Prevalence and epidemiological characteristics of methicillin-resistant Staphylococcus aureus isolated from cattle in Bangalore India as a part of the One Health approach.

In India, limited studies are available on the epidemiological aspects of methicillin-resistant Staphylococcus aureus (MRSA) infections in both animal and human settings. Herein, we investigated the prevalence, antimicrobial resistance profile and molecular characteristics of MRSA isolates recovered from cattle using the One Health approach. Out of 66 mecA-positive staphylococci, species-specific multiplex PCR detected 24 % (n=16) of isolates as MRSA. Maximum antibiotic resistance was seen against cloxacillin (94 %, n=15) and least for enrofloxacin and cephalothin (each 13 %, n=2). Overall, 13 % (n=2) of MRSA isolates were multidrug-resistant. Molecular characterization by SCCmec typing identified 88 % (n=14) of MRSA isolates as type V. Twelve isolates (75 %) belonged to novel spa-type t17242, of which 67 % (n=8) belonged to agr type I. MLST analysis revealed ST 1687 (50 %, n=8) as the most predominant sequence type. Circulation of different MRSA clones among the cattle populace offers a risk of transmission to humans through direct contact, food chain or environmental contamination. Thus, continuous monitoring of MRSA strains is imperative for early diagnosis and for establishing effective treatment strategies to restrain the disease burden caused by MRSA infections.

Novel pneumococcal capsule type 33E results from the inactivation of glycosyltransferase WciE in vaccine type 33F.

The polysaccharide (PS) capsule is essential for immune evasion and virulence of Streptococcus pneumoniae. Existing pneumococcal vaccines are designed to elicit anticapsule antibodies; however, the effectiveness of these vaccines is being challenged by the emergence of new capsule types or variants. Herein, we characterize a newly discovered capsule type, 33E, that appears to have repeatedly emerged from vaccine type 33F via an inactivation mutation in the capsule glycosyltransferase gene, wciE. Structural analysis demonstrated that 33E and 33F share an identical repeat unit backbone [→5)-ß-D-Galf2Ac-(1→3)-ß-D-Galp-(1→3)-α-D-Galp-(1→3)-ß-D-Galf-(1→3)-ß-D-Glcp-(1→], except that a galactose (α-D-Galp) branch is present in 33F but not in 33E. Though the two capsule types were indistinguishable using conventional typing methods, the monoclonal antibody Hyp33FM1 selectively bound 33F but not 33E pneumococci. Further, we confirmed that wciE encodes a glycosyltransferase that catalyzes the addition of the branching α-D-Galp and that its inactivation in 33F strains results in the expression of the 33E capsule type. Though 33F and 33E share a structural and antigenic similarity, our pilot study suggested that immunization with a 23-valent pneumococcal PS vaccine containing 33F PS did not significantly elicit cross-opsonic antibodies to 33E. New conjugate vaccines that target capsule type 33F may not necessarily protect against 33E. Therefore, studies of new conjugate vaccines require knowledge of the newly identified capsule type 33E and reliable pneumococcal typing methods capable of distinguishing it from 33F.

Estimation of baseline IgG antibody levels to 23 pneumococcal vaccine-type capsular polysaccharides in healthy vaccine naïve Indian adults.

Since immunological responses to pneumococcal vaccines are assessed by a fold-increase in antibody levels relative to pre-immunization levels, it is therefore critical to determine baseline antibody levels to establish putative threshold as a measure of normal response. Herein, for the first time, we measured baseline IgG antibody levels in 108 healthy unvaccinated Indian adults using WHO-recommended ELISA. Median baseline IgG concentration ranged between 0.54 µg/mL to 12.35 µg/mL. Highest levels of baseline capsule polysaccharide (cPS)-specific IgG were found against types 14, 19A, and 33F. Whereas, lowest baseline IgG levels were observed against types 3, 4, and 5. Overall, ∼79% of study population had median baseline IgG levels ≥1.3 µg/mL against 74% of cPS's. Substantial baseline antibody levels in unvaccinated adults were observed. The study would be critical in bridging gaps in baseline immunogenicity data and may offer a valuable foundation for evaluating immune response of Indian adults to pneumococcal vaccination.

Discovery and Characterization of Pneumococcal Serogroup 36 Capsule Subtypes, Serotypes 36A and 36B.

