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Invasive non-typhoidal Salmonella (iNTS) disease is an important cause of morbidity and mortality in African countries. However, the incidence in Indian subcontinent remains poorly documented. This study has assessed the incidence of iNTS in India with a perspective on its AMR profiles and serovar distribution for a period of 21 years from 2000 to 2020 from a tertiary care centre in South India.A total of 461 iNTS isolates were subjected to serotyping and antimicrobial susceptibility testing (AST). A subset of isolates was genotyped by multi locus sequence typing (MLST) and results were compared to serotyping to predict the accuracy.Overall, 461 iNTS isolates were characterised mostly comprising of S. Typhimurium (49.2%) and S. Enteritidis (28.8%). Proportion of isolates resistant to first line antibiotics such as ampicillin, chloramphenicol and trimethoprim/sulphamethoxazole were 6.7%, 1.7% and 3.6% respectively. Isolates resistant to third generation cephalosporin are at a gradual rise while decreased susceptibility to quinolones was most common. The incidence of iNTS infection was maximum in the age group ofgt;15 years. MLST analysis showed discrepancies in assigning the serovars by serotyping as three S. Saintpaul were identified as S. Typhimurium.The clinical epidemiology, serovar distribution and antimicrobial susceptibility patterns of invasive Salmonella isolates from India suggest that there is only a small burden of iNTS disease. However the gradual emergence of AMR in iNTS isolates indicates serious risk for public health warranting the importance enhanced molecular surveillance.

Background Following a relatively mild first wave of coronavirus disease 2019 (COVID-19) in India, a deadly second wave of the pandemic overwhelmed the healthcare system due to the emergence of fast-transmitting SARS-CoV-2 genetic variants. The emergence and spread of the B.1.617.2/Delta variant considered to be driving the devastating second wave of COVID-19 in India. Currently, the Delta variant has rapidly overtaken the previously circulating variants to become the dominant strain. Critical mutations in the spike/RBD region of these variants have raised serious concerns about the virus's increased transmissibility and decreased vaccine effectiveness. As a result, significant scientific and public concern has been expressed about the impact of virus variants on COVID-19 vaccines. Objectives The purpose of this article is to provide an additional explanation in the context of the evolutionary trajectory of SARS-CoV-2 variants in India, the vaccine-induced immune response to the variants of concern (VOC), and various vaccine deployment strategies to rapidly increase population immunity. Content Phylogenetic analysis of SARS-CoV-2 isolates circulating in India suggests the emergence and spread of B.1.617 variant. The immunogenicity of currently approved vaccines indicates that the majority of vaccines elicit an antibody response and some level of protection. According to current data, vaccines in the pre-fusion configuration (2p substitution) have an advantage in terms of nAb titer, but the duration of vaccine-induced immunity, as well as the role of T cells and memory B cells in protection, remain unknown. Since vaccine efficacy on virus variants is one of the major factors to be considered for achieving herd immunity, existing vaccines need to be improved or effective next-generation vaccines should be developed to cover the new variants of the virus.

Background: Klebsiella pneumoniae (Kp), a common multidrug-resistant pathogen, causes a wide spectrum of nosocomial infections with high rates of morbidity and mortality. The emergence of pan drug-...

Background Recent reports have established the emergence and dissemination of extensively drug resistant (XDR) H58 Salmonella Typhi clone in Pakistan. In India where typhoid fever is endemic, only sporadic cases of ceftriaxone resistant S. Typhi are reported. This study aimed at elucidating the phylogenetic evolutionary framework of ceftriaxone resistant S. Typhi isolates from India to predict their potential dissemination. Methods Five ceftriaxone resistant S. Typhi isolates from three tertiary care hospitals in India were sequenced on an Ion Torrent Personal Genome Machine (PGM). A core genome single-nucleotide-polymorphism (SNP) based phylogeny of the isolates in comparison to the global collection of MDR and XDR S. Typhi isolates was built. Two of five isolates were additionally sequenced using Oxford Nanopore MinION to completely characterize the plasmid and understand its transmission dynamics within Enterobacteriaceae. Results Comparative genomic analysis and detailed plasmid characterization indicate that while in Pakistan (4.3.1 lineage I) the XDR trait is associated with blaCTX-M-15 gene on IncY plasmid, in India (4.3.1 lineage II), the ceftriaxone resistance is due to short term persistence of resistance plasmids such as IncX3 (blaSHV-12) or IncN (blaTEM-1B + blaDHA-1). Conclusion Considering the selection pressure exerted by the extensive use of ceftriaxone in India, there are potential risks for the occurrence of plasmid transmission events in the predominant H58 lineages. Therefore, continuous monitoring of S. Typhi lineages carrying plasmid-mediated cephalosporin resistant genes is vital not just for India but also globally., Highlights • S. Typhi to develop cephalosporin resistance by acquiring diverse plasmids from other Enterobacteriaceae. • Independent acquisition of drug-resistant plasmids such as IncX3 and IncN with genes encoding beta-lactamases in H58 lineage II of S. Typhi. • A short-term persistence of drug-resistant plasmids in H58 lineage II can be the reason for the sporadic cases cephalosporin resistant S. Typhi in India. • Plasmid acquisition and maintenance of cephalosporin resistant S. Typhi appears to be specific to the phylogenetic lineage. • Critical strategies in monitoring and control of cephalosporin resistant S. Typhi is needed to tackle further public health crisis.

