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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.

We report an outbreak of typhoid fever between April and June 2019 in the Surveillance for Enteric Fever in India cohort, a pediatric cohort from four contiguous semi-urban settlements of Vellore in South India. This cohort of children 6 months to 15 years of age was under surveillance from October 2017 to December 2019. A clustering of typhoid cases in the cohort was noted with reference to time, place, and person. The overall typhoid attack rate in the cohort was 0.9%, with the highest attack rate of 1.7% being documented in one of the four areas. The rate of hospitalization and complications in children who were typhoid positive during the outbreak was 28% and 2%, respectively. Given the background of suboptimal water, sanitation, and hygiene, and the risk of typhoid fever outbreaks in these settings, it is imperative that a typhoid vaccine be considered for introduction as a pragmatic preventive approach.

Paratyphoid fever caused by S. Paratyphi A is endemic in parts of South Asia and Southeast Asia. The proportion of enteric fever cases caused by S. 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 of S. 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 global S. Paratyphi A collection into seven lineages (A-G), and the present study isolates were distributed in lineages A, C and F. Our analysis highlights that the genome degradation events and gene acquisitions or losses are key molecular events in the evolution of new S. Paratyphi A lineages/sub-lineages. A total of 10 hypothetically disrupted coding sequences (HDCS) or pseudogenes-forming mutations possibly associated with the emergence of lineages were identified. The 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 have identified six characteristic missense mutations associated with lipopolysaccharide (LPS) biosynthesis genes of S. Paratyphi A, however, these mutations confer only a low structural impact and possibly have minimal impact on vaccine effectiveness. Since S. 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 monitoring by means of genomic surveillance would be constantly needed to avoid S. Paratyphi A serovar becoming a public health threat similar to the S. Typhi of today.

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.

Background: \ud The emergence of increasingly antimicrobial-resistant Salmonella enterica serovar Typhi (S Typhi) threatens to undermine effective treatment and control. Understanding where antimicrobial resistance in S Typhi is emerging and spreading is crucial towards formulating effective control strategies.\ud \ud Methods: \ud In this genomic epidemiology study, we sequenced the genomes of 3489 S Typhi strains isolated from prospective enteric fever surveillance studies in Nepal, Bangladesh, Pakistan, and India (between 2014 and 2019), and combined these with a global collection of 4169 S Typhi genome sequences isolated between 1905 and 2018 to investigate the temporal and geographical patterns of emergence and spread of antimicrobial-resistant S Typhi. We performed non-parametric phylodynamic analyses to characterise changes in the effective population size of fluoroquinolone-resistant, extensively drug-resistant (XDR), and azithromycin-resistant S Typhi over time. We inferred timed phylogenies for the major S Typhi sublineages and used ancestral state reconstruction methods to estimate the frequency and timing of international and intercontinental transfers.\ud \ud Findings: \ud Our analysis revealed a declining trend of multidrug resistant typhoid in south Asia, except for Pakistan, where XDR S Typhi emerged in 2016 and rapidly replaced less-resistant strains. Mutations in the quinolone-resistance determining region (QRDR) of S Typhi have independently arisen and propagated on at least 94 occasions, nearly all occurring in south Asia. Strains with multiple QRDR mutations, including triple mutants with high-level fluoroquinolone resistance, have been increasing in frequency and displacing strains with fewer mutations. Strains containing acrB mutations, conferring azithromycin resistance, emerged in Bangladesh around 2013 and effective population size of these strains has been steadily increasing. We found evidence of frequent international (n=138) and intercontinental transfers (n=59) of antimicrobial-resistant S Typhi, followed by local expansion and replacement of drug-susceptible clades.\ud \ud Interpretation: \ud Independent acquisition of plasmids and homoplastic mutations conferring antimicrobial resistance have occurred repeatedly in multiple lineages of S Typhi, predominantly arising in south Asia before spreading to other regions.\ud \ud Funding: \ud Bill & Melinda Gates Foundation.

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: Aminoglycoside resistance is a growing challenge, and it is commonly mediated by the aminoglycoside-modifying enzymes (AMEs), followed by 16S rRNA methyl transferase. Plazomicin, a novel aminoglycoside agent approved by the Food and Drug Administration for complicated urinary tract infections is proven to overcome resistance mediated by AMEs but not due to 16S rRNA methyl transferase (16SRMTases). We undertook this study to predict the efficacy of plazomicin in India based on the antimicrobial resistance profile derived from whole-genome sequencing (WGS). Methodology: A total of 386 clinical isolates of Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii subjected to WGS were screened for aminoglycoside-resistance mechanisms such as AMEs and 16SRMTases and its association with carbapenemases. Results: AMEs was present in all E. coli, A. baumannii and in 90% of K. pneumoniae. In addition, up to 47% of E. coli and 38% of K. pneumoniae co-carried 16SRMTases with AMEs genes. However, A. baumannii showed 87% of isolates co-harbouring 16SRMTase. bla NDM, bla Oxa-48-like and bla Oxa-23-like were the most predominant carbapenemases in E. coli, K. pneumoniae and A. baumannii, respectively. Notably, 48% of NDM-producing E. coli and 35% of Oxa-48-like producing K. pneumoniae were identified to co-harbour AMEs + RMTAses, where plazomicin may not be useful. Conclusion: Overall, 53%, 62% and 14% of carbapenemase-producing E. coli, K. pneumoniae and A. baumannii harbours only AMEs, indicating the role of plazomicin use. Plazomicin can be used both for ESBLs as “carbapenem-sparing agent” and carbapenemase producers as “colistin-sparing agent.” For optimal use, it is essential to know the molecular epidemiology of resistance in a given geographical region where plazomicin empirical therapy is considered.

