Emayella augustorita, New Member of Pasteurellaceae, Isolated from Blood Cultures of Septic Patient.

We report discovery of a new bacterial genus and species of the family Pasteurellaceae by using phylogenetic and metabolic analysis. The bacterium, Emayella augustorita, was isolated from blood cultures of a patient in France diagnosed with an adenocarcinoma of the intestines and who was treated with a biliary prosthesis placement.

Sedimentation field-flow fractionation for rapid phenotypic antimicrobial susceptibility testing: a pilot study.

BACKGROUND: The increase in antibiotic resistance is a major public health issue. The development of rapid antimicrobial susceptibility testing (AST) methods is becoming a priority to ensure early and appropriate antibiotic therapy. OBJECTIVES: To evaluate sedimentation field-flow fractionation (SdFFF) as a method for performing AST in less than 3 h. METHODS: SdFFF is based on the detection of early biophysical changes in bacteria, using a chromatographic-type technology. One hundred clinical Escherichia coli strains were studied. A calibrated bacterial suspension was incubated for 2 h at 37°C in the absence (untreated) or presence (treated) of five antibiotics used at EUCAST breakpoint concentrations. Bacterial suspensions were then injected into the SdFFF machine. For each E. coli isolate, retention times and elution profiles of antibiotic-treated bacteria were compared with retention times and elution profiles of untreated bacteria. Algorithms comparing retention times and elution profiles were used to determine if the strain was susceptible or resistant. Performance evaluation was done according to CLSI and the ISO standard 20776-2:2021 with broth microdilution used as the reference method. RESULTS: AST results from SdFFF were obtained in less than 3 h. SdFFF showed high categorical agreement (99.8%), sensitivity (99.5%) and specificity (100.0%) with broth microdilution. Results for each antimicrobial were also in agreement with the ISO 20776-2 recommendations, with sensitivity and specificity of ≥95.0%. CONCLUSIONS: This study showed that SdFFF can be used as a rapid, accurate and reliable phenotypic AST method with a turnaround time of less than 3 h.

Prevalence and Genomic Investigation of Salmonella Isolates Associated with Watermelons and Their Environmental Reservoirs in Bejaia, Algeria.

This study was conducted in Bejaia, Algeria, to determine the presence of Salmonella in fresh watermelon (n = 105), soil (n = 23), and irrigation water samples (n = 17) collected from two different farms. After isolation, antimicrobial susceptibility testing, serotype determination, multilocus sequence typing, antimicrobial resistance genes detection, and whole genome sequencing were performed. Twenty watermelon samples (19%) were contaminated with Salmonella, but none were found in the soil or irrigation water. Among the 20 Salmonella isolates, 2 serovars were identified (Salmonella Liverpool and Salmonella Anatum), belonging to sequence types ST1959 and ST64, respectively. Ten Salmonella isolates showed significant resistance to nalidixic acid, ofloxacin, and ciprofloxacin but were susceptible to all other antibiotics. The coexistence of point mutations (parC:p.T57S) in Quinolone Resistance-Determining Regions and the qnrB19 gene may contribute to quinolone resistance. The study identified 164 virulence genes in the Salmonella isolates. Our study found Salmonella in fresh watermelon during the preharvest season in Bejaia, Algeria. Our study indicates a relatively high prevalence of Salmonella on watermelon samples before harvest. Although we cannot directly compare our results with previous studies, it is crucial to recognize that the absence of comprehensive comparative data underscores the need for further research and surveillance.

Characterization of Pseudomonas aeruginosa resistance to ceftolozane-tazobactam due to ampC and/or ampD mutations observed during treatment using semi-mechanistic PKPD modeling.

