Time to intestinal clearance of carbapenemase-producing Enterobacterales in hospital patients: a longitudinal retrospective observational cohort study.

BACKGROUND: Intestinal clearance of carbapenemase-producing Enterobacterales (CPE-IC) is a cornerstone to discontinue isolation precautions for CPE patients in hospitals. This study aimed to evaluate the time to spontaneous CPE-IC and identify its potential associated risk factors. METHODS: This retrospective cohort study was carried out between January 2018 and September 2020 on all patients in a 3200-bed teaching referral hospital with confirmed CPE intestinal carriage. CPE-IC was defined as at least three consecutive CPE-negative rectal swab cultures without a subsequent positive result. A survival analysis was performed to determine the median time to CPE-IC. A multivariate Cox model was implemented to explore the factors associated with CPE-IC. RESULTS: A total of 110 patients were positives for CPE, of whom 27 (24.5%) achieved CPE-IC. Median time to CPE-IC was 698 days. Univariate analysis showed that female sex (P=0.046), multiple CPE-species in index cultures (P=0.005), Escherichia coli or Klebsiella spp. (P=0.001 and P=0.028, respectively) were significantly associated with the time to CPE-IC. Multivariate analysis highlighted that identification of E. coli carbapenemase-producing or CPEs harbouring ESBL genes in index culture extended the median time to CPE-IC, respectively (adjusted hazard ratio (aHR) = 0.13 (95% confidence interval: 0.04-0.45]; P=0.001 and aHR = 0.34 (95% confidence interval: 0.12-0.90); P=0.031). CONCLUSION: Intestinal decolonization of CPE can take several months to years to occur. Carbapenemase-producing E. coli are likely to play a key role in delaying intestinal decolonization, probably through horizontal gene transfer between species. Therefore, discontinuation of isolation precautions in CPE-patients should be considered with caution.

Identification of clinically relevant Corynebacterium strains by Api Coryne, MALDI-TOF-mass spectrometry and molecular approaches.

We evaluated the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) for the identification of 97 Corynebacterium clinical in comparison to identification strains by Api Coryne and MALDI-TOF-MS using 16S rRNA gene and hypervariable region of rpoB genes sequencing as a reference method. C. striatum was the predominant species isolated followed by C. amycolatum. There was an agreement between Api Coryne strips and MALDI-TOF-MS identification in 88.65% of cases. MALDI-TOF-MS was unable to differentiate C. aurimucosum from C. minutissimum and C. minutissimum from C. singulare but reliably identify 92 of 97 (94.84%) strains. Two strains remained incompletely identified to the species level by MALDI-TOF-MS and molecular approaches. They belonged to Cellulomonas and Pseudoclavibacter genus. In conclusion, MALDI-TOF-MS is a rapid and reliable method for the identification of Corynebacterium species. However, some limits have been noted and have to be resolved by the application of molecular methods.

Foodborne transmission of sorbitol-fermenting Escherichia coli O157:[H7] via ground beef: an outbreak in northern France, 2011.

Sorbitol-fermenting Escherichia coli O157:[H7] is a particularly virulent clone of E. coli O157:H7 associated with a higher incidence of haemolytic uraemic syndrome and a higher case fatality rate. Many fundamental aspects of its epidemiology remain to be elucidated, including its reservoir and transmission routes and vehicles. We describe an outbreak of sorbitol-fermenting E. coli O157:[H7] that occurred in France in 2011. Eighteen cases of paediatric haemolytic uraemic syndrome with symptom onset between 6 June and 15 July 2011 were identified among children aged 6 months to 10 years residing in northern France. A strain of sorbitol-fermenting E. coli O157:[H7] stx2a eae was isolated from ten cases. Epidemiological, microbiological and trace-back investigations identified multiply-contaminated frozen ground beef products bought in a supermarket chain as the outbreak vehicle. Strains with three distinct pulsotypes that were isolated from patients, ground beef preparations recovered from patients' freezers and from stored production samples taken at the production plant were indistinguishable upon molecular comparison. This investigation documents microbiologically confirmed foodborne transmission of sorbitol-fermenting of E. coli O157 via beef and could additionally provide evidence of a reservoir in cattle for this pathogen.

