Immunization with a whole cell vaccine reduces pneumococcal nasopharyngeal density and shedding, and middle ear infection in mice.

Pneumococcal Conjugate Vaccines (PCVs) have substantially reduced the burden of disease caused by Streptococcus pneumoniae (the pneumococcus). However, protection is limited to vaccine serotypes, and when administered to children who are colonized with pneumococci at the time of vaccination, immune responses to the vaccine are blunted. Here, we investigate the potential of a killed whole cell pneumococcal vaccine (WCV) to reduce existing pneumococcal carriage and mucosal disease when given therapeutically to infant mice colonized with pneumococci. We show that a single dose of WCV reduced pneumococcal carriage density in an antibody-dependent manner. Therapeutic vaccination induced robust immune responses to pneumococcal surface antigens CbpA, PspA (family 1) and PiaA. In a co-infection model of otitis media, a single dose of WCV reduced pneumococcal middle ear infection. Lastly, in a two-dose model, therapeutic administration of WCV reduced nasal shedding of pneumococci. Taken together, our data demonstrate that WCV administered in colonized mice reduced pneumococcal density in the nasopharynx and the middle ear, and decreased shedding. WCVs would be beneficial in low and middle-income settings where pneumococcal carriage in children is high.

Spatial-temporal and phylogenetic analyses of epidemiologic data to help understand the modes of transmission of endemic typhoid fever in Samoa.

Salmonella enterica serovar Typhi (S. Typhi) is either widely distributed or proximally transmitted via fecally-contaminated food or water to cause typhoid fever. In Samoa, where endemic typhoid fever has persisted over decades despite water quality and sanitation improvements, the local patterns of S. Typhi circulation remain unclear. From April 2018-June 2020, epidemiologic data and GPS coordinates were collected during household investigations of 260 acute cases of typhoid fever, and 27 asymptomatic shedders of S. Typhi were detected among household contacts. Spatial and temporal distributions of cases were examined using Average Nearest Neighbor and space-time hotspot analyses. In rural regions, infections occurred in sporadic, focal clusters contrasting with persistent, less clustered cases in the Apia Urban Area. Restrictions to population movement during nationwide lockdowns in 2019-2020 were associated with marked reductions of cases. Phylogenetic analyses of isolates with whole genome sequences (n = 186) revealed one dominant genotype 3.5.4 (n = 181/186) that contains three Samoa-exclusive sub-lineages: 3.5.4.1, 3.5.4.2, and 3.5.4.3. Variables of patient sex, age, and geographic region were examined by phylogenetic groupings, and significant differences (p<0.05) associated genetically-similar isolates in urban areas with working ages (20-49 year olds), and in rural areas with age groups typically at home (<5, 50+). Isolates from asymptomatic shedders were among all three sub-lineages. Whole genome sequencing provided evidence of bacterial genetic similarity, which corroborated 10/12 putative epidemiologic linkages among cases and asymptomatic shedders, as well as 3/3 repeat positives (presumed relapses), with a median of one single nucleotide polymorphism difference. These findings highlight various patterns of typhoid transmission in Samoa that differ between urban and rural regions as well as genomic subtypes. Asymptomatic shedders, detectable only through household investigations, are likely an important reservoir and mobile agent of infection. This study advances a "Samoan S. Typhi framework" that supports current and future typhoid surveillance and control efforts in Samoa.

Persistence of Rare Salmonella Typhi Genotypes Susceptible to First-Line Antibiotics in the Remote Islands of Samoa.

