Malika Gouali

Intercontinental dissemination of azithromycin-resistant shigellosis through sexual transmission: a cross-sectional study

BACKGROUND Shigellosis is an acute, severe bacterial colitis that, in high-income countries, is typically associated with travel to high-risk regions (Africa, Asia, and Latin America). Since the 1970s, shigellosis has also been reported as a sexually transmitted infection in men who have sex with men (MSM), in whom transmission is an important component of shigellosis epidemiology in high-income nations. We aimed to use sophisticated subtyping and international sampling to determine factors driving shigellosis emergence in MSM linked to an outbreak in the UK. METHODS We did a large-scale, cross-sectional genomic epidemiological study of shigellosis cases collected from 29 countries between December, 1995, and June 8, 2014. Focusing on an ongoing epidemic in the UK, we collected and whole-genome sequenced clinical isolates of Shigella flexneri serotype 3a from high-risk and low-risk regions, including cases associated with travel and sex between men. We examined relationships between geographical, demographic, and clinical patient data with the isolate antimicrobial susceptibility, genetic data, and inferred evolutionary relationships. FINDINGS We obtained 331 clinical isolates of S flexneri serotype 3a, including 275 from low-risk regions (44 from individuals who travelled to high-risk regions), 52 from high-risk regions, and four outgroup samples (ie, closely related, but genetically distinct isolates used to determine the root of the phylogenetic tree). We identified a recently emerged lineage of S flexneri 3a that has spread intercontinentally in less than 20 years throughout regions traditionally at low risk for shigellosis via sexual transmission in MSM. The lineage had acquired multiple antimicrobial resistance determinants, and prevailing sublineages were strongly associated with resistance to the macrolide azithromycin. Eight (4%) of 206 isolates from the MSM-associated lineage were obtained from patients who had previously provided an isolate; these serial isolations indicated atypical infection patterns (eg, reinfection). INTERPRETATION We identified transmission-facilitating behaviours and atypical course(s) of infection as precipitating factors in shigellosis-affected MSM. The intercontinental spread of antimicrobial-resistant shigella through established transmission routes emphasises the need for new approaches to tackle the public health challenge of sexually transmitted infections in MSM. FUNDING Wellcome Trust (grant number 098051).

Comparative Genomics of Recent Shiga Toxin-Producing Escherichia coli O104:H4: Short-Term Evolution of an Emerging Pathogen

ABSTRACT The large outbreak of diarrhea and hemolytic uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli O104:H4 in Europe from May to July 2011 highlighted the potential of a rarely identified E. coli serogroup to cause severe disease. Prior to the outbreak, there were very few reports of disease caused by this pathogen and thus little known of its diversity and evolution. The identification of cases of HUS caused by E. coli O104:H4 in France and Turkey after the outbreak and with no clear epidemiological links raises questions about whether these sporadic cases are derived from the outbreak. Here, we report genome sequences of five independent isolates from these cases and results of a comparative analysis with historical and 2011 outbreak isolates. These analyses revealed that the five isolates are not derived from the outbreak strain; however, they are more closely related to the outbreak strain and each other than to isolates identified prior to the 2011 outbreak. Over the short time scale represented by these closely related organisms, the majority of genome variation is found within their mobile genetic elements: none of the nine O104:H4 isolates compared here contain the same set of plasmids, and their prophages and genomic islands also differ. Moreover, the presence of closely related HUS-associated E. coli O104:H4 isolates supports the contention that fully virulent O104:H4 isolates are widespread and emphasizes the possibility of future food-borne E. coli O104:H4 outbreaks. IMPORTANCE In the summer of 2011, a large outbreak of bloody diarrhea with a high rate of severe complications took place in Europe, caused by a previously rarely seen Escherichia coli strain of serogroup O104:H4. Identification of subsequent infections caused by E. coli O104:H4 raised questions about whether these new cases represented ongoing transmission of the outbreak strain. In this study, we sequenced the genomes of isolates from five recent cases and compared them with historical isolates. The analyses reveal that, in the very short term, evolution of the bacterial genome takes place in parts of the genome that are exchanged among bacteria, and these regions contain genes involved in adaptation to local environments. We show that these recent isolates are not derived from the outbreak strain but are very closely related and share many of the same disease-causing genes, emphasizing the concern that these bacteria may cause future severe outbreaks. In the summer of 2011, a large outbreak of bloody diarrhea with a high rate of severe complications took place in Europe, caused by a previously rarely seen Escherichia coli strain of serogroup O104:H4. Identification of subsequent infections caused by E. coli O104:H4 raised questions about whether these new cases represented ongoing transmission of the outbreak strain. In this study, we sequenced the genomes of isolates from five recent cases and compared them with historical isolates. The analyses reveal that, in the very short term, evolution of the bacterial genome takes place in parts of the genome that are exchanged among bacteria, and these regions contain genes involved in adaptation to local environments. We show that these recent isolates are not derived from the outbreak strain but are very closely related and share many of the same disease-causing genes, emphasizing the concern that these bacteria may cause future severe outbreaks.