Paratyphoid fever caused byS. Paratyphi A is endemic in parts of Asia and Sub-Saharan Africa. The proportion of enteric fever cases caused byS. Paratyphi A has substantially increased, yet only limited data is available on the population structure and genetic diversity of this serovar. We examined the phylogenetic distribution and evolutionary trajectory ofS. Paratyphi A isolates collected as part of the Indian enteric fever surveillance study “Surveillance of Enteric Fever in India (SEFI).” In the study period (2017-2020),S. Paratyphi A comprised 17.6% (441/2503) of total enteric fever cases in India, with the isolates highly susceptible to all the major antibiotics used for treatment except fluoroquinolones. Phylogenetic analysis clustered the globalS. Paratyphi A collection into seven lineages (A-G), and the present study isolates were distributed in lineages A, C and F. Our analysis documented that the genome degradation events and gene acquisitions or losses play a major role in the evolution of newS. Paratyphi A lineages/sub-lineages. A total of 10 pseudogene-forming mutations possibly associated with the emergence of lineages were identified. Pan-genome analysis identified the insertion of P2/PSP3 phage and acquisition of IncX1 plasmid during the selection in 2.3.2/2.3.3 and 1.2.2 genotypes, respectively. We also identified that the six characteristic missense mutations associated with the lipopolysaccharide (LPS) biosynthesis genes ofS. Paratyphi A confer only a low structural impact and would therefore have minimal impact on vaccine effectiveness. SinceS. Paratyphi A is human restricted, high levels of genetic drift are not expected unless these bacteria transmit to naive hosts. However, public-health investigation and intervention by means of genomic surveillance would be continually needed to avoidS. Paratyphi A serovar becoming a public health threat similar to theS. Typhi of today.

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.

BackgroundSystematic studies to estimate the disease burden of typhoid and paratyphoid in India are limited. Therefore, a multicenter study on the Surveillance of Enteric Fever in India was carried out to estimate the incidence, clinical presentation, and antimicrobial resistance (AMR) trend. The data presented here represent the national burden of AMR in Salmonella Typhi and Salmonella Paratyphi A.MethodsAntimicrobial susceptibility testing was performed for S. Typhi and S. Paratyphi A (n = 2373) isolates collected prospectively during a 2-year period from November 2017 to January 2020.ResultsOf 2373 Salmonella isolates, 2032 (85.6%) were identified as S. Typhi and 341 (14.4%) were S. Paratyphi A. Approximately 2% of S. Typhi were multidrug-resistant (MDR), whereas all 341 (100%) of S. Paratyphi A isolates were sensitive to the first-line antimicrobials. Among 98% of ciprofloxacin nonsusceptible isolates, resistance (minimum inhibitory concentration [MIC] >0.5 µg/mL) was higher in S. Typhi (37%) compared with S. Paratyphi A (20%). Azithromycin susceptibility was 99.9% and 100% with a mean MIC of 4.98 μg/mL for S. Typhi and 7.39 μg/mL for S. Paratyphi A respectively. Ceftriaxone was the only agent that retained 100% susceptibility. Moreover, beta-lactam/beta-lactamase inhibitors showed potent in vitro activity against the study isolates.ConclusionsData obtained from this systematic surveillance study confirms the declining trend of MDR Salmonella isolates from India. The higher prevalence of ciprofloxacin nonsusceptibility enforces to limit its use and adhere to the judicious usage of azithromycin and ceftriaxone for enteric fever management.

