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Abstract Invasive non-typhoidal Salmonella (iNTS) disease manifesting as bloodstream infection with high mortality is responsible for a huge public health burden in sub-Saharan Africa. Salmonella enterica serovar Typhimurium (S. Typhimurium) is the main cause of iNTS disease in Africa. By analysing whole genome sequence data from 1303 S. Typhimurium isolates originating from 19 African countries and isolated between 1979 and 2017, here we show a thorough scaled appraisal of the population structure of iNTS disease caused by S. Typhimurium across many of Africa’s most impacted countries. At least six invasive S. Typhimurium clades have already emerged, with ST313 lineage 2 or ST313-L2 driving the current pandemic. ST313-L2 likely emerged in the Democratic Republic of Congo around 1980 and further spread in the mid 1990s. We observed plasmid-borne as well as chromosomally encoded fluoroquinolone resistance underlying emergences of extensive-drug and pan-drug resistance. Our work provides an overview of the evolution of invasive S. Typhimurium disease, and can be exploited to target control measures.

Abstract Background Bacterial meningitis caused by non-typhoid Salmonella can be a fatal condition which is more common in low and middle-income countries. Case presentation We report the case of a Salmonella meningitis in a Belgian six-month old male infant. The first clinical examination was reassuring, but after a few hours, his general state deteriorated. A blood test and a lumbar puncture were therefore performed. The cerebrospinal fluid analysis was compatible with a bacterial meningitis which was later identified by the NRC (National Reference Center) as Salmonella enterica serovar Durban. Conclusions In this paper, we present the clinical presentation, genomic typing, and probable sources of infection for an unusually rare serovar of Salmonella. Through an extended genomic analysis, we established its relationship to historical cases with links to Guinea.

Invasive non-typhoidal Salmonella (iNTS) (serotypes Typhimurium and Enteritidis) are major causes of bloodstream infections in sub-Saharan Africa, but their reservoir is unknown. Aiming to demonstrate human carriers as a reservoir, we assessed an iNTS disease endemic rural community (Kikonka health area, Democratic Republic of the Congo) for intestinal carriage of iNTS. After a census, healthy subjects from randomly selected households provided three successive stool samples for Salmonella culture. We next compared the stool isolates for genetic relatedness with time and health area-matched blood culture isolates obtained from hospitalized patients by multiple locus variable-number tandem repeat analysis (MLVA) and performed whole genome sequencing (WGS) on a subset of stool and blood isolates. Among 2,354 eligible subjects, 2,234 (94.9%) consented and provided at least one stool sample, and 2,219 (94.3%) provided three stool samples. The cumulative proportion of Salmonella carriers after 3 days was 4.4% (n = 98). S. Typhimurium and Enteritidis were found in 26 and 3 carriers, respectively, representing 1.3% (29 out of 2,234) of participants living in 6.0% (26 out of 482) of households. MLVA types of all 26 S. Typhimurium stool isolates matched with the corresponding MLVA types of blood isolates. The MLVA type of one out of three Enteritidis stool isolates matched the single MLVA type of the five Enteritidis blood isolates. WGS analysis of S. Typhimurium (n = 20) and S. Enteritidis (n = 4) isolates revealed Typhimurium multilocus sequence type (ST)313 Lineage 2 and Enteritidis ST11 Central/Eastern African and Outlier clades and confirmed the MLVA clustering. More than three-quarters of Typhimurium isolates showed combined multidrug resistance, ceftriaxone resistance, and fluoroquinolone non-susceptibility. In conclusion, the present study demonstrated iNTS carriage among healthy community members, with stool isolates that were genetically similar to blood culture isolates obtained in patients from the same community. These findings contribute to the evidence of a human reservoir of iNTS.

IntroductionInvasive non-typhoidal Salmonella (iNTS), mainly Salmonella Typhimurium and Salmonella Enteritidis, causes a severe burden in sub-Saharan Africa; however, its reservoir (animal or environmental) is unclear. The present study assessed healthy household members of index patients for intestinal carriage of Salmonella.MethodsIndex patients were admitted to the University Hospital of Kisangani (DR Congo), and Salmonella was grown from blood cultures. Household members were asked to provide three stool samples for culture for Salmonella. Salmonella Typhimurium and S. Enteritidis isolates from index patients, and household members were assessed for genetic relatedness using the multiple-locus variable number of tandem repeat analysis (MLVA), and the multilocus sequence type (ST) was determined by whole genome sequencing.ResultsBetween May 2016 and January 2020, 22 households were visited. The index patient serotypes were Typhimurium, Enteritidis, Typhi, and Paratyphi C; II:42:r:-; and I:7:y:- (n = 8, 7, 5, and each 1, respectively). The median (range) delay between the index patient and household sampling was 25 days (2 days to 7.3 months); 203 household members provided at least one stool sample. In all, 15 (7.3%) Salmonella carriers were found in nine of 22 households. For one index patient, the household comprised S. Typhimurium in four household members, including the index patient, sampled 27 days after bloodstream infection; the MLVA types of these five isolates were similar. They belonged to ST313 lineage 2 and were closely related [0–1 allelic distance (AD) among the stool isolates and eight AD with the blood culture isolate]. In another household, the stool culture of the index patient (obtained 67 days after bloodstream infection) grew S. Enteritidis of the same MLVA type; both isolates belonged to the ST11 Central/Eastern African clade and were closely related (three AD).DiscussionThe present study provides evidence of household clustering of S. Typhimurium ST313 and intestinal carriage of iNTS several weeks after bloodstream infection.