[MALDI-TOF mass spectrometry to identify clinical bacterial isolates: evaluation in a teaching hospital in Lille]. / Identification bactérienne par spectrométrie de masse de type MALDI-TOF: évaluation au CHU de Lille.

PURPOSE: The aim of our study was to evaluate the capacity of MALDI-TOF mass spectrometry to identify clinical bacterial isolates, as compared to the automated identification system Vitek 2 (bioMérieux) used routinely in a teaching hospital. METHODS: Three hundred and sixty-two strains representing 178 species from the laboratory collection were analysed by a Microflex spectrometer (Bruker Daltonics) and Vitek 2. Discrepancies between MALDI-TOF and Vitek 2 identifications were investigated by genetic identification (rrS, sodA, rpoB), considered as a reference. RESULTS: Among the 362 isolates, 264 (73%) were consistently identified by Vitek 2 and Microflex. Taking into account genetic identification, we found that 44 (44.9%) of the 98 remaining isolates were correctly identified by mass spectrometry but not by Vitek 2. Conversely, 33 isolates (33.7%) were correctly identified by Vitek 2, but not by Microflex. The genetic identification of the 21 remaining isolates (21,4%) did not match either Vitek 2 or Microflex results. CONCLUSION: The performances of MALDI-TOF mass spectrometry for bacterial identification correspond to those of a reference automated identification system.

Molecular diversity of porcine and human isolates of Pasteurella multocida.

AIMS: To examine the variability among Pasteurella multocida strains isolated from pigs (nasal, tonsil and lung specimens) and humans in France. METHODS AND RESULTS: The genetic diversity of 117 French isolates of P. multocida, obtained from pigs (n = 101) and humans (n = 16) and three reference strains, was evaluated by pulsed-field gel electrophoresis (PFGE) after macrorestriction with ApaI. Sixty-four patterns were detected. The genetic relationships revealed five clusters (Aa1, Aa2, Aa3, Ab and B). The pig isolates obtained from pneumonic lungs and nasal cavities were clustered in groups Ab and Aa1, respectively (P < 0.05). Up to four different PFGE patterns were detected in the same farm. Isolates producing dermonecrotic toxins were clustered only in group Aa1, suggesting that the toxigenic isolates were more genetically homogenous than the others. Conversely, cluster Aa3 was significantly associated with human isolates even if the human isolates are spread over most of the clusters. CONCLUSIONS: Pasteurella multocida strains were genetically diverse, but pig and human isolates were significantly clustered in distinct phylogenetic groups. SIGNIFICANCE AND IMPACT OF THE STUDY: The discrimination index was >0.95 in both populations of human and pig isolates. Therefore, ApaI-PFGE seems to be a useful tool for epidemiological tracing of P. multocida infections.

[Legionella and legionellosis: of water, bacteria and men]. / Légionelles et légionelloses: de l'eau, des bactéries et des hommes.

Legionella species are natural dwellers of stagnant waters. Inhalation of contaminated aerosols may result in pneumonia, with a noteworthy mortality rate (20%). However, such infections are infrequent, when compared to the rate of human exposition to Legionella. Legionellosis apparently occurs in patients in which respiratory tract macrophages allow bacterial replication, especially that of Legionella pneumophila serogroup 1. Following a silent incubation period, large amounts of bacteria are released, resulting in a strong inflammatory response responsible for the severity of symptoms. The outcome depends on quick establishment of antibiotic therapy and early diagnosis is therefore necessary. Nowadays, the risk of acquiring legionellosis raises passionate discussions, in which the presence of Legionella in man-made water systems is often assimilated with the disease itself. Significant efforts are being made to detect and monitor the amount of Legionella in potentially contaminant environments. However, the prevention of legionellosis also requires that research efforts for a better understanding of the virulence mechanisms of infective strains are carried out.

[ATP-dependant proteolysis and bacterial pathogenesis]. / Protéolyse ATP-dépendante et virulence bactérienne.