Funder: National Institute for Health Research (NIHR), Funder: Bill and Melinda Gates Foundation, Enteric fever infections remain a significant public health issue, with up to 20 million infections per year. Increasing rates of antibiotic resistant strains have rendered many first-line antibiotics potentially ineffective. Genotype 4.3.1 (H58) is the main circulating lineage of S. Typhi in many South Asian countries and is associated with high levels of antibiotic resistance. The emergence and spread of extensively drug resistant (XDR) typhoid strains has increased the need for a rapid molecular test to identify and track these high-risk lineages for surveillance and vaccine prioritisation. Current methods require samples to be cultured for several days, followed by DNA extraction and sequencing to determine the specific lineage. We designed and evaluated the performance of a new multiplex PCR assay, targeting S. Paratyphi A as well as the H58 and XDR lineages of S. Typhi on a collection of bacterial strains. Our assay was 100% specific for the identification of lineage specific S. Typhi and S. Paratyphi A, when tested with a mix of non-Typhi Salmonella and non-Salmonella strains. With additional testing on clinical and environmental samples, this assay will allow rapid lineage level detection of typhoid of clinical significance, at a significantly lower cost to whole-genome sequencing. To our knowledge, this is the first report of a SNP-based multiplex PCR assay for the detection of lineage specific serovars of Salmonella Typhi.

Helicobacter pylori is a ubiquitous bacterium and contributes significantly to the burden of chronic gastritis, peptic ulcers, and gastric cancer across the world. Adaptive phenotypes and virulence factors in H. pylori are heterogeneous and dynamic. However, limited information is available about the molecular nature of antimicrobial resistance phenotypes and virulence factors of H. pylori strains circulating in India. In the present study, we analyzed the whole genome sequences of 143 H. pylori strains, of which 32 are isolated from two different regions (eastern and southern) of India. Genomic repertoires of individual strains show distinct region-specific signatures. We observed lower resistance phenotypes and genotypes in the East Indian (Kolkata) H. pylori isolates against amoxicillin and furazolidone antibiotics, whereas higher resistance phenotypes to metronidazole and clarithromycin. Also, at molecular level, a greater number of AMR genes were observed in the east Indian H. pylori isolates as compared to the southern Indian isolates. From our findings, we suggest that metronidazole and clarithromycin antibiotics should be used judicially in the eastern India. However, no horizontally acquired antimicrobial resistance gene was observed in the current H. pylori strains. The comparative genome analysis shows that the number of genes involved in virulence, disease and resistance of H. pylori isolated from two different regions of India is significantly different. Single-nucleotide polymorphisms (SNPs) based phylogenetic analysis distinguished H. pylori strains into different clades according to their geographical locations. Conditionally beneficial functions including antibiotic resistance phenotypes that are linked with faster evolution rates in the Indian isolates.

We investigated the evolution, phylogeny and antimicrobial resistance of Vibrio cholerae O1 isolates (VCO1) from Ghana. Outbreak and environmental sources of VCO1 were characterized, whole-genome sequenced and compared to globally available seventh pandemic (7P) strains of V. cholerae at SNP resolution. Final analyses included 636 isolates. Novel Ghanaian isolates clustered into three distinct clades (clades 1, 2 and 3) in wave 3 of the 7P lineage. The closest relatives of our novel Ghanaian isolates were from Benin, Cameroon, Togo, Niger and Nigeria. All novel Ghanaian isolates were multi-drug resistant. Environmental isolates clustered into clade 2, despite being isolated years later, showing the possibility of persistence and re-emergence of older clades. A lag phase of several years from estimated introduction to reported cases suggests pathogen persistence in the absence of reported cholera cases. These results highlight the importance of deeper surveillance for understanding transmission routes between bordering countries and planning tailored vaccination campaigns in an effort to eradicate cholera.

Cholera is a life-threatening infectious disease that remains an important public health issue in several low and middle-income countries. In 1992, a newly identified O139 Vibrio cholerae temporarily displaced the O1 serogroup. No study has been able to answer why the potential eighth cholera pandemic (8CP) causing V. cholerae O139 emerged so successfully and then died out. We conducted a genomic study, including 330 O139 isolates, covering emergence of the serogroup in 1992 through to 2015. We noted two key genomic evolutionary changes that may have been responsible for the disappearance of genetically distinct but temporally overlapping waves (A-C) of O139. Firstly, as the waves progressed, a switch from a homogenous toxin genotype in wave-A to heterogeneous genotypes. Secondly, a gradual loss of antimicrobial resistance (AMR) with the progression of waves. We hypothesize that these two changes contributed to the eventual epidemiological decline of O139.

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: 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.

Antimicrobial resistance is on the rise across the globe. Increasing incidence of infections due to carbapenem resistance organisms is becoming difficult to treat, due to the limited availability of therapeutic agents. Very few agents such as colistin, fosfomycin, tigecycline and minocycline are widely used, despite its toxicity. However, with the availability of novel antimicrobials, beta-lactam/beta-lactamase inhibitor-based and non-beta-lactam-based agents could be of great relief. This review covers three important aspects which include (i) current management of carbapenem-resistant infections, (ii) determination of specific types of carbapenemases produced by multidrug-resistant and extensively drug-resistant Gram-negative pathogens and (iii) the currently available novel beta-lactam/beta-lactamase inhibitors and non-beta-lactam-based agents' laboratory findings, clinical outcome and implications.

Tigecycline is a reserve antibiotic increasingly used for the treatment of multidrug-resistant bacteria, especially Klebsiella pneumoniae and Acinetobacter baumannii. At present, there are concerns regarding the testing and interpretation of tigecycline susceptibility to bugs such as K. pneumoniae and A. baumannii, which limit clinicians in appropriate usage. Use of appropriate method for testing such as broth microdilution is essential. In addition, tigecycline susceptibility testing is a challenge due to inconsistent results from various antimicrobial susceptibility testing automated platforms. There is a great need to define a suitable methodology along with interpretive criteria, especially for K. pneumoniae and A. baumannii. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Food and Drug Administration (FDA) breakpoints show wide variation and are defined for different set of organisms. Non-species-related pharmacokinetic/pharmacodynamic (PK/PD) breakpoints defined by the EUCAST can be used for organisms such as K. pneumoniae and A. baumannii.

Diphtheria is a respiratory disease caused by the bacterium Corynebacterium diphtheriae. Although the development of a toxin-based vaccine in the 1930s has allowed a high level of control over the disease, cases have increased in recent years. Here, we describe the genomic variation of 502 C. diphtheriae isolates across 16 countries and territories over 122 years. We generate a core gene phylogeny and determine the presence of antimicrobial resistance genes and variation within the tox gene of 291 tox+ isolates. Numerous, highly diverse clusters of C. diphtheriae are observed across the phylogeny, each containing isolates from multiple countries, regions and time of isolation. The number of antimicrobial resistance genes, as well as the breadth of antibiotic resistance, is substantially greater in the last decade than ever before. We identified and analysed 18 tox gene variants, with mutations estimated to be of medium to high structural impact., Cases of diphtheria have increased in recent years. Here, the authors analyse the genomes of 502 Corynebacterium diphtheriae isolates across 16 countries and territories over 122 years, describing an increase in antimicrobial resistance genes and identifying toxin variants.

