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The emergence of multi drug resistant clone CC320 serotype19F/19A and their capsular (cps) antigenic variants due to selective pressures such as vaccine had been reported worldwide. Hence, it is important to identify the prevalent clones, sequence types and cps variants of serotype 19F/19A in India, where PCV13 has been recently introduced. Multi-locus sequence typing (MLST) was performed for all (n = 21) invasive S. pneumoniae isolates of serotype 19A (n = 5) and 19F (n = 16) collected between the years 2012 and 2018 from children less than 5 years. The genome characterization by whole genome sequencing for the Sequence types (STs) 320 and 271(n = 7) were performed and compared with another six Indian WGSs of similar STs available from the GPS platform. The predominant STs in the serotype 19F/19A study isolates were of CC320: ST 320, 236 and 271, associated with PMEN clone Taiwan19F-14. The WGSs of CC320 study isolates showed high genomic similarity to the Taiwan19F-14 clone, and the penicillin binding protein (PBP) amino acid sequence similarity was 100% for PBP1A, 93% for PBP 2B and 2X. Whilst PBP comparison with other global MDR ST320 strains revealed that the ST320 clones in India are of low-level penicillin resistance. The presence of a few ST320/19A/19F invasive isolates with high similarity to the Taiwan clone suggests slow and gradual expansion of Taiwan19F-14 associated CC320 clones in India. Since serotype 19F/19A is covered by PCV13 vaccine, the expansion of 19F/19A cones with non-PCV13 vaccine serotype in India should be monitored.

Penicillin-binding proteins are the primary targets for beta lactam drugs, which are main stay of treatment for Streptococcus pneumoniae. The emergence of increased penicillin resistance in meninge...

Linezolid resistance has increasingly been described in coagulase negative staphylococci (CoNS) in recent years. Here, we describe the molecular mechanism of linezolid resistance in Staphylococcus haemolyticus using whole genome sequencing. Three S. haemolyticus isolates (VB5326, VB19458, and VB840) carried G2576T mutation at the domain V of the 23S rRNA. In addition, VB5326 and VB19458 carried the cfr gene in the chromosome. The presence of cfr gene, in combination with G2576T mutation in 23S rRNA, resulted in a high linezolid Minimum inhibitory concentration (MIC) of > 256 µg/ml. Three mutations, including D471E, I527M, and S532N, in rpoB contributed to an increased rifampicin MIC of 32 µg/ml. Subsequent development of linezolid and rifampicin resistance in S. haemolyticus is worrisome and greatly limits clinical management.

Objective:Resistance to colistin, a last resort antibiotic, has emerged in India. We investigated colistin-resistantKlebsiella 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 846K. pneumoniaeisolates, 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.

Salmonella Typhimurium ST313 is known to cause invasive disease in sub Saharan African (sSA) countries while the same sequence type is often associated with gastro-intestinal infections in the UK and Brazil. Although S. Typhimurium has been frequently isolated from human samples in India, the prevalence and invasive nature of infection of ST313 is currently unknown. The present study elucidates the phenotypic and genotypic characteristics of S. Typhimurium strain B3589 that belongs to ST313. The isolate was subjected to serotyping and antimicrobial susceptibility test to understand its phenotypical characteristics. Whole genome sequencing and comparative genomic analysis was carried out to provide an insight into S. Typhimurium ST313 lineage in India. The results suggests antibiotic resistance against aminoglycoside was associated with the presence of aminoglycoside modifying enzymes aac(6′)-Ia in the genome. Phylogenetic analysis revealed the India-ST313 isolates are genotypically distinct from the known African, UK and Brazilian ST313 lineages. The isolate possess the characteristic prophage gene repertoire except BTP-5. The presence of BTP-1 and more importantly bstA virulence gene has been the distinguishable feature of strain B3589 among other non-African isolates. In addition the genome degradation of African ST313 lineage-2 was not conserved in the Indian ST313 isolates. Fewer genome degradation events as well as the absence of plasmid mediated MDR locus suggest the Indian ST313 isolates are of low risk. The identification of ST313 isolates in India reveals the previously unknown characteristics of ST313 S. Typhimurium isolated from India.