Background The steady increase in the proportion of Non-typhoidal Salmonella (NTS) infections in humans represents a major health problem worldwide. The current study investigated the serovar distribution and antimicrobial susceptibility trends of NTS isolated from faecal samples during the period 2000–2018. Methods Faecal specimens of patients were cultured according to standard lab protocol. The isolates were serotyped and antimicrobial susceptibility testing (AST) were performed according to CLSI guidelines. Results A total of 1436 NTS isolates were obtained from faeces samples mostly comprising of S. Typhimurium (27.3%), S. Weltevreden (13%), S. Bareilly (11%), S. Newport (4.2%), S. Cholerasuis (4%), S. Infantis (3.4%), and S. Enteritidis (2.4%). Resistance to nalidixic acid (26%) was most common among the tested NTS, followed by ampicillin (18.5%), cotrimoxazole (13.5%), ciprofloxacin (12%), ceftriaxone (6.3%) and chloramphenicol (3.6%). Multidrug resistance was observed in 5% of NTS isolates with the highest rate (10.52%) in 2014. The incidence of NTS infection was maximum in children Conclusions Based on this study, the faecal NTS isolates have high resistance rates against first line antimicrobial agents except chloramphenicol. The gradual but consistent increase in resistance to fluoroquinolones, third generation cephalosporins and macrolide may restrict future treatment options. Hence periodic monitoring of NTS infections, serotype distribution and antimicrobial resistance trend is recommended.

We report here the draft genome sequence of two rare

Objectives Elizabethkingia spp. are Gram-negative, glucose-non-fermenting bacilli that are ubiquitous in natural environments such as soil, plant and water sources. Besides environmental sources, the bacterium can be found in hospital environments, particularly medical equipment and reagents. Here we report the draft genome sequence of an Elizabethkingia anophelis isolate from a blood culture. Methods Genomic DNA of E. anophelis strain BP8467 was sequenced on an Ion Torrent PGM platform and the reads were assembled de novo using SPAdes v.5.0.0. The draft genome was annotated using the Prokaryotic Genome Annotation Pipeline (PGAP) v.4.9. Genetic determinants of antimicrobial resistance as well as virulence factors were identified using computational tools. Results The assembled draft genome is 3 859 105 bp in length with a G + C content of 35.62% distributed in 30 contigs. Presence of the blaBlaB and blaGOB-4 genes associated with resistance to carbapenems was identified. In addition, genes conferring resistance to other β-lactams (blaCME-1), aminoglycosides [ant(6)-I] and chloramphenicol (catB) were also detected. Antimicrobial susceptibility testing showed that the isolate was susceptible to levofloxacin, trimethoprim/sulfamethoxazole, tetracycline and rifampicin. Conclusion The presence of a multidrug-resistant isolate harbouring diverse antimicrobial resistance genes along with numerous virulence factors suggests the risk associated with Elizabethkingia spp. infections. This genome analysis provides insights into the antimicrobial resistance and pathogenicity mechanisms of multidrug-resistant E. anophelis that can help in the management of Elizabethkingia spp. infections in the future.

Recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and subsequent containment procedures have impacted the world as never seen before. Therefore, there is considerable curiosity about the genome evolution related to the origin, transmission and vaccine impact of this virus. We have analysed genome sequences of SARS-CoV-2 isolated from Indian patients to gain an in-depth understanding of genomic evolution and transmission in India. Phylogenetic analysis and mutation profiling revealed major lineages being evolved by characteristic mutations. As the mutation frequency in spike protein is comparatively lesser, the candidate vaccines expected to have wide coverage worldwide including 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.

The prime goal of molecular epidemiology is to identify the origin and evolution of pathogens, which can potentially influence the public health worldwide. Traditional methods provide limited information which is not sufficient for outbreak investigation and studying transmission dynamics. The recent advancement of next-generation sequencing had a major impact on molecular epidemiological studies. Currently, whole-genome sequencing (WGS) has become the gold standard typing method, especially for clinically significant pathogens. Here, we aimed to describe the application of appropriate molecular typing methods for global antimicrobial resistance surveillance system pathogens based on the level of discrimination and epidemiological settings. This shows that sequence-based methods such as multi-locus sequence typing (MLST) are widely used due to cost-effectiveness and database accessibility. However, WGS is the only method of choice for studying Escherichia coli and Shigella spp. WGS is shown to have higher discrimination than other methods in typing Klebsiella pneumoniae, Acinetobacter baumannii and Salmonella spp. due to its changing accessory genome content. For Gram positives such as Streptococcus pneumoniae, WGS would be preferable to understand the evolution of the strains. Similarly, for Staphylococcus aureus, combination of MLST, staphylococcal protein A or SCCmec typing along with WGS could be the choice for epidemiological typing of hospital- and community-acquired strains. This review highlights that combinations of different typing methods should be used to get complete information since no one standalone method is sufficient to study the varying genome diversity.

