Plasmids are widely involved in the dissemination of characteristics within bacterial communities. Their genomic content can be assessed by high-throughput sequencing of the whole plasmid fraction of an environment, the plasmidome. In this study, we analyzed the plasmidome of a biofilm formed in the effluents of the teaching hospital of Clermont-Ferrand (France). Our analysis discovered >350 new complete plasmids, with a length ranging from 1219 to 40,193 bp. Forty-two plasmid incompatibility (Inc) groups were found among all the plasmid contigs. Ten large plasmids, described here in detail, were reconstructed from plasmid contigs, seven of which carried antibiotic resistance genes. Four plasmids potentially confer resistance to numerous families of antibiotics, including carbapenems, aminoglycosides, colistin, and chloramphenicol. Most of these plasmids were affiliated to Proteobacteria, a phylum of Gram-negative bacteria. This study therefore illustrates the composition of an environmental mixed biofilm in terms of plasmids and antibiotic resistance genes.

The World Health Organization Global Action Plan recommends integrated surveillance programs as crucial strategies for monitoring antibiotic resistance. Although several national surveillance programs are in place for clinical and veterinary settings, no such schemes exist for monitoring antibiotic-resistant bacteria in the environment. In this transnational study, we developed, validated, and tested a low-cost surveillance and easy to implement approach to evaluate antibiotic resistance in wastewater treatment plants (WWTPs) by targeting cefotaxime-resistant (CTX-R) coliforms as indicators. The rationale for this approach was: i) coliform quantification methods are internationally accepted as indicators of fecal contamination in recreational waters and are therefore routinely applied in analytical labs; ii) CTX-R coliforms are clinically relevant, associated with extended-spectrum β-lactamases (ESBLs), and are rare in pristine environments. We analyzed 57 WWTPs in 22 countries across Europe, Asia, Africa, Australia, and North America. CTX-R coliforms were ubiquitous in raw sewage and their relative abundance varied significantly (<0.1% to 38.3%), being positively correlated (p < 0.001) with regional atmospheric temperatures. Although most WWTPs removed large proportions of CTX-R coliforms, loads over 103 colony-forming units per mL were occasionally observed in final effluents. We demonstrate that CTX-R coliform monitoring is a feasible and affordable approach to assess wastewater antibiotic resistance status.

Ventilator-associated pneumonia (VAP) is the most frequent hospital-acquired infections in intensive care units (ICU). In the bundle of care to prevent the VAP, the oral care is very important strategies, to decrease the oropharyngeal bacterial colonization and presence of causative bacteria of VAP. In view of the paucity of medical economics studies, our objective was to determine the cost of implementing this oral care program for preventing VAP.

•Oral chlorhexidine and tooth brushing improved oral health scores in ICU patients.•Increased caregiver satisfaction with an easy and simple oral care protocol.•Decreased VAP rates with an oral chlorhexidine and tooth brushing protocol.

Discharge of antimicrobial residues and resistant bacteria in hospital effluents is supposed to have strong impacts on the spread of antibiotic resistant bacteria in the environment. This study aimed to characterize the effluents of the Gabriel Montpied teaching hospital, Clermont-Ferrand, France, by simultaneously measuring the concentration of ciprofloxacin and of biological indicators resistant to this molecule in biofilms formed in the hospital effluent and by comparing these data to ciprofloxacin consumption and resistant bacterial isolates of the hospital. Determination of the measured environmental concentration of ciprofloxacin by spot sampling and polar organic chemical integrative (POCIS) sampling over 2 weeks, and comparison with predicted environmental concentrations produced a hazard quotient >1, indicating a potential ecotoxicological risk. A negative impact was also observed with whole hospital effluent samples using the Tetrahymena pyriformis biological model. During the same period, biofilms were formed within the hospital effluent, and analysis of ciprofloxacin-resistant isolates indicated that Gamma-Proteobacteria were numerous, predominantly Aeromonadaceae (69.56%) and Enterobacteriaceae (22.61%). Among the 115 isolates collected, plasmid-mediated fluoroquinolone-resistant genes were detected, with mostly aac(6')-lb-cr and qnrS. In addition, 60% of the isolates were resistant to up to six antibiotics, including molecules mostly used in the hospital (aminosides and third-generation cephalosporins). In parallel, 1247 bacteria isolated from hospitalized patients and resistant to at least one of the fluoroquinolones were collected. Only 5 of the 14 species identified in the effluent biofilm were also found in the clinical isolates, but PFGE typing of the Gram-negative isolates found in both compartments showed there was no clonality among the strains. Altogether, these data confirm the role of hospital loads as sources of pollution for wastewater and question the role of environmental biofilms communities as efficient shelters for hospital-released resistance genes.

