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BackgroundKlebsiella pneumoniae is a World Health Organization high-priority antibiotic-resistant pathogen. However, little is known about Klebsiella lineages circulating in Nigeria.MethodsWe performed whole-genome sequencing (WGS) of 141 Klebsiella isolated between 2016 and 2018 from clinical specimens at 3 antimicrobial-resistance (AMR) sentinel surveillance tertiary hospitals in southwestern Nigeria. We conducted in silico multilocus sequence typing; AMR gene, virulence gene, plasmid, and K and O loci profiling; as well as phylogenetic analyses, using publicly available tools and Nextflow pipelines.ResultsPhylogenetic analysis revealed that the majority of the 134 K. pneumoniae and 5 K. quasipneumoniae isolates from Nigeria characterized are closely related to globally disseminated multidrug-resistant clones. Of the 39 K. pneumoniae sequence types (STs) identified, the most common were ST307 (15%), ST5241 (12%), ST15 (~9%), and ST25 (~6%). ST5241, 1 of 10 novel STs detected, is a single locus variant of ST636 carrying dfrA14, tetD, qnrS, and oqxAB resistance genes. The extended-spectrum β-lactamase (ESBL) gene blaCTX_M-15 was seen in 72% of K. pneumoniae genomes, while 8% encoded a carbapenemase. No isolate carried a combination of carbapenemase-producing genes. Four likely outbreak clusters from 1 facility, within STs 17, 25, 307, and 5241, were ESBL but not carbapenemase-bearing clones.ConclusionsThis study uncovered known and novel K. pneumoniae lineages circulating in 3 hospitals in Southwest Nigeria that include multidrug-resistant ESBL producers. Carbapenemase-producing isolates remain uncommon. WGS retrospectively identified outbreak clusters, pointing to the value of genomic approaches in AMR surveillance for improving infection prevention and control in Nigerian hospitals.

BackgroundKlebsiella pneumoniae is a critically important pathogen in the Philippines. Isolates are commonly resistant to at least two classes of antibiotics, yet mechanisms and spread of its resistance are not well studied.MethodsA retrospective sequencing survey was performed on carbapenem-, extended spectrum beta-lactam- and cephalosporin-resistant Klebsiella pneumoniae isolated at 20 antimicrobial resistance (AMR) surveillance sentinel sites from 2015-2017. We characterized 259 isolates using biochemical methods, antimicrobial susceptibility testing, and whole genome sequencing (WGS). Known AMR mechanisms were identified. Potential outbreaks were investigated by detecting clusters from epidemiologic, phenotypic and genome-derived data.ResultsPrevalent AMR mechanisms detected include blaCTX-M-15 (76.8%) and blaNDM-1 (37.5%). An epidemic IncFII(Yp) plasmid carrying blaNDM-1 was also detected in 46 isolates from 6 sentinel sites and 14 different sequence types (ST). This plasmid was also identified as the main vehicle of carbapenem resistance in 2 previously unrecognized local outbreaks of ST348 and ST283 at 2 different sentinel sites. A third local outbreak of ST397 was also identified but without the IncFII(Yp) plasmid. Isolates in each outbreak site showed identical STs, K- and O-loci, and similar resistance profiles and AMR genes. All outbreak isolates were collected from blood of children aged ConclusionWGS provided an in-depth understanding of the epidemiology of AMR in the Philippines, which was not possible with only phenotypic and epidemiologic data. The identification of three previously unrecognized Klebsiella outbreaks highlights the utility of WGS in outbreak detection, as well as its importance in public health and in implementing infection control programs.summaryWhole genome sequencing identified three distinct previously unrecognized local outbreaks in a retrospective study in the Philippines, along with an epidemic plasmid carrying antimicrobial resistance genes, highlighting its importance in antimicrobial resistance surveillance, outbreak detection and infection control.

Background Antimicrobial resistance (AMR) is a global health threat affecting treatment outcome in animals and humans. A pre-requisite for development of AMR reduction strategies is knowledge of antimicrobial use patterns, and how these affect resistance development. The aim of this study was to determine antimicrobial usage (AMU) and whether such usage was associated with AMR in Salmonella from poultry farms in Northwest Nigeria. Results Fifteen (37%) of antimicrobial products observed contained compounds that are of highest priority and critically important for human medicine. Broilers chicken consumed higher (28 ± 14 mg/kg active ingredients) amounts of antimicrobials compared to layers (13 ± 8 mg/kg) per week (p = 0.0009). Surprisingly, chickens raised under backyard system consumed higher amounts of antimicrobials (34 ± 7 mg/kg) than poultry in other systems (p = 0.02). High levels of resistance to tetracycline (58%), sulphonamides (65%), ciprofloxacin (46%) and gentamicin (42%) correlated with high farm level usage of these antimicrobials, and there was a strong correlation (r = 0.9) between farm usage and resistance of isolates to the same antimicrobials (p = 0.03). Conclusion High AMU, including use of highest priority critically important antimicrobials was observed at poultry farms in Northwest Nigeria. AMU correlated with high levels of resistance. Communication of prudent use of antimicrobials to farmers and regulation to obtain reduction in AMU should be a priority.

In this Supplement, we detail outputs of the National Institute for Health Research Global Health Research Unit on Genomic Surveillance of Antimicrobial Resistance project, covering practical implementation of whole-genome sequencing across our consortium, which consists of laboratories in Colombia, India, Nigeria, and the Philippines.

Objective Plasmids are key to antimicrobial resistance transmission among enteric bacteria. It is becoming increasingly clear that resistance genes alone do not account for the selective advantage of plasmids and bacterial strains that harbor them. Deletion of a 32 Kb fitness-conferring region of pMB2, a conjugative resistance plasmid, produced a hyper-autoaggregation phenotype in laboratory Escherichia coli. This study sought to determine the genetic basis for hyper-autoaggregation conferred by the pMB2-derived mini-plasmid. Results The 32 Kb fragment deleted from pMB2 included previously characterized nutrient acquisition genes as well as putative transposase and integrase genes, a 272 bp papB/ pefB-like gene, and several open-reading frames of unknown function. We cloned the papB/ pefB paralogue and found it sufficient to temper the hyper-autoaggregation phenotype. Hyper-autoaggregation conferred by the mini-plasmid did not occur in a fim-negative background. This study has identified and characterized a gene capable of down-regulating host adhesins and has shown that trans-acting papB/pefB paralogues can occur outside the context of an adhesin cluster. This plasmid-mediated modification of a bacterial host’s colonization program may optimize horizontal transfer of the mobile element bearing the genes.