Streptococcus pneumoniae can produce a wide breadth of antigenically diverse capsule types, a fact that poses a looming threat to the success of vaccines that target pneumococcal polysaccharide (PS) capsule. Yet, many pneumococcal capsule types remain undiscovered and/or uncharacterized. Prior sequence analysis of pneumococcal capsule synthesis (cps) loci suggested the existence of capsule subtypes among isolates identified as "serotype 36" according to conventional capsule typing methods. We discovered these subtypes represent two antigenically similar but distinguishable pneumococcal capsule serotypes, 36A and 36B. Biochemical analysis of their capsule PS structure reveals that both have the shared repeat unit backbone [→5)-α-d-Galf-(1→1)-d-Rib-ol-(5→P→6)-ß-d-ManpNAc-(1→4)-ß-d-Glcp-(1→] with two branching structures. Both serotypes have a ß-d-Galp branch to Ribitol. Serotypes 36A and 36B differ by the presence of a α-d-Glcp-(1→3)-ß-d-ManpNAc or α-d-Galp-(1→3)-ß-d-ManpNAc branch, respectively. Comparison of the phylogenetically distant serogroup 9 and 36 cps loci, which all encode this distinguishing glycosidic bond, revealed that the incorporation of Glcp (in types 9N and 36A) versus Galp (in types 9A, 9V, 9L, and 36B) is associated with the identity of four amino acids in the cps-encoded glycosyltransferase WcjA. Identifying functional determinants of cps-encoded enzymes and their impact on capsule PS structure is key to improving the resolution and reliability of sequencing-based capsule typing methods and discovering novel capsule variants indistinguishable by conventional serotyping methods.

Ficolin-2 Lectin Complement Pathway Mediates Capsule-Specific Innate Immunity Against Invasive Pneumococcal Disease.

Reports conflict regarding which lectin-microbial ligand interactions elicit a protective response from the lectin pathway (LP) of complement. Using fluorescent microscopy, we demonstrate the human lectin ficolin-2 binds to Streptococcus pneumoniae serotype 11A capsule polysaccharide dependent on the O-acetyltransferase gene wcjE. This triggers complement deposition and promotes opsonophagocytosis of encapsulated pneumococci. Even partial loss of ficolin-2 ligand expression through wcjE mutation abrogated bacterial killing. Ficolin-2 did not interact with any pneumococcal non-capsule structures, including teichoic acid. We describe multiple 11A clonal derivatives expressing varying degrees of wcjE-dependent epitopes co-isolated from single blood specimens, likely representing microevolutionary shifts towards wcjE-deficient populations during invasive pneumococcal disease (IPD). We find epidemiological evidence of wcjE impairing pneumococcal invasiveness, supporting that the LP's ficolin-2 axis provides innate, serotype-specific serological protection against IPD. The fact that the LP is triggered by only a few discrete carbohydrate ligands emphasizes the need to reevaluate its impact in a glycopolymer-specific manner.

Occurrence and characterization of genetic determinants of ß-lactam-resistance in Escherichia coli clinical isolates.

ß-lactamase mediated resistance in Escherichia coli is a significant problem that requires immediate attention. Herein, we aim to characterize and understand the dynamics of the genetic determinants of ß-lactam resistance (i.e. ESBL, AmpC, and MBL) in E. coli. Out of 203 E. coli isolates, genetic determinants of ß-lactam resistance were identified in 50% (n = 101) of isolates. ESBL, AmpC, and MBL resistance determinants were detected in 78%, 40%, and 18% of isolates, respectively with blaCTX-M group 4 (48%), blaCMY (40%), and blaSIM (33%) as the most prevalent ß-lactam resistance genes. Among these isolates, 45% harbored plasmid replicon types, with L/M (40%) and Y (33%) as the most dominant replicon types. Integrons were detected in 40% of such isolates, with Class-1 and Class-3 representing 62% and 55%, respectively. Overall, we observed high rate of genetic determinants of ß-lactam-resistance in E. coli isolates recovered from patients in clinical settings. The co-occurrence of antimicrobial resistance genes and mobile genetic elements in a high percentage of isolates is a major concern and relates to complex resistance mechanisms. To combat the serious threat of antimicrobial resistance, it is imperative to develop strategies for robust surveillance and understand the molecular basis of resistance acquisition and transmission.

