The Arginine Deiminase Operon Is Responsible for a Fitness Trade-Off in Extended-Spectrum-ß-Lactamase-Producing Strains of Escherichia coli.

We previously identified an operon involved in an arginine deiminase (ADI) pathway (arc operon) on a CTX-M-producing plasmid from an O102-ST405 strain of Escherichia coli As the ADI pathway was shown to be involved in the virulence of various Gram-positive bacteria, we tested whether the ADI pathway could be involved in the epidemiological success of extended-spectrum-ß-lactamase (ESBL)-producing E. coli strains. We studied two collections of human E. coli isolated in France (n = 493) and England (n = 1,509) and show that the prevalence of the arc operon (i) is higher in ESBL-producing strains (12.1%) than in nonproducers (2.5%), (ii) is higher in CTX-M-producing strains (16%) than in other ESBL producers (3.5%), and (iii) increased over time in ESBL-producing strains from 0% before 2000 to 43.3% in 2011 to 2012. The arc operon, found in strains from various phylogenetic backgrounds, is carried by IncF plasmids (85%) or chromosomes (15%) in regions framed by numerous insertion sequences, indicating multiple arrivals. Competition experiments showed that the arc operon enhances fitness of the strain in vitro in lysogeny broth with arginine. In vivo competition experiments showed that the arc operon is advantageous for the strain in a mouse model of urinary tract infection (UTI), whereas it is a burden in a mouse model of intestinal colonization. In summary, we have identified a trait linked to CTX-M-producing strains that is responsible for a trade-off between two main E. coli lifestyles, UTI and gut commensalism. This trait alone cannot explain the wide spread of ESBLs in E. coli but merits epidemiological surveillance.

Strain-specific impact of the high-pathogenicity island on virulence in extra-intestinal pathogenic Escherichia coli.

In order to clarify the role of the high-pathogenicity island (HPI) in the experimental virulence of Escherichia coli, we constructed different deletion mutants of the entire HPI and of three individual genes (irp2, fyuA and ybtA), encoding for three main functions within the HPI. Those mutants were constructed for three phylogroup B2 strains (536-STc127, CFT073-STc73, and NU14-STc95), representative of the main B2 subgroups causing extra-intestinal infections. Transcriptional profiles obtained for the selected HPI genes irp2, fyuA and ybtA revealed similar patterns for all strains, both under selective iron-deplete conditions and in intracellular bacterial communities in vitro, with a high expression of irp2. Deletion of irp2 and ybtA abrogated yersiniabactin production, whereas the fyuA knockout was only slightly impaired for siderophore synthesis. The experimental virulence of the strains was then tested in amoeba Dictyostelium discoideum and mouse septicaemia models. No effect of any HPI mutant was observed for the two more virulent strains 536 and CFT073. In contrast, the virulence of the less virulent NU14 strain was dramatically diminished by the complete deletion of the HPI and irp2 gene whereas a lesser reduction in virulence was observed for the fyuA and ybtA deletion mutants. The two experimental virulence models gave similar results. It appears that the role of the HPI in experimental virulence is depending on the genetic background of the strains despite similar inter-strain transcriptional patterns of HPI genes, as well as of the functional class of the studied gene. Altogether, these data indicate that the intrinsic extra-intestinal virulence in the E. coli species is multigenic, with epistatic interactions between the genes.

Prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae infections detected by real-time PCR among individuals reporting sexual assaults in the Paris, France area.

BACKGROUND: Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) are the most common bacteria involved in sexually transmitted infections (STIs). In France, combined screening for CT and NG using nucleic acid amplification tests is recommended in populations that are considered at risk. However, no data have been published about victims of sexual assaults. The aim of this retrospective study was to assess the usefulness of real-time PCR for the rapid detection of CT and NG genomic DNA, and the prevalence of CT/NG infections in a sample of sexual assault victims examined at a department of forensic medicine. METHODS: Between July 2012 and July 2013, 326 adults and adolescents aged over 12 years reported a sexual assault and they were referred to the Department of Forensic Medicine for a medical and forensic examination. Secretions from urogenital (cervix or vagina), anorectal, or pharyngeal sites were collected for CT/NG assays. RESULTS: CT and NG were detected in 48/326 (15%) and 16/326 patients (5%), respectively, where 10 (3%) had a CT/NG co-infection. Among 48 patients with CT infection, 13 (27.1%) patients had co-occurring genital and anorectal infections. For the pharyngeal sites, 3/21 men (14.3%) were NG-positive. CONCLUSIONS: Given the high prevalence of CT and NG infections, systematic screening of both pathogens at the time of forensic examination should provide an opportunity for the early treatment of diagnosed STIs.

