Prevalence and risk factors of toxigenic Clostridioides difficile asymptomatic carriage in 11 French hospitals.

Clostridioides difficile infection (CDI) incidence has increased over the last 20 years. Studies suggest that asymptomatic carriers may be an important reservoir of C. difficile in healthcare settings. We conducted a point prevalence study to estimate the toxigenic C. difficile asymptomatic carriage rate and the associated risk factors in patients >3 years old. Between September 16, 2019 and January 15, 2020, all patients hospitalized in 11 healthcare facilities in the Paris urban area were included in the study. They were screened on the day of the survey for toxigenic C. difficile carriage by rectal swab and interviewed. Isolates were characterized by PCR ribotyping and multiplex PCR targeting toxin genes. A logistic regression model was used to determine the risk factors associated with toxigenic C. difficile asymptomatic carriage using uni- and multivariate analysis in the subpopulation of patients >3 years old. During the study period, 2,389 patients were included and screened. The median age was 62 years (interquartile range 35-78 years) and 1,153 were male (48.3%). Nineteen patients had a previous CDI (0.9%). Overall, 185/2389 patients were positive for C. difficile (7.7%), including 93 toxigenic strains (3.9%): 77 (82.8%) were asymptomatic (prevalence 3.2%) whereas 12 (12.9%) were diarrheic. Prevalences of toxigenic C. difficile were 3.5% in patients >3 years old and 7.0% in ≤3 years old subjects, respectively. Toxigenic strains mainly belonged to PCR ribotypes 106 (n = 14, 15.0%), 014 (n = 12, 12.9%), and 020 (n = 10, 10.8%). Among toxigenic strains, 6 (6.4%) produced the binary toxin. In multivariate analysis, two factors were positively associated with toxigenic C. difficile asymptomatic carriage in patients >3 years old: multidrug-resistant organisms co-carriage [adjusted Odd Ratio (aOR) 2.3, CI 95% 1.2-4.7, p = 0.02] and previous CDI (aOR 5.8, CI 95% 1.2-28.6, p = 0.03). Conversely, consumption of raw milk products were associated with reduced risk of toxigenic C. difficile colonization (aOR 0.5, CI 95% 0.2-0.9, p = 0.01). We showed that there was a low prevalence of asymptomatic toxigenic C. difficile carriage in hospitalized patients. Consumption of raw milk prevents toxigenic C. difficile colonization, probably due to the barrier effect of milk-associated bacteria.

Comparison of the New VITEK MS PRIME System with the Matrix-Assisted Laser Desorption Ionization Biotyper Microflex LT for the Identification of Microorganisms.

Background: Matrix-assisted laser desorption ionization (MALDI) is the cornerstone of bacterial identification. The performance of a new MALDI time-of-flight mass spectrometry VITEK MS PRIME (VMS-P) system was compared with that of the MALDI Biotyper Microflex LT (MBT) system, which is routinely used in our laboratory. Methods: Sixteen bacterial and yeast reference strains cultured in 20 different media were analyzed over 10 consecutive rounds using both systems. Bacterial and yeast isolates from the routine workflow were processed using both systems. Microcolonies were identified after a 4-hour agar subculture from positive blood culture bottles, without extraction. Results: To determine the repeatability based on the reference strains, 1,190 spots were processed using each system. Correct identification was achieved for 94.0% (MBT) and 98.4% (VMS-P; P<0.01) of spots. Among these, 83.0% (MBT) and 100.0% (VMS-P) were identified with a high degree of confidence. For 1,214 spots from routine isolates, species identification was achieved for 90.0% (MBT) and 91.4% (VMS-P; P=0.26) of spots. For 69.8% (MBT) and 87.4% (VMS-P) of the spots, identification was achieved with a high degree-of-confidence score. When identification was performed using both systems, the agreement between them was 97.9%. The identification of microcolonies from positive blood culture bottles was achieved for 55.5% (MBT) and 70.2% (VMS-P; P=0.01) of spots. Conclusions: The MBT and VMS-P systems perform similarly in routine daily practice. The new VMS-P system shows high repeatability, better confidence scores for identification, and promising ability to identify microcolonies.