For decades, the remote island nation of Samoa (population ~200,000) has faced endemic typhoid fever despite improvements in water quality, sanitation, and economic development. We recently described the epidemiology of typhoid fever in Samoa from 2008 to 2019 by person, place, and time; however, the local Salmonella enterica serovar Typhi (S. Typhi) population structure, evolutionary origins, and genomic features remained unknown. Herein, we report whole genome sequence analyses of 306 S. Typhi isolates from Samoa collected between 1983 and 2020. Phylogenetics revealed a dominant population of rare genotypes 3.5.4 and 3.5.3, together comprising 292/306 (95.4%) of Samoan versus 2/4934 (0.04%) global S. Typhi isolates. Three distinct 3.5.4 genomic sublineages were identified, and their defining polymorphisms were determined. These dominant Samoan genotypes, which likely emerged in the 1970s, share ancestry with other 3.5 clade isolates from South America, Southeast Asia, and Oceania. Additionally, a 106-kb pHCM2 phenotypically cryptic plasmid, detected in a 1992 Samoan S. Typhi isolate, was identified in 106/306 (34.6%) of Samoan isolates; this is more than double the observed proportion of pHCM2-containing isolates in the global collection. In stark contrast with global S. Typhi trends, resistance-conferring polymorphisms were detected in only 15/306 (4.9%) of Samoan S. Typhi, indicating overwhelming susceptibility to antibiotics that are no longer effective in most of South and Southeast Asia. This country-level genomic framework can help local health authorities in their ongoing typhoid surveillance and control efforts, as well as fill a critical knowledge gap in S. Typhi genomic data from Oceania. IMPORTANCE In this study, we used whole genome sequencing and comparative genomics analyses to characterize the population structure, evolutionary origins, and genomic features of S. Typhi associated with decades of endemic typhoid fever in Samoa. Our analyses of Samoan isolates from 1983 to 2020 identified a rare S. Typhi population in Samoa that likely emerged around the early 1970s and evolved into sublineages that are presently dominant. The dominance of these endemic genotypes in Samoa is not readily explained by genomic content or widespread acquisition of antimicrobial resistance. These data establish the necessary framework for future genomic surveillance of S. Typhi in Samoa for public health benefit.

Point-of-Care Ultrasound by Nonexpert Operators Demonstrates High Sensitivity and Specificity in Detecting Gallstones: Data from the Samoa Typhoid Fever Control Program.

Approximately 90% of chronic typhoid carriers with persistent Salmonella enterica serovar Typhi (S. Typhi) gallbladder infection have gallstones. In Samoa, where typhoid fever has been endemic for many decades, risk factors predisposing to the development of gallstones are increasing among adults. The Samoa Typhoid Fever Control Program dispatches a "Typhoid Epidemiologic SWAT Team" to perform a household investigation of every blood culture-confirmed case of acute typhoid fever. Investigations include screening household contacts to detect chronic carriers. Following limited training, two nonexpert ultrasound operators performed point-of-care ultrasound (POCUS) on 120 Samoan adults from August to September 2019 to explore the feasibility of POCUS to detect individuals with gallstones during household investigations and community screenings. POCUS scans from 120 Samoan adults in three cohorts (28 food handlers, two typhoid cases and their 18 household contacts, and 72 attendees at an ambulatory clinic) were reviewed by a board-certified radiologist who deemed 96/120 scans (80%) to be interpretable. Compared with the radiologist (gold standard), the nonexpert operators successfully detected 6/7 Samoans with gallstones (85.7% sensitivity) and correctly identified 85/89 without gallstones (95.5% specificity). The proportion (24/120) of uninterpretable scans from this pilot that used minimally trained clinicians (who are neither radiologists nor ultrasound technicians) indicates the need for additional training of POCUS operators. Nevertheless, this pilot feasibility study engenders optimism that in the Samoan setting nonexperts can be trained to use POCUS to diagnose cholelithiasis, thereby helping (along with stool cultures and Vi serology) to identify possible chronic S. Typhi carriers.

Re-evaluation of a Neonatal Mouse Model of Infection With Enterotoxigenic Escherichia coli.

Enterotoxigenic E. coli (ETEC) is a common cause of diarrhea in children in low- and middle-income countries, and in travelers to these countries. ETEC is also an important cause of morbidity and premature mortality in piglets, calves, goat kids and lambs. The major virulence determinants of ETEC are enterotoxins and colonization factors, which enable the pathogen to colonize the small intestine and deliver enterotoxins, such as the heat-stable enterotoxins, STp and STh, to epithelial cells. Because most ETEC strains are host-specific, there are few convenient animal models to investigate the pathogenesis of ETEC infections or to evaluate specific anti-ETEC interventions, such as drugs and vaccines. An exception is ETEC strains bearing F41 pili, which mediate intestinal colonization of various young animals, including neonatal mice, to cause disease and in some cases death. In this study, we used the archetypal F41-producing bovine ETEC strain, B41 (O101:NM; K99, F41, STp) to validate and further explore the contribution of F41 and STp to bacterial virulence. By using targeted gene deletion and trans-complementation studies, augmented by whole genome sequencing, and in vitro and animal studies of virulence, we established that F41 mediates colonization of the mouse intestine and is essential for bacterial virulence. In addition, we showed for the first time that STp is as important as F41 for virulence. Together, these findings validate the use of neonatal mice to study the pathogenesis of F41-bearing ETEC and to investigate possible specific anti-ETEC interventions including vaccines that target heat-stable enterotoxins.

Tenacious Endemic Typhoid Fever in Samoa.