Background: Pneumococcal pneumonia is one of the major causes of mortality in children less than 5 years in Asia, especially in India. Available PCVs have less serotype coverage in India compared to western countries. Moreover, the baseline pneumococcal serotype and sequence type data is limited and available data doesn't represent the entire India. With this background we aimed to characterize invasive and carriage isolates of S. pneumoniae from a tertiary care hospital in South India. Materials and Methods: A total of 221 S. pneumoniae isolates, invasive (n=138) and carriage (n=83) between the time period of 2012-2018 were included. Isolates was identified and confirmed using standard laboratory protocols. Serotyping was performed by Customized sequential multiplex PCR and MLST as described in www.pubmlst.org. Results: The major serotypes were 19F, 6B, 14, 6A and 19A and the sequence types (ST) were ST63, 236 and 230. Predominant STs in invasive was ST 63 whereas in carriage were ST4894 and 1701. High level ST diversity in carriage was observed. Majority of the STs were SLVs or DLVs of previously reported STs or PMEN clones. Phylogenetic analyses of the STs revealed gradual expansion of three PMEN CCs CC320, 63 and 230. Conclusion: The vaccine serotypes were the predominant ones found to be associated with IPD, PMEN clones, new STs and antimicrobial resistance. Accordingly, PCV13 is expected to provide invasive serotype coverage of 75% in Indian children less than 5 years. This study provides baseline serotype and sequence type data prior to the introduction of PCV in South India.

Shigella is the major cause of bacillary dysentery worldwide, especially in developing countries. There are several virulence factors essential for the organism to be virulent which are generally present in the virulence plasmid and on chromosomal pathogenicity islands. The present study was undertaken to determine the virulence gene profile of Shigella spp isolated from a clinical specimen and to study their significant association with common clinical symptoms and antimicrobial resistance. Sixty Shigella whole genome sequences, including 22 S. flexneri, 14 S. sonnei, 17 S. boydii and 7 S. dysenteriae were analyzed for the presence of virulence genes. The gene found predominantly in this study were ipaH (90%) followed by sigA (83%), and lpfA (78%) respectively. The virulence genes were significantly higher in S. flexneri, particularly in serotype 2 compared to S. sonnei. Interestingly, a significant association was observed between sigA gene and fever whereas sepA and sigA were found to be associated with diarrhea. Among the studied Shigella isolates, the presence of virulence genes was found higher in isolates resistant to more than three antibiotic classes. The present work revealed the varying incidence of virulence determinants among different Shigella serogroups and shows their contribution to disease severity.

Background: Non-typhoidal Salmonella (NTS) infection is a serious public health problem globally. Although NTS infections are self-limited, antimicrobial therapy is recommended for severe infections and immunocompromised patients. Antimicrobial resistance (AMR) in these pathogens further limits its therapeutic options. Here, we report an incidence of ceftriaxone resistance in NTS over the past 9 years in a southern Indian region. Materials and Methods: Molecular mechanisms of resistance in ceftriaxone-resistant NTS have been tested by both phenotypic and molecular methods. Minimum inhibitory concentration was determined by the E-test and broth microdilution method. AMR gene markers of β-lactamases such as AmpCs (blaMOX, blaCMY, blaDHA, blaFOX, blaACC and blaACT) and extended-spectrum β-lactamases (ESBLs) (blaSHV, blaTEM, blaVEB, blaPER, blaCTXM-1like,blaCTXM-2like, blaCTXM-8like, blaCTXM-9like and blaCTXM-25like) were screened. The presence of IncH12 and IncI1 plasmid was also analysed. Results: The study reports a 5% prevalence of ceftriaxone resistance in NTS. The most common serogroup was Salmonella Group B followed by Salmonella Group E and Salmonella group C1/C2. The occurrence of blaCTX-M-1, blaTEM, blaCMY and blaSHV genes was observed in 54%, 54%, 48% and 3% of the isolates, respectively. Interestingly, few isolates carried dual resistance genes (ESBLs and AmpCs). IncH12 and IncI1 plasmid was identified in isolates carrying ESBL and AmpC genes, respectively. Conclusion: This study shows that ceftriaxone resistance is mainly mediated by β-lactamases such as ESBL and AmpC. As the incidence of ceftriaxone resistance is rising gradually over the years, it is imperative to monitor the AMR in this species.