Background Typhoid fever caused by Salmonella Typhi is a major public health concern in low-/middle-income countries. A recent study of 1900 global S. Typhi indicated that South Asia might be the site of the original emergence of the most successful and hypervirulent clone belonging to the 4.3.1 genotype. However, this study had limited samples from India. Methods We analyzed 194 clinical S. Typhi, temporal representatives from those isolated from blood and bone marrow cultures in southern India, over 26 years (1991–2016). Antimicrobial resistance (AMR) testing was performed for most common clinical agents. Whole-genome sequencing and SNP-level analysis was conducted. Comparative genomics of Vellore isolates was performed to infer transmission and AMR events. Results We identified multidrug-resistance (MDR)–associated clade 4.3.1 as the dominant genotype. We detected 4.3.1 S. Typhi as early as 1991, the earliest to be reported form India, and the majority were fluoroquinolone resistant and not MDR. MDR was not detected at all in other genotypes circulating in Vellore. Comparison with global S. Typhi showed 2 Vellore subgroups (I and II) that were phylogenetically highly related to previously described South Asia (subgroup I, II) and Southeast Asia (subgroup II) clades. Conclusions 4.3.1 S. Typhi has dominated in Vellore for 2 decades. Our study would assist public health agencies in better tracking of transmission and persistence of this successful clade in India and globally. It informs clinicians of the AMR pattern of circulating clone, which would add confidence to their prophylactic/treatment decision making and facilitate efficient patient care., The molecular epidemiology of Salmonella Typhi circulating in India since early 1990s and its dominance is reported. An in-depth AMR profiling of the major drug-resistant lineage, 4.3.1, would help in management and for public health agencies in planning intervention strategies.

Shigella is the second leading cause of bacterial diarrhea worldwide. This has been categorized as a priority pathogen among enteric bacteria by the Global Antimicrobial Resistance Surveillance System (GLASS) of the World Health Organization (WHO). Recently, S. sonnei seems to be replacing S. flexneri in low- and middle-income countries undergoing economic development. Antimicrobial resistance in S. flexneri and S. sonnei is a growing international concern, specifically with the international dominance of the multidrug-resistant (MDR) lineage. Genomic studies focusing on S. flexneri and S. sonnei in India remain largely unexplored. This study provides information on the introduction and expansion of drug-resistant Shigella strains in India for the first time by comparing the genome sequences of S. flexneri and S. sonnei isolates from India with the publicly available genomes of global strains. The study discusses the key differences between the two dominant species of Shigella at the genomic level to understand the evolutionary trends and genome dynamics of emerging and existing resistance clones. The present work demonstrates evidence for the long-term persistence of all PGs of S. flexneri and the recent dominance of a ciprofloxacin-resistant S. sonnei lineage in India., Shigella is the second leading cause of bacterial diarrhea worldwide. Recently, Shigella sonnei seems to be replacing Shigella flexneri in low- and middle-income countries undergoing economic development. Despite this, studies focusing on these species at the genomic level remain largely unexplored. Here, we compared the genome sequences of S. flexneri and S. sonnei isolates from India with the publicly available genomes of global strains. Our analysis provides evidence for the long-term persistence of all phylogenetic groups (PGs) of S. flexneri and the recent dominance of the ciprofloxacin-resistant S. sonnei lineage in India. Within S. flexneri PGs, the majority of the study isolates belonged to PG3 within the predominance of serotype 2. For S. sonnei, the current pandemic involves globally distributed multidrug-resistant (MDR) clones that belong to Central Asia lineage III. The presence of such epidemiologically dominant lineages in association with stable antimicrobial resistance (AMR) determinants results in successful survival in the community. IMPORTANCE Shigella is the second leading cause of bacterial diarrhea worldwide. This has been categorized as a priority pathogen among enteric bacteria by the Global Antimicrobial Resistance Surveillance System (GLASS) of the World Health Organization (WHO). Recently, S. sonnei seems to be replacing S. flexneri in low- and middle-income countries undergoing economic development. Antimicrobial resistance in S. flexneri and S. sonnei is a growing international concern, specifically with the international dominance of the multidrug-resistant (MDR) lineage. Genomic studies focusing on S. flexneri and S. sonnei in India remain largely unexplored. This study provides information on the introduction and expansion of drug-resistant Shigella strains in India for the first time by comparing the genome sequences of S. flexneri and S. sonnei isolates from India with the publicly available genomes of global strains. The study discusses the key differences between the two dominant species of Shigella at the genomic level to understand the evolutionary trends and genome dynamics of emerging and existing resistance clones. The present work demonstrates evidence for the long-term persistence of all PGs of S. flexneri and the recent dominance of a ciprofloxacin-resistant S. sonnei lineage in India.

Clathrin mediated endocytosis is the primary pathway for receptor and cargo internalization in eukaryotic cells. It is characterized by a polyhedral clathrin lattice that coats budding membranes. The mechanism and control of lattice assembly, curvature, and vesicle formation at the plasma membrane has been a matter of longstanding debate. Here, we use platinum replica and cryo electron microscopy and tomography to present a global structural framework of the pathway. We determine the shape and size parameters common to clathrin-mediated endocytosis. We show that clathrin sites maintain a constant surface area during curvature across multiple cell lines. Flat clathrin is present in all cells and spontaneously curves into coated pits without additional energy sources or recruited factors. Finally, we attribute curvature generation to loosely connected and pentagon-containing flat lattices that can rapidly curve when a flattening force is released. Together, these data present a new universal mechanistic model of clathrin-mediated endocytosis.

