Hybrid Plasmids Encoding Antimicrobial Resistance and Virulence Traits Among Hypervirulent Klebsiella pneumoniae ST2096 in India.

Background: Hypervirulent variants of Klebsiella pneumoniae (HvKp) were typically associated with a broadly antimicrobial susceptible clone of sequence type (ST) 23 at the time of its emergence. Concerningly, HvKp is now also emerging within multidrug-resistant (MDR) clones, including ST11, ST15, and ST147. MDR-HvKp either carry both the virulence and resistance plasmids or carry a large hybrid plasmid coding for both virulence and resistance determinants. Here, we aimed to genetically characterize a collection of MDR-HvKp ST2096 isolates haboring hybrid plasmids carrying both antimicrobial resistance (AMR) and virulence genes. Methods: Nine K. pneumoniae ST2096 isolated over 1 year from the blood sample of hospitalized patients in southern India that were MDR and suspected to be HvKp were selected. All nine isolates were subjected to short-read whole-genome sequencing; a subset (n = 4) was additionally subjected to long-read sequencing to obtain complete genomes for characterization. Mucoviscosity assay was also performed for phenotypic assessment. Results: Among the nine isolates, seven were carbapenem-resistant, two of which carried blaNDM-5 on an IncFII plasmid and five carried blaOXA-232 on a ColKP3 plasmid. The organisms were confirmed as HvKp, with characteristic virulence genes (rmpA2, iutA, and iucABCD) carried on a large (~320 kbp) IncFIB-IncHI1B co-integrate. This hybrid plasmid also carried the aadA2, armA, blaOXA-1, msrE, mphE, sul1, and dfrA14 AMR genes in addition to the heavy-metal resistance genes. The hybrid plasmid showed about 60% similarity to the IncHI1B virulence plasmid of K. pneumoniae SGH10 and ~70% sequence identity with the first identified IncHI1B pNDM-MAR plasmid. Notably, the hybrid plasmid carried its type IV-A3 CRISPR-Cas system which harbored spacer regions against traL of IncF plasmids, thereby preventing their acquisition. Conclusion: The convergence of virulence and AMR is clinically concerning in K. pneumoniae. Our data highlight the role of hybrid plasmids carrying both AMR and virulence genes in K. pneumoniae ST2096, suggesting that MDR-HvKp is not confined to selected clones; we highlight the continued emergence of such genotypes across the species. The convergence is occurring globally amidst several clones and is of great concern to public health.

Genomic insights of optrA-carrying linezolid-resistant Enterococcus faecium using hybrid assembly: first report from India.

OBJECTIVES: Linezolid resistance in Enterococcus faecium is emerging worldwide. In this study, we aimed to characterise two linezolid-resistant E. faecium isolates using whole-genome sequencing. METHODS: Antimicrobial susceptibility testing was performed by the broth microdilution method. A hybrid assembly approach of IonTorrent and MinION sequencing reads was used to generate the complete genome of linezolid-resistant E. faecium isolates VB3025 and VB3240. RESULTS: VB3025 and VB3240 had minimum inhibitory concentration (MICs) for linezolid of 1024 µg/mL and 512 µg/mL, respectively. In addition, VB3025 was found to be resistant to both vancomycin and teicoplanin, while VB3240 was susceptible to these antibiotics. A hybrid assembly approach was used to generate the complete genome of VB3025 and VB3240 isolates harbouring the optrA gene. Notably, VB3025 carried two copies of optrA (chromosomal and plasmid), while in VB3240 optrA was identified on the chromosome. Interestingly, the plasmid pVB3025_2 co-carried the resistance gene clusters aph(3)-IIIa-sat4-ant(6)-Ia-ermB, the vanHAX operon and a copy of the optrA gene. Moreover, the optrA gene inserted into a Tn554 transposon carrying the ermA gene was identified in both VB3025 and VB3240 isolates. Furthermore, mutation analysis revealed the presence of a G2592T mutation in the 23S rRNA of both isolates. CONCLUSION: This is the first study reporting optrA-positive linezolid-resistant E. faecium from India. A novel plasmid co-carrying vancomycin and linezolid resistance determinants highlights the threat for potential dissemination.