BACKGROUND: Typhoid fever has been endemic on the island nation of Samoa (2016 population, 195 979) since the 1960s and has persisted through 2019, despite economic development and improvements in water supply and sanitation. METHODS: Salmonella enterica serovar Typhi isolates from the 2 hospitals with blood culture capability and matched patient demographic and clinical data from January 2008 through December 2019 were analyzed. Denominators to calculate incidence by island, region, and district came from 2011 and 2016 censuses and from 2017-2019 projections from Samoa's Bureau of Statistics. Data were analyzed to describe typhoid case burden and incidence from 2008 to 2019 by time, place, and person. RESULTS: In sum, 53-193 blood culture-confirmed typhoid cases occurred annually from 2008 to 2019, without apparent seasonality. Typhoid incidence was low among children age < 48 months (17.6-27.8/105), rose progressively in ages 5-9 years (54.0/105), 10-19 years (60.7-63.4/105), and 20-34 years (61.0-79.3/105), and then tapered off; 93.6% of cases occurred among Samoans < 50 years of age. Most typhoid cases and the highest incidence occurred in Northwest Upolu, but Apia Urban Area (served by treated water supplies) also exhibited moderate incidence. The proportion of cases from short-cycle versus long-cycle transmission is unknown. Samoan S. Typhi are pansusceptible to traditional first-line antibiotics. Nevertheless, enhanced surveillance in 2019 detected 4 (2.9%) deaths among 140 cases. CONCLUSIONS: Typhoid has been endemic in Samoa in the period 2008-2019. Interventions, including mass vaccination with a Vi-conjugate vaccine coadministered with measles vaccine are planned.

Neonatal BCG Vaccination Reduces Interferon-γ Responsiveness to Heterologous Pathogens in Infants From a Randomized Controlled Trial.

BACKGROUND: BCG vaccination has beneficial nonspecific (heterologous) effects that protect against nonmycobacterial infections. We have previously reported that BCG vaccination at birth alters in vitro cytokine responses to heterologous stimulants in the neonatal period. This study investigated heterologous responses in 167 infants in the same trial 7 months after randomization. METHODS: A whole-blood assay was used to interrogate in vitro cytokine responses to heterologous stimulants (killed pathogens) and Toll-like receptor (TLR) ligands. RESULTS: Compared to BCG-naive infants, BCG-vaccinated infants had increased production of interferon gamma (IFN-γ) and monokine induced by gamma interferon (MIG) (CXCL9) in response to mycobacterial stimulation and decreased production of IFN-γ in response to heterologous stimulation and TLR ligands. Reduced IFN-γ responses were attributable to a decrease in the proportion of infants who mounted a detectable IFN-γ response. BCG-vaccinated infants also had increased production of MIG (CXCL9) and interleukin-8 (IL-8), and decreased production of IL-10, macrophage inflammatory protein-1α (MIP-1α), and MIP-1ß, the pattern of which varied by stimulant. IL-1Ra responses following TLR1/2 (Pam3CYSK4) stimulation were increased in BCG-vaccinated infants. Both sex and maternal BCG vaccination status influenced the effect of neonatal BCG vaccination. CONCLUSIONS: BCG vaccination leads to changes in IFN-γ responsiveness to heterologous stimulation. BCG-induced changes in other cytokine responses to heterologous stimulation vary by pathogen.

Diarrhoeal disease and subsequent risk of death in infants and children residing in low-income and middle-income countries: analysis of the GEMS case-control study and 12-month GEMS-1A follow-on study.

