Genome analysis of third-generation cephalosporin-resistant Escherichia coli and Salmonella species recovered from healthy and diseased food-producing animals in Europe.

The animal reservoir of Enterobacterales producing Extended-Spectrum-ß-Lactamases (ESBL) and plasmid-borne cephalosporinases (pAmpC) is a global concern. Using genome data, we analyzed a population of Escherichia coli and Salmonella species resistant to third-generation cephalosporins (3GC-R) recovered from healthy food animals (HA) and diseased food animals (DA) across Europe. Among the isolates collected from HA (n = 4,498) and DA (n = 833) in up to twelve European countries, 62 (1.4%) and 45 (5.4%) were 3GC-R, respectively. The genomes of these 3GC-R 107 isolates were sequenced to identify blaESBL and blaAmpC, sequence types (STs), virulence-associated genes, and Salmonella serovars. We also assessed their population structure using core genome multilocus sequence typing. The 78 3GC-R Escherichia coli originated from poultry (n = 27), swine (n = 26), and cattle (n = 25). Almost all (n = 77; 98.7%) harbored at least one blaESBL or blaAmpC, with blaCTX-M-1 predominating. We identified 51 STs, with ST10 and ST101 being the most frequent. The population of 3GC-R E. coli was polyclonal. The 29 3GC-R Salmonella spp. were mostly retrieved from healthy broiler (96.5%). blaCMY-2 dominated in this population. We found two clusters of CMY-2-producing Salmonella spp. in Germany: one with 15 isolates of S. Heidelberg isolates and another with six S. Minnesota, all of them with blaCMY-2. Our results confirm the low prevalence of 3GC-R E. coli and Salmonella spp. in HA and DA. blaCTX-M-1 was dominating in a highly diverse population of E. coli. 3GC-R E.coli isolated from HA and DA were genetically unrelated, with high clonal diversity suggesting multiple origins of contamination. This contrasted with the clonal population of 3GC-R Salmonella spp. in which blaCMY-2 dominated through two dominant serovars in this collection.

Persistent contamination of a hospital hot water network by Legionellapneumophila.

OBJECTIVES: We assessed the contamination with Legionella pneumophila (Lp) of the hot water network (HWN) of a hospital, mapped the risk of contamination, and evaluated the relatedness of isolates. We further validated phenotypically the biological features that could account for the contamination of the network. METHODS: We collected 360 water samples from October 2017 to September 2018 in 36 sampling points of a HWN of a building from a hospital in France. Lp were quantified and identified with culture-based methods and serotyping. Lp concentrations were correlated with water temperature, date and location of isolation. Lp isolates were genotyped by pulsed-field gel electrophoresis and compared to a collection of isolates retrieved in the same HWN two years later, or in other HWN from the same hospital. RESULTS: 207/360 (57.5%) samples were positive with Lp. In the hot water production system, Lp concentration was negatively associated with water temperature. In the distribution system, the risk of recovering Lp decreased when temperature was >55 °C (p < 10-3), the proportion of samples with Lp increased with distance from the production network (p < 10-3), and the risk of finding high loads of Lp increased 7.96 times in summer (p = 0.001). All Lp isolates (n = 135) were of serotype 3, and 134 (99.3%) shared the same pulsotype which is found two years later (Lp G). In vitro competition experiments showed that a 3-day culture of Lp G on agar inhibited the growth of a different pulsotype of Lp (Lp O) contaminating another HWN of the same hospital (p = 0.050). We also found that only Lp G survived to a 24h-incubation in water at 55 °C (p = 0.014). CONCLUSION: We report here a persistent contamination with Lp of a hospital HWN. Lp concentrations were correlated with water temperature, season, and distance from the production system. Such persistent contamination could be due to biotic parameters such as intra-Legionella inhibition and tolerance to high temperature, but also to the non-optimal configuration of the HWN that prevented the maintenance of high temperature and optimal water circulation.

Hospital-diagnosed infections with Escherichia coli clonal group ST131 are mostly acquired in the community.

