Central role of the ramAR locus in the multidrug resistance in ESBL-Enterobacterales.

The aim of this study was to evaluate the proportion of resistance to a temocillin, tigecycline, ciprofloxacin, and chloramphenicol phenotype called t2c2 that resulted from mutations within the ramAR locus among extended-spectrum ß-lactamases-Enterobacterales (ESBL-E) isolated in three intensive care units for 3 years in a French university hospital. Two parallel approaches were performed on all 443 ESBL-E included: (i) the minimal inhibitory concentrations of temocillin, tigecycline, ciprofloxacin, and chloramphenicol were determined and (ii) the genomes obtained from the Illumina sequencing platform were analyzed to determine multilocus sequence types, resistomes, and diversity of several tetR-associated genes including ramAR operon. Among the 443 ESBL-E strains included, isolates of Escherichia coli (n = 194), Klebsiella pneumoniae (n = 122), and Enterobacter cloacae complex (Ecc) (n = 127) were found. Thirty-one ESBL-E strains (7%), 16 K. pneumoniae (13.1%), and 15 Ecc (11.8%) presented the t2c2 phenotype in addition to their ESBL profile, whereas no E. coli presented these resistances. The t2c2 phenotype was invariably reversible by the addition of Phe-Arg-ß-naphthylamide, indicating a role of resistance-nodulation-division pumps in these observations. Mutations associated with the t2c2 phenotype were restricted to RamR, the ramAR intergenic region (IR), and AcrR. Mutations in RamR consisted of C- or N-terminal deletions and amino acid substitutions inside its DNA-binding domain or within key sites of protein-substrate interactions. The ramAR IR showed nucleotide substitutions involved in the RamR DNA-binding domain. This diversity of sequences suggested that RamR and the ramAR IR represent major genetic events for bacterial antimicrobial resistance.IMPORTANCEMorbimortality caused by infectious diseases is very high among patients hospitalized in intensive care units (ICUs). A part of these outcomes can be explained by antibiotic resistance, which delays the appropriate therapy. The transferable antibiotic resistance gene is a well-known mechanism to explain the high rate of multidrug resistance (MDR) bacteria in ICUs. This study describes the prevalence of chromosomal mutations, which led to additional antibiotic resistance among MDR bacteria. More than 12% of Klebsiella pneumoniae and Enterobacter cloacae complex strains presented mutations within the ramAR locus associated with a dysregulation of an efflux pump called AcrAB-TolC and a porin: OmpF. These dysregulations led to an increase in antibiotic output notably tigecycline, ciprofloxacin, and chloramphenicol associated with a decrease of input for beta-lactam, especially temocillin. Mutations within transcriptional regulators such as ramAR locus played a major role in antibiotic resistance dissemination and need to be further explored.

Temocillin Resistance in the Enterobacter cloacae Complex Is Conferred by a Single Point Mutation in BaeS, Leading to Overexpression of the AcrD Efflux Pump.

The Enterobacter cloacae complex (ECC) has become a major opportunistic pathogen with antimicrobial resistance issues. Temocillin, an "old" carboxypenicillin that is remarkably stable toward ß-lactamases, has been used as an alternative for the treatment of multidrug-resistant ECC infections. Here, we aimed at deciphering the never-investigated mechanisms of temocillin resistance acquisition in Enterobacterales. By comparative genomic analysis of two clonally related ECC clinical isolates, one susceptible (Temo_S [MIC of 4 mg/L]) and the other resistant (Temo_R [MIC of 32 mg/L]), we found that they differed by only 14 single-nucleotide polymorphisms, including one nonsynonymous mutation (Thr175Pro) in the two-component system (TCS) sensor histidine kinase BaeS. By site-directed mutagenesis in Escherichia coli CFT073, we demonstrated that this unique change in BaeS was responsible for a significant (16-fold) increase in temocillin MIC. Since the BaeSR TCS regulates the expression of two resistance-nodulation-cell division (RND)-type efflux pumps (namely, AcrD and MdtABCD) in E. coli and Salmonella, we demonstrated by quantitative reverse transcription-PCR that mdtB, baeS, and acrD genes were significantly overexpressed (15-, 11-, and 3-fold, respectively) in Temo_R. To confirm the role of each efflux pump in this mechanism, multicopy plasmids harboring mdtABCD or acrD were introduced into either Temo_S or the reference strain E. cloacae subsp. cloacae ATCC 13047. Interestingly, only the overexpression of acrD conferred a significant increase (from 8- to 16-fold) of the temocillin MIC. Altogether, we have shown that temocillin resistance in the ECC can result from a single BaeS alteration, likely resulting in the permanent phosphorylation of BaeR and leading to AcrD overexpression and temocillin resistance through enhanced active efflux.

