Multiplexed real-time PCR for the detection and differentiation of Klebsiella pneumoniae O-antigen serotypes.

Klebsiella pneumoniae has emerged as a global health threat due to its role in the spread of antimicrobial resistance and because it is a frequent cause of hospital-acquired infections and neonatal sepsis. Capsular and lipopolysaccharide (LPS) O-antigen polysaccharide surface antigens are major immunogens that are useful for strain classification and are candidates for vaccine development. We have developed real-time PCR reagents for molecular serotyping, subtyping, and quantitation of the most prevalent LPS O-antigen types (i.e., O1, O2, O3, and O5) of Klebsiella pneumoniae. We describe two applications for this O-typing assay: for screening culture isolates and for direct typing of Klebsiella pneumoniae present in stool samples. We find 100% concordance between the results of the O-typing assay and whole-genome sequencing of 81 culture isolates, and >90% agreement in O-typing performed directly on specimens of human stool, with disagreement arising primarily from a lack of sensitivity of the culture-based comparator method. Additionally, we find evidence for mixed O-type populations at varying levels of abundance in direct tests of stool from a hospitalized patient population. Taken together, these results demonstrate that this novel O-typing assay can be a useful tool for K. pneumoniae epidemiologic and vaccine studies.IMPORTANCEKlebsiella pneumoniae is an important opportunistic pathogen. The gastrointestinal (GI) tract is the primary reservoir of K. pneumoniae in humans, and GI carriage is believed to be a prerequisite for invasive infection. Knowledge about the dynamics and duration of GI carriage has been hampered by the lack of tools suitable for detection and strain discrimination. Real-time PCR is particularly suited to the higher-throughput workflows used in population-based studies, which are needed to improve our understanding of carriage dynamics and the factors influencing K. pneumoniae colonization.

Association between chlorine-treated drinking water, the gut microbiome, and enteric pathogen burden in young children in Haiti: An observational study.

OBJECTIVE: The effects of sanitation and hygiene interventions on the gut microbiome and enteric pathogen burden are not well understood. We measured the association between free chlorine residue (FCR) levels in drinking water, microbiome composition, and stool enteric pathogens in infants and young children in Haiti. METHODS: FCR levels were measured in household drinking water and enteric pathogen burden was evaluated using multiplex RT-PCR of stool among 131 children from one month to five years of age living in Mirebalais, Haiti. Microbiome profiling was performed using metagenomic sequencing. RESULTS: Most individuals lived in households with undetectable FCR measured in the drinking water (112/131, 86%). Detection of enteric pathogen DNA in stool was common and did not correlate with household water FCR. The infant microbiome in households with detectable FCR demonstrated reduced richness (fewer total number of species, P = 0.04 Kruskall-Wallis test) and less diversity by Inverse Simpson measures (P = 0.05) than households with undetectable FCR. Infants in households with a detectable FCR were more likely to have abundant Bifidobacterium. Using in vitro susceptibility testing, we found that some Bifidobacterium species were resistant to chlorine. CONCLUSIONS: FCR in household drinking water did not correlate with enteric pathogen burden in our study.

Vibrio cholerae O1 experiences mild bottlenecks through the gastrointestinal tract in some but not all cholera patients.

Vibrio cholerae O1 causes the diarrheal disease cholera, and the small intestine is the site of active infection. During cholera, cholera toxin is secreted from V. cholerae and induces a massive fluid influx into the small intestine, which causes vomiting and diarrhea. Typically, V. cholerae genomes are sequenced from bacteria passed in stool, but rarely from vomit, a fluid that may more closely represents the site of active infection. We hypothesized that V. cholerae O1 population bottlenecks along the gastrointestinal tract would result in reduced genetic variation in stool compared to vomit. To test this, we sequenced V. cholerae genomes from 10 cholera patients with paired vomit and stool samples. Genetic diversity was low in both vomit and stool, consistent with a single infecting population rather than coinfection with divergent V. cholerae O1 lineages. The amount of single-nucleotide variation decreased from vomit to stool in four patients, increased in two, and remained unchanged in four. The variation in gene presence/absence decreased between vomit and stool in eight patients and increased in two. Pangenome analysis of assembled short-read sequencing demonstrated that the toxin-coregulated pilus operon more frequently contained deletions in genomes from vomit compared to stool. However, these deletions were not detected by PCR or long-read sequencing, indicating that interpreting gene presence or absence patterns from short-read data alone may be incomplete. Overall, we found that V. cholerae O1 isolated from stool is genetically similar to V. cholerae recovered from the upper intestinal tract. IMPORTANCE: Vibrio cholerae O1, the bacterium that causes cholera, is ingested in contaminated food or water and then colonizes the upper small intestine and is excreted in stool. Shed V. cholerae genomes from stool are usually studied, but V. cholerae isolated from vomit may be more representative of where V. cholerae colonizes in the upper intestinal epithelium. V. cholerae may experience bottlenecks, or large reductions in bacterial population sizes and genetic diversity, as it passes through the gut. Passage through the gut may select for distinct V. cholerae mutants that are adapted for survival and gut colonization. We did not find strong evidence for such adaptive mutations, and instead observed that passage through the gut results in modest reductions in V. cholerae genetic diversity, and only in some patients. These results fill a gap in our understanding of the V. cholerae life cycle, transmission, and evolution.

