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Objectives: This study investigated antimicrobial susceptibility and genomic profiling of S. enterica isolated from bloodstream infections at a tertiary referral hospital in Lusaka, Zambia, 2018-2019.Method: This was a prospective hospital-based study involving routine blood culture samples submitted to the microbiology laboratory at the University Teaching Hospital. Identification of S. enterica and determination of antimicrobial susceptibility profiles was achieved through conventional and automated methods. Whole-genome sequencing (WGS) was conducted, and the sequence data outputs were processed for species identification, serotype determination, multilocus sequence typing (MLST) profile determination, identification of antimicrobial resistance determinants, and phylogeny.Results: Seventy-six Salmonella enterica were isolated and 64 isolates underwent WGS. Salmonella Typhi (72%) was the most prevalent serotype. Notable was the occurrence of invasive non-typhoidal Salmonella Typhimurium ST313 (3%), resistance to cephalosporins (4%) and ciprofloxacin (5%), multidrug resistance (46%), and reduced susceptibility to ciprofloxacin (30%) and imipenem (3%). Phylogenetic cluster analysis showed multiple Salmonella serovars with a wide range of genetic diversity.Conclusion: The genetic diversity of Salmonella Typhi, high prevalence of multidrug resistance, and the emergence of ciprofloxacin and cephalosporin resistance warrants improved hygiene and water and sanitation provision, continued surveillance to apprise antibiograms and inform policy, and the introduction of the typhoid conjugate vaccine.

2AbstractThe antimicrobial resistance ofNeisseria gonorrhoeato all classes of current available antibiotics is a global concern. National surveillance programmes monitoring the susceptibility profiles ofNeisseria gonorrhoeaehardly exist in resource constraint settings. Therefore, little is known about the antimicrobial susceptibility profile and associated genetic resistance mechanisms ofN. gonorrhoeaein Madagascar. We report susceptibility data ofN. gonorrhoeaeisolates obtained by the medical laboratory of the Institut Pasteur de Madagascar, from 2014 -2020. In addition, we present data on the antimicrobial resistance mechanisms and antimicrobial resistance profile of a subset of isolates (N=46), including all isolates available of 2020. Over the study period, ceftriaxone resistant isolates exceeding the threshold of 5% in 2017 and 2020, were reported. Of the subset of re-tested isolates, all were found susceptible to ceftriaxone, azithromycin, and spectinomycin. Conversely, all isolates were resistant to ciprofloxacin and the majority was also resistant to penicillin and tetracycline. None of the isolates carried the mosaicpenAgene and chromosomal mutations associated to the antibiotic resistance ingyrA, parC, penA, ponA, porBandmtrRgenes were detected. The high rate of resistance to Penicillin and Tetracycline is explained by the presence of β-lactamaseblaTEMandtetMgenes, respectively, a plasmid mediated resistance. We found a high number of circulating multilocus sequence types. Almost half of them were new types, and one of them was among the four most predominant sequence types. Our report provides a detailed dataset obtained through phenotypic and genotypic methods which will serve as baseline for future surveillance ofN. gonorrhoeaein our setting, and Madagascar.1.5RepositoriesThis Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the Accession BioProject PRJNA929018.3Impact statementNeisseria gonorrhoeaeis becoming increasingly resistant to all classes of antibiotics available for infections treatment.Resource constraint settings encounter difficulties in implementing surveillance of the antimicrobial susceptibility ofN. gonorrhoeae. We report here antimicrobial susceptibility results from gonorrhoea among patients consulting a medical laboratory in Antananarivo, Madagascar in 2014–2020. We used whole-genome sequencing to identify resistance mechanisms in a subset of isolates including all viable isolates of 2020.We report multilocus sequence types and discuss phenotypes and genotypes according to the phylogenetic analysis of the isolates. The susceptibility results are the first in a decade to be reported. We set the baseline to study further the evolution and transmission ofN. gonorrhoeaeresistance mechanisms and genotypes in general. Our report will enable improving surveillance ofNeisseria gonorrhoeaein Madagascar and Africa.Overall, it will contribute to the global, regional, and national surveillance ofN. gonorrhoeae. In addition, it may set a benchmark for implementation in other settings facing barriers implementing phenotypic resistance surveillance ofN. gonorrhoeae.4Data summaryThe source code of the Tormes pipeline used in this study is also available on Github:https://github.com/nmquijada/tormes.The SRA sequences have been deposited in the NCBI SRA database under accession numbers SRR23260223 and SRR23260193.The genome assemblies can be accessed using the accession numbers: SAMN32949360 - SAMN32949405.The MLST genes of all isolates can be accessed through this link:https://doi.org/10.5281/zenodo.7581537The Perl scripts used for a quality filtering of the assemblies which is to remove small contigs can be accessed through the following link:https://github.com/Alainnoah/Remove-Small-Contigs-Draft-Assemblies.Genomic analysis with metadata in Pathogenwatch could be accessed with this link:https://pathogen.watch/collection/f7t4wjtjlybh-mdgwhofaA visualization of genomic epidemiology of our isolates with Microreact can be accessed with this link:https://microreact.org/project/7FADQYwhm5h1zJyJTynM6R-unnamed-projectA supplementary material in an Excel sheet summarizing the lists of plasmids used for alignment (Table S1), the statistics of the sequences (Table S2), and the minimum inhibitory concentration values ofN. gonorrhoeaeisolates and their antimicrobial resistance genetic mechanisms (Table S3) is available through this link:https://doi.org/10.6084/m9.figshare.21973511