Summary: Background: The Invasive Respiratory Infection Surveillance (IRIS) Consortium was established to assess the impact of the COVID-19 pandemic on invasive diseases caused by Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, and Streptococcus agalactiae. We aimed to analyse the incidence and distribution of these diseases during the first 2 years of the COVID-19 pandemic compared to the 2 years preceding the pandemic. Methods: For this prospective analysis, laboratories in 30 countries and territories representing five continents submitted surveillance data from Jan 1, 2018, to Jan 2, 2022, to private projects within databases in PubMLST. The impact of COVID-19 containment measures on the overall number of cases was analysed, and changes in disease distributions by patient age and serotype or group were examined. Interrupted time-series analyses were done to quantify the impact of pandemic response measures and their relaxation on disease rates, and autoregressive integrated moving average models were used to estimate effect sizes and forecast counterfactual trends by hemisphere. Findings: Overall, 116 841 cases were analysed: 76 481 in 2018–19, before the pandemic, and 40 360 in 2020–21, during the pandemic. During the pandemic there was a significant reduction in the risk of disease caused by S pneumoniae (risk ratio 0·47; 95% CI 0·40–0·55), H influenzae (0·51; 0·40–0·66) and N meningitidis (0·26; 0·21–0·31), while no significant changes were observed for S agalactiae (1·02; 0·75–1·40), which is not transmitted via the respiratory route. No major changes in the distribution of cases were observed when stratified by patient age or serotype or group. An estimated 36 289 (95% prediction interval 17 145–55 434) cases of invasive bacterial disease were averted during the first 2 years of the pandemic among IRIS-participating countries and territories. Interpretation: COVID-19 containment measures were associated with a sustained decrease in the incidence of invasive disease caused by S pneumoniae, H influenzae, and N meningitidis during the first 2 years of the pandemic, but cases began to increase in some countries towards the end of 2021 as pandemic restrictions were lifted. These IRIS data provide a better understanding of microbial transmission, will inform vaccine development and implementation, and can contribute to health-care service planning and provision of policies. Funding: Wellcome Trust, NIHR Oxford Biomedical Research Centre, Spanish Ministry of Science and Innovation, Korea Disease Control and Prevention Agency, Torsten Söderberg Foundation, Stockholm County Council, Swedish Research Council, German Federal Ministry of Health, Robert Koch Institute, Pfizer, Merck, and the Greek National Public Health Organization.

The timely differentiation of the AviPro Salmonella VAC T and VAC E strains from the wild-type Salmonella enterica ser. Typhimurium and ser. Enteritidis isolates is crucial for effectively monitoring veterinary isolates. Currently, the distinction between field and vaccine strains has been conducted routinely via phenotypic antimicrobial resistance testing since the vaccines were first introduced more than 20 years ago, and the differentiation based on the antimicrobial resistance profile is still a valid and well-established method. However, an alternative method was sought for those laboratories that prefer a PCR-based method for logistic and/or operational reasons. In this study, we developed two triplex Real-Time PCR reactions that targeted conserved and specific mutations and, therefore, enabled the reliable differentiation of field and vaccine strains. To validate the effectiveness of both assays, we extensively tested them on a dataset consisting of 405 bacterial strains. The results demonstrated a 100% sensitivity and specificity for distinguishing both Salmonella enterica ser. Typhimurium and Enteritidis, although a confirmed culture is required.

Background: Nontyphoidal Salmonella serovars predominantly cause gastrointestinal infections. However, other clinical presentations, including urogenital infections, have been reported, although they are rather rare. Case presentation: This case is about a 33-year-old woman diagnosed with Salmonella enterica serovar Hvittingfoss (S. Hvittingfoss) bacteremia and endometritis six days post uterine aspiration in the context of a missed abortion. She had traveled to Indonesia two weeks prior to the positive blood and cervical culture. She never developed gastrointestinal symptoms but was found to carry S. Hvittingfoss in her stool sample. The patient was successfully treated with a seven-day course of iv ciprofloxacin. Conclusions: S. Hvittingfoss is a rare serovar that has caused a few outbreaks of foodborne infections in Asia, the United States, and Australia. To the best of our knowledge, this is the first reported case of Salmonella urogenital infection caused by this serovar. Salmonella as a cause of urogenital infections is rare but not uncommon. Therefore, it should be considered in identifying members of the Enterobacterales among urogenital flora in cases of severe urogenital infections, especially when other cultures remain negative.