BACKGROUND: The Global Enteric Multicenter Study (GEMS) was a 3-year case-control study that measured the burden, aetiology, and consequences of moderate-to-severe diarrhoea (MSD) in children aged 0-59 months. GEMS-1A, a 12-month follow-on study, comprised two parallel case-control studies, one assessing MSD and the other less-severe diarrhoea (LSD). In this report, we analyse the risk of death with each diarrhoea type and the specific pathogens associated with fatal outcomes. METHODS: GEMS was a prospective, age-stratified, matched case-control study done at seven sites in Africa and Asia. Children aged 0-59 months with MSD seeking care at sentinel health centres were recruited along with one to three randomly selected matched community control children without diarrhoea. In the 12-month GEMS-1A follow-on study, children with LSD and matched controls, in addition to children with MSD and matched controls, were recruited at six of the seven sites; only cases of MSD and controls were enrolled at the seventh site. We compared risk of death during the period between enrolment and one follow-up household visit done about 60 days later (range 50-90 days) in children with MSD and LSD and in their respective controls. Approximately 50 pathogens were detected using, as appropriate, classic bacteriology, immunoassays, gel-based PCR and reverse transcriptase PCR, and quantitative real-time PCR (qPCR). Specimens from a subset of GEMS cases and controls were also tested by a TaqMan Array Card that compartmentalised probe-based qPCR for 32 enteropathogens. FINDINGS: 223 (2·0%) of 11 108 children with MSD and 43 (0·3%) of 16 369 matched controls died between study enrolment and the follow-up visit at about 60 days (hazard ratio [HR] 8·16, 95% CI 5·69-11·68, p<0·0001). 12 (0·4%) of 2962 children with LSD and seven (0·2%) of 4074 matched controls died during the follow-up period (HR 2·78, 95% CI 0·95-8·11, p=0·061). Risk of death was lower in children with dysenteric MSD than in children with non-dysenteric MSD (HR 0·20, 95% CI 0·05-0·87, p=0·032), and lower in children with LSD than in those with non-dysenteric MSD (HR 0·29, 0·14-0·59, p=0·0006). In children younger than 24 months with MSD, infection with typical enteropathogenic Escherichia coli, enterotoxigenic E coli encoding heat-stable toxin, enteroaggregative E coli, Shigella spp (non-dysentery cases), Aeromonas spp, Cryptosporidium spp, and Entamoeba histolytica increased risk of death. Of 61 deaths in children aged 12-59 months with non-dysenteric MSD, 31 occurred among 942 children qPCR-positive for Shigella spp and 30 deaths occurred in 1384 qPCR-negative children (HR 2·2, 95% CI 1·2-3·9, p=0·0090), showing that Shigella was strongly associated with increased risk of death. INTERPRETATION: Risk of death is increased following MSD and, to a lesser extent, LSD. Considering there are approximately three times more cases of LSD than MSD in the population, more deaths are expected among children with LSD than in those with MSD. Because the major attributable LSD-associated and MSD-associated pathogens are the same, implementing vaccines and rapid diagnosis and treatment interventions against these major pathogens are rational investments. FUNDING: Bill & Melinda Gates Foundation.

Effect of a pneumococcal whole cell vaccine on influenza A-induced pneumococcal otitis media in infant mice.

The pneumococcus remains a common cause of otitis media (OM) despite the widespread introduction of pneumococcal conjugate vaccines. In mice, a pneumococcal whole cell vaccine (WCV) induces serotype-independent protection against pneumococcal colonisation and invasive disease via TH17- and antibody-mediated immunity, respectively. We investigated the effect of WCV on influenza A-induced pneumococcal OM in an infant mouse model. C57BL/6 mice were immunised subcutaneously with a single dose of WCV or adjuvant at 6 days of age, infected with pneumococci (EF3030 [serotype 19F] or PMP1106 [16F]) at 12 days of age, and given influenza A virus (A/Udorn/72/307 [H3N2], IAV) at 18 days of age to induce pneumococcal OM. Pneumococcal density in middle ear and nasopharyngeal tissues was determined 6 and 12 days post-virus. Experiments were repeated in antibody (B6.µMT-/-)- and CD4+ T-cell-deficient mice to investigate the immune responses involved. A single dose of WCV did not prevent the development of pneumococcal OM, nor accelerate pneumococcal clearance compared with mice receiving adjuvant alone. However, WCV reduced the density of EF3030 in the middle ear at 6 days post-viral infection (p = 0.022), and the density of both isolates in the nasopharynx at 12 days post-viral infection (EF3030, p = 0.035; PMP1106, p = 0.011), compared with adjuvant alone. The reduction in density in the middle ear required antibodies and CD4+ T cells: WCV did not reduce EF3030 middle ear density in B6.µMT-/- mice (p = 0.35) nor in wild-type mice given anti-CD4 monoclonal antibody before and after IAV inoculation (p = 0.91); and WCV-immunised CD4+ T cell-deficient GK1.5 mice had higher levels of EF3030 in the middle ear than their adjuvant-immunised counterparts (p = 0.044). A single subcutaneous dose of WCV reduced pneumococcal density in the middle ears of co-infected mice in one of two strains tested, but did not prevent OM from occurring in this animal model.

Mycobacteria-Specific Mono- and Polyfunctional CD4+ T Cell Profiles in Children With Latent and Active Tuberculosis: A Prospective Proof-of-Concept Study.