Antibody responses in Klebsiella pneumoniae bloodstream infection: a cohort study.

Background: Klebsiella pneumonia (Kpn) is the fourth leading cause of infection-related deaths globally, yet little is known about human antibody responses to invasive Kpn. In this study, we sought to determine whether the O-specific polysaccharide (OPS) antigen, a vaccine candidate, is immunogenic in humans with Kpn bloodstream infection (BSI). We also sought to define the cross-reactivity of human antibody responses among structurally related Kpn OPS subtypes and to assess the impact of capsule production on OPS-targeted antibody binding and function. Methods: We measured plasma antibody responses to OPS (and MrkA, a fimbrial protein) in a cohort of patients with Kpn BSI and compared these with controls, including a cohort of healthy individuals and a cohort of individuals with Enterococcus BSI. We performed flow cytometry to measure the impact of Kpn capsule production on whole cell antibody binding and complement deposition, utilizing patient isolates with variable levels of capsule production and isogenic capsule-deficient strains derived from these isolates. Findings: We enrolled 69 patients with Kpn BSI. Common OPS serotypes accounted for 57/69 (83%) of infections. OPS was highly immunogenic in patients with Kpn BSI, and peak OPS-IgG antibody responses in patients were 10 to 30-fold higher than antibody levels detected in healthy controls, depending on the serotype. There was significant cross-reactivity among structurally similar OPS subtypes, including the O1v1/O1v2, O2v1/O2v2 and O3/O3b subtypes. Physiological amounts of capsule produced by both hyperencapsulated and non-hyperencapsulated Kpn significantly inhibited OPS-targeted antibody binding and function. Interpretation: OPS was highly immunogenic in patients with Kpn BSI, supporting its potential as a candidate vaccine antigen. The strong cross-reactivity observed between similar OPS subtypes in humans with Kpn BSI suggests that it may not be necessary to include all subtypes in an OPS-based vaccine. However, these observations are tempered by the fact that capsule production, even in non-highly encapsulated strains, has the potential to interfere with OPS antibody binding. This may limit the effectiveness of vaccines that exclusively target OPS. Funding: National Institute of Allergy and Infectious Diseases at the National Institutes of Health. Research in Context: Evidence before this study: Despite the potential of O-specific polysaccharide (OPS) as a vaccine antigen against Klebsiella pneumoniae (Kpn), the immunogenicity of OPS in humans remains largely unstudied, creating a significant knowledge gap with regard to vaccine development. A search of PubMed for publications up to March 18, 2024, using the terms " Klebsiella pneumoniae " and "O-specific polysaccharide" or "O-antigen" or "lipopolysaccharide" revealed no prior studies addressing OPS antibody responses in humans with Kpn bloodstream infections (BSI). One prior study 1 evaluated antibody response to a single lipopolysaccharide (which contains one subtype of OPS) in humans with invasive Kpn infection; however, in this study OPS typing of the infecting strains and target antigen were not described. Added value of this study: Our investigation into OPS immunogenicity in a human cohort marks a significant advance. Analyzing plasma antibody responses in 69 patients with Kpn BSI, we found OPS to be broadly immunogenic across all the types and subtypes examined, and there was significant cross-reactivity among structurally related OPS antigens. We also demonstrated that Kpn capsule production inhibit OPS antibody binding and the activation of complement on the bacterial surface, even in classical Kpn strains expressing lower levels of capsule.Implications of all the available evidence: While the immunogenicity and broad cross-reactivity of OPS in humans with Kpn BSI suggests it is a promising vaccine candidate, the obstruction of OPS antibody binding and engagement by physiologic levels of Kpn capsule underscores the potential limitations of an exclusively OPS-antigen based vaccine for Kpn. Our study provides insights for the strategic development of vaccines aimed at combating Kpn infections, an important antimicrobial resistant pathogen.

Cholera toxin and O-specific polysaccharide immune responses after oral cholera vaccination with Dukoral in different age groups of Bangladeshi participants.

