The importance of naturally attenuated SARS-CoV-2in the fight against COVID-19.

The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.

High susceptibility of MDR and XDR Gram-negative pathogens to biphenyl-diacetylene-based difluoromethyl-allo-threonyl-hydroxamate LpxC inhibitors.

OBJECTIVES: Inhibitors of uridine diphosphate-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC, which catalyses the first, irreversible step in lipid A biosynthesis) are a promising new class of antibiotics against Gram-negative bacteria. The objectives of the present study were to: (i) compare the antibiotic activities of three LpxC inhibitors (LPC-058, LPC-011 and LPC-087) and the reference inhibitor CHIR-090 against Gram-negative bacilli (including MDR and XDR isolates); and (ii) investigate the effect of combining these inhibitors with conventional antibiotics. METHODS: MICs were determined for 369 clinical isolates (234 Enterobacteriaceae and 135 non-fermentative Gram-negative bacilli). Time-kill assays with LPC-058 were performed on four MDR/XDR strains, including Escherichia coli producing CTX-M-15 ESBL and Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii producing KPC-2, VIM-1 and OXA-23 carbapenemases, respectively. RESULTS: LPC-058 was the most potent antibiotic and displayed the broadest spectrum of antimicrobial activity, with MIC90 values for Enterobacteriaceae, P. aeruginosa, Burkholderia cepacia and A. baumannii of 0.12, 0.5, 1 and 1 mg/L, respectively. LPC-058 was bactericidal at 1× or 2× MIC against CTX-M-15, KPC-2 and VIM-1 carbapenemase-producing strains and bacteriostatic at ≤4× MIC against OXA-23 carbapenemase-producing A. baumannii. Combinations of LPC-058 with ß-lactams, amikacin and ciprofloxacin were synergistic against these strains, albeit in a species-dependent manner. LPC-058's high efficacy was attributed to the presence of the difluoromethyl-allo-threonyl head group and a linear biphenyl-diacetylene tail group. CONCLUSIONS: These in vitro data highlight the therapeutic potential of the new LpxC inhibitor LPC-058 against MDR/XDR strains and set the stage for subsequent in vivo studies.

Psychrobacter sanguinis: an unusual bacterium for nosocomial meningitis.

We report the first case of postneurosurgical meningitis due to Psychrobacter sanguinis, identified only by 16S rRNA analysis. Psychrobacter spp. usually live in deep sea environments and cold habitats. Despite a strict questioning of the patient and the medical staff, we did not find the source of this bacterium.

Helicobacter pylori urease and flagellin alter mucin gene expression in human gastric cancer cells.

BACKGROUND: Helicobacter pylori (Hp), which is one of the causative agents in human gastric adenocarcinoma, is known to interact with mucous gel and alter mucin gene expression. The aim of this work was to study, using an in vitro model of cell infection, the effects of urease, flagellin, and CagA virulence factors on the regulation of the four 11p15 mucin genes (MUC2, MUC5AC, MUC5B, and MUC6). METHODS: KATO-III and AGS gastric cancer cells were infected for 1, 3 or 6 h with Hp wild-type strains (ATCC 43504, N6, and SS1) or corresponding isogenic mutants deficient for urease subunit B, flagellin subunit A, and CagA. mRNA levels of MUC2, MUC5B, MUC5AC and MUC6 were assessed by RT-PCR, and functional activity of their promoters was measured by transient transfection assays. RESULTS: Infection of KATO-III cells with Hp wild-type strains resulted in an early (at 1 h) transient expression of MUC2, MUC5AC, and MUC6 mRNA concomitant with those of interleukin (IL)-1ß, IL-8, and TNF-α cytokines. In these cells, the UreB(-) isogenic mutant induced strong activation of MUC5AC expression, and UreB-responsive elements were located in the -486/-1 region of the promoter. FlaA(-) and CagA(-) mutants had no effect on mucin gene mRNA levels in KATO-III cells. In AGS cells, Hp-responsive elements were identified in all promoters, and overexpression of NF-κB induced upregulation of MUC5AC promoter activity when infected with the UreB(-) isogenic mutant. CONCLUSION: These results indicate that Hp infection of gastric cancer cells alters 11p15 mucin gene transcription and that MUC5AC downregulation is mediated by urease virulence factor.

Ruminococcus gnavus: an unusual pathogen in septic arthritis.

