Comparison of pulsed-field gel electrophoresis and whole-genome-sequencing-based typing confirms the accuracy of pulsed-field gel electrophoresis for the investigation of local Pseudomonas aeruginosa outbreaks.

AIM: To determine whether pulsed-field gel electrophoresis (PFGE) accurately recognizes isolates belonging to clusters defined by techniques based on whole-genome sequencing (WGS) using Pseudomonas aeruginosa as a model. METHODS: We selected 65 isolates of ST395 P. aeruginosa isolated in seven European hospitals between 1998 and 2012. Isolates were typed by PFGE and sequenced by WGS. A core genome multi-locus sequence typing (cgMLST) analysis based on 3831 genes was performed with a homemade pipeline. FINDINGS: PFGE identified eight pulsotypes and cgMLST differentiated nine clusters and nine singletons. Five cgMLST clusters and pulsotypes (31/65 isolates) coincided perfectly. Isolates without evident epidemiological links grouped by PFGE were separated by cgMLST (16/65 isolates) differentiating cities, suggesting that PFGE should be kept for the investigation of local outbreaks. Importantly, hypermutator isolates still shared the pulsotype with their parents (16/65 isolates), whereas they were not recognized by cgMLST. This shows that PFGE was less affected than WGS-based typing by the accelerated genetic drift that occurs in epidemic P. aeruginosa. CONCLUSIONS: although WGS-based typing has logically become the new reference standard, we show here that the PFGE can be used with confidence for the investigation of local outbreaks caused by P. aeruginosa.

Clonal complex 398 methicillin-susceptible Staphylococcus aureus bloodstream infections are associated with high mortality.

Within the last decade, methicillin-resistant Staphylococcus aureus belonging to clonal complex 398 (CC398) has become a worldwide threat associated with livestock. More recently, methicillin-susceptible S. aureus (MSSA) belonging to CC398 have been increasingly reported as a cause of invasive infections in patients without livestock contact. We investigated risk factors associated with CC398 bloodstream infections (BSIs) compared with non-CC398 BSIs with a case-control study in a French university Hospital. From January 2010 to December 2014, nonduplicate Staphylococcus aureus (SA) isolates responsible for BSIs in adult patient were typed to identify those belonging to CC398. Each adult patient with a CC398 SA BSI (cases) was matched with 2 non-CC398 SA BSI controls randomly selected on the basis of the time at risk, the unit of hospitalization and susceptibility to methicillin. We retrospectively extracted the clinical information from electronic medical records and used conditional logistic regression for univariate and multivariate analyses. We identified 67 CC398 isolates among the 770 SA responsible for BSI in adult patients. All CC398 isolates were susceptible to methicillin. The proportion of CC398 among MSSA increased steadily from 4.6% in 2010 to 15.1% in 2013 and then stabilized at 13.8% in 2014. Factors significantly associated with CC398 MSSA BSIs were healthcare-associated infection (odds ratio (OR) 3.02, 95% confidence interval (CI) 1.19-7.63), history of neurologic disease (OR 2.51, 95% CI 1.13-5.65) and 30-day mortality (OR 2.44, 95% CI 1.23-4.85).

Acquisition of carbapenem-resistant Gram-negative bacilli in intensive care unit: predictors and molecular epidemiology.

OBJECTIVES: We had for aim to determine the risk factors for acquiring carbapenem-intermediate or -resistant Gram-negative bacilli (CR-GNB) in an intensive care unit (ICU) and to identify the resistance mechanisms involved. PATIENTS AND METHODS: We conducted an observational prospective cohort study during 6 months in medical and surgical ICUs of the Besançon Teaching Hospital. Patients with acquired CR-GNB were patients whose cultures (screening or diagnosis) became positive more than 48h after admission to the ICU. The risk factors for ICU-acquired CR-GNB were determined by multivariate logistic regression. CR-GNB isolates were typed by pulsed-field gel electrophoresis (PFGE) and screened for resistance mechanisms with phenotypic and genotypic tests. RESULTS: Twenty-three of the 347 included patients had acquired a CR-GNB. The multivariate analysis revealed significant associations between this acquisition and the duration of previous treatments with piperacillin-tazobactam (adjusted odds ratio [aOR], 1.13, P=0.02) and aminoglycosides (aOR, 1.62; P=0.005), but not with carbapenems. The CR-GNB strains were identified as Pseudomonas aeruginosa (n=10), Stenotrophomonas maltophilia (n=7), and Enterobacter cloacae (n=6). No acquired carbapenemase-producing strain was identified. PFGE typing identified 1 multiple clone among P. aeruginosa isolates (4 patients), whereas for the other bacteria, all the strains were different. CONCLUSION: Our study results suggest that the strategy to prevent the emergence and spread of CR-GNB should not be limited to the sole restriction of carbapenem use in ICU settings.

Hospital cross-transmission of extended-spectrum ß-lactamase producing Escherichia coli and Klebsiella pneumoniae.