A double ampC (AmpCG183D) and ampD (AmpDH157Y) genes mutations have been identified by whole genome sequencing in a Pseudomonas aeruginosa (PaS) that became resistant (PaR) in a patient treated by ceftolozane/tazobactam (C/T). To precisely characterize the respective contributions of these mutations on the decreased susceptibility to C/T and on the parallel increased susceptibility to imipenem (IMI), mutants were generated by homologous recombination in PAO1 reference strain (PAO1- AmpCG183D, PAO1-AmpDH157Y, PAO1-AmpCG183D/AmpDH157Y) and in PaR (PaR-AmpCPaS/AmpDPaS). Sequential time-kill curve experiments were conducted on all strains and analyzed by semi-mechanistic PKPD modeling. A PKPD model with adaptation successfully described the data, allowing discrimination between initial and time-related (adaptive resistance) effects of mutations. With PAO1 and mutant-derived strains, initial EC50 values increased by 1.4, 4.1, and 29-fold after AmpCG183D , AmpDH157Y and AmpCG183D/AmpDH157Y mutations, respectively. EC50 values were increased by 320, 12.4, and 55-fold at the end of the 2 nd experiment. EC50 of PAO1-AmpCG183D/AmpDH157Y was higher than that of single mutants at any time of the experiments. Within the PaR clinical background, reversal of AmpCG183D, and AmpDH157Y mutations led to an important decrease of EC50 value, from 80.5 mg/L to 6.77 mg/L for PaR and PaR-AmpCPaS/AmpDPaS, respectively. The effect of mutations on IMI susceptibility mainly showed that the AmpCG183D mutation prevented the emergence of adaptive resistance. The model successfully described the separate and combined effect of AmpCG183D and AmpDH157Y mutations against C/T and IMI, allowing discrimination and quantification of the initial and time-related effects of mutations. This method could be reproduced in clinical strains to decipher complex resistance mechanisms.

Sedimentation Field-Flow Fractionation: A Diagnostic Tool for Rapid Antimicrobial Susceptibility Testing.

Conventional antimicrobial susceptibility testing (AST) methods require 24-48 h to provide results, creating the need for a probabilistic antibiotic therapy that increases the risk of antibiotic resistance emergence. Consequently, the development of rapid AST methods has become a priority. Over the past decades, sedimentation field-flow fractionation (SdFFF) has demonstrated high sensitivity in early monitoring of induced biological events in eukaryotic cell populations. This proof-of-concept study aimed at investigating SdFFF for the rapid assessment of bacterial susceptibility to antibiotics. Three bacterial species were included (Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) with two panels of antibiotics tailored to each bacterial species. The results demonstrate that SdFFF, when used in "Hyperlayer" elution mode, enables monitoring of antibiotic-induced morphological changes. The percentage variation of the retention factor (PΔR) was used to quantify the biological effect of antibiotics on bacteria with the establishment of a threshold value of 16.8% to differentiate susceptible and resistant strains. The results obtained with SdFFF were compared to that of the AST reference method, and a categorical agreement of 100% was observed. Overall, this study demonstrates the potential of SdFFF as a rapid method for the determination of antibiotic susceptibility or resistance since it is able to provide results within a shorter time frame than that needed for conventional methods (3-4 h vs 16-24 h, respectively), enabling earlier targeted antibiotic therapy. Further research and validation are necessary to establish the effectiveness and reliability of SdFFF in clinical settings.

Metagenomics for the microbiological diagnosis of hospital-acquired pneumonia and ventilator-associated pneumonia (HAP/VAP) in intensive care unit (ICU): a proof-of-concept study.

BACKGROUND: Hospital-acquired and ventilator-associated-pneumonia (HAP/VAP) are one of the most prevalent health-care associated infections in the intensive care unit (ICU). Culture-independent methods were therefore developed to provide faster route to diagnosis and treatment. Among these, metagenomic next-generation sequencing (mNGS) has shown considerable promise. METHODS: This proof-of-concept study describes the technical feasibility and evaluates the clinical validity of the mNGS for the detection and characterization of the etiologic agents causing hospital-acquired and ventilator-associated pneumonia. We performed a prospective study of all patients with HAP/VAP hospitalized in our intensive care unit for whom a bronchoalveolar lavage (BAL) was performed between July 2017 and November 2018. We compared BAL fluid culture and mNGS results of these patients. RESULTS: A total of 32 BAL fluids were fully analyzed. Of these, 22 (69%) were positive by culture and all pathogens identified were also reported by mNGS. Among the culture-positive BAL samples, additional bacterial species were revealed by mNGS for 12 patients, raising the issue of their pathogenic role (colonization versus coinfection). Among BALF with culture-negative test, 5 were positive in mNGS test. CONCLUSIONS: This study revealed concordant results for pneumonia panel pathogens between mNGS and culture-positive tests and identified additional pathogens potentially implicated in pneumonia without etiologic diagnosis by culture. mNGS has emerged as a promising methodology for infectious disease diagnoses to support conventional methods. Prospective studies with real-time mNGS are warranted to examine the impact on antimicrobial decision-making and clinical outcome.