Medical devices can be contaminated by microbial biofilm which causes nosocomial infections. One of the strategies for the prevention of such microbial adhesion is to modify the biomaterials by creating micro or nanofeatures on their surface. This study aimed (1) to nanostructure acrylonitrile-butadiene-styrene (ABS), a polymer composing connectors in perfusion devices, using Anodic Alumina Oxide templates, and to control the reproducibility of this process; (2) to characterize the physico-chemical properties of the nanostructured surfaces such as wettability using captive-bubble contact angle measurement technique; (3) to test the impact of nanostructures on Staphylococcus epidermidis biofilm development. Fabrication of Anodic Alumina Oxide molds was realized by double anodization in oxalic acid. This process was reproducible. The obtained molds present hexagonally arranged 50 nm diameter pores, with a 100 nm interpore distance and a length of 100 nm. Acrylonitrile-butadiene-styrene nanostructures were successfully prepared using a polymer solution and two melt wetting methods. For all methods, the nanopicots were obtained but inside each sample their length was different. One method was selected essentially for industrial purposes and for better reproducibility results. The flat ABS surface presents a slightly hydrophilic character, which remains roughly unchanged after nanostructuration, the increasing apparent wettability observed in that case being explained by roughness effects. Also, the nanostructuration of the polymer surface does not induce any significant effect on Staphylococcus epidermidis adhesion.

OBJECTIVES:To characterize a CTX-M-15-producing Klebsiella pneumoniae isolate that was identified during an outbreak involving 16 patients who had undergone endoscopic retrograde cholangiopancreatography between December 2008 and August 2009. The strain was also detected in one endoscope used for these examinations.METHODS:Disc diffusion assays, MICs and isoelectric focusing were used to characterize the plasmidic CTX-M-15 β-lactamase. PCRs were used to check for the presence of genes associated with virulence or antibiotic resistance. Antibiotic tolerance tests and plasmid transfer were carried out in both planktonic and biofilm conditions.RESULTS:The strain belonged to sequence type 14 and to the virulent capsular serotype K2, but produced little glucuronic acid. It contained a 62.5 kb conjugative plasmid carrying the bla(CTX-M-15), bla(OXA-1) and aac(6')-Ib-cr genes and harboured few virulence genes (uge, wabG, kfu and mrkD). The strain was highly resistant to cefotaxime (MIC 516 mg/L) and the presence of this antibiotic at sub-MIC concentrations enhanced biofilm formation. The isolate was susceptible to ofloxacin (MIC 2 mg/L), but the bactericidal effect of this antibiotic was greater in planktonic cultures and 6 h old biofilm than in 24 or 48 h old biofilms. The K. pneumoniae strain was notable for its ability to transfer its plasmid, especially in biofilm conditions, in which the rate of plasmid transfer was about 0.5/donor.CONCLUSIONS:These findings demonstrate the ability of this strain to survive in a hospital environment and to transfer its extended-spectrum β-lactamase-encoding plasmid.

