In Vivo Emergence of Dual Resistance to Rifampin and Levofloxacin in Osteoarticular Cutibacterium avidum.

Cutibacterium avidum is an emerging causative agent of orthopedic device-related infections (ODRIs). There are no guidelines for the antimicrobial treatment of C. avidum ODRI, but oral rifampin is frequently used in combination with a fluoroquinolone following intravenous antibiotics. We describe the in vivo emergence of combined resistance to rifampin and levofloxacin in a C. avidum strain isolated from a patient with early-onset ODRI treated with debridement, antibiotic treatment, and implant retention (DAIR) using rifampin combined with levofloxacin as the oral treatment. Whole-genome sequencing of C. avidum isolates before and after antibiotic exposure confirmed strain identity and identified new mutations in rpoB and gyrA, leading to amino acid substitutions previously reported to be associated with resistance to rifampin (S446P) and fluoroquinolones (S101L), respectively, in other microbial agents, in the posttherapy isolate. Aside from the molecular insights reported here, this study highlights potential limitations of the combination of oral rifampin and levofloxacin in patients undergoing a DAIR procedure for C. avidum ODRI and the potential need to evaluate specific optimal therapy for emerging ODRI pathogens. IMPORTANCE In this study, we report for the first time the in vivo emergence of dual resistance to levofloxacin and rifampin in C. avidum isolated from a patient who received both antibiotics orally in the setting of a salvage debridement and implant retention of an ODRI. Aside from the molecular insights reported here, this study highlights potential limitations of the combination of oral rifampin and levofloxacin in patients undergoing these surgical procedures and the potential need to evaluate specific optimal therapy for emerging ODRI pathogens.

IgA Serological Response for the Diagnosis of Mycobacterium abscessus Infections in Patients with Cystic Fibrosis.

The immunoglobulin A (IgA) status of cystic fibrosis (CF) patients, presenting with or without a non-tuberculous mycobacterial (NTM) infection, has to date not been fully elucidated toward two antigenic preparations previously described. We have chosen to determine the clinical values of an IgA ELISA for the diagnosis of NTM and/or Mycobacterium abscessus infections in CF patients. One hundred and 73 sera from CF patients, comprising 33 patients with M. abscessus positive cultures, and 31 non-CF healthy controls were assessed. IgA levels were evaluated by indirect ELISAs using a surface antigenic extract named TLR2eF for TLR2 positive extract and a recombinant protein, the phospholipase C (rMAB_0555 or rPLC). These assays revealed a sensitivity of 52.6% (95% CI = 35.8% to 69%) and 42.1% (95% CI = 26.3% to 59.2%) using TLR2eF and rPLC, respectively, and respective specificities of 92.6% (95% CI = 87.5% to 96.1%) and 92% (95% CI = 86.7% to 95.7%) for samples culture positive for M. abscessus. Overall sensitivity and specificity of 66.7% and 85.4%, respectively, were calculated for IgA detection in M. abscessus-culture positive CF patients, when we combine the results of the two used antigens, thus demonstrating the efficiency in detection of positive cases for these two antigens with IgA isotype. CF patients with a positive culture for M. abscessus had the highest IgA titers against TLR2eF and rPLC. The diagnosis of NTM infections, including those due to M. abscessus, can be improved by the addition of an IgA serological assay, especially when cultures, for example, are negative. Based on these promising results, a serological follow-up of a larger number of patients should be performed to determine if the IgA response may be correlated with an active/acute infection state or a very recent infection. IMPORTANCE Mycobacterium abscessus is currently the most frequently isolated rapid growing mycobacterium in human pathology and the major one involved in lung infections. It has recently emerged as responsible for severe pulmonary infections in patients with cystic fibrosis (CF) or those who have undergone lung transplantation. In addition, it represents the most antibiotic resistant mycobacterial species. However, despite its increasing clinical importance, very little is known about the use of M. abscessus parietal compounds and the host response. This has led to the development of serological tests to measure the antibody response in infected patients, and potentially to link this to the culture of respiratory samples. Herein, we describe an important analysis of the serological IgA response from CF patients, and we demonstrate the full diagnostic usefulness of this assay in the diagnosis of NTM infections, and more particularly M. abscessus, in CF patients.

Non-Legionella pneumophila serogroup 1 pneumonia: Diagnosis of a nosocomial legionellosis with the Biofire Pneumonia plus panel.