Evolution of the incidence of COVID-19 during the first five waves in residents and professionals of nursing homes in Normandy, France.

BACKGROUND: Older adults living in nursing homes (NH) paid a heavy price to the COVID-19 pandemic, despite early and often drastic prevention measures. AIMS: To study the characteristics and the impact of the pandemic on NH residents and professionals over 2 years. METHODS: Cross-sectional study of COVID-19 clusters among residents and/or professionals in NH, from March 2020 to February 2022, in Normandy, France. We used data from the French mandatory reporting system, and cross-correlation analysis. RESULTS: The weekly proportion of NH with clusters was strongly correlated with population incidence (r > 0.70). Attack rates among residents and professionals were significantly lower in period 2 (vaccination rate in residents ≥ 50%) compared with periods 1 (waves 1 and 2) and 3 (Omicron variant ≥ 50%). Among residents, mortality and case fatality rates decreased drastically during periods 2 and 3. CONCLUSION: Our study provides figures on the evolution of the pandemic in NH.

A 10-year microbiological study of Pseudomonas aeruginosa strains revealed the circulation of populations resistant to both carbapenems and quaternary ammonium compounds.

Pseudomonas aeruginosa is one of the leading causes of healthcare-associated infections. For this study, the susceptibility profiles to antipseudomonal antibiotics and a quaternary ammonium compound, didecyldimethylammonium chloride (DDAC), widely used as a disinfectant, were established for 180 selected human and environmental hospital strains isolated between 2011 and 2020. Furthermore, a genomic study determined resistome and clonal putative relatedness for 77 of them. During the ten-year study period, it was estimated that 9.5% of patients' strains were resistant to carbapenems, 11.9% were multidrug-resistant (MDR), and 0.7% were extensively drug-resistant (XDR). Decreased susceptibility (DS) to DDAC was observed for 28.0% of strains, a phenotype significantly associated with MDR/XDR profiles and from hospital environmental samples (p < 0.0001). According to genomic analyses, the P. aeruginosa population unsusceptible to carbapenems and/or to DDAC was diverse but mainly belonged to top ten high-risk clones described worldwide by del Barrio-Tofiño et al. The carbapenem resistance appeared mainly due to the production of the VIM-2 carbapenemase (39.3%) and DS to DDAC mediated by MexAB-OprM pump efflux overexpression. This study highlights the diversity of MDR/XDR populations of P. aeruginosa which are unsusceptible to compounds that are widely used in medicine and hospital disinfection and are probably distributed in hospitals worldwide.

Antimicrobial resistance and genetic diversity of Klebsiella pneumoniae strains from different clinical sources in horses.

Introduction: Klebsiella pneumoniae is a major cause of infections and reproductive disorders among horses, ranked in recent French studies as the sixth most frequently isolated bacterial pathogen in equine clinical samples. The proportion of multidrug-resistant (MDR) K. pneumoniae is therefore significant in a context where MDR K. pneumoniae strains are considered a major global concern by the World Health Organization. Methods: In this study, we used a genomic approach to characterize a population of 119 equine K. pneumoniae strains collected by two laboratories specialized in animal health in Normandy (France). We describe the main antibiotic resistance profiles and acquired resistance genes, and specify the proportion of virulence-encoding genes carried by these strains. The originality of our panel of strains lies in the broad collection period covered, ranging from 1996 to 2020, and the variety of sample sources: necropsies, suspected bacterial infections (e.g., genital, wound, allantochorion, and umbilical artery samples), and contagious equine metritis analyses. Results: Our results reveal a remarkable level of genomic diversity among the strains studied and we report the presence of 39% MDR and 9% hypervirulent strains (including 5% that are both MDR and hypervirulent). Discussion: These findings clearly emphasize the importance of improving the surveillance of K. pneumoniae in routine equine diagnostic tests to detect high-risk MDR-hypervirulent Klebsiella pneumoniae strains. The circulation of these worrisome strains reveals that they are not being detected by the simple K1, K2, and K5 serotype approach currently implemented in the French horse-breeding sector.