Faecal carriage of multidrug-resistant bacteria and associated risk factors: results from a point prevalence study.

OBJECTIVES: Since 2003, incidences of carbapenemase-producing Gram-negative bacilli (CP-GNB) and vancomycin-resistant Enterococcus faecium (VRE) have steadily increased in France. We therefore conducted a point prevalence study to estimate carriage rates of CP-GNB, VRE and ESBL-producing Enterobacterales (ESBL-PE) and associated risk factors. METHODS: Between September 2019 and January 2020, all inpatients hospitalized on a given day in 11 teaching hospitals in the Paris urban area were eligible. Patient interviews and rectal swab screening results were recorded by dedicated nurses. The swabs were plated onto selective chromogenic media and processed using the GeneXpert® system. RESULTS: Of 2396 patients, 364 (15.2%) yielded at least one multiresistant bacterial isolate, including 29 CP-GNB carriers (1.2%), 13 VRE carriers (0.5%) and 338 ESBL-PE carriers (14%). In 15 patients (4.4% of ESBL-PE carriers and 36.6% of CP-GNB/VRE carriers), concomitant CP-GNB/VRE and ESBL-PE carriage was observed. In 7/29 CP-GNB and 7/13 VRE carriers, carbapenemase production and vanA in the screening samples was only detected with Xpert® tests. The OXA-48 gene was predominant in 13/34 CP-GNB isolates from 29 carriers. From the 338 ESBL-PE carriers, 372 isolates were recovered, mainly Escherichia coli (61.2%). Among 379 children, 1.1% carried a CP-GNB/VRE strain, and 12.4% carried an ESBL strain. Previous hospitalization outside mainland France, previous antimicrobial treatment and previous ESBL-PE carriage were the main risk factors associated with CP-GNB and/or VRE carriage. CONCLUSIONS: The low CP-GNB and VRE prevalence likely reflects the French policy to limit intrahospital spread of CP-GNB and VRE strains.

Appropriateness of empirical antibiotic prescription for bloodstream infections in an emergency department from 2006 to 2018: impact of the spread of ESBL-producing Enterobacterales.

The spread of ESBL producers in the community may impact the management of patients with bloodstream infections (BSI) involving Enterobacterales in emergency departments. Thus, from 2006 to 2018, data for all BSI episodes involving Enterobacterales from the emergency department of a French teaching hospital were retrospectively included. Antimicrobial susceptibility test results and empirical antibiotic regimens were recorded. Treatment was considered as appropriate if all isolates were susceptible in vitro to at least one prescribed antibiotic. A total of 1369 BSI episodes in 1321 patients was included. Urinary tract infection was the main source of BSI (61%). The prevalence of ESBL producers increased from zero to 9.2/100 Enterobacterales BSI cases (p < 0.001), mainly Escherichia coli (6.9 cases/100 BSI in 2018); and no Klebsiella. Third-generation cephalosporins (3GC) were used most frequently (71.8%) and their use as monotherapy increased during the study period (p < 0.001). The rate of appropriate treatment decreased from 95.8 to 89.2% (p = 0.023). Appropriateness of treatment was greater using two drugs vs one (97.3% vs 89.3%, p < 0.001). Treatments with 3GC were appropriate in 92% and 98.3%, when used alone or with another antibiotic, respectively (p < 0.001). Among inappropriate treatments, 45% concerned 3GC, with 74.6% of them attributable to ESBL production. The spread of ESBL producers in the community had a direct impact on the rate of inappropriate empirical treatment. Local antimicrobial resistance monitoring is required to optimize the management of BSI in emergency departments.

Assessment of version 2.5 of QMAC-dRAST for rapid antimicrobial susceptibility testing with reduced sample-to-answer turnaround time and an integrated expert system.