Anatomical Site-Specific Carbohydrate Availability Impacts Streptococcus pneumoniae Virulence and Fitness during Colonization and Disease.

Streptococcus pneumoniae colonizes the nasopharynx asymptomatically but can also cause severe life-threatening disease. Importantly, stark differences in carbohydrate availability exist between the nasopharynx and invasive disease sites, such as the bloodstream, which most likely impact S. pneumoniae's behavior. Herein, using chemically defined medium (CDM) supplemented with physiological levels of carbohydrates, we examined how anatomical site-specific carbohydrate availability impacted S. pneumoniae physiology and virulence. S. pneumoniae cells grown in CDM modeling the nasopharynx (CDM-N) had reduced metabolic activity and a lower growth rate, demonstrated mixed acid fermentation with marked H2O2 production, and were in a carbon-catabolite repression (CCR)-derepressed state versus S. pneumoniae cells grown in CDM modeling blood (CDM-B). Using transcriptome sequencing (RNA-seq), we determined the transcriptome for the S. pneumoniae wild-type (WT) strain and its isogenic CCR-deficient mutant in CDM-N and CDM-B. Genes with altered expression as a result of changes in carbohydrate availability or catabolite control protein deficiency, respectively, were primarily involved in carbohydrate metabolism, but also encoded established virulence determinants, such as polysaccharide capsule and surface adhesins. We confirmed that anatomical site-specific carbohydrate availability directly influenced established S. pneumoniae virulence traits. S. pneumoniae cells grown in CDM-B formed shorter chains, produced more capsule, were less adhesive, and were more resistant to macrophage killing in an opsonophagocytosis assay. Moreover, growth of S. pneumoniae in CDM-N or CDM-B prior to the challenge of mice impacted relative fitness in a colonization model and invasive disease model, respectively. Thus, anatomical site-specific carbohydrate availability alters S. pneumoniae physiology and virulence, in turn promoting anatomical site-specific fitness.

Effect of Oral Streptococci Expressing Pneumococcus-like Cross-Reactive Capsule Types on World Health Organization Recommended Pneumococcal Carriage Detection Procedure.

BACKGROUND: Carriage studies are fundamental to assessing the effects of pneumococcal vaccines. Because a large proportion of oral streptococci carry homologues of pneumococcal genes, non-culture-based detection and serotyping of upper respiratory tract (URT) samples can be problematic. In the current study, we investigated whether culture-free molecular methods could differentiate pneumococci from oral streptococci carried by adults in the URT. METHODS: Paired nasopharyngeal (NP) and oropharyngeal (OP) samples were collected from 100 older adults twice a month for 1 year. Extracts from the combined NP + OP samples (n = 2400) were subjected to lytA real-time polymerase chain reaction (PCR). Positive samples were subjected to pure culture isolation, followed by species confirmation using multiple approaches. Multibead assays and whole-genome sequencing were used for serotyping. RESULTS: In 20 of 301 combined NP + OP extracts with positive lytA PCR results, probable pneumococcus-like colonies grew, based on colony morphology and biochemical tests. Multiple approaches confirmed that 4 isolates were Streptococcus pneumoniae, 3 were Streptococcus pseudopneumoniae, 12 were Streptococcus mitis, and 1 were Streptococcus oralis. Eight nonpneumococcal strains carried pneumococcus-like cps loci (approximate size, 18-25 kb) that showed >70% nucleotide identity with their pneumococcal counterparts. While investigating the antigenic profile, we found that some S. mitis strains (P066 and P107) reacted with both serotype-specific polyclonal (type 39 and FS17b) and monoclonal (Hyp10AG1 and Hyp17FM1) antisera, whereas some strains (P063 and P074) reacted only with polyclonal antisera (type 5 and FS35a). CONCLUSION: The extensive capsular overlap suggests that pneumococcal vaccines could reduce carriage of oral streptococci expressing cross-reactive capsules. Furthermore, direct use of culture-free PCR-based methods in URT samples has limited usefulness for carriage studies.

Capsule Promotes Intracellular Survival and Vascular Endothelial Cell Translocation during Invasive Pneumococcal Disease.