2.AbstractSalmonella Enteritidis is one of the most commonly reported serovars of non-typhoidal Salmonella causing human disease and is responsible for both gastroenteritis and invasive non-typhoidal Salmonella (iNTS) disease worldwide. Whole-genome sequence (WGS) comparison of Salmonella Enteritidis isolates from across the world have identified three distinct clades, named Global Epidemic, Central/East African and West African, all of which have been implicated in epidemics: the Global Epidemic clade was linked to poultry-associated gastroenteritis, while the two African clades were related to iNTS disease. However, the distribution and epidemiology of these clades across Africa is poorly understood because identification of these clades currently requires whole genome sequencing capacity. Here, we report a sensitive, time- and cost-effective real-time PCR assay capable of differentiating between the Salmonella Enteritidis clades to facilitate surveillance and to inform public health responses.3.Impact statementChallenges in the diagnosis and treatment of invasive Salmonella Enteritidis (S. Enteritidis) bloodstream infections in sub–Saharan Africa are responsible for a case fatality rate of approximately 15% (12). It is important to identify distinct clades of S. Enteritidis in diagnostic laboratories in the African setting to determine whether particular outbreaks are associated with different health outcomes. Here, we have described the development of a high-quality molecular classification assay for the clade-typing of S. Enteritidis that is ideal for use in public health laboratories in resource-limited settings.

Introduction In 2009 and 2010, more than 6,000 cholera cases were recorded during these outbreaks with more than 80% of cases recorded in Lusaka province. After a five-year break, in 2016 an outbreak occurred in Lusaka, causing more than 1,000 cases of cholera. This study will strengthen the epidemiological information on the changing characteristics of the cholera outbreaks, for treatment, prevention and control of the disease. Methods This was a laboratory-based descriptive cross-sectional study conducted at the University Teaching Hospital in Lusaka, Zambia. A total of 83 V. cholerae O1 isolates were characterised by biochemical testing, serotyping, antimicrobial susceptibility testing, and macrorestriction analysis using Pulsed-Field Gel Electrophoresis. Results Macrorestriction analysis of the isolates demonstrated high genetic diversity among the isolates with 16 different patterns. The largest pattern comprised 9 isolates while the smallest one had 1 isolate. 2009 and 2010 isolates were highly resistant to nalidixic acid and cotrimoxazole, but highly sensitive to azithromycin and ampicillin. Of the fifty-two isolates from the 2016 cholera outbreak, 90% (47) were sensitive to cotrimoxazole, 94% (49) to tetracycline, and 98% (51) to azithromycin, while 98% (51) were resistant to nalidixic acid and 31(60%) to ampicillin. Conclusion macrorestriction analysis demonstrated high genetic diversity among the V. cholerae O1 strains, suggesting that these isolates were probably not from a similar source. This study also revealed the emergence of multidrug resistance among the 2016 V. cholerae outbreak isolates but were susceptible to cotrimoxazole, tetracycline, and azithromycin, which can be used for treatment of the cholera cases.

Introduction: Salmonella is well recognized as an aetiological agent of gastrointestinal and diarrhoeal disease. Salmonella enterica serotype Enteritidis (Salmonella Enteritidis) is one of the commonest serotypes associated with foodborne illness. In South Africa, we compared Salmonella Enteritidis strains isolated from humans with gastroenteritis and strains isolated from captive wild animals, between June 2011 and July 2012. Methodology: Bacteria were phenotypically characterized using standard microbiological techniques. Genotypic relatedness of isolates was investigated by pulsed-field gel electrophoresis (PFGE) analysis. Results: a diversity of 27 PFGE patterns amongst 196 human non-invasive isolates was shown; two PFGE patterns predominated and accounted for 74% of all human isolates. Human isolates showed a 12% prevalence rate for nalidixic acid resistance. Animal isolates from 5 different sources were investigated. With the exception of an isolate from a ground hornbill, all animal isolates (jaguar, crocodile, lion and poultry) showed PFGE pattern matches to a human isolate. Animal isolates showed susceptibility to all antimicrobial agents tested, with the exception of nalidixic acid resistance in isolates from the lion and poultry source. Conclusions: Our data showed similarities between Salmonella Enteritidis strains isolated from humans and captive wild animals, suggesting a probable common source for strains from humans and animals.

We report a technique useful for transformation experiments involving bacteria naturally competent for DNA transformation. It allows the selection of antibiotic-susceptible transformants following the transformation of a resistant strain with an antibiotic susceptibility gene. We show the effectiveness of this technique through the selection of penicillin-susceptible (MIC, 0.03 microg/ml) transformants following the transformation of a penicillin-resistant (MIC, 16 microg/ml) pneumococcal strain with a penicillin-susceptibility gene.