A retrospective and regional approach assessing the genomic diversity of Salmonella Dublin.

From a historically rare serotype, Salmonella enterica subsp. enterica Dublin slowly became one of the most prevalent Salmonella in cattle and raw milk cheese in some regions of France. We present a retrospective genomic analysis of 480 S. Dublin isolates to address the context, evolutionary dynamics, local diversity and the genesis processes of regional S. Dublin outbreaks events between 2015 and 2017. Samples were clustered and assessed for correlation against metadata including isolation date, isolation matrices, geographical origin and epidemiological hypotheses. Significant findings can be drawn from this work. We found that the geographical distance was a major factor explaining genetic groups in the early stages of the cheese production processes (animals, farms) while down-the-line transformation steps were more likely to host genomic diversity. This supports the hypothesis of a generalised local persistence of strains from animal to finished products, with occasional migration. We also observed that the bacterial surveillance is representative of diversity, while targeted investigations without genomics evidence often included unrelated isolates. Combining both approaches in phylogeography methods allows a better representation of the dynamics, of outbreaks.

Professional practice for COVID-19 risk reduction among health care workers: A cross-sectional study with matched case-control comparison.

BACKGROUND: Health care workers (HCWs) are particularly exposed to COVID-19 and therefore it is important to study preventive measures in this population. AIM: To investigate socio-demographic factors and professional practice associated with the risk of COVID-19 among HCWs in health establishments in Normandy, France. METHODS: A cross-sectional and 3 case-control studies using bootstrap methods were conducted in order to explore the possible risk factors that lead to SARS-CoV2 transmission within HCWs. Case-control studies focused on risk factors associated with (a) care of COVID-19 patients, (b) care of non COVID-19 patients and (c) contacts between colleagues. PARTICIPANTS: 2,058 respondents, respectively 1,363 (66.2%) and 695 (33.8%) in medical and medico-social establishments, including HCW with and without contact with patients. RESULTS: 301 participants (14.6%) reported having been infected by SARS-CoV2. When caring for COVID-19 patients, HCWs who declared wearing respirators, either for all patient care (ORa 0.39; 95% CI: 0.29-0.51) or only when exposed to aerosol-generating procedures (ORa 0.56; 95% CI: 0.43-0.70), had a lower risk of infection compared with HCWs who declared wearing mainly surgical masks. During care of non COVID-19 patients, wearing mainly a respirator was associated with a higher risk of infection (ORa 1.84; 95% CI: 1.06-3.37). An increased risk was also found for HCWs who changed uniform in workplace changing rooms (ORa 1.93; 95% CI: 1.63-2.29). CONCLUSION: Correct use of PPE adapted to the situation and risk level is essential in protecting HCWs against infection.

Salmonella enterica subsp. enterica Welikade: guideline for phylogenetic analysis of serovars rarely involved in foodborne outbreaks.

BACKGROUND: Salmonella spp. is a major foodborne pathogen with a wide variety of serovars associated with human cases and food sources. Nevertheless, in Europe a panel of ten serovars is responsible for up to 80% of confirmed human cases. Clustering studies by single nucleotide polymorphism (SNP) core-genome phylogenetic analysis of outbreaks due to these major serovars are simplified by the availability of many complete genomes in the free access databases. This is not the case for outbreaks due to less common serovars, such as Welikade, for which no reference genomes are available. In this study, we propose a method to solve this problem. We propose to perform a core genome MLST (cgMLST) analysis based on hierarchical clustering using the free-access EnteroBase to select the most suitable genome to use as a reference for SNP phylogenetic analysis. In this study, we applied this protocol to a retrospective analysis of a Salmonella enterica serovar Welikade (S. Welikade) foodborne outbreak that occurred in France in 2016. Finally, we compared the cgMLST and SNP analyses. SNP phylogenetic reconstruction was carried out considering the effect of recombination events identified by the ClonalFrameML tool. The accessory genome was also explored by phage content and virulome analyses. RESULTS: Our findings revealed high clustering concordance using cgMLST and SNP analyses. Nevertheless, SNP analysis allowed for better assessment of the genetic distance among strains. The results revealed epidemic clones of S. Welikade circulating within the poultry and dairy sectors in France, responsible for sporadic and non-sporadic human cases between 2012 and 2019. CONCLUSIONS: This study increases knowledge on this poorly described serovar and enriches public genome databases with 42 genomes from human and non-human S. Welikade strains, including the isolate collected in 1956 in Sri Lanka, which gave the name to this serovar. This is the first genomic analysis of an outbreak due to S. Welikade described to date.