We report a nosocomial case of Legionella pneumophila pneumonia caused by a serogroup 10 strain diagnosed with the Biofire® Pneumonia plus panel. Molecular investigations of the environment of the patient allowed us to identify the source of contamination.

Serological biomarkers for the diagnosis of Mycobacterium abscessus infections in cystic fibrosis patients.

BACKGROUND: Culture conditions sometimes make it difficult to detect non-tuberculous mycobacteria (NTM), particularly Mycobacterium abscessus, an emerging cystic fibrosis (CF) pathogen. The diagnosis of NTM positive cases not detected by classical culture methods might benefit from the development of a serological assay. METHODS: As part of a diagnostic accuracy study, a total of 173 sera CF-patients, including 33 patients with M. abscessus positive cultures, and 31 non-CF healthy controls (HC) were evaluated. Four M. abscessus antigens were used separately, comprising two surface extracts (Interphase (INP) and a TLR2 positive extract (TLR2eF)) and two recombinant proteins (rMAB_2545c and rMAB_0555 also known as the phospholipase C (rPLC)). RESULTS: TLR2eF and rPLC were the most efficient antigens to discriminate NTM-culture positive CF-patients from NTM-culture negative CF-patients. The best clinical values were obtained for the detection of M. abscessus-culture positive CF-patients; with sensitivities for the TLR2eF and rPLC of 81.2% (95% CI:65.7-92.3%) and 87.9% (95% CI:71.9-95.6%) respectively, and specificities of 88.9% (95% CI:85.3-94.8%) and 84.8% (95% CI:80.6-91.5%) respectively. When considering as positive all sera, giving a positive response in at least one of the two tests, and, as negative, all sera negative for both tests, we obtained a sensitivity of 93.9% and a specificity of 80.7% for the detection of M. abscessus-culture positive CF-patients. CONCLUSION: High antibody titers against TLR2eF and rPLC were obtained in M. abscessus-culture positive CF-patients, allowing us to consider these serological markers as potential tools in the detection of CF-patients infected with M. abscessus.

Cutibacterium acnes clonal complexes display various growth rates in blood culture vials used for diagnosing orthopedic device-related infections.

OBJECTIVES: Blood culture bottles (BCBs) are commonly used for the diagnosis of infections associated with orthopedic devices. Although Cutibacterium acnes is an important pathogen in orthopedics, relatively little is known about its growth characteristics in BCBs. This prompted us to analyze the influence of bacterial genotype and clinical significance on time-to-detection (TTD) in BCBs. METHODS: We reviewed 59 cases of orthopedic device-related infections in which at least one intraoperative specimen yielded a pure C. acnes culture from anaerobic BCBs (BD Bactec Lytic/10 Anaerobic/F; Lytic-Ana) and/or solid media. A strain was considered infectant if the same genotype was present in two or more intraoperative samples. From these cases, we isolated a total of 72 unique C. acnes strains belonging to four multilocus sequence type clonal complexes (CCs): CC18, CC28, CC36 and CC53. Growth rate and TTD in Lytic-Ana BCB were studied under experimental conditions (inoculation of standard inoculum) and in clinical samples (inoculation of periprosthetic tissue samples). RESULTS: Median TTD values were shorter for CC53 compared to other CCs under experimental conditions (69 vs. 103 h; p < 0.001) and from clinical specimens (70 vs. 200 h; p = 0.02). Infectant strains had a shorter median TTD compared to contaminant strains in a clinical situation, while the difference was not observed under experimental conditions. CONCLUSIONS: The detection dynamics of C. acnes in Lytic-Ana BCBs were associated with genotype. Thus, TTD not only reflects the bacterial load in clinical samples, but may also reflect the intrinsic properties of the clonal complex of C. acnes.

Bloodstream infections in older population: epidemiology, outcome, and impact of multidrug resistance.