Abstract Background Salmonella and E. coli O157 are common causes of foodborne diseases. Evisceration and de-hiding steps can lead to carcass contamination during slaughter operation. In Ethiopia, information on the association between the presence of these pathogens in the rectal content and/or on the hide of cattle and their presence on the carcass is lacking. Methods The aim of this study was to assess the sources of beef carcass contamination with Salmonella and E. coli O157 during slaughter. Rectal contents and hide- and carcass-swabs (from three sites: foreleg, brisket and hind leg) were collected from 70 beef cattle at two small scale slaughterhouses. Isolates were genotyped by the Pulsed Field Gel Electrophoresis method and tested for resistance against 14 microbial drugs. Results Salmonella was detected at equal proportions (7.1%) in rectal content samples and hide swabs. E. coli O157 was detected in 8.6% of the rectal contents and 4.3% of the hide swabs. The proportion of contaminated carcasses was 8.6% for Salmonella and 7.1% for E. coli O157. Genetic linkage between the Salmonella and E. coli O157 isolates from the rectal contents and/or hides and carcasses were observed only in a few cases (2 and 1 carcasses, respectively) indicating the limited direct transfer of the pathogens from the feces and/or hide to the carcass during slaughter. Most carcasses became positive by cross contamination. All the S. Typhimurium isolates (n = 8) were multidrug resistant being resistant to ampicillin, chloramphenicol, sulfamethoxazole and tetracycline. The two S. Dublin isolates were resistant to colistin. All E. coli O157 isolates were susceptible to the antimicrobials tested. Conclusion The results indicated that cross contamination may be an important source for carcass contamination.

BackgroundInvasive non-typhoidal Salmonella (iNTS-mainly serotypes Enteritidis and Typhimurium) are major causes of bloodstream infections in children in sub-Saharan Africa, but their reservoir remains unknown. We assessed iNTS carriage in rats in an urban setting endemic for iNTS carriage and compared genetic profiles of iNTS from rats with those isolated from humans.Methodology/principal findingsFrom April 2016 to December 2018, rats were trapped in five marketplaces and a slaughterhouse in Kisangani, Democratic Republic of the Congo. After euthanasia, blood, liver, spleen, and rectal content were cultured for Salmonella. Genetic relatedness between iNTS from rats and humans-obtained from blood cultures at Kisangani University Hospital-was assessed with multilocus variable-number tandem repeat (VNTR) analysis (MLVA), multilocus sequence typing (MLST) and core-genome MLST (cgMLST). 1650 live-capture traps yielded 566 (34.3%) rats (95.6% Rattus norvegicus, 4.4% Rattus rattus); 46 (8.1%) of them carried Salmonella, of which 13 had more than one serotype. The most common serotypes were II.42:r:- (n = 18 rats), Kapemba (n = 12), Weltevreden and Typhimurium (n = 10, each), and Dublin (n = 8). Salmonella Typhimurium belonged to MLST ST19 (n = 7 rats) and the invasive ST313 (n = 3, isolated from deep organs but not from rectal content). Sixteen human S. Typhimurium isolates (all ST313) were available for comparison: MLVA and cgMLST revealed two distinct rat-human clusters involving both six human isolates, respectively, i.e. in total 12/16 human ST313 isolates. All ST313 Typhimurium isolates from rats and humans clustered with the ST313 Lineage 2 isolates and most were multidrug resistant; the remaining isolates from rats including S. Typhimurium ST19 were pan-susceptible.ConclusionThe present study provides evidence of urban rats as potential reservoirs of S. Typhimurium ST313 in an iNTS endemic area in sub-Saharan Africa.

Abstract Background Neisseria meningitidis has rarely been described as an agent of necrotic soft tissue infection. Case presentation We report a case of a septic shock with necrotizing cellulitis due to Neisseria meningitidis serogroup W, treated by urgent extensive surgical debridement followed by skin grafts. The invasive meningococcal disease occurred together with a complement deficiency, possibly acquired after bypass surgery that took place 1 year before. Conclusions Necrotic tissue infections should be considered part of the invasive meningococcal diseases spectrum and should prompt clinicians to look for complement deficiencies. Gastric bypass surgery associated malnutrition may be implicated but further verification is needed.

Summary: Background: Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis, which are typically transmitted via respiratory droplets, are leading causes of invasive diseases, including bacteraemic pneumonia and meningitis, and of secondary infections subsequent to post-viral respiratory disease. The aim of this study was to investigate the incidence of invasive disease due to these pathogens during the early months of the COVID-19 pandemic. Methods: In this prospective analysis of surveillance data, laboratories in 26 countries and territories across six continents submitted data on cases of invasive disease due to S pneumoniae, H influenzae, and N meningitidis from Jan 1, 2018, to May, 31, 2020, as part of the Invasive Respiratory Infection Surveillance (IRIS) Initiative. Numbers of weekly cases in 2020 were compared with corresponding data for 2018 and 2019. Data for invasive disease due to Streptococcus agalactiae, a non-respiratory pathogen, were collected from nine laboratories for comparison. The stringency of COVID-19 containment measures was quantified using the Oxford COVID-19 Government Response Tracker. Changes in population movements were assessed using Google COVID-19 Community Mobility Reports. Interrupted time-series modelling quantified changes in the incidence of invasive disease due to S pneumoniae, H influenzae, and N meningitidis in 2020 relative to when containment measures were imposed. Findings: 27 laboratories from 26 countries and territories submitted data to the IRIS Initiative for S pneumoniae (62 837 total cases), 24 laboratories from 24 countries submitted data for H influenzae (7796 total cases), and 21 laboratories from 21 countries submitted data for N meningitidis (5877 total cases). All countries and territories had experienced a significant and sustained reduction in invasive diseases due to S pneumoniae, H influenzae, and N meningitidis in early 2020 (Jan 1 to May 31, 2020), coinciding with the introduction of COVID-19 containment measures in each country. By contrast, no significant changes in the incidence of invasive S agalactiae infections were observed. Similar trends were observed across most countries and territories despite differing stringency in COVID-19 control policies. The incidence of reported S pneumoniae infections decreased by 68% at 4 weeks (incidence rate ratio 0·32 [95% CI 0·27–0·37]) and 82% at 8 weeks (0·18 [0·14–0·23]) following the week in which significant changes in population movements were recorded. Interpretation: The introduction of COVID-19 containment policies and public information campaigns likely reduced transmission of S pneumoniae, H influenzae, and N meningitidis, leading to a significant reduction in life-threatening invasive diseases in many countries worldwide. Funding: Wellcome Trust (UK), Robert Koch Institute (Germany), Federal Ministry of Health (Germany), Pfizer, Merck, Health Protection Surveillance Centre (Ireland), SpID-Net project (Ireland), European Centre for Disease Prevention and Control (European Union), Horizon 2020 (European Commission), Ministry of Health (Poland), National Programme of Antibiotic Protection (Poland), Ministry of Science and Higher Education (Poland), Agencia de Salut Pública de Catalunya (Spain), Sant Joan de Deu Foundation (Spain), Knut and Alice Wallenberg Foundation (Sweden), Swedish Research Council (Sweden), Region Stockholm (Sweden), Federal Office of Public Health of Switzerland (Switzerland), and French Public Health Agency (France).