Performance of the Cepheid Methicillin-Resistant Staphylococcus aureus/S. aureus Skin and Soft Tissue Infection PCR Assay on Respiratory Samples from Mechanically Ventilated Patients for S. aureus Screening during the Phase 2 Double-Blind SAATELLITE Study.

We investigated the performance of the Xpert methicillin-resistant Staphylococcus aureus (MRSA)/S. aureus skin and soft tissue (SSTI) quantitative PCR (qPCR) assay in SAATELLITE, a multicenter, double-blind, phase 2 study of suvratoxumab, a monoclonal antibody (MAb) targeting S. aureus alpha-toxin, for reducing the incidence of S. aureus pneumonia. The assay was used to detect methicillin-susceptible S. aureus (MSSA) and MRSA in lower respiratory tract (LRT) samples from mechanically ventilated patients. LRT culture results were compared with S. aureus protein A (spa) gene cycle threshold (CT) values. Receiver operating characteristic (ROC) and Youden index were used to determine the CT cutoff for best separation of culture-S. aureus-negative and S. aureus-positive patients. Of 720 screened subjects, 299 (41.5%) were S. aureus positive by qPCR, of whom 209 had culture data: 162 (77.5%) were S. aureus positive and 47 (22.5%) were S. aureus negative. Culture results were negatively affected by antibiotic use and cross-laboratory variability. An inverse linear correlation was observed between CT values and quantitative S. aureus culture results. A spa CT value of 29 (≈2 × 103 CFU/mL) served as the best cutoff for separation between culture-negative and culture-positive samples. The associated area under the ROC curve was 83.8% (95% confidence interval [CI], 78 to 90%). Suvratoxumab provided greater reduction in S. aureus pneumonia or death than placebo in subjects with low S. aureus load (CT ≥ 29; relative risk reduction [RRR], 50.0%; 90% CI, 2.7 to 74.4%) versus the total study population (RRR, 25.2%; 90% CI, -4.3 to 46.4%). The qPCR assay was easy to perform, sensitive, and standardized and provided better sensitivity than conventional culture for S. aureus detection. Quantitative PCR CT output correlated with suvratoxumab efficacy in reducing S. aureus pneumonia incidence or death in S. aureus-colonized, mechanically ventilated patients.

Integron detection for prediction of trimethoprim/sulfamethoxazole susceptibility in children with Enterobacterales urinary tract infections.

OBJECTIVES: In some countries, third-generation cephalosporins (3GCs) serve as first-line therapy in children with urinary tract infections (UTIs). However, their use may contribute to the emergence of antibiotic resistance, notably among Gram-negative bacteria (GNB). Integrons are bacterial genetic elements involved in antibiotic resistance in GNB. Their absence is associated with >97% susceptibility to trimethoprim/sulfamethoxazole in adults infected with GNB. The objective of this study was to examine the value of integron detection directly from urine samples as a predictive marker of resistance to trimethoprim/sulfamethoxazole in children with GNB-related UTIs. METHODS: Children admitted to the Limoges University Hospital's paediatric emergency department between February 2018 and March 2019 with a suspicion of UTI were eligible for the study. Only confirmed cases presenting a positive urine culture with unique GNB were retained for further study analyses. Integrons were detected directly from urines using real-time PCR. RESULTS: The data of 72 patients were analysed and integrons were detected in 15 urine samples. The negative predictive value of integron detection for resistance to trimethoprim/sulfamethoxazole was 100% as all of the GNB (all were Enterobacterales) isolated from patients with no integrons detected in their urine samples were susceptible to trimethoprim/sulfamethoxazole. CONCLUSIONS: The detection of integrons in cases of paediatric patients with suspected UTI could help limit 3GC empirical use and empower an empirical first-line strategy better tailored to the needs of each patient.

