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Background: Enteroaggregative Escherichia coli (EAEC) is a predominant but neglected enteric pathogen implicated in infantile diarrhoea and nutrient malabsorption. There are no non-antibiotic approaches to dealing with persistent infection by these exceptional colonizers, which form copious biofilms. We screened the Medicines for Malaria Venture Pathogen Box for chemical entities that inhibit EAEC biofilm formation. Methodology: We used EAEC strains, 042 and MND005E in a medium-throughput crystal violet-based antibiofilm screen. Hits were confirmed in concentration-dependence, growth kinetic and time course assays and activity spectra were determined against a panel of 25 other EAEC strains. Antibiofilm activity against isogenic EAEC mutants, molecular docking simulations and comparative genomic analysis were used to identify the mechanism of action of one hit. Principal findings: In all, five compounds (1.25%) reproducibly inhibited biofilm accumulation by at least one strain by 30–85% while inhibiting growth by under 10%. Hits exhibited potent antibiofilm activity at concentrations at least 10-fold lower than those reported for nitazoxanide, the only known EAEC biofilm inhibitor. Reflective of known EAEC heterogeneity, only one hit was active against both screen isolates, but three hits showed broad antibiofilm activity against a larger panel of strains. Mechanism of action studies point to the EAEC anti-aggregation protein (Aap), dispersin, as the target of compound MMV687800. Conclusions: This study identified five compounds, not previously described as anti-adhesins or Gram-negative antibacterials, with significant EAEC antibiofilm activity. Molecule, MMV687800 targets the EAEC Aap. In vitro small-molecule inhibition of EAEC colonization opens a way to new therapeutic approaches against EAEC infection. Author summary: Diarrhoea accounts for over half a million deaths in children under five annually. It additionally contributes to childhood malnutrition as well as growth and development deficiencies, particularly in low-income countries. Enteroaggregative Escherichia coli (EAEC) causes diarrhoea that is often persistent and can also contribute to growth deficiencies in young children. EAEC is a neglected pathogen that is often resistant to antimicrobial drugs. Small molecules that block EAEC colonization may hold the key to interfering with EAEC disease without promoting antimicrobial resistance. We screened the Medicines for Malaria Ventures Pathogen Box for chemicals that can interfere with EAEC biofilm formation, a key colonization indicator. Our screen identified five biofilm-inhibiting molecules that did not interfere with bacterial viability and therefore are unlikely to exert strong pressure for resistance. Molecular biology and computational investigations point to the EAEC anti-aggregative protein, also known as dispersin, as a possible target for one of these hit molecules. Optimizing EAEC antibiofilm hits will create templates that can be employed for resolving EAEC diarrhoea and related infections. [ABSTRACT FROM AUTHOR]

Health research in low-income settings must prioritize sustainability to truly impact target diseases in the long term. Here, I satirically summarize how biomedical investigators from high-income countries can collaboratively work to (not) accomplish this. Health research in low‐income settings must prioritize sustainability in order to truly impact target diseases in the long term; this Perspective article satirically summarizes how biomedical investigators from high-income countries can collaboratively work to (not) accomplish this. [ABSTRACT FROM AUTHOR]

Salmonella is an important human pathogen and poultry products constitute an important source of human infections. This study investigated prevalence; identified serotypes based on whole genome sequence, described spatial distribution of Salmonella serotypes and predicted risk factors that could influence the prevalence of Salmonella infection in commercial poultry farms in Nigeria. A cross sectional approach was employed to collect 558 pooled shoe socks and dust samples from 165 commercial poultry farms in North West Nigeria. On-farm visitation questionnaires were administered to obtain information on farm management practices in order to assess risk factors for Salmonella prevalence. Salmonella was identified by culture, biotyping, serology and polymerase chain reaction (PCR). PCR confirmed isolates were paired-end Illumina- sequenced. Following de novo genome assembly, draft genomes were used to obtain serotypes by SeqSero2 and SISTR pipeline and sequence types by SISTR and Enterobase. Risk factor analysis was performed using the logit model. A farm prevalence of 47.9% (CI95 [40.3–55.5]) for Salmonella was observed, with a sample level prevalence of 15.9% (CI95 [12.9–18.9]). Twenty-three different serotypes were identified, with S. Kentucky and S. Isangi as the most prevalent (32.9% and 11%). Serotypes showed some geographic variation. Salmonella detection was strongly associated with disposal of poultry waste and with presence of other livestock on the farm. Salmonella was commonly detected on commercial poultry farms in North West Nigeria and S. Kentucky was found to be ubiquitous in the farms. [ABSTRACT FROM AUTHOR]