Ruminococcus gnavus is an anaerobic Gram positive coccus that can be found in the gastrointestinal tract of animals and humans. We report a case of septic arthritis caused by R. gnavus that was identified by mass spectrometry and confirmed by 16S rRNA sequencing.

CHROMagar Yersinia, a new chromogenic agar for screening of potentially pathogenic Yersinia enterocolitica isolates in stools.

CHROMagar Yersinia (CAY) is a new chromogenic medium for the presumptive detection of virulent Yersinia enterocolitica in stools. Based on a comparative analysis of 1,494 consecutive stools from hospitalized patients, CAY was found to be just as sensitive as the reference medium (cefsulodin-irgasan-novobiocin agar) but was significantly more specific and had a very low false-positive rate. CAY reduces the workload (and thus costs) for stool analysis and can therefore be recommended for routine laboratory use.

Evaluation of a new molecular test, the BD Max Cdiff, for detection of toxigenic Clostridium difficile in fecal samples.

A new molecular assay detecting toxigenic Clostridium difficile, the BD Max Cdiff (Becton, Dickinson), was evaluated with 360 diarrheal feces samples. It exhibited high sensitivity (97.7%) and specificity (99.7%). The positive (97.7%) and negative (99.7%) predictive values of this test allow an accurate answer within 2 h.

Proficiency of PCR in hospital settings for nonculture diagnosis of invasive meningococcal infections.

BACKGROUND: Meningococcal meningitis requires rapid diagnosis and immediate management which is enhanced by the use of PCR for the ascertainment of these infections. However, its use is still restricted to reference laboratories. METHODS: We conducted an inter-laboratory study to assess the implementation and the performance of PCR in ten French hospital settings in 2010. RESULTS: Our data are in favour of this implementation. Although good performance was obtained in identifying Neisseria meningitidis positive samples, the main issue was reported in identifying other species (Streptococcus pneumoniae and Haemophilus influenzae) which are also involved in bacterial meningitis cases. CONCLUSIONS: Several recommendations are required and, mainly, PCR should target the major etiological agents (N. meningitidis, S. pneumonia, and H. influenzae) of acute bacterial meningitis. Moreover, PCR should predict the most frequent serogroups of Neisseria meningitidis according to local epidemiology.

Chemical extraction versus direct smear for MALDI-TOF mass spectrometry identification of anaerobic bacteria.

In the present study, two pre-analytic processes for mass spectrometric bacterial identification were compared: the time-consuming reference method, chemical extraction, and the direct smear technique directly using cultured colonies without any further preparation. These pre-analytic processes were compared in the identification of a total of 238 strains of anaerobic bacteria representing 34 species. The results showed that 218/238 strains were identified following chemical extraction, 185 identifications (77.7%) were secured to both genus and species [log(score) > 2.0] whereas 33 identifications (14%) were secured to genus only [log(score) between 1.7 and 2.0]. Following direct smear, 207/238 anaerobic bacteria were identified, 158 identifications (66.4%) were secured to both genus and species [log(score) > 2.0] whereas 49 identifications were secured to genus only [log(score) between 1.7 and 2.0]. Twenty strains were not identified [log(score) < 1.7] by MALDI-TOF MS following chemical extraction whereas 31 strains were not identified with the direct smear technique. Although direct smear led to a significant decrease of the log(score) values for the Clostridium genus and the Gram positive anaerobic bacteria (GPAC) group (p < 0.0001, Wilcoxon test), identification to both species and genus were not changed. However these differences were not statistically significant (p = 0.1, Chi square). Therefore, MALDI-TOF MS identification following the direct smear technique appears to both non-inferior to the reference method and relevant for anaerobic bacteria identification.

Comparison of the Xpert MTB/RIF test with an IS6110-TaqMan real-time PCR assay for direct detection of Mycobacterium tuberculosis in respiratory and nonrespiratory specimens.