OBJECTIVES: We had for objective to measure the incidence and the clonal diversity of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum ß-lactamases (ESBL) in order to assess the role of patient stay in amplification of the phenomenon, in our teaching hospital. MATERIAL AND METHODS: We measured the quarterly incidence rates of E. coli and K. pneumoniae producing or not producing ESBL in clinical samples between 1999 and 2010. The incidence of ESBL-producing isolates was season-adjusted. We determined the pulsotype of and identified the ESBL in all non-redundant strains isolated between 2009 and 2010. RESULTS: The incidence for 1000 hospitalization days increased from 0.00 to 0.44 for ESBL-producing E. coli, from 0.012 to 0.24 for ESBL-producing K. pneumoniae, from 1999 to 2010. Fifty-three different clones of E. coli were identified among the 61 genotyped isolates. The 28 K. pneumoniae isolates genotyped clustered into 11 different clones, among which one major epidemic clone that included 18 isolates. Respectively 66 and 75% of E. coli and K. pneumoniae isolates produced a CTX-M group 1 ESBL. CONCLUSION: The hospital seems to play a different role in the amplification of ESBL according to the producing species (K. pneumoniae or E. coli). ESBL-producing E. coli seem to have a limited cross-transmission within the hospital and seem to be added to non-producers. Conversely, ESBL-producing K. pneumoniae seem to be cross-transmitted within the hospital and to replace non-producers.

Alternatives to carbapenems in ESBL-producing Escherichia coli infections.

OBJECTIVES: The authors had for objective to assess the activity of a wide panel of antibiotics on extended-spectrum-ß-lactamase producing Escherichia coli isolates (ESBL-Ec), because of the sharp increase of their frequency, leading to an increased use of carbapenems. MATERIAL AND METHODS: We selected 100 ESBL-Ec in which ESBLs were identified by PCR and sequencing, between 2009 and 2010. We determined the MICs of amoxicillin-clavulanate, piperacillin-tazobactam, temocillin, mecillinam, cefoxitin, cefotaxime, ceftazidime, aztreonam, tigecycline, nitrofurantoin, and fosfomycin using reference methods. The susceptibility profiles were defined according to EUCAST 2012 recommendations. RESULTS: Fosfomycin, nitrofurantoin, and pivmecillinam were active against more than 90% of isolates and remain excellent choices for the oral treatment of urinary tract infections (UTIs). Temocillin and piperacillin-tazobactam are also good candidates for the treatment of pyelonephritis or bloodstream infections. Only 27, 23, and 8% of isolates were susceptible to ceftazidime, cefepime, and cefotaxime, respectively. CONCLUSION: Our study results prove that in many cases, there are non-carbapenem alternatives for the treatment of ESBL-Ec infections.

Multilevel modelling of the prevalence of hospitalized patients infected with Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the leading nosocomial pathogens. The question of the respective contribution of endogenous and exogenous sources remains controversial. In this study, we shed new light on this issue by means of a multilevel logistic regression analysis which allowed a simultaneous investigation of factors associated with prevalence of patients infected with P. aeruginosa at two levels: patient and healthcare facility (HCF) in the eastern regions of France. A total of 25 533 in-patients from 51 HCFs were included in the analysis. The overall prevalence was 0·37% (range 0-1·65%). Multilevel modelling estimated that <14% of total variability of the outcome variable was explained by differences between HCFs and that after adjusting for patient-level variables, which explained 52% of HCF-level variance, the latter became non-significantly different from zero. A compositional effect (patient factors), rather than a contextual effect (ecological factors), explains heterogeneity of the prevalence of patients infected with P. aeruginosa in the eastern HCFs of France.

Molecular epidemiology of multidrug-resistant Pseudomonas aeruginosa in a French university hospital.

The aim of this study was to assess the incidence and molecular epidemiology of multidrug-resistant (MDR) Pseudomonas aeruginosa in our university hospital. Analysis included antimicrobial susceptibility profiling, bla gene identification and pulsed-field gel electrophoresis (PFGE). During the one-year study, 654 patients had at least one sample that tested positive for P. aeruginosa, of whom 38 (5.8%) were colonised or infected with an MDR isolate, giving an incidence of 0.1 patient per 1000 patient-days. The 38 non-duplicate isolates yielded 12 different PFGE patterns, three of which included isolates from four patients and one of which included isolates from 15 patients. Two isolates produced acquired extended-spectrum ß-lactamase (one OXA-14 and one OXA-28). Genotyping showed that cross-transmission was responsible for about 70% of MDR P. aeruginosa cases although spatio-temporal analysis failed to demonstrate when this might have occurred for most cases. The major epidemic and the three main micro-epidemic clones were already present in our hospital with a more susceptible phenotype. It is likely that some P. aeruginosa clones are endemic in our hospital and that, within these clones, MDR isolates emerge under antibiotic pressure. Our results indicate that cross-transmission plays a major role in the spread of MDR P. aeruginosa and suggest that priority should be given to the improvement of standard hygienic precautions.