Michelle Thouverez

Most Multidrug-Resistant Pseudomonas aeruginosa Isolates from Hospitals in Eastern France Belong to a Few Clonal Types

ABSTRACT This study aimed to determine the genetic diversity of clinical multidrug-resistant Pseudomonas aeruginosa. We used pulsed-field gel electrophoresis and multilocus sequence typing to analyze 187 strains isolated in different French hospitals. To illustrate the diversity of resistance mechanisms to antibiotics in a given clone, we identified β-lactamases with an extended spectrum by using phenotypic and genotypic methods. Typing results showed that the majority of our multidrug-resistant isolates belong to a few clonal types (ST235, ST111, and ST175) that are already spreading worldwide. These successful international clones sporadically produced extended-spectrum β-lactamase-encoding genes but mostly became extensively resistant to β-lactams after derepression of intrinsic resistance mechanisms (i.e., AmpC cephalosporinase). Our results indicate that cross-transmission plays a major role in the spread of multidrug-resistant P. aeruginosa in hospital settings.

Discriminatory power and usefulness of pulsed- field gel electrophoresis in epidemiological studies of Pseudomonas aeruginosa

We assessed the discriminatory power of pulsed-field gel electrophoresis (PFGE) for the analysis of DNA restriction fragment length polymorphism (RFLP) in Pseudomonas aeruginosa. We determined DraI PFGE-RFLP of DNA of unrelated clinical and environmental strains, and clinical strains isolated from two intensive care units of the Besançon University Hospital. The typeability and reproducibility was 100%. The discriminatory index was 0.998, and the DNA patterns were stable in vitro and in vivo. There was a very low correlation between PFGE-RFLP and traditional typing methods. The typeability, reproducibility, the high discriminatory power and the stability of PFGE-RFLP make this a valuable method to be used in conjunction with serotyping. Further standardization and quantitative interpretation are possible and should lead to this technique becoming a library typing system.

Nasal carriage of Staphylococcus aureus and cross-contamination in a surgical intensive care unit: efficacy of mupirocin ointment

A six month prospective study was carried out in a surgical intensive care unit (SICU) of a university hospital to assess the incidence and routes of exogenous colonization by Staphylococcus aureus. A total of 157 patients were included in the study. One thousand one hundred and eleven specimens (nasal, surgical wound swabs, tracheal secretions obtained on admission and once a week thereafter, and all clinical specimens) were collected over a four month period from patients without nasal decontamination (A). They were compared with 729 specimens collected over a two month period from patients treated with nasal mupirocin ointment (B). All S. aureus strains were typed by restriction fragment length polymorphism (RFLP) pulsed-field gel electrophoresis after SmaI macrorestriction. The nasal colonization rates on admission were 25.5 and 32.7% in groups A and B, respectively. Thirty-one untreated patients (31.3%) and three patients (5.1%) treated with nasal ointment, acquired the nasal S. aureus in the SICU (P = 0.00027). Nasal carriers were more frequently colonized in the bronchopulmonary tract (Bp) and surgical wound (Sw) (62%) than patients who were not nasal carriers (14%) (P < 0.00001). The patterns were identical for nasal, Bp and Sw strains from the same patient. RFLP analysis characterized seven epidemic strains of methicillin-resistant S. aureus (MRSA) which colonized 60% of group A and 9% of group B patients (P < 0.00001). The bronchopulmonary tract infection rate was reduced in group B (P = 0.032). In conclusion, in an SICU, nasal carriage of S. aureus appeared to be the source of endogenous and cross-colonization. The use of nasal mupirocin ointment reduced the incidence of Bp and Sw colonization, as well as the MRSA infection rate.

Association of private isolation rooms with ventilator-associated Acinetobacter baumanii pneumonia in a surgical intensive-care unit.

OBJECTIVE To determine the rates and routes of Acinetobacter baumanii colonization and pneumonia among ventilated patients in a surgical intensive-care unit (SICU) before and after architectural modifications. DESIGN A nonsequential study comparing two groups of patients. All isolates from systematic and clinical samples were genotyped by pulsed-field gel electrophoresis (PFGE). Records of patients hospitalized during the first and second periods were reviewed and findings were compared. Between the two periods, the SICU was remodeled from enclosed isolation rooms and open rooms to only enclosed isolation rooms with handwashing facilities in each room. SETTING AND PATIENTS All patients hospitalized and mechanically ventilated for more than 48 hours in the 15-bed SICU of the University Hospital of Besançon (France). RESULTS For the first and second periods, the rates of colonization were, respectively, 28.1% and 5.0% of patients (P < 10(-7); relative risk [RR], 2.23; 95% confidence interval [CI95], 1.8-2.75) and the specific rates of bronchopulmonary (BP) colonization were, respectively, 9.1 and 0.5 per 1,000 days of mechanical ventilation (P < 10(-5). Seven major PFGE isolate types were identified, 4 of which were isolated from 44 of the 47 colonized or infected patients. Logistic regression analysis showed that colonization was not associated with patient characteristics. CONCLUSION Conversion from open rooms to isolation rooms may help control nosocomial BP tract acquisition of A baumanii in mechanically ventilated patients hospitalized in an SICU.

