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.

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.

Temporal effects of infection control practices and the use of antibiotics on the incidence of MRSA.

BACKGROUND: Meticillin-resistant Staphylococcus aureus (MRSA) has spread throughout the world and has become highly endemic worldwide. The need for implementing MRSA control strategies is no longer a matter of debate. AIM: To determine the temporal association between various infection control practices, the use of antibiotics and the incidence of MRSA in a 1200-bed French university hospital. METHODS: A multi-variate time-series analysis, based on monthly data from a nine-year period (January 2000-December 2008), was performed in a 1200-bed French university hospital to determine the temporal association between different variables and the incidence of MRSA. MRSA colonization pressure, infection control practices and use of antibiotics were considered in the analysis. FINDINGS: Time-series analysis showed a positive significant relationship between the incidence of hospital-acquired MRSA (HA-MRSA) and MRSA colonization pressure, the use of antibiotics (fluoroquinolones, macrolides and aminoglycosides) and the use of gloves. Conversely, a global negative correlation was observed between the incidence of HA-MRSA and the use of alcohol-based hand rub. Overall, the model explained 40.5% of the variance in the monthly incidence of MRSA. CONCLUSION: This study showed that admission of patients with MRSA, the use of antibiotics and infection control practices contributed to the incidence of HA-MRSA. This suggests that efforts should be focused on high compliance with hand disinfection. These results also raise concerns about the use of gloves when caring for patients with MRSA.

Mupirocin resistance is not an inevitable consequence of mupirocin use.

The rate of mupirocin resistance in meticillin-resistant Staphylococcus aureus (MRSA) in Besançon University Hospital is low with a decreasing trend, from 10% in 2004 to 3% in 2009. This trend in resistance paralleled mupirocin consumption. Genotyping results showed that this decrease was not linked to a change in MRSA clones. It appears that the way in which the mupirocin is used, rather than the volume, plays a role in the emergence of resistance and that its cautious use is likely to maintain the mupirocin resistance at a low level, thus preserving its efficacy.

Longitudinal analysis and genotyping of infant dominant bifidobacterial populations.

Bifidobacterial population dynamics were investigated by the longitudinal analysis of the dominant population isolated from the feces of young infants. After molecular identification and fingerprinting comparison, clone identity of the consecutive strains belonging to the same species for one individual was performed by pulsed-field gel electrophoresis. The results, obtained from 15 individuals sampled four times over a five-week period suggested a turnover of the dominant bifidobacteria in the population not only at the species but also at its species representative levels. This study provides new insights of the in vivo dynamics of commensal bifidobacteria. It highlights the need to take into consideration the fluctuation of bifidobacterial populations that may occur in one individual in order to investigate reliably the impact of dietary components, such as probiotics or prebiotics, on the intestinal ecosystem.

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.

[Is surveillance of Pseudomonas aeruginosa carriage in intensive care units useful?]. / Faut-il procéder à un dépistage du portage de Pseudomonas aeruginosa dans les services de réanimation ?

OBJECTIVE: To evaluate the potential interest of screening of Pseudomonas aeruginosa on admission and during hospitalisation in intensive care units patients. METHOD: A retrospective study was carried out in two adult ICU of the University-Hospital of Besançon in 2007. P. aeruginosa screening was performed on admission and once a week during ICU stay. Clinical samples positive with P. aeruginosa were collected. RESULTS: Among the 754 patients included, 146 had a screening sample positive giving an average incidence of 19.4 per 100 patients. Thirty-five were imported and 111 ICU-acquired. Sixty-one patients had at least one positive clinical sample, that is an incidence 8.1 cases per 100 admitted patients. Sensibility, specificity, positive and negative predictive values of screening as an indicator of subsequent infection were 54.1%, 86.9%, 26.6% and 95.6%, respectively. CONCLUSION: Screening samples are necessary to assess P. aeruginosa endemicity in intensive care units. The high negative predictive value of screening suggests that use of specifics anti-Pseudomonas antimicrobials could be reduced. However, the benefit of this strategy remains to be evaluated.

[Nosocomial infections caused by Pseudomonas aeruginosa: Exogenous or endogenous origin of this bacterium?]. / Infections nosocomiales à Pseudomonas aeruginosa : origine exogène ou endogène de la bactérie responsable?