Background India is among the nations reporting substantial healthcare burden linked to pneumococcal infections. Nafithromycin is a novel lactone ketolide antibiotic, which recently entered Phase 3 development in India for the indication of community-acquired bacterial pneumonia (CABP). Objectives To assess the in vitro activity of nafithromycin against serotyped invasive and non-invasive Streptococcus pneumoniae isolates, collected from nine medical centres across India. Methods A total of 534 isolates of S. pneumoniae were collected during 2015–20 and serotyped as per CDC protocol. A subset of erythromycin-non-susceptible S. pneumoniae (n = 200) was screened for the presence of erm(B) and mef(A/E) genes. A subset of MDR isolates (n = 54) were also subjected to MLST. The MICs of antibiotics were determined by the reference agar-dilution method (CLSI). Susceptibilities of the comparators were interpreted as per CLSI criteria. Results Fifty-nine distinct serotypes were identified among the 534 isolates. Among erythromycin-non-susceptible isolates, erm(B) and mef(A/E) genes were found in 49% and 59% strains respectively, while MLST showed clonal diversity. Azithromycin (67.6% non-susceptible) and clindamycin (31.8% non-susceptible) showed limited activity. Penicillin (for non-meningitis) or quinolone non-susceptibility was low ( Conclusions Indian pneumococcal isolates show poor susceptibilities to macrolides, in concordance with the global trend. Nafithromycin overcomes erm as well as mef-mediated macrolide resistance mechanisms expressed individually or concurrently in S. pneumoniae. This study supports continued clinical development of nafithromycin for pneumococcal infections including CABP.

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.

Non-typhoidal Salmonella (NTS) are foodborne pathogens that are responsible for self-limiting gastroenteritis in humans. The present study aims at the molecular characterisation and comparative genomics of Salmonella enterica serovar Senftenberg strain P5558 isolated from the pus samples of a patient suffering from stump infection. The isolate was subjected to serotyping and antimicrobial susceptibility test to understand the phenotypical characteristics. Whole genome sequencing (WGS) was carried out and comparative genomics using computational tools showed the antimicrobial resistance and virulence gene profile of the isolates from the genome sequence data. Typing experiments confirmed that the isolate belong to S. Senftenberg with sequence type ST14. Resistance against β-lactams is associated with the presence of blaTEM-1, blaOXA-9, blaCMY-2 and blaNDM-1 genes. Similarly resistance to aminoglycoside was associated with five aminoglycoside modifying enzymes aac(6')-Ia, aac(6')-Ib, aph(3')-Ib, aph(6')-Ib and ant(3'')-Ia, sulfonamide with sul-1 and sul-2 and chloramphenicol with florR gene. Substitutions in gyrA (S83Y, D87G) and parC (S80I) genes found to be the reason for fluoroquinolone resistance. The plasmid profiling showed the isolate has four resistance plasmids in which plasmid p5558-NDM (IncA/C) harbours major resistance genes including blaNDM-1 and blaCMY-2. Determination of virulence gene profile revealed that the genome carries all major Salmonella pathogenicity islands and virulence factors. From our findings it is clear that the isolate possess characteristic pathogenicity islands (SPI 1-6, 13, 14), major virulence factors and acquired resistance genes. Comparative analysis suggests the evolution and distribution of the MDR gene encoding plasmids in NTS.

Background\ud Multidrug-resistant (MDR) E. coli with extended-spectrum β-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.\ud \ud Methods\ud 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.\ud \ud Results\ud 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).\ud \ud Conclusions\ud 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.

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 β-lactamases with specific Inc groups in this study.

Purpose: Pneumococcal meningitis is a life-threatening infection, requiring prompt diagnosis and effective treatment. Penicillin resistance in pneumococcal infections is a concern. Here, we present the antibiotic susceptibility profile of pneumococcal meningeal isolates from January 2008 to August 2016 to elucidate treatment guidelines for pneumococcal meningitis. Materials and Methods: Invasive pneumococcal isolates from all age groups, were included in this study. Minimum inhibitory concentrations for the isolates were identified by agar dilution technique and VITEK System 2. Serotyping of isolates was done by co-agglutination technique. Results: Out of 830 invasive pneumococcal isolates, 167 (20.1%) isolates were from meningeal infections. Cumulative penicillin resistance in pneumococcal meningitis was 43.7% and cefotaxime non-susceptibility was 14.9%. Penicillin resistance amongst meningeal isolates in those younger than 5 years, 5–16 years of age and those aged 16 years and older was 59.7%, 50% and 27.3%, respectively, with non-susceptibility to cefotaxime in the same age groups being 18%, 22.2% and 10.4%. Penicillin resistance amongst pneumococcal meningeal isolates increased from 9.5% in 2008 to 42.8% in 2016, whereas cefotaxime non-susceptibility increased from 4.7% in 2008 to 28.5% in 2016. Serotypes 14, 19F, 6B, 6A, 23F, 9V and 5 were the most common serotypes causing meningitis, with the first five accounting for over 75% of resistant isolates. Conclusions: The present study reports increasing penicillin resistance and cefotaxime non-susceptibility to pneumococcal meningitis in our setting. This highlights the need for empiric therapy with third-generation cephalosporins and vancomycin for all patients with meningitis while awaiting results of culture and susceptibility testing.