OBJECTIVES:The aim of the study was to assess whether an isolated positive catheter culture is predictive of a subsequent bloodstream infection in intensive care unit patients.DESIGN:Retrospective clinical study between 2000 and 2007.SETTING:Intensive care unit of a university hospital.SUBJECTS:All arterial, central venous, and dialysis catheters yielding selected pathogenic microorganisms from isolated positive catheter cultures. Positive catheter culture was defined by a catheter tip culture performed with the Brun-Buisson technique yielding ≥103 colony-forming units/mL; isolated positive catheter culture by a positive catheter culture without concomitant bloodstream infection due to the microorganism of the positive catheter culture evidenced within 48 hrs before or after catheter removal; and subsequent bloodstream infection by a bloodstream infection developing between 48 hrs and 30 days after catheter removal and due to a selected pathogenic microorganism of an isolated positive catheter culture. Active antibiotic therapy was active if at least one of the antibiotics administered was effective against the selected pathogenic microorganism of the positive catheter culture.INTERVENTION:None.MEASUREMENT AND MAIN RESULTS:The end point of the study was the ratio of the number of subsequent bloodstream infections to that of selected pathogenic microorganisms isolated from positive catheter culture 30 days after catheter removal. A total of 138 isolated positive catheter cultures for 149 selected pathogenic micro-organisms was included in the study. Only two cases (1.3%) of subsequent bloodstream infection were evidenced, one resulting from Escherichia coli and the other from Staphylococcus epidermidis. The incidence of subsequent bloodstream infection did not differ with regard to the presence or absence of active antibiotics at catheter removal: zero of 23 vs. two of 121 (p = 1), respectively.CONCLUSIONS:Our results suggest that the risk of subsequent bloodstream infection in intensive care unit patients when the Brun-Buisson technique is used to define isolated positive catheter culture is low.

Background. Interdialytic locking of catheters with antimicrobial agents is frequently used for preventing catheter-related infections, often associated with biofilm formation. We determined the bactericidal effect of 60% ethanol (ETOH) versus a 46.7% trisodium citrate (TSC) solution on biofilm embedded in silicone catheters.Methods. Four- and 24-h biofilms of Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans established in a micro-fermentor were exposed to ETOH and TSC for up to 24 h and the number of remaining viable microorganisms was determined.Results. ETOH 60% was significantly more effective than 46.7% TSC in rapidly eradicating sessile cells from all microorganisms tested. A 20-min ETOH 60% treatment completely eradicated the Gram-negative bacilli and C. albicans biofilms, which initially contained up to 108 and 105 cells, respectively. Gram-positive cocci biofilms only showed a significant 2.6-4.3 log reduction in the initial viable counts after 20 min of ETOH 60% treatment, with eradication occurring after 30 min. Confocal laser scanning microscopy observation of ETOH-treated biofilm showed sparse cells with respiratory activity. TSC 46.7% eradicated none of the tested microorganisms. In contrast, ETOH 60% totally eradicated planktonic cells, whereas TSC had significant bactericidal activity against K. pneumoniae, P. aeruginosa and C. albicans after 20 min, 1 and 24 h, respectively, but none on the Staphylococcus species.Conclusions. This in vitro study demonstrates the superior antimicrobial activity of ETOH 60% in contrast to TSC 46.7% in eradicating biofilm formed on a silicon catheter. Hence, ethanol-based solution shows promise as a catheter lock solution.

Background: Human adenovirus (HAdV) infections, while common in infancy and childhood, occur rarely in the neonatal period but may be fatal.Objectives: To describe a transmission of HAdV from a patient with fatal pneumonia to heath-care workers that could be considered as a model of respiratory virus transmission in a care unit.Study design: Case report with virologic studies.Results: A 10-day-old neonate developed pneumonia with acute respiratory distress, external pulmonary bleeding and coagulopathy and died 36 h after admission of multivisceral failure. An adenovirus was isolated from pulmonary biopsy and detected by PCR in blood and respiratory secretions. Ten days later, three members of medical staff in charge of this infant, who used neither masks nor glasses for close patient contact, developed keratoconjunctivitis. Molecular analysis of the infant's and one of the pediatrician's isolates identified a species D HAdv and showed 100% identity, thereby demonstrating viral transmission.Conclusion: In view of the serious outcome, HAdV infections should be considered in the differential diagnosis of pneumonia in neonates. This case illustrates the epidemic potential of viruses with respiratory transmission and underlines the importance of complying with standard precautions to prevent viral spread in routine practice. (C) 2009 Elsevier B.V. All rights reserved.