Bloodstream infections (BSIs) among older patients are frequent with high rate of mortality. Infections with multidrug-resistant organisms (MDRO) are associated with higher mortality than with susceptible microorganisms. We aimed to evaluate the prevalence of MDRO infection during BSI in older population and the factors associated with unfavorable outcome. This study is a retrospective cohort of all BSI episodes occurring among older patients (> 65yo), from April 1, 2010, to December 31, 2016, in a French university hospital for geriatric medicine. A total of 255 BSI episodes were analyzed. Mean age was 86.3±6.5 years, and sex ratio (M/F) was 0.96. Main comorbidities were orthopedic device (26.7%), active neoplasia (24.3%), and diabetes mellitus (18.4%). Main primary sites of infection were urinary tract infections (56.9%), respiratory tract infections (10.6%), intra-abdominal infections (7.1%), and skin and soft tissue infections (4.7%). Main bacteria identified were Escherichia coli (45.1%), Staphylococcus aureus (14.1%), enterococci (10.7%), coagulase-negative staphylococci (CoNS) (5.5%), and streptococci (5.1%). MDROs were involved in 17.2% of BSI (gram-negative bacilli: 9.0%; CoNS: 4.3%; and methicillin-resistant S. aureus (MRSA): 3.9%). The main factor associated with MDRO BSI was colonization with MDRO (OR=6.29; 95%CI=2.9-14.32). Total mortality was 18.4% and significantly higher in case of initial severity (OR=3.83; 95%CI=1.75-8.38), healthcare-associated infection (OR=5.29; 95%CI=1.11-25.30), and MRSA BSI (OR=9.16; 95%CI=1.67-50.16). BSI due to MDRO is frequent in older population and is strongly associated with carriage of MDRO. Healthcare-associated BSI, severe episodes, and BSI due to MRSA are associated with unfavorable outcome. In these cases, a broad-spectrum antibiotic should be promptly initiated.

Clinical Cutaneous Features of Patients Infected With SARS-CoV-2 Hospitalized for Pneumonia: A Cross-sectional Study.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a current pandemic worldwide. This virus can reach all organs and disturbs the immune system, leading to a cytokine storm in severe forms. We aimed to report cutaneous features among coronavirus disease 2019 (COVID-19) hospitalized patients. METHODS: We performed a cross-sectional study on 1 given day among all patients hospitalized in acute care for COVID-19 and included all patients with cutaneous features. Follow-up 48 hours later was obtained. RESULTS: Among 59 adult patients hospitalized on the day of the study in an infectious diseases ward for SARS-CoV-2 infection who were confirmed by molecular assay and/or radiological findings (computed tomography scan), 40 were included. Several cutaneous manifestations were found: macular exanthema (80%), face edema (32%), livedo (13%), urticarial rash (8%), purpura (5%), oral lichenoid lesions (33%), and conjunctivitis (18%). Cutaneous biopsy was performed in 17 patients. Histological findings showed mast cell hyperplasia (100%), superficial perivascular infiltrate of lymphocytes (94%), and superficial edema (47%) consistent with capillary leak. CONCLUSIONS: Various dermatological signs can be encountered during COVID-19. A macular rash was the most frequent. All cutaneous features could be related to a vascular leak process.

A TLR2-Activating Fraction From Mycobacterium abscessus Rough Variant Demonstrates Vaccine and Diagnostic Potential.

Mycobacterium abscessus is a prevalent pathogenic mycobacterium in cystic fibrosis (CF) patients and one of the most highly drug resistant mycobacterial species to antimicrobial agents. It possesses the property to transition from a smooth (S) to a rough (R) morphotype, thereby influencing the host innate immune response. This transition from the S to the R morphotype takes place in patients with an exacerbation of the disease and a persistence of M. abscessus. We have previously shown that the exacerbation of the Toll-like receptor 2 (TLR2)-mediated inflammatory response, following this S to R transition, is essentially due to overproduction of bacilli cell envelope surface compounds, which we were able to extract by mechanical treatment and isolation by solvent partition in a fraction called interphase. Here, we set up a purification procedure guided by bioactivity to isolate a fraction from the R variant of M. abscessus cells which exhibits a high TLR2 stimulating activity, referred to as TLR2-enriched fraction (TLR2eF). As expected, TLR2eF was found to contain several lipoproteins and proteins known to be stimuli for TLR2. Vaccination with TLR2eF showed no protection toward an M. abscessus aerosol challenge, but provided mild protection in ΔF508 mice and their FVB littermates when intravenously challenged by M. abscessus. Interestingly however, antibodies against TLR2eF compounds were detected during disease in CF patients. In conclusion, we show the potential for compounds in TLR2eF as vaccine and diagnostic candidates, in order to enhance diagnosis, prevent and/or treat M. abscessus-related infections.