Phylogenetic, virulence and antibiotic resistance characteristics of commensal strain populations of Escherichia coli from community subjects in the Paris area in 2010 and evolution over 30 years.

It is important to study commensal populations of Escherichia coli because they appear to be the reservoir of both extra-intestinal pathogenic E. coli and antibiotic resistant strains of E. coli. We studied 279 dominant faecal strains of E. coli from 243 adults living in the community in the Paris area in 2010. The phylogenetic group and subgroup [sequence type complex (STc)] of the isolates and the presence of 20 virulence genes were determined by PCR assays. The O-types and resistance to 18 antibiotics were assessed phenotypically. The B2 group was the most frequently recovered (34.0 %), followed by the A group (28.7 %), and other groups were more rare. The most prevalent B2 subgroups were II (STc73), IV (STc141), IX (STc95) and I (STc131), with 22.1, 21.1, 16.8 and 13.7 %, respectively, of the B2 group strains. Virulence factors (VFs) were more common in B2 group than other strains. One or more resistances were found in 125 strains (44.8 % of the collection) but only six (2.2 % of the collection) were multiresistant; no extended-spectrum beta-lactamase-producing strain was isolated. The C phylogroup and clonal group A strains were the most resistant. No trade-off between virulence and resistance was evidenced. We compared these strains with collections of strains gathered under the same conditions 30 and 10 years ago. There has been a parallel and linked increase in the frequency of B2 group strains (from 9.4 % in 1980, to 22.7 % in 2000 and 34.0 % in 2010) and of VFs. Antibiotic resistance also increased, from 22.6 % of strains resistant to at least one antibiotic in 1980, to 31.8 % in 2000 and 44.8 % in 2010; resistance to streptomycin, however, remained stable. Commensal human E. coli populations have clearly evolved substantially over time, presumably reflecting changes in human practices, and particularly increasing antibiotic use.

Emergence of Antimicrobial-Resistant Escherichia coli of Animal Origin Spreading in Humans.

In the context of the great concern about the impact of human activities on the environment, we studied 403 commensal Escherichia coli/Escherichia clade strains isolated from several animal and human populations that have variable contacts to one another. Multilocus sequence typing (MLST) showed a decrease of diversity 1) in strains isolated from animals that had an increasing contact with humans and 2) in all strains that had increased antimicrobial resistance. A specific B1 phylogroup clonal complex (CC87, Institut Pasteur schema nomenclature) of animal origin was identified and characterized as being responsible for the increased antimicrobial resistance prevalence observed in strains from the environments with a high human-mediated antimicrobial pressure. CC87 strains have a high capacity of acquiring and disseminating resistance genes with specific metabolic and genetic determinants as demonstrated by high-throughput sequencing and phenotyping. They are good mouse gut colonizers but are not virulent. Our data confirm the predominant role of human activities in the emergence of antimicrobial resistance in the environmental bacterial strains and unveil a particular E. coli clonal complex of animal origin capable of spreading antimicrobial resistance to other members of microbial communities.

Interactions between genotype and environment drive the metabolic phenotype within Escherichia coli isolates.

To gain insights into the adaptation of the Escherichia coli species to different environments, we monitored protein abundances using quantitative proteomics and measurements of enzymatic activities of central metabolism in a set of five representative strains grown in four contrasted culture media including human urine. Two hundred and thirty seven proteins representative of the genome-scale metabolic network were identified and classified into pathway categories. We found that nutrient resources shape the general orientation of metabolism through coordinated changes in the average abundances of proteins and in enzymatic activities that all belong to the same pathway category. For example, each culture medium induces a specific oxidative response whatever the strain. On the contrary, differences between strains concern isolated proteins and enzymes within pathway categories in single environments. Our study confirms the predominance of genotype by environment interactions at the proteomic and enzyme activity levels. The buffering of genetic variation when considering life-history traits suggests a multiplicity of evolutionary strategies. For instance, the uropathogenic isolate CFT073 shows a deregulation of iron demand and increased oxidative stress response.