Objective We compared handwashing (HW) and alcohol-based handrub (ABH) for skin tolerance, acceptability, and hand hygiene compliance in health-care workers (HCWs) in a multicentric study. Design and setting Before/after study over two periods (P1 and P2) in seven ICUs. Monitoring of methicillin-resistant Staphylococcus aureus (MRSA) acquisition in two ICUs (U4 and U6). Subjects and patients All HCWs of the seven ICUs, and the patients of U4 and U6 without MRSA carriage on admission. Interventions Handwashing was available in P1 and P2. ABH was introduced after P1 and available throughout P2. HCWs completed questionnaires on skin tolerance in P1 and P2, and on acceptability in P2. Compliance with hand hygiene was assessed in P1 and P2 using a standardized method. ICU-acquired MRSA colonization/infection in U4 and U6 was recorded. Measurements and main results Hand erythema was reported during P1 and P2 by 21.7 and 11% of HCWs, respectively ( P = 0.01), itching by 15.8 and 7.1% ( P = 0.02), oozing by 0.8 and 1.1% ( P = 0.82), and bleeding by 4.2 and 1.1% ( P = 0.1). ABH was considered easier and quicker than HW ( P < 0.01), but overall acceptability did not differ between the two. Compliance increased from 51% (334/647) in P1 to 60% (251/418) in P2, P < 0.01. No significant reduction in MRSA colonization/infection between P1 and P2 ( P = 0.30) was observed, but the study was underpowered. Conclusions Self-report questionnaires show that when ABH is available in ICUs, fewer unpleasant skin sensations are reported. ABH is considered to be easier and quicker to use than HW and compliance is improved after glove removal.

Introduction Hand hygiene is one of the cornerstones of the prevention of health care-associated infection, but health care worker (HCW) compliance with good practices remains low. Alcohol-based handrub is the new standard for hand hygiene action worldwide and usually requires a system change for its successful introduction in routine care. Product acceptability by HCWs is a crucial step in this process. Methods We conducted a prospective intervention study to compare the impact on HCW compliance of a liquid (study phase I) versus a gel (phase II) handrub formulation of the same product during daily patient care. All staff (102 HCWs) of the medical intensive care unit participated. Compliance with hand hygiene was monitored by a single observer. Skin tolerance and product acceptability were assessed using subjective and objective scoring systems, self-report questionnaires, and biometric measurements. Logistic regression was used to estimate the association between predictors and compliance with the handrub formulation as the main explanatory variable and to adjust for potential risk factors. Results Overall compliance (phases I and II) with hand hygiene practices among nurses, physicians, nursing assistants, and other HCWs was 39.1%, 27.1%, 31.1%, and 13.9%, respectively (p = 0.027). Easy access to handrub improved compliance (35.3% versus 50.6%, p = 0.035). Nurse status, working on morning shifts, use of the gel formulation, and availability of the alcohol-based handrub in the HCW's pocket were independently associated with higher compliance. Immediate accessibility was the strongest predictor. Based on self-assessment, observer assessment, and the measurement of epidermal water content, the gel performed significantly better than the liquid formulation.

Catheter colonization is commonly used as an end point in studies on central venous catheter (CVC) infections. This study aimed at comparing the rates of catheter colonization in patients according to the administration of antibiotics at removal.

INTRODUCTION:Hand hygiene is one of the cornerstones of the prevention of health care-associated infection, but health care worker (HCW) compliance with good practices remains low. Alcohol-based handrub is the new standard for hand hygiene action worldwide and usually requires a system change for its successful introduction in routine care. Product acceptability by HCWs is a crucial step in this process.METHODS:We conducted a prospective intervention study to compare the impact on HCW compliance of a liquid (study phase I) versus a gel (phase II) handrub formulation of the same product during daily patient care. All staff (102 HCWs) of the medical intensive care unit participated. Compliance with hand hygiene was monitored by a single observer. Skin tolerance and product acceptability were assessed using subjective and objective scoring systems, self-report questionnaires, and biometric measurements. Logistic regression was used to estimate the association between predictors and compliance with the handrub formulation as the main explanatory variable and to adjust for potential risk factors.RESULTS:Overall compliance (phases I and II) with hand hygiene practices among nurses, physicians, nursing assistants, and other HCWs was 39.1%, 27.1%, 31.1%, and 13.9%, respectively (p = 0.027). Easy access to handrub improved compliance (35.3% versus 50.6%, p = 0.035). Nurse status, working on morning shifts, use of the gel formulation, and availability of the alcohol-based handrub in the HCW's pocket were independently associated with higher compliance. Immediate accessibility was the strongest predictor. Based on self-assessment, observer assessment, and the measurement of epidermal water content, the gel performed significantly better than the liquid formulation.CONCLUSION:Facilitated access to an alcohol-based gel formulation leads to improved compliance with hand hygiene and better skin condition in HCWs.