Measurement of Serum Anti-staphylococcal Antibodies Increases Positive Predictive Value of Preoperative Aspiration for Hip Prosthetic Joint Infection.

BACKGROUND: Preoperative synovial fluid culture is pivotal in the early diagnosis of prosthetic joint infection (PJI) but may yield false-positive and false-negative results. We evaluated the predictive value of synovial fluid culture results combined with the measurement of serum anti-staphylococcal antibodies (SASA). QUESTIONS/PURPOSES: (1) For hip and knee PJI, does combining positive SASA results with preoperative synovial culture results improve the positive predictive value (PPV) of preoperative synovial fluid culture alone? (2) Does combining preoperative synovial fluid culture results with a positive cell count and differential result increase the PPV of preoperative synovial fluid culture alone? (3) What proportion of isolated organisms exhibit concordance in antibiotic susceptibility: preoperative aspiration versus intraoperative isolates? METHODS: A prospective study was conducted at two French reference centers that manage bone and joint infections and included 481 adult patients who had a revision or resection arthroplasty between June 25, 2012 and June 23, 2014. Exclusion criteria including no serum sample available for immunoassay, the lack of microbiological documentation, and the absence of preoperative aspiration reduced the patient number to 353. Seven patients with an undetermined SASA result were excluded from the analysis. We also excluded patients with PJI involving more than one Staphylococcus species (polystaphylococcal infection) and those in whom more than one Staphylococcus species was recovered from the preoperative synovial fluid culture (polystaphylococcal synovial fluid culture). In total, 340 patients were included in the analysis (no infection, 67% [226 of 340]; staphylococcal infection, 21% [71 of 340]; other infection, 13% [43 of 340]). The preoperative synovial fluid analysis included a cell count and differential and bacterial culture. SASAs were measured using a multiplex immunoassay. The diagnosis of PJI was determined using the Infectious Diseases Society of America (IDSA) criteria [] and intraoperative tissue culture at the time of revision surgery was used as the gold standard (at least one positive intraoperative sample for a "virulent" organism (such as S. aureus) or two positive samples for a "non-virulent" (for example S. epidermidis). RESULTS: SASA increased the PPV compared with synovial fluid culture alone (92% [95% CI 82 to 97] versus 79% [95% CI 68 to 87]; p = 0.04); when stratified by site, an increase in PPV was seen in hip infections (100% [95% CI 89 to 100] versus 77% [95% CI 63 to 88]; p = 0.01) but not in knee infections (84% [95% CI 66 to 95] versus 80% [95% CI 64 to 91]; p = 0.75). A positive cell count and differential result increased the PPV of staphylococcal synovial fluid cultures compared with synovial fluid culture alone (86% [95% CI 70 to 95] versus 79% [95% CI 68 to 87]; p = 0.36); when stratified by site, no difference in hip and knee infections was observed (86% [95% CI 67 to 96] versus 77% [95% CI 63 to 88]; p = 0.42) and 86% [95% CI 70 to 95] versus 80% [95% CI 64 to 91]; p = 0.74). CONCLUSION: SASA measurement improves the predictive value of synovial fluid cultures of the hip for all staphylococcal organisms, including coagulase-negative staphylococci, but the PPV of SASA plus synovial fluid culture it is not superior to the PPV of synovial fluid cell count/differential plus synovial culture for the knee. LEVEL OF EVIDENCE: Level III, diagnostic study.

Mycobacterium abscessus virulence traits unraveled by transcriptomic profiling in amoeba and macrophages.

Free-living amoebae are thought to represent an environmental niche in which amoeba-resistant bacteria may evolve towards pathogenicity. To get more insights into factors playing a role for adaptation to intracellular life, we characterized the transcriptomic activities of the emerging pathogen Mycobacterium abscessus in amoeba and murine macrophages (Mϕ) and compared them with the intra-amoebal transcriptome of the closely related, but less pathogenic Mycobacterium chelonae. Data on up-regulated genes in amoeba point to proteins that allow M. abscessus to resist environmental stress and induce defense mechanisms, as well as showing a switch from carbohydrate carbon sources to fatty acid metabolism. For eleven of the most upregulated genes in amoeba and/or Mϕ, we generated individual gene knock-out M. abscessus mutant strains, from which ten were found to be attenuated in amoeba and/or Mϕ in subsequence virulence analyses. Moreover, transfer of two of these genes into the genome of M. chelonae increased the intra-Mϕ survival of the recombinant strain. One knock-out mutant that had the gene encoding Eis N-acetyl transferase protein (MAB_4532c) deleted, was particularly strongly attenuated in Mϕ. Taken together, M. abscessus intra-amoeba and intra-Mϕ transcriptomes revealed the capacity of M. abscessus to adapt to an intracellular lifestyle, with amoeba largely contributing to the enhancement of M. abscessus intra-Mϕ survival.