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.

Colistin resistance in Acinetobacter baumannii is mediated by multiple mechanisms. Recently, mutations within pmrAB two component system and overexpression of eptA due to upstream insertion of ISAba1 play a major role.To characterize colistin resistance mechanisms among the clinical isolates of A. baumannii in India. A total of 224 clinical isolates of A. baumannii collected from 2016 to 2019 were included in this study. Mutations within lipid A biosynthesis and pmrAB genes were characterized by Whole Genome Shotgun sequencing. Twenty eight complete genomes were further characterized for insertional inactivation of lpx genes and the association of ISAba1-eptA using hybrid assembly approach. Non-sysnonymous mutations like M12I in pmrA, A138T and A444V in pmrB and E117K in lpxD were identified. Four of the five colistin resistant A.baumannii isolates had insertion of ISAba1 upstream eptA. No mcr genes were identified.Overall, the present study highlights the diversity of colistin resistance mechanisms in A. baumannii. ISAba1-driven eptA overexpression could be responsible for colistin resistance among Indian isolates of colistin resistant A. baumannii.

The currently ongoing COVID-19 pandemic caused by SARS-CoV-2 has accounted for millions of infections and deaths across the globe. Genome sequences of SARS-CoV-2 are being published daily in public databases and the availability of this genome datasets has allowed unprecedented access into the mutational patterns of SARS-CoV-2 evolution. We made use of the same genomic information for conducting phylogenetic analysis and identifying lineage-specific mutations. The catalogued lineage defining mutations were analysed for their stabilizing or destabilizing impact on viral proteins. We recorded persistence of D614G, S477N, A222V V1176F variants and a global expansion of the PANGOLIN variant B.1. In addition, a retention of Q57H (B.1.X), R203K/G204R (B.1.1.X), T85I (B.1.2-B.1.3), G15S+T428I (C.X) and I120F (D.X) variations was observed. Overall, we recorded a striking balance between stabilizing and destabilizing mutations, therefore well-maintained protein structures. With selection pressures in the form of newly developed vaccines and therapeutics to mount soon in coming months, the task of mapping of viral mutations and recording of their impact on key viral proteins would be crucial to pre-emptively catch any escape mechanism that SARS-CoV-2 may evolve for.STUDY IMPORTANCEAs large numbers of the SARS CoV-2 genome sequences are shared in publicly accessible repositories, it enables scientists a detailed evolutionary analysis since its initial isolation in Wuhan, China. We investigated the evolutionarily associated mutational diversity overlaid on the major phylogenetic lineages circulating globally, using 513 representative genomes. We detailed phylogenetic persistence of key variants facilitating global expansion of the PANGOLIN variant B.1, including the recent, fast expanding, B.1.1.7 lineage. The stabilizing or destabilizing impact of the catalogued lineage defining mutations on viral proteins indicates their possible involvement in balancing the protein function and structure. A clear understanding of this mutational profile is of high clinical significance to catch any vaccine escape mechanism, as the same proteins make crucial components of vaccines recently approved and in development. In this direction, our study provides an imperative framework and baseline data upon which further analysis could be built as newer variants of SARS-CoV-2 continue to appear.

