Francis Yesurajan Inbanathan

Molecular Mechanisms of Colistin Resistance in Klebsiella pneumoniae Causing Bacteremia from India—A First Report

Colistin has long been a reserve drug used for the treatment of carbapenem resistant Klebsiella pneumoniae. Carbapenem resistance in K. pneumoniae has been increasing and is as high as 44% in India. Although a reserve agent, with rise in rates of resistance to carbapenems, the usage of colistin has increased over the years leading to slow emergence of resistance. Colistin resistance is mainly mediated by the alteration in the LPS of bacterial outer membrane with the addition of L-Ara4-N and PEtN molecules. These alterations are mediated by mutations in several genes involved in lipidA modifications and most commonly mutations in mgrB gene has been reported. Recently there is emergence of plasmid mediated resistance due to mcr-1 and mcr-2 genes which poses a threat for the rapid global spread. This study aims at characterizing eight colistin resistant K. pneumoniae from bacteremia by whole genome sequencing. Eight K. pneumoniae were isolated from blood culture during 2013 and 2014 at the Department of Clinical Microbiology, Christian Medical College, India. Antimicrobial susceptibility testing was performed and minimum inhibitory concentration (MIC) was determined for colistin and polymyxin B by broth-micro dilution method. Whole genome sequencing was performed using Ion Torrent and the genome of all eight isolates was analyzed. The eight isolates were resistant to all the antimicrobials expect tigecycline. MIC of colistin and polymyxin B were ranged from 4 to 1024 μg/ml and 0.5 to 2048 μg/ml respectively. Multiple mutations were observed in the chromosomal genes involved in lipid A modifications. mcr-1 and mcr-2 gene was absent in all the isolates. The most significant were mutations in mgrB gene. Among the eight isolates, four, three and one were belonged to sequence types ST 231, ST14 and ST147 respectively. Seven isolates had blaOXA−48 like, one co-expressed blaNDM−1 and blaOXA−48 like genes leading to carbapenem resistance. Overall, multiple numbers of alterations have been observed. This includes silent mutations, point mutations, insertions and/or deletions. Mutations in mgrB gene is responsible for resistance to colistin in this study. Due to emergence of resistance to reserve drugs, there is a need for combination therapies for carbapenem resistant K. pneumoniae and colistin must be judiciously used.

Whole-Genome Shotgun Sequencing of Cephalosporin-Resistant Salmonella enterica Serovar Typhi

ABSTRACT Typhoid is one of the leading causes of mortality in developing countries. Here, we report the draft genome sequences of four Salmonella enterica serovar Typhi strains isolated from bloodstream infections in a tertiary care hospital. The sequence data indicate genomes of ~4.5 Mb for all isolates, with one plasmid in each.

Accurate differentiation of Escherichia coli and Shigella serogroups: challenges and strategies

Shigella spp. and Escherichia coli are closely related; both belong to the family Enterobacteriaceae. Phenotypically, Shigella spp. and E. coli share many common characteristics, yet they have separate entities in epidemiology and clinical disease, which poses a diagnostic challenge. We collated information for the best possible approach to differentiate clinically relevant E. coli from Shigella spp. We found that a molecular approach is required for confirmation. High discriminatory potential is seen with whole genome sequencing analysed for k-mers and single nucleotide polymorphism. Among these, identification using single nucleotide polymorphism is easy to perform and analyse, and it thus appears more promising. Among the nonmolecular methods, matrix-assisted desorption ionization–time of flight mass spectrometry may be applicable when data analysis is assisted with advanced analytic tools.

Multi locus sequence typing of Burkholderia pseudomallei isolates from India unveils molecular diversity and confers regional association in Southeast Asia

Objectives Burkholderia pseudomallei, the causative agent for melioidosis, has become a public health problem in India and across the world. Melioidosis can be difficult to diagnose because of the inconsistent clinical presentations of the disease. This study aims to determine the genetic diversity among the clinical isolates of B. pseudomaelli from India in order to establish a molecular epidemiology and elucidate the Southeast Asian association. Methods Molecular typing using multi locus sequence typing was performed on thirty one archived B. pseudomallei clinical isolates, previously characterised from specimens obtained from patients admitted to the Christian Medical College & Hospital, Vellore from 2015 to 2016. Further investigations into the genetic heterogeneity and evolution at a regional and global level were performed using insilico tools. Results Multi locus sequence typing (MLST) of the isolates from systemic and localized forms of melioidosis, including blood, pus, tissue, and urine specimens, revealed twenty isolates with novel sequence types and eleven with previously reported sequence types. High genetic diversity was observed using MLST with a strong association within the Southeast Asian region. Conclusions Molecular typing of B. pseudomallei clinical isolates using MLST revealed high genetic diversity and provided a baseline molecular epidemiology of the disease in India with a strong Southeast Asian association of the strains. Future studies should focus on whole genome based Single-Nucleotide-Polymorphism (SNP) which has the advantage of a high discriminatory power, to further understand the novel sequence types reported in this study.