Proteolysis plays an central role in key metabolic pathways and cellular adaptation to environmental changes. It modulates the activity of regulatory peptides and eliminates misfolded or damaged proteins such as those generated by stress exposure. In eucaryotic cells ATP- dependent proteolysis is carried out by the 26S proteasome whose substrates are identified by ubiquitin tags. Conversely, bacteria possess several tagging systems and different ATP- dependent proteases. Bacterial ATP-dependent proteases carry distinct chaperone-ATPase and peptidase activities, either on the same molecule or on separate subunits. Although unrelated, all ATP-dependent proteases function according to a similar multistep scheme, from the docking of a substrate by the ATPase region to its proteolysis by the peptidase. Major bacterial ATP- dependent proteases include FtsH, Lon, HslUV and the Clp proteases. Clp proteases are multimeric complexes assembled into a structure centered on the proteolytic component ClpP. They are essential for quick adaptation to stress and regulate important developmental processes. Clp-mediated proteolysis is also required for disease progression and virulence of several bacterial pathogens, favoring survival in the host or modulating the activity of genuine virulence factors.

[Human pasteurellosis: diagnosis, treatment and precautions]. / Le diagnostic, le traitement et la prévention des pasteurelloses humaines.

The circumstances of diagnosis of human pasteurellosis are reviewed. The diagnosis is usually suspected for animal bite or scratch wounds. Conversely, in other infections the diagnosis is only based on bacteriological data. Phenotypic misidentification of Pasteurellaceae from clinical material is common. The phenotypic criteria of identification of the six species of human pathogen Pasteurella are presented. We emphasise that bite wound specimens have to be cultured for aerobic and anaerobic bacteria and yield an average of 5 bacterial isolates per culture. Antibiotic therapy relies upon amino-penicillins or cephalosporins, although b-lactamase producing strains are scarce. Fluoroquinolones can be an alternative for systemic infections. Molecular typing unequivocally points out the risk of transmission from pets to humans. Immunocompromised persons have to be made aware of precautions.

Stress-induced ClpP serine protease of Listeria monocytogenes is essential for induction of listeriolysin O-dependent protective immunity.

The stress-induced protease ClpP is required for virulence of the facultative intracellular pathogen Listeria monocytogenes. We previously found that in the absence of ClpP, the virulence of this pathogen was strongly reduced, mainly due to the decreased production of functional listeriolysin O (LLO), a major immunodominant virulence factor promoting intracellular growth. In this work, a clpP deletion mutant of L. monocytogenes was used to study the generation of anti-Listeria protective immunity. We found that ClpP is required for the intracellular growth of L. monocytogenes in resident macrophages in vivo. Mice infected with doses as high as 10(6) clpP mutant bacteria were not protected against a lethal challenge of wild-type bacteria and did not develop any detectable LLO-specific cytolytic T cells or antibodies, suggesting that the amount of LLO produced in infected mice under these conditions was too low to induce a specific immune response. However, in contrast to the results obtained with a mutant with a disrupted hly gene, this lack of protection was overcome by inoculation of very high infecting doses of clpP mutant bacteria (5 x 10(8)), thus producing sufficient amounts of LLO to stimulate anti-Listeria immunity. The role of ClpP was confirmed by showing that anti-Listeria immunity was restored in mice infected with a clpP-complemented mutant. These results indicate that the stress-induced serine protease ClpP is a potential target for modulating the presentation of protective antigens such as LLO and thereby the immune response against L. monocytogenes.

Contribution of Mn-cofactored superoxide dismutase (SodA) to the virulence of Streptococcus agalactiae.

Superoxide dismutases convert superoxide anions to molecular oxygen and hydrogen peroxide, which, in turn, is metabolized by catalases and/or peroxidases. These enzymes constitute one of the major defense mechanisms of cells against oxidative stress and hence play a role in the pathogenesis of certain bacteria. We previously demonstrated that group B streptococci (GBS) possess a single Mn-cofactored superoxide dismutase (SodA). To analyze the role of this enzyme in the pathogenicity of GBS, we constructed a sodA-disrupted mutant of Streptococcus agalactiae NEM316 by allelic exchange. This mutant was subsequently cis complemented by integration into the chromosome of pAT113/Sp harboring the wild-type sodA gene. The SOD specific activity detected by gel analysis in cell extracts confirmed that active SODs were present in the parental and complemented strains but absent in the sodA mutant. The growth rates of these strains in standing cultures were comparable, but the sodA mutant was extremely susceptible to the oxidative stress generated by addition of paraquat or hydrogen peroxide to the culture medium and exhibited a higher mutation frequency in the presence of rifampin. In mouse bone marrow-derived macrophages, the sodA mutant showed an increased susceptibility to bacterial killing by macrophages. In a mouse infection model, after intravenous injection the survival of the sodA mutant in the blood and the brain was markedly reduced in comparison to that of the parental and complemented strains whereas only minor effects on survival in the liver and the spleen were observed. These results suggest that SodA plays a role in GBS pathogenesis.