Vaccination is important to prevent cholera. There are limited data comparing anti-O-specific polysaccharide (OSP) and anti-cholera toxin-specific immune responses following oral whole-cell with cholera toxin B-subunit (WC-rBS) vaccine (Dukoral, Valneva) administration in different age groups. An understanding of the differences is relevant because young children are less well protected by oral cholera vaccines than older children and adults. We compared responses in 50 adults and 49 children (ages 2 to <18) who were administered two doses of WC-rBS at a standard 14-day interval. All age groups had significant IgA and IgG plasma-blast responses to the OSP and cholera toxin B-subunit (CtxB) antigens that peaked 7 days after vaccination. However, in adults and older children (ages 5 to <18), antibody responses directed at the OSP antigen were largely IgA and IgG, with a minimal IgM response, while younger children (ages 2 to <5) mounted significant increases in IgM with minimal increases in IgA and IgG antibody responses 30 days after vaccination. In adults, anti-OSP and CtxB memory B-cell responses were detected after completion of the vaccination series, while children only mounted CtxB-specific IgG memory B-cell responses and no OSP-memory B-cell responses. In summary, children and adults living in a cholera endemic area mounted different responses to the WC-rBS vaccine, which may be a result of more prior exposure to Vibrio cholerae in older participants. The absence of class-switched antibody responses and memory B-cell responses to OSP may explain why protection wanes more rapidly after vaccination in young children compared to older vaccinees.IMPORTANCEVaccination is an important strategy to prevent cholera. Though immune responses targeting the OSP of V. cholerae are believed to mediate protection against cholera, there are limited data on anti-OSP responses after vaccination in different age groups, which is important as young children are not well protected by current oral cholera vaccines. In this study, we found that adults mounted memory B-cell responses to OSP, which were not seen in children. Adults and older children mounted class-switched (IgG and IgA) serum antibody responses to OSP, which were not seen in young children who had only IgM responses to OSP. The lack of class-switched antibody responses and memory B-cell responses to OSP in younger participants may be due to lack of prior exposure to V. cholerae and could explain why protection wanes more rapidly after vaccination in young children.

Diversity of Vibrio cholerae O1 through the human gastrointestinal tract during cholera.

Vibrio cholerae O1 causes the diarrheal disease cholera, and the small intestine is the site of active infection. During cholera, cholera toxin is secreted from V. cholerae and induces a massive fluid influx into the small intestine, which causes vomiting and diarrhea. Typically, V. cholerae genomes are sequenced from bacteria passed in stool, but rarely from vomit, a fluid that may more closely represents the site of active infection. We hypothesized that the V. cholerae O1 population bottlenecks along the gastrointestinal tract would result in reduced genetic variation in stool compared to vomit. To test this, we sequenced V. cholerae genomes from ten cholera patients with paired vomit and stool samples. Genetic diversity was low in both vomit and stool, consistent with a single infecting population rather than co-infection with divergent V. cholerae O1 lineages. The number of single nucleotide variants decreased between vomit and stool in four patients, increased in two, and remained unchanged in four. The number of genes encoded in the V. cholerae genome decreased between vomit and stool in eight patients and increased in two. Pangenome analysis of assembled short-read sequencing demonstrated that the toxin-coregulated pilus operon more frequently contained deletions in genomes from vomit compared to stool. However, these deletions were not detected by PCR or long-read sequencing, indicating that interpreting gene presence or absence patterns from short-read data alone may be incomplete. Overall, we found that V. cholerae O1 isolated from stool is genetically similar to V. cholerae recovered from the upper intestinal tract.

Appearance of tolerance-induction and non-inflammatory SARS-CoV-2 spike-specific IgG4 antibodies after COVID-19 booster vaccinations.

Background: Understanding the characteristics of the humoral immune responses following COVID-19 vaccinations is crucial for refining vaccination strategies and predicting immune responses to emerging SARS-CoV-2 variants. Methods: A longitudinal analysis of SARS-CoV-2 spike receptor binding domain (RBD) specific IgG antibody responses, encompassing IgG subclasses IgG1, IgG2, IgG3, and IgG4 was performed. Participants received four mRNA vaccine doses (group 1; n=10) or two ChAdOx1 nCoV-19 and two mRNA booster doses (group 2; n=19) in Bangladesh over two years. Results: Findings demonstrate robust IgG responses after primary Covishield or mRNA doses; declining to baseline within six months. First mRNA booster restored and surpassed primary IgG responses but waned after six months. Surprisingly, a second mRNA booster did not increase IgG levels further. Comprehensive IgG subclass analysis showed primary Covishield/mRNA vaccination generated predominantly IgG1 responses with limited IgG2/IgG3, Remarkably, IgG4 responses exhibited a distinct pattern. IgG4 remained undetectable initially but increased extensively six months after the second mRNA dose, eventually replacing IgG1 after the 3rd/4th mRNA doses. Conversely, initial Covishield recipients lack IgG4, surged post-second mRNA booster. Notably, mRNA-vaccinated individuals displayed earlier, robust IgG4 levels post first mRNA booster versus Covishield counterparts. IgG1 to IgG4 ratios decreased with increasing doses, most pronounced with four mRNA doses. This study highlights IgG response kinetics, influenced by vaccine type and doses, impacting immunological tolerance and IgG4 induction, shaping future vaccination strategies. Conclusions: This study highlights the dynamics of IgG responses dependent on vaccine type and number of doses, leading to immunological tolerance and IgG4 induction, and shaping future vaccination strategies.