The sensitivities of the Xpert MTB/RIF test and an in-house IS6110-based real-time PCR using TaqMan probes (IS6110-TaqMan assay) for the detection of Mycobacterium tuberculosis complex (MTBC) DNA were compared by use of 117 clinical specimens (97 culture positive and 20 culture negative for MTBC) that were frozen in sediment. The 97 clinical specimens included 60 respiratory and 37 nonrespiratory specimens distributed into 36 smear-positive and 61 smear-negative specimens. Among the 97 culture-positive specimens, 4 had rifampin-resistant isolates. Both methods were highly specific and exhibited excellent sensitivity (100%) with smear-positive specimens. The sensitivity of the Xpert MTB/RIF test with the whole smear-negative specimens was more reduced than that of the IS6110-TaqMan assay (48 versus 69%, P = 0.005). Both methods exhibited similar sensitivities with smear-negative respiratory specimens, but the Xpert MTB/RIF test had lower sensitivity with smear-negative nonrespiratory specimens than the IS6110-TaqMan assay (37 versus 71%, P = 0.013). Finally, the sensitivities of the Xpert MTB/RIF test and the IS6110-TaqMan assay were 79% and 84%, respectively, with respiratory specimens and 53% and 78%, respectively (P = 0.013), with nonrespiratory specimens. The Xpert MTB/RIF test correctly detected the rifampin resistance in smear-positive specimens but not in the one smear-negative specimen. The Xpert MTB/RIF test is a simple rapid method well adapted to a routine laboratory that appeared to be as sensitive as the IS6110-TaqMan assay with respiratory specimens but less sensitive with paucibacillary specimens, such as smear-negative nonrespiratory specimens.

Relative contribution of three main virulence factors in Pseudomonas aeruginosa pneumonia.

OBJECTIVE: The pathogenesis and the outcome of Pseudomonas aeruginosa ventilator-acquired pneumonia depend on the virulence factors displayed by the bacteria as well as the host response. Thus, quorum sensing, lipopolysaccharide, and type 3 secretion system have each individually been shown to be important virulence systems in laboratory reference strains. However, the relative contribution of these three factors to the in vivo pathogenicity of clinically relevant strains has never been studied. We analyzed the virulence of 56 nonclonal Pseudomonas aeruginosa strains isolated from critically ill patients with ventilator-acquired pneumonia. To avoid the variation of human immune response, we used a murine model of pneumonia. The aim was to determine which virulence factor was the most important. SETTING: Research laboratory of a university. SUBJECTS: Male adult BALB/c mice. INTERVENTIONS: In vitro, the phenotype of each strain was established as to the expression of quorum sensing-regulated factors (elastase and pyocyanin), type 3 secretion system exotoxin secretion (Exotoxin U, S and/or T, or "nonsecreting"), and lipopolysaccharide O-antigen serotype. Strain pathogenicity was evaluated in vivo in a mouse model of acute pneumonia through lung injury assessment by measuring alveolar-capillary barrier permeability to proteins, lung wet/dry weight ratio, and bacterial dissemination. Associations were then sought between virulence system phenotypes and levels of lung injury. MEASUREMENTS AND MAIN RESULTS: In univariate analysis, elastase production, O11 serotype, and type 3 secretion system exotoxin secretion were associated with increased lung injury and exotoxin U was linked to an increase risk of bacteremia. In multivariate analysis, we observed that type 3 secretion system exotoxin secretion and to a lesser degree elastase production were associated with increased lung injury. CONCLUSION: In a murine model of pneumonia, our data suggest that type 3 secretion system and elastase are the most important virulence factors in clinically relevant P. aeruginosa strains.

First case of postaneurysmal prosthetic vascular infection due to a nonsuperantigenic Yersinia pseudotuberculosis strain.

Among Yersinia spp., Y. enterocolitica is the species most frequently isolated from infected aneurysms. This report describes the first case of postaneurysmal prosthetic vascular infection due to a superantigen-negative Yersinia pseudotuberculosis strain, showing a potential affinity of this species for endovascular tissue.

Isolation of Helcococcus kunzii from plantar phlegmon in a vascular patient.

Helcococcus kunzii has previously been considered to belong to the normal skin flora of podiatry patients. Here, H. kunzii was isolated in abundance from a pus specimen collected by incision and drainage of plantar phlegmon. This fastidious Gram-positive species was unambiguously identified with the colorimetric VITEK 2 GP card identification system. This suggests that this phenotypic identification system is able to identify promptly H. kunzii, which should be considered a potential pathogen.

Molecular diagnosis of a bilateral panuveitis due to Borrelia burgdorferi sensu lato by cerebral spinal fluid analysis.

The present paper describes a case of bilateral panuveitis due to Borrelia burgdorferi sensu lato diagnosed by a PCR approach using cerebral spinal fluid. Since the culture of B. burgdorferi takes a long time to grow and the accuracy of serological tests is doubtful in patients, the PCR method of amplifying a B. burgdorferi flagellin could be suitable to make a positive diagnosis in a case of atypical clinical history of Lyme disease.