IntroductionThe surveillance of human salmonellosis in Belgium is dependent on the referral of human Salmonella isolates to the National Reference Center (NRC). Knowledge of current diagnostic practices and the coverage of the national Salmonella surveillance system are important to correctly interpret surveillance data and trends over time, to estimate the true burden of salmonellosis in Belgium, and to evaluate the appropriateness of implementing whole-genome sequencing (WGS) at this central level.MethodsThe coverage of the NRC was defined as the proportion of all diagnosed human Salmonella cases in Belgium reported to the NRC and was assessed for 2019 via a survey among all licensed Belgian medical laboratories in 2019, and for 2016-2020 via a capture-recapture study using the Sentinel Network of Laboratories (SNL) as the external source. In addition, the survey was used to assess the impact of the implementation of culture-independent diagnostic tests (CIDTs) at the level of peripheral laboratory sites, as a potential threat to national public health surveillance programs.ResultsThe coverage of the NRC surveillance system was estimated to be 83% and 85%, based on the results of the survey and on the two-source capture-recapture study, respectively. Further, the results of the survey indicated a limited use of CIDTs by peripheral laboratories in 2019.ConclusionGiven the high coverage and the limited impact of CIDTs on the referral of isolates, we may conclude that the NRC can confidently monitor the epidemiological situation and identify outbreaks throughout the country. These findings may guide the decision to implement WGS at the level of the NRC and may improve estimates of the true burden of salmonellosis in Belgium.

BACKGROUND:Non-typhoidal Salmonella (NTS) serotypes Typhimurium and Enteritidis are a major cause of bloodstream infections in children in sub-Saharan Africa but their reservoir is unknown. We compared pairs of NTS blood and stool isolates (with the same NTS serotype recovered in the same patient) for genetic similarity. METHODS:Between November 2013 and April 2017, hospital-admitted children (29 days to 14 years) with culture-confirmed NTS bloodstream infections were enrolled in a cross-sectional study at Kisantu Hospital, DR Congo. Stool cultures for Salmonella were performed on a subset of enrolled children, as well as on a control group of non-febrile hospital-admitted children. Pairs of blood and stool NTS isolates were assessed for genetic similarity by multiple-locus variable-number of tandem repeats (MLVA) and genomics analysis. RESULTS:A total of 299 children with NTS grown from blood cultures (Typhimurium 68.6%, Enteritidis 30.4%, other NTS 1.0%) had a stool sample processed; in 105 (35.1%) of them NTS was detected (Typhimurium 70.5%, Enteritidis 25.7%, other NTS 3.8%). A total of 87/105 (82.9%) pairs of blood and stool NTS isolates were observed (representing 29.1% of the 299 children). Among 1598 controls, the proportion of NTS stool excretion was 2.1% (p < 0.0001). MLVA types among paired isolates were identical in 82/87 (94.3%) pairs (27.4% of the 299 children; 61/66 (92.4%) in Typhimurium and 21/21 (100%) in Enteritidis pairs). Genomics analysis confirmed high genetic similarity within 41/43 (95.3%) pairs, showing a median SNP difference of 1 (range 0-77) and 1 (range 0-4) for Typhimurium and Enteritidis pairs respectively. Typhimurium and Enteritidis isolates belonged to sequence types ST313 lineage II and ST11 respectively. CONCLUSION:Nearly 30% of children with NTS bloodstream infection showed stool excretion of an NTS isolate with high genetic similarity, adding to the evidence of humans as a potential reservoir for NTS.