Background: Current immune-based TB tests, including the tuberculin skin test (TST) and interferon-gamma release assays (IGRA), have significant limitations, including the inability to distinguish between latent TB infection (LTBI) and active TB. Few biomarkers with the potential to discriminate between these two infection states have been identified. Objective: To determine whether functional profiling of mycobacteria-specific T cells can distinguish between TB-infected and -uninfected children, and simultaneously discriminate between LTBI and active TB. Methods: One hundred and forty-nine children with suspected active TB or risk factors for LTBI were recruited at the Royal Children's Hospital Melbourne. Whole-blood stimulation assays, using ESAT-6, CFP-10, PPD, and heat-killed M. tuberculosis as stimulants, were done, followed by intracellular cytokine staining and flow cytometric analysis. Results: Eighty-two participants in the well-defined diagnostic categories 'uninfected individuals' (asymptomatic, TST 0 mm / IGRA-; n = 61), LTBI (asymptomatic, TST ≥10 mm / IGRA+, normal chest radiograph; n = 15), or active TB [microbiologically-confirmed (n = 3) or fulfilling stringent criteria (n = 3)] were included in the final analysis. The proportions of mycobacteria-specific single-positive TNF-α+ and double-positive IFN-γ+/TNF-α+ CD4+ T cells were significantly higher in participants with active TB than in those with LTBI and uninfected individuals. Additionally, the frequency of IL-17-expressing CD4+ T cells, predominately with single-positive IL-17+ and double-positive IL-2+/IL-17+ phenotypes, was higher in participants with active TB than in the other two groups. Conclusions: The frequencies and functional profiles of mycobacteria-specific CD4+ T cells differ significantly both between TB-infected and TB-uninfected children, and between LTBI and active TB. Although confirmation in further studies will be required, these findings indicate that functional profiling of mycobacteria-specific CD4+ T cells could potentially be exploited for novel immune-based TB assays that enable the distinction between infection states based on a blood sample alone.

The incidence, aetiology, and adverse clinical consequences of less severe diarrhoeal episodes among infants and children residing in low-income and middle-income countries: a 12-month case-control study as a follow-on to the Global Enteric Multicenter Study (GEMS).

BACKGROUND: Diarrheal diseases remain a leading cause of illness and death among children younger than 5 years in low-income and middle-income countries. The Global Enteric Multicenter Study (GEMS) has described the incidence, aetiology, and sequelae of medically attended moderate-to-severe diarrhoea (MSD) among children aged 0-59 months residing in censused populations in sub-Saharan Africa and south Asia, where most child deaths occur. To further characterise this disease burden and guide interventions, we extended this study to include children with episodes of less-severe diarrhoea (LSD) seeking care at health centres serving six GEMS sites. METHODS: We report a 1-year, multisite, age-stratified, matched case-control study following on to the GEMS study. Six sites (Bamako, Mali; Manhiça, Mozambique; Basse, The Gambia; Mirzapur, Bangladesh; Kolkata, India; and Bin Qasim Town, Karachi, Pakistan) participated in this study. Children aged 0-59 months at each site who sought care at a sentinel hospital or health centre during a 12-month period were screened for diarrhoea. New (onset after ≥7 diarrhoea-free days) and acute (onset within the previous 7 days) episodes of diarrhoea in children who had sunken eyes, whose skin lost turgor, who received intravenous hydration, who had dysentery, or who were hospitalised were eligible for inclusion as MSD. The remaining new and acute diarrhoea episodes among children who sought care at the same health centres were considered LSD. We aimed to enrol the first eight or nine eligible children with MSD and LSD at each site during each fortnight in three age strata: infants (aged 0-11 months), toddlers (aged 12-23 months), and young children (aged 24-59 months). For each included case of MSD or LSD, we enrolled one to three community control children without diarrhoea during the previous 7 days. From patients and controls we collected clinical and epidemiological data, anthropometric measurements, and faecal samples to identify enteropathogens at enrolment, and we performed a follow-up home visit about 60 days later to ascertain vital status, clinical outcome, and interval growth. Primary outcomes were to characterise, for MSD and LSD, the pathogen-specific attributable risk and population-based incidence values, and to assess the frequency of adverse clinical consequences associated with these two diarrhoeal syndromes. FINDINGS: From Oct 31, 2011, to Nov 14, 2012, we recruited 2368 children with MSD, 3174 with LSD, and one to three randomly selected community control children without diarrhoea matched to cases with MSD (n=3597) or LSD (n=4236). Weighted adjusted population attributable fractions showed that most attributable cases of MSD and LSD were due to rotavirus, Cryptosporidium spp, enterotoxigenic Escherichia coli encoding heat-stable toxin (with or without genes encoding heat-labile enterotoxin), and Shigella spp. The attributable incidence per 100 child-years for LSD versus MSD, by age stratum, for rotavirus was 22·3 versus 5·5 (0-11 months), 9·8 versus 2·9 (12-23 months), and 0·5 versus 0·2 (24-59 months); for Cryptosporidium spp was 3·6 versus 2·3 (0-11 months), 4·3 versus 0·6 (12-23 months), and 0·3 versus 0·1 (24-59 months); for enterotoxigenic E coli encoding heat-stable toxin was 4·2 versus 0·1 (0-11 months), 5·2 versus 0·0 (12-23 months), and 1·1 versus 0·2 (24-59 months); and for Shigella spp was 1·0 versus 1·3 (0-11 months), 3·1 versus 2·4 (12-23 months), and 0·8 versus 0·7 (24-59 months). Participants with both MSD and LSD had significantly more linear growth faltering than controls at follow-up. INTERPRETATION: Inclusion of participants with LSD markedly expands the population of children who experience adverse clinical and nutritional outcomes from acute diarrhoeal diseases. Since MSD and LSD have similar aetiologies, interventions targeting rotavirus, Shigella spp, enterotoxigenic E coli producing heat-stable toxin, and Cryptosporidium spp might substantially reduce the diarrhoeal disease burden and its associated nutritional faltering. FUNDING: Bill & Melinda Gates Foundation.