Contribution of the anaerobic blood culture vial for the recovery of Candida glabrata: A retrospective multicentric study.

Although Candida spp are aerobic microorganisms, some Candida strains, mainly Candida glabrata, can be recovered from anaerobic blood culture vials. We assessed the contribution of the anaerobic vials for the diagnosis of candidemia, especially for C. glabrata. We conducted a multicenter retrospective study including eight university or regional hospitals. A single episode of monomicrobial candidemia per patient was included from September 1st, 2016, to August 31st, 2019. The characteristics of all aerobic and anaerobic blood culture vials sampled within 2 h before and after the first positive blood culture vials were recorded (type of vials, result, and for positive vials time-to-positivity and Candida species). Overall, 509 episodes of candidemia were included. The main species were C. albicans (55.6%) followed by C. glabrata (17.1%), C. parapsilosis (4.9%), and C. tropicalis (4.5%). An anaerobic vial was positive in 76 (14.9%) of all episodes of which 56 (73.8%) were due to C. glabrata. The number of C. glabrata infections only positive in anaerobic vials was 1 (2.6%), 1 (11.1%), and 15 (37.5%) with the BACT/ALERT 3D the BACT/ALERT VIRTUO and the BACTEC FX instrument, respectively (P < 0.01). The initial positivity of an anaerobic vial was highly predictive of the isolation of C. glabrata with the BACTEC FX (sensitivity of 96.8%). C. glabrata time-to-positivity was shorter in anaerobic vial than aerobic vial with all instruments. Anaerobic blood culture vials improve the recovery of Candida spp mainly C. glabrata. This study could be completed by further analyses including mycological and pediatric vials. LAY SUMMARY: Although Candida spp are aerobic microorganisms, C. glabrata is able to grow in anaerobic conditions. In blood culture, the time-to-positivity of C. glabrata is shorter in anaerobic than aerobic vials. Only the anaerobic vial was positive in up to 15 (37.5%) C. glabrata bloodstream infections.

Routine use of 16S rRNA PCR and subsequent sequencing from blood samples in septic shock: about two case reports of Capnocytophaga canimorsus infection in immunocompetent patients.

BACKGROUND: Capnocytophaga canimorsus infection happens frequently in immunosuppressed patients with reported domestic animal bites. Clinical presentation ranges from simple cellulitis to fulminant septic shock with disseminated intravascular coagulopathy, with an overall mortality of 30%. Conventional blood culture is often negative as this is a slow-growing pathogen. Nevertheless, the increasing use of 16S rRNA gene amplification and Sanger sequencing allows a much more rapid diagnostic confirmation. We present two case reports where 16S rRNA gene sequencing helped to diagnose Capnocytophaga canimorsus infection. CASE PRESENTATION: Case 1: A 53-year-old man with a history of non-cirrhotic chronic alcohol consumption was admitted to the intensive care unit (ICU) for septic shock and disseminated intravascular coagulopathy (DIC) of unknown origin. Blood cultures remained negative and a 16S rRNA PCR was performed leading to the identification of Capnocytophaga Canimorsus on day 4. Targeted antibiotic therapy with ceftriaxone for 14 days lead to overall recovery. Afterwards, the patient recalled a dog bite 2 days before hospitalization with a punctiform necrotic wound localized on a finger, which was not obvious at admission. Case 2: A 38-year-old man arrived to the emergency department for acute alcohol intoxication and history of a dog bite 2 days before. At admission, septic shock with purpura fulminans was diagnosed and required ICU hospitalization, invasive mechanical ventilation, vasopressor support and renal replacement therapy due to the rapid clinical deterioration. In the context of septic shock with purpura fulminans, DIC and recent dog bite, the diagnosis of Capnocytophaga canimorsus septic shock was suspected, and early confirmed by 16S rRNA PCR coupled to Sanger sequencing on day 2. Blood cultures became only positive for Capnocytophaga canimorsus 5 days after admission. Ceftriaxone alone was infused for 10 days in total, and the patient was discharged from the ICU on day 25. CONCLUSIONS: 16S rRNA gene PCR proves an important diagnostic tool when facing a sepsis of unknown origin. In these two cases of septic shock related to Capnocytophaga canimorsus, initial blood cultures remained negative at 24 h, whereas the diagnosis was achieved by 16S rRNA PCR sequencing performed from blood samples obtained at admission.