Benefits of Rapid Molecular Diagnosis of Chlamydia Trachomatis and Neisseria Gonorrhoeae Infections in Women Attending Family Planning Clinics.

We evaluated the benefits of on-demand systematic screening for Chlamydia trachomatis and Neisseria gonorrhoeae using the Xpert CT/NG assay in 589 women attending family planning clinics. The sexually transmitted infection prevalence was 16.5% with 15.1% C. trachomatis and 3.1% N. gonorrhoeae infections. The on-demand test allowed for a quicker management of patients at high risk for sexually transmitted infections.

Quantitative analysis of commensal Escherichia coli populations reveals host-specific enterotypes at the intra-species level.

The primary habitat of the Escherichia coli species is the gut of warm-blooded vertebrates. The E. coli species is structured into four main phylogenetic groups A, B1, B2, and D. We estimated the relative proportions of these phylogroups in the feces of 137 wild and domesticated animals with various diets living in the Ile de France (Paris) region by real-time PCR. We distinguished three main clusters characterized by a particular abundance of two or more phylogroups within the E. coli animal commensal populations, which we called "enterocolitypes" by analogy with the enterotypes defined in the human gut microbiota at the genus level. These enterocolitypes were characterized by a dominant (>50%) B2, B1, or A phylogroup and were associated with different host species, diets, and habitats: wild and herbivorous species (wild rabbits and deer), domesticated herbivorous species (domesticated rabbits, horses, sheep, and cows), and omnivorous species (boar, pigs, and chickens), respectively. By analyzing retrospectively the data obtained using the same approach from 98 healthy humans living in Ile de France (Smati et al. 2013, Appl. Environ. Microbiol. 79, 5005-5012), we identified a specific human enterocolitype characterized by the dominant and/or exclusive (>90%) presence of phylogroup B2. We then compared B2 strains isolated from animals and humans, and revealed that human and animal strains differ regarding O-type and B2 subgroup. Moreover, two genes, sfa/foc and clbQ, were associated with the exclusive character of strains, observed only in humans. In conclusion, a complex network of interactions exists at several levels (genus and intra-species) within the intestinal microbiota.

The rpoS gene is predominantly inactivated during laboratory storage and undergoes source-sink evolution in Escherichia coli species.

The rpoS gene codes for an alternative RNA polymerase sigma factor, which acts as a general regulator of the stress response. Inactivating alleles of rpoS in collections of natural Escherichia coli isolates have been observed at very variable frequencies, from less than 1% to more than 70% of strains. rpoS is easily inactivated in nutrient-deprived environments such as stab storage, which makes it difficult to determine the true frequency of rpoS inactivation in nature. We studied the evolutionary history of rpoS and compared it to the phylogenetic history of bacteria in two collections of 82 human commensal and extraintestinal E. coli strains. These strains were representative of the phylogenetic diversity of the species and differed only by their storage conditions. In both collections, the phylogenetic histories of rpoS and of the strains were congruent, indicating that horizontal gene transfer had not occurred at the rpoS locus, and rpoS was under strong purifying selection, with a ratio of the nonsynonymous mutation rate (Ka) to the synonymous substitution rate (Ks) substantially smaller than 1. Stab storage was associated with a high frequency of inactivating alleles, whereas almost no amino acid sequence variation was observed in RpoS in the collection studied directly after isolation of the strains from the host. Furthermore, the accumulation of variations in rpoS was typical of source-sink dynamics. In conclusion, rpoS is rarely inactivated in natural E. coli isolates within their mammalian hosts, probably because such strains rapidly become evolutionary dead ends. Our data should encourage bacteriologists to freeze isolates immediately and to avoid the use of stab storage.

Virulence patterns in a murine sepsis model of ST131 Escherichia coli clinical isolates belonging to serotypes O25b:H4 and O16:H5 are associated to specific virotypes.