The polysaccharide capsule that surrounds Streptococcus pneumoniae (Spn) is one of its most important virulence determinants, serving to protect against phagocytosis. To date, 100 biochemical and antigenically distinct capsule types, i.e., serotypes, of Spn have been identified. Yet how capsule influences pneumococcal translocation across vascular endothelial cells (VEC), a key step in the progression of invasive disease, was unknown. Here, we show that despite capsule being inhibitory of Spn uptake by VEC, capsule enhances the escape rate of internalized pneumococci and thereby promotes translocation. Upon investigation, we determined that capsule protected Spn against intracellular killing by VEC and H2O2-mediated killing in vitro. Using a nitroblue tetrazolium reduction assay and nuclear magnetic resonance (NMR) analyses, purified capsule was confirmed as having antioxidant properties which varied according to serotype. Using an 11-member panel of isogenic capsule-switch mutants, we determined that serotype affected levels of Spn resistance to H2O2-mediated killing in vitro, with killing resistance correlated positively with survival duration within VEC, rate of transcytosis to the basolateral surface, and human attack rates. Experiments with mice supported our in vitro findings, with Spn producing oxidative-stress-resistant type 4 capsule being more organ-invasive than that producing oxidative-stress-sensitive type 2 capsule during bacteremia. Capsule-mediated protection against intracellular killing was also observed for Streptococcus pyogenes and Staphylococcus aureus. We conclude that capsular polysaccharide plays an important role within VEC, serving as an intracellular antioxidant, and that serotype-dependent differences in antioxidant capabilities impact the efficiency of VEC translocation and a serotype's potential for invasive disease. IMPORTANCE Streptococcus pneumoniae (Spn) is the leading cause of invasive disease. Importantly, only a subset of the 100 capsule types carried by Spn cause the majority of serious infections, suggesting that the biochemical properties of capsular polysaccharide are directly tied to virulence. Here, we describe a new function for Spn's capsule-conferring resistance to oxidative stress. Moreover, we demonstrate that capsule promotes intracellular survival of pneumococci within vascular endothelial cells and thereby enhances bacterial translocation across the vasculature and into organs. Using isogenic capsule-switch mutants, we show that different capsule types, i.e., serotypes, vary in their resistance to oxidative stress-mediated killing and that resistance is positively correlated with intracellular survival in an in vitro model, organ invasion during bacteremia in vivo, and epidemiologically established pneumococcal attack rates in humans. Our findings define a new role of capsule and provide an explanation for why certain serotypes of Spn more frequently cause invasive pneumococcal disease.

Streptococcus pneumoniae genomic datasets from an Indian population describing pre-vaccine evolutionary epidemiology using a whole genome sequencing approach.

Globally, India has a high burden of pneumococcal disease, and pneumococcal conjugate vaccine (PCV) has been rolled out in different phases across the country since May 2017 in the national infant immunization programme (NIP). To provide a baseline for assessing the impact of the vaccine on circulating pneumococci in India, genetic characterization of pneumococcal isolates detected prior to introduction of PCV would be helpful. Here we present a population genomic study of 480 Streptococcus pneumoniae isolates collected across India and from all age groups before vaccine introduction (2009-2017), including 294 isolates from pneumococcal disease and 186 collected through nasopharyngeal surveys. Population genetic structure, serotype and antimicrobial susceptibility profile were characterized and predicted from whole-genome sequencing data. Our findings revealed high levels of genetic diversity represented by 110 Global Pneumococcal Sequence Clusters (GPSCs) and 54 serotypes. Serotype 19F and GPSC1 (CC320) was the most common serotype and pneumococcal lineage, respectively. Coverage of PCV13 (Pfizer) and 10-valent Pneumosil (Serum Institute of India) serotypes in age groups of ≤2 and 3-5 years were 63-75 % and 60-69 %, respectively. Coverage of PPV23 (Merck) serotypes in age groups of ≥50 years was 62 % (98/158). Among the top five lineages causing disease, GPSC10 (CC230), which ranked second, is the only lineage that expressed both PCV13 (serotypes 3, 6A, 14, 19A and 19F) and non-PCV13 (7B, 13, 10A, 11A, 13, 15B/C, 22F, 24F) serotypes. It exhibited multidrug resistance and was the largest contributor (17 %, 18/103) of NVTs in the disease-causing population. Overall, 42 % (202/480) of isolates were penicillin-resistant (minimum inhibitory concentration ≥0.12 µg ml-1) and 45 % (217/480) were multidrug-resistant. Nine GPSCs (GPSC1, 6, 9, 10, 13, 16, 43, 91, 376) were penicillin-resistant and among them six were multidrug-resistant. Pneumococci expressing PCV13 serotypes had a higher prevalence of antibiotic resistance. Sequencing of pneumococcal genomes has significantly improved our understanding of the biology of these bacteria. This study, describing the pneumococcal disease and carriage epidemiology pre-PCV introduction, demonstrates that 60-75 % of pneumococcal serotypes in children ≤5 years are covered by PCV13 and Pneumosil. Vaccination against pneumococci is very likely to reduce antibiotic resistance. A multidrug-resistant pneumococcal lineage, GPSC10 (CC230), is a high-risk clone that could mediate serotype replacement.