Background: Antimicrobial resistance among enteric bacteria in Africa is increasingly mediated by integrons on horizontally acquired genetic elements. There have been recent reports of such elements in invasive pathogens across Africa, but very little is known about the faecal reservoir of integron-borne genes. Methods and findings: We screened 1098 faecal Escherichia coli isolates from 134 mother-child pairs for integron cassettes by PCR using primers that anneal to the 5’ and 3’ conserved ends of the cassette regions and for plasmid replicons. Genetic relatedness of isolates was determined by flagellin and multi-locus sequence typing. Integron cassettes were amplified in 410 (37.5%) isolates and were significantly associated with resistance to trimethoprim and multiple resistance. Ten cassette combinations were found in class 1 and two in class 2 integrons. The most common class 1 cassette configurations were single aadA1 (23.4%), dfrA7 (18.3%) and dfrA5 (14.4%). Class 2 cassette configurations were all either dfrA1-satI-aadA1 (n = 31, 7.6%) or dfrA1-satI (n = 13, 3.2%). A dfr cassette was detected in 294 (31.1%) of trimethoprim resistant strains and an aadA cassette in 242 (23%) of streptomycin resistant strains. Strains bearing integrons carried a wide range of plasmid replicons of which FIB/Y (n = 169; 41.2%) was the most frequently detected. Nine isolates from five different individuals carried the dfrA17-aadA5-bearing ST69 clonal group A (CGA). The same integron cassette combination was identified from multiple distinct isolates within the same host and between four mother-child pairs. Conclusions: Integrons are important determinants of resistance in faecal E. coli. Plasmids in integron-containing strains may contribute to dispersing resistance genes. There is a need for improved surveillance for resistance and its mechanisms of dissemination and persistence and mobility of resistance genes in the community and clinical settings. [ABSTRACT FROM AUTHOR]

Enteroaggregative E. coli (EAEC) is an established diarrhoeagenic pathotype. The association with virulence gene content and ability to cause disease has been studied but little is known about the population structure of EAEC and how this pathotype evolved. Analysis by Multi Locus Sequence Typing of 564 EAEC isolates from cases and controls in Bangladesh, Nigeria and the UK spanning the past 29 years, revealed multiple successful lineages of EAEC. The population structure of EAEC indicates some clusters are statistically associated with disease or carriage, further highlighting the heterogeneous nature of this group of organisms. Different clusters have evolved independently as a result of both mutational and recombination events; the EAEC phenotype is distributed throughout the population of E. coli. [ABSTRACT FROM AUTHOR]

The plasmid-encoded quinolone resistance gene qnrS1 was recently found to be commonly associated with ciprofloxacin resistance in Nigeria. We mapped the qnrS1 gene from an Escherichia coli isolate obtained in Nigeria to a 43.5 Kb IncX2 plasmid. The plasmid, pEBG1, was sufficient to confer ciprofloxacin non-susceptibility, as well as tetracycline and trimethoprim resistance, on E. coli K-12. Deletion analysis confirmed that qnrS1 accounted for all the ciprofloxacin non-suceptibility conferred by pEBG1 and tetracycline and trimethoprim resistance could be attributed to tetAR and dfrA14 genes respectively. While it contained a complete IncX conjugation system, pEBG1 was not self-transmissible likely due to an IS3 element inserted between the pilX5 and pilX6 genes. The plasmid was however efficiently mobilizable. pEBG1 was most similar to another qnrS1-bearing IncX2 plasmid from Nigeria, but both plasmids acquired qnrS1 independently and differ in their content of other resistance genes. Screening qnrS1–positive isolates from other individuals in Nigeria revealed that they carried neither pEBG1 nor pNGX2-QnrS1 but that IncX plasmids were prevalent. This study demonstrates that the IncX backbone is a flexible platform that has contributed to qnrS1 dissemination in Nigeria. [ABSTRACT FROM AUTHOR]