Identification of plasmids by PCR based replicon typing in bacteremic Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae is a notorious pathogen with plasmid mediated resistance to all classes of antibiotics. It is important to determine the plasmid profile coding for resistance genes. Plasmid profile varies among geographical regions and tracking the types helps in determining the MDR and XDR K. pneumoniae spread especially in hospital setting. Aim of the present study was to determine the plasmid profile and types among bacteraemic K. pneumoniae. MATERIALS AND METHODS: Ninety consecutive K. pneumoniae collected over a period of three months from blood cultures were characterised by PCR for plasmid profile. Inc plasmid types were determined by PCR based replicon typing (PBRT) and carbapenemases were determined by multiplex PCR. For a subset of isolates hybrid assemblies were developed by sequencing with Ion Torrent and MinIon. RESULTS: Overall, PBRT showed 29% of isolates carried four plasmids including IncHI1B, IncFIA, IncFII(K) and IncR. The most common type of plasmid was IncHI1B (93%) followed by IncFIIK (89%) and IncR (82%). IncFIA was predominant among carbapenem resistant isolates. Almost all plasmids identified in K. pneumoniae were AMR plasmids, except two isolates which had virulence plasmids. IncX3 plasmid observed in this study was previously reported to be self-disseminating. Furthermore, the hybrid genome sequencing revealed complete structural arrangements of plasmids, which would be missed in short-read sequencing. NDM and OXA48-like were co-produced in 59% of the carbapenem resistant isolates. BlaOXA-232 was present on ColKP3; aac(6')-lb3 and rmtF on IncFIB. CONCLUSION: Diverse plasmid profile among the successive K. pneumoniae isolates indicates the transfer of resistance genes through different types of plasmids. IncHI1B, IncFIA, IncFIIK and IncR were the prevalent plasmid types. Hybrid assembly revealed blaOXA-232 was present on ColKP3 unlike global reports of IncL/M. Hybrid assemblies provide better plasmid structure that long and short read assemblies. There was no significant association of ß-lactamases with specific Inc groups in this study.

First Indian report on genome-wide comparison of multidrug-resistant Escherichia coli from blood stream infections.

BACKGROUND: Multidrug-resistant (MDR) E. coli with extended-spectrum ß-lactamases (ESBLs) is becoming endemic in health care settings around the world. Baseline data on virulence and antimicrobial resistance (AMR) of specific lineages of E. coli circulating in developing countries like India is currently lacking. METHODS: Whole-genome sequencing was performed for 60 MDR E. coli isolates. The analysis was performed at single nucleotide polymorphism (SNP) level resolution to identify the presence of their virulence and AMR genes. RESULTS: Genome comparison revealed the presence of ST-131 global MDR and ST410 as emerging-MDR clades of E. coli in India. AMR gene profile for cephalosporin and carbapenem resistance differed between the clades. Genotypes blaCTX-M-15 and blaNDM-5 were common among cephalosporinases and carbapenemases, respectively. For aminoglycoside resistance, rmtB was positive for 31.7% of the isolates, of which 95% were co-harboring carbapenemases. In addition, the FimH types and virulence gene profile positively correlated with the SNP based phylogeny, and also revealed the evolution of MDR clones among the study population with temporal accumulation of SNPs. The predominant clone was ST167 (blaNDM lineage) followed by ST405 (global clone ST131 equivalent) and ST410 (fast spreading high risk clone). CONCLUSIONS: This is the first report on the whole genome analysis of MDR E. coli lineages circulating in India. Data from this study will provide public health agencies with baseline information on AMR and virulent genes in pathogenic E. coli in the region.

Multiple importations and transmission of colistin-resistant Klebsiella pneumoniae in a hospital in northern India.

OBJECTIVE: Resistance to colistin, a last resort antibiotic, has emerged in India. We investigated colistin-resistant Klebsiella pneumoniae(ColR-KP) in a hospital in India to describe infections, characterize resistance of isolates, compare concordance of detection methods, and identify transmission events. DESIGN: Retrospective observational study. METHODS: Case-patients were defined as individuals from whom ColR-KP was isolated from a clinical specimen between January 2016 and October 2017. Isolates resistant to colistin by Vitek 2 were confirmed by broth microdilution (BMD). Isolates underwent colistin susceptibility testing by disk diffusion and whole-genome sequencing. Medical records were reviewed. RESULTS: Of 846 K. pneumoniae isolates, 34 (4%) were colistin resistant. In total, 22 case-patients were identified. Most (90%) were male; their median age was 33 years. Half were transferred from another hospital; 45% died. Case-patients were admitted for a median of 14 days before detection of ColR-KP. Also, 7 case-patients (32%) received colistin before detection of ColR-KP. All isolates were resistant to carbapenems and susceptible to tigecycline. Isolates resistant to colistin by Vitek 2 were also resistant by BMD; 2 ColR-KP isolates were resistant by disk diffusion. Moreover, 8 multilocus sequence types were identified. Isolates were negative for mobile colistin resistance (mcr) genes. Based on sequencing analysis, in-hospital transmission may have occurred with 8 case-patients (38%). CONCLUSIONS: Multiple infections caused by highly resistant, mcr-negative ColR-KP with substantial mortality were identified. Disk diffusion correlated poorly with Vitek 2 and BMD for detection of ColR-KP. Sequencing indicated multiple importation and in-hospital transmission events. Enhanced detection for ColR-KP may be warranted in India.

Whole-Genome Shotgun Sequencing of the First Observation of Neisseria meningitidis Sequence Type 6928 in India.

Neisseria meningitidis is one of the leading global causes of bacterial meningitis. Here, we discuss the draft genome sequences of two N. meningitidis strains, isolated from bloodstream infections in two pediatric patients at a tertiary care hospital in South India. The sequence data indicate that strains VB13856 and VB15548 encode genomes of ~2.09 Mb in size with no plasmids.