Structural, Genetic, and Serological Elucidation of Streptococcus pneumoniae Serogroup 24 Serotypes: Discovery of a New Serotype, 24C, with a Variable Capsule Structure.

Pneumococcal capsules are important in pneumococcal pathogenesis and vaccine development. Although conjugate vaccines have brought about a significant reduction in invasive pneumococcal disease (IPD) caused by vaccine serotypes, the relative serotype prevalence has shifted with the dramatic emergence of serotype 24F in some countries. Here, we describe 14 isolates (13 IPD and 1 non-IPD) expressing a new capsule type, 24C, which resembles 24F but has a novel serological profile. We also describe the antigenic, biochemical, and genetic basis of 24F and 24C and the related serotypes 24A and 24B. Structural studies show that 24B, 24C, and 24F have identical polysaccharide backbones [ß-Ribf-(1→4)-α-Rhap-(1→3)-ß-GlcpNAc-(1→4)-ß-Rhap-(1→4)-ß-Glcp] but with different side chains, as follows: 24F has arabinitol-phosphate and 24B has ribitol-phosphate. 24C has a mixture of 24F and 24B repeating units, with the ratio of ribitol to arabinitol being strain dependent. In contrast, the 24A capsule has a backbone without ß-Ribf but with arabinitol-phosphate and phosphocholine side chains. These structures indicate that factor-sera 24d and 24e recognize arabinitol and ribitol, respectively, which explains the serology of serogroup 24, including those of 24C. The structures can be genetically described by the bispecificity of wcxG, which is capable of transferring arabinitol or ribitol when arabinitol is limiting. Arabinitol is likely not produced in 24B but is produced in reduced amounts in 24C due to various mutations in abpA or abpB genes. Our findings demonstrate how pneumococci modulate their capsule structure and immunologic properties with small genetic changes, thereby evading host immune responses. Our findings also suggest a potential for new capsule types within serogroup 24.

A New Pneumococcal Capsule Type, 10D, is the 100th Serotype and Has a Large cps Fragment from an Oral Streptococcus.

Streptococcus pneumoniae (pneumococcus) is a major human pathogen producing structurally diverse capsular polysaccharides. Widespread use of highly successful pneumococcal conjugate vaccines (PCVs) targeting pneumococcal capsules has greatly reduced infections by the vaccine types but increased infections by nonvaccine serotypes. Herein, we report a new and the 100th capsule type, named serotype 10D, by determining its unique chemical structure and biosynthetic roles of all capsule synthesis locus (cps) genes. The name 10D reflects its serologic cross-reaction with serotype 10A and appearance of cross-opsonic antibodies in response to immunization with 10A polysaccharide in a 23-valent pneumococcal vaccine. Genetic analysis showed that 10D cps has three large regions syntenic to and highly homologous with cps loci from serotype 6C, serotype 39, and an oral streptococcus strain (S. mitis SK145). The 10D cps region syntenic to SK145 is about 6 kb and has a short gene fragment of wciNα at the 5' end. The presence of this nonfunctional wciNα fragment provides compelling evidence for a recent interspecies genetic transfer from oral streptococcus to pneumococcus. Since oral streptococci have a large repertoire of cps loci, widespread PCV usage could facilitate the appearance of novel serotypes through interspecies recombination.IMPORTANCE The polysaccharide capsule is essential for the pathogenicity of pneumococcus, which is responsible for millions of deaths worldwide each year. Currently available pneumococcal vaccines are designed to elicit antibodies to the capsule polysaccharides of the pneumococcal isolates commonly causing diseases, and the antibodies provide protection only against the pneumococcus expressing the vaccine-targeted capsules. Since pneumococci can produce different capsule polysaccharides and therefore reduce vaccine effectiveness, it is important to track the appearance of novel pneumococcal capsule types and how these new capsules are created. Herein, we describe a new and the 100th pneumococcal capsule type with unique chemical and serological properties. The capsule type was named 10D for its serologic similarity to 10A. Genetic studies provide strong evidence that pneumococcus created 10D capsule polysaccharide by capturing a large genetic fragment from an oral streptococcus. Such interspecies genetic exchanges could greatly increase diversity of pneumococcal capsules and complicate serotype shifts.