OBJECTIVE: To assess the performance of the new rapid antimicrobial susceptibility testing (AST) QMAC-dRAST V2.5 system. METHODS: ASTs were performed using QMAC-dRAST-V2.5 and a disk diffusion method, directly from positive blood bottles with Gram-negative bacteria. Discrepancies between the results obtained using the two methods were categorized into very major errors (VME, S with dRAST vs. R with disk diffusion), major errors (ME, R vs. S, respectively), minor errors (mE, S vs. I or I vs. R, respectively), and very minor errors (Vme, I vs. S or R vs. I, respectively). For each AST, results were recorded after 4, 5, and 6h of incubation. RESULTS: From 106 bacteremia, 1416 individual AST results were obtained. Overall agreement between results using the two methods was 91%, ranging from 76.9% to 99.1% depending upon the antibiotic, with 128 errors, i.e. 14/1416 (1%) VME, 59/1416 (4.2%) ME, 25/1416 (1.8%) mE and 30/1416 (2.1%) Vme. VMEs were encountered for Klebsiellasp and Serratia marcescens isolates with low-level piperacillin and amikacin resistance, respectively. Using the integrated QMAC-dRAST-V2.5 expert system, all 14 VMEs and 3 mEs were eliminated, leading to 92.2% categorical agreement. After 45min of pre-incubation in the QMAC-dRAST-V2.5 device, 22.2% of the 1416 AST results were obtained after 4h, an additional 31.4% after 5h and a further 46.3% after 6h. CONCLUSION: QMAC-dRAST-V2.5 is an optimized version of QMAC-dRAST V2.0, particularly with respect to utilization of an expert system and reduced TAT according to the antibiotic tested.

Comparative evaluation of the QMAC-dRAST V2.0 system for rapid antibiotic susceptibility testing of Gram-negative blood culture isolates.

To comparatively evaluate the performance of the rapid antimicrobial susceptibility testing (AST) system QMAC-dRAST V2.0 and of standard disk diffusion in agar, AST was performed directly from 100 positive blood culture bottles with Gram-negative bacilli. AST results provided by QMAC-dRAST showed 92.9% agreement with disk diffusion method results. Discrepancies observed between results obtained with QMAC-dRAST and disk diffusion method conducted to 10 very major errors (0.8%, S with QMAC-dRAST vs R with disk diffusion method), 40 major errors (3.2%, R vs S, respectively), 15 minor errors (1.2%, S vs I or I vs R, respectively) and 23 very minors errors (1.8%, I vs S or R vs I, respectively). For very major and major errors, in only 36% of the cases did the repeat QMAC-dRAST confirm the initial result, whereas a repeat AST using disk diffusion method confirmed the initial result in 92% of cases. AST results obtained using microdilution in liquid medium confirmed those obtained with QMAC-dRAST and disk diffusion method in 4% and 89%, respectively. Repeatability and reproducibility tests performed on QMAC-dRAST using reference strains showed 94% to 100% of R/S/I categorical agreement.

In vitro activity of ß-lactams in combination with avibactam against multidrug-resistant Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Achromobacter xylosoxidans isolates from patients with cystic fibrosis.

The in vitro activity of anti-pseudomonal ß-lactams in combination with avibactam was evaluated against 54 multidrug-resistant non-fermenting Gram-negative bacilli isolated from cystic fibrosis patients. Avibactam increased and/or restored the antibacterial activities of ceftazidime and aztreonam against Pseudomonas aeruginosa and Stenotrophomonas maltophilia, respectively. No ß-lactam-avibactam combination was active against Achromobacter xylosoxidans.

In Vitro Activity of Ceftolozane-Tazobactam against Multidrug-Resistant Nonfermenting Gram-Negative Bacilli Isolated from Patients with Cystic Fibrosis.

Ceftolozane-tazobactam was tested against 58 multidrug-resistant nonfermenting Gram-negative bacilli (35 Pseudomonas aeruginosa, 11 Achromobacter xylosoxydans, and 12 Stenotrophomonas maltophilia isolates) isolated from cystic fibrosis patients and was compared to ceftolozane alone, ceftazidime, meropenem, and piperacillin-tazobactam. Ceftolozane-tazobactam was the most active agent against P. aeruginosa but was inactive against A. xylosoxydans and S. maltophilia In time-kill experiments, ceftolozane-tazobactam had complete bactericidal activity against 2/6 clinical isolates (33%).