Prevalence, quantification and antimicrobial resistance of Campylobacter spp. on chicken neck-skins at points of slaughter in 5 major cities located on 4 continents

Quantitative data on Campylobacter contamination of food are lacking, notably in developing countries. We assessed Campylobacter contamination of chicken neck-skins at points of slaughter in 5 major cities in Africa (Dakar in Senegal, Yaounde in Cameroon), Oceania (Noumea in New Caledonia), the Indian Ocean (Antananarivo in Madagascar) and Asia (Ho Chi Minh City (HCMC) in Vietnam. One hundred and fifty slaughtered chickens were collected in each of the 5 major cities from semi-industrial abattoirs or markets (direct slaughter by the seller), and 65.5% (491/750) were found to be Campylobacter-positive. Two cities, Yaounde and Noumea, demonstrated high prevalence Campylobacter detection rates (92.7% and 96.7% respectively) in contrast with HCMC (15.3%). Four species were identified among 633 isolates, namely C. jejuni (48.3%), C. coli (37.3%), C. lari (11.7%) and C. upsaliensis (1%). HCMC was the only city with C. lari isolation as was Antananarivo for C. upsaliensis. C. coli was highly prevalent only in Yaounde (69.5%). Among the 491 samples positive in Campylobacter detection, 329 were also positive with the enumeration method. The number of Campylobacter colony-forming units (CFU) per gram of neck-skin in samples positive in enumeration was high (mean of the log(10): 3.2 log(10) CFU/g, arithmetic mean: 7900CFU/g). All the cities showed close enumeration means except HCMC with a 1.81 log(10) CFU/g mean for positive samples. Semi-industrial abattoir was linked to a significant lower count of Campylobacter contamination than direct slaughter by the seller (p=0.006). On 546 isolates (546/633, 86.3%) tested for antibiotic susceptibility, resistance to erythromycin, ampicillin and ciprofloxacin was observed for respectively 11%, 19% and 50%. HCMC was the city where antibiotic resistant rates were the highest (95%, p=0.014). Considering the 329 positive chickens in Campylobacter enumeration, the mean number of resistant isolates to at least 2 different antibiotic families (19.8%), may be estimated ca. 1500CFU/g; the corresponding mean of the log(10) would be 2.5 log(10)CFU/g. As chickens are sold at slaughter and brought directly at home to be cooked, these data suggest a high probability of cross-contamination. A substantial proportion of isolates are drug-resistant, which could lead to potential public health issues. Health authorities should consider measures to reduce Campylobacter contamination of chicken during farming and at slaughter, and to provide appropriate food hygiene education. Further studies are needed in particular to investigate food-handling practices in domestic kitchens.

Evaluation of CHROMagar STEC and STEC O104 Chromogenic Agar Media for Detection of Shiga Toxin-Producing Escherichia coli in Stool Specimens

