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Introduction: Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae are the most prominent bacterial species resistant to almost all commonly used antibiotics. Carbapenem is one of the last resort drugs for treating such emerging multidrug-resistant bacteria. This study aimed to detect carbapenem-resistant blaNDM-1 gene in ESBL producing E. coli and K. pneumoniae isolates. Methodology: A total of 190 E. coli and 350 K. pneumoniae isolates were screened for extended spectrumβ-lactamase (ESBL), carbapenemase and metallo β-lactamase (MBL) production via double-disk synergy test (DDST), modified Hodge test and combined-disk diffusion method. The blaNDM-1 gene was detected by PCR and confirmed via Sanger sequencing method. Results: Of the 540 isolates tested, 71.8% were found to be multidrug-resistant. Overall rate of ESBL-positive isolates were 57.89% E. coli and 31.42% K. pneumoniae. Among ESBL positive isolates, 49.09% E. coli and 40% K. pneumoniae were positive for carbapenemase production whereas MBL production was detected in 29% E. coli and 22% K. pneumoniae isolates. In MBL positive isolates, (37%) E. coli and (40%) K. pneumoniae isolates harboured blaNDM-1 gene. The pair-wise DNA was aligned with the NDM-1 sequence from GenBank. The alignment score was 243 and the blast nucleotide sequencing results showed 97% sequence similarity with the sequences in GenBank for the blaNDM-1 gene. Conclusions: The blaNDM-1 gene was found to be the most prevalent in urine samples. There is a dire need to conduct screening tests in hospitals and communities to find out the exact prevalence of the blaNDM-1 spread in our population.

Background: The presence of plasmid mediated mcr-1 gene in multidrug resistant Gram-negative bacteria poses a serious public health concern in today’s world. Objectives: The present study was aimed to detect the presence of plasmid mediated mcr-1 encoding resistance to colistin in multiple drug resistant (MDR) E. coli and K. pneumoniae isolates. Methods: A total 180 clinical isolates of E. coli (n=120) and K. pneumoniae (n=60) were isolated from different clinical specimens i.e. urine, blood, stool and pus, from diagnostic labs of two major public sector tertiary care hospitals in Peshawar, Pakistan. MDR profile of these isolates was assessed through Kirby-Baur disc diffusion method. All isolates were screened for colistin resistance by dilution methods. Colistin resistant isolates were subjected to PCR for mcr-1 detection and confirmation was done by Sanger sequencing method. Results: Overall 83.3% (100/120) E. coli and 93.3% (56/60) K. pneumoniae were detected as MDR. Colistin resistance was found in 23.3% (28/120) E. coli and 40% (24/60) K. pneumoniae isolates whereas mcr-1 gene was detected in 10 out of 52 colistin resistant isolates including six E. coli and four K. pneumoniae isolates. Minimum inhibitory concentrations (MICs) of colistin in these ten mcr-1 positive isolates ranged from 4µg/ml to 16µg/ml. All mcr-1 positive isolates showed 99% sequence similarity when compared with other present sequences in GenBank. Conclusion: Hence, our study confirms the presence of mcr-1 mediated colistin resistance in the studied area. Therefore, urgently larger scale surveillance studies are recommended to investigate prevalence of mcr-1 mediated colistin resistance and to prevent its further spread in the area.

Background: Colistin is the last-resort antibiotic available to date against Multiple-drug-resistant (MDR) bacteria, particularly carbapenem-resistant Enterobacteriaceae (CRE) harboring the NDM 1 and KPC 2 genes. Objectives: The current study was designed to investigate extended-spectrum β-lactamase (ESBL) production, colistin resistance, and the presence of mcr-1 in Klebsiella pneumoniae isolated from urine samples. Methods: A total of 298 clinical isolates of K. pneumoniae were collected for seven months in 2017 from the main labs of three government tertiary care hospitals in Pakistan. The ESBL activity of the isolates was assessed by the Double Disc Synergy test (DDST). All the ESBL-producing isolates were phenotypically screened for colistin resistance by dilution methods. Colistin-resistant isolates were subjected to PCR for mcr-1 detection. The confirmation was done by the Sanger sequencing method. Results: Out of 298 K. pneumoniae isolates, 35 (11.7%) isolates showed ESBL activity. They were phenotypically screened for colistin resistance. Four (11.4%) colistin-resistant isolates out of 35 (11.7%) showed the minimum inhibitory concentrations (MICs) ranging from 4 mg/L to 8 mg/L. The mcr-1 gene was detected in all four colistin-resistant isolates via specific primers/PCR and confirmed by Sanger sequencing, showing 99% sequence similarity with the mcr-1 gene in GenBank. The sequence was submitted to NCBI GenBank, and an accession number was assigned. Conclusions: The presence of the mcr-1 gene in ESBL-producing bacteria isolated from human urine samples highlights the urgent need for surveillance studies on a larger scale to overcome the inappropriate use of colistin-containing formulations and prevent further spread of resistance to this antibiotic.

Purpose The multiple-drug resistant Escherichia coli are among the deadliest pathogens causing life-threatening infections. This study was planned to determine the molecular epidemiology of mcr-1, bla KPC-2 , and bla NDM-1 harboring clinically isolated E. coli from Pakistan. Methods In total, 545 strains of E. coli from clinical samples were collected from June 2018 to September 2019. All the isolates were screened for colistin-resistance, extended-spectrum-β-lactamases (ESBL), and carbapenemases through the micro-dilution method, Double-Disk-Synergy-Test (DDST), and Modified-Hodge-Test (MHT). The detection, sequence-typing, conjugal transfer, S1-PFGE, plasmid-replicon-typing, and southern-blotting for mcr , ESBL, and carbapenemase-encoding genes were performed. Findings A total of four (0.73%) colistin-resistant strains carrying alongside mcr-1 and bla CTX-M-15 genes, three of these strains also had the bla TEM-1 gene. The presence of ESBL genes was detected in 139 (25.5%) isolates harboring bla CTXM-15 (74.82%), bla TEM (34.53%), bla SHV (28.06%) and bla OXA-1 (28.78%). In 129 carbapenemase-producers, 35.83% possessed bla NDM-1 , 26.67% bla KPC-2 , 8.3% bla OXA-48 , 25% bla VIM-1 , and 20.83% bla IMP-1 genes. The sequence typing revealed that mcr-1 harboring isolates belonged to ST405, ST117, and ST156. Fifty percent of bla KPC-2 and 48.83% of bla NDM-1 were found on ST131 and ST1196, respectively. Two rare types of STs, ST7584, and ST8671 were also identified in this study. The mcr-1 gene was located on Incl2 (60-kb) plasmid. The bla KPC-2 was present on (140-kb) IncH12, (100-kb) IncN, (90-kb) Incl1, while bla NDM-1 was located on (70-kb) IncFIIK, (140-kb) IncH12, (100-kb) IncN, (60-kb) IncA/C, and (45-kb) IncFII plasmids, which were successfully trans-conjugated. Among the plasmid types, the Incl1 carrying bla KPC-2 , IncH12 harboring bla KPC-2 and bla NDM-1 , and IncFIIK carrying bla NDM-1 ...