Background: The current millennium has seen a steep rise in the number, size and case-fatalities of cholera outbreaks in many African countries. Over 40,000 cases of cholera were reported from Nigeria in 2010. Variants of Vibrio cholerae O1 El Tor biotype have emerged but very little is known about strains causing cholera outbreaks in West Africa, which is crucial for the implementation of interventions to control epidemic cholera. Methodology/Principal Findings: V. cholerae isolates from outbreaks of acute watery diarrhea in Nigeria from December, 2009 to October, 2010 were identified by standard culture methods. Fifteen O1 and five non-O1/non-O139 strains were analyzed; PCR and sequencing targeted regions associated with virulence, resistance and biotype were performed. We also studied genetic interrelatedness among the strains by multilocus sequence analysis and pulsed-field gel electrophoresis. The antibiotic susceptibility was tested by the disk diffusion method and E-test. We found that multidrug resistant atypical El Tor strains, with reduced susceptibility to ciprofloxacin and chloramphenicol, characterized by the presence of the SXT element, and gyrASer83Ile/parCSer85Leu alleles as well CTX phage and TCP cluster characterized by rstRElTor, ctxB-7 and tcpACIRS alleles, respectively, were largely responsible for cholera outbreaks in 2009 and 2010. We also identified and characterized a V. cholerae non-O1/non-O139 lineage from cholera-like diarrhea cases in Nigeria. Conclusions/Significance: The recent Nigeria outbreaks have been determined by multidrug resistant atypical El Tor and non-O1/non-O139 V. cholerae strains, and it seems that the typical El Tor, from the beginning of seventh cholera pandemic, is no longer epidemic/endemic in this country. This scenario is similar to the East Africa, Asia and Caribbean countries. The detection of a highly virulent, antimicrobial resistant lineage in Nigeria is worrisome and points to a need for vaccine-based control of the disease. This study has also revealed the putative importance of non-O1/non-O139 V. cholerae in diarrheal disease in Nigeria. Author Summary: Cholera is acute watery diarrhoea, severely dehydrating, caused by Vibrio cholerae, a bacterium ubiquitous in aquatic environments. Cholera is a global threat, particularly, in areas where sanitary conditions, such as drinking water and sewage, are not available. Seven cholera pandemics, all originating in Asia, occurred. The ongoing pandemic, the 7th, has been caused by V. cholerae El Tor biotype. Recently, El Tor has undergone genetic changes and the strains being referred to as "atypical" El Tor are rapidly replacing the original El Tor in many areas. The atypical El Tor is characterized by multi-antibiotic resistance and changes in the major virulence determinants. Cholera caused by atypical strains may be more clinically severe. In Africa, cholera outbreaks are occurring with increasing frequency and severity, as demonstrated by the recent major outbreaks in Nigeria, Angola, Mozambique and Zimbabwe. Here, we performed a comprehensive characterization of V. cholerae isolated from different recent outbreaks in Nigeria. Our results show that cholera outbreaks in Nigeria are driven by atypical El Tor strains, as worldwide. [ABSTRACT FROM AUTHOR]