Background Pulmonary veno-occlusive disease (PVOD) and pulmonary arterial hypertension (PAH) share an overlapping disease phenotype. Hence it is necessary to distinguish them. Case presentation Our 14-year-old female patient admitted with progressive shortness of breath, dizziness, and fatigue even after minimal physical activity was clinically suspected for PAH, based on her previous history. Her chest computed tomography artery reported the presence of PVOD triad features - subpleural thickened septal lines, ground-glass nodules/opacities and mediastinal lymphadenopathy. Because of her weak physical stature, a lung biopsy was not performed; however, the genetic testing identified a novel heterozygous EIF2AK4 mutation at c.4833_4836dup (p.Q1613Kfs*10) - the dominant susceptible factor driving PVOD. Combination of genetic testing and computed tomography artery facilitated us to distinguish PVOD from PAH. Her disease symptoms advanced aggressively so that she died even before the lung transplantation, which was less than 6 months from the onset of disease symptoms. Conclusion This case report highlights that novel EIF2AK4 mutation at [c.4833_4836dup (p.Q1613Kfs*10)] would predict an aggressive phenotype of PVOD. Hence, we conclude that a genetic test identifying EIF2AK4 mutation would serve as a tool for the early diagnosis of PVOD, circumventing lung biopsy.

Emerging multidrug-resistant (MDR) nosocomial pathogens are a great threat. Polymyxins, an old class of cationic polypeptide antibiotic, are considered as last-resort drugs in treating infections caused by MDR Gram-negative bacteria. Increased use of polymyxins in treating critically ill patients necessitates routine polymyxin susceptibility testing. However, susceptibility testing both of colistin and polymyxin B (PMB) is challenging. In this review, currently available susceptibility testing methods are briefly discussed. The multicomponent composition of colistin and PMB significantly influences susceptibility testing. In addition, poor diffusion in the agar medium, adsorption to microtitre plates and the synergistic effect of the surfactant polysorbate 80 with polymyxins have a great impact on the performance of susceptibility testing methods This review also describes recently identified chromosomal resistance mechanisms, including modification of lipopolysaccharide (LPS) with 4-amino-4-deoxy-l-arabinose (L-Ara4-N) and phosphoethanolamine (pEtN) resulting in alteration of the negative charge, as well as the plasmid-mediated colistin resistance determinants mcr-1, mcr-1.2, mcr-2 and mcr-3.

Background: The emergence of antibiotic resistance among bacterial pathogens in the hospital and community has increased the concern to the health-care providers due to the limited treatment options. Surveillance of antimicrobial resistance (AMR) in frequently isolated bacterial pathogens causing severe infections is of great importance. The data generated will be useful for the clinicians to decide empiric therapy on the local epidemiological resistance profile of the antimicrobial agents. This study aims to monitor the distribution of bacterial pathogen and their susceptibility pattern to the commonly used antimicrobial agents. Materials and Methods: This study includes Gram-negative bacilli collected from intra-abdominal, urinary tract and respiratory tract infections during 2014–2016. Isolates were collected from seven hospitals across India. All the study isolates were characterised up to species level, and minimum inhibitory concentration was determined for a wide range of antimicrobials included in the study panel. The test results were interpreted as per standard Clinical Laboratory Standards Institute guidelines. Results: A total of 2731 isolates of gram-negative bacteria were tested during study period. The most frequently isolated pathogens were 44% of Escherichia coli (n = 1205) followed by 25% of Klebsiella pneumoniae (n = 676) and 11% of Pseudomonas aeruginosa (n = 308). Among the antimicrobials tested, carbapenems were the most active, followed by amikacin and piperacillin/tazobactam. The rate of extended-spectrum beta-lactamase (ESBL)-positive isolates were ranged from 66%–77% in E. coli to 61%–72% in K. pneumoniae, respectively. Overall, colistin retains its activity in > 90% of the isolates tested and appear promising. Conclusion: Increasing rates of ESBL producers have been noted, which is alarming. Further, carbapenem resistance was also gradually increasing, which needs much attention. Overall, this study data show that carbapenems, amikacin and colistin continue to be the best agents available to treat drug-resistant infections. Thus continuous monitoring of susceptibility profile of the clinically important Gram-negative pathogens is of great importance to guide effective antimicrobial therapy.

Testing for antimicrobial interactions has gained popularity in the last decade due to the increasing prevalence of drug-resistant organisms and limited options for the treatment of these infections. In vitro combination testing provides information, on which two or more antimicrobials can be combined for a good clinical outcome. Amongst the various in vitro methods of drug interactions, time-kill assay (TKA), checkerboard (CB) assay and E-test-based methods are most commonly used. Comparative performance of these methods reveals the TKA as the most promising method to detect synergistic combinations followed by CB assay and E-test. Various combinations of antimicrobials have been tested to demonstrate synergistic activity. Promising results were obtained for the combinations of meropenem plus colistin and rifampicin plus colistin against Acinetobacter baumannii, colistin plus carbapenem and carbapenem plus fluoroquinolones against Pseudomonas aeruginosa and colistin/polymyxin B plus rifampicin/meropenem against Klebsiella pneumoniae. Antagonism was detected in only few instances. The presence of synergy or antagonism with a combination seems to correlate with minimum inhibitory concentration of the agent and molecular mechanism involved in the resistance. Further studies need to be conducted to assess the utility of in vitro testing to predict clinical outcome and direct therapy for drug-resistant organisms.

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

Carbapenem-resistantEnterobacteriaceaeconferred by New Delhi metallo-b-lactamase (NDM-1) resistance mechanism are endemic in India and Southeast Asia. An understanding of risk factors for NDM-1 infections is necessary to guide prevention strategies. We performed a retrospective case-control study of patients admitted at Christian Medical College Hospital, Vellore, India between May 2010 and August 2014 withKlebsiella pneumoniaeblood stream infection (BSI). We compared patients with BSI caused by NDM-1 producing strains to two control groups: BSI with other multidrug resistant (MDR) strains and BSI with pan-susceptible strains. The study groups were assessed for risk factors for the outcomes: (1) infection with any MDR strain compared to pan-susceptible; and, (2) infection with NDM-1 strain as compared with other MDR and (3) Mortality. A total of 101 patients with BSI with NDM-1 producingKlebsiella pneumoniaewere matched to two groups of controls: 112 with non-NDM-1 MDR strains and 101 with pan-susceptible strains. Medical (OR 10.4) and neonatal (OR 0.7) ICU admission, central venous catheter placement (CVC, OR 7.4) predicted MDR BSI. Prior carbapenem use (OR 8.4) and CVC (OR 4.8) predicted acquisition of an NDM-1 strain. Significant predictors for mortality included ICU stay (OR 3.0), mechanical ventilation (OR 3.2), female gender (OR 2.2), diabetes (OR 0.4). CVC placement, prior carbapenem use and ICU admission were significantly associated with BSI with NDM-1 producing and other MDR strains.