Evidence for Induction of Integron-Based Antibiotic Resistance by the SOS Response in a Clinical Setting

Bacterial resistance to β-lactams may rely on acquired β-lactamases encoded by class 1 integron-borne genes. Rearrangement of integron cassette arrays is mediated by the integrase IntI1. It has been previously established that integrase expression can be activated by the SOS response in vitro, leading to speculation that this is an important clinical mechanism of acquiring resistance. Here we report the first in vivo evidence of the impact of SOS response activated by the antibiotic treatment given to a patient and its output in terms of resistance development. We identified a new mechanism of modulation of antibiotic resistance in integrons, based on the insertion of a genetic element, the gcuF1 cassette, upstream of the integron-borne cassette bla OXA-28 encoding an extended spectrum β-lactamase. This insertion creates the fused protein GCUF1-OXA-28 and modulates the transcription, the translation, and the secretion of the β-lactamase in a Pseudomonas aeruginosa isolate (S-Pae) susceptible to the third generation cephalosporin ceftazidime. We found that the metronidazole, not an anti-pseudomonal antibiotic given to the first patient infected with S-Pae, triggered the SOS response that subsequently activated the integrase IntI1 expression. This resulted in the rearrangement of the integron gene cassette array, through excision of the gcuF1 cassette, and the full expression the β-lactamase in an isolate (R-Pae) highly resistant to ceftazidime, which further spread to other patients within our hospital. Our results demonstrate that in human hosts, the antibiotic-induced SOS response in pathogens could play a pivotal role in adaptation process of the bacteria.

Wastewater Treatment Plants Release Large Amounts of Extended-Spectrum β-Lactamase–Producing Escherichia coli Into the Environment

BACKGROUND The determinants of the spread of extended-spectrum β-lactamase-producing Escherichia coli (ESBLEC) in the community remain unclear. To evaluate its dissemination in the environment, we analyzed the ESBLEC population throughout an urban wastewater network. METHODS Samples were collected weekly, over a 10-week period, from 11 sites throughout the wastewater network of Besançon city (France). Total E. coli and ESBLEC loads were determined for each sample. As a control, we analyzed 51 clinical ESBLEC isolates collected at our hospital. We genotyped both environmental and clinical ESBLEC by pulsed-field gel electrophoresis and multilocus sequence typing and identified their blaESBL genes by sequencing. RESULTS The E. coli load was higher in urban wastewater than in hospital wastewater (7.5 × 10(5) vs 3.5 × 10(5) CFU/mL, respectively). ESBLEC was recovered from almost all the environmental samples and accounted for 0.3% of total E. coli in the untreated water upstream from the wastewater treatment plant (WWTP). The ESBLEC load was higher in hospital wastewater than in community wastewater (27 × 10(3) vs 0.8 × 10(3) CFU/mL, respectively). Treatment by the WWTP eliminated 98% and 94% of total E. coli and ESBLEC, respectively. The genotyping revealed considerable diversity within both environmental and clinical ESBLEC and the overrepresentation of some clonal complexes. Most of the sequence types displayed by the clinical isolates were also found in the environment. CTX-M enzymes were the most common enzymes whatever the origin of the isolates. CONCLUSIONS The treatment at the WWTP led to the relative enrichment of ESBLEC. We estimated that >600 billion of ESBLEC are released into the river Doubs daily and the sludge produced by the WWTP, used as fertilizer, contains 2.6 × 10(5) ESBLEC per gram.

Tracking Down Antibiotic-Resistant Pseudomonas aeruginosa Isolates in a Wastewater Network

The Pseudomonas aeruginosa-containing wastewater released by hospitals is treated by wastewater treatment plants (WWTPs), generating sludge, which is used as a fertilizer, and effluent, which is discharged into rivers. We evaluated the risk of dissemination of antibiotic-resistant P. aeruginosa (AR-PA) from the hospital to the environment via the wastewater network. Over a 10-week period, we sampled weekly 11 points (hospital and urban wastewater, untreated and treated water, sludge) of the wastewater network and the river upstream and downstream of the WWTP of a city in eastern France. We quantified the P. aeruginosa load by colony counting. We determined the susceptibility to 16 antibiotics of 225 isolates, which we sorted into three categories (wild-type, antibiotic-resistant and multidrug-resistant). Extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) were identified by gene sequencing. All non-wild-type isolates (n = 56) and a similar number of wild-type isolates (n = 54) were genotyped by pulsed-field gel electrophoresis and multilocus sequence typing. Almost all the samples (105/110, 95.5%) contained P. aeruginosa, with high loads in hospital wastewater and sludge (≥3×106 CFU/l or/kg). Most of the multidrug-resistant isolates belonged to ST235, CC111 and ST395. They were found in hospital wastewater and some produced ESBLs such as PER-1 and MBLs such as IMP-29. The WWTP greatly reduced P. aeruginosa counts in effluent, but the P. aeruginosa load in the river was nonetheless higher downstream than upstream from the WWTP. We conclude that the antibiotic-resistant P. aeruginosa released by hospitals is found in the water downstream from the WWTP and in sludge, constituting a potential risk of environmental contamination.

