Circulation of Third-Generation Cephalosporin Resistant Salmonella Typhi in Mumbai, India.

We report the persistent circulation of third-generation cephalosporin resistant Salmonella Typhi in Mumbai, linked to the acquisition and maintenance of a previously characterized IncX3 plasmid carrying the ESBL gene blaSHV-12 and the fluoroquinolone resistance gene qnrB7 in the genetic context of a triple mutant also associated with fluoroquinolone resistance.

High-Resolution Genomic Profiling of Carbapenem-Resistant Klebsiella pneumoniae Isolates: A Multicentric Retrospective Indian Study.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a threat to public health in India because of its high dissemination, mortality, and limited treatment options. Its genomic variability is reflected in the diversity of sequence types, virulence factors, and antimicrobial resistance (AMR) mechanisms. This study aims to characterize the clonal relationships and genetic mechanisms of resistance and virulence in CRKP isolates in India. MATERIALS AND METHODS: We characterized 344 retrospective K. pneumoniae clinical isolates collected from 8 centers across India collected in 2013-2019. Susceptibility to antibiotics was tested with VITEK 2. Capsular types, multilocus sequence type, virulence genes, AMR determinants, plasmid replicon types, and a single-nucleotide polymorphism phylogeny were inferred from their whole genome sequences. RESULTS: Phylogenetic analysis of the 325 Klebsiella isolates that passed quality control revealed 3 groups: K. pneumoniae sensu stricto (n = 307), K. quasipneumoniae (n = 17), and K. variicola (n = 1). Sequencing and capsular diversity analysis of the 307 K. pneumoniae sensu stricto isolates revealed 28 sequence types, 26 K-locus types, and 11 O-locus types, with ST231, KL51, and O1V2 being predominant. blaOXA-48-like and blaNDM-1/5 were present in 73.2% and 24.4% of isolates, respectively. The major plasmid replicon types associated with carbapenase genes were IncF (51.0%) and Col group (35.0%). CONCLUSION: Our study documents for the first time the genetic diversity of K and O antigens circulating in India. The results demonstrate the practical applicability of genomic surveillance and its utility in tracking the population dynamics of CRKP. It alerts us to the urgency for longitudinal surveillance of these transmissible lineages.

Overcoming Data Bottlenecks in Genomic Pathogen Surveillance.

Performing whole genome sequencing (WGS) for the surveillance of antimicrobial resistance offers the ability to determine not only the antimicrobials to which rates of resistance are increasing, but also the evolutionary mechanisms and transmission routes responsible for the increase at local, national, and global scales. To derive WGS-based outputs, a series of processes are required, beginning with sample and metadata collection, followed by nucleic acid extraction, library preparation, sequencing, and analysis. Throughout this pathway there are many data-related operations required (informatics) combined with more biologically focused procedures (bioinformatics). For a laboratory aiming to implement pathogen genomics, the informatics and bioinformatics activities can be a barrier to starting on the journey; for a laboratory that has already started, these activities may become overwhelming. Here we describe these data bottlenecks and how they have been addressed in laboratories in India, Colombia, Nigeria, and the Philippines, as part of the National Institute for Health Research Global Health Research Unit on Genomic Surveillance of Antimicrobial Resistance. The approaches taken include the use of reproducible data parsing pipelines and genome sequence analysis workflows, using technologies such as Data-flo, the Nextflow workflow manager, and containerization of software dependencies. By overcoming barriers to WGS implementation in countries where genome sampling for some species may be underrepresented, a body of evidence can be built to determine the concordance of antimicrobial sensitivity testing and genome-derived resistance, and novel high-risk clones and unknown mechanisms of resistance can be discovered.

Assessment of in vitro colistin susceptibility of carbapenem-resistant clinical Gram-negative bacterial isolates using four commercially available systems & Whole-genome sequencing: A diagnostic accuracy study.

AIM: To analyze the diagnostic utility of commercially available platforms and Whole-genome sequencing (WGS) for accurate determination of colistin susceptibility test results. MATERIAL & METHODS: An exploratory diagnostic accuracy study was conducted in which sixty carbapenem-resistant Gram-negative bacteria were subjected to identification and AST using MALDI-TOF MS & MicroScan walkaway 96 Plus. Additional AST was performed using the BD Phoenix system and Mikrolatest colistin kit. The test isolates were subjected to Vitek-2 and WGS at CRL, Bengaluru. RESULTS: There was no statistically significant agreement between the colistin susceptibility results obtained by WGS, with those of commercial phenotypic platforms. The MicroScan 96 Plus had the highest sensitivity (31 %) & NPV (77 %), and the BD Phoenix system had the highest specificity (97 %) and PPV (50 %), respectively, for determining colistin resistance. CONCLUSION: The utility of WGS as a tool in AMR surveillance and validation of phenotypic AST methods should be explored further.

An insight into whole genome sequencing data of methicillin-resistant Staphylococcus aureus circulating in a teaching hospital in North India.