First molecular characterization of related cases of healthcare-associated infections involving multidrug-resistant Enterococcus faecium vanA in Algeria.

PURPOSE: Vancomycin-resistant Enterococcus (VRE) faecium (VREfm) are highly resistant bacteria emerging worldwide and rarely studied using molecular tools in Algeria since their first report in 2006. The aim of the study was to investigate healthcare-associated infections (HAIs) involving the first VRE in Batna University Hospital, Algeria, and characterize isolates using molecular tools. PATIENTS AND METHODS: Medical charts were reviewed for patients with VREfm. van genes were detected by multiplex polymerase chain reaction (PCR), and strains were characterized by automated repetitive sequence-based PCR (rep-PCR), multiplex rep-PCR, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). RESULTS: During a 6-month period, VREfm infections occurred in four patients hospitalized in three wards. The four isolates were E. faecium vanA belonging to the hospital-adapted clonal complex 17. PCR-based methods did not discriminate the isolates but MLST and PFGE delineated a subgroup of three VREfm of identical pulsotype and sequence type (ST) 80 (yet identified for five isolates in the international PubMLST database) while the fourth isolate was of ST789 (not previously identified for a VREfm) and displayed an unrelated pulsotype. The three genotypically related isolates were recovered in patients who underwent surgery in the same department, suggesting an outbreak for which the source and route of transmission remained unidentified. CONCLUSION: This first molecular epidemiology study of VRE in Algeria was useful in delimiting an outbreak involving three of the four HAI cases and revealed rarely encountered genotypes. Considering the threat and burden of VRE infections worldwide, particularly in the USA, and the late emergence in Algeria, our study supports the urgent need for improved and early adequate infection control measures to avoid VRE spread in North African hospitals.

A case of mitral endocarditis due to Campylobacter fetus subsp. fetus.

This report describes a patient presenting mitral native endocarditis due to Campylobacter fetus subsp. fetus, which was revealed by syncope and identified using 16S ribosomal RNA gene sequencing. This gene sequencing method has become the preferred approach to identifying the new emerging pathogens when discrepancies exist between phenotypical tests.

Performance and economic evaluation of the molecular detection of pathogens for patients with severe infections: the EVAMICA open-label, cluster-randomised, interventional crossover trial.

PURPOSE: Microbiological diagnosis (MD) of infections remains insufficient. The resulting empirical antimicrobial therapy leads to multidrug resistance and inappropriate treatments. We therefore evaluated the cost-effectiveness of direct molecular detection of pathogens in blood for patients with severe sepsis (SES), febrile neutropenia (FN) and suspected infective endocarditis (SIE). METHODS: Patients were enrolled in a multicentre, open-label, cluster-randomised crossover trial conducted during two consecutive periods, randomly assigned as control period (CP; standard diagnostic workup) or intervention period (IP; additional testing with LightCycler®SeptiFast). Multilevel models used to account for clustering were stratified by clinical setting (SES, FN, SIE). RESULTS: A total of 1416 patients (907 SES, 440 FN, 69 SIE) were evaluated for the primary endpoint (rate of blood MD). For SES patients, the MD rate was higher during IP than during CP [42.6% (198/465) vs. 28.1% (125/442), odds ratio (OR) 1.89, 95% confidence interval (CI) 1.43-2.50; P < 0.001], with an absolute increase of 14.5% (95% CI 8.4-20.7). A trend towards an association was observed for SIE [35.4% (17/48) vs. 9.5% (2/21); OR 6.22 (0.98-39.6)], but not for FN [32.1% (70/218) vs. 30.2% (67/222), P = 0.66]. Overall, turn-around time was shorter during IP than during CP (22.9 vs. 49.5 h, P < 0.001) and hospital costs were similar (median, mean ± SD: IP €14,826, €18,118 ± 17,775; CP €17,828, €18,653 ± 15,966). Bootstrap analysis of the incremental cost-effectiveness ratio showed weak dominance of intervention in SES patients. CONCLUSION: Addition of molecular detection to standard care improves MD and thus efficiency of healthcare resource usage in patients with SES. ClinicalTrials.gov registration number: NCT00709358.

Against all odds: molecular confirmation of an implausible case of bone tuberculosis.

We report the use of typing based on a variable number of tandem repeats of genetic elements called "mycobacterial interspersed repetitive units" to clarify a puzzling situation involving a patient with an exceptional case of spondylodiskitis that initially led to the suspicion of a possible event of laboratory cross-contamination with Mycobacterium tuberculosis.