OBJECTIVE To report the percentage of non-vaccine pneumococcal serotypes and their antibiotic susceptibility pattern in children with invasive peumococcal disease. METHODS Invasive pneumococcal isolates of children

For accurate and earlier detection of invasive pneumococcal serogroup/serotypes from India, we have rearranged the African sequence of multiplex PCR provided by the Centers for Disease Control and Prevention, USA. This modified approach can successfully be adapted for earlier serotype detection of 95% of the pneumococcal strains prevalent in India.

Purpose: To investigate the epidemiology of invasive pneumococcal disease (IPD), prevalent serotypes, and pattern of antimicrobial resistance (AMR) in Indian adults. Methods: Prospective laboratory based surveillance of IPD was carried out in >18 years age group between January 2007 and July 2017, from a tertiary care hospital in South India. All Streptococcus pneumoniae culture positives from blood, CSF and sterile body fluids were characterized to identify the serotypes and AMR. Results: A total of 408 IPD cases were characterized in this study. The overall case fatality rate in this study was 17.8% (95% confidence interval (CI): 14.1, 22.4). Pneumonia (39%), meningitis (24.3%), and septicaemia (18.4%) were the most common clinical conditions associated with IPD. Serotypes 1, 3, 5, 19F, 8, 14, 23F, 4, 19A and 6B were the predominant serotypes in this study. Penicillin non-susceptibility was low with 6.4% Conclusion: Serotype data from this study helped in accurate estimation of pneumococcal conjugate vaccine-13 and pneumococcal polysaccharide vaccine-23 protective coverage against serotypes causing IPD in India as 58.7% (95% CI: 53.8, 63.4) and 67.4% (95% CI: 62.7, 71.8) respectively. Penicillin non-susceptibility in meningeal IPD cases is 27.4%. Empirical therapy for meningeal IPD must be cephalosporin in combination with vancomycin since cefotaxime non-susceptibility in meningeal IPD is 9.9% Keywords: Invasive pneumococcal disease, Adults, India, Vaccine coverage, Penicillin resistance

Objectives Neisseria meningitidis is an important causative agent of meningitis and/or sepsis with high morbidity and mortality. Baseline genome data on N. meningitidis, especially from developing countries such as India, are lacking. This study aimed to investigate the whole genome sequences of N. meningitidis isolates from a tertiary care centre in India. Methods Whole-genome sequencing was performed using an Ion Torrent™ Personal Genome Machine™ (PGM) with 400-bp chemistry. Data were assembled de novo using SPAdes Genome Assembler v.5.0.0.0. Sequence annotation was performed through PATRIC, RAST and the NCBI PGAAP server. Downstream analysis of the isolates was performed using the Center for Genomic Epidemiology databases for antimicrobial resistance genes and sequence types. Virulence factors and CRISPR were analysed using the PubMLST database and CRISPRFinder, respectively. Results This study reports the whole genome shotgun sequences of eight N. meningitidis isolates from bloodstream infections. The genome data revealed two novel sequence types (ST12777 and ST12778), along with ST11, ST437 and ST6928. The virulence profile of the isolates matched their sequence types. All isolates were negative for plasmid-mediated resistance genes. Conclusions To the best of our knowledge, this is the first report of ST11 and ST437 N. meningitidis isolates in India along with two novel sequence types (ST12777 and ST12778). These results indicate that the sequence types circulating in India are diverse and require continuous monitoring. Further studies strengthening the genome data on N. meningitidis are required to understand the prevalence, spread, exact resistance and virulence mechanisms along with serotypes.