High prevalence of infections in non-COVID-19 patients admitted to the Emergency Department with severe lymphopenia.

BACKGROUND: In the Emergency Department (ED), early and accurate recognition of infection is crucial to prompt antibiotic therapy but the initial presentation of patients is variable and poorly characterized. Lymphopenia is commonly associated with bacteraemia and poor outcome in intensive care unit patients. The objective of this retrospective study was to assess the prevalence of community-acquired infection in a cohort of unselected patients admitted to the ED with undifferentiated symptoms and severe lymphopenia. METHODS: This is a retrospective single-center study conducted over a 1 year-period before the COVID-19 pandemic. Consecutive adult patients admitted to the ED with severe lymphopenia (lymphocyte count < 0.5 G/L) were studied. Patients with hematological or oncological diseases, HIV infection, hepato-cellular deficiency, immunosuppression, or patients over 85 years old were excluded. Diagnoses of infection were validated by an independent adjudication committee. The association between various parameters and infection was assessed using a multivariate logistic regression analysis. RESULTS: Of 953 patients admitted to the ED with severe lymphopenia, 245 were studied (148 men; mean age: 63 ± 19 years). Infection was confirmed in 159 patients (65%) (bacterial: 60%, viral: 30%, other: 10%). Only 61 patients (25%) were referred to the ED for a suspected infection. In the univariate analysis, SIRS criteria (OR: 5.39; 95%CI: 3.04-9.70; p < 0.001) and temperature ≥ 38.3 °C (OR: 10.95; 95%CI: 5.39-22.26; p < 0.001) were strongly associate with infection. In the multivariate analysis, only SIRS criteria (OR: 2.4; 95%CI: 1.48-3.9; p < 0.01) and fever (OR: 3.35; 95%CI: 1.26-8.93; p = 0.016) were independently associated with infection. CONCLUSIONS: The prevalence of underlying infection is high in patients admitted to the ED with lymphopenia, irrespective of the reason for admission. Whether lymphopenia could constitute a valuable marker of underlying infection in this clinical setting remains to be confirmed prospectively in larger cohorts. TRIAL REGISTRATION: No registration required as this is a retrospective study.

Safety, efficacy, and pharmacokinetics of gremubamab (MEDI3902), an anti-Pseudomonas aeruginosa bispecific human monoclonal antibody, in P. aeruginosa-colonised, mechanically ventilated intensive care unit patients: a randomised controlled trial.

BACKGROUND: Ventilator-associated pneumonia caused by Pseudomonas aeruginosa (PA) in hospitalised patients is associated with high mortality. The effectiveness of the bivalent, bispecific mAb MEDI3902 (gremubamab) in preventing PA nosocomial pneumonia was assessed in PA-colonised mechanically ventilated subjects. METHODS: EVADE (NCT02696902) was a phase 2, randomised, parallel-group, double-blind, placebo-controlled study in Europe, Turkey, Israel, and the USA. Subjects ≥ 18 years old, mechanically ventilated, tracheally colonised with PA, and without new-onset pneumonia, were randomised (1:1:1) to MEDI3902 500, 1500 mg (single intravenous dose), or placebo. The primary efficacy endpoint was the incidence of nosocomial PA pneumonia through 21 days post-dose in MEDI3902 1500 mg versus placebo, determined by an independent adjudication committee. RESULTS: Even if the initial sample size was not reached because of low recruitment, 188 subjects were randomised (MEDI3902 500/1500 mg: n = 16/87; placebo: n = 85) between 13 April 2016 and 17 October 2019. Out of these, 184 were dosed (MEDI3902 500/1500 mg: n = 16/85; placebo: n = 83), comprising the modified intent-to-treat set. Enrolment in the 500 mg arm was discontinued due to pharmacokinetic data demonstrating low MEDI3902 serum concentrations. Subsequently, enrolled subjects were randomised (1:1) to MEDI3902 1500 mg or placebo. PA pneumonia was confirmed in 22.4% (n = 19/85) of MEDI3902 1500 mg recipients and in 18.1% (n = 15/83) of placebo recipients (relative risk reduction [RRR]: - 23.7%; 80% confidence interval [CI] - 83.8%, 16.8%; p = 0.49). At 21 days post-1500 mg dose, the mean (standard deviation) serum MEDI3902 concentration was 9.46 (7.91) µg/mL, with 80.6% (n = 58/72) subjects achieving concentrations > 1.7 µg/mL, a level associated with improved outcome in animal models. Treatment-emergent adverse event incidence was similar between groups. CONCLUSIONS: The bivalent, bispecific monoclonal antibody MEDI3902 (gremubamab) did not reduce PA nosocomial pneumonia incidence in PA-colonised mechanically ventilated subjects. Trial registration Registered on Clinicaltrials.gov ( NCT02696902 ) on 11th February 2016 and on EudraCT ( 2015-001706-34 ) on 7th March 2016.