Escherichia coli bloodstream infections are typically attributed to a limited number of lineages that carry virulence factors associated with invasion and, in recent years, invasive E. coli are increasingly multiply antimicrobial resistant. In Nigeria, E. coli is a common cause of bloodstream infections but the identity of circulating clones is largely unknown and surveillance of their antimicrobial resistance has been limited. We verified and whole genome-sequenced 68 bloodstream E. coli isolates recovered between 2016 and 2018 at three sentinel sites in southwestern Nigeria and susceptibility tested 67 of them. Resistance to antimicrobials commonly used in Nigeria was high, with 67(100%), 62 (92.5%), 53 (79%) and 37(55%) showing resistance to trimethoprim, ampicillin, ciprofloxacin and aminoglycosides, respectively. All the isolates were susceptible to carbapenems and colistin. The strain set included isolates from globally disseminated high risk clones including those belonging to ST12 (n=2), ST131 (n=12) and ST648 (n = 4). Twenty-three (33.82%) of the isolates clustered within two clades. The first of these consisted of ST131 strains, comprised of O16:H5 and O25:H4 sub-lineages. The second was an ST10-ST167 complex clade comprised of strains carrying capsular genes that may have originated in Klebsiella. We additionally determined that four temporally-associated ST90 strains from one sentinel were closely related enough to suggest that at least some of them represented a retrospectively detected outbreak cluster. Our data demonstrate that a broad repertoire of invasive E. coli isolates cause bloodstream infections in southwest Nigeria. In addition to pandemic lineages, particularly ST131, these include a previously undescribed lineage carrying an O-antigen cluster previously only reported from Klebsiella. Genomic surveillance is valuable for tracking these and other clones and for outbreak identification.Data SummaryPhylogenetic tree, clinical data, and epidemiological data were visualized using Microreact (https://microreact.org/project/hmj3KwxS1dmmFPCKFx6qeA-invasive-escherichia-coli-sw-nigeria-2016-2018). All the sequence data have been deposited in the ENA under the project ID PRJEB29739 (https://www.ebi.ac.uk/ena/browser/view/PRJEB29739). Accessions can be found in Table S6.

Background The relative contribution of bacterial infections to febrile disease is poorly understood in many African countries due to diagnostic limitations. This study screened pediatric and adult patients attending 4 healthcare facilities in Ibadan, Nigeria, for bacteremia and malaria parasitemia. Methods Febrile patients underwent clinical diagnosis, malaria parasite testing, and blood culture. Bacteria from positive blood cultures were isolated and speciated using biochemical and serological methods, and Salmonella subtyping was performed by polymerase chain reaction. Antimicrobial susceptibility was tested by disk diffusion. Results A total of 682 patients were recruited between 16 June and 16 October 2017; 467 (68.5%) were Conclusions The study demonstrates that bacteria were commonly recovered from febrile patients with or without malaria in this location. Focused and extended epidemiological studies are needed for the introduction of typhoid conjugate vaccines that have the potential to prevent a major cause of severe community-acquired febrile diseases in our locality.

BackgroundKlebsiella species, including the notable pathogen K. pneumoniae, are increasingly associated with antimicrobial resistance (AMR). Genome-based surveillance can inform interventions aimed at controlling AMR. However, its widespread implementation requires tools to streamline bioinformatic analyses and public health reporting.MethodsWe developed the web application Pathogenwatch, which implements analytics tailored to Klebsiella species for integration and visualization of genomic and epidemiological data. We populated Pathogenwatch with 16,537 public Klebsiella genomes to enable contextualization of user genomes. We demonstrated its features with 1,636 genomes from four low- and middle-income countries (LMICs) participating in the NIHR Global Health Research Unit (GHRU) on AMR.ResultsUsing Pathogenwatch, we found that GHRU genomes were dominated by a small number of epidemic drug-resistant clones of K. pneumoniae. However, differences in their distribution were observed (e.g. ST258/512 dominated in Colombia, ST231 in India, ST307 in Nigeria, ST147 in the Philippines). Phylogenetic analyses including public genomes for contextualization enabled retrospective monitoring of their spread. In particular, we identified hospital outbreaks, detected introductions from abroad, and uncovered clonal expansions associated with resistance and virulence genes. Assessment of loci encoding O-antigens and capsule in K. pneumoniae, which represent possible vaccine candidates, showed that three O-types (O1-O3) represented 88.9% of all genomes, whereas capsule types were much more diverse.ConclusionsPathogenwatch provides a free, accessible platform for real-time analysis of Klebsiella genomes to aid surveillance at local, national and global levels. We have improved representation of genomes from GHRU participant countries, further facilitating ongoing surveillance.40-word summaryPathogenwatch is a free web-application for analysis of Klebsiella genomes to aid surveillance at local, national and global levels. We improved the representation of genomes from middle-income countries through the Global Health Research Unit on AMR, further facilitating ongoing surveillance.FUNDINGThis work was supported by Official Development Assistance (ODA) funding from the National Institute of Health Research [grant number 16_136_111].This research was commissioned by the National Institute of Health Research using Official Development Assistance (ODA) funding. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health.CONFLICT OF INTERESTThe authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

IntroductionKlebsiella pneumoniae is a World Health Organization high-priority antibiotic-resistant pathogen. However, little is known about the population structure and evolution of Klebsiella circulating in Nigeria.MethodsWe performed whole genome sequencing (WGS) of 141 Klebsiella isolated between 2016 and 2018 from clinical specimens at 3 antimicrobial-resistance (AMR) sentinel surveillance tertiary hospitals in southwestern Nigeria. We conducted in silico multilocus sequence typing, AMR gene, virulence gene, plasmid, and K and O loci profiling, as well as phylogenetic analyses, using publicly available tools and Nextflow pipelines.ResultsPhylogenetic analysis revealed that the majority of the 134 K. pneumoniae and 5 K. quasipneumoniae isolates from Nigeria characterized are closely related to globally disseminated multidrug-resistant clones. Of the 39 K. pneumoniae sequence types (STs) identified, the most common were ST307 (15%), ST5241 (12%), ST15 (~9%), and ST25 (~6%). ST5241, one of 10 novel STs detected, is a single locus variant of ST636 carrying dfrA14, tetD, qnrS, and oqxAB resistance genes. The extended-spectrum β lactamase (ESBL) gene blaCTX_M-15 was seen in 72 % of K. pneumoniae genomes, while 8% encoded a carbapenemase. Four likely outbreak clusters from one facility, within STs 17, 25, 307, and 5241, were ESBL but not carbapenemase-bearing clones.ConclusionThis study uncovered known and novel K. pneumoniae lineages circulating in Nigeria that include multidrug-resistant ESBL producers. Carbapenemase-producing isolates remain uncommon. WGS retrospectively identified outbreak clusters, pointing to the value of genomic approaches in AMR surveillance for improving infection prevention and control in Nigerian hospitals.summaryWe performed whole genome sequencing (WGS) of 141 Klebsiella isolated in 2016-2018 at 3 antimicrobial-resistance (AMR) sentinel surveillance tertiary hospitals in southwestern Nigeria. This study uncovered known and novel K. pneumoniae lineages circulating in Nigeria that include multidrug-resistant ESBL producers.FUNDINGThis work was supported by Official Development Assistance (ODA) funding from the National Institute of Health Research [16/136/111: NIHR Global Health Research Unit on Genomic Surveillance of Antimicrobial Resistance].This research was commissioned by the National Institute of Health Research using Official Development Assistance (ODA) funding. INO is an African Research Leader supported by the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement that is also part of the EDCTP2 program supported by the European Union. The funders had no role in the content, crafting or submission of this paper. The views expressed in this publication are those of the authors and not necessarily those of the funders or their affiliates.CONFLICT OF INTERESTThe authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