Molecular basis for different levels of tet(M) expression in Streptococcus pneumoniae clinical isolates.

Seventy-four unrelated clinical isolates of Streptococcus pneumoniae harboring the tet(M) gene were studied. Seven strains with low tetracycline (Tc) MICs (0.25 to 0.5 µg/ml) were found to harbor truncated tet(M) alleles that were inactivated by different frameshift mutations. In contrast, five strains bore deletions in the tet(M) promoter region, among which four displayed increased Tc MICs (16 to 64 µg/ml). The same promoter mutations were detected in Tc-resistant mutants selected in vitro from various susceptible strains. Sequence analysis revealed that these deletions might impede the formation of the transcriptional attenuator located immediately upstream of tet(M). Expression in Enterococcus faecalis of a tet(M) reporter gene transcribed from these promoter mutants conferred a level of Tc resistance similar to that observed in the parental S. pneumoniae strains. These results show that different levels of Tc susceptibility found in clinical isolates of S. pneumoniae can be explained by frameshift mutations within tet(M) and by alterations of the upstream transcriptional attenuator.

Robustness of two MALDI-TOF mass spectrometry systems for bacterial identification.

MALDI-TOF-MS systems (Microflex-Bruker Daltonics/BioTyper™ and Axima-Assurance-Shimadzu/SARAMIS-AnagnosTec) were assessed for bacterial identification. Focusing on bacteria difficult to identify routinely, 296 strains were identified by molecular biology techniques as gold standard. MALDI-TOF-MS identification provided correct results at genus and species level for 94.9%, 83.4% and 83.8%, 65.9% with Biotyper and Saramis respectively.

Accuracy of MIC determination for Streptococcus pneumoniae using the Sirscan2000automatic MIC determination system.

The Sirscan2000automatic MIC determination (SIR-MD) system is a new system for MIC determination based on the automatic detection of growth of bacteria spotted onto agar medium using a camera scan. To evaluate its accuracy, 3608 Streptococcus pneumoniae clinical isolates yielding 18,165 MICs were tested in parallel with the SIR-MD and a standard interpretive antibiogram procedure. The overall percent agreement between the 2 methods within 1 log(2) dilution was 86.9%. After exclusion of the 11.8% noninterpretable results, errors in the deduction of susceptibilities were very major in 0.03%, major in 0.2%, and minor in 1.3%.

Impact of electronic alerts on isolation precautions for patients with multidrug-resistant bacteria.

BACKGROUND: Health care workers' compliance with isolation precautions for patients colonized or infected with multidrug-resistant bacteria (MRB) is low. METHODS: In a 750-bed, acute care university hospital with a patient information system covering the entire hospital, a database that included all patients with MRB was created and was merged daily with the admission-discharge-transfer application. An electronic alert was generated for all new cases of MRB and for all transfers between wards and all readmissions of patients with MRB. Two successive interventions were implemented based on this alert system. First, alerts were dispatched to medical and staff members in charge of infection control in each ward with requests to order isolation precautions for the patients. Second, alerts were dispatched to the infection control team, who directly ordered implementation of isolation precautions in electronic nursing records. Five audits during a 3-year period were performed to evaluate their effect on health care workers' compliance with isolation precautions. RESULTS: Awareness of the MRB status for the nurses in charge of the patients statistically significantly increased from 24.0% at baseline to 59.4% at 1 year after the first intervention. This proportion improved to 93.1% at 1 year after the second intervention. Similarly, the implementation of isolation precautions statistically significantly increased from 15.0% at baseline to 50.5% at 1 year after the first intervention and then to 90.2% at 1 year after the second intervention. CONCLUSION: A computer alert system can lead to effective and lasting improvement in the implementation of isolation precautions for patients with MRB in health care institutions.

Relevance of routine use of the anaerobic blood culture bottle.