Genome-wide analysis of mammary gland shows modulation of transcriptome landscape with alternative splice variants in Staphylococcus aureus mastitis in mice.

Epidemiological mapping shows Staphylococcus aureus to be the leading mastitis causing pathogen in India with diverse genetic lineages circulating in the dairy cattle population. We previously reported that endemic clonal strains of S. aureus isolated from subclinical mastitis lead to specific alteration of epigenetic modulators resulting in deviating immune response in intramammary infection mouse model. However, the extent of transcriptome modulation and associated alternative splicing in S. aureus mastitis is poorly understood. Hence, to gain a deeper insight of the extent of modulation of transcriptome landscape, we expanded the study here using high throughput, paired-end RNA sequencing analysis of the mouse mammary gland inoculated with three strains of S. aureus (SA1, SA2, and SA3) possessing specific genotype, virulence and enterotoxin traits. Overall, we detected 35,878 transcripts in S. aureus inoculated mammary gland, 23% more than those annotated in the reference genome. Expression of 20,756 transcripts was > 1 fragment per kilobase of transcript per million mapped fragments and 25.95% of multi-exonic genes were alternatively spliced. We noted Alternative Splicing (AS) events for > 100 immune-related genes. S. aureus infection quantitatively altered AS events in mice mammary gland. Collectively, the majority of differentially expressed significant genes clustered into immune-associated, cell adhesion and metabolic process categories. We observed AS events for 379 transcripts of genes putatively encoding several splicing associated proteins and transcription factors besides inflammatory mediators. The present analysis provides new insights into global transcriptome landscape and AS events in host-defense related genes in response to S. aureus intramammary infection, suggesting the need for studies focusing on multi-target and/or network therapeutics approach to combat mastitis.

Pneumococci Can Become Virulent by Acquiring a New Capsule From Oral Streptococci.

Pneumococcal conjugate vaccines have been successful, but their use has increased infections by nonvaccine serotypes. Oral streptococci often harbor capsular polysaccharide (PS) synthesis loci (cps). Although this has not been observed in nature, if pneumococcus can replace its cps with oral streptococcal cps, it may increase its serotype repertoire. In the current study, we showed that oral Streptococcus strain SK95 and pneumococcal strain D39 both produce structurally identical capsular PS, and their genetic backgrounds influence the amount of capsule production and shielding from nonspecific killing. SK95 is avirulent in a well-established in vivo mouse model. When acapsular pneumococcus was transformed with SK95 cps, the transformant became virulent and killed all mice. Thus, cps from oral Streptococcus strains can make acapsular pneumococcus virulent, and interspecies cps transfer should be considered a potential mechanism of serotype replacement. Our findings, along with publications from the US Centers for Disease Control and Prevention, highlight potential limitations of the 2013 World Health Organization criterion for studying pneumococcal serotypes carried without isolating bacteria. We show that an oral streptococcal strain, SK95, and a pneumococcal strain, D39, both produce chemically identical capsular PS. We also show that transferring SK95 cps into noncapsulated, avirulent pneumococcus gave it the capacity for virulence in a mouse model.

Streptococcus pneumoniae Acquisition and Carriage in Vaccine Naïve Indian Children with HIV and their Parents: A Longitudinal Household Study.