SHV-type extended-spectrum beta-lactamase in a Shigella flexneri clinical isolate.

A Shigella flexneri isolate resistant to oxyimino-cephalosporins was recovered from a stool sample of a 16 month-old Algerian child hospitalized in Paris, France. This isolate harboured an SHV-2 beta-lactamase gene located on a c. 80 kb self-transferable plasmid. This is the first report of an Ambler class A extended-spectrum beta-lactamase from Shigella spp.

Optimization of green fluorescent protein expression vectors for in vitro and in vivo detection of Listeria monocytogenes.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is a useful reporter molecule for monitoring in vivo gene expression in eukaryotic and prokaryotic cells. We constructed a series of GFP vectors for in situ detection of the intracellular pathogen Listeria monocytogenes. The gfp-mutl gene, which encodes a red-shifted GFP, was transcriptionally fused to a strong L. monocytogenes promoter and inserted into various Escherichia coli-Listeria shuttle vectors: i) the integrative monocopy plasmid pAT113; ii) the low copy number plasmid pTCV-Exl; iii) the high copy number plasmid pAT18. Listeria cells harboring pNF6 and pNF7, constructed from pAT113 and pTCV-Exl, respectively, gave low fluorescence intensities, and were optically detected in cultured macrophages, but not in tissue sections. The fluorescence of Listeria with the pAT18 derivative pNF8 was about 40 times greater than that with pNF6 and 15 times greater than that with pNF7. Listeria cells harboring pNF8 were readily detected in both cultured macrophages and tissue sections. Constructed GFP vectors did not affect the virulence of L. monocytogenes in a murine model of infection.

The ClpP serine protease is essential for the intracellular parasitism and virulence of Listeria monocytogenes.

We identified the stress-induced ClpP of Listeria monocytogenes and demonstrated its crucial role in intracellular survival of this pathogen. ClpP is a 21.6 kDa protein belonging to a family of proteases highly conserved in prokaryotes and eukaryotes. A clpP-deleted mutant enabled us to demonstrate that ClpP is involved in proteolysis and is required for growth under stress conditions. Intramacrophage survival of this mutant was strongly restricted, thus resulting in loss of virulence for the mouse. The activity of listeriolysin O, a major virulence factor implicated in bacterial escape from phagosomes of macrophages, was much reduced in the clpP mutant under stress conditions. Direct evidence for the role of ClpP in the intracellular parasitism was obtained by showing that virulence and haemolytic activity were fully restored by complementation of the mutant. These results suggest that ClpP is involved in the rapid adaptive response of intracellular pathogens during the infectious process.

Evaluation of CHROMagar Staph. aureus, a new chromogenic medium, for isolation and presumptive identification of Staphylococcus aureus from human clinical specimens.

CHROMagar Staph. aureus (CSA) is a new chromogenic medium for presumptive identification of Staphylococcus aureus as mauve colonies after 24 h of incubation. We conducted a preliminary study with 100 S. aureus and 45 coagulase-negative Staphylococcus (CoNS) stock isolates plated on CSA. All S. aureus isolates yielded mauve colonies after 24 h of incubation at 37 degrees C, while CoNS isolates grew as blue, white, or beige colonies. Culture on CSA was then prospectively compared to a conventional laboratory method, i.e. , culture on 5% horse blood agar (HBA), catalase test, and latex agglutination test (HBA-catalase-latex), for isolation and presumptive identification of S. aureus from 2,000 consecutive clinical samples. Among the 310 S. aureus isolates recovered by at least one of the two methods, 296 grew as typical mauve colonies on CSA, while only 254 yielded catalase-positive, latex-positive colonies on HBA. The sensitivity of CSA was significantly higher than that of the conventional method (95.5 and 81.9%, respectively; P < 0.001) and allowed the recovery of important clinical isolates that were undetected on blood agar. The specificities of the two methods were not significantly different, although that of CSA was slightly higher (99.4% versus 98.9% for HBA-catalase-latex; P = 0. 08). On the basis of its excellent sensitivity and specificity, ease of identification of positive colonies, and absence of complementary testing, CSA can be recommended as a routine plating medium for presumptive identification of S. aureus in clinical specimens.