Escherichia coli sequence type (ST)131 is an emerging disseminated public health threat implicated in multidrug-resistant extraintestinal infections worldwide. Although the majority of ST131 isolates belong to O25b:H4 serotype, new variants with different serotypes, STs using the discriminative multilocus sequence typing scheme of Pasteur Institute, and virulence-gene profiles (virotypes) have been reported with unknown implications on the pattern of spread, persistence and virulence. The aim of the present study was to compare virulence in a mouse subcutaneous sepsis model of representative ST131 clinical isolates belonging to 2 serotypes (O25b:H4, O16:H5) and nine virotypes and subtypes (A, B, C, D1, D2, D3, D4, D5 and E). Fourteen out of the 23 ST131 isolates tested (61%) killed 90 to 100% of mice challenged, and 18 of 23 (78%) at least 50%. Interestingly, different virulence patterns in association with virotypes were observed, from highly rapid lethality (death in less than 24 h) to low final lethality (death at 7 days) but with presence of an acute inflammation. This is the first study to assess virulence of ST131 isolates belonging to serotype O16:H5, which exhibited virotype C. In spite of their low virulence-gene score, O16:H5 isolates did not show significant differences in final lethality compared with highly virulent O25b:H4 isolates of virotypes A, B and C, but killed mice less rapidly. Significant differences were found, however, between virotypes A, B, C (final lethality ≥80% of mice challenged) and virotypes D, E. Particularly unexpected was the low lethality of the newly assigned virotype E taking into account that it exhibited high virulence-gene score, and the same clonotype H30 as highly virulent O25b:H4 isolates of virotypes A, B and C. In vivo virulence diversity reported in this study would reflect the genetic variability within ST131 clonal group evidenced by molecular typing.

Commensal Escherichia coli strains in Guiana reveal a high genetic diversity with host-dependant population structure.

We undertook a large-scale epidemiological survey of commensal Escherichia coli in Trois-Sauts, an isolated village located in the south of French Guiana where human population exchanges are restricted and source of antibiotics controlled. Stools from 162 Wayampi Amerindians and rectal swabs from 33 human associated and 198 wild animals were collected in the close proximity of the village. The prevalence of E. coli was decreasing from humans (100%) to human associated (64%) and wild (45%) animals. A clear genetic structure between these three E. coli populations was observed with human strains belonging very rarely to B2 phylogroup (3.7%), exhibiting few virulence genes and bacteriocins but being antibiotic resistant whereas wild animal strains were characterized by 46.1% of B2 phylogroup belonging, with very unique and infrequent sequence types, numerous extraintestinal genes and bacteriocins but no antibiotic resistance; the human-associated animal strains being intermediate. Furthermore, an unexpected genetic diversity was observed among the strains, as the housekeeping gene nucleotide diversity per site of the Trois-Sauts's strains was higher than the one of reference strains representative of the known species diversity. The existence of such E. coli structured phylogenetic diversity within various hosts of a single localization has never been reported.

Real-time PCR for quantitative analysis of human commensal Escherichia coli populations reveals a high frequency of subdominant phylogroups.

Escherichia coli is divided into four main phylogenetic groups, which each exhibit ecological specialization. To understand the population structure of E. coli in its primary habitat, we directly assessed the relative proportions of these phylogroups from the stools of 100 healthy human subjects using a new real-time PCR method, which allows a large number of samples to be studied. The detection threshold for our technique was 0.1% of the E. coli population, i.e., 10(5) CFU/g of feces; in other methods based on individual colony analysis, the threshold is 10%. One, two, three, or four phylogenetic groups were simultaneously found in 21%, 48%, 21%, and 8% of the subjects, respectively. Phylogroups present at a threshold of less than 10% of the population were found in 40% of the subjects, revealing high within-individual diversity. Phylogroups A and B2 were detected in 74% and 70% of the subjects, respectively; phylogroups B1 and D were detected in 36% and 32%, respectively. When phylogroup B2 was dominant, it tended not to cooccur with other phylogroups. In contrast, other phylogroups were present when phylogroup A was dominant. These data indicate a complex pattern of interactions between the members of a single species within the human gut and identify a reservoir of clones that are present at a low frequency. The presence of these minor clones could explain the fluctuation in the composition of the E. coli microbiota within single individuals that may be seen over time. They could also constitute reservoirs of virulent and/or resistant strains.