Through numerous reports of Pseudomonas aeruginosa outbreaks linked to transmission from environmental reservoirs, infection control practitionners have a univocal picture of its epidemiology: it is an opportunistic pathogen responsible of major outbreaks in intensive care units (ICU) with a major role played by the water network. The objective of this review was to answer to three questions: what is the part of hospital acquisition of P. aeruginosa? What is the part of outbreaks on incidence of hospital-acquired infections? What is the part of environment as a reservoir for transmission? Genotyping of hospital-acquired P. aeruginosa isolates allows us to define the endogenous or exogenous source of the infection and replace the concept of imported/acquired infection. If 80% of infections could be considered as acquired in ICU, the proportion of infections from exogenous source could be estimated at 50%. Even in a context of major outbreak, the epidemic clone represents 20% of the patients colonized and among these patients, only 50% are recognized with clinical samples. Some studies show that water fittings are a major source of P. aeruginosa in ICU. Other reports demonstrate a weak epidemiological link between environmental and clinical strains. Finally, despite the fact that the relative contributions of endogenous and exogenous sources to P. aeruginosa acquisition are not well established, we can assume that the epidemiological pattern of P. aeruginosa infection and colonization is not univocal and may vary both between ICU and within ICU depending on the period considered.

Molecular epidemiology of Enterobacter cloacae in a neonatal department: a 2-year surveillance study.

A surveillance program was implemented to investigate the molecular epidemiology of Enterobacter cloacae in neonatal units. This program ran for 2 years and involved screening for E. cloacae intestinal colonization of all infants at admission and weekly thereafter. In addition, mothers whose children were admitted to neonatal units were also screened. Pulsed-field gel electrophoresis analysis was used to establish genetic relationships between strains and to identify mother-to-child transmission. During the survey period, 166 (22.6%) of the 735 included children had E. cloacae intestinal colonization, and 29 (3.9%) patients gave clinical samples positive for E. cloacae. Genotyping revealed 90 different pulsotypes in the 199 clinical and screening isolates from neonates, including three major epidemic clones. Mother-to-child transmission of E. cloacae was directly responsible for 8.8% of intestinal colonization of the neonates. This surveillance program reveals a major contribution of patient-to-patient transmission and the rarity of mother-to-child transmission in the spread of E. cloacae in neonates. This highlights the importance of good compliance with infection control procedures by health-care workers.

Are antibiotic-resistant Pseudomonas aeruginosa isolated from hospitalised patients recovered in the hospital effluents?

Previous reports have studied the presence of antibiotic-resistant Pseudomonas aeruginosa strains in hospital wastewater without determination of their clonal relationship with the clinical strains of this species. The objectives of this study were to quantify the presence of P. aeruginosa in wastewater of our hospital, to determine their antibiotic-resistance profile and to potentially trace clinical antibiotic-resistant strains from patients to wastewater. Specimens were taken at the end of the wastewater network of our hospital just before the reject in the collective network of the town. Two specimens were taken each Monday during 12 weeks. All P. aeruginosa isolates recovered from hospitalised patients during the study period were collected. Genotyping of both clinical and wastewater isolates was determined by using pulsed-field gel electrophoresis (PFGE). The antibiotic-resistance profile of wastewater isolates was different from that of clinical isolates. The mechanisms involved in antibiotic resistance were different according to the origin of the isolates (wastewater versus human isolates). There was no common PFGE pattern in antibiotic-resistant P. aeruginosa from humans and wastewater. This study suggests that the risk of spread of antibiotic resistance in hospital wastewater is limited.

[Are isolation measures needed in the hospital management of Acinetobacter baumannii colonizations-infections?]. / L'isolement septique des patients colonisés-infectés par Acinetobacter baumannii est-il totalement superflu, utile ou indispensable?