Antimicrobial resistance is a global public health concern, and resistance genes in Salmonella, especially those located on mobile genetic elements, are part of the problem. This study used phenotypic and genomic methods to identify antimicrobial resistance and resistance genes, as well as the plasmids that bear them, in Salmonella isolates obtained from poultry in Nigeria. Seventy-four isolates were tested for susceptibility to eleven commonly used antimicrobials. Plasmid reconstruction and identification of resistance and virulence genes were performed with a draft genome using in silico approaches in parallel with plasmid extraction. Phenotypic resistance to ciprofloxacin (50.0%), gentamicin (48.6%), nalidixic acid (79.7%), sulphonamides (71.6%) and tetracycline (59.5%) was the most observed. Antibiotic resistance genes (ARGs) detected in genomes corresponded well with these observations. Commonly observed ARGs included sul1, sul2, sul3, tet (A), tet (M), qnrS1, qnrB19 and a variety of aminoglycoside-modifying genes, in addition to point mutations in the gyrA and parC genes. Multiple ARGs were predicted to be located on IncN and IncQ1 plasmids of S. Schwarzengrund and S. Muenster, and most qnrB19 genes were carried by Col (pHAD28) plasmids. Seventy-two percent (19/24) of S. Kentucky strains carried multidrug ARGs located in two distinct variants of Salmonella genomic island I. The majority of strains carried full SPI-1 and SPI-2 islands, suggesting full virulence potential This work was part-supported by an African Research Leader Award to INO from the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement that is also part of the EDCTP2 programme supported by the European Union. VG acknowledges Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia for her post-doctoral grant (Grant Number ED481B-2018/018) SI

Background Diarrhoeagenic Escherichia coli (DEC) infections are common in children in low-middle income countries (LMICs). However, detecting the various DEC pathotypes is complex as they cannot be differentiated by classical microbiology. We developed four multiplex real-time PCR assays were to detect virulence markers of six DEC pathotypes; specificity was tested using DEC controls and other enteric pathogens. PCR amplicons from the six E. coli pathotypes were purified and amplified to be used to optimize PCR reactions and to calculate reproducibility. After validation, these assays were applied to clinical samples from healthy and diarrhoeal Vietnamese children and associated with clinical data. Results The multiplex real-time PCRs were found to be reproducible, and specific. At least one DEC variant was detected in 34.7% (978/2815) of the faecal samples from diarrhoeal children; EAEC, EIEC and atypical EPEC were most frequent Notably, 41.2% (205/498) of samples from non-diarrhoeal children was positive with a DEC pathotype. In this population, only EIEC, which was detected in 34.3% (99/289) of diarrhoeal samples vs. 0.8% (4/498) non-diarrhoeal samples (p Conclusions This approach revealed high carriage rates of DEC pathotypes among Vietnamese children. We describe a novel diagnostic approach for DEC, which provides baseline data for future surveillance studies assessing DEC burden in LMICs.

While advances in genomic sequencing have highlighted significant strain variability between and within Salmonella serovars, only a few protein variants have been directly related to evolutionary adaptation for survival, such as host specificity or differential virulence. The current study investigated whether allelic variation of the Salmonella adhesin/invasin PagN influences bacterial interaction with their receptors. The Salmonella enterica, subspecies enterica serovar Typhi (S. Typhi) allelic variant of PagN was found to bind significantly better to different enterocytes as well as to the extracellular matrix protein laminin than did the major Salmonella enterica, subspecies enterica serovar Typhimurium (S. Typhimurium) allele. The two alleles differed at amino acid residues 49 and 109 in two of the four predicted PagN surface loops, and residue substitution analysis revealed that a glutamic acid at residue 49 increased the adhesive and invasive properties of S. Typhi PagN. PagN sequence comparisons from 542 Salmonella strains for six representative S. enterica serovars and S. diarizonae further supported the role of glutamic acid at residues 49 and 109 in optimizing adhesion to cells and laminin, as well as for cell invasion. In summary, this study characterized unique residues in allelic variants of a virulence factor that participates in the colonization and invasive properties of different Salmonella stains, subspecies and serovars.

In settings with limited resources and a wide range of possible etiologies, molecular technologies offer an effective solution for infectious disease diagnostics, because they are agile, fast and flexible. Health systems that routinely use molecular diagnostics will achieve economies of scale, maximize limited expertize and rapidly respond to new threats.

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.

Antimicrobial resistance is rapidly expanding, in a large part due to mobile genetic elements. We screened 94 fecal fluoroquinolone-resistant Escherichia coli isolates from Nigeria for six plasmid-mediated quinolone resistance (PMQR) genes. Sixteen isolates harbored at least one of the PMQR genes and four were positive for aac-6-Ib-cr. In one strain, aac-6-Ib-cr was mapped to a 125 Kb self-transmissible IncFII plasmid, pMB2, which also bears blaCTX-M-15, seven other functional resistance genes and multiple resistance pseudogenes. Laboratory strains carrying pMB2 grew faster than isogenic strains lacking the plasmid in both rich and minimal media. We excised a 32 Kb fragment containing transporter genes and several open-reading frames of unknown function. The resulting 93 Kb mini-plasmid conferred slower growth rates and lower fitness than wildtype pMB2. Trans-complementing the deletion with the cloned sitABCD genes confirmed that they accounted for the growth advantage conferred by pMB2 in iron-depleted media. pMB2 is a large plasmid with a flexible resistance region that contains loci that can account for evolutionary success in the absence of antimicrobials. Ancillary functions conferred by resistance plasmids can mediate their retention and transmissibility, worsening the trajectory for antimicrobial resistance and potentially circumventing efforts to contain resistance through restricted use.

Antimicrobial resistance is rapidly expanding, in a large part due to mobile genetic elements. We screened 94 fecal fluoroquinolone-resistantEscherichia coliisolates from Nigeria for six plasmid-mediated quinolone resistance (PMQR) genes. Sixteen isolates harbored at least one of the PMQR genes and four were positive foraac-6-Ib-cr. In one strain,aac-6-Ib-crwas mapped to a 125 Kb self-transmissible IncFII plasmid, pMB2, which also bearsblaCTX-M-15, seven other functional resistance genes and multiple resistance pseudogenes. We hypothesized that pMB2 had been selected by antimicrobials and that its large size would confer a growth disadvantage. However, laboratory strains carrying pMB2 grew at least as fast as isogenic strains lacking the plasmid in both rich and minimal media. We excised a 32 Kb fragment containing thesitABCDand another putative transporter,pefB, apapBhomolog, and several open-reading frames of unknown function. The resulting 93 Kb mini-plasmid conferred slower growth rates and lower fitness than wildtype pMB2. Trans-complementing the deletion with the clonedsitABCDgenes confirmed that they accounted for the growth advantage conferred by pMB2 in iron-depleted media. The mini-plasmid additionally conferred autoaggregation and was less transmissible and both phenotypes could be complemented with apefBclone. pMB2 is a large plasmid with a flexible resistance region that contains multiple loci that can account for evolutionary success in the absence of antimicrobials. Ancillary functions conferred by resistance plasmids can mediate their retention and transmissibility, worsening the trajectory for antimicrobial resistance and potentially circumventing efforts to contain resistance through restricted use.