CtsR controls class III heat shock gene expression in the human pathogen Listeria monocytogenes.

Stress proteins play an important role in virulence, yet little is known about the regulation of stress response in pathogens. In the facultative intracellular pathogen Listeria monocytogenes, the Clp ATPases, including ClpC, ClpP and ClpE, are required for stress survival and intracellular growth. The first gene of the clpC operon of L. monocytogenes encodes a homologue of the Bacillus subtilis CtsR repressor of stress response genes. An L. monocytogenes ctsR-deleted mutant displayed enhanced survival under stress conditions (growth in the presence of 2% NaCl or at 42 degrees C), but its level of virulence in the mouse was not affected. The virulence of a wild-type strain constitutively expressing CtsR is significantly attenuated, presumably because of repression of the stress response. Regulation of the L. monocytogenes clpC, clpP and clpE genes was investigated using transcriptional fusions in B. subtilis as a host. The L. monocytogenes ctsR gene was placed under the control of an inducible promoter, and regulation by CtsR and heat shock was demonstrated in vivo in B. subtilis. The purified CtsR protein of L. monocytogenes binds specifically to the clpC, clpP and clpE regulatory regions, and the extent of the CtsR binding sites was defined by DNase I footprinting. Our results demonstrate that this human pathogen possesses a CtsR regulon controlling class III heat shock genes, strikingly similar to that of the saprophyte B. subtilis. This is the first description of a stress response regulatory gene in a pathogen.

[Molecular epidemiology of strains of Staphylococcus aureus resistant to methicillin, sensitive to aminoglycosides]. / Epidémiologie moléculaire de souches de staphylocoque doré résistantes à la méticilline, sensibles aux aminosides.

Twenty methicillin-resistant Staphylococcus aureus (MRSA) isolates surprisingly susceptible to all aminoglycosides, macrolides, sulfonamides and tetracycline were recovered from 20 elderly patients (mean age, 77) hospitalized in 4 neighbouring facilities between 1996 and 1998. Molecular typing of the isolates performed by restriction fragment length polymorphism of the coagulase gene (PCR-RFLP) and pulsed-field gel electrophoresis (PFGE) of Sma I macrorestriction fragments of total DNA, demonstrated the existence of distinct bacterial clones. Epidemic spreading was demonstrated for at least 2 clones, while others were responsible for sporadic cases. Most of the isolates of this study appeared to be genetically related to strains of MRSA resistant to aminoglycosides and macrolides included as controls, suggesting that multiresistant MRSA may have evolved recently in a manner that resulted in greater susceptibility to certain antibiotics.

[Inquiry into the incidence of nosocomial infections and evaluation of the transmission of methicillin-resistant Staphylococcus aureus in an orthopedic surgical unit]. / Enquête d'incidence des infections nosocomiales et évaluation du portage de Staphylococcus aureus résistant à la méticilline dans un service de chirurgie orthopédique.

Nosocomial infections are an important cause of morbidity and mortality. Methicillin resistant Staphylococcus aureus (MRSA) is often the severe causal agent in this kind of infections. In order to evaluate risk factors for nosocomial infections and nasal MRSA carriage, an incidence study was carried out on patients hospitalized in an orthopaedic surgery department in Boucicaut Hospital (Paris). This study was carried out over a five month period. Data of all the patients who stayed more than two days in the unit were collected in medical and nursing records. Nasal swab specimens were taken at the admission of each patient included in order to screen nasal MRSA carriers. Statistical analysis were performed using Epi Info software version 6.0. A total of 451 patients were included in the study. Nosocomial infections incidence rate was 11.5%. Risk factor significantly associated with nosocomial infection was high wound containation classes III and IV (Altemeier). Incidence rate of MRSA carriage was 3.1%. A previous hospitalization in a general hospital 6 months before an admission at Boucicaut Hospital was the only risk factor identified. According to this, these patients, when they are admitted, are proposed to be preventely isolated awaiting their microbiological results.