BACKGROUND:Little is known about vascular access infections in patients with acute renal failure.METHODS:We prospectively compared infection rates of dialysis catheters (DCs) and central venous catheters (CVCs) in patients in the intensive care unit treated with renal replacement therapy for acute renal failure. The same insertion and maintenance procedures were used for CVCs and DCs. To circumvent the allocation bias caused by severity of patient condition, only patients with both types of catheters were included.RESULTS:A total of 150 CVCs and 130 DCs were analyzed in 99 patients with a mean Simplified Acute Physiology Score II of 67 +/- 21. The major cause of acute renal failure was sepsis (62%). Hospital mortality was 62%. Mean catheter duration was shorter for DCs (6.7 +/- 4.4 days) than CVCs (7.8 +/- 4.9 days; P = 0.03). There was no difference between CVCs and DCs in cumulative incidence of catheter colonization (quantitative catheter cultures > or = 10(3) colony-forming units/mL; 4.7% versus 6.2%; P = 0.58) or incidence density of catheter colonization per 1,000 catheter days (5.9 versus 9.1; P = 0.44, respectively). There also was no difference between CVCs and DCs in cumulative incidence and incidence density regardless of whether catheters were placed at the internal jugular (P = 0.34 and P = 0.23) or femoral site (P = 0.57 and P = 0.80), respectively. Three cases of CVC-related bacteremia (the same microorganism responsible for both catheter colonization and blood culture result) were recorded, but none with DC use.CONCLUSION:When severity of patient condition is controlled for, epidemiological characteristics of colonization in CVCs and DCs are similar if similar infection control measures are used for insertion and maintenance.

Objectives > To evaluate a strategy based on screening and isolation at admission to a department of infectious diseases during on epidemic of vancomycin-resistant Enterococcus (VRE) at the University Hospital of Clermont-Ferrand.Methods > Systematic screening for VRE by anal swabs begon on November 15, 2004. Patients were isolated on admission if (a) they hod been hospitalized more than 24 h in on at-risk deportment of our hospital at (b) they hod received a course of wide-spectrum anti-microbial therapy for longer than 48 h in the three months preceding admission. Patients hospitalized in our deportment were screened weekly if they were treated with wide-spectrum antibiotics, had a urinary catheter left in place for one week, or were neutropenic.Results > Through May 15, 2005, 12 (3.5%) of 341 swabs were found to be positive for VRE eight were detected on admission and four during hospitalization. In all, 87 patients were isolated on admission. A case-control study confirmed that the criteria for patient isolation were indeed risk factors for VRE. Isolation was well accepted when it was clearly explained. No new case has been detected since March 2005.Conclusion > An isolation strategy based on known risk factors for VRE with systematic screening on admission appears to be on effective way to control an outbreak of VRE, perhaps in port because it helps to keep the medical staff alert to this problem. Isolation is well tolerated as long as it is explained clearly