ABSTRACT The performance of CHROMagar STEC and CHROMagar STEC O104 (CHROMagar Microbiology, Paris, France) media for the detection of Shiga toxin-producing Escherichia coli (STEC) was assessed with 329 stool specimens collected over 14 months from patients with suspected STEC infections (June 2011 to August 2012). The CHROMagar STEC medium, after an enrichment broth step, allowed the recovery of the STEC strain from 32 of the 39 (82.1%) Shiga toxin-positive stool specimens, whereas the standard procedure involving Drigalski agar allowed the recovery of only three additional STEC strains. The isolates that grew on CHROMagar STEC medium belonged to 15 serotypes, including the prevalent non-sorbitol-fermenting (NSF) O157:H7, O26:H11, and O104:H4 serotypes. The sensitivity, specificity, and positive and negative predictive values for the CHROMagar STEC medium were between 89.1% and 91.4%, 83.7% and 86.7%, 40% and 51.3%, and 98% and 98.8%, respectively, depending on whether or not stx-negative eae-positive E. coli was considered atypical enteropathogenic E. coli (EPEC) or STEC that had lost Shiga toxin genes during infection. In conclusion, the good performance of CHROMagar STEC agar medium, in particular, the high negative predictive value, and its capacity to identify NSF O157:H7 as well as common non-O157 STEC may be useful for clinical bacteriology, public health, and reference laboratories; it could be used in addition to a method targeting Shiga toxins (detection of stx genes by PCR, immunodetection of Shiga toxins in stool specimens, or Vero cell cytotoxicity assay) as an alternative to O157 culture medium. This combined approach should allow rapid visualization of both putative O157 and non-O157 STEC colonies for subsequent characterization, essential for real-time surveillance of STEC infections and investigations of outbreaks.

Species-wide whole genome sequencing reveals historical global spread and recent local persistence in Shigella flexneri

Shigella flexneri is the most common cause of bacterial dysentery in low-income countries. Despite this, S. flexneri remains largely unexplored from a genomic standpoint and is still described using a vocabulary based on serotyping reactions developed over half-a-century ago. Here we combine whole genome sequencing with geographical and temporal data to examine the natural history of the species. Our analysis subdivides S. flexneri into seven phylogenetic groups (PGs); each containing two-or-more serotypes and characterised by distinct virulence gene complement and geographic range. Within the S. flexneri PGs we identify geographically restricted sub-lineages that appear to have persistently colonised regions for many decades to over 100 years. Although we found abundant evidence of antimicrobial resistance (AMR) determinant acquisition, our dataset shows no evidence of subsequent intercontinental spread of antimicrobial resistant strains. The pattern of colonisation and AMR gene acquisition suggest that S. flexneri has a distinct life-cycle involving local persistence. DOI: http://dx.doi.org/10.7554/eLife.07335.001

Enteric Bacterial Pathogens in Children with Diarrhea in Niger: Diversity and Antimicrobial Resistance

Background Although rotavirus is the leading cause of severe diarrhea among children in sub-Saharan Africa, better knowledge of circulating enteric pathogenic bacteria and their antimicrobial resistance is crucial for prevention and treatment strategies. Methodology/Principal Findings As a part of rotavirus gastroenteritis surveillance in Maradi, Niger, we performed stool culture on a sub-population of children under 5 with moderate-to-severe diarrhea between April 2010 and March 2012. Campylobacter, Shigella and Salmonella were sought with conventional culture and biochemical methods. Shigella and Salmonella were serotyped by slide agglutination. Enteropathogenic Escherichia coli (EPEC) were screened by slide agglutination with EPEC O-typing antisera and confirmed by detection of virulence genes. Antimicrobial susceptibility was determined by disk diffusion. We enrolled 4020 children, including 230 with bloody diarrhea. At least one pathogenic bacterium was found in 28.0% of children with watery diarrhea and 42.2% with bloody diarrhea. Mixed infections were found in 10.3% of children. EPEC, Salmonella and Campylobacter spp. were similarly frequent in children with watery diarrhea (11.1%, 9.2% and 11.4% respectively) and Shigella spp. were the most frequent among children with bloody diarrhea (22.1%). The most frequent Shigella serogroup was S. flexneri (69/122, 56.5%). The most frequent Salmonella serotypes were Typhimurimum (71/355, 20.0%), Enteritidis (56/355, 15.8%) and Corvallis (46/355, 13.0%). The majority of putative EPEC isolates was confirmed to be EPEC (90/111, 81.1%). More than half of all Enterobacteriaceae were resistant to amoxicillin and co-trimoxazole. Around 13% (46/360) Salmonella exhibited an extended-spectrum beta-lactamase phenotype. Conclusions This study provides updated information on enteric bacteria diversity and antibiotic resistance in the Sahel region, where such data are scarce. Whether they are or not the causative agent of diarrhea, bacterial infections and their antibiotic resistance profiles should be closely monitored in countries like Niger where childhood malnutrition pre-disposes to severe and invasive infections.

Global phylogeography and evolutionary history of Shigella dysenteriae type 1.