Antimicrobial Resistance and Genetic Diversity of Pseudomonas aeruginosa Strains Isolated from Equine and Other Veterinary Samples.

Pseudomonas aeruginosa is one of the leading causes of healthcare-associated infections in humans. This bacterium is less represented in veterinary medicine, despite causing difficult-to-treat infections due to its capacity to acquire antimicrobial resistance, produce biofilms, and persist in the environment, along with its limited number of veterinary antibiotic therapies. Here, we explored susceptibility profiles to antibiotics and to didecyldimethylammonium chloride (DDAC), a quaternary ammonium widely used as a disinfectant, in 168 P. aeruginosa strains isolated from animals, mainly Equidae. A genomic study was performed on 41 of these strains to determine their serotype, sequence type (ST), relatedness, and resistome. Overall, 7.7% of animal strains were resistant to carbapenems, 10.1% presented a multidrug-resistant (MDR) profile, and 11.3% showed decreased susceptibility (DS) to DDAC. Genomic analyses revealed that the study population was diverse, and 4.9% were ST235, which is considered the most relevant human high-risk clone worldwide. This study found P. aeruginosa populations with carbapenem resistance, multidrug resistance, and DS to DDAC in equine and canine isolates. These strains, which are not susceptible to antibiotics used in veterinary and human medicine, warrant close the setting up of a clone monitoring, based on that already in place in human medicine, in a one-health approach.

Whole Genome Analysis Detects the Emergence of a Single Salmonella enterica Serovar Chester Clone in Japan's Kanto Region.

In Japan's Kanto region, the number of Salmonella enterica serovar Chester infections increased temporarily between 2014 and 2016. Concurrently with this temporal increase in the Kanto region, S. Chester isolates belonging to one clonal group were causing repetitive outbreaks in Europe. A recent study reported that the European outbreaks were associated with travelers who had been exposed to contaminated food in Morocco, possibly seafood. Because Japan imports a large amount of seafood from Morocco, we aimed to establish whether the temporal increase in S. Chester infections in the Kanto region was associated with imported Moroccan seafood. Short sequence reads from the whole-genome sequencing of 47 S. Chester isolates from people in the Kanto region (2014-2016), and the additional genome sequences from 58 isolates from the European outbreaks, were analyzed. The reads were compared with the complete genome sequence from a S. Chester reference strain, and 347 single nucleotide polymorphisms (SNPs) were identified. These SNPs were used in this study. Cluster and Bayesian cluster analyses showed that the Japanese and European isolates fell into two different clusters. Therefore, Φ PT and I A S values were calculated to evaluate genetic differences between these clusters. The results revealed that the Japanese and European isolates were genetically distinct populations. Our root-to-tip analysis showed that the Japanese isolates originating from one clone had accumulated mutations, suggesting that an emergence of this organism occurred. A minimum spanning tree analysis demonstrated no correlation between genetic and geographical distances in the Japanese isolates, suggesting that the emergence of the serovar in the Kanto region did not involve person-to-person contact; rather, it occurred through food consumption. The d N /d S ratio indicated that the Japanese strain has evolved under positive selection pressure. Generally, a population of bacterial clones in a reservoir faces negative selection pressure. Therefore, the Japanese strain must have existed outside of any reservoir during its emergence. In conclusion, S. Chester isolates originating from one clone probably emerged in the Kanto region via the consumption of contaminated foods other than imported Moroccan seafood. The emerging strain may have not established a reservoir for survival in the food supply chain resulting in its disappearance after 2017.

Healthcare associated coronavirus disease 2019 among health care workers in Normandy, France: a multi-center study.