PURPOSE: To provide an insight into the Whole-genome sequencing (WGS) data of MRSA strains circulating in a teaching hospital in north India. METHODS: An exploratory study was conducted in which fifty non-repetitive MRSA isolates obtained from pus samples of inpatients from July 2018 to February 2019 were subjected to preliminary identification (ID) and antibiotic susceptibility testing (AST) at our centre. These isolates were later sent to Central Research Laboratory, India for further testing using the VITEK-2 compact system followed by WGS. Only eighteen isolates were eventually considered for final analysis and the rest (n â€‹= â€‹32) were excluded due to various technical reasons. RESULTS: The WGS confirmed MRSA isolates predominantly belonged to CC22 (56.25%) and CC30 (31.25%). The CC22 MRSA strains carried SCCmec types IVa (77.8%) & IVc (22.2%) and belonged to spa types t005 (44.4%), t4584 (22.2%), t11808 (11.1%), t1328 (11.1%) and t309 (11.1%), respectively. The MRSA isolates of CC30 carried SCCmec types IVa (60%), IVg (20%) & V (20%) and belonged to spa types t021 (80%) & t2575 (20%), respectively. One MRSA isolate carried a novel SCCmec type V. The luk-PV and tsst-1 genes were present in 93.75% and 33.33% of MRSA isolates, respectively. The concordance between the phenotypic and genotypic AST results was 100% for Beta-lactams, Fluoroquinolones, Tetracyclines & Lipoglycopeptides, respectively. CONCLUSIONS: Through this study, we intend to embark upon a relatively newer avenue of clinical-genomic surveillance of nosocomial bacterial isolates like MRSA, which would help us improve the existing infection control and antibiotic stewardship practices in our hospital.

Novel multidrug-resistant sublineages of Staphylococcus aureus clonal complex 22 discovered in India.

Staphylococcus aureus is a major pathogen in India causing community and nosocomial infections, but little is known about its molecular epidemiology and mechanisms of resistance in hospital settings. Here, we use whole-genome sequencing (WGS) to characterize 478 S. aureus clinical isolates (393 methicillin-resistant Staphylococcus aureus (MRSA) and 85 methicilin-sensitive Staphylococcus aureus (MSSA) collected from 17 sentinel sites across India between 2014 and 2019. Sequencing results confirmed that sequence type 22 (ST22) (142 isolates, 29.7%), ST239 (74 isolates, 15.48%), and ST772 (67 isolates, 14%) were the most common clones. An in-depth analysis of 175 clonal complex (CC) 22 Indian isolates identified two novel ST22 MRSA lineages, both Panton-Valentine leukocidin+, both resistant to fluoroquinolones and aminoglycosides, and one harboring the the gene for toxic shock syndrome toxin 1 (tst). A temporal analysis of 1797 CC22 global isolates from 14 different studies showed that the two Indian ST22 lineages shared a common ancestor in 1984 (95% highest posterior density [HPD]: 1982-1986), as well as evidence of transmission to other parts of the world. Moreover, the study also gives a comprehensive view of ST2371, a sublineage of CC22, as a new emerging lineage in India and describes it in relationship with the other Indian ST22 isolates. In addition, the retrospective identification of a putative outbreak of multidrug-resistant (MDR) ST239 from a single hospital in Bangalore that persisted over a period of 3 years highlights the need for the implementation of routine surveillance and simple infection prevention and control measures to reduce these outbreaks. To our knowledge, this is the first WGS study that characterized CC22 in India and showed that the Indian clones are distinct from the EMRSA-15 clone. Thus, with the improved resolution afforded by WGS, this study substantially contributed to our understanding of the global population of MRSA. IMPORTANCE The study conducted in India between 2014 and 2019 presents novel insights into the prevalence of MRSA in the region. Previous studies have characterized two dominant clones of MRSA in India, ST772 and ST239, using whole-genome sequencing. However, this study is the first to describe the third dominant clone, ST22, using the same approach. The ST22 Indian isolates were analyzed in-depth, leading to the discovery of two new sublineages of hospital-acquired Staphylococcus aureus in India, both carrying antimicrobial resistance genes and mutations, which limit treatment options for patients. One of the newly characterized sublineages, second Indian cluster, carries the tsst-1 virulence gene, increasing the risk of severe infections. The geographic spread of the two novel lineages, both within India and internationally, could pose a global public health threat. The study also sheds light on ST2371 in India, a single-locus variant of ST22. The identification of a putative outbreak of MDR ST239 in a single hospital in Bangalore emphasizes the need for routine surveillance and simple infection prevention and control measures to reduce these outbreaks. Overall, this study significantly contributes to our understanding of the global population of MRSA, thanks to the improved resolution afforded by WGS.

Molecular characterisation of carbapenem-resistant Klebsiella pneumoniae clinical isolates: preliminary experience from a tertiary care teaching hospital in the Himalayas.

BACKGROUND: There is a lack of whole-genome sequencing (WGS) data on multidrug-resistant (MDR) bacteria from the Uttarakhand region of India. The aim of this study was to generate WGS data of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates recovered from patients in Uttarakhand's tertiary care centre. METHODS: A cross-sectional study included 29 MDR K. pneumoniae test isolates obtained from various clinical samples submitted to the bacteriology laboratory for culture and sensitivity testing from July 2018 to August 2019. After preliminary identification and antibiotic susceptibility testing, these isolates were subjected to WGS. RESULTS: A total of 27 of 29 isolates were CRKP. ST14 was the most common sequence type (n=8 [29.6%]). Carbapenem resistance was mainly encoded by OXA-48-like genes (21/27 [77.8%]). All isolates had a varied arsenal of resistance genes to different antibiotic classes. KL2 (9/27 [33.3%]) and KL51 (8/27 [29.6%]) were dominant K loci types. O1 and O2 together accounted for 88.9% (n=27) of CRKP isolates. Genes encoding yersiniabactin (ybt) and aerobactin (iuc) were identified in 88.9% (24/27) and 29.6% (8/27) of isolates. The predominant plasmid replicons present were ColKP3 (55.5%), IncFII(K) (51.8%) and IncFIB(pQil) (44.4%). CONCLUSIONS: This study emphasises the need for continued genomic surveillance of MDR bacteria that could be instrumental in developing treatment guidelines based on integrating phenotypic and molecular methods.