Background: Antimicrobial resistance is a growing international problem. We observed a 50% increase in the prevalence of trimethoprim resistance among fecal Escherichia coli from healthy Nigerian students between 1998 and 2005, a trend to increase that continued in 2009. Methods and Findings: A PCR-based screen revealed that 131 (43.1%) of isolates obtained in Nigeria in 2005 and 2009 carried integron-borne dfrA cassettes. In the case of 67 (51.1%) of these isolates, the cassette was a class 1-integron-borne dfrA7 gene, which has been reported at high prevalence from E. coli isolates from other parts of Africa. Complete sequencing of a 27 Kb dfrA7-bearing plasmid from one isolate located the dfrA7 gene within a Tn21-type transposon. The transposon also contained an IS26-derived bla/sul/str element, encoding resistance to b-lactams, sulphonamides and streptomycin, and mercury resistance genes. Although the plasmid backbone was only found in 12 (5.8%) of trimethoprimresistant isolates, dfrA7 and other transposon-borne genes were detected in 14 (16.3%) and 32 (26.3%) of trimethoprim resistant isolates collected in Nigeria in 2005 and 2009, respectively. Additionally, 37 (19.3%) of trimethoprim-resistant E. coli isolates collected between 2006 and 2008 from Ghana were positive for the dfrA7 and a transposon marker, but only 4 (2.1%) harbored the plasmid backbone. Conclusions: Our data point to transposition as a principal mechanism for disseminating dfrA7 among E. coli from Nigeria and Ghana. On-going intensive use of the affordable broad-spectrum antibacterials is likely to promote selective success of a highly prevalent transposable element in West Africa. [ABSTRACT FROM AUTHOR]

Background: Enteroaggregative Escherichia coli (EAEC) are defined by their stacked-brick adherence pattern to human epithelial cells. There is no all-encompassing genetic marker for EAEC. The category is commonly implicated in diarrhea but research is hampered by perplexing heterogeneity. Methodology/Principal Findings: To identify key EAEC lineages, we applied multilocus sequence typing to 126 E. coli isolates from a Nigerian case-control study that showed aggregative adherence in the HEp-2 adherence assay, and 24 other EAEC strains from diverse locations. EAEC largely belonged to the A, B1 and D phylogenetic groups and only 7 (4.6%) isolates were in the B2 cluster. As many as 96 sequence types (STs) were identified but 60 (40%) of the EAEC strains belong to or are double locus variants of STs 10, 31, and 394. The remainder did not belong to predominant complexes. The most common ST complex, with predicted ancestor ST10, included 32 (21.3%) of the isolates. Significant age-related distribution suggests that weaned children in Nigeria are at risk for diarrhea from of ST10-complex EAEC. Phylogenetic group D EAEC strains, predominantly from ST31- and ST394 complexes, represented 38 (25.3%) of all isolates, include genome-sequenced strain 042, and possessed conserved chromosomal loci. Conclusions/Significance: We have developed a molecular phylogenetic framework, which demonstrates that although grouped by a shared phenotype, the category of 'EAEC' encompasses multiple pathogenic lineages. Principal among isolates from Nigeria were ST10-complex EAEC that were associated with diarrhea in children over one year and ECOR D strains that share horizontally acquired loci. [ABSTRACT FROM AUTHOR]