Hypervirulent K. pneumoniae (HvKp) is typically associated with ST23 clone; however, hvKp is also emerging from clones ST11, ST15 and ST147, which are also multi-drug resistant (MDR). Here, we aimed to characterise nine novel MDR hvKp isolates harbouring mosaic plasmids simultaneously carrying antimicrobial resistance (AMR) and virulence genes. Nine HvKp isolates obtained from hospitalised patients in southern India were characterized for antimicrobial susceptibility and hypervirulence phenotypes. All nine hvKp isolates were subjected to whole genome sequencing (WGS) using Ilumina HiSeq2500 and a subset of four were sequenced using Oxford Nanopore MinION. 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 virulence determinants were encoded in a mosaic plasmid (∼320 Kbp) that formed as a result of its insertion in a IncFIB-IncHI1B plasmid co-integrate. The mosaic plasmid carried AMR genes (aadA2, armA, blaOXA-1, msrE, mphE, sul1 and dfrA14) in addition to rmpA2, iutA and iucABCD virulence genes. Interestingly the mosaic plasmid carried its own type IV-A3 CRISPR-cas system that is likely able to target the acquisition of IncF plasmid with the help of a traL spacer. The convergence of virulence and AMR is the biggest threat among invasive K. pneumoniae infections. However, increasing reports of the presence of mosaic plasmid carrying both AMR and virulence genes suggests MDR-hvKp isolates are no longer confined to selected clones and the containment of such isolates is very challenging.IMPORTANCEKlebsiella pneumoniae is an opportunistic pathogen that commonly associated with hospital-acquired infections in the urinary tract, respiratory tract, lung, wound sites. The organism has gained notoriety by acquiring additional genetic traits to become either hypervirulent (HV) phenotype or multidrug resistant (MDR) phenotype. Though the infections by both these phenotypes were very challenging to treat, the MDR K. pneumonia (MDR-Kp) were remained in the hospital settings while HV K. pneumonia (hvKp) strains were mostly originated from the community settings. In a recent turn of events, the evolution of MDR-Kp and hvKp has converged as both clones found to carry both MDR plasmids and virulence plasmid. These convergent strains are challenging to treat and is associated with higher mortality rate. As the recent hvKp isolates harbour mosaic plasmid encoding both AMR and virulence determinants there is a need to investigate the evolution of these pathogens. The significance of our research is in characterising the novel mosaic plasmid identified in MDR-hvKp isolates that belong sequence type (ST) 2096. Tracking the possible evolution pathway of MDR-hvKPs would greatly help in the proper surveillance and management of this superbugs.RepositoriesThe whole genome sequences of the present study isolates have been deposited in GenBank, NCBI, with accession numbers CP053765 - CP053770, CP053771 – CP053780, CP058798-CP058806, JAARNO010000001.1 - JAARNO010000005.1, JAAQSG000000000, JAARNJ000000000, JAARMH000000000 and JAAQTC000000000

Azithromycin is increasingly being used for the treatment of shigellosis despite a lack of interpretative guidelines and with limited clinical evidence. The present study determined azithromycin susceptibility and correlated this with macrolide-resistance genes in Shigella spp. isolated from stool specimens in Vellore, India. The susceptibility of 332 Shigella isolates to azithromycin was determined using the disc diffusion method. Of these, 31 isolates were found to be azithromycin resistant. The azithromycin minimum inhibitory concentration (MIC) was determined using the broth microdilution method. In addition, isolates were screened for mphA and ermB genes using conventional PCR. Furthermore, an isolate that was positive for resistance genes was subjected to complete genome analysis, and was analysed for mobile genetic elements. The azithromycin MIC for the 31 resistant Shigella isolates ranged between 2 and 16 mg l−1. PCR results showed that a single isolate of Shigella sonnei carried a mphA gene. Complete genome analysis revealed integration of an IncFII plasmid into the chromosome of S. sonnei , which was also found to carry the following resistance genes: sul1, bla DHA1, qnrB4, mphA, tetR. Mutations in the quinolone-resistance-determining region (QRDR) were also observed. Additionally, prophages, insertion sequences and integrons were identified. The novel finding of IncFII plasmid integration into the chromosome of S. sonnei highlights the potential risk of Shigella spp. becoming resistance to azithromycin in the future. These suggests that it is imperative to monitor Shigella susceptibility and to study the resistance mechanism of Shigella to azithromycin considering the limited treatment choices for shigellosis.

BackgroundIn recent years, the emergence of multidrug resistant hypervirulent K. pneumoniae (MDR hvKp) isolates poses severe therapeutic challenge to global public health. The present study used the complete genome sequence of two MDR hvKp isolates belonging to ST23 to characterize the phylogenetic background and plasmid diversity.MethodsTwo hvKp isolates from patients with bacteremia were sequenced using Ion Torrent PGM and Oxford Nanopore MinION platforms and assembled by hybrid genome assembly approach. Comparative genomics approaches were used to investigate the population structure, evolution, virulence, and antimicrobial resistance of MDR hvKp strains.ResultsThe study isolates exhibited typical features of hvKp phenotypes associated with ST23. The convergence of multidrug resistance and hypervirulence were attributed by the presence of multiple plasmids including a 216 kb virulence plasmid and MDR plasmids belonging to IncA/C2, IncFIB, IncX3, and ColKP3 groups. The insertion of catA1 gene into virulence plasmid was observed along with genetic factors such as aerobactin, salmochelin, and rmpA2 that confer hvKp’s hypervirulent phenotype. The core genome single nucleotide polymorphism (SNP) phylogenetic analyses of the isolates showed the evolution of ST23 hvKp was predominantly driven by ICEKp acquisitions.ConclusionTo the best of our knowledge, this is the first report of MDR hvKp isolates of ST23 with insertion of catA1 gene into the virulence plasmid which presents the possibility of hotspot integration sites on the plasmids to aid acquisition of AMR genes. ST23 is no longer confined to susceptible strains of hvKp. Our findings emphasize the need for more studies on recombinant events, plasmid transmission dynamics and evolutionary process involving hvKp.