The global spread of antimicrobial resistance (AMR) and lack of novel alternative treatments have been declared a global public health emergency by WHO. The greatest impact of AMR is experienced in resource-poor settings, because of lack of access to alternative antibiotics and because the prevalence of multidrug-resistant bacterial strains may be higher in low-income and middle-income countries (LMICs). Intelligent surveillance of AMR infections is key to informed policy decisions and public health interventions to counter AMR. Molecular surveillance using whole-genome sequencing (WGS) can be a valuable addition to phenotypic surveillance of AMR. WGS provides insights into the genetic basis of resistance mechanisms, as well as pathogen evolution and population dynamics at different spatial and temporal scales. Due to its high cost and complexity, WGS is currently mainly carried out in high-income countries. However, given its potential to inform national and international action plans against AMR, establishing WGS as a surveillance tool in LMICs will be important in order to produce a truly global picture. Here, we describe a roadmap for incorporating WGS into existing AMR surveillance frameworks, including WHO Global Antimicrobial Resistance Surveillance System, informed by our ongoing, practical experiences developing WGS surveillance systems in national reference laboratories in Colombia, India, Nigeria and the Philippines. Challenges and barriers to WGS in LMICs will be discussed together with a roadmap to possible solutions.

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.

Due to therapeutic challenges, hospital-acquired infections (HAIs) caused by Acinetobacter baumannii (HA-AB), particularly carbapenem-resistant strains (HA-CRAB) pose a serious health threat to patients worldwide. This systematic review sought to summarize recent data on the incidence and prevalence of HA-AB and HA-CRAB infections in the WHO-defined regions of Europe (EUR), Eastern Mediterranean (EMR) and Africa (AFR). A comprehensive literature search was performed using MEDLINE, EMBASE and GMI databases (01/2014-02/2019). Random-effects meta-analyses were performed to determine the pooled incidence of HA-AB and HA-CRAB infections as well as the proportions of A. baumannii among all HAIs. 24 studies from 3,340 records were included in this review (EUR: 16, EMR: 6, AFR: 2). The pooled estimates of incidence and incidence density of HA-AB infection in intensive care units (ICUs) were 56.5 (95% CI 33.9-92.8) cases per 1,000 patients and 4.4 (95% CI 2.9-6.6) cases per 1,000 patient days, respectively. Five studies conducted at a hospital-wide level or in specialized clinical departments/wards (ICU + non-ICU patients) showed HA-AB incidences between 0.85 and 5.6 cases per 1,000 patients. For carbapenem-resistant A. baumannii infections in ICUs, the pooled incidence and incidence density were 41.7 (95% CI 21.6-78.7) cases per 1,000 patients and 2.1 (95% CI 1.2-3.7) cases per 1,000 patient days, respectively. In ICUs, A. baumannii and carbapenem-resistant A. baumannii strains accounted for 20.9% (95% CI 16.5-26.2%) and 13.6% (95% CI 9.7-18.7%) of all HAIs, respectively. Our study highlights the persistent clinical significance of hospital-acquired A. baumannii infections in the studied WHO regions, particularly in ICUs.

Background The World Health Assembly adopted the Global Action Plan on Antimicrobial Resistance, which includes improving the knowledge base through surveillance and research. Noteworthily, the World Health Organization has advocated a Global Antimicrobial Resistance Surveillance System to address the plan's surveillance objective, with most African countries enrolling in or after 2017. Aim The aim of this article was to review prior data on antimicrobial resistance of Vibrio cholerae from sub-Saharan Africa with a view for future control and intervention strategies. Methods We used the Preferred Reporting Items for Systematic Review and Meta-Analysis (or 'PRISMA') guidelines to search the PubMed and African Journals Online databases, as well as additional articles provided by the Nigeria Centre for Disease Control, for articles reporting on the antibiotic susceptibility of V. cholerae between January 2000 and December 2017. Results We identified 340 publications, of which only 25 (reporting from 16 countries within the sub-Saharan African region) were eligible. The majority (20; 80.0%) of the cholera toxigenic V. cholerae isolates were of the serogroup O1 of the El Tor biotype with Ogawa and Inaba serotypes predominating. Resistance was predominantly documented to trimethoprim-sulphamethoxazole (50% of the studies), ampicillin (43.3% of the studies), chloramphenicol (43.3% of the studies) and streptomycin (30% of the studies). Resistance mechanisms were reported in 40% of the studies. Conclusion Our results demonstrate a documented antimicrobial resistance of V. cholerae to multiple antibiotic classes, including cell wall active agents and antimetabolites with evidence of phenotypic/genotypic resistance to fluoroquinolones.

Enteroaggregative Escherichia coli (EAEC) bacteria are exceptional colonizers that are associated with diarrhea. The genome of EAEC strain 042, a diarrheal pathogen validated in a human challenge study, encodes multiple colonization factors. Notable among them are aggregative adherence fimbriae (AAF/II) and a secreted antiaggregation protein (Aap). Deletion of aap is known to increase adherence, autoaggregation, and biofilm formation, so it was proposed that Aap counteracts AAF/II-mediated interactions. We hypothesized that Aap sterically masks heat-resistant agglutinin 1 (Hra1), an integral outer membrane protein recently identified as an accessory colonization factor. We propose that this masking accounts for reduced in vivo colonization upon hra1 deletion and yet no colonization-associated phenotypes when hra1 is deleted in vitro . Using single and double mutants of hra1 , aap , and the AAF/II structural protein gene aafA , we demonstrated that increased adherence in aap mutants occurs even when AAF/II proteins are genetically or chemically removed. Deletion of hra1 together with aap abolishes the hyperadherence phenotype, demonstrating that Aap indeed masks Hra1. The presence of all three colonization factors, however, is necessary for optimal colonization and for rapidly building stacked-brick patterns on slides and cultured monolayers, the signature EAEC phenotype. Altogether, our data demonstrate that Aap serves to mask nonstructural adhesins such as Hra1 and that optimal colonization by EAEC is mediated through interactions among multiple surface factors. IMPORTANCE Enteroaggregative Escherichia coli (EAEC) bacteria are exceptional colonizers of the human intestine and can cause diarrhea. Compared to other E. coli pathogens, little is known about the genes and pathogenic mechanisms that differentiate EAEC from harmless commensal E. coli . EAEC bacteria attach via multiple proteins and structures, including long appendages produced by assembling molecules of AafA and a short surface protein called Hra1. EAEC also secretes an antiadherence protein (Aap; also known as dispersin) which remains loosely attached to the cell surface. This report shows that dispersin covers Hra1 such that the adhesive properties of EAEC seen in the laboratory are largely produced by AafA structures. When the bacteria colonize worms, dispersin is sloughed off, or otherwise removed, such that Hra1-mediated adherence occurs. All three factors are required for optimal colonization, as well as to produce the signature EAEC stacked-brick adherence pattern. Interplay among multiple colonization factors may be an essential feature of exceptional colonizers.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe infantile diarrhea in developing countries. Previous research has focused on the diversity of the EPEC virulence plasmid, whereas less is known regarding the genetic content and distribution of antibiotic resistance plasmids carried by EPEC. A previous study demonstrated that in addition to the virulence plasmid, reference EPEC strain B171 harbors a second, larger plasmid that confers antibiotic resistance. To further understand the genetic diversity and dissemination of antibiotic resistance plasmids among EPEC strains, we describe the complete sequence of an antibiotic resistance plasmid from EPEC strain B171. The resistance plasmid, pB171_90, has a completed sequence length of 90,229 bp, a GC content of 54.55%, and carries protein-encoding genes involved in conjugative transfer, resistance to tetracycline ( tetA ), sulfonamides ( sulI ), and mercury, as well as several virulence-associated genes, including the transcriptional regulator hha and the putative calcium sequestration inhibitor ( csi ). In silico detection of the pB171_90 genes among 4,798 publicly available E. coli genome assemblies indicates that the unique genes of pB171_90 ( csi and traI ) are primarily restricted to genomes identified as EPEC or enterotoxigenic E. coli . However, conserved regions of the pB171_90 plasmid containing genes involved in replication, stability, and antibiotic resistance were identified among diverse E. coli pathotypes. Interestingly, pB171_90 also exhibited significant similarity with a sequenced plasmid from Shigella dysenteriae type I. Our findings demonstrate the mosaic nature of EPEC antibiotic resistance plasmids and highlight the need for additional sequence-based characterization of antibiotic resistance plasmids harbored by pathogenic E. coli .