Colonization factors among enterotoxigenic Escherichia coli isolates from children with moderate-to-severe diarrhea and from matched controls in the Global Enteric Multicenter Study (GEMS).

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) encoding heat-stable enterotoxin (ST) alone or with heat-labile enterotoxin (LT) cause moderate-to-severe diarrhea (MSD) in developing country children. The Global Enteric Multicenter Study (GEMS) identified ETEC encoding ST among the top four enteropathogens. Since the GEMS objective was to provide evidence to guide development and implementation of enteric vaccines and other interventions to diminish diarrheal disease morbidity and mortality, we examined colonization factor (CF) prevalence among ETEC isolates from children age <5 years with MSD and from matched controls in four African and three Asian sites. We also assessed strength of association of specific CFs with MSD. METHODOLOGY/PRINCIPAL FINDINGS: MSD cases enrolled at healthcare facilities over three years and matched controls were tested in a standardized manner for many enteropathogens. To identify ETEC, three E. coli colonies per child were tested by polymerase chain reaction (PCR) to detect genes encoding LT, ST; confirmed ETEC were examined by PCR for major CFs (Colonization Factor Antigen I [CFA/I] or Coli Surface [CS] antigens CS1-CS6) and minor CFs (CS7, CS12, CS13, CS14, CS17, CS18, CS19, CS20, CS21, CS30). ETEC from 806 cases had a single toxin/CF profile in three tested strains per child. Major CFs, components of multiple ETEC vaccine candidates, were detected in 66.0% of LT/ST and ST-only cases and were associated with MSD versus matched controls by conditional logistic regression (p≤0.006); major CFs detected in only 25.0% of LT-only cases weren't associated with MSD. ETEC encoding exclusively CS14, identified among 19.9% of 291 ST-only and 1.5% of 259 LT/ST strains, were associated with MSD (p = 0.0011). No other minor CF exhibited prevalence ≥5% and significant association with MSD. CONCLUSIONS/SIGNIFICANCE: Major CF-based efficacious ETEC vaccines could potentially prevent up to 66% of pediatric MSD cases due to ST-encoding ETEC in developing countries; adding CS14 extends coverage to ~77%.

Dynamics of antimicrobial resistance in intestinal Escherichia coli from children in community settings in South Asia and sub-Saharan Africa.

The dynamics of antimicrobial resistance (AMR) in developing countries are poorly understood, especially in community settings, due to a sparsity of data on AMR prevalence and genetics. We used a combination of phenotyping, genomics and antimicrobial usage data to investigate patterns of AMR amongst atypical enteropathogenic Escherichia coli (aEPEC) strains isolated from children younger than five years old in seven developing countries (four in sub-Saharan Africa and three in South Asia) over a three-year period. We detected high rates of AMR, with 65% of isolates displaying resistance to three or more drug classes. Whole-genome sequencing revealed a diversity of known genetic mechanisms for AMR that accounted for >95% of phenotypic resistance, with comparable rates amongst aEPEC strains associated with diarrhoea or asymptomatic carriage. Genetic determinants of AMR were associated with the geographic location of isolates, not E. coli lineage, and AMR genes were frequently co-located, potentially enabling the acquisition of multi-drug resistance in a single step. Comparison of AMR with antimicrobial usage data showed that the prevalence of resistance to fluoroquinolones and third-generation cephalosporins was correlated with usage, which was higher in South Asia than in Africa. This study provides much-needed insights into the frequency and mechanisms of AMR in intestinal E. coli in children living in community settings in developing countries.