Serial twofold dilution methods are most commonly used to identify the antimicrobial activities of antibiotics. This can be achieved by different methods like broth dilution or agar dilution. Though these methods are simple, they can be influenced by various experimental factors and result in discrepancy. The following protocol has been validated for Acinetobacter species, including A. baumannii. It is important to include appropriate control strains to determine the minimum inhibitory concentration values and to compare the experiment results.

Acinetobacter baumannii is considered to be an important nosocomial pathogen responsible for various outbreaks that have resulted in a need for effective epidemiological typing methods. Different typing methods are available for A. baumannii epidemiological studies. Currently, the phenotypic typing methods are not being used and replaced by various molecular methods. In this chapter, two important epidemiological typing methods, pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST), are discussed.

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.

Background Recently, in India, there has been a shift from NDM to OXA48-like carbapenemases. OXA-181 and OXA-232 are the frequently produced variants of OXA48-like carbapenemases. OXA48-like carbapenemases are also known to be carried on transposons such as Tn1999, Tn1999.2 and it is also associated with IS1R carried on Tn1999. In India, there are no previous reports studying the association of mobile genetic elements (MGEs) with OXA48-like carbapenemases. The present study was aimed at determining the genetic backbone of OXA48-like carbapenemases to determine the role of MGEs in its transfer and to investigate the Inc plasmid type carrying blaOXA48-like. Results A total of 49 carbapenem resistant K. pneumoniae which included 25 isolates from South India and 24 isolates from North India, were included in the study. Whole genome sequencing using Ion Torrent PGM was performed to study the isolates. OXA-232 was present in 35 isolates (71%). In 19 isolates (39%), blaOXA48-like was associated with MGEs. Insertion sequences such as ISX4, IS1, IS3, ISKpn1, ISKpn26, ISKpn25, ISSpu2, ISKox1, IS4321R, ISEc36, and ISPa38; and transposons such as TnAs3 and Tn2, were present. Isolates from northern and southern India belonging to same sequence type (ST) had diverse genetic backbone for blaOXA48-like. ST14 isolates from north had IS5 and Tn3 families while from south they had IS1, IS5 and IS630 families. ST231 from north had IS5, IS6 and Tn3 families with blaOXA-232 while from south, IS1, IS3 and IS5 families were observed; with ISKpn26 being present among isolates from both the regions. blaOXA48-like was predominantly found on ColKP3 plasmid. ST231 was the predominant ST in 22 isolates (45%). Conclusion OXA-232 is the predominant variant of OXA48-like carbapenemase with ST231 being the commonest ST of OXA48-like carbapenemase producing K. pneumoniae in India. Diverse MGEs have been associated with both blaOXA-232 and blaOXA-181 which contribute to their spread. The MGEs in the present study are different from those reported earlier. There is no clonal expansion of blaOXA48-like producing K. pneumoniae since diverse STs were observed. Monitoring the genetic backbone of OXA48-like carbapenemase is essential to better understand the transmission dynamics of XDR K. pneumoniae.

Infections due to Pseudomonas aeruginosa is a major health concern, especially hospital-acquired infections, in critically ill individuals. Antimicrobial resistance (AMR) increases the morbidity and mortality rates associated with pseudomonal infections. In this review, we aim to address two major aspects of P. aeruginosa. The first part of the review will focus on the burden of AMR and its prevailing mechanisms seen in India, while the second part will focus on the challenges and approaches in the management with special emphasis on the role of newer antimicrobial agents.

Pseudomonas aeruginosa is one of the most common opportunistic pathogens that cause severe infections in humans. The burden of carbapenem resistance is particularly high and is on the rise. Very little information is available on the molecular mechanisms and its clonal types of carbapenem-resistant P. aeruginosa seen in Indian hospitals. This study was undertaken to monitor the β-lactamase profile and to investigate the genetic relatedness of the carbapenemase-producing (CP) P. aeruginosa collected across different hospitals from India.A total of 507 non-duplicate, carbapenem-resistant P. aeruginosa isolated from various clinical specimens collected during 2014-2017 across seven Indian hospitals were included. Conventional multiplex polymerase chain reaction for the genes encoding beta-lactamases such as extended-spectrum beta-lactamase (ESBL) and carbapenemase were screened. A subset of isolates (n = 133) of CP P. aeruginosa were genotyped by multilocus sequence typing (MLST) scheme.Of the total 507 isolates, 15%, 40% and 20% were positive for genes encoding ESBLs, carbapenemases and ESBLs + carbapenemases, respectively, whilst 25% were negative for the β-lactamases screened. Amongst the ESBL genes, blaThis is the first large study from India to report the baseline data on the molecular resistance mechanisms and its association with genetic relatedness of CP P. aeruginosa circulating in Indian hospitals. bla

Antimicrobial resistance (AMR) is a major public health concern across the globe, and it is increasing at an alarming rate. Multiple classes of antimicrobials have been used for the treatment of infectious diseases. Rise in the AMR limits its use and hence the prerequisite for the newer agents to combat drug resistance. Among the infections caused by Gram-negative organisms, beta-lactams are one of the most commonly used agents. However, the presence of diverse beta-lactamases hinders its use for therapy. To overcome these enzymes, beta-lactamase inhibitors are being discovered. The aim of this document is to address the burden of AMR in India and interventions to fight against this battle. This document addresses and summarises the following: The current scenario of AMR in India (antimicrobial susceptibility, resistance mechanisms and molecular epidemiology of common pathogens); contentious issues in the use of beta-lactam/beta-lactamase inhibitor as an carbapenem sparing agent; role of newer beta-lactam/beta-lactamase inhibitor agents with its appropriateness to Indian scenario and; the Indian Council of Medical Research interventions to combat drug resistance in terms of surveillance and infection control as a national response to AMR. This document evidences the need for improved national surveillance system and country-specific newer agents to fight against the AMR.