Multiplex Antibody Measurement for Post-treatment Follow-up of Staphylococcal Prosthetic Joint Infection: A Diagnostic Pilot Study.

Introduction: Multiplex-antibody detection has been recently proposed for the noninvasive diagnosis of staphylococcal prosthetic joint infection (PJI). We evaluated this approach for the post-treatment follow-up of patients. Methods: Nineteen cases of staphylococcal PJI were prospectively followed for one year after treatment. The IgG response against eight staphylococcal antigens was measured before surgery and one year post-surgery using Luminex technology (Austin, TX, USA); median fluorescence intensity values determined for each antigen were transformed into a "Total Response Index" (TRI). Results: Patients (11 women/8 men) had a mean (SD) age of 72.2 (12.4) years. Site of prosthesis was the knee (n=10), the hip (n=8) and the shoulder (n=1). Ten patients were infected by S. epidermidis, six by S. aureus, and three by S. lugdunensis. TRI values at one year were significantly lower than pre-surgery values (mean [SD]: 5.9 [1.8] versus 8.1 [3.4], p=0.02) and decreased, on average, by 21.2%. TRI values markedly increased in two patients. One patient had a relapse of S. aureus PJI at five months post-surgery, with a 37% increase of the TRI. The other had septic failure three months after revision for S. lugdunensis PJI; all intraoperative samples remained culture-negative, but the TRI increased by 51% and the antibody profile showed a marked change, suggesting a reinfection with another staphylococcal species. Conclusion: Multiplex-antibody measurement may be useful for the follow-up of staphylococcal PJI and may help to detect septic failure involving organisms targeted by the assay.

Lsr2 Is an Important Determinant of Intracellular Growth and Virulence in Mycobacterium abscessus.

Mycobacterium abscessus, a pathogen responsible for severe lung infections in cystic fibrosis patients, exhibits either smooth (S) or rough (R) morphotypes. The S-to-R transition correlates with inhibition of the synthesis and/or transport of glycopeptidolipids (GPLs) and is associated with an increase of pathogenicity in animal and human hosts. Lsr2 is a small nucleoid-associated protein highly conserved in mycobacteria, including M. abscessus, and is a functional homolog of the heat-stable nucleoid-structuring protein (H-NS). It is essential in Mycobacterium tuberculosis but not in the non-pathogenic model organism Mycobacterium smegmatis. It acts as a master transcriptional regulator of multiple genes involved in virulence and immunogenicity through binding to AT-rich genomic regions. Previous transcriptomic studies, confirmed here by quantitative PCR, showed increased expression of lsr2 (MAB_0545) in R morphotypes when compared to their S counterparts, suggesting a possible role of this protein in the virulence of the R form. This was addressed by generating lsr2 knock-out mutants in both S (Δlsr2-S) and R (Δlsr2-R) variants, demonstrating that this gene is dispensable for M. abscessus growth. We show that the wild-type S variant, Δlsr2-S and Δlsr2-R strains were more sensitive to H2O2 as compared to the wild-type R variant of M. abscessus. Importantly, virulence of the Lsr2 mutants was considerably diminished in cellular models (macrophage and amoeba) as well as in infected animals (mouse and zebrafish). Collectively, these results emphasize the importance of Lsr2 in M. abscessus virulence.

Molecular Typing of Multiple Isolates Is Essential to Diagnose Cutibacterium acnes Orthopedic Device-related Infection.

Cutibacterium acnes orthopedic device-related infections (ODRIs) range from obvious infections to solely culture-based diagnoses. Multilocus sequence typing of multiple isolates from the same procedure revealed that most cases with normal C-reactive protein levels that were classified as C. acnes ODRI would be considered contaminations when accounting for genotypic data.

Mycobacterium bolletii Lung Disease in Cystic Fibrosis.