Together with plague, smallpox and typhus, epidemics of dysentery have been a major scourge of human populations for centuries1. A previous genomic study concluded that Shigella dysenteriae type 1 (Sd1), the epidemic dysentery bacillus, emerged and spread worldwide after the First World War, with no clear pattern of transmission2. This is not consistent with the massive cyclic dysentery epidemics reported in Europe during the eighteenth and nineteenth centuries1,3,4 and the first isolation of Sd1 in Japan in 18975. Here, we report a whole-genome analysis of 331 Sd1 isolates from around the world, collected between 1915 and 2011, providing us with unprecedented insight into the historical spread of this pathogen. We show here that Sd1 has existed since at least the eighteenth century and that it swept the globe at the end of the nineteenth century, diversifying into distinct lineages associated with the First World War, Second World War and various conflicts or natural disasters across Africa, Asia and Central America. We also provide a unique historical perspective on the evolution of antibiotic resistance over a 100-year period, beginning decades before the antibiotic era, and identify a prevalent multiple antibiotic-resistant lineage in South Asia that was transmitted in several waves to Africa, where it caused severe outbreaks of disease.

Role of Deoxyribose Catabolism in Colonization of the Murine Intestine by Pathogenic Escherichia coli Strains

ABSTRACT We previously suggested that the ability to metabolize deoxyribose, a phenotype encoded by the deoK operon, is associated with the pathogenic potential of Escherichia coli strains. Carbohydrate metabolism is thought to provide the nutritional support required for E. coli to colonize the intestine. We therefore investigated the role of deoxyribose catabolism in the colonization of the gut, which acts as a reservoir, by pathogenic E. coli strains. Molecular and biochemical characterization of 1,221 E. coli clones from various collections showed this biochemical trait to be common in the E. coli species (33.6%). However, multivariate analysis evidenced a higher prevalence of sugar-metabolizing E. coli clones in the stools of patients from countries in which intestinal diseases are endemic. Diarrhea processes frequently involve the destruction of intestinal epithelia, so it is plausible that such clones may be positively selected for in intestines containing abundant DNA, and consequently deoxyribose. Statistical analysis also indicated that symptomatic clinical disorders and the presence of virulence factors specific to extraintestinal pathogenic E. coli were significantly associated with an increased risk of biological samples and clones testing positive for deoxyribose. Using the streptomycin-treated-mouse model of intestinal colonization, we demonstrated the involvement of the deoK operon in gut colonization by two pathogenic isolates (one enteroaggregative and one uropathogenic strain). These results, indicating that deoxyribose availability promotes pathogenic E. coli growth during host colonization, suggest that the acquisition of this trait may be an evolutionary step enabling these pathogens to colonize and persist in the mammalian intestine.

Enterohemorrhagic Escherichia coli Hybrid PathotypeO80:H2 as a New Therapeutic Challenge

This emerging clonal group harbors the extraintestinal virulence–associated plasmid pS88 and can induce invasive infections and death.

Prevalence of Shiga toxin-producing Shigella species isolated from French travellers returning from the Caribbean: an emerging pathogen with international implications

Shiga toxins (Stxs) are potent cytotoxins that inhibit host cell protein synthesis, leading to cell death. Classically, these toxins are associated with intestinal infections due to Stx-producing Escherichia coli or Shigella dysenteriae serotype 1, and infections with these strains can lead to haemolytic-uraemic syndrome. Over the past decade, there has been increasing recognition that Stx is produced by additional Shigella species. We recently reported the presence and expression of stx genes in Shigella flexneri 2a clinical isolates. The toxin genes were carried by a new stx-encoding bacteriophage, and infection with these strains correlated with recent travel to Haiti or the Dominican Republic. In this study, we further explored the epidemiological link to this region by utilizing the French National Reference Centre for Escherichia coli, Shigella and Salmonella collection to survey the frequency of Stx-producing Shigella species isolated from French travellers returning from the Caribbean. Approximately 21% of the isolates tested were found to encode and produce Stx. These isolates included strains of S. flexneri 2a, S. flexneri Y, and S. dysenteriae 4. All of the travellers who were infected with Stx-producing Shigella had recently travelled to Haiti, the Dominican Republic, or French Guiana. Furthermore, whole genome sequencing showed that the toxin genes were encoded by a prophage that was highly identical to the phage that we identified in our previous study. These findings demonstrate that this new stx-encoding prophage is circulating within that geographical area, has spread to other continents, and is capable of spreading to multiple Shigella serogroups.