The worldwide spread of E. coli ST131 has significantly contributed to the dissemination of E. coli producing extended-spectrum ß-lactamases (ESBL). In a French University hospital, we assessed the molecular features of ESBL-producing E. coli and identified risk factors in patients for colonization or infection with E. coli ST131. Over a 2-year period (2015-2017), each patient with at least one clinical isolate or one screening isolate positive with ESBL-producing E. coli were included (n = 491). The ST131 clonal group accounted for 17.5% (n = 86) of all ESBL-producing E. coli and represented 57.3% isolates of phylogroup B2. FimH-based sub-typing showed that 79.1% (68/86) of ST131 isolates were fimH30, among which 67.6% (n = 46), 20.6% (n = 14) and 11.8% (n = 8) isolates harbored genes encoding the ESBL CTX-M-15, CTX-M-27, and CTX-M-14, respectively. The multivariate analysis identified two factors independently associated with ST131 ESBL-producing E. coli isolates: infection (Odds ratio [OR] = 1.887, 95% confidence interval [CI]: 1.143-3.115; p = 0.013) and community acquisition (OR = 2.220, 95% CI: 1.335-3.693; p = 0.002). In conclusion, our study confirmed the predominance of ST131 clonal group among ESBL-producing E. coli and the difficulty to identify common risk factors associated with carriage of this pandemic clonal group.

ESBL-producing Klebsiella pneumoniae in a University hospital: Molecular features, diffusion of epidemic clones and evaluation of cross-transmission.

The worldwide spread of Klebsiella pneumoniae producing extended-spectrum ß-lactamase (ESBL-Kp) is a significant threat. Specifically, various pandemic clones of ESBL-Kp are involved in hospital outbreaks and caused serious infections. In that context, we assessed the phenotypic and molecular features of a collection of ESBL-Kp isolates in a French university hospital and evaluated the occurrence of potential cross-transmissions. Over a 2-year period (2017-2018), 204 non-duplicate isolates of ESBL-Kp were isolated from clinical (n = 118, 57.8%) or screening (n = 86, 42.2%) sample cultures. These isolates were predominantly resistant to cotrimoxazole (88.8%) and ofloxacin (82.8%) but remained susceptible to imipenem (99.3%) and amikacin (93.8%). CTX-M-15 was the most frequent ESBL identified (83.6%). Multilocus sequence typing and pulse-field gel electrophoresis analysis showed an important genetic variability with 41 sequence types (ST) and 50 pulsotypes identified, and the over representation of the international epidemic clones ST307 and ST405. An epidemiological link attesting probable cross-transmission has been identified for 16 patients clustered in 4 groups during the study period. In conclusion, we showed here the dissemination of pandemic clones of ESBL-Kp in our hospital on a background of clonal diversity.

Epidemiology and risk factors of Staphylococcus aureus CC398 bone and joint infections.

BACKGROUND: A particular ability of the Staphylococcus aureus clonal complex 398 (CC398) to cause bone and joint infections (BJI) remains questionable, since some studies have described high prevalence of MSSA CC398 in prosthetic joint infection (PJI) and diabetic foot ostemolyelitis (DFO). Here, we described the long-term epidemiology of CC398 among S. aureus isolated from BJI and identified risk factors associated with CC398. METHODS: We included all bone and joint samples with S. aureus-positive culture in our university hospital between January 2010 and December 2017. Logistic regression was used for univariate and multivariate analysis. RESULTS: We identified 124 CC398 isolates among the 958 BJI-associated S. aureus. The proportion of CC398 among S. aureus increased steadily from 4% in 2010 to 26% in 2017. Only 4 isolates of CC398 were resistant to methicillin. The distribution of BJI types due to CC398 and non CC398 isolates was similar. In multivariate analysis, age (p = 0.034, OR = 3.9), McCabe score (p = 0.005, OR = 5) and inoculation mechanism (p = 0.020, OR = 3.7) were associated with PJI-related CC398. The year of infection (p < 0.001, OR = 1.6), Charlson's score (p = 0.001, OR = 1.5) and grade 4 (severe) of the International Working Group of the Diabetic Foot classification (p < 0.001, OR = 8.5) were associated with DFO-related CC398. CONCLUSION: We highlighted here the emergence and spread of CC398-MSSA in BJI. Patients with comorbidities are at high risk of CC398 MSSA PJI and DFO. The spread of CC398 in the community and hospital settings remains unclear and further epidemiological studies are needed to identify the determinants of its success.