Integrons, a predictive biomarker for antibiotic resistance in acute sepsis: the IRIS study.

BACKGROUND: Considering the increase in MDR Gram-negative bacteria (GNB), the choice of empirical antibiotic therapy is challenging. In parallel, use of broad-spectrum antibiotics should be avoided to decrease antibiotic selection pressure. Accordingly, clinicians need rapid diagnostic tools to narrow antibiotic therapy. Class 1-3 integrons, identified by intI1-3 genes, are genetic elements that play a major role in antibiotic resistance in GNB. OBJECTIVES: The objective of the IRIS study was to evaluate the negative and positive predictive values (NPVs and PPVs, respectively) of intI1-3 as markers of antibiotic resistance. METHODS: The IRIS study was an observational cross-sectional multicentre study that enrolled adult subjects with suspected urinary tract or intra-abdominal infections. intI1-3 were detected directly from routinely collected biological samples (blood, urine or intra-abdominal fluid) using real-time PCR. A patient was considered 'MDR positive' if at least one GNB, expressing acquired resistance to at least two antibiotic families among ß-lactams, aminoglycosides, fluoroquinolones and/or co-trimoxazole, was isolated from at least one biological sample. RESULTS: Over a 2 year period, 513 subjects were enrolled and 409 had GNB documentation, mostly Enterobacterales. intI1 and/or intI2 were detected in 31.8% of patients and 24.4% of patients were considered 'MDR positive'. The NPV of intI1 and/or intI2 as a marker of acquired antibiotic resistances was estimated at 92.8% (89.1%-95.5%). The NPVs for first-line antibiotics were all above 92%, notably >96% for resistance to third-generation cephalosporins. CONCLUSIONS: The IRIS study strongly suggests that the absence of intI1 and intI2 in biological samples from patients with GNB-related infections is predictive of the absence of acquired resistances.

Early and specific targeted mass spectrometry-based identification of bacteria in endotracheal aspirates of patients suspected with ventilator-associated pneumonia.

Rapid and reliable pathogen identification is compulsory to confirm ventilator-associated pneumonia (VAP) in order to initiate appropriate antibiotic treatment. In the present proof of concept, the effectiveness of rapid microorganism identification with a targeted bottom-up proteomics approach was investigated in endotracheal aspirate (ETA) samples of VAP patients. To do so, a prototype selected-reaction monitoring (SRM)-based assay was developed on a triple quadrupole mass spectrometer tracking proteotypic peptide surrogates of bacterial proteomes. Through the concurrent monitoring of 97 species-specific peptides, this preliminary assay was dimensioned to characterize the occurrence of six most frequent bacterial species responsible for over more than 65% of VAP. Assay performance was subsequently evaluated by analyzing early and regular 37 ETA samples collected from 15 patients. Twenty-five samples were above the significant threshold of 105 CFU/mL and five samples showed mixed infections (both pathogens ≥ 105 CFU/mL). The targeted proteomics assay showed 100% specificity for Acinetobacter baumannii, Escherichia coli, Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae. No false bacterial identification was reported and no interference was detected arising from the commensal flora. The overall species identification sensitivity was 19/25 (76%) and was higher at the patient level (84.6%). This successful proof of concept provides a rational to broaden the panel of bacteria for further clinical evaluation.