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.

C. Roper et al. report the disturbing dissemination of pyrimethamine-resistant Plasmodia (the parasite that causes malaria) from Asia to Africa (“Intercontinental spread of pyrimethamine-resistant malaria,” Brevia, 20 Aug., p. [1124][1]). They recommend that travelers moving between Southeast

The heat-resistant agglutinin 1 (Hra1) is an integral outer membrane protein found in strains of Escherichia coli that are exceptional colonizers. Hra1 from enteroaggregative E. coli strain 042 is sufficient to confer adherence to human epithelial cells and to cause bacterial autoaggregation. Hra1 is closely related to the Tia invasin, which also confers adherence, but not autoaggregation. Here, we have demonstrated that Hra1 mediates autoaggregation by self-association and we hypothesize that at least some surface-exposed amino acid sequences that are present in Hra1, but absent in Tia, represent autoaggregation motifs. We inserted FLAG tags along the length of Hra1 and used immune-dot blots to verify that four in silico-predicted outer loops were indeed surface exposed. In Hra1 we swapped nine candidate motifs in three of these loops, ranging from one to ten amino acids in length, to the corresponding sequences in Tia. Three of the motifs were required for Hra1-mediated autoaggregation. The database was searched for other surface proteins containing these motifs; the GGXWRDDXK motif was also present in a surface-exposed region of Rck, a Salmonella enterica serotype Typhimurium complement resistance protein. Cloning and site-specific mutagenesis demonstrated that Rck can confer weak, GGXWRDDXK-dependent autoaggregation by self-association. Hra1 and Rck appear to form heterologous associations and GGXWRDDXK is required on both molecules for Hra1-Rck association. However, a GGYWRDDLKE peptide was not sufficient to interfere with Hra1-mediated autoaggregation. In the present study, three autoaggregation motifs in an integral outer membrane protein have been identified and it was demonstrated that at least one of them works in the context of a different cell surface.

In recent years, the frequency of cholera epidemics across Africa has increased significantly with thousands of people dying each year. However, there still exists a lack of information concerning the Vibrio cholerae O1 lineages driving early and contemporary epidemics since the seventh cholera pandemic started in the continent. This compromises the understanding of the forces determining the epidemiology of cholera in Africa and its control. This study aimed to analyze a collection of V. cholerae O1 strains from the beginning of the seventh cholera pandemic in Ghana and to compare them with recent isolates to understand the evolution of the cholera epidemic in Ghana. V. cholerae O1 strains were characterized by means of Multilocus Sequence Analysis (MLSA), genes from the virulence core genome (VCG), and genes related to the choleragenic phenotype. Our results revealed two major clusters of Ghanaian V. cholerae O1 strains, El Tor and Amazonia/Ghana. Concerning the virulence genes, all strains harbored the set of VCG and most were positive for VSP-II genomic island. The ctxB gene of the contemporary strains was characterized as Altered El Tor. The strains from 1970 to 1980 were susceptible to all antibiotics tested, except for the Amazonia/Ghana cluster that was resistant to aminoglycosides and carried the class 2 integron with the sat2-aadA1 arrangement. This study showed that distinct V. cholerae O1 were the determinants of cholera outbreaks in Ghana. Thus, in endemic regions, such as Africa, cholera can be caused by various V. cholerae O1 genotypes.

Enterohaemorrhagic E. coli (EHEC) particularly O157:H7 (Sequence type 11 complex), is the best documented and most well-known of E. coli that cause diarrhoea. The importance of EHEC lies in the severity of disease. Outbreaks can infect thousands of people causing bloody diarrhoea and haemolytic uremic syndrome (HUS) that in turn can result in protracted illness or even death. The ability of EHEC to colonise the human gut is normally associated with the presence of genes from another group of diarrhoeagenic E. coli, the enteropathogenic E. coli (EPEC), via the locus of enterocyte effacement. However, the massive outbreak in Germany was caused by an EHEC which had acquired virulence genes from yet another group of diarrhoeagenic E. coli, the enteroaggregative E. coli (EAEC). In reality EAEC is probably the most common bacterial cause of diarrhoea but is not identified in most diagnostic laboratories. This outbreak emphasises the importance of being able to detect all diarrhoeagenic E. coli and not to focus on E. coli O157:H7 alone. Routine surveillance systems for EAEC, a once ignored global pathogen, would go a long way to reaching this goal. This review describes methods for identifying non-O157 EHEC and describes the key genetic features of EHEC and EAEC. Our aim is to provide information for laboratories and policy makers which enables them to make informed decisions about the best methods available for detecting newly emergent strains of diarrhoeagenic E. coli.

Diarrhea is a leading cause of illness and death, particularly in developing countries. Enteropathogenic Escherichia coli, enterotoxigenic E. coli (ETEC), enteroinvasive E. coli, enterohemorrhaghic E. coli (EHEC), enteroaggregative E. coli (EAEC) and diffusely adherent E. coli (DAEC) have been associated with diarrheal disease in different parts of Africa, particularly among young children, HIV-positive individuals, and visitors from abroad. Each of these E. coli pathotypes uses distinct mechanisms, which are only partially understood, to colonize infected hosts and produce diarrhea. All known diarrheagenic E. coli pathotypes have been reported from diverse locations within Africa but the true burden from these pathogens is unknown because very few studies seeking these organisms with discriminatory methodology have been performed. Recent reports implicate ETEC and EAEC in a considerable proportion of childhood and travellers' diarrheas and suggest that some sub-types of these categories may have greater epidemiological significance than others. The significant contribution of EHEC to bloody diarrhea and hemolytic uremic syndrome is underappreciated because diagnostic capacity for this pathotype is generally inferior to that for confounders such as Shigella and Entamoeba. Recent studies in Africa have revealed the worrisome emergence of antimicrobial resistance and high asymptomatic carriage rates for diarrheagenic E. coli but bacterial and host factors that predispose to disease, as well as non-human reservoirs, are largely unknown. Future diarrheal disease research needs to focus on broadening the repertoire of pathogens sought in epidemiological surveys to include multiple categories of diarrheagenic E. coli while building capacity to detect these pathogens in local reference laboratories.