BACKGROUND:Clinical observations and animal studies have suggested that Salmonella intestinal carriage is promoted by concurrent Schistosoma infection. The present study assessed association of Salmonella intestinal carriage and Schistosoma mansoni infection among individuals in a Schistosoma endemic area in sub-Saharan Africa. METHODS:From November 2015 to March 2016, a cross-sectional community-wide study was conducted in Kifua II, a rural village in Kongo Central Province, Democratic Republic of Congo. Stool samples were collected and analyzed for Salmonella intestinal carriage (culture) and Schistosoma mansoni infection (Kato Katz microscopy with determination of egg load). Salmonella Typhimurium and Enteritidis isolates were assessed for genetic similarity with blood culture isolates obtained during the same period in a neighboring hospital using multi-locus variable-numbers tandem repeat analysis (MLVA). RESULTS:A total of 1,108 participants were included (median age 15 years (IQR: 7-36), male-to-female ratio of 1:1.1). The overall prevalence of Schistosoma mansoni infection and non-typhoidal Salmonella carriage was 51.2% (95% CI: 48.2-54.1) and 3.4% (95% CI: 2.5-4.7) respectively, with 2.2% (95% CI: 1.5-3.2) of participants coinfected. The proportion of Salmonella carriage tended to be higher among Schistosoma mansoni infected participants compared to non-infected participants but this difference did not reach statistical significance (4.2% versus 2.6%, p = 0.132). However, the proportion of Salmonella carriage among participants with a heavy Schistosoma mansoni infection was significantly higher compared to those with a light and moderate infection (8.7% versus 3.2%, p = 0.012) and compared to Schistosoma mansoni negatives (8.7% versus 2.6%, p = 0.002). The 38 Salmonella isolates comprised five and four Enteritidis and Typhimurium serotypes respectively, the majority of them had MLVA types identical or similar to those observed among blood culture isolates. CONCLUSION:Salmonella intestinal carriage was associated with a heavy intensity of Schistosoma mansoni infection. Further studies are needed to address causation.

Whole genome sequencing is increasingly recognized as the most informative approach for characterization of bacterial isolates. Success of the routine use of this technology in public health laboratories depends on the availability of well-characterized and verified data analysis methods. However, multiple subtyping workflows are now often being used for a single organism, and differences between them are not always well described. Moreover, methodologies for comparison of subtyping workflows, and assessment of their performance are only beginning to emerge. Current work focuses on the detailed comparison of WGS-based subtyping workflows and evaluation of their suitability for the organism and the research context in question. We evaluated the performance of pipelines used for subtyping of Neisseria meningitidis, including the currently widely applied cgMLST approach and different SNP-based methods. In addition, the impact of the use of different tools for detection and filtering of recombinant regions and of different reference genomes were tested. Our benchmarking analysis included both assessment of technical performance of the pipelines and functional comparison of the generated genetic distance matrices and phylogenetic trees. It was carried out using replicate sequencing datasets of high- and low-coverage, consisting mainly of isolates belonging to the clonal complex 269. We demonstrated that cgMLST and some of the SNP-based subtyping workflows showed very good performance characteristics and highly similar genetic distance matrices and phylogenetic trees with isolates belonging to the same clonal complex. However, only two of the tested workflows demonstrated reproducible results for a group of more closely related isolates. Additionally, results of the SNP-based subtyping workflows were to some level dependent on the reference genome used. Interestingly, the use of recombination-filtering software generally reduced the similarity between the gene-by-gene and SNP-based methodologies for subtyping of N. meningitidis. Our study, where N. meningitidis was taken as an example, clearly highlights the need for more benchmarking comparative studies to eventually contribute to a justified use of a specific WGS data analysis workflow within an international public health laboratory context.

BACKGROUND:Salmonella Typhimurium and Enteritidis are major causes of bloodstream infection in children in sub-Saharan Africa. This study assessed evidence for their zoonotic versus human reservoir. METHODS:Index patients were children with blood culture confirmed Salmonella infection recruited during a microbiological surveillance study in Nanoro, rural Burkina between May 2013 and August 2014. After consent, their households were visited. Stool from household members and livestock (pooled samples per species) as well as drinking water were cultured for Salmonella. Isolates with identical serotype obtained from index patient and any household sample were defined as "paired isolates" and assessed for genetic relatedness by multilocus variable number tandem-repeat analysis (MLVA) and whole-genome sequencing (WGS). RESULTS:Twenty-nine households were visited for 32/42 (76.2%) eligible index patients: two households comprised two index patients each, and in a third household the index patient had a recurrent infection. Among the 32 index patients, serotypes were Salmonella Typhimurium (n = 26), Salmonella Enteritidis (n = 5) and Salmonella Freetown (n = 1). All Typhimurium isolates were sequence type (ST)313. Median delay between blood culture sampling and household visits was 13 days (range 6-26). Salmonella was obtained from 16/186 (8.6%) livestock samples (13 serotypes) and 18/290 (6.2%) household members (9 serotypes). None of the water samples yielded Salmonella. Paired Salmonella Typhimurium isolates were obtained from three households representing four index patients. MLVA types were identical in two pairs and similar in the third (consisting of two index patients and one household member). WGS showed a strong genetic relatedness with 0 to 2 core genome SNPs difference between pairs on a household level. Livestock samples did not yield any Salmonella Typhimurium or Salmonella Enteritidis, and the latter was exclusively obtained from blood culture. Other serotypes shared by human and/or livestock carriers in the same household were Salmonella Derby, Drac, Tennessee and Muenster. CONCLUSIONS/SIGNIFICANCE:The current study provides further evidence of a human reservoir for invasive non-Typhoidal Salmonella (iNTS) in sub-Saharan Africa.