A very high incidence of typhoid was described in studies conducted in urban locations on the Indian subcontinent at the end of the twentieth century. Despite their availability, licensed immunogenic conjugate typhoid vaccines have not been introduced in the national immunization program, in part, because of a lack of understanding of where and for whom prevention is most necessary. Uncertainty regarding the burden of disease is based on the lack of reliable, recent estimates of culture-confirmed typhoid and an observed trend of low isolations of Salmonella Typhi and fewer complications at large referral hospitals in India. In this article, we examine the trends of S. Typhi isolation at three large tertiary care centers across India over 15 years and describe trends of recognized risk factors for typhoid from published literature. There appears to be a decline in the isolation of S. Typhi in blood cultures, which is more apparent in the past 5 years. These trends are temporally related to economic improvement, female literacy, and the use of antibiotics such as cephalosporins and azithromycin. The analysis of trends of culture-confirmed typhoid may not accurately capture the typhoid incidence trends if antibiotic use confounds the burden of disease presenting to larger facilities. Emerging antimicrobial resistance may result in a resurgence of disease if the underlying incidence and transmission of typhoid are not adequately addressed through public health approaches.

Aminoglycosides are important agents used for treating drug-resistant infections. The current dosing regimen of aminoglycosides does not achieve sufficient serum level concentration for the infected bacterial pathogen interpreted as susceptible based on laboratory testing. Minimum inhibitory concentration was determined for nearly 2000 isolates of Enterobacteriaceae and Pseudomonas aeruginosa by broth microdilution method. Results were interpreted based on CLSI and EUCAST interpretative criteria and the inconsistencies in the susceptibility profile were noted. This study provides insights into the inconsistencies existing in the laboratory interpretation and the corresponding clinical success rates. This urges the need for revising clinical breakpoints for amikacin, to resolve under dosing leading to clinical failure.

The main purpose of this study was to establish 'Antimicrobial Resistance Surveillance Network' in India and to monitor the antimicrobial susceptibility profile of clinical isolates to establish a national network across the country for monitoring antimicrobial resistance in Salmonella.This study was conducted at All India Institute of Medical Sciences, nodal centre with clinical isolates of Salmonellae collected from four centres across India, which included Christian Medical College, Vellore; Postgraduate Institute of Medical Education and Research, Chandigarh and Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry. Total 20% of the selected strains from each centre were characterised for molecular studies which included molecular mechanism of fluoroquinolones resistance and multiple locus sequence type.A total of 622 Salmonellae were received from all centres during January 2014 to December 2015. Out of these 622 isolates, 380 were Salmonella Typhi, 162 were Salmonella Paratyphi A and 7 were S. Paratyphi B isolated from blood and 73 were other Salmonella serotypes. Multiple drug resistance (resistant to ampicillin, chloramphenicol and co-trimoxazole) was less than 3% in S. Typhi. In S. Paratyphi A, chloramphenicol and co-trimoxazole susceptibility was 100% and 99%, respectively, whereas ampicillin susceptibility was 86% (139/161). Ciprofloxacin and nalidixic acid susceptibility was 15% (24/162) and 1% (2/162) from all centres. S. Paratyphi B was isolated from 7 patients. All isolates were third-generation cephalosporin sensitive. The most common mutations found were at codon 83 and at codon 87. We did not find any mutation in acrR gene. Efflux pump and qnr genes were not found in any isolate tested. All 86 S. Typhi isolates clustered into two sequence types - ST1 and ST2. Out of these 86 isolates, 70 S. Typhi were ST1 and 16 were ST2. All S. Paratyphi A was clustered in ST85 and ST129 on the basis of mutation in sucA gene. Out of 27 S. Paratyphi A, 13 were grouped into ST85 and 14 were grouped into ST129.Enteric fever is one such infection which poses challenges in antimicrobial resistance. Hence, continuous surveillance is important to track bacterial resistance and to treat infections in a cost-effective manner.

Carbapenemase-mediated carbapenem resistance is a major concern across the world. Rapid detection of carbapenemase-producing organisms is of great importance in clinical settings. However, it is essential to have a test with good sensitivity and specificity. The aim of the study was to compare the performance of RAPIDEC® CARBA NP and modified carbapenem inactivation method (mCIM) recommended by Clinical and Laboratory Standards Institute guideline 2017. A total of ninety carbapenem resistant Escherichia coli and Klebsiella pneumoniae have been tested. The presence of various carbapenemases was screened by conventional multiplex polymerase chain reaction. RAPIDEC® CARBA NP detected 90%, whereas mCIM detected 99% of the study isolates tested. Although RAPIDEC® CARBA NP is a rapid test, the sensitivity is reduced for blaOxa-48Likedetection; while mCIM could pick up blaOxa-48Likeenzymes with excellent sensitivity. Further, organisms producing low carbapenemase activity enzymes, thickness of the inoculum and the disc potency are likely to influence the test results of mCIM with an overnight delay.

Carbapenem-resistant Enterobacteriaceae (CRE) are an increasing cause of nosocomial infection in hospitalized children worldwide. Few studies have investigated risk factors for mortality in children with CRE bloodstream infection (BSI). Data are particularly scarce in areas where NDM and OXA carbapenemases predominate. Here, we investigate mortality rates, clinical and microbiologic risk factors for mortality in 50 pediatric patients with CRE BSI in India.Children younger than 17 years old with meropenem-resistant Klebsiella pneumoniae or Escherichia coli isolated from blood culture in 2014 and 2015 were identified from laboratory records. Clinical records were systematically reviewed for each child to establish mortality at 30 days and clinical details. Bacterial isolates were subjected to meropenem E test and multiplex polymerase chain reaction to determine carbapenemase gene. Data were analyzed to establish clinical and bacterial risk factors for mortality.All CRE BSI were hospital-acquired or associated with healthcare. A total of 84% of children had an underlying comorbidity and 46% had a malignancy. K. pneumoniae was the most common bacteria isolated; NDM was the most common carbapenemase gene detected. The mortality rate was 52%. Significant risk factors for mortality included intensive care admission, intubation, inotropic support and respiratory source. Failure to clear bacteremia and a minimum inhibitory concentration8 mg/L for the isolate was associated with a statistically significant increase in mortality. Mortality rates were significantly lower when two or more effective drugs were used in combination.CRE BSI affects children with multiple comorbidities and repeated admissions to hospital. The mortality rate is high; combination therapy may be beneficial.