Enteroaggregative Escherichia coli (EAEC) is an important cause of acute and persistent diarrhea. The defining stacked brick adherence pattern of Peruvian EAEC isolate 042 has previously been attributed to aggregative adherence fimbriae II (AAF/II), which confer aggregative adherence on laboratory E. coli strains. EAEC strains also show exceptional autoaggregation and biofilm formation, other phenotypes that have hitherto been ascribed to AAF/II. We report that EAEC 042 carries the heat-resistant agglutinin ( hra1 ) gene, also known as hek , which encodes an outer membrane protein. Like AAF/II, the cloned EAEC 042 hra1 gene product is sufficient to confer autoaggregation, biofilm formation, and aggregative adherence on nonadherent and nonpathogenic laboratory E. coli strains. However, an 042 hra1 deletion mutant is not deficient in these phenotypes compared to the wild type. EAEC strain 042 produces a classic honeycomb or stacked brick pattern of adherence to epithelial cells. Unlike wild-type 042, the hra1 mutant typically does not form a tidy stacked brick pattern on HEp-2 cells in culture, which is definitive for EAEC. Moreover, the hra1 mutant is significantly impaired in the Caenorhabditis elegans slow kill colonization model. Our data suggest that the exceptional colonization of strain 042 is due to multiple factors and that Hra1 is an accessory EAEC colonization factor.

Objectives: Vibrio cholerae O1 from a 2006 outbreak in Accra were commonly resistant to multiple antimicrobials and, in particular, to trimethoprim/sulfamethoxazole, drugs commonly used in the treatment of cholera. We sought to determine the genetic basis for trimethoprim/sulfamethoxazole resistance in outbreak isolates. Methods: Twenty-seven isolates from the outbreak were screened by PCR and sequencing for class 1 and 2 integrons and for the SXT element. Results: Twenty-one of the 27 isolates examined, all from the Accra metropolitan area, carried both SXT, an integrated chromosomal element, and a class 2 integron bearing dfrA1, sat and aadA1 cassettes. All these isolates had identical random amplification of polymorphic DNA profiles and two of them also carried a class 1 integron. Conclusions: Most strains characterized carried multiple elements conferring resistance to trimethoprim. This suggests that trimethoprim/sulfamethoxazole should not be used empirically in cholera treatment.

Fluoroquinolones came into widespread use in African countries in the early 2000s, after patents for the first generation of these drugs expired. By that time, quinolone antibacterial agents had been used intensively worldwide and resistant lineages of many bacterial species had evolved. We sought to understand which Gram negative enteric pandemic lineages have been reported from Africa, as well as the nature and transmission of any indigenous resistant clones. A systematic review of articles indexed in the Medline and AJOL literature databases was conducted. We report on the findings of 43 eligible studies documenting local or pandemic fluoroquinolone-resistant enteric clones in sub-Sahara African countries. Most reports are of invasive non-typhoidal Salmonella and Escherichia coli lineages and there have been three reports of cholera outbreaks caused by fluoroquinolone-resistant Vibrio cholerae O1. Fluoroquinolone-resistant clones have also been reported from commensals and animal isolates but there are few data for non-Enterobacteriaceae and almost none for difficult-to-culture Campylobacter spp. Fluoroquinolone-resistant lineages identified in African countries were universally resistant to multiple other classes of antibacterial agents. Although as many as 972 non-duplicate articles refer to fluoroquinolone resistance in enteric bacteria from Africa, most do not report on subtypes and therefore information on the epidemiology of fluoroquinolone-resistant clones is available from only a handful of countries in the subcontinent. When resistance is reported, resistance mechanisms and lineage information is rarely investigated. Insufficient attention has been given to molecular and sequence-based methods necessary for identifying and tracking resistant clones in Africa and more research is needed in this area.

Background Diarrhoeagenic Escherichia coli (DEC) pathotypes are among the most common bacterial causes of morbidity and mortality in young children. These pathogens are not sought routinely and capacity for their detection is limited in Africa. We investigated the distribution and dissemination of DEC in 126 children paired with their mothers in a Nigerian community. Methods A total of 861 E. coli were isolated from 126 children with diarrhoea and their mothers. Antimicrobial susceptibility of each isolate was determined by Kirby-Bauer disc diffusion technique. All the isolates were screened for DEC markers by multiplex PCR. Genetic relatedness of DEC strains was determined by flagellin typing and Insertion element 3 (IS3)-based PCR. Results DEC were identified from 35.7 % of individuals with the most common pathotype being shiga toxin-producing E. coli (42, 16.7 %). Identical pathotypes were found in 13 (10.3 %) of the mother-child pairs and in three of these strains from mothers and their children showed identical genetic signatures. Over 90 % of DEC isolates were resistant to ampicillin, sulphonamide, tetracycline, streptomycin or trimethoprim, but only 9 (7.2 %) were ciprofloxacin resistant Conclusion The data suggest that healthy mothers are asymptomatic reservoirs of multiply-resistant strains that are pathogenic in their children and there are instances in which identical strains are found in mother-child pairs.

Summary This study was designed to investigate the physical properties and antimicrobial activities of the essential oil hydrodistilled from leaves of Lippia multiflora (lippia oil) in order to develop the oil to a pharmaceutical standard. The values of the physical parameters of the oil were weight-per-millilitre: 0.8762 ± 0.0041 g, refractive index: 1.468 ± 0.001, optical rotation range: from +46.55° to +57.60°, residue on evaporation: 3.85 ± 0.96%, pH: 4.75 ± 0.24, kinematic viscosity: 5.46 ± 0.02 cSt and surface tension: 29.20 ± 0.04 dyn/cm at 28 °C. Lippia oil was soluble in 1 volume of ethanol (80%) at 28 °C, with the solution becoming opalescence on addition of 4 or more volumes of the ethanol. The oil is highly volatile at temperatures above 37.7 °C. While fungi were more susceptible to the lippia oil than bacteria, the oil was found to be more effective against Gram-positive bacteria than Gram-negative bacteria, with clinical isolates showing more resistance than type strains. The potential application of the physical properties and antimicrobial activities of lippia oil in its formulation as a drug product is discussed.

Enteroaggregative Escherichia coli (EAEC) strains are etiologic agents of acute and persistent diarrhea. In this study, the results of phenotypic assays suggested that EAEC strains possess specialized iron acquisition systems. Genes required for the synthesis ( iucA ) or transport ( fepC ) of siderophores, and genes encoding siderophore ( fyuA , ireA , and iroN ) or heme transport ( chu ) receptors or hemoglobin proteases ( pic and hbp ), were sought in EAEC strains which have been characterized with respect to known virulence genes and phylogeny. The chuA , iucA , fyuA , fepC , and pic genes were detected in 33, 76.2, 85.7, 33, and 61.9% of these EAEC strains, respectively, and the other genes were absent. The majority of EAEC strains possessed genes encoding multiple iron transport systems, and there was no phylogenetic correlation in the distribution of the majority of these loci, as is typical for EAEC. The notable exceptions were chuA and fepC (which is associated with the prrA-modA-fepC pathogenicity island); these genes were restricted to the EAEC2 and DAEC2 phylogenetic groups, which could represent pathogenic subsets. When collections of EAEC strains isolated during case-control studies in Nigeria and Brazil were examined, no association of the presence of either chuA or iucA alone with diarrhea was seen, but both genes together were present in significantly more strains from cases than from controls in the Nigerian collection ( P < 0.05). It is possible that the presence of both genes marks at least some virulent strains. The data also demonstrate geographical variation in the association of iron utilization genes with disease in EAEC.