BackgroundRecent reports have established the emergence and dissemination of extensively drug resistant (XDR) H58 Salmonella Typhi clone in Pakistan. In India where typhoid fever is endemic, only sporadic cases of ceftriaxone resistant S. Typhi are reported. This study aimed at elucidating the phylogenetic evolutionary framework of ceftriaxone resistant S. Typhi isolates from India to predict their potential dissemination in endemic regions.MethodsFive ceftriaxone resistant S. Typhi isolates from three tertiary care hospitals in India were sequenced on an Ion Torrent Personal Genome Machine (PGM). A core genome single-nucleotide-polymorphism (SNP) based phylogeny of the isolates in comparison to the global collection of MDR and XDR S. Typhi isolates was built. Two of five isolates were additionally sequenced using Oxford Nanopore MinION to completely characterize the plasmid and understand its transmission dynamics within Enterobacteriaceae.ResultsComparative genomic analysis and detailed plasmid characterization indicate that while in Pakistan (4.3.1 lineage I) the XDR trait is associated with blaCTX-M-15 gene on IncY plasmid, in India (4.3.1 lineage II), the ceftriaxone resistance is due to short term adaptation of resistance plasmids such as IncX3 or IncN.ConclusionSince the bacterial acquisition of smaller resistance plasmids such as IncX3 or IncN from other Enterobacteriaceae can be much faster than the larger IncY plasmids, the rapid expansion of these genotypically novel XDR S. Typhi could potentially cause large outbreaks. Therefore, continuous monitoring of S. Typhi lineages carrying cephalosporin resistance on IncX3 or IncN plasmids is vital not just for India but globally.ImportanceGenomic analysis of cephalosporin resistant S. Typhi isolated from India indicates the potential of S. Typhi to develop cephalosporin resistance by acquiring diverse plasmids from other Enterobacteriaceae. We identified the occurrence of independent acquisition of drug-resistant plasmids such as IncX3 and IncN with genes encoding beta-lactamases in H58/4.3.1.2 lineage. A short term adaptation of drug-resistant plasmids in H58/4.3.1.2 lineage can be the reason for the sporadic cases cephalosporin resistant S. Typhi in India. However, the IncY plasmid acquired by isolates that belong to H58/4.3.1.1 lineage appeared to be well adapted as observed in XDR S. Typhi outbreak in Pakistan. Plasmid acquisition and maintenance of cephalosporin resistant S. Typhi appears to be specific to the phylogenetic lineage as lineages differ in compensating the initial cost imposed by the plasmid. The stable maintenance of these resistance plasmids without a fitness cost, are determinant in understanding the future spread of cephalosporin resistance in S. Typhi. Therefore, critical strategies in monitoring and control of cephalosporin resistant S. Typhi is needed to tackle further public health crisis.

Traditional serotyping based on the phenotypic variation of O- and H-antigen remains as the gold-standard for the identification and classification of Salmonella isolates for last 70 years. Although this classification is a globally recognized nomenclature, huge diversity of Salmonella serotypes have made the serovar identification to be very complex. Seven gene multilocus sequence typing (MLST) on the other hand can provide serovar prediction as well as the evolutionary origin between the serovars. In this study non typhoidal Salmonella (NTS) strains (n = 45) isolated from clinical samples (blood, faeces and pus) were identified by traditional phenotypic serotyping and biochemical testing. All the tested Salmonella isolates were designated as serovar Typhimurium based on phenotyping. However, by MLST 60% (27/45) of the isolates were S. Typhimurium, 35.5% (16/45) were S. Agona (ST13), 2.2% (1/45) were S. Kentucky (ST198) and 2.2% (1/45) were S. Saintpaul (ST27). MLST analysis assigned S. Typhimurium isolates as ST36 (18/127), ST19 (7/27) and ST313 (2/27). Mismatches in serovar designation between MLST database and phenotypic serotyping can be due to the misinterpretation of phenotypic serotyping as the antigenic structures of S. Typhimurium, S. Agona differs by a surface antigen. MLST based phylogeny of study isolates showed clustering according to sequence types. Concordance between MLST based sequence type and phenotypic serotype is important to provide insights into genetic population structure of Salmonella.