Control of Enterobacteriaceae producing extended-spectrum beta-lactamase in intensive care units: rectal screening may not be needed in non-epidemic situations.

OBJECTIVE To evaluate the usefulness of screening cultures in the control of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in intensive care units (ICUs). DESIGN A 4-year retrospective study. SETTING Two adult ICUs of a university-affiliated public hospital in France. RESULTS A total of 7,777 specimens were analyzed and 28 (0.97%) of 2,883 screened patients had a positive result on a screening test, among the 3,678 admitted patients. Thirteen of these 28 patients were only carriers; 4 were carriers and then were colonized or infected 2, 2, 3, and 8 days later, respectively; and 11 were colonized or infected before a screening test was positive. Cluster analysis showed that the occurrence of ESBL-producing Enterobacteriaceae cross-transmission within both ICUs was limited to 9 cases. Thus, most cases (19 of 28) were probably imported. Surveillance cultures failed to detect 9 of the 19 cases. CONCLUSION The low prevalence of ESBL-producing Enterobacteriaceae carriers on admission (0.45%) and the relative ineffectiveness of our screening test to detect imported cases suggest that systematic detection of ESBL-producing Enterobacteriaceae in ICU patients is not cost-effective and that the use of clinical cultures may be sufficient to control ESBL-producing Enterobacteriaceae in non-epidemic situations.

Epidemiological study of variations in β-lactam antibiotic susceptibility of Pseudomonas aeruginosa in two intensive care units

A six-month prospective study was carried out in the medical and surgical intensive care units (ICUs) at Besançon University Hospital to assess the frequency and risk factors for beta-lactam-resistant isolates of Pseudomonas aeruginosa. Clinical samples were screened for P. aeruginosa, and four antibiograms were distinguished using imipenem and ceftazidime, namely: fully susceptible (SS), imipenem-resistant (RS), ceftazidime-resistant (SR), and resistant to both (RR). DraI restriction fragment length polymorphism of isolates from different patients or with different resistance patterns but the same serotype was assessed by pulsed-field gel electrophoresis. One hundred and twenty-one isolates were obtained from 50 of 281 patients, 60.3% were fully susceptible. 19.8% imipenem-resistant, 13.2% ceftazidime-resistant, and 6.6% resistant to both. Antibiotic-resistance was independent of serotype. Twenty-two of 32 imipenem-resistant isolates from six patients were of the same DNA type, and six other isolates from four patients were of a second DNA type. On only one occasion did a clonally defined strain develop imipenem resistance. By contrast ceftazidime-resistant strains had differing DNA types, but had been originally ceftazidime-susceptible in seven of 12 patients. Reversion of imipenem resistant strains to susceptibility occurred in one patient, and of ceftazidime-resistant strains in five patients. Case-control studies identified prior antibiotic therapy as a risk factor in colonization with resistant strains. Resistance to imipenem followed imipenem therapy, and resistance to ceftazidime followed use of weakly anti-pseudomonal beta-lactam antibiotics. The major route of spread of imipenem-resistant strains was cross-colonization. Thus, assuming appropriate isolation, a carbapenem should be preferred to an extended-spectrum cephalosporin to treat pseudomonas infections in ICU patients.

Molecular Epidemiology of Carbapenem Non-Susceptible Acinetobacter baumannii in France

Carbapenem-resistant Acinetobacter baumannii have emerged globally. The objective of this study was to investigate the epidemiology, clonal diversity and resistance mechanisms of imipenem non-susceptible A. baumannii isolates in France. Between December 2010 and August 2011, 132 notifications were collected, including 37 outbreaks corresponding to 242 cases (2 to 55 per cluster). Multilocus sequence typing, pulsed-field gel electrophoresis (PFGE) and characterisation of carbapenemase-encoding genes were performed on 110 non-repetitive isolates. Gene bla OXA-23 was the most frequently detected (82%), followed by bla OXA-24 (11%) and bla OXA-58 (7%). Eleven sequence types (ST) were distinguished, among which sequence types ST1, ST2 (64%), ST20, ST25, ST85 and ST107. Isolates from epidemiological clusters had the same ST and resistance genes, indicating probable transmission within centres. In contrast, PFGE types of isolates differed among centres, arguing against transmission among centers. This study provides the first epidemiological snapshot of the population of A. baumannii with reduced susceptibility to carbapenems from France, and further underlines the predominance of international clones.