Antibacterial activity of rhodomyrtone on Clostridium difficile vegetative cells and spores in vitro.

The increasing incidence and severity of diarrhoea and colitis caused by Clostridium difficile, together with a high rate of relapse following treatment with currently recommended antimicrobials, calls for novel interventions for C. difficile infection (CDI). Rhodomyrtone, a bioactive compound derived from the leaves of the rose myrtle (Rhodomyrtus tomentosa) has demonstrated antibacterial activity against several Gram-positive bacteria. This study compared the in vitro antimicrobial activity of rhodomyrtone on C. difficile with that of vancomycin, a recommended agent for the treatment of CDI. Determination of the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of rhodomyrtone and vancomycin for ten C. difficile isolates showed that the MICs of rhodomyrtone for C. difficile vegetative cells (0.625-2.5 mg/L) were comparable with that of vancomycin (1.25 mg/L), but the MBCs of rhodomyrtone (1.25-5 mg/L) were significantly lower than those for vancomycin (5 mg/L to ˃40 mg/L; P < 0.001). Time-kill assays showed rapid bactericidal activity for rhodomyrtone, with ≥99% killing within 4 h. Rhodomyrtone was also four-fold more potent than vancomycin in inhibiting C. difficile spore outgrowth. Transmission electron microscopy of rhodomyrtone-treated C. difficile revealed cell lysis and evidence of defective cell division and spore formation. These studies indicate that rhodomyrtone should be further investigated as a potential treatment for CDI.

Neonatal BCG Vaccination Influences Cytokine Responses to Toll-like Receptor Ligands and Heterologous Antigens.

Background: BCG vaccination is associated with a reduction in all-cause infant mortality in high-mortality settings. The underlying mechanisms remain uncertain, but long-term modulation of the innate immune response (trained immunity) may be involved. Methods: Whole-blood specimens, collected 7 days after randomization from 212 neonates enrolled in a randomized trial of neonatal BCG vaccination, were stimulated with killed pathogens and Toll-like receptor (TLR) ligands to interrogate cytokine responses. Results: BCG-vaccinated infants had increased production of interleukin 6 (IL-6) in unstimulated samples and decreased production of interleukin 1 receptor antagonist, IL-6, and IL-10 and the chemokines macrophage inflammatory protein 1α (MIP-1α), MIP-1ß, and monocyte chemoattractant protein 1 (MCP-1) following stimulation with peptidoglycan (TLR2) and R848 (TLR7/8). BCG-vaccinated infants also had decreased MCP-1 responses following stimulation with heterologous pathogens. Sex and maternal BCG vaccination status interacted with neonatal BCG vaccination. Conclusions: Neonatal BCG vaccination influences cytokine responses to TLR ligands and heterologous pathogens. This effect is characterized by decreased antiinflammatory cytokine and chemokine responses in the context of higher levels of IL-6 in unstimulated samples. This supports the hypothesis that BCG vaccination modulates the innate immune system. Further research is warranted to determine whether there is an association between these findings and the beneficial nonspecific (heterologous) effects of BCG vaccine on all-cause mortality.

Corrigendum: In silico serotyping of E. coli from short read data identifies limited novel O-loci but extensive diversity of O:H serotype combinations within and between pathogenic lineages.

[This corrects the article DOI: 10.1099/mgen.0.000064.].

Control of Virulence Gene Expression by the Master Regulator, CfaD, in the Prototypical Enterotoxigenic Escherichia coli Strain, H10407.

Enterotoxigenic Escherichia coli (ETEC) is the most common bacterial cause of diarrhea in children in developing countries, as well as in travelers to these countries. To cause disease, ETEC needs to produce a series of virulence proteins including enterotoxins, colonization factors and secretion pathways, which enable this pathogen to colonize the human small intestine and deliver enterotoxins to epithelial cells. Previously, a number of studies have demonstrated that CfaD, an AraC-like transcriptional regulator, plays a key role in virulence gene expression by ETEC. In this study, we carried out a transcriptomic analysis of ETEC strain, H10407, grown under different conditions, and determined the complete set of genes that are regulated by CfaD. In this way, we identified a number of new target genes, including rnr-1, rnr-2, etpBAC, agn43, flu, traM and ETEC_3214, whose expression is strongly activated by CfaD. Using promoter-lacZ reporters, primer extension and electrophoretic mobility shift assays, we characterized the CfaD-mediated activation of several selected target promoters. We also showed that the gut-associated environmental signal, sodium bicarbonate, stimulates CfaD-mediated upregulation of its virulence target operons. Finally, we screened a commercial small molecule library and identified a compound (CH-1) that specifically inhibited the regulatory function of CfaD, and by 2-D analoging, we identified a second inhibitor (CH-2) with greater potency.