Epidemiological Investigation and Successful Management of Burkholderia cepacia Outbreak in Neuro-Trauma Intensive Care Unit

OBJECTIVE The detailed epidemiological and molecular characterization of an outbreak of Burkholderia cepacia at a neurotrauma intensive care unit of a level 1 trauma centre is described. The stringent infection control interventions taken to successfully curb this outbreak are emphasized. METHODS The clinical and microbiological data for those patients who had more than one blood culture that grew B. cepacia were reviewed. Bacterial identification and antimicrobial susceptibility testing was done using automated Vitek 2 systems. Prospective surveillance, environmental sampling, and multilocus sequence typing (MLST) were performed for extensive source tracking. Intensive infection control measures were taken to further control the hospital spread. RESULTS Out of a total 48 patients with B. cepacia bacteraemia, 15 (31%) had central line-associated blood stream infections. Two hundred and thirty-one environmental samples were collected and screened, and only two water samples grew B. cepacia with similar phenotypic characteristics. The clinical strains characterized by MLST typing were clonal. However, isolates from the water represented a novel strain type (ST-1289). Intensive terminal cleaning, disinfection of the water supply, and the augmentation of infection control activities were done to curb the outbreak. A subsequent reduction in bacteraemia cases was observed. CONCLUSION Early diagnosis and appropriate therapy, along with the rigorous implementation of essential hospital infection control practices is required for successful containment of this pathogen and to curb such an outbreak.

Multiple mutations in lipid-A modification pathway & novel fosA variants in colistin-resistant Klebsiella pneumoniae

Aim: To investigate antimicrobial resistance mechanisms in a cluster of colistin-resistant Klebsiella pneumoniae. Methods: Antimicrobial susceptibility was tested by disk diffusion and broth microdilution. Whole-genome sequencing and genome analysis were performed. Results: The eight colistin-resistant K. pneumoniae isolates belonged to three different clones (ST11, 14 and 231). The eptA and arnT genes from lipid modification pathway had novel (R157S in arnT and Q319R in eptA) and rare mutations (V39L, R152H, S260L and A279G in eptA). Several substitutions were also identified in mgrB, pmrB, phoP and phoQ genes. The mcr genes were absent in all isolates. Isolates had variants from existing classes of fosA gene. Conclusion: Complex combination of mutations might have led to colistin resistance, which suggests that continuous surveillance of molecular mechanisms is required.

Whole genome shotgun sequences of Streptococcus pyogenes causing acute pharyngitis from India

Streptococcus pyogenes, belonging to group A streptococcus (GAS), causes over 600 million infections annually being a predominant human pathogen. Lack of genomic data on GAS from India is one limitation to understand its virulence and antimicrobial resistance determinants. The genome of GAS isolates from clinical samples collected at Navi Mumbai, India was sequenced and annotated. Sequencing was performed on Ion Torrent PGM platform. The size of annotated S. pyogenes genomes ranged from ~1.69 to ~1.85 Mb with coverage of 38× to 189×. Most of the isolates had msr(D) and mef(A), and four isolates had erm(B) gene for macrolide resistance. The genome harboured multiple virulence factors including exotoxins in addition to phage elements in all GAS genomes. Four isolates belonged to sequence type ST28, 7 were identified as ST36 and 1 as ST55.

Whole genome shotgun sequencing of Indian strains of Streptococcus agalactiae

Group B streptococcus is known as a leading cause of neonatal infections in developing countries. The present study describes the whole genome shotgun sequences of four Group B Streptococcus (GBS) isolates. Molecular data on clonality is lacking for GBS in India. The present genome report will add important information on the scarce genome data of GBS and will help in deriving comparative genome studies of GBS isolates at global level. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession numbers NHPL00000000 – NHPO00000000.

First Report on a Cluster of Colistin-Resistant Klebsiella pneumoniae Strains Isolated from a Tertiary Care Center in India: Whole-Genome Shotgun Sequencing

ABSTRACT Klebsiella pneumoniae is a nosocomial pathogen with clinical importance due to its increasing resistance to carbapenems and colistin. Here, we report the genome sequences of eight colistin-resistant K. pneumoniae strains which might help in understanding the molecular mechanism of the species. The sequence data indicate genomes of ~5.2 to 5.4 Mb, along with several plasmids.

Novel Observations in 11 Heteroresistant Vancomycin-Intermediate Methicillin-Resistant Staphylococcus aureus Strains from South India

ABSTRACT We report here the draft genome sequences of 11 heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) strains from bloodstream infection. All strains harbor mutations in vraSR, graSR, walKR, and/or tcaRAB and are often implicated as the frequently mutated candidate genes in hVISA phenotypes.