Abstract The phylogenetically closely related Shigella species and enteroinvasive Escherichia coli (EIEC) are responsible for millions of episodes of bacterial dysenteriae worldwide. Given its distinct epidemiology and public health relevance, only Shigellae are subject to mandatory reporting and follow‐up by public health authorities. However, many clinical laboratories struggle to differentiate non‐EIEC, EIEC, and Shigella in their current workflows, leading to inaccuracies in surveillance and rising numbers of misidentified E. coli samples at the National Reference Centre (NRC). In this paper, we describe two novel tools to enhance Shigella surveillance. First, we developed a low‐cost Luminex‐based multiplex assay combining five genetic markers for species identification with 11 markers for serotype prediction for S. sonnei and S. flexneri isolates. Using a test panel of 254 clinical samples, this assay has a sensitivity of 100% in differentiation of EIEC/Shigella pathotype from non‐EIEC strains, and 68.7% success rate in distinction of Shigella and EIEC. A novel, and particularly successful marker was a Shigella‐specific deletion in the spermidine acetyltransferase gene speG, reflecting its metabolic decay. For Shigella serotype prediction, the multiplex assay scored a sensitivity and specificity of 96.6% and 98.4%, respectively. All discrepancies were analyzed with whole‐genome sequencing and shown to be related to causative mutations (stop codons, indels, and promoter mutations) in glycosyltransferase genes. This observation spurred the development of an in silico workflow which extracts the Shigella serotype from Next‐Generation Sequencing (NGS) data, taking into account gene functionality. Both tools will be implemented in the workflow of the NRC, and will play a major role in the shift from phenotypic to genotyping‐based surveillance of shigellosis in Belgium.

Despite being a well-established research method, the use of whole-genome sequencing (WGS) for routine molecular typing and pathogen characterization remains a substantial challenge due to the required bioinformatics resources and/or expertise. Moreover, many national reference laboratories and centers, as well as other laboratories working under a quality system, require extensive validation to demonstrate that employed methods are “fit-for-purpose” and provide high-quality results. A harmonized framework with guidelines for the validation of WGS workflows does currently, however, not exist yet, despite several recent case studies highlighting the urgent need thereof. We present a validation strategy focusing specifically on the exhaustive characterization of the bioinformatics analysis of a WGS workflow designed to replace conventionally employed molecular typing methods for microbial isolates in a representative small-scale laboratory, using the pathogen Neisseria meningitidis as a proof-of-concept. We adapted several classically employed performance metrics specifically toward three different bioinformatics assays: resistance gene characterization (based on the ARG-ANNOT, ResFinder, CARD, and NDARO databases), several commonly employed typing schemas (including, among others, core genome multilocus sequence typing), and serogroup determination. We analyzed a core validation dataset of 67 well-characterized samples typed by means of classical genotypic and/or phenotypic methods that were sequenced in-house, allowing to evaluate repeatability, reproducibility, accuracy, precision, sensitivity, and specificity of the different bioinformatics assays. We also analyzed an extended validation dataset composed of publicly available WGS data for 64 samples by comparing results of the different bioinformatics assays against results obtained from commonly used bioinformatics tools. We demonstrate high performance, with values for all performance metrics >87%, >97%, and >90% for the resistance gene characterization, sequence typing, and serogroup determination assays, respectively, for both validation datasets. Our WGS workflow has been made publicly available as a “push-button” pipeline for Illumina data at https://galaxy.sciensano.be to showcase its implementation for non-profit and/or academic usage. Our validation strategy can be adapted to other WGS workflows for other pathogens of interest and demonstrates the added value and feasibility of employing WGS with the aim of being integrated into routine use in an applied public health setting.

Shigellosis is an acute enteric infection caused mainly by the species Shigella flexneri and Shigella sonnei. Since surveillance of these pathogens indicated an increase in ciprofloxacin-resistant samples collected in Belgium between 2013 and 2018, a subset of 148 samples was analyzed with whole genome sequencing (WGS) to investigate their dispersion and underlying genomic features associated with ciprofloxacin resistance. A comparison between observed phenotypes and WGS-based resistance prediction to ciprofloxacin revealed perfect correspondence for all samples. Core genome multi-locus sequence typing and single nucleotide polymorphism-typing were used for phylogenomic investigation to characterize the spread of these infections within Belgium, supplemented with data from international reference collections to place the Belgian isolates within their global context. For S. flexneri, substantial diversity was observed with ciprofloxacin-resistant isolates assigned to several phylogenetic groups. Besides travel-related imports, several clusters of highly similar Belgian isolates could not be linked directly to international travel suggesting the presence of domestically circulating strains. For S. sonnei, Belgian isolates were all limited to lineage III, and could often be traced back to travel to countries in Asia and Africa, sometimes followed by domestic circulation. For both species, several clusters of isolates obtained exclusively from male patients were observed. Additionally, we illustrated the limitations of conventional serotyping of S. flexneri, which was impacted by serotype switching. This study contributes to a better understanding of the spread of shigellosis within Belgium and internationally, and highlights the added value of WGS for the surveillance of this pathogen.