INTRODUCTION: In the early phase of the coronavirus disease (COVID-19) epidemic in France, knowledge of SARS-COV-2 characteristics was limited, and personal protective equipment (PPE) was lacking. Thus, health care workers (HCWs) were exposed to nosocomial transmission. METHODS: A multicenter regional descriptive study of fifty-two heath care facilities covering 30,533 HCWs in western Normandy, France, from March 3 to March 27, 2020, before the incidence threshold of 10/100,000 inhabitants was crossed in the study area. The incidence rate of COVID-19 in HCWs, the attack rates and the serial interval distribution of nosocomial transmission were computed. Demographic characteristics of HCWs, contacts with index cases, and the use of personal protective equipment were collected by a structured questionnaire. RESULTS: The incidence rate of COVID-19 in HCWs was 2.7‰. Among 19 situations (13 clusters >2 cases), 10 were HCW-HCW and 9 patient-HCW transmission, the global attack rate was 13.7% (95% confidence interval, 10.6%-17.3%), and 68 HCWs were involved (10 index cases, with 58 secondary cases). Exposure of secondary cases was only in the presymptomatic phase of the index case in 29% of cases, 48% for HCW-HCW and 10% for patient-HCW transmission (P<0.001). The mean serial interval was 5.1 days (95% CI, 4.2-5.9 days). Preventative measures were not optimal. CONCLUSIONS: Our investigation demonstrated that HCWs who were not assigned to the care of COVID-19 patients were not prepared for the arrival of this particularly insidious new virus, which spread rapidly from an often asymptomatic colleague or patient.

Cefepime use: A need for antimicrobial stewardship.

OBJECTIVES: Unlike other 3GCs, Cefepime is a cephalosporin that has, in animal model studies, shown a low risk of selecting resistant mutants. It also enables carbapenems to be saved in treatment of Pseudomonasaeruginosa and the CESP group (Citrobacter, Enterobacter, Serratia and Providencia, as well as the genus Klebsiellaaerogenes, Morganella and Hafnia), consequently producing cephalosporinase. We aimed to determine whether its prescription in a French teaching hospital met criteria for proper use. PATIENTS AND METHODS: We conducted a retrospective study of proper cefepime use between March 1st, 2018 and February 28th, 2019, to assess indication, antimicrobial stewardship, dosing schedule, microbiological documentation, reevaluation, and treatment duration. Prescriptions were then compared to local guidelines established from international literature. RESULTS: Out of 142 cefepime prescriptions, 97.2% were prescribed as validated according to indication. The duration of the documented treatments matched the guidelines for 56.5% of patients, dosage was adapted to the indication for 77.4% and to kidney function for 97.2%. Bacteriological documentation was performed in all cases and an antibiogram was generated in 99.2% of cases. The treatment was reassessed between 48 and 72h and between the 7th and 10th day for 44.2% and 60.9% of the prescriptions respectively. The antimicrobial stewardship team managed half of the prescriptions. Only 13.4% of prescriptions met all criteria for proper use. CONCLUSION: Notwithstanding a highly sizable majority of validated indications, a very small proportion of cefepime prescriptions met all the criteria for proper use. In the context of increased cefepime consumption, which is favored by its increased place in the latest recommendations published in 2019, proper use of cefepime prescriptions needs to be more effectively promoted.

Intrauterine infection caused by nontyphoidal Salmonella: a literature review.

OBJECTIVES: Although nontyphoidal Salmonella infections have a prevalence of 0.2-1.8%. It is mostly described in veterinary medicine; it could be responsible for severe intra-amniotic infections in humans. The objective of this review is to describe the clinical and microbiological aspects of intrauterine infection (IUI) caused by nontyphoidal Salmonella. METHODS: We reported a case analysis and subsequently conducted a systematic literature review of IUI caused by nontyphoidal Salmonella between 1966 and 2018. RESULTS: In literature nine cases have been reported, and were confirmed by the identification of a nontyphoidal Salmonella in the biological samples. Our review reveals severe clinical presentations in pregnant women. Indeed, sepsis, spontaneous abortions, and fatal outcomes for fetuses were described in 90, 60, and 80% of the cases, respectively. The major clinical symptoms were in majority acute, with high fever, abdominal pain, metrorrhagia, and premature membranes ruptures. Nulliparity is a risk factor and the prognosis depends on the pregnancy stage. All mothers received antibiotics and their outcomes were favorable. CONCLUSIONS: Nontyphoidal Salmonella infections can be responsible for severe pregnancy complications. Considering the severe neonatal prognosis, in case of a history of diarrhea and/or sepsis, a search for this pathogen should be considered, and a preventive strategy could be discussed during pregnancy.