OBJECTIVES: To investigate the difference in pneumococcal carriage, acquisition, antibiotic resistance profiles and serotype distribution, in human immunodeficiency virus (HIV) affected and unaffected families. METHODS: A prospective cohort study was conducted in children with and without HIV in West Bengal from March 2012 through August 2014, prior to 13-valent pneumococcal conjugate vaccine (PCV-13) immunization. One thousand four hundred forty one nasopharyngeal swabs were collected and cultured at five-time points from children and their parents for pneumococcal culture, and serotyping by Quellung method. RESULTS: One hundred twenty five HIV infected children and their parents, and 47 HIV uninfected children and their parents participated. Two hundred forty pneumococcal isolates were found. In children under 6 y, the point prevalence of colonization was 31% in children living with HIV (CLH) and 32% in HIV uninfected children (HUC), p = 0.6. The most common vaccine type (VT) serotypes were 6A, 6B and 19A. All isolates from parents and 71% from children in the HIV uninfected cohort were PCV-13 representative, compared to 33% of isolates from CLH and their parents. Acquisition rate in children was 1.77 times that of parents (OR = 1.77, 95%CI: 1.18-2.65). The HIV status of child or parent did not affect acquisition. Isolates from CLH were more frequently resistant to multiple antibiotics (p = 0.02). CONCLUSIONS: While the rate of pneumococcal carriage and acquisition did not differ between CLH and HUC, HIV affected families had exposure to a wider range of serotypes including non-vaccine type serotypes and antibiotic resistant serotypes, than HIV unaffected families.

Dissemination and characterisation of Escherichia coli producing extended-spectrum ß-lactamases, AmpC ß-lactamases and metallo-ß-lactamases from livestock and poultry in Northeast India: A molecular surveillance approach.

OBJECTIVES: The aim of this study was to identify and characterise probable extended-spectrum ß-lactamase (ESBL)-, AmpC lactamase- and/or metallo-ß-lactamase (MBL)-producing Escherichia coli variants circulating in the livestock and poultry environment to establish their epidemiological significance, genetic diversity, antimicrobial resistance (AMR) trends and virulence. METHODS: The culture method and E. coli-specific multiplex PCR identified 78 E. coli strains from faecal samples of healthy livestock and poultry. The antibiogram was determined by the disk diffusion and minimum inhibitory concentration (MIC) methods. Antimicrobial-resistant E. coli isolates were screened for the presence of ESBL, AmpC and MBL genes. Isolates were further characterised by plasmid replicon typing, integron assay and virulence gene analysis. Genetic diversity was assessed by random amplification of polymorphic DNA (RAPD) analysis and multilocus sequence typing (MLST). RESULTS: ESBL (CTX-M group 1, CTX-M group 4, TEM), AmpC (EBC, FOX, CMY, DHA) and MBL (IMP, SIM) resistance determinants were identified in 75%, 19% and 6% of isolates, respectively. Nine plasmid replicon types were distributed among resistant E. coli strains, with the most common plasmid replicon types being L/M and Y. Integrons were detected in 19% of E. coli isolates. RAPD analysis categorised the E. coli isolates into three clusters. MLST revealed seven different sequence types (STs), with ST10 being the most common. CONCLUSIONS: This study demonstrated a high prevalence of animals carrying potential ESBL- and AmpC-producing E. coli and emphasises the need for rigorous surveillance in the animal sector to identify critical control points conducive to prevent the rapid dissemination of AMR.

Molecular detection and typing of methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci isolated from cattle, animal handlers, and their environment from Karnataka, Southern Province of India.

BACKGROUND AND AIM: Methicillin-resistant staphylococci are among the emerging pathogens which have become a threat to both human and animal health. The present investigation intended to examine the occurrence and the molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MRCoNS) recovered from cattle, its handlers, and their environment. MATERIALS AND METHODS: A total of 666 specimens were subjected to culture method and genus-specific polymerase chain reaction (PCR) for the identification of Staphylococcus. Methicillin resistance was substantiated by PCR identification of mecA and mecC resistance determinants. Species-specific identification of mecA positive isolates was conducted by multiplex PCR. The unidentified species were deciphered by 16S rRNA gene sequencing approach. The mecA positive isolates were further characterized by staphylococcal cassette chromosome mec (SCCmec) typing and multilocus sequence typing (MLST). RESULTS: Duplex PCR identified 728 Staphylococcus isolates, of which 66 (9%) were positive for mecA gene. MRSA constituted 24% of the total mecA positive isolates. Among MRCoNS, Staphylococcus epidermidis (42%), and Staphylococcus haemolyticus (11%) were the most common species identified. Overall, 47% of the mecA positive isolates belonged to SCCmec type V. MLST analysis showed eight different sequence types (STs) among MRSA isolates of which five were novel STs. Among methicillin-resistant S. epidermidis, 19 different STs were found, of which nine novel STs were detected. CONCLUSION: The increase in the prevalence of mecA positive staphylococci, especially MRCoNS in cattle is a great concern in view of their transmission potential. Hence, continuous monitoring and molecular characterization of methicillin-resistant staphylococci should be elucidated in human and animal sectors so as to prevent the spread of these resistant pathogens.