In the present study, bacterial magnetosomes were used as a carrier molecule to couple with lemon grass extract (LGE) for the prevention of microbial biofilm in wound dressing material. Magnetosomes were extracted from Magnetospirillum sp. VITRJS-1 and characterised by microscopic and X-ray diffraction analysis. The phytochemical analyses of the extract showed the presence of bioactive compounds that are reported for antimicrobial and anti-inflammatory activity. Gas chromatography–mass spectrometry analysis revealed the presence of antimicrobial citral and fernesal compounds. The extract was conjugated onto the magnetosomes and confirmed by Fourier transform infrared spectroscopy analysis. The prepared magnetosome–lemon grass extract (MLGE) was evaluated for its antibiofilm property against the biofilm-forming pathogens Bacillus subtilis, Pseudomonas, Escherichia coli, Klebsiella and Staphylococcus aureus on wound dressing material by plate assay. The results indicated that the number of colonies formed was considerably reduced in MLGE coated wound dressing compared with that of LGE and control. Furthermore, SEM analysis displayed that the MLGE drastically reduced the spread of biofilm formation. Thus, MLGE coated wound dressings are effective in preventing the microbial biofilm formation, and further investigation on animal models will enable its use commercially.

Highlights • Lipase was immobilized on magnetosome via glutaraldehyde, confirmed in FTIR and SEM. • Release of p-nitro phenol confirmed the enzyme activity of immobilize lipase (88 %). • The optimal pH (7) and temperature (40 °C) was standardised for activity. • The immobilized lipase stored at 4 °C retained higher activity (87 %) than free lipase after 30 days. • Immobilized lipase removed vegetable oil stain, better cleaning efficiency than free lipase., Lipase was immobilized onto bacterial magnetosomes using glutaraldehyde cross-linking and confirmed by Fourier transform infrared spectrometry (FT-IR) and Scanning electron microscopy (SEM). Enzyme activity of immobilised lipase as well as free lipase was estimated by the release of p-nitro phenol due to the hydrolysis of p-nitro phenyl acetate (pNPA). The immobilisation yield of lipase onto magnetosome was found to be 88 %. The optimal pH (7) and temperature (40 °C) for activity was standardised and found to be similar to free lipase. The stored immobilized lipase maintained higher activity even after 30 days at a temperature of 4 °C whereas compared to free lipase. Immobilized lipase found to have removed vegetable oil stain and showed higher cleaning efficiency when compared to free lipase. The results suggest that bacterial magnetosome displays great potential as a support material for the immobilization of industrial enzymes such as lipase.

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 bla(TEM-1), bla(OXA-9), bla(CMY-2) and bla(NDM-1) genes. Similarly resistance to aminoglycoside was associated with five aminoglycoside modifying enzymes aac(6ʹ)-Ia, aac(6ʹ)-Ib, aph(3ʹ)-Ib, aph(6ʹ)-Ib and ant(3ʹʹ)-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 bla(NDM-1) and bla(CMY-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.

Oxford Nanopore MinION sequencing technology has been gaining immense importance in identification of pathogen and antimicrobial resistance, though with 10-15% error rate. Short read technologies generates high accurate genome but with multiple fragments of genome. This study proposes a novel workflow to reduce the indels resulted from MinION long read sequencing by overlaying short read sequences from IonTorrent in the clinical isolates. Best of both techniques were employed which generated highly accurate-single chromosomal microbial genomes with increase in completeness of genomes from 44.5%, 30% and 43% to 98.6%, 98.6% and 96.6% for P. aeruginosa, A. veronii and B. pertussis respectively. To the best of our knowledge, this is the first study to generate a hybrid of IonTorrent and MinION reads to obtain single chromosomal genomes. This would enable to precisely infer both structural arrangement of genes and SNP based analysis for phylogenetic information.