Spontaneous community-acquired PVL-producing Staphylococcus aureus mediastinitis in an immunocompetent adult - a case report.

BACKGROUND: Mediastinitis caused by hematogenous spread of an infection is rare. We report the first known case of community-acquired mediastinitis from hematogenous origin in an immunocompetent adult. This rare invasive infection was due to Panton-Valentine Leucocidin-producing (PVL+) methicillin-susceptible Staphylococcus aureus (MSSA). CASE PRESENTATION: A 22-year-old obese man without other medical history was hospitalized for febrile precordial chest pain. He reported a cutaneous back abscess 3 weeks before. CT-scan was consistent with mediastinitis and blood cultures grew for a PVL+ MSSA. Intravenous clindamycin (600 mg t.i.d) and cloxacillin (2 g q.i.d.), secondary changed for fosfomycin (4 g q.i.d.) because of a related toxidermia, was administered. Surgical drainage was performed and confirmed the presence of a mediastinal abscess associated with a fistula between the mediastinum and right pleural space. All local bacteriological samples also grew for PVL+ MSSA. In addition to clindamycin, intravenous fosfomycin was switched to trimethoprim-sulfamethoxazole after 4 weeks for a total of 10 weeks of antibiotics. CONCLUSIONS: We present the first community-acquired mediastinitis of hematogenous origin with PVL+ MSSA. Clinical evolution was favorable after surgical drainage and 10 weeks of antibiotics. The specific virulence of MSSA PVL+ strains played presumably a key role in this rare invasive clinical presentation.

Molecular basis of macrolide-lincosamide-streptogramin (MLS) resistance in Finegoldia magna clinical isolates.

Of 69 clinical isolates of Finegoldia magna tested, 36% presented high-level MICs of erythromycin (>256 µg/ml), harboring erm(A) (n = 20) or erm(B) (n=5). Of nine isolates exhibiting an inducible resistance phenotype to macrolides-lincosamides-streptogramins B, four (44%) were susceptible with a potential risk of treatment failure due to emergence of resistant mutants.

Proteae: a reservoir of class 2 integrons?

OBJECTIVES: Our aim was to confirm with a large panel of clinical isolates that class 2 integrons are highly prevalent in Proteae and to analyse their genetic characteristics. METHODS: Proteae (Proteus spp., Morganella spp. and Providencia spp.) isolates were collected from clinical samples during 2013 at Limoges University Hospital, France. The presence of class 1, 2 and 3 integrons was investigated by quantitative PCR. The presence of a stop codon in the intI2 gene was determined by Sanger sequencing. The gene cassette arrays of class 2 integrons were determined by PCR-RFLP and Sanger sequencing or next-generation sequencing when needed. RESULTS: Of the 327 Proteae collected, 103 (31.5%) harboured a class 2 integron and 45 (13.8%) a class 1 integron. No class 3 integrons were detected. One functional IntI2 integrase was detected in a Morganella morganii isolate. Six different gene cassette arrays were detected. Four had already been described in the literature: dfrA1-sat2-aadA1 (72 isolates), dfrA1-catB2-sat2-aadA1 (17), sat2-aadA1 (6) and lnu(F), dfrA1, aadA1 (1). We identified two new gene cassette arrays: (i) a new variant of the dfrA1 gene cassette (one isolate; the one with the functional IntI2); and (ii) the array dfrA1-gcu115-sat2 harbouring the new gcu115 gene cassette with two ORFs encoding proteins of unknown functions (five isolates). CONCLUSIONS: We showed a high frequency of class 2 integrons, as well as a diversity of gene cassette arrays, among Proteae. This work highlights that the Proteae tribe plays an important role as a reservoir of class 2 integrons.

Community-acquired Staphylococcus aureus bacteriuria: a warning microbiological marker for infective endocarditis?