Background & objectives: Klebsiella pneumoniae (KP), a common cause of invasive infections, is often extensively drug resistant in India. At present, studies on resistance mechanism and clonal relationship of KP from India are limited. The present study was undertaken to determine the resistance mechanism and clonal relationship of colistin-resistant isolates obtained from various specimens. Carbapenemases were also determined since the isolates were carbapenem resistant. Methods: Sixty five isolates from blood, exudates and respiratory specimens collected between 2016 and 2017 were studied. Colistin minimum inhibitory concentration (MIC) was performed by broth-micro dilution method. Multiplex PCR was carried out to determine carbapenemases. Targeted sequencing was performed to determine mutations in mgrB, phoP, phoQ and multilocus sequence typing was performed to determine the prevalent clones. Results: Colistin MIC ranged from 4 to 256 μg/ml. SHV, TEM and CTX-M were co-produced in 60 per cent and OXA48-like in 71 per cent. Thirteen isolates had mutations in mgrB. Mutations included a premature stop codon at 21st amino acid, the presence of insertion sequences such as IS903, IS Kpn 14 and ISK pn 26; and elongation of mgrB. Novel mutations were also observed among phoP and phoQ genes. Colistin resistance due to mcr genes was absent. Fifteen clonal types were seen with ST231, ST14 and ST2096 being predominant. Interpretation & conclusions: This study revealed the changing trend of carbapenem resistance mechanism predominantly to OXA48-like from NDM. Known mgrB mutations and novel mutations in phoP and phoQ were detected. There was no plasmid-mediated colistin resistance. ST14 and ST231 were international clones associated with carbapenem resistance. Colistin-resistant KP was of diverse clones with predominantly ST231, ST14 and ST2096.

This record review of 82 children with Down Syndrome (DS) between April 2004 and March 2014 who had thyroid dysfunction, showed that majority (76, 92.6%) had subclinical hypothyroidism. Of the 60 patients who underwent radionuclide scan, 63.3% had a normal gland; the rest exhibited only impaired tracer uptake. Ultrasonograms done in 20 patients showed reduction of thyroid gland size in 3 (15%) patients only.

Darier's disease (DD) is an inherited autosomal-dominant skin disorder characterized histologically by loss of adhesion between keratinocytes. DD is typically caused by mutations in sarcoendoplasmic reticulum Ca2+-ATPase isoform 2 (SERCA2), a major regulator of intracellular Ca2+ homeostasis in the skin. However, a defined role for SERCA2 in regulating intercellular adhesion remains poorly understood. We found that diminution of SERCA2 function by pharmacological inhibition or siRNA silencing in multiple human epidermal-derived cell lines was sufficient to disrupt desmosome assembly and weaken intercellular adhesive strength. Specifically, SERCA2-deficient cells exhibited up to a 60% reduction in border translocation of desmoplakin (DP), the desmosomal cytolinker protein necessary for intermediate filament (IF) anchorage to sites of robust cell-cell adhesion. In addition, loss of SERCA2 impaired the membrane translocation of protein kinase C α (PKCα), a known regulator of DP-IF association and desmosome assembly, to the plasma membrane by up to 70%. Exogenous activation of PKCα in SERCA2-deficient cells was sufficient to rescue the defective DP localization, desmosome assembly, and intercellular adhesive strength to levels comparable to controls. Our findings indicate that SERCA2-deficiency is sufficient to impede desmosome assembly and weaken intercellular adhesive strength via a PKCα-dependent mechanism, implicating SERCA2 as a novel regulator of PKCα signaling.—Hobbs, R. P., Amargo, E. V., Somasundaram, A., Simpson, C. L., Prakriya, M., Denning, M. F., Green, K. J. The calcium ATPase SERCA2 regulates desmoplakin dynamics and intercellular adhesive strength through modulation of PKCα signaling.

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Enteropathogenic Escherichia coli (EPEC) are a major cause of infant diarrhoea in developing countries and a significant public health issue in industrialized countries. Currently there are no simple tests available for the diagnosis of EPEC. Serology of O-antigens is widely used routinely in many laboratories throughout the world, even though it has been known for many years to be an unreliable indicator of EPEC virulence. We have developed a simple, low-cost immunodiagnostic test based on the EspA filament, an essential virulence factor of EPEC and the related enterohaemorrhagic E. coli (EHEC). Using recombinant proteins of the five major variants of EspA as immunogens, we raised a panel of three monoclonal antibodies in mice that detects all variants of the native target in bacterial cultures. The antibodies proved suitable for application in sandwich-type assays, including ELISA and lateral flow immunoassays (LFI). Prototypes for both assays were specific for EPEC and EHEC strains when tested against a panel of control micro-organisms. We have also developed a simple, affordable culture medium, A/E medium, which optimizes expression of EspA allowing improved sensitivity of detection compared with standard Dulbecco’s modified Eagle’s medium. Together these reagents form the basis of robust, informative tests for EPEC for use especially in developing countries but also for routine screening in any clinical laboratory.

We describe the pathogens and their antimicrobial profile causing blood stream infections in children over a 4-year period. The commonest pathogens were: non-fermenting Gram negative bacilli other than Pseudomonas, Salmonella species, Escherichia coli, Staphylococcus aureus and Klebsiella species. High rates of drug-resistance were observed.

Ca(2+) release-activated Ca(2+) (CRAC) channels are activated through a mechanism wherein depletion of intracellular calcium stores results in the aggregation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca(2+) sensor, and Orai1, the CRAC channel protein, at overlapping sites in the ER and plasma membranes (PMs). The redistribution of CRAC channels is driven through direct STIM1-Orai1 binding, an important event that not only controls gating, but also regulates Orai1 ion selectivity. Orai1 harbours two STIM1 binding sites, one each on the intracellular C- and N-termini. Previous studies have proposed modular functions for these sites, with the C-terminal site thought to regulate STIM1-Orai1 binding and trapping of Orai1 at the ER-PM junctions, and the N-terminal site mediating gating. However, here we find that a variety of mutations in the N-terminal site impair the binding of Orai1 to STIM1 and to the soluble CRAC activation domain (CAD). Gating could be restored in several N- and C-terminal point mutants by directly tethering the minimal STIM1 activation domain (S) to Orai1 (Orai1-SS channels), indicating that loss of gating in these mutants by full-length STIM1 results from insufficient ligand binding. By contrast, gating could not be restored in mutant Orai1-SS channels carrying more drastic deletions that removed the STIM1 binding sites (1-85, 73-85, or 272-279 Orai1), suggesting that STIM1 binding to both sites is essential for channel activation. Moreover, analysis of ion selectivity indicated that the molecular requirements for gating and modulation of ion selectivity are similar, yet substantively different from those for Orai1 puncta formation, suggesting that ion selectivity and gating are mechanistically coupled in CRAC channels. Our results indicate that the C- and N-terminal STIM1 binding sites are both essential for multiple aspects of Orai1 function including STIM1-Orai1 association, Orai1 trapping, and channel activation.