To identify the major source of Salmonella contamination in a pig slaughterhouse, samples were collected from the clean and unclean area and Salmonella isolates were further typed. Carcasses entering the clean area showed a Salmonella contamination rate of 96.7% in the oral cavity and 55.0% in the rectum content samples. Evisceration seemed not to be critical as the contamination rate of the carcasses was similar before (16.7%) and after (18.3%) this slaughter step. In the unclean area, a limited number of oral cavity samples were positive after bleeding, while a dramatic increase of positives was observed after dehairing. Salmonella was detected in up to 0.01 mL of the recycled water collected from the dehairing machine. Genotyping of Salmonella isolates showed that similar pulsotypes were present in the oral cavity and recycled water. Based on these observations it can be concluded that the recycled water used in the dehairing machine was the major source for the carcass contamination in this slaughterhouse.

[This corrects the article DOI: 10.1371/journal.pone.0117950.].

Whole genome sequencing represents a promising new technology for subtyping of bacterial pathogens. Besides the technological advances which have pushed the approach forward, the last years have been marked by considerable evolution of the whole genome sequencing data analysis methods. Prior to application of the technology as a routine epidemiological typing tool, however, reliable and efficient data analysis strategies need to be identified among the wide variety of the emerged methodologies. In this work, we have compared three existing SNP-based subtyping workflows using a benchmark dataset of 32 Salmonella enterica subsp. enterica serovar Typhimurium and serovar 1,4,[5],12:i:- isolates including five isolates from a confirmed outbreak and three isolates obtained from the same patient at different time points. The analysis was carried out using the original (high-coverage) and a down-sampled (low-coverage) datasets and two different reference genomes. All three tested workflows, namely CSI Phylogeny-based workflow, CFSAN-based workflow and PHEnix-based workflow, were able to correctly group the confirmed outbreak isolates and isolates from the same patient with all combinations of reference genomes and datasets. However, the workflows differed strongly with respect to the SNP distances between isolates and sensitivity towards sequencing coverage, which could be linked to the specific data analysis strategies used therein. To demonstrate the effect of particular data analysis steps, several modifications of the existing workflows were also tested. This allowed us to propose data analysis schemes most suitable for routine SNP-based subtyping applied to S. Typhimurium and S. 1,4,[5],12:i:-. Results presented in this study illustrate the importance of using correct data analysis strategies and to define benchmark and fine-tune parameters applied within routine data analysis pipelines to obtain optimal results.

We present the results of a longitudinal surveillance study (1995-2014) on fluoroquinolone resistance (FQ-R) among Belgian non-invasive Streptococcus pneumoniae isolates (n = 5,602). For many years, the switch to respiratory fluoroquinolones for the treatment of (a)typical pneumonia had no impact on FQ-R levels. However, since 2011 we observed a significant decrease in susceptibility towards ciprofloxacin, ofloxacin and levofloxacin with peaks of 9.0%, 6.6% and 3.1% resistant isolates, respectively. Resistance to moxifloxacin arised sporadically, and remained

Surveillance of Salmonella enterica subsp. enterica serovar Enteritidis is generally considered to benefit from molecular techniques like multiple-locus variable-number of tandem repeats analysis (MLVA), which allow early detection and confinement of outbreaks. Here, a surveillance study, including phage typing, antimicrobial susceptibility testing and MLVA on 1,535 S. Enteritidis isolates collected between 2007 and 2012, was used to evaluate the added value of MLVA for public health surveillance in Belgium. Phage types PT4, PT8, PT21, PT1, PT6, PT14b, PT28 and PT13 dominate the Belgian S. Enteritidis population. The isolates of S. Enteritidis were most frequently susceptible to all antibiotics tested. 172 different MLVA profiles were detected, of which 9 frequent profiles included 67.2% of the S. Enteritidis population. During a serial passage experiment on selected isolates to investigate the in vitro stability of the 5 MLVA loci, no variations over time were observed indicating that the MLVA profiles were stable. The MLVA profile of isolates originating from different outbreaks in the Democratic Republic of the Congo (DRC) between 2010 and 2011 were distinct from any of the MLVA profiles found in Belgian isolates throughout the six year observational period and demonstrates that MLVA improves public health surveillance of S. Enteritidis. However, MLVA should be complemented with other subtyping methods when investigating outbreaks is caused by the most common MLVA profile.

Invasive meningococcal disease (IMD) is a major cause of bacterial meningitides and septicaemia. This study shows the results of the laboratory-based surveillance of IMD in Belgium over the period 1997-2012.The results are based on microbiological and molecular laboratory surveillance of 2997 clinical isolates of N. meningitides received by the Belgian Meningococcal Reference Centre (BMRC) over the period 1997-2012.Serogroup B has always been a major cause of meningococcal disease in Belgium, with P3.4 as most frequent serotype till 2008, while an increase in non-serotypable strains has been observed in the last few years. Clonal complexes cc-41/44 and cc-269 are most frequently observed in serogroup B strains. In the late nineties, the incidence of serogroup C disease increased considerably and peaked in 2001, mainly associated with phenotypes C:2a:P1.5,2, C:2a:P1.5 and C:2a:P1.2 (ST-11/ET-37 clonal complex). The introduction of the meningococcal C conjugate vaccine has been followed by an 88% significant decrease in serogroup C disease from 2001 to 2004 nationally, yet sharper in Flanders (92%) compared to Wallonia (77%). Since 2008 a difference in incidence of serogroup C was observed in Flanders (0-0.1/100,000) versus Wallonia (0.1-0.3/100,000).This study showed the change in epidemiology and strain population over a 16 years period spanning an exhaustive vaccination campaign and highlights the influence of regional vaccination policies with different cohorts sizes on short and long-term IMD incidences.