Increasing incidence of bloodstream infections due to Staphylococcus aureus clonal complex 398 in a French hospital between 2010 and 2017.

The epidemiology of Staphylococcus aureus is changing and several surveillances worldwide have evidenced an increasing incidence of S. aureus bloodstream infections (BSIs). Here, we described the long-term epidemiology of the emergent clonal group CC398 among S. aureus isolated from BSIs in our French university hospital between 2010 and 2017. Each patient with at least one blood culture positive with S. aureus during the study period was included (N = 1455). Cefoxitin susceptibility was determined using the disk diffusion method according to EUCAST recommendations. CC398 isolates were first screened from the whole S. aureus collection with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) typing method confirmed by a CC398-specific PCR. In our hospital, the incidence of hospital- and community-acquired BSIs due to S. aureus and MSSA increased in parallel between 2010 and 2017 while that of BSIs with MRSA decreased. The prevalence of CC398 isolates among S. aureus from BSIs increased from 3.6 in 2010 to 20.2% in 2017 (p < 0.05). CC398-MRSA emerged but remains very sparse. Our data suggested that CC398-MSSA disseminates in the community. We showed here the emergence and the diffusion of CC398-MSSA, a subclone associated with invasive infections, in our hospital. The monitoring of this particular human-adapted S. aureus clone is needed and genomic studies will have to identify the determinants of its diffusion.

Fourier-Transform InfraRed Spectroscopy Can Quickly Type Gram-Negative Bacilli Responsible for Hospital Outbreaks.

The typing of epidemic bacterial pathogens in hospitals relies on DNA-based, expensive, and time-consuming techniques, that are often limited to retrospective studies. However, the quick identification of epidemic pathogens in the routine of the microbiology laboratories would expedite infection control procedures that limit the contamination of new patients. IR Biotyper (Bruker Daltonics GmbH) is a new typing machine based on Fourier-transform infrared (FTIR) spectroscopy which generates spectra, aiming at typing the micro-organisms within 3 h. This technique discriminates the isolates by exploring the differences of the surface cell polysaccharides. In this work, we evaluated the ability of the FTIR spectroscopy to recognize Gram-negative bacilli clones responsible for hospital outbreaks. Isolates of Pseudomonas aeruginosa (n = 100), Klebsiella pneumoniae (n = 16), Enterobacter cloacae (n = 23), and Acinetobacter baumannii (n = 20) were typed by the reference methods Multi-Locus Sequence Typing (defining sequence types - STs) along with or without pulsed field gel electrophoresis (PFGE) (defining pulsotypes), and by FTIR spectroscopy. The congruence of FTIR spectroscopy clustering was compared to those of MLST and PFGE by Adjusted Rand index and Adjusted Wallace coefficient. We found that FTIR spectroscopy accurately clustered P. aeruginosa, K. pneumoniae, and E. cloacae isolates belonging to the same ST. The performance of the FTIR spectroscopy was slightly lower for A. baumannii. Furthermore, FTIR spectroscopy also correctly clustered P. aeruginosa isolates having a similar pulsotype. Overall, the IR Biotyper can quickly (in less than 3 h) detect the spread of clones of P. aeruginosa, K. pneumoniae, E. cloacae, and A. baumannii. The use of this technique by clinical microbiology laboratories may help to tackle the spread of epidemic clones by the quick implementation of infection control measures.

Occurrence of VIM-4 metallo-ß-lactamase-producing Pseudomonas aeruginosa in an Algerian hospital.