BACKGROUND: Urinary tract infection (UTI) is frequently diagnosed in the Emergency Department (ED). Staphylococcus aureus (SA) is an uncommon isolate in urine cultures (0.5-6% of positive urine cultures), except in patients with risk factors for urinary tract colonization. In the absence of risk factors, community-acquired SA bacteriuria may be related to deep-seated SA infection including infective endocarditis. We hypothesized that SA bacteriuria could be a warning microbiological marker of unsuspected infective endocarditis in the ED. METHODS: This is a retrospective chart review of consecutive adult patients between December 2005 and February 2018. All patients admitted in the ED with both SA bacteriuria (104 CFU/ml SA isolated from a single urine sample) and SA bacteremia, without risk factors for UT colonization (i.e., < 1 month UT surgery, UT catheterization) were analyzed. Diagnosis of infective endocarditis was based on the Duke criteria. RESULTS: During the study period, 27 patients (18 men; median age: 61 [IQR: 52-73] years) were diagnosed with community-acquired SA bacteriuria and had subsequently documented bacteremia and SA infective endocarditis. Only 5 patients (18%) had symptoms related to UT infection. Median delay between ED admission and SA bacteriuria identification was significantly shorter than that between ED admission and the diagnosis of infective endocarditis (1.4 ± 0.8 vs. 4.3 ± 4.2 days: p = 0.01). Mitral and aortic valves were most frequently involved by infective endocarditis (93%). Mortality on day 60 reached 56%. CONCLUSIONS: This study suggests that community-acquired SA bacteriuria should warn the emergency physician about a potentially associated left-sided infective endocarditis in ED patients without risk factors for UT colonization.

Diversity of Class 1 Integron Gene Cassette Rearrangements Selected under Antibiotic Pressure.

UNLABELLED: Integrons are bacterial genetic elements able to capture and express genes contained within mobile gene cassettes. Gene cassettes are expressed via a Pc promoter and can be excised from or integrated into the integron by integrase IntI. Although the mechanisms of gene cassette integration and excision are well known, the kinetics and modes of gene cassette shuffling leading to new gene cassette arrays remain puzzling. It has been proposed that under antibiotic selective pressure, IntI-mediated rearrangements can generate integron variants in which a weakly expressed gene cassette moves closer to Pc, thus leading to higher-level resistance. To test this hypothesis, we used an integron with four gene cassettes, intI1-aac(6')-Ib-dfrA15-aadA1-catB9, and applied selective pressure with chloramphenicol, resistance to which is encoded by catB9. Experiments were performed with three different Pc variants corresponding to three IntI1 variants. All three integrases, even when not overexpressed, were able to bring catB9 closer to Pc via excision of the dfrA15 and aadA1 gene cassettes, allowing their host bacteria to adapt to antibiotic pressure and to grow at high chloramphenicol concentrations. Integrase IntI1(R32_H39), reported to have the highest recombination activity, was able, when overexpressed, to trigger multiple gene cassette rearrangements. Although we observed a wide variety of rearrangements with catB9 moving closer to Pc and leading to higher chloramphenicol resistance, "cut-and-paste" relocalization of catB9 to the first position was not detected. Our results suggest that gene cassette rearrangements via excision are probably less cost-effective than excision and integration of a distal gene cassette closer to Pc. IMPORTANCE: Integrons are bacterial genetic elements able to capture and express gene cassettes. Gene cassettes are expressed via a Pc promoter; the closer they are to Pc, the more strongly they are expressed. Gene cassettes can be excised from or integrated into the integron by integrase IntI. The kinetics and modes of gene cassette shuffling, leading to new gene cassette arrays remain puzzling. We used an integron with 4 antibiotic resistance gene cassettes and applied selective pressure with the antibiotic for which resistance was encoded by cassette 4. All IntI variants were able to bring cassette 4 closer to Pc. Rearrangements occur via excision of the previous gene cassettes instead of cut-and-paste relocalization of the fourth gene cassette.

Non-O1/Non-O139 Vibrio cholerae Avian Isolate from France Cocarrying the bla(VIM-1) and bla(VIM-4) Genes.

We describe here a non-O1/non-O139 Vibrio cholerae isolate producing both VIM-1 and VIM-4 carbapenemases. It was isolated from a yellow-legged gull in southern France. The blaVIM genes were part of a class 1 integron structure located in an IncA/C plasmid. This study emphasizes the presence of carbapenemase genes in wildlife microbiota.