Comparison of conventional molecular and whole-genome sequencing methods for subtyping Salmonella enterica serovar Enteritidis strains from Tunisia.

We sought to determine the relative value of conventional molecular methods and whole-genome sequencing (WGS) for subtyping Salmonella enterica serovar Enteritidis recovered from 2000 to 2015 in Tunisia and to investigate the genetic diversity of this serotype. A total of 175 Salmonella Enteritidis isolates were recovered from human, animal, and foodborne outbreak samples. Pulsed-field gel electrophoresis (PFGE), multiple locus variable-number tandem repeat analysis (MLVA), and whole-genome sequencing were performed. Eight pulsotypes were detected for all isolates with PFGE (DI = 0.518). Forty-five Salmonella Enteritidis isolates were selected for the MLVA and WGS techniques. Eighteen MLVA profiles were identified and classified into two major clusters (DI = 0.889). Core genome multilocus typing (cgMLST) analysis revealed 16 profiles (DI = 0.785). Whole-genome analysis indicated 660 single-nucleotide polymorphism (SNP) divergences dividing these isolates into 43 haplotypes (DI = 0.997). The phylogenetic tree supported the classification of Salmonella Enteritidis isolates into two distinct lineages subdivided into five clades and seven subclades. Pairwise SNP differences between the isolates ranged between 302 and 350. We observed about 311 SNP differences between the two foodborne outbreaks, while only less or equal to 4 SNP differences within each outbreak. SNP-based WGS typing showed an excellent discriminatory power comparing with the conventional methods such as PFGE and MLVA. Besides, we demonstrate the added value of WGS as a complementary subtyping method to discriminate outbreak from non-outbreak isolates belonging to common subtypes. It is important to continue the survey of Salmonella Enteritidis lineages in Tunisia using WGS.

Antimicrobial Resistance in Wildlife in Guadeloupe (French West Indies): Distribution of a Single bla CTX-M-1/IncI1/ST3 Plasmid Among Humans and Wild Animals.

Limited data are available on the contribution of wildlife to the spread of antibacterial resistance. We determined the prevalence of resistance to antibiotics in Escherichia coli isolates collected from wild animals in 2013 and 2014 and the genetic basis for resistance to third-generation cephalosporin in Guadeloupe. We recovered 52 antibiotic-resistant (AR) E. coli strains from 48 of the 884 (5.4%) wild animals tested (46 iguanas, 181 birds, 289 anoles, and 368 rodents at 163 sampling sites). Rodents had higher rates of carriage (n = 38, 10.3%) than reptiles and birds (2.4% and 1.1%, respectively, p < 0.001). A significant association (p < 0.001) was found between the degree of anthropization and the frequency of AR E. coli carriage for all species. The carriage rate of ciprofloxacin- and cefotaxime-resistant isolates was 0.7% (6/884) and 1.5% (13/884), respectively. Most (65.4%) AR E. coli were multi-drug resistant, and the prevalence of extended-spectrum beta-lactamase (ESBL)-producing E. coli was low (n = 7, 0.8%) in all species. Eight ESBL-producing E. coli were recovered, two genetically unrelated isolates being found in one bird. These isolates and 20 human invasive ESBL E. coli isolates collected in Guadeloupe during the same period were investigated by whole genome sequencing. bla CTX-M-1 was the only ESBL gene shared by three animal classes (humans, n = 2; birds, n = 2; rodents, n = 2). The bla CTX-M-1 gene and most of the antimicrobial resistance genes were present in a large conjugative IncI1 plasmid that was highly similar (>99% nucleotide identity) to ESBL-carrying plasmids found in several countries in Europe and in Australia. Although the prevalence of ESBL-producing E. coli isolates was very low in wild animals, it is of concern that the well-conserved IncI1 plasmid-carrying bla CTX-M-1 is widespread and occurs in various E. coli strains from animals and humans.

ramR Deletion in an Enterobacter hormaechei Isolate as a Consequence of Therapeutic Failure of Key Antibiotics in a Long-Term Hospitalized Patient.