Dear Sir: A recent article by Lindsay and others1 reported that stool quantitative polymerase chain reaction (qPCR), with a 14,000 copy number cutoff, identified more cases of Shigella infection than conventional and widely used culture methods. The authors suggested that there may be a significant underestimation of the contribution of Shigella to diarrheal disease because of the limits of culture and have made a similar claim in a previous but smaller study.2 The target gene for Shigella used in the study was ipaH, one of the most robust and widely used Shigella targets. As some of the authors remarked in a previous article, enteroinvasive Escherichia coli (EIEC) strains also possess ipaH.2 Indeed, this target is applied for both Shigella and EIEC when the two pathotypes are not delineated. It is therefore possible, or indeed probable, that the qPCR methodology has uncovered a hidden burden due to EIEC rather than—or with—missed Shigella cases. EIEC strains are less commonly sought overall than Shigella since they cannot be reliably delineated biochemically from commensal E. coli by biochemical and serological testing alone.3 They are genetically more heterogeneous than Shigella and only detectable by invasion assays and molecular tests.4 The Global Enteric Multicenter Study from which these findings emanate sought a wider range of pathogens than most, including some diarrheagenic E. coli, but not EIEC.5 Therefore, the only presumptive data pointing to EIEC from that study are the ipaH qPCR data. We note with particular interest from the report of Lindsay and others1 that the proportion of “shigellae” that were uncultured, as determined from qPCR data, varied by site. There might be technical, stability, host biology, or coinfection explanations for this finding. However, a more plausible explanation is that EIEC are more prevalent at the African sites where Shigella culture was less sensitive than in Bangladesh where most “Shigella” infections were culture positive. This presumption is backed up by the finding by Lindsay and others1 that the distribution of Shigella serotypes varied among the sites. The greater diversity of EIEC lineages may additionally explain why culture-negative ipaH-positive cases were more common in older children. Shigella and EIEC are both E. coli pathotypes, both are known to produce dysentery (and therefore require antimicrobial chemotherapy) and to have low infection doses. In spite of these similarities in virulence, and the close relationships between some EIEC and Shigella lineages,6 harder-to-detect EIEC are often overlooked in epidemiological studies and routine diagnosis are presumed to be less significant in the absence of a robust evidence base.2,3 If EIEC accounts for some or all of the excess detection produced by ipaH qPCR, the epidemiological data reported in the article by Lindsay and others1 suggest that it is as important to detect EIEC as Shigella since nutritional status and risk factors were similar in culture and ipaH qPCR-positive cases. Perhaps the most important and completely unstated finding of the study is that alternatives to culture are essential for capturing the burden from all ipaH-positive pathogens, and more importantly delineating EIEC from Shigella. It is critical to highlight that the findings of the Lindsay and others1 study point to epidemiological importance of EIEC, particularly at African sites, to properly inform future studies and vaccine development.

Stool specimens from 113 adult outpatients with diarrhea in southwestern Nigeria and 63 controls were examined for bacterial and parasitic enteric pathogens. Enterohemorrhagic Escherichia coli (EHEC) ( P < 0.02), enteroaggregative E. coli (EAEC) ( P < 0.02), and Entamoeba histolytica ( P < 0.0002) were significantly associated with diarrhea. Salmonella , Shigella , nontoxigenic Vibrio cholerae , other categories of diarrheagenic E. coli , as well as a variety of helminths were recovered more frequently from the stools of patients than from the stools of controls but did not show a significant association with disease. Multiple pathogens were recovered from 36.3% of specimens, and bloody diarrhea was commonly associated with E. histolytica and diarrheagenic E. coli infections. The majority of EHEC isolates were non-O157 strains that carried the stx 2 gene. Of the 23 EHEC-infected patients, 12 (52.2%) presented during the 10th week of the study. EHEC strains isolated within this cluster were more likely to hybridize with the enterohemolysin gene probe, to be nonmotile and sorbitol positive, and to fail to agglutinate O157 antisera. Pulsed-field gel electrophoresis demonstrated that the only strains with Xba I profiles that occurred more than once were isolated during the 10th and 11th weeks of the study, suggesting an outbreak. The study has demonstrated that E. histolytica , EHEC, and EAEC are important diarrheal pathogens within the study area and that sporadic and epidemic EHEC infections occur in developing as well as developed countries. Routine surveillance for diarrheagenic E. coli , even only at the tertiary-care level, would be useful in identifying outbreaks and assist in identifying environmental reservoirs and transmission routes.

The properties of 23 cell-detaching Escherichia coli strains that were isolated from stool specimens in Nigeria are described. Common properties of the strains included the presence of genes encoding α-hemolysin (100%), pyelonephritis-associated pili (100%), and cytotoxic necrotizing factor 1 (70%) as well as lactose negativity (70%) and multiple antibiotic resistance (74%). Antibiotic resistance was shown in most cases to be transferable and associated with the presence of class 1 integrons. Phenotypic properties and pulsed-field gel electrophoresis analysis demonstrated that the majority of the strains, particularly multiply resistant, lactose-negative O4:H40 strains, were closely related. Multiply-resistant cell-detaching E. coli strains may represent an important reservoir for antibiotic resistance genes.

Enteroaggregative Escherichia coli (EAEC) are an increasingly important cause of diarrhoea. E. coli belonging to this category cause watery diarrhoea, which is often persistent and can be inflammatory. EAEC have been implicated in sporadic diarrhoea in children and adults, in both developing and developed countries, and have been identified as the cause of several outbreaks worldwide. EAEC are defined by their ability to adhere to epithelial cells in a characteristic "stacked-brick" pattern but are otherwise highly heterogeneous. Genes that could contribute to the pathogenicity of EAEC encode adhesins, toxins, and other factors, all of which are only partially conserved. Practicable tools are needed to improve diagnosis and identify risk factors. EAEC-infected individuals can be treated with fluoroquinolones but there is a need to examine alternative treatment protocols.

Aims: To evaluate the bacteriological quality of skin moisturizing products in the South-west part of Nigeria and study factors predisposing their bacterial contamination under tropical conditions. Methods and Results: Viable counts for bacteria exceeded 103 cfu ml–1or cfu g–1 in 8 (16·3%) commercially available creams and lotions at time of purchase. Escherichia coli (8), Pseudomonas spp. (7), Staphylococcus spp. (9) and Bacillus spp. (6) were the most commonly recovered bacteria. Following use by volunteers, the proportion of E. coli and other Gram-negative organisms recovered increased. Organic matter, particularly in the absence of preservatives, enhanced survival and growth of bacteria in creams stored under ambient tropical conditions during challenge experiments. Conclusions: Contaminated products are relatively uncommon but some products present a potential health hazard because they are unable to suppress the growth of organisms of likely faecal origin during use. Significance and Impact of the Study: Quality assurance during manufacture, pack size, preservative evaluation, organic matter and water content were identified as factors to be considered during the development of creams and lotions for use in tropical developing countries.

