5, 3%), Achromobacter xylosoxidans (n=5, 3%), Proteus mirabilis (n=5, 3%) and Elizabethkingia meningoseptica (n=1, 0.7%). Most of the isolates were isolated from urine 37% (56/151) and blood 28% (42/151) and from other sources such as pus (7%), bronchial secretion (2%), cerebrospinal fluid (1%), pulmonary secretion (1%), bile fluid (5%) and unknown (19%).
Antibiotic susceptibility studies
Table 1 summarizes the antibiotic susceptibility pattern of all the isolates tested against eight different antibiotics. MIC for meropenem showed that 107/151 (71%) isolates were resistant (fig.1), whereas 128 (84.7%) isolates were meropenem- resistant by the disk-diffusion method. For imipenem, 68% (n=103) were resistant by micro-broth dilution method whereas 83% (n=125) resistant by the disk-diffusion method. MIC50 and MIC90 values for meropenem were 16 mg/L and 8 mg/L respectively and for imipenem MIC50 = 8 mg/L and MIC90 = 8 mg/L.
Distribution of carbapenemase resistance genes
The distribution of beta-lactamase resistance genes among 151 Gram-negative isolates is summarized in Table 2. Of the 57 E. coli, 32 isolates carried carbapenemase and five E. coli isolates carried more than one carbapenem resistance genes. Among the K. pneumoniae, 19/40 carried the studied genes and one isolate was positive for both blaNDM and blaOXA-48-like. Carbapenem resistance genes were detected in 71/151 by PCR and 10 isolates had more than one gene. The most prevalent resistance gene was blaNDM-1 (n=22), blaOXA-48-like (n=21), blaGES-1 (n=11), blaGES-9 (n=8), blaOXA-23-like (n=7), blaOXA-51-like (n=9) and blaIMP-1 (n=3). The beta-lactamase genes blaKPC, blaVIM, blaBIC, blaGIM, blaDIM, blaSIM and blaAIM were not detected in the isolates. Sequencing of genes showed that all the amplified NDM genes were NDM-1, OXA-48-like genes were OXA-181 and IMP genes were IMP-1.
Identification of E. coli pathotypes and Klebsiella serotypes
The E. coli pathotyping results showed that, of the 57 E. coli isolates tested 12 were Enteropathogenic (EPEC), 9 Enteroaggregative (EAEC), 8 Enterohemorrhagic (EHEC), 3 Enterotoxigenic (ETEC), 1 Enteroinvasive (EIEC) and 24 unclassified E. coli (Table 3). Of the 12 EPEC isolates 3 were positive for NDM-1, 2 for OXA-181 and one for OXA-23; among 8 EHEC isolates NDM-1 was detected in 2 isolates, OXA-181 in one isolate and GES-1 plus GES-9 in one isolate; among 9 EAEC isolates GES-1 was found in 1 isolate, OXA-23 in 1 and OXA-23 along with GES-9 in one isolate; among 3 ETEC isolates 1 isolate carried NDM-1 and one EIEC isolate carried NDM-1 among with OXA-181. The virulence genes found in E. coli isolates included eaeA (n=20), LT (n=3), aggR (n=6), astA (n=5) and VirA (n=1). 24 E. coli isolates did not belong to any of the tested pathotypes.
Of the 45 Klebsiella species, 14 belonged to K1 serotype, 11 were K2, none of the isolates were of K5 serotypes and 20 were of unknown serotypes (Table 3). Of the 14 K. pneumoniae, NDM-1 (n=1), OXA-181 (n=4) and OXA-51 (n=1) was detected in K1 serotypes. Among 11 K. pneumoniae, OXA-181 (n=1) and GES-9 (n=1) were detected in K2 serotypes.
Plasmid incompatibility typing and conjugation
Plasmid DNA was isolated from 70 isolates which carried resistance genes (Table 4). The plasmids ranged from 10-100 kb in size. In total, of the 151 isolates studied 70 isolates carried resistance genes of which 11 were plasmid-borne and 59 were chromosomal. Of the 37 E. coli isolates, 32 isolates carried resistance genes of which 6 were plasmid-borne. Among 40 K. pneumoniae, only 19 carried resistance genes of which 3 were associated with plasmids. In E. cloacae, one isolate carried blaNDM-1 on plasmid and in P. mirabilis; one isolate carried plasmid-borne blaIMP-1. Plasmid incompatibility/replicon (inc/rep) typing results showed that the plasmids were belonging to Inc/rep types: IncX, IncA/C, IncFIA-FIB and IncFIIA (Table 4). E. coli isolates harboured blaNDM-1 genes in IncX (EC10), IncA/C (EC21) and IncFIA-FIB (EC29) type plasmids whereas blaOXA-48-like genes were associated with IncFIIA (EC39) and IncFIA-FIB (EC29), and blaGES-1/9 genes with IncFIA-FIB (EC47) type plasmid.
K. pneumoniae isolates harboured blaNDM-1 genes in IncFIA-FIB (KP10) and blaGES-1, blaOXA-23/51-like genes in IncA/C (KP31 and KP39) type plasmids. One E. cloacae isolate harboured blaNDM-1 gene in IncFIIA (EL3) type plasmid and one P. mirabilis isolate harboured blaIMP-1 gene in IncFIA-FIB (PM5) type plasmid.
Overall, 6 E. coli (EC10, 21, 29, 39, 44, 47) isolates, 3 K. pneumoniae isolates (KP10, 31, 39), one E. cloacae isolate (EL3) and one P. mirabilis isolate (PM5) carried resistance genes on plasmid of the identified inc/rep types. All the 6 E. coli isolates (EC10, 21, 29, 39, 44, 47) were found to transfer resistance plasmids to susceptible E. coli AB1157. Inter-generic transfer of NDM-1 was observed in one K. pneumoniae isolate (KP10) in which blaNDM-1 harbouring plasmid IncFIA-FIB was transferrable to E. coli AB1157 (Table 4).