Are Escherichia coli Pathotypes Still Relevant in the Era of Whole-Genome Sequencing?

The empirical and pragmatic nature of diagnostic microbiology has given rise to several different schemes to subtype E.coli, including biotyping, serotyping, and pathotyping. These schemes have proved invaluable in identifying and tracking outbreaks, and for prognostication in individual cases of infection, but they are imprecise and potentially misleading due to the malleability and continuous evolution of E. coli. Whole genome sequencing can be used to accurately determine E. coli subtypes that are based on allelic variation or differences in gene content, such as serotyping and pathotyping. Whole genome sequencing also provides information about single nucleotide polymorphisms in the core genome of E. coli, which form the basis of sequence typing, and is more reliable than other systems for tracking the evolution and spread of individual strains. A typing scheme for E. coli based on genome sequences that includes elements of both the core and accessory genomes, should reduce typing anomalies and promote understanding of how different varieties of E. coli spread and cause disease. Such a scheme could also define pathotypes more precisely than current methods.

Investigation of Streptococcus salivarius-mediated inhibition of pneumococcal adherence to pharyngeal epithelial cells.

BACKGROUND: Pneumococcal adherence to the nasopharyngeal epithelium is a critical step in colonisation and disease. The probiotic bacterium, Streptococcus salivarius, can inhibit pneumococcal adherence to epithelial cells in vitro. We investigated the mechanism(s) of inhibition using a human pharyngeal epithelial cell line (Detroit 562) following pre-administration of two different strains of S. salivarius. RESULTS: Whilst the bacteriocin-encoding megaplasmids of S. salivarius strains K12 and M18 were essential to prevent pneumococcal growth on solid media, they were not required to inhibit pneumococcal adherence. Experiments testing S. salivarius K12 and two pneumococcal isolates (serotypes 19F and 6A) showed that inhibition of 19F may involve S. salivarius-mediated blocking of pneumococcal binding sites: a negative correlation was observed between adherence of K12 and 19F, and no inhibition occurred when K12 was prevented from contacting epithelial cells. K12-mediated inhibition of adherence by 6A may involve additional mechanisms, since no correlation was observed between adherence of K12 and 6A, and K12 could inhibit 6A adherence in the absence of cell contact. CONCLUSIONS: These results suggest that S. salivarius employs several mechanisms, including blocking pneumococcal binding sites, to reduce pneumococcal adherence to pharyngeal epithelial cells. These findings extend our understanding of how probiotics may inhibit pneumococcal adherence and could assist with the development of novel strategies to prevent pneumococcal colonisation in the future.

Early gut colonization by Bifidobacterium breve and B. catenulatum differentially modulates eczema risk in children at high risk of developing allergic disease.

BACKGROUND: An altered compositional signature and reduced diversity of early gut microbiota are linked to development of allergic disease. We investigated the relationship between dominant Bifidobacterium species during the early post-natal period and subsequent development of allergic disease in the first year of life. METHODS: Faecal samples were collected at age 1 week, 1 month and 3 months from 117 infants at high risk of allergic disease. Bifidobacterium species were analysed by quantitative PCR and terminal restriction fragment length polymorphism. Infants were examined at 3, 6 and 12 months, and skin prick test was performed at 12 months. Eczema was diagnosed according to the UK Working Party criteria. RESULTS: The presence of B. catenulatum at 3 months was associated with a higher risk of developing eczema (ORadj = 4.5; 95% CI: 1.56-13.05, padj = 0.005). Infants colonized with B. breve at 1 week (ORadj = 0.29; 95% CI: 0.09-0.95, padj = 0.04) and 3 months (ORadj = 0.15; 95% CI: 0.05-0.44, padj = 0.00001) had a reduced risk of developing eczema. Furthermore, the presence of B. breve at 3 months was associated with a lower risk of atopic sensitization at 12 months (ORadj = 0.38; 95% CI: 0.15-0.98, padj = 0.05). B. breve colonization patterns were influenced by maternal allergic status, household pets and number of siblings. CONCLUSIONS: Temporal variations in Bifidobacterium colonization patterns early in life are associated with later development of eczema and/or atopic sensitization in infants at high risk of allergic disease. Modulation of the early microbiota may provide a means to prevent eczema in high-risk infants.