Fitness, stress resistance, and extraintestinal virulence in Escherichia coli.

The extraintestinal virulence of Escherichia coli is dependent on numerous virulence genes. However, there is growing evidence for a role of the metabolic properties and stress responses of strains in pathogenesis. We assessed the respective roles of these factors in strain virulence by developing phenotypic assays for measuring in vitro individual and competitive fitness and the general stress response, which we applied to 82 commensal and extraintestinal pathogenic E. coli strains previously tested in a mouse model of sepsis. Individual fitness properties, in terms of maximum growth rates in various media (Luria-Bertani broth with and without iron chelator, minimal medium supplemented with gluconate, and human urine) and competitive fitness properties, estimated as the mean relative growth rate per generation in mixed cultures with a reference fluorescent E. coli strain, were highly diverse between strains. The activity of the main general stress response regulator, RpoS, as determined by iodine staining of the colonies, H2O2 resistance, and rpoS sequencing, was also highly variable. No correlation between strain fitness and stress resistance and virulence in the mouse model was found, except that the maximum growth rate in urine was higher for virulent strains. Multivariate analysis showed that the number of virulence factors was the only independent factor explaining the virulence in mice. At the species level, growth capacity and stress resistance are heterogeneous properties that do not contribute significantly to the intrinsic virulence of the strains.

[When should request a stool culture?]. / Quand faut-il demander une coproculture ?

A stool culture needs a medical prescription and is justified only in a case of clearly defined acute diarrhea. The clinical context must be suggestive of a bacterial etiology. The detection of the bacterial agents has to follow a well-defined strategy to optimize a rigorous prescription. Standardized stool culture is for patients treated in medical community practice and for patients hospitalized for less than 3 days. Clostridium difficile toxin testing is systematically performed in case of nosocomial diarrhea. Additional bacterial detections are limited to defined indications.

Antibiotic resistance plasmids spread among natural isolates of Escherichia coli in spite of CRISPR elements.

Clustered, regularly interspaced, short palindromic repeats (CRISPRs) are implicated in defence against foreign DNA in various archaeal and bacterial species. They have also been associated with a slower spread of antibiotic resistance. However, experimental and evolutionary studies raise doubts about the role of CRISPRs as a sort of immune system in Escherichia coli. We studied a collection of 263 natural E. coli isolates from human and animal hosts, representative of the phylogenetic and lifestyle diversity of the species and exhibiting various levels of plasmid-encoded antibiotic resistance. We characterized the strains in terms of CRISPRs, performed replicon typing of the plasmids and tested for class 1 integrons to explore the possible association between CRISPRs and the absence of plasmids and mobile antibiotic resistance determinants. We found no meaningful association between the presence/absence of the cas genes, reflecting the activity of the CRISPRs, and the presence of plasmids, integrons or antibiotic resistance. No CRISPR in the collection contained a spacer that matched an antibiotic resistance gene or element involved in antibiotic resistance gene mobilization, and 79.8% (210/263) of the strains lacked spacers matching sequences in the 2282 plasmid genomes available. Hence, E. coli CRISPRs do not seem to be efficient barriers to the spread of plasmids and antibiotic resistance, consistent with what has been reported for phages, and contrary to reports concerning other species.

Postsurgical meningitis due to multiresistant Acinetobacter baumannii successfully treated with high doses of ampicillin/sulbactam combined with rifampicin and fosfomycin.

We report a case of postsurgical meningitis caused by multiresistant Acinetobacter baumannii successfully treated with high doses of ampicillin/sulbactam combined with rifampicin and fosfomycin.

Antibiotic resistance plasmids spread among natural isolates of Escherichia coli in spite of CRISPR elements.