UNLABELLED: Until 2001, the infection control department of the Besançon university hospital (France) recommended isolation precautions for all patients colonized-infected by Acinetobacter baumannii (Ab) whatever the antibiotic susceptibility of the strain. These systematic isolation procedures were given up at the beginning of 2002 since the number of colonized-infected patients remained stable from 1998 to 2001. OBJECTIVE: The aim of this study was to evaluate the impact of this decision on the risk of Ab infection. METHOD: Incidence rates of colonization-infections that were observed during years 2002 and 2003 were compared with expected incidence rate, based on data from 1998-2001 period. Infection control practices and antibiotics consumption were evaluated for each ward of hospitalisation. Genotyping made it possible to determine diversity of clones inside each unit and the whole hospital. RESULTS: The expected incidence per 1000 patients-days was 0.22 in comparison with observed data in 2002, 0.34 (CI(95%) [0.28-0.42]), and in 2003, 0.53 (CI(95%) [0.45-0.63]). The expected number of Ab bloodstream infections, about two per year compared with the observed numbers in 2002 and 2003 respectively seven and 17. The number of unit with more than three cases per year increased from seven in 1999 to 18 in 2003. Antibiotics consumption did not change significantly. CONCLUSION: Genotyping results show the importance of cross-transmission in these units. Finally, observed results suggest that some measures of isolation precautions in addition to standard precautions are needed to prevent outbreaks of Ab.

[Increased susceptibility to non-beta-lactam antimicrobial agents of MRSA isolates: relationship between genotype and antibiotype]. / Retour vers la sensibilité aux antibiotiques des souches de Staphylococcus aureus résistant à la méticilline (SARM) : relation entre génotype and antibiotype.

The objective of our study was to investigate the relationship between molecular epidemiology and antibiotic susceptibility of MRSA during a four-year period. In this aim, we determined the antibiotype of all MRSA identified during a yearly period of 3 months and typed 50 consecutive non-replicate MRSA isolates of each year. We also recorded antibiotic use. Susceptibility rates to gentamicin, tobramycin and ofloxacin remained stable, respectively, 95, 16 and 4%. In contrast, the proportion of MRSA susceptible to erythromycin progressively increased from 7.0% to 32.5% (P < 0.001). PFGE analysis of genomic DNA from the 200 isolates revealed 15 different clones. We identified two epidemic clones, which contained 150 (clone A) and 28 isolates (clone C), respectively. The proportion of isolates belonging to clone A decreased during the study from 86% to 66%. Conversely, clone C increased from 4% to 22%. The increase of erythromycin-susceptibility within MRSA was caused by the emergence of clone C. Non-epidemic strains were more frequently susceptible to ofloxacin (31.8% vs. 1.1%) and tobramycin (45.4% vs. 16.8%) than epidemic strains. Antimicrobial use had not significantly varied during the study. The proportion of beta-lactams, fluoroquinolones, macrolides and aminosides was 71.8, 13.9, 5.0 and 3.8% of the total antibiotic use, respectively. In our hospital, MRSA isolates became more susceptible to antimicrobial of minor use. The selection pressure exerted by beta-lactams and fluoroquinolones was in favor of the spread of strains resistant to these both major antibiotic classes.

Emergence of Enterobacter cloacae as a common pathogen in neonatal units: pulsed-field gel electrophoresis analysis.

In the first week of December 2002, three infants hospitalized in the neonatal department of our hospital had blood cultures positive with Enterobacter cloacae. Screening cultures and genotyping showed that 10 of 25 screened patients also carried E. cloacae and that nine isolates belonged to the same clone as that responsible for all three bacteraemias. This epidemic cluster was limited to one of the two units of the department. Surveillance of both units continued until the end of March 2003; 51 of 159 neonates screened were colonized with E. cloacae, 38 out of 80 (47.5%) in the premature unit (PU) and 13 out of 79 (16.4%) in the paediatric intensive care unit (PICU). Pulsed-field gel electrophoresis (PFGE) analysis of 130 available isolates revealed 30 different pulsotypes, including 24 unique pulsotypes from individual patients and six from multiple patients. Antibiotic (particularly beta-lactam) use did not significantly vary from 1999 to 2003. The consumption of alcohol-based hand rub (four-fold higher in the PICU than in the PU) and nurse-to-patient ratio (1:2 in the PICU and 1:4 in the PU) might explain the higher cross-transmission rate in the PU. Finally, despite an epidemiological survey, we failed to identify the causes of the emergence of E. cloacae in our neonatology units. However, improved hygiene practices combined with restriction of admission led to the progressive disappearance of the epidemic strain. The increasing importance of this type of unit and the dramatic consequences of infections emphasize the need for additional research on the constitution of the flora of newborns and the mode of acquisition Gram-negative multi-resistant bacteria.

Relationship between glycopeptide use and decreased susceptibility to teicoplanin in isolates of coagulase-negative staphylococci.