Comparison of CHROMagar Salmonella medium and hektoen enteric agar for isolation of salmonellae from stool samples.

CHROMagar Salmonella (CAS), a new chromogenic medium, was retrospectively compared to Hektoen enteric agar (HEA) with 501 Salmonella stock isolates and was then prospectively compared to HEA for the detection and presumptive identification of Salmonella spp. with 508 stool samples before and after enrichment. All stock cultures (100%), including cultures of H2S-negative isolates, yielded typical mauve colonies on CAS, while 497 (99%) isolates produced typical lactose-negative, black-centered colonies on HEA. Following overnight incubation at 37 degreesC, a total of 20 Salmonella strains were isolated from the 508 clinical samples. Sensitivities for primary plating and after enrichment were 95% (19 isolates) and 100% (20 isolates), respectively, for CAS and 80% (16 isolates) and 100% (20 isolates), respectively, for HEA. The specificity of CAS (88.9%) was significantly higher than that of HEA (78.5%; P < 0.0001). On the basis of its good sensitivity and specificity, CAS medium can be recommended for use for primary plating when human stool samples are screened for Salmonella spp.

Salmonella enteritidis: AmpC plasmid-mediated inducible beta-lactamase (DHA-1) with an ampR gene from Morganella morganii.

DHA-1, a plasmid-mediated cephalosporinase from a single clinical Salmonella enteritidis isolate, conferred resistance to oxyimino-cephalosporins (cefotaxime and ceftazidime) and cephamycins (cefoxitin and moxalactam), and this resistance was transferable to Escherichia coli HB101. An antagonism was observed between cefoxitin and aztreonam by the diffusion method. Transformation of the transconjugant E. coli strain with plasmid pNH5 carrying the ampD gene (whose product decreases the level of expression of ampC) resulted in an eightfold decrease in the MIC of cefoxitin. A clone with the same AmpC susceptibility pattern with antagonism was obtained, clone E. coli JM101(pSAL2-ind), and its nucleotide sequence was determined. It contained an open reading frame with 98. 7% DNA sequence identity with the ampC gene of Morganella morganii. DNA sequence analysis also identified a gene upstream of ampC whose sequence was 97% identical to the partial sequence of the ampR gene (435 bp) from M. morganii. The gene encoded a protein with an amino-terminal DNA-binding domain typical of transcriptional activators of the LysR family. Moreover, the intercistronic region between the ampC and ampR genes was 98% identical to the corresponding region from M. morganii DNA. AmpR was shown to be functional by enzyme induction and a gel mobility-shift assay. An ampG gene was also detected in a Southern blot of DNA from the S. enteritidis isolate. These findings suggest that this inducible plasmid-mediated AmpC type beta-lactamase, DHA-1, probably originated from M. morganii.

Nosocomial outbreak of Klebsiella pneumoniae producing SHV-5 extended-spectrum beta-lactamase, originating from a contaminated ultrasonography coupling gel.

Klebsiella pneumoniae resistant to ceftazidime was isolated from six adult women and two neonates hospitalized between July and November 1993 in the Department of Obstetrics and Gynecology of Boucicaut Hospital (Paris, France). The epidemiological investigation revealed a notably short delay (less than 48 h) between admission and contamination of the six adults and peripartum transmission to the neonates. The only environmental source of ceftazidime-resistant K. pneumoniae was the ultrasonography coupling gel used in the emergency room. Phenotypic (biotyping and antibiotyping) and genotypic (plasmid profile and pulsed-field gel electrophoresis) analysis of all the clinical isolates indicated the spread of a single strain. It produced SHV-5 and TEM-1 beta-lactamases, as demonstrated by isoelectric focusing and gene sequencing. The risk of cross-contamination in ultrasonography procedures is usually low and had not been associated so far with bacteria producing an extended-spectrum beta-lactamase (ESBL). Furthermore, this is the first time an epidemic of an SHV-5 ESBL-producing member of the family Enterobacteriaceae has been reported from a French hospital.