Heavy metal contamination of surface water bodies and ground water has been a major problem around the world. Calcium-based adsorbents are effective but cannot be separated easily after the treatment. Magnetosomes are biogenic magnetite synthesised as highly ordered chain-like structures by magnetotactic bacteria. In this study, we have prepared magnetically controlled calcite microcrystals using magnetosomes for the adsorption experiment. The ability of magnetic calcite as adsorbent was investigated for the removal of Cr (III) and Ni (II) ions from synthetic solution. Critical parameters, such as the effect of pH, temperature, contact time, initial ion concentration, and adsorbent dose, were optimised in comparison with calcite, magnetosomes, and activated carbon for maximum metal ion removal. The study showed that equilibrium was established in 1 h for both Cr (III) and Ni (II) at a pH of 6.0 and 8.0, respectively. The adsorption process follows pseudo-second-order reaction kinetics, along with Langmuir and Freundlich adsorption isotherms. The thermodynamics of adsorption of both metal ions on magnetic calcite showed that the adsorption was spontaneous and endothermic in nature. Magnetically controlled calcite crystals successfully removed Cr (III) and Ni (II) from collected tannery effluent and separated from the solution by applying magnetic field. Maximum removal of chromium and nickel (94 and 84%) by magnetic calcite is similar to calcite crystals but higher than magnetosomes and activated carbon. The results indicated that magnetic calcite could be used as an alternative adsorbent for removing heavy metals from tannery effluent. Bacterial magnetosomes coated with calcite can adsorb heavy metals from industrial waste while being easily recoverable with a magnet. The tanning industry produces large volumes of wastewater contaminated with heavy metals, and these toxins require removal prior to waste discharge into the environment. Calcium carbonate adsorbs heavy metals with high efficiency, but is difficult to recover from treated water, lowering water quality. A team led by Krish Suthindhiran at VIT University in India combine environmentally-innocent magnetosomes—magnetic iron-based minerals enveloped by lipid membranes and produced by magnetotactic bacteria—with a calcium carbonate coating to achieve materials that not only remove Cr(III) and Ni(II) from tannery effluents with efficiencies of up to 94% and 84%, but are easily separated post-treatment by application of a magnetic field.

Perchlorate reducing bacteria (PRB) reduces perchlorate to chlorate (ClO3-), which in turn reduced to chlorite (ClO2-) and ultimately to chloride (Cl−). Magnetospirillum strains are reported to use chlorate/perchlorate as electron acceptors. This study describes the perchlorate reducing property of Magnetospirillum sp. VITRJS5 isolated from freshwater sediment collected at Chelur freshwater lake, Kerala, India. The strain was microaerophile and phylogenetically related to a Magnetospirillum sp., a member of the α-subclass of the class Proteobacteria. The placement of the isolate in genus Magnetospirillum has further confirmed by the presence of four key magnetosome membrane genes. PCR amplification and phylogenetic analysis of central metabolic genes such as nifH (nitrogenase) and cbbM (type II RubisCo) displayed the highest similarity (97% and 81% respectively) with Magnetospirillum sp. BB-1 The growth kinetic parameters of the isolate were studied with acetate as electron donor in batch experiments. Monods substrate utilisation model has been established with oxygen, nitrate and perchlorate as electron acceptors separately. The maximum specific growth rate (µmax) and half saturation constant (ksconc) for the bacterium varied while utilizing different electron acceptors. The maximum specific growth rate was 0.226, 0.190 and 0.096 per hr and half velocity constant Ks was 25.09, 33.36 and 65.37 mg acetate/L for oxygen, nitrate and perchlorate respectively. The reduction of perchlorate has been analysed using kinetic studies of the substrate uptake by the bacteria and half velocity constant Ks was found to be 52.8 mg/L. The results indicate that the strain VITRJS5 effectively reduces perchlorate by using it as an electron acceptor.

Geomagnetism aided navigation has been demonstrated by certain organisms which allows them to identify a particular location using magnetic field. This attractive technique to recognize the course was earlier exhibited in numerous animals, for example, birds, insects, reptiles, fishes and mammals. Magnetotactic bacteria (MTB) are one of the best examples for magnetoreception among microorganisms as the magnetic mineral functions as an internal magnet and aid the microbe to move towards the water columns in an oxic-anoxic interface (OAI). The ability of MTB to biomineralize the magnetic particles (magnetosomes) into uniform nano-sized, highly crystalline structure with uniform magnetic properties has made the bacteria an important topic of research. The superior properties of magnetosomes over chemically synthesized magnetic nanoparticles made it an attractive candidate for potential applications in microbiology, biophysics, biochemistry, nanotechnology and biomedicine. In this review article, the scope of MTB, magnetosomes and its challenges in research and industrial application have been discussed in brief. This article mainly focuses on the application based on the magnetotactic behaviour of MTB and magnetosomes in different areas of modern science.