INTRODUCTION: Pseudomonas aeruginosa is one of the most common nosocomial pathogens, known with a wide resistance to antimicrobials. Carbapenemases producing Pseudomonas aeruginosa is a growing global public health concern as this pathogen is easily transmissible among patients. Metallo-Beta-lactamases is the most important class of these carbapenemases with their broad-spectrum resistance profile. This study was conducted to investigate the prevalence of MBL-producing P. aeruginosa collected in an Algerian hospital. METHODOLOGY: All Metallo-ß-lactamase (MBL)-producing P. aeruginosa isolates recovered from patients during a 2 years period (2015-2016) were studied using a combination of phenotypic and molecular typing methods (susceptibility testing, molecular characterization of carbapenemase-encoding genes, multi-locus sequence typing and pulsed-field gel electrophoresis). RESULTS: A total of twenty-six MBL producing P. aeruginosa of 188 isolates were investigated. The burns unit ranked in the first position of the majority of identified cases with 73.07%. About 73.07% of total MBL isolates were mainly isolated from pus samples. The studied isolates were subjected to the molecular typing, in which 4 different Dra1-PFGE patterns and 3 sequences type were assigned (ST244, ST381, and ST1076), and all isolates were revealed positive for VIM-4. CONCLUSIONS: We report the third description of blaVIM-4 in Algeria indicating the emergence and spread of carbapenemase-encoding genes among P. aeruginosa in the hospital environment.

Rapid antibiotic susceptibility testing on blood cultures using MALDI-TOF MS.

Antibiotic resistance is a major public health problem requiring the early optimization of antibiotic prescriptions. Matrix-Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS) has been shown to accurately identify bacteria from positive blood culture. Here, we developed a rapid detection of Escherichia coli resistance to amoxicillin (AMX) and cefotaxime (CTX) from positive blood culture based on MALDI-TOF MS. Potential sparing of broad-spectrum antibiotics was also evaluated. We tested 103 E. coli-positive blood cultures. Blood cultures were subculture 1-hour in antibiotic-free rich liquid media before further incubation with and without AMX for 2.5 h or CTX for 2 h. Protein extracts associated with an internal control were spotted on a MALDI-TOF target and spectra were analyzed with the MBT-ASTRA prototype software (Bruker Daltonik GmbH, Bremen, Germany). Bacterial growth ratio was calculated from the AUC spectra obtained in the presence and absence of the antibiotic and compared to a threshold which classified E. coli as susceptible or resistant. Results were interpreted with MICs determined using agar dilution method as reference technique. MBT-ASTRA recognized 95% and 84% of the AMX- and CTX-susceptible isolates, respectively. Overall, quantitative analysis of mass spectra allows susceptibility testing within 4 hours after the positivity of blood culture with E. coli. At the first report of positive blood culture, MALDI-TOF MS would then provide the prescribers with the bacterial identification and the susceptibility to AMX and CTX, thus limiting the use of broad-spectrum compounds.

mcr-1-like detection in commensal Escherichia coli and Salmonella spp. from food-producing animals at slaughter in Europe.

We evaluate here the presence of the mcr-1-like and mcr-2 genes in Escherichia coli and Salmonella spp. isolated from healthy food-producing animals at slaughter between 2002 and 2014 in Europe. Isolates were retrieved from cattle, pig and chicken from 11 European countries of production. The susceptibility to colistin and antibiotics used in human medicine was determined by agar dilution. Colistin-resistant isolates were PCR-screened for mcr genes. mcr-positive isolates were typed by Pulsed-Field Gel Electrophoresis (PFGE) and Multi-Locus Sequence Typing. Among the 10,206 E. coli and 1774 Salmonella spp. isolated from cattle, pigs and chickens, 148 E. coli and 92 Salmonella spp. isolates were resistant to colistin. We found mcr-1-like gene in 68 (0.7%) E. coli and 2 (0.1%) Salmonella isolates whereas none of the isolates tested positive for mcr-2. MCR-1-like-positive E. coli were isolated from 2008 to 2014 in chicken (n=44, 1.2%) and pigs (n=24, 0.7%). The presence of mcr-1-like varied from 0 to 4.0% depending on the year and the animal species. mcr-1-like-positive isolates came from animals originating from Germany (n=38), Spain (n=23), The Netherlands (n=5), and France (n=4). They were distributed in 63 different PFGE types and 37 different STs, with ST10 being the most prevalent. The two mcr-1-like-positive Salmonella spp. were isolated from France and Germany from a pig and a chicken, respectively. mcr-1-like gene is present in food-producing animals at slaughter in European countries with the highest occurrence in chickens. The high clonal diversity of E. coli underlines the evidence for horizontal transfer of mcr-1-like genes.