Understanding the contribution of different diarrhoeagenic Escherichia coli pathotypes to disease burden is critical to mapping risk and informing vaccine development. Targeting select virulence genes by PCR is the diagnostic approach of choice in high-burden, least-resourced African settings. We compared the performance of a commonly-used multiplex protocol to whole genome sequencing (WGS). PCR was applied to 3,815 E. coli isolates from 120 children with diarrhoea and 357 healthy controls. Three or more isolates per specimen were also Illumina-sequenced. Following quality assurance, ARIBA and Virulencefinder database were used to identify virulence targets. Root cause analysis of deviant PCR results was performed by examining target sensitivity using BLAST, Sanger sequencing false-positive amplicons, and identifying lineages prone to false-positivity using in-silico multilocus sequence typing and a Single Nucleotide Polymorphism phylogeny constructed using IQTree. The sensitivity and positive predictive value of PCR compared to WGS ranged from 0-77.8% while specificity ranged from 74.5-94.7% for different pathotypes. WGS identified more enteroaggregative E. coli (EAEC), fewer enterotoxigenic E. coli (ETEC) and none of the Shiga toxin-producing E. coli detected by PCR, painting a considerably different epidemiological picture. Use of the CVD432 target resulted in EAEC under-detection, and enteropathogenic E. coli eae primers mismatched more recently described intimin alleles common in our setting. False positive ETEC were over-represented among West Africa-predominant ST8746 complex strains. PCR precision varies with pathogen genome so primers optimized for use in one part of the world may have noticeably lower sensitivity and specificity in settings where different pathogen lineages predominate., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Akinlabi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Antimicrobials are only indicated in acute childhood diarrhea if it is invasive or persistent. Rapid screening for invasive diarrhea can therefore inform treatment decisions but pathogen identification by culture is slow, expensive and cumbersome. This study aimed to assess the diagnostic utility of stool microscopy and immunochromatographic fecal occult blood test (FOBT) kits for identifying invasive or potentially invasive diarrhea in Ibadan, Nigeria. Fecal specimens from 46 children under 5 years old with diarrhea, collected as part of ongoing case-control studies, were subjected to stool microscopy for erythrocytes and leucocytes, and FOBT using the innovator's product and four locally procurable generic immunochromatographic kits, each according to manufacturers' instructions. Stool specimens were cultured for enteric bacterial pathogens using standard procedures. Presumptive pathogen isolates were identified biochemically and by PCR, and then confirmed by whole genome sequencing. Shigella, enteroinvasive Escherichia coli and Yersinia, pathogens that invariably cause invasive diarrhea, were detected in five of 46 specimens. Occult blood detection by microscopy was 55.6% sensitive and 78.4% specific, while the innovator's FOBT product was respectively 62.5% and 81.6% sensitive and specific compared to strict invasive pathogen recovery. Microscopy and FOBT testing were less sensitive in identifying specimens that contained pathogens that do not always elicit invasive diarrhea. Generic FOBT tests compared well with the innovator's product. Microscopy and FOBT testing have some value for delineating likely invasive diarrheas. They could inform treatment and serve as early warning indicators for dysentery outbreaks in resource limited settings. Inexpensive, generic FOBT kits that are locally procurable in Nigeria performed as well as the innovator's product., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kwasi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Salmonellosis causes significant morbidity and mortality in Africa. Information on lineages of invasive Salmonella circulating in Nigeria is sparse., Methods: Salmonella enterica isolated from blood (n = 60) and cerebrospinal fluid (CSF, n = 3) between 2016 and 2020 from five tertiary hospitals in southwest Nigeria were antimicrobial susceptibility-tested and Illumina-sequenced. Genomes were analysed using publicly-available bioinformatic tools., Results: Isolates and sequence types (STs) from blood were S. Typhi [ST1, n = 1 and ST2, n = 43] and invasive non-typhoidal Salmonella (iNTS) (S. Enteritidis [ST11, n = 7], S. Durham [ST10, n = 2], S. Rissen [ST8756, n = 2], S. Chester [ST2063, n = 1], S. Dublin [ST10, n = 1], S. Infantis [ST603, n = 1], S. Telelkebir [ST8757, n = 1] and S. Typhimurium [ST313, n = 1]). S. Typhi ST2 (n = 2) and S. Adabraka ST8757 (n = 1) were recovered from CSF. Most S. Typhi belonged to genotype 3.1.1 (n = 44), carried an IncY plasmid, had several antibiotic resistance genes (ARGs) including blaTEM-1 (n = 38), aph(6)-Id (n = 32), tet(A) (n = 33), sul2 (n = 32), dfrA14 (n = 30) as well as quinolone resistance-conferring gyrA_S83Y single-nucleotide polymorphisms (n = 37). All S. Enteritidis harboured aph(3")-Ib, blaTEM-1, catA1, dfrA7, sul1, sul2, tet(B) genes, and a single ARG, qnrB19, was detected in S. Telelkebir. Typhoidal toxins cdtB, pltA and pltB were detected in S. Typhi, Rissen, Chester, and Telelkebir., Conclusion: Most invasive salmonelloses in southwest Nigeria are vaccine-preventable infections due to multidrug-resistant, West African dominant S. Typhi lineage 3.1.1. Invasive NTS serovars, including some harbouring typhoidal toxin or resistance genes, represented a third of the isolates emphasizing the need for better diagnosis and surveillance., Competing Interests: The authors have declared that no competing interests exist.