Draft Genome Sequence of Multidrug-Resistant Escherichia coli NIVEDI-P44, Isolated from a Chicken Fecal Sample in Northeast India.

We report here the draft genome sequence of a multidrug-resistant Escherichia coli strain (NIVEDI-P44) isolated from a chicken fecal sample. The estimated genome size is 4.76 Mb, with a G+C content of 50.65%. The genome harbors multiple antibiotic resistance genes, blaDHA-1, mph(A), strA, strB, dfrA14, sul-2, tet(A), and qnrS1.

Impact of Hajj on the S. pneumoniae carriage among Indian pilgrims during 2016- a longitudinal molecular surveillance study.

BACKGROUND: The population flow dynamics of Hajj increases the probability of pneumococcal acquisition and amplification among Hajis. This multi-site longitudinal molecular surveillance study was designed to assess the impact and potential variations of pneumococcal carriage in a single cohort of pre and post-Hajj pilgrims from India. METHOD: A total of 3228 pre and post-Hajj, nasopharyngeal and oropharyngeal swabs were collected from 807 pilgrims with an interval of 40 ±â€¯5 days. The carriage was detected by culture and qmPCR. Quellung test, mPCR-FAF, PCRseqTyping, and MLST was used for typing. Antibiogram was performed by MIC method. RESULTS: An increased incidence of pneumococcal carriage was detected in post Hajj cohort by qmPCR (19% vs 21.8%) (p-value = 0.0487) and culture (6.5% vs 8.2%) (p-value = 0.0645). Fragment analysis could identify multiple serotype carriage in 76 pilgrims. Increase in drug resistance was also observed in post-hajj cohort for Tetracycline (29% vs 51%), Erythromycin (26% vs 46%) and Levofloxacin (6% vs 17%). Multidrug resistant strains in post Hajj group was 32% compared to 11% in pre Hajj group (p-value = 0.0002). CONCLUSION: Our results confirm high acquisition rate of multidrug-resistant S. pneumoniae in Hajj pilgrims and highlight its potential spread to home countries upon their return. Surveillance studies are needed to evaluate modifiable factors associated with carriage.

Nasopharyngeal Pneumococcal Colonization and Impact of a Single Dose of 13-Valent Pneumococcal Conjugate Vaccine in Indian Children With HIV and Their Unvaccinated Parents.

BACKGROUND: Human immunodeficiency virus (HIV) infection increases risk of invasive disease from Streptococcus pneumoniae. Pneumococcal conjugate vaccines (PCV) prevent invasive disease and acquisition of vaccine type (VT) pneumococcus in the nasopharynx. OBJECTIVE: To look at the safety and impact of one dose of PCV13 on acquisition of VT pneumococcal carriage in Indian children with HIV. METHOD: We conducted a cohort study in families of HIV-infected children (CLH) and families of HIV-uninfected children (HUC) in West Bengal. All children received one dose of PCV13. Nasopharyngeal swabs were collected from children and parents at baseline and 2 months after vaccination. RESULT: One hundred and fifteen CLH and 47 HUC received one dose of PCV13. Fifty-eight percent of CLH were on antiretroviral therapy (ART), and the median nadir CD4 count was 287. There were no significant adverse events in either group. HUC had more VT colonization than CLH-55% versus 23% of all pneumococcal isolates. HIV infection doubled the risk of nonvaccine serotype colonization (P = 0.03). There was no difference in acquisition of VT isolates in CLH (4.4%) and HUC (4.5%) post-PCV13; however, older CLH (>5 years) had decreased clearance of VT strains. ART made no difference in pneumococcal colonization at baseline or after PCV13; however, CLH with higher nadir CD4 counts before starting ART were less likely to have VT colonization post-PCV13 (prevalence ratio, 0.2; 95% confidence interval: 0.1-0.5). CONCLUSION: While there was no difference in acquisition of VT nasopharyngeal carriage of pneumococcus in CLH and HUC after one dose of PCV13, earlier access to ART may impact response to PCV13 in CLH.