Microwave, oven, and oil roasting of almonds were used to promote almond flavor and color formation. Raw pasteurized almonds were roasted in a microwave for 1 to 3 min, in an oven at 177 °C for 5, 10, 15, and 20 min; and at 135 and 163 °C for 20 min, and in oil at 135, 163, and 177 °C for 5 min and 177 °C for 10 min. Volatile compounds were quantified in the headspace of ground almonds, both raw and roasted, by selected ion flow tube mass spectrometry. Strong correlations were found between L value, chroma, and 5-(hydroxy methyl)-2- furfural; and were independent of roasting method. Raw almonds had lower concentrations of most volatiles than roasted almonds. Conditions that produced color equivalent to commercial samples were 2 min in the microwave, 5 min at 177 °C in the oven, and 5 min at 135 °C in oil. Microwave heating produced higher levels of most volatiles than oven and oil roasting at commercial color. Sensory evaluation indicated that microwave-roasted almonds had the strongest aroma and were the most preferred. Oil-roasted almonds showed significantly lower levels of volatiles than other methods, likely due to loss of these volatiles into the oil. Alcohols such as benzyl alcohols and strecker aldehydes including benzaldehyde and methional were at higher concentrations than other volatiles in roasted almonds. The oxidation of lipids to form alkanals such as nonanal and degradation of sugars to form furan type compounds was also observed. The Maillard reaction contributed to the formation of more of the total volatiles in almonds than the lipid oxidation reaction. Practical Application: The level of 5-(hydroxy methyl)-2- furfural (HMF), color, volatile profile, and sensory perception can be used to develop the best roasting method, time, and temperature for almonds. The rate of color development and the production of volatiles differ under different roasting conditions. Based on the color, volatile, and sensory assessments of the 3 almonds, the use of microwave technology as a process for roasting almonds reduces processing time and leads to an almond product with better flavor than oven or oil roasting.

Ca2+ is a ubiquitous signaling messenger mediating many essential cellular functions such as excitability, exocytosis and transcription. Among the different pathways by which cellular Ca2+ signals are generated, the entry of Ca2+ through store-operated Ca2+ release-activated Ca2+ (CRAC) channels has emerged as a widespread mechanism for regulating Ca2+ signaling in many eukaryotic cells. CRAC channels are implicated in the physiology and pathophysiology of numerous cell types, underlie several disease processes including a severe combined immunodeficiency syndrome, and have emerged as major targets for drug development. Although little was known of the molecular mechanisms of CRAC channels for several decades, the discovery of Orai1 as a prototypic CRAC channel pore-subunit, and the identification of STIM1 as the ER Ca2+ sensor, have led to rapid progress in our understanding of many aspects of CRAC channel behavior. This review examines the molecular features of the STIM and Orai proteins that regulate the activation and conduction mechanisms of CRAC channels.

Activation of Ca(2+) release-activated Ca(2+) channels by depletion of intracellular Ca(2+) stores involves physical interactions between the endoplasmic reticulum Ca(2+) sensor, STIM1, and the channels composed of Orai subunits. Recent studies indicate that the Orai3 subtype, in addition to being store-operated, is also activated in a store-independent manner by 2-aminoethyldiphenyl borate (2-APB), a small molecule with complex pharmacology. However, it is unknown whether the store-dependent and -independent activation modes of Orai3 channels operate independently or whether there is cross-talk between these activation states. Here we report that in addition to causing direct activation, 2-APB also regulates store-operated gating of Orai3 channels, causing potentiation at low doses and inhibition at high doses. Inhibition of store-operated gating by 2-APB was accompanied by the suppression of several modes of Orai3 channel regulation that depend on STIM1, suggesting that high doses of 2-APB interrupt STIM1-Orai3 coupling. Conversely, STIM1-bound Orai3 (and Orai1) channels resisted direct gating by high doses of 2-APB. The rate of direct 2-APB activation of Orai3 channels increased linearly with the degree of STIM1-Orai3 uncoupling, suggesting that 2-APB has to first disengage STIM1 before it can directly gate Orai3 channels. Collectively, our results indicate that the store-dependent and -independent modes of Ca(2+) release-activated Ca(2+) channel activation are mutually exclusive: channels bound to STIM1 resist 2-APB gating, whereas 2-APB antagonizes STIM1 gating.

Ca(2+) entry through store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels initiates key functions such as gene expression and exocytosis of inflammatory mediators. Activation of CRAC channels by store depletion involves the redistribution of the ER Ca(2+) sensor, stromal interaction molecule 1 (STIM1), to peripheral sites where it co-clusters with the CRAC channel subunit, Orai1. However, how STIM1 communicates with the CRAC channel and initiates the subsequent events culminating in channel opening is unclear. Here, we show that redistribution of STIM1 and Orai1 occurs in parallel with a pronounced increase in fluorescence resonance energy transfer (FRET) between STIM1 and Orai1, supporting the idea that activation of CRAC channels occurs through physical interactions with STIM1. Co-expression of Orai1-CFP and Orai1-YFP results in a high degree of FRET in resting cells, indicating that Orai1 exists as a multimer. However, store depletion triggers molecular rearrangements in Orai1 resulting in a decline in Orai1-Orai1 FRET. The decline in Orai1-Orai1 FRET is not seen in the absence of STIM1 co-expression and is abolished in Orai1 mutants with impaired STIM1 interaction. Both the STIM1-Orai1 interaction as well as the molecular rearrangements in Orai1 are altered by two powerful modulators of CRAC channel activity: extracellular Ca(2+) and 2-APB. These studies identify a STIM1-dependent conformational change in Orai1 during the activation of CRAC channels and reveal that STIM1-Orai1 interaction and the downstream Orai1 conformational change can be independently modulated to fine-tune CRAC channel activity.