Genome changes are central to the adaptation of bacteria, especially under antibiotic pressure. The aim of this study was to report phenotypic and genomic adaptations undergone by an Enterobacter hormaechei clinical strain that became highly resistant to key antimicrobials during a 4-month period in a patient hospitalized in an intensive care unit (ICU). All six clinical E. hormaechei strains isolated in one ICU-hospitalized patient have been studied. MICs regarding 17 antimicrobial molecules have been measured. Single nucleotide polymorphisms (SNPs) were determined on the sequenced genomes. The expression of genes involved in antibiotic resistance among Enterobacter cloacae complex strains were determined by reverse transcription-quantitative PCR (qRT-PCR). All the strains belonged to sequence type 66 and were distant by a maximum of nine SNPs. After 3 months of hospitalization, three strains presented a significant increase in MICs for ceftazidime, cefepime, temocillin, ertapenem, tigecycline, ciprofloxacin, and chloramphenicol. Those resistant strains did not acquire additional antibiotic resistance genes but harbored a 16-bp deletion in the ramR gene. This deletion led to upregulated expression of RamA, AcrA, AcrB, and TolC and downregulated expression of OmpF. The ΔramR mutant harbored the same phenotype as the resistant clinical strains regarding tigecycline, chloramphenicol, and ciprofloxacin. The increased expression of RamA due to partial deletion in the ramR gene led to a cross-resistance phenotype by an increase of antibiotic efflux through the AcrAB-TolC pump and a decrease of antibiotic permeability by porin OmpF. ramR appears to be an important adaptative trait for E. hormaechei strains.

Source Attribution Study of Sporadic Salmonella Derby Cases in France.

Salmonella enterica subsp. enterica serovar Derby is one of the most frequent causes of gastroenteritis in humans. In Europe, this pathogen is one of the top five most commonly reported serovars in human cases. In France, S. Derby has been among the ten most frequently isolated serovars in humans since the year 2000. The main animal hosts of this serovar are pigs and poultry, and white meat is the main source of human contamination. We have previously shown that this serovar is polyphyletic and that three distinct genetic lineages of S. Derby cohabit in France. Two of them are associated with pork and one with poultry. In this study, we conducted a source attribution study based on single nucleotide polymorphism analysis of a large collection of 440 S. Derby human and non-human isolates collected in 2014-2015, to determine the contribution of each lineage to human contamination. In France, the two lineages associated with pork strains, and corresponding to the multilocus sequence typing (MLST) profiles ST39-ST40 and ST682 were responsible for 94% of human contaminations. Interestingly, the ST40 profile is responsible for the majority of human cases (71%). An analysis of epidemiologic data and the structure of the pork sector in France allowed us to explain the spread and the sporadic pattern of human cases that occurred in the studied period.

Genetic diversity of clinical Salmonella enterica serovar Typhimurium in a university hospital of south Tunisia, 2000-2013.

Typhimurium is one of the main Salmonella serovar responsible for non-typhoidal gastro-enteritis in Tunisia. Here, we aimed to assess the genetic diversity of 88 clinical Salmonella Typhimurium strains recovered during 14 years from 2000 to 2013. Phage typing, CRISPR polymorphisms (CRISPOL), pulsed-field gel electrophoresis (PFGE), multi-locus variable-number tandem repeat analysis (MLVA) and Whole genome sequencing (WGS) were used to study the relatedness and spatio-temporal evolution of Salmonella Typhimurium populations (Typhimurium (n = 81), monophasic (n = 3) and nonmotile (n = 4) variants). Seven-locus MLST from whole genome assemblies showed that all isolates, except one, belonged to ST19. The isolates were divided into 10 definitive phage (DT) types, dominated by DT104-L (39.8%), DT41 (14.8%), DT116 (11.4%) and DT120 (5.7%). Fifty-seven MLVA patterns (DI, 0.978) were obtained compared to 11 different CRISPOL types and 15 PFGE types (DI,0.845). For cgMLST analysis, 20 profiles were found. A total of 3056 SNPs were identified from the whole genome of the 88 Salmonella Typhimurium isolates. These SNPs resolved these isolates into 86 SNP haplotypes. The phylogeny result allocated most Salmonella Typhimurium isolates into four distinct clades and seven subclades. Genetic diversity between the four clades ranged in the order of 249 to 720 nucleotide changes. The prevalent phage type DT104L formed a major clade on the phylogenetic tree. Pairwise SNP differences between the strains of this clade ranged between 0 and 59. SNP-based WGS typing seems to be the most valuable molecular markers for studying the evolutionary relationships of homogeneous serovar Typhimurium isolates.