To investigate the relationship between glycopeptide use and decreased susceptibility to teicoplanin in coagulase-negative staphylococci (CNS) isolates, data on teicoplanin susceptibility and glycopeptide use from existing microbiology laboratory and pharmacy databases were collected for the period between July 2000 and March 2001. Pooled data for the entire study period were first used to analyse associations. Univariate analysis showed that the incidence of CNS with decreased susceptibility to teicoplanin was significantly correlated with the use of glycopetides, particularly with vancomycin use. This association was confirmed by multivariate analysis. This study suggests that variations in antimicrobial resistance are related to variations in antimicrobial use in the model of CNS with decreased susceptibility to teicoplanin, thus confirming the usefulness of restricting antimicrobial prescribing as a means of controlling resistance.

Microbiological and epidemiological studies of Enterococcus faecium resistant to amoxycillin in a university hospital in eastern France.

Over 3 years, during six 1-month periods of surveillance, 69 cases of Enterococcus faecium colonisation or infection were detected in a university hospital in eastern France. Thirty-two cases involved strains resistant to amoxycillin (crude incidence of 0.21/1000 patient-days). The risk of infection with E. faecium was higher if the patient was hospitalised in a haematology ward and/or treated with cephalosporins. Amoxycillin-resistant isolates (AmRE) were isolated from different wards and time periods, and none of the characteristics studied were associated significantly with amoxycillin resistance. Amoxycillin-sensitive and -resistant isolates were characterised by pulsed-field gel electrophoresis. Three epidemic patterns were identified which contained 87.5% (28/32) of the AmRE isolates, indicating that clonal spread was responsible, at least partially, for the high incidence of AmRE in this hospital.

[E. coli: resistance to quinolones and beta-lactams of clinical strains isolated in the Franche-Comté region of France]. / Escherichia coli: résistance aux quinolones et aux beta-lactamines des souches cliniques isolées en France-Comté.

AIM OF THE STUDY: Numerous European studies have reported an increase of resistance to quinolones among E. coli. We conducted a regional study to update our knowledge on this evolution. MATERIALS AND METHODS: We evaluated the resistance phenotype and genotype of 115 clinical strains of E. coli. We collected data on individual treatment with fluoroquinolones, and the evolution of the use of these antimicrobial agents. RESULTS: Resistance to nalidixic acid and ciprofloxacin was 13.0 and 6.9, respectively. The frequency of resistance increased from 1999 to 2001, from 7.5% to 13.0% for nalidixic acid and from 5.4% to 6.9% for fluoroquinolones. Resistance to quinolones was significantly associated to beta-lactams resistance and was slightly higher for nosocomial isolates compared to community-acquired isolates. Previous treatment with fluoroquinolones was the major risk factor associated to E. coli resistance. From 1997 to 2001, fluoroquinolones use has increased in our hospital and particularly in the community. Analysis of molecular epidemiology shows a large clonal diversity among E. coli isolates. CONCLUSION: This study confirms the evolution through resistance to quinolones of E. coli isolates. This observation is not due to dissemination of resistant clonal strains and the selective pressure exerted by fluoroquinolones influences this evolution. Therapeutic alternatives, surveillance, and restriction of fluoroquinolones use are needed to control this spread of resistance.

Is pulsed-field gel electrophoresis a valuable tool to identify nosocomial cases of Legionella pneumophila disease?

Pulsed-field gel electrophoresis (PFGE) was used to type Legionella pneumophila isolates collected from the water systems of Besançon University Hospital and other hospitals in Franche-Comté region between January 2001 and December 2002 and compare them with patient isolates to identify hospital-acquired pneumonia. Genomic DNA was digested with SfiI and subjected to PFGE in a clamped, homogeneous electric field apparatus. Two of 11 Legionella infections were hospital-acquired. Both were with the same type, also present in the ward water. An environmental strain isolated from the water system of Vesoul Hospital, exhibited DNA pattern 6, also found in three patients with community-acquired pneumonia, who had never been in Vesoul hospital and lived in different towns located >60 km away. Patient 11, who lived in Besançon, was infected with a DNA pattern 12 strain. This patient had never been to the Besançon swimming pool, from which a similar strain was collected. Subtype matching of patient and environmental isolates should be interpreted with caution, and it is important to combine a molecular typing method with sound epidemiological data to ensure that the most stringent criteria are used to determine whether a hospital-reservoir is responsible for nosocomial pneumonia.