Clustered, regularly interspaced, short palindromic repeats (CRISPRs) are implicated in defence against foreign DNA in various archaeal and bacterial species. They have also been associated with a slower spread of antibiotic resistance. However, experimental and evolutionary studies raise doubts about the role of CRISPRs as a sort of immune system in Escherichia coli. We studied a collection of 263 natural E. coli isolates from human and animal hosts, representative of the phylogenetic and lifestyle diversity of the species and exhibiting various levels of plasmid-encoded antibiotic resistance. We characterized the strains in terms of CRISPRs, performed replicon typing of the plasmids and tested for class 1 integrons to explore the possible association between CRISPRs and the absence of plasmids and mobile antibiotic resistance determinants. We found no meaningful association between the presence/absence of the cas genes, reflecting the activity of the CRISPRs, and the presence of plasmids, integrons or antibiotic resistance. No CRISPR in the collection contained a spacer that matched an antibiotic resistance gene or element involved in antibiotic resistance gene mobilization, and 79.8 % (210/263) of the strains lacked spacers matching sequences in the 2282 plasmid genomes available. Hence, E. coli CRISPRs do not seem to be efficient barriers to the spread of plasmids and antibiotic resistance, consistent with what has been reported for phages, and contrary to reports concerning other species.

Animal and human pathogenic Escherichia coli strains share common genetic backgrounds.

Escherichia coli is a versatile species encompassing both commensals of the digestive tracts of many vertebrates, including humans, and pathogenic strains causing various intra- and extraintestinal infections. Despite extensive gene flow between strains, the E. coli species has a globally clonal population structure, consisting of distinct phylogenetic groups. Little is known about the relationships between phylogenetic groups and host specificity. We therefore used multilocus sequence typing (MLST) to investigate phylogenetic relationships and evaluated the virulence gene content of 35 E. coli strains representative of the diverse diseases encountered in domestic animals. We compared these strains with a panel of 101 human pathogenic and 98 non-human and human commensal strains representative of the phylogenetic and pathovar diversity of this species. A global factorial analysis of correspondence indicated that extraintestinal infections were caused mostly by phylogenetic group B2 strains, whereas intraintestinal infections were caused mostly by phylogenetic group A/B1/E strains, with strains responsible from extraintestinal or intraintestinal infections having specific virulence factors. It was not possible to distinguish between strains of human and animal origin. A detailed phylogenetic analysis of the MLST data showed that numerous pathogenic animal and human strains are very closely related, and had a number of virulence genes in common. However, a set of specific adhesins was identified in animal non-B2 group strains of all pathotypes. In conclusion, human and animal pathogenic strains share common genetic backgrounds, but non-B2 strains of different origins seem to have different sets of adhesins that could be involved in host specificity.

Molecular and evolutionary bases of within-patient genotypic and phenotypic diversity in Escherichia coli extraintestinal infections.

Although polymicrobial infections, caused by combinations of viruses, bacteria, fungi and parasites, are being recognised with increasing frequency, little is known about the occurrence of within-species diversity in bacterial infections and the molecular and evolutionary bases of this diversity. We used multiple approaches to study the genomic and phenotypic diversity among 226 Escherichia coli isolates from deep and closed visceral infections occurring in 19 patients. We observed genomic variability among isolates from the same site within 11 patients. This diversity was of two types, as patients were infected either by several distinct E. coli clones (4 patients) or by members of a single clone that exhibit micro-heterogeneity (11 patients); both types of diversity were present in 4 patients. A surprisingly wide continuum of antibiotic resistance, outer membrane permeability, growth rate, stress resistance, red dry and rough morphotype characteristics and virulence properties were present within the isolates of single clones in 8 of the 11 patients showing genomic micro-heterogeneity. Many of the observed phenotypic differences within clones affected the trade-off between self-preservation and nutritional competence (SPANC). We showed in 3 patients that this phenotypic variability was associated with distinct levels of RpoS in co-existing isolates. Genome mutational analysis and global proteomic comparisons in isolates from a patient revealed a star-like relationship of changes amongst clonally diverging isolates. A mathematical model demonstrated that multiple genotypes with distinct RpoS levels can co-exist as a result of the SPANC trade-off. In the cases involving infection by a single clone, we present several lines of evidence to suggest diversification during the infectious process rather than an infection by multiple isolates exhibiting a micro-heterogeneity. Our results suggest that bacteria are subject to trade-offs during an infectious process and that the observed diversity resembled results obtained in experimental evolution studies. Whatever the mechanisms leading to diversity, our results have strong medical implications in terms of the need for more extensive isolate testing before deciding on antibiotic therapies.