Can MALDI-TOF Mass Spectrometry Reasonably Type Bacteria?

Bacterial typing is crucial to tackle the spread of bacterial pathogens but current methods are time-consuming and costly. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been recently integrated into the microbiology laboratory workflow for a quick and low-cost microbial species identification. Independent research groups have successfully redirected the original function of this technology from their primary purpose to discriminate subgroups within pathogen species. However, identical bacterial subgroups could be identified by unrelated peaks by independent methods, thus limiting their robustness and exportability. We propose several guidelines that could improve the performance of MALDI-TOF MS-based typing methods for use as a first-line epidemiological tool.

Trends of extended-spectrum ß-lactamase-producing Escherichia coli sequence type 131 and its H30 subclone in a French hospital over a 15-year period.

Sequence type 131 (ST131) is a predominant lineage among extraintestinal pathogenic Escherichia coli. It plays a major role in the worldwide dissemination of E. coli producing extended-spectrum ß-lactamases (ESBLs). Here we describe the long-term epidemiology of this clonal group in a French university hospital, where the incidence of ESBL-producing E. coli has increased from 0.018 case per 1000 patient-days in the year 2000 to 0.50 case per 1000 patient-days in 2014. The first of the 141 ST131 isolates was recovered in 2006, and the ST131 clonal group accounted for 18.1% of total ESBL-producing E. coli over the whole period (2000-2014). Subclonal typing showed that 75.9% (107/141) of ST131 isolates were H30, of which 81.3% (87/107) were H30-Rx. The large majority (137/141) of ESBLs produced were of the CTX-M group, with 94 CTX-M-15, 19 CTX-M-1, 10 CTX-M-27, 8 CTX-M-14 and four other CTX-M types (n = 6). Pulsed-field gel electrophoresis (PFGE) analysis showed high diversity, which increased during the course of the study. The 141 ST131 isolates clustered in 53 pulsotypes (PTs), with 2 dominant PTs (PT14 and PT13) with 36 and 17 isolates, respectively. These findings showed that ST131 was a predominant clone among ESBL-producing E. coli in our hospital, even though it only accounted for <20%. Moreover, ST131 should be regarded not as a unified entity but as a cluster of distinct clonal subsets even if the increase in resistance within ST131 has a strong clonal basis, being attributable mainly to the spread of C1/H30-R and C2/H30-Rx clades.

Matrix-assisted laser desorption ionization-time of flight Mass spectrometry can detect Staphylococcus aureus clonal complex 398.

Within the last decade methicillin-resistant Staphylococcus aureus belonging to 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. It appears therefore necessary to implement a convenient tool for the surveillance this emerging pathogen. We evaluated the MALDI-TOF MS as a tool for rapid detection of S. aureus CC398. We used 626 S. aureus isolates characterized by a CC398-specific PCR, to constitute independent training (300 isolates including 60 isolates CC398) and validation sets (326 isolates including 82 isolates CC398). Fifteen peak biomarkers of CC398 were identified from the mass spectra of the training set. Ninety four % (307 of 326) of strains of the validation set were well assigned with an overall sensitivity of 93% and a specificity of 95%. Six CC398 and 13 non-CC398 isolates were misclassified. With MALDI-TOF MS, clinical laboratories could rapidly detect S. aureus CC398 associated with a higher mortality in hospitalized patients.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry identifies Pseudomonas aeruginosa high-risk clones.

We show here that matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) accurately and quickly identified the five high-risk clones of Pseudomonas aeruginosa sequence type 111 (ST111), ST175, ST235, ST253, and ST395. The use of this screening technique by clinical microbiology laboratories may tackle the spread of high-risk clones by the quick implementation of hygiene control procedures for relevant patients.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry assigns Escherichia coli to the phylogroups A, B1, B2 and D.

