Accurate identification and epidemiological characterization of Burkholderia cepacia complex : an update

Bacteria belonging to the Burkholderia cepacia complex (Bcc) are among the most important pathogens isolated from cystic fibrosis (CF) patients and in hospital acquired infections (HAI). Accurate identification of Bcc is questionable by conventional biochemical methods. Clonal typing of Burkholderia is also limited due to the problem with identification. Phenotypic identification methods such as VITEK2, protein signature identification methods like VITEK MS, Bruker Biotyper, and molecular targets such as 16S rRNA, recA, hisA and rpsU were reported with varying level of discrimination to identify Bcc. rpsU and/or 16S rRNA sequencing, VITEK2, VITEK MS and Bruker Biotyper could discriminate between Burkholderia spp. and non-Burkholderia spp. Whereas, Bcc complex level identification can be given by VITEK MS, Bruker Biotyper, and 16S rRNA/rpsU/recA/hisA sequencing. For species level identification within Bcc hisA or recA sequencing are reliable. Identification of Bcc is indispensable in CF patients and HAI to ensure appropriate antimicrobial therapy

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

The influence of biofilms on carbapenem susceptibility and patient outcome in device associated K. pneumoniae infections : insights Into phenotype vs genome-wide analysis and correlation

Klebsiella pneumoniae is one of the leading causes of nosocomial infections. Carbapenem-resistant K. pneumoniae are on the rise globally. The biofilm forming ability of K. pneumoniae further complicates patient management. There is still a knowledge gap on the association of biofilm formation with patient outcome and carbapenem susceptibility, which is investigated in present study. K. pneumoniae isolates from patients admitted in critical care units with catheters and ventilators were included. K. pneumoniae (n = 72) were subjected to 96-well plate biofilm formation assay followed by MBEC assay for subset of strong biofilm formers. Whole genome sequencing and a core genome phylogenetic analysis in comparison with global isolates were performed. Phenotypic analyses showed a positive correlation between biofilm formation and carbapenem resistance. Planktonic cells observed to be susceptible in vitro exhibited higher MICs in biofilm structure, hence MICs cannot be extrapolated for treatment. The biofilm forming ability had a significant association with morbidity/mortality. Infections by stronger biofilm forming pathogens significantly (p < 0.05) resulted in fewer “average days alive” for the patient (3.33 days) in comparison to those negative for biofilms (11.33 days). Phylogenetic analysis including global isolates revealed clear association of sequence types with genes for biofilm formation and carbapenem resistance. Known hypervirulent clone-ST23 with wcaG, magA, rmpA, rmpA2, and wzc with lack of mutation for hyper-capsulation might be poor biofilm formers. ST15, ST16, ST307, and ST258 (reported global high-risk clones) were wcaJ negative indicating the high potential of biofilm forming capacity. Genes wabG and treC for CPS, bcsA and pgaC for adhesins, luxS for quorum sensing were common in all clades in addition to genes for aerobactin (iutA), allantoin (allS), type I and III fimbriae (fimA, fimH, and mrkD) and pili (pilQ and ecpA). This study is the first of its kind to compare genetic features of antimicrobial resistance with a spectrum covering most of the genetic factors for K. pneumoniae biofilm. These results highlight the importance of biofilm screening to effectively manage nosocomial infections by K. pneumoniae. Further, data obtained on epidemiology and associations of biofilm and resistance genetic factors will serve to enhance our understanding on biofilm mechanisms in K. pneumoniae

First report on a cluster of colistin-resistant Klebsiella pneumoniae strains isolated from a tertiary care center in India : whole-genome shotgun sequencing

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

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. Keywords: 16S rRNA, k-mer, MALDI-TOF MS, single nucleotide polymorphism, whole genome sequencin

First Indian report of IncX3 plasmid carrying blaNDM-7 in Escherichia coli from bloodstream infection: potential for rapid dissemination

Enterobacteriaceae with blaNDM-7 is only infrequently observed. Self-transmissible plasmids carrying the blaNDM gene increase the dissemination of carbapenem resistance in developing countries. This study investigates the whole genome sequence of a blaNDM-7-positive Escherichia coli. The isolate was an extended-spectrum β-lactamase producer by combined disc diffusion test and carbapenemase producer by CarbaNP method. Sequencing results revealed the isolate as E. coli ST-167 with IncX3 plasmid carrying blaNDM-7 in addition to blaTEM-1 and blaCMY-42 genes. The identification of IncX3-blaNDM-7 combination is the first report in India where blaNDM-7 is known to cause higher resistance to carbapenems compared to its variants

Plasmid profiles among some ESKAPE pathogens in a tertiary care centre in south India

Background & objectives: Plasmid has led to increase in resistant bacterial pathogens through the exchange of antimicrobial resistance (AMR) genetic determinants through horizontal gene transfer. Baseline data on the occurrence of plasmids carrying AMR genes are lacking in India. This study was aimed to identify the plasmids associated with AMR genetic determinants in ESKAPE pathogens. Methods: A total of 112 ESKAPE isolates including Escherichia coli (n=37), Klebsiella pneumoniae (n=48, including 7 pan-drug susceptible isolates), Acinetobacter baumannii (n=8), Pseudomonas aeruginosa (n=1) and Staphylococcus aureus (n=18) were analyzed in the study. Isolates were screened for antimicrobial susceptibility and whole genome sequencing of isolates was performed using Ion Torrent (PGM) sequencer. Downstream data analysis was done using PATRIC, ResFinder, PlasmidFinder and MLSTFinder databases. All 88 whole genome sequences (WGS) were deposited at GenBank. Results: Most of the study isolates showed resistant phenotypes. As analyzed from WGS, the isolates included both known and unknown sequence types. The plasmid analysis revealed the presence of single or multiple plasmids in the isolates. Plasmid types such as IncHI1B(pNDM-MAR), IncFII(pRSB107), IncFIB(Mar), IncFIB(pQil), IncFIA, IncFII(K), IncR, ColKP3 and ColpVC were present in K. pneumoniae. In E. coli, IncFIA, IncFII, IncFIB, Col(BS512), IncL1, IncX3 and IncH were present along with other types. S. aureus harboured seven different plasmid groups pMW2 (rep 5), pSAS1 (rep 7), pDLK1 (rep 10), pUB110 (rep US12), Saa6159 (rep 16), pKH12 (rep 21) and pSA1308 (rep 21). The overall incidence of IncF type plasmids was 56.5 per cent followed by Col type plasmids 18.3 per cent and IncX 5.3 per cent. Other plasmid types identified were <5 per cent. Interpretation & conclusions: Results from the study may serve as a baseline data for the occurrence of AMR genes and plasmids in India. Information on the association between phenotypic and genotypic expression of AMR was deciphered from the data. Further studies on the mechanism of antibiotic resistance dissemination are essential for enhancing clinical lifetime of antibiotics