Using the BacT/Alert automated system, we conducted a 1-year retrospective study on blood cultures, focusing on the relevance of routine use of the anaerobic bottle. The rate of patients with positive blood cultures was 19.7%. Among these, 13.5% had a positive anaerobic bottle in the absence of any aerobic bottle, and 2/3 of these grew with nonobligate anaerobes. These patients were hospitalized in 20 out of 26 wards of the hospital group. For 65.4% of the monomicrobial-positive blood cultures growing Enterobacteriaceae, the anaerobic bottle detected growth earlier than the corresponding aerobic bottle. These data suggest that, in our institution, the use of an anaerobic bottle is still relevant.

Activities of different fluoroquinolones against Bacillus anthracis mutants selected in vitro and harboring topoisomerase mutations.

Three sets of mutants of Bacillus anthracis resistant to fluoroquinolones were selected on ciprofloxacin and moxifloxacin in a stepwise manner from a nalidixic acid-resistant but fluoroquinolone-susceptible plasmidless strain harboring a Ser85Leu GyrA mutation. A high level of resistance to fluoroquinolones could be obtained in four or five selection steps. In each case, ParC was the secondary target. However, in addition to the GyrA mutation, expression of high-level resistance required (i) in the first set of mutants, active drug efflux associated with a mutation in the QRDR of ParC; (ii) in the second set, two mutations in the QRDR of ParC associated with a mutation in GyrB; and (iii) in the third set, two QRDR mutations, one in ParC and one in GyrA. Interestingly, several selection steps occurred without obvious mutations in the QRDR of any topoisomerase, thereby implying the existence of other resistance mechanisms. Among the fluoroquinolones tested, garenoxacin showed the best activity.

In vitro activities of garenoxacin (BMS-284756) against Streptococcus pneumoniae, viridans group streptococci, and Enterococcus faecalis compared to those of six other quinolones.

The activity of garenoxacin, a new quinolone, was determined in comparison with other quinolones against different strains of S. pneumoniae, viridans group streptococci (VGS), and Enterococcus faecalis. Strains were quinolone-susceptible clinical isolates and quinolone-resistant strains with defined mechanisms of resistance obtained from either clinical isolates or derivatives of S. pneumoniae R6. Clinical quinolone-susceptible strains of S. pneumoniae, VGS and E. faecalis showed garenoxacin MICs within a range of 0.03 microg/ml to 0.25 micro g/ml. Garenoxacin MICs increased two- to eightfold when one mutation was present in the ParC quinolone resistance-determining region (QRDR), fourfold when one mutation was present in the GyrA QRDR (S. pneumoniae), 8- to 64-fold when two or three mutations were associated in ParC and GyrA QRDR, and 2,048-fold when two mutations were present in both the GyrA and ParC QRDRs (Streptococcus pneumoniae). Increased active efflux had a moderate effect on garenoxacin MICs for S. pneumoniae and VGS. Against S. pneumoniae, garenoxacin behaved like moxifloxacin and sparfloxacin, being more affected by a single gyrA mutation than by a single parC mutation. Although garenoxacin was generally two- to fourfold more active than moxifloxacin against the different wild-type or mutant strains of S. pneumoniae, VGS, and E. faecalis, it was two- to fourfold less active than gemifloxacin. At four times the respective MIC for each strain, the bactericidal effect of garenoxacin, observed at 6 h for S. pneumoniae and at 24 h for S. oralis and E. faecalis, was not influenced by the presence of mutation either in the ParC or in both the ParC and GyrA QRDRs.

In vitro bactericidal activities of linezolid in combination with vancomycin, gentamicin, ciprofloxacin, fusidic acid, and rifampin against Staphylococcus aureus.

The in vitro activities of linezolid were determined alone and in combination with vancomycin, ciprofloxacin, gentamicin, fusidic acid, or rifampin against five methicillin-susceptible Staphylococcus aureus (MSSA) and five methicillin-resistant S. aureus (MRSA) strains. Similar responses were obtained against MSSA and MRSA. When combined with fusidic acid, gentamicin, or rifampin, linezolid prevented selection of resistant mutants but showed no synergy. When linezolid was combined with vancomycin and ciprofloxacin, a slight antagonism was observed. While the combination with linezolid may reduce the emergence of mutants resistant to the associated drugs, the absence of synergy, especially in the case of vancomycin and ciprofloxacin, does not argue in favor of such combinations.