Escherichia coli classification into phylogroups reflects the diversity of their pathogenicity and their ecological niche, B2 isolates being the most virulent among extra-intestinal strains. MALDI-TOF MS allows a quick, automated, simple and inexpensive bacterial identification. We evaluated the MALDI-TOF MS as a tool for E. coli phylogroup differentiation. We used 656 E. coli isolates, previously assigned to phylogroup A, B1, B2, and D by multiplex PCR, to constitute independent training and validation sets. We then defined two phylogrouping strategies, both validated on spectra obtained by the 'direct transfer method'. The first strategy used the MALDI Biotyper software (Bruker Daltonik) that identified a single peak shift between isolates of phylogroup B2 and those of groups A, B1 and D. It accurately classified 89% of the isolates. The second strategy used the ClinProTools software (Bruker Daltonik) and was based on three successive models. The model 1 adequately differentiated 92% of phylogroup B2-isolates from those belonging to phylogroups A, B1, D. The model 2 adequately discriminated 87% of phylogroup D-isolates from those of phylogroups A and B1. The model 3 correctly sorted 69% of A and B1-isolates. We concluded that clinical laboratories could routinely and very quickly assign E. coli isolates to phylogroups with MALDI-TOF MS. These methods could (i) expedite the detection of the most virulent strains belonging to phylogroup B2 and (ii) be a first-line tool to monitor the epidemiology of extra-intestinal pathogenic E. coli.

Rapid, sensitive and specific detection of OXA-48-like-producing Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The emergence of Enterobacteriaceae producing the class D carbapenemase OXA-48 has become a public health concern, particularly in southern Europe and in the Mediterranean area. The existing MALDI-TOF MS assays that detect carbapenemase activity have disregarded the Enterobacteriaceae producing OXA-48-like enzymes. Here, we assessed the ability of an optimized MALDI-TOF MS imipenem hydrolysis assay to detect OXA-48-like-producing Enterobacteriaceae. We tested 372 non duplicate isolates of Enterobacteriaceae from 9 species, among which 92 harbored a blaOXA-48-like gene and 180 were non-susceptible to ertapenem. The vast majority (98.9%) of the OXA-48-like producers were detected and 97.8% of the ertapenem non-susceptible isolates were correctly classified. From the agar plate to the final result, the turnaround time of the test was 90min. This quick, easy and accurate MALDI-TOF MS test can expedite the implementation of hygiene measures to prevent dissemination of OXA-48-like-producing Enterobacteriaceae and limit outbreaks.

Wastewater treatment plants release large amounts of extended-spectrum ß-lactamase-producing Escherichia coli into the environment.

BACKGROUND: The determinants of the spread of extended-spectrum ß-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.

Validation of an automated blood culture system for sterility testing of cell therapy products.

BACKGROUND AIMS: Automated blood culture systems are widely used for the detection of microorganisms in cell therapy products. However, they are not validated by the manufacturers for this purpose. The aim of this study was to assess the ability of the Bactec system (Becton-Dickinson, Le Pont-De-Claix, France) to detect the microorganisms that could contaminate cell therapy products. METHODS: Three types of vials and conditions were tested: Plus Aerobic/F and Anaerobic/F media incubated at 35°C and Mycosis IC/F medium incubated at 30°C. All vials were incubated 10 days. We tested 18 microorganisms, including slow growers and some with fastidious nutritional requirements (10 bacteria, four yeasts, four filamentous fungi), each with four inocula (10-10(4) colony-forming units) performed in quintuplicate. RESULTS: The combination of Plus Aerobic/F and Plus Anaerobic/F vials detected all the tested pathogenic bacteria, all the tested Gram-positive skin commensal or environmental bacteria, all the tested yeasts, and three of four tested filamentous fungi. The addition of the Mycosis IC/F vial extended the range of detected microorganisms to one fungal environmental contaminant. Two bacterial environmental contaminants were not detected by our method. Low inocula of the skin contaminant Propionibacterium acnes were detected only after 7 days of incubation. CONCLUSIONS: These data suggest that (i) the prolongation of the incubation time of Plus Aerobic/F and Plus Anaerobic/F vials from 7 to 10 days and (ii) the use of Mycosis IC/F medium make minor contributions in the sterility testing of cell therapy products. We have validated the Bactec method using aerobic and anaerobic vials incubated 7 days at 35°C.