OBJECTIVES:To evaluate a strategy based on screening and isolation at admission to a department of infectious diseases during an epidemic of vancomycin-resistant Enterococcus (VRE) at the University Hospital of Clermont-Ferrand.METHODS:Systematic screening for VRE by anal swabs began on November 15, 2004. Patients were isolated on admission if (a) they had been hospitalized more than 24 h in an at-risk department of our hospital or (b) they had received a course of wide-spectrum antimicrobial therapy for longer than 48 h in the three months preceding admission. Patients hospitalized in our department were screened weekly if they were treated with wide-spectrum antibiotics, had a urinary catheter left in place for one week, or were neutropenic.RESULTS:Through May 15, 2005, 12 (3.5%) of 341 swabs were found to be positive for VRE: eight were detected on admission and four during hospitalization. In all, 81 patients were isolated on admission. A case-control study confirmed that the criteria for patient isolation were indeed risk factors for VRE. Isolation was well accepted when it was clearly explained. No new case has been detected since March 2005.CONCLUSION:An isolation strategy based on known risk factors for VRE with systematic screening on admission appears to be an effective way to control an outbreak of VRE, perhaps in part because it helps to keep the medical staff alert to this problem. Isolation is well tolerated as long as it is explained clearly.

Objective:To compare the rates of positive quantitative culture (PQC) of arterial catheter (AC) and central venous catheter (CVC) tips and of CVC- and AC-related bacteremia in intensive care unit patients undergoing placement of both ACs and CVCs. Design:Prospective, descriptive survey. To control for a difference in the severity of patients having an AC or CVC, only patients having both an AC and a CVC were included. Setting:An adult, nine-bed medical/surgical intensive care unit at a university teaching hospital. Subjects:The analysis included 308 CVCs and 299 ACs inserted in 212 severely ill patients, with a mean ± sd Simplified Acute Physiology Score II of 52 ± 22 and an intensive care unit mortality of 33% (69 of 212). Interventions:None. Measurements and Main Results:The same insertion and maintenance procedures were used for both types of catheter. A PQC was defined by a catheter tip culture yielding ≥10 colony forming units/mL. Catheter-related bacteremia was defined by a PQC and a blood culture positive for the same microorganism. The cumulative incidence (PQCs/number of catheters inserted) was 9.4% (29/308) for CVCs and 7.7% (23/299) for ACs (p = .44). Incidence density (PQCs/1,000 catheter days) was 12.0 for CVCs versus 9.3 for ACs. At the femoral site, there was no significant difference between CVCs and ACs in the cumulative incidences and incidence densities of PQCs. Two instances of catheter-related bacteremia were observed, one involving a CVC and one involving an AC. Conclusions:Among severely ill patients with both CVCs and ACs, the epidemiology of PQCs of CVCs and ACs is comparable when the same infection control measures are used for the insertion and maintenance of both types of catheters.

BACKGROUND:The efficacy of systemic antibiotics on the treatment of ventilator-associated infectious maxillary sinusitis (VAIMS) is debated. The objective of this study was to determine the etiologic diagnosis of VAIMS in patients receiving antibiotics.METHODS:Patients mechanically ventilated for more than or equal to 72 h, who had persistent fever while on antibiotics for more than or equal to 48 h, underwent computed tomography scan followed by transnasal puncture of involved maxillary sinuses. VAIMS was defined as follows: fever greater than or equal to 38 degrees C, radiographic signs (air fluid level or opacification of maxillary sinuses on computed tomography scan), and a quantitative culture of sinus aspirate yielding more than or equal to 103 colony-forming units/ml.RESULTS:Twenty-four patients had radiographic signs of sinusitis. The mean +/- SD prior durations of mechanical ventilation and antibiotic exposure were 9.5 +/- 4.7 days and 6 +/- 4 days, respectively. Six unilateral and nine bilateral VAIMS were diagnosed in 15 patients. The median number of etiologic organisms per patient was two (range, one to four). The bacteriologic cultures yielded gram-positive bacteria (n = 21), gram-negative bacteria (n = 22), and yeasts (n = 5). Forty percent of causative agents were susceptible to the antibiotics prescribed. Seven patients with VAIMS developed 10 concomitant infections: ventilator-associated pneumonia (n = 5), urinary tract infection (n = 3), catheter infections (n = 2). In all cases of ventilator-associated pneumonia, the implicated agents were the causative agents of VAIMS.CONCLUSION:In VAIMS patients on antibiotics, quantitative cultures of sinus aspirates may contribute to establish the diagnosis. The frequent recovery of microorganisms susceptible to the antimicrobial treatment administered suggests that therapy of VAIMS with systemic antibiotics may not be sufficient.