Enteropathogenic Escherichia coli (EPEC) strains that carry the EPEC adherence factor (EAF) plasmid were screened for the presence of different EAF sequences, including those of the plasmid-encoded regulator ( per ). Considerable variation in gene content of EAF plasmids from different strains was seen. However, bfpA , the gene encoding the structural subunit for the bundle-forming pilus, bundlin, and per genes were found in 96.8% of strains. Sequence analysis of the per operon and its promoter region from 15 representative strains revealed that it is highly conserved. Most of the variation occurs in the 5′ two-thirds of the perA gene. In contrast, the C-terminal portion of the predicted PerA protein that contains the DNA-binding helix-turn-helix motif is 100% conserved in all strains that possess a full-length gene. In a minority of strains including the O119:H2 and canine isolates and in a subset of O128:H2 and O142:H6 strains, frameshift mutations in perA leading to premature truncation and consequent inactivation of the gene were identified. Cloned perA , - B , and - C genes from these strains, unlike those from strains with a functional operon, failed to activate the LEE1 operon and bfpA transcriptional fusions or to complement a per mutant in reference strain E2348/69. Furthermore, O119, O128, and canine strains that carry inactive per operons were deficient in virulence protein expression. The context in which the perABC operon occurs on the EAF plasmid varies. The sequence upstream of the per promoter region in EPEC reference strains E2348/69 and B171-8 was present in strains belonging to most serogroups. In a subset of O119:H2, O128:H2, and O142:H6 strains and in the canine isolate, this sequence was replaced by an IS 1294 -homologous sequence.

The development of antibiotic-resistant (AR) bacteria in any country is of global importance. After their initial selection and local dissemination, AR bacteria can be transferred across international borders by human travelers, animal and insect vectors, agricultural products, and surface water. The sources and routes of importation of strains of AR bacteria are most often unknown or undetected, because many bacteria carrying resistance genes do not cause disease, and routine surveillance often does not detect them. Control of international dissemination of AR bacteria depends on methods to reduce selection pressure for the development of such bacteria and improved surveillance to detect their subsequent spread.

Diarrhoeagenic Escherichia coli (DEC) were sought in stool specimens from 72 adults and children aged over 3 years, who presented with diarrhoea at a hospital in Accra, Ghana, and 72 matched controls. Only diffusely-adherent E. coli were significantly associated with disease in these older individuals (P=0.029). We additionally tested 53 specimens from infants among whom DEC were collectively associated with disease (P=0.012). Enteropathogenic, enterotoxigenic and enteroaggregative E. coli, the most commonly isolated pathotypes from infants with diarrhoea, were frequently recovered from healthy adults. Asymptomatic carriage of DEC by older individuals in Accra may place young children at risk for diarrhoea.

In a study carried out in small-town and rural primary health care centers in southwestern Nigeria, 330 Escherichia coli strains isolated from 187 children with diarrhea and 144 apparently healthy controls were examined for virulence traits. Based on the results of colony blot hybridization, strains were categorized as enteropathogenic E. coli (1.8%), enterotoxigenic E. coli (2.4%), enteroinvasive E. coli (1.2%), enterohemorrhagic E. coli (0.6%), enteroaggregative E. coli (10.3%), diffusely adherent E. coli (7.9%), cell-detaching E. coli (6.9%), and cytolethal distending toxin-producing E. coli (0.9%). E. coli strains that hybridized with a Shiga toxin gene probe but lacked other characteristics usually present in enterohemorrhagic E. coli constituted 8.4% of the isolates. Ninety-seven E. coli isolates adhered to HEp-2 cells in an aggregative fashion but did not hybridize with any of the probes employed in the study. Overall the pathotypes, apart from cytolethal distending toxin-producing E. coli , were recovered both from children with diarrhea and from children without diarrhea, though to a lower extent from the healthy children. All diarrheagenic E. coli strains were associated with diarrhea ( P < 0.02). Heat-stable-enterotoxin-producing enterotoxigenic E. coli showed significant association with diarrhea ( P < 0.02), as did strains that demonstrated aggregative adherence to HEp-2 cells ( P < 0.04), but not those that hybridized with the CVD432 enteroaggregative probe.

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.

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.

We report on an 8-year-old patient with septicaemia unresponsive to therapy for five weeks. Undetected, extended-spectrum β-lactamase (ESBL) production by the infecting Klebsiella strain was regarded as responsible for treatment failure. Intravenously administered imipenem during the sixth week led to sustained resolution of fever. Resource-limited hospitals can incur prohibitive costs from ESBL-producer infections because of diagnostic limitations and consequent treatment failure involving prolonged supportive therapy.

Heat-resistant agglutinin 1 (Hra1) is an accessory colonization factor of enteroaggregative Escherichia coli (EAEC) strain 042. Tia, a close homolog of Hra1, is an invasin and adhesin that has been described in enterotoxigenic E. coli . We devised a PCR-restriction fragment length polymorphism screen for the associated genes and found that they occur among 55 (36.7%) of the enteroaggregative E. coli isolates screened, as well as lower proportions of enterotoxigenic, enteropathogenic, enterohemorrhagic, and commensal E. coli isolates. Overall, 25%, 8%, and 3% of 150 EAEC strains harbored hra1 alone, tia alone, or both genes, respectively. One EAEC isolate, 60A, produced an amplicon with a unique restriction profile, distinct from those of hra1 and tia . We cloned and sequenced the full-length agglutinin gene from strain 60A and have designated it hra2 . The hra2 gene was not detected in any of 257 diarrheagenic E. coli isolates in our collection but is present in the genome of Salmonella enterica serovar Heidelberg strain SL476. The cloned hra2 gene from strain 60A, which encodes a predicted amino acid sequence that is 64% identical to that of Hra1 and 68% identical to that of Tia, was sufficient to confer adherence on E. coli K-12. We constructed an hra2 deletion mutant of EAEC strain 60A. The mutant was deficient in adherence but not autoaggregation or invasion, pointing to a functional distinction from the autoagglutinin Hra1 and the Tia invasin. Hra1, Tia, and the novel accessory adhesin Hra2 are members of a family of integral outer membrane proteins that confer different colonization-associated phenotypes.

Enteroaggregative Escherichia coli (EAEC) strains are important diarrheal pathogens. EAEC strains are defined by their characteristic stacked-brick pattern of adherence to epithelial cells but show heterogeneous virulence and have different combinations of adhesin and toxin genes. Pathoadaptive deletions in the lysine decarboxylase ( cad ) genes have been noted among hypervirulent E. coli subtypes of Shigella and enterohemorrhagic E. coli . To test the hypothesis that cad deletions might account for heterogeneity in EAEC virulence, we developed a Caenorhabditis elegans pathogenesis model. Well-characterized EAEC strains were shown to colonize and kill C. elegans , and differences in virulence could be measured quantitatively. Of 49 EAEC strains screened for lysine decarboxylase activity, 3 tested negative. Most notable is isolate 101-1, which was recovered in Japan, from the largest documented EAEC outbreak. EAEC strain 101-1 was unable to decarboxylate lysine in vitro due to deletions in cadA and cadC , which, respectively, encode lysine decarboxylase and a transcriptional activator of the cadAB genes. Strain 101-1 was significantly more lethal to C. elegans than control strain OP50. Lethality was attenuated when the lysine decarboxylase defect was complemented from a multicopy plasmid and in single copy. In addition, restoring lysine decarboxylase function produced derivatives of 101-1 deficient in aggregative adherence to cultured human epithelial cells. Lysine decarboxylase inactivation is pathoadapative in an important EAEC outbreak strain, and deletion of cad genes could produce hypervirulent EAEC lineages in the future. These results suggest that loss, as well as gain, of genetic material can account for heterogeneous virulence among EAEC strains.