Serratia plymuthica strain RVH1, initially isolated from an industrial food processing environment, displays potent antimicrobial activity towards a broad spectrum of Gram-positive and Gram-negative bacterial pathogens. Isolation and subsequent structure determination of bioactive molecules led to the identification of two polyamino antibiotics with the same molecular structure as zeamine and zeamine II as well as a third, closely related analogue, designated zeamine I. The gene cluster encoding the biosynthesis of the zeamine antibiotics was cloned and sequenced and shown to encode FAS, PKS as well as NRPS related enzymes in addition to putative tailoring and export enzymes. Interestingly, several genes show strong homology to the pfa cluster of genes involved in the biosynthesis of long chain polyunsaturated fatty acids in marine bacteria. We postulate that a mixed FAS/PKS and a hybrid NRPS/PKS assembly line each synthesize parts of the backbone that are linked together post-assembly in the case of zeamine and zeamine I. This interaction reflects a unique interplay between secondary lipid and secondary metabolite biosynthesis. Most likely, the zeamine antibiotics are produced as prodrugs that undergo activation in which a nonribosomal peptide sequence is cleaved off.

Surveillance of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) is generally considered to benefit from molecular techniques like multiple-locus variable-number of tandem repeats analysis (MLVA), which allow earlier detection and confinement of outbreaks. Here, a surveillance study, including phage typing, antimicrobial susceptibility testing and the in Europe most commonly used 5-loci MLVA on 1,420 S. Typhimurium isolates collected between 2010 and 2012 in Belgium, was used to evaluate the added value of MLVA for public health surveillance. Phage types DT193, DT195, DT120, DT104, DT12 and U302 dominate the Belgian S. Typhimurium population. A combined resistance to ampicillin, streptomycin, sulphonamides and tetracycline (ASSuT) with or without additional resistances was observed for 42.5% of the isolates. 414 different MLVA profiles were detected, of which 14 frequent profiles included 44.4% of the S. Typhimurium population. During a serial passage experiment on selected isolates to investigate the in vitro stability of the 5 MLVA loci, variations over time were observed for loci STTR6, STTR10, STTR5 and STTR9. This study demonstrates that MLVA improves public health surveillance of S. Typhimurium. However, the 5-loci MLVA should be complemented with other subtyping methods for investigation of possible outbreaks with frequent MLVA profiles. Also, variability in these MLVA loci should be taken into account when investigating extended outbreaks and studying dynamics over longer periods.

The increasing antibiotic resistance in bacterial populations requires alternatives for classical treatment of infectious diseases and therefore drives the renewed interest in phage therapy. Methicillin resistant Staphylococcus aureus (MRSA) is a major problem in health care settings and live-stock breeding across the world. This research aims at a thorough microbiological, genomic, and proteomic characterization of S. aureus phage ISP, required for therapeutic applications. Host range screening of a large batch of S. aureus isolates and subsequent fingerprint and DNA microarray analysis of the isolates revealed a substantial activity of ISP against 86% of the isolates, including relevant MRSA strains. From a phage therapy perspective, the infection parameters and the frequency of bacterial mutations conferring ISP resistance were determined. Further, ISP was proven to be stable in relevant in vivo conditions and subcutaneous as well as nasal and oral ISP administration to rabbits appeared to cause no adverse effects. ISP encodes 215 gene products on its 138,339 bp genome, 22 of which were confirmed as structural proteins using tandem electrospray ionization-mass spectrometry (ESI-MS/MS), and shares strong sequence homology with the 'Twort-like viruses'. No toxic or virulence-associated proteins were observed. The microbiological and molecular characterization of ISP supports its application in a phage cocktail for therapeutic purposes.

We describe the small-scale, laboratory-based, production and quality control of a cocktail, consisting of exclusively lytic bacteriophages, designed for the treatment of Pseudomonas aeruginosa and Staphylococcus aureus infections in burn wound patients. Based on successive selection rounds three bacteriophages were retained from an initial pool of 82 P. aeruginosa and 8 S. aureus bacteriophages, specific for prevalent P. aeruginosa and S. aureus strains in the Burn Centre of the Queen Astrid Military Hospital in Brussels, Belgium. This cocktail, consisting of P. aeruginosa phages 14/1 (Myoviridae) and PNM (Podoviridae) and S. aureus phage ISP (Myoviridae) was produced and purified of endotoxin. Quality control included Stability (shelf life), determination of pyrogenicity, sterility and cytotoxicity, confirmation of the absence of temperate bacteriophages and transmission electron microscopy-based confirmation of the presence of the expected virion morphologic particles as well as of their specific interaction with the target bacteria. Bacteriophage genome and proteome analysis confirmed the lytic nature of the bacteriophages, the absence of toxin-coding genes and showed that the selected phages 14/1, PNM and ISP are close relatives of respectively F8, phiKMV and phage G1. The bacteriophage cocktail is currently being evaluated in a pilot clinical study cleared by a leading Medical Ethical Committee.