BACKGROUND: The cystic fibrosis (CF) pathogen, Mycobacterium abscessus complex, covers three subspecies: M. abscessus, M. massiliense, and M. bolletii. There are no clinical outcome data concerning M. bolletii. Our aim was to characterize M. bolletii lung infections in patients with CF. METHODS: We included patients with M. bolletii lung infection recorded between 1994 and 2012 in France. Data were collected from the CF registry, medical records, and questionnaires submitted to the CF primary physician. Strains were typed by multilocus sequence typing analysis. RESULTS: Fifteen cases were identified in nine CF centers. Nine patients (60%) presented with nontuberculous mycobacterial pulmonary disease. Follow-up of 13 patients showed a trend to more rapid decline in FEV1 in the first year of colonization (-9.4%; SD 19.3) in comparison with noninfected control subjects (-2.3%; SD 12.1) (P = .16). Twelve patients were treated, and 11 received oral macrolides. Treatment-induced eradication occurred in five patients (41.7%). Four patients died (26.7%), including one patient with fatal nontuberculous mycobacterial pulmonary disease. Inducible macrolide resistance was demonstrated in all strains. Patients always harbored unique strains. CONCLUSIONS: Our study reports the largest study cohort of CF patients infected with M. bolletii. M. bolletii infection affects both children and young adults, is most often symptomatic, and may be fatal. Macrolide-based therapies have poor effectiveness. There is no evidence of patient-to-patient transmission.

Monomicrobial bone and joint infection due to Corynebacterium striatum: literature review and amoxicillin-rifampin combination as treatment perspective.

Corynebacterium striatum is a ubiquitous colonizer of human skin and mucous membranes. It is increasingly involved in infections, especially with prosthetic devices or in immunocompromised individuals. Microbiological diagnosis is challenging and bacterial resistance is a major concern. We performed a retrospective study of monomicrobial bone and joint infections (BJI) due to C. striatum in two referral centers from April 2012 to July 2017. We collected the patients' clinical and microbiological characteristics and outcomes. We also performed a literature review of BJI due to C. striatum. We identified 12 cases (nine prosthetic joint infections, one osteosynthetic device infection, one non-union, and one arthritis) in 11 patients, five of which were immunocompromised. Microbiological diagnosis was performed with prolonged culture media. Ten out of 12 strains were susceptible to aminopenicillin, a drug class not recommended for testing by the EUCAST/CASFM guidelines, and 8/12 patients were treated with amoxicillin-rifampicin. The cure rate was 8/12, after a median follow-up period of 487.5 days (IQR 140.3-1348.5). Twelve cases of BJI due to C. striatum were previously reported. Among them, 5/12 patients were immunocompromised, 3/12 cases were acute BJI, and 2/12 were device-related infections. The diagnosis was performed by PCR in one case, and 10/12 patients were treated with glycolipopeptides, with a cure rate of 11/12. We report the largest cohort of monomicrobial BJI with C. striatum. Determination of aminopenicillin susceptibility is essential since it is frequently active in our experience, even in BJI. The cure rate of this infection seems high.

Prospective Whole-Genome Sequencing in Tuberculosis Outbreak Investigation, France, 2017-2018.

During June 2017-April 2018, active tuberculosis with Beijing SIT1 isolates was diagnosed in 14 persons living in 4 distant cities in France. Whole-genome sequencing indicated that these patients belonged to a single transmission chain. Whole-genome sequencing-based laboratory investigations enabled prompt tracing of linked cases to improve tuberculosis control.

In-Host Adaptation of Salmonella enterica Serotype Dublin during Prosthetic Hip Joint Infection.

Genome degradation has been central to the adaptation of Salmonella enterica serotypes to their hosts throughout evolution. We witnessed the patho-adaptation of a strain of Salmonella Dublin (a cattle-adapted serotype) to a human host during the course of a recurrent prosthetic hip joint infection evolving over several years.

MmpL8MAB controls Mycobacterium abscessus virulence and production of a previously unknown glycolipid family.

Mycobacterium abscessus is a peculiar rapid-growing Mycobacterium (RGM) capable of surviving within eukaryotic cells thanks to an arsenal of virulence genes also found in slow-growing mycobacteria (SGM), such as Mycobacterium tuberculosis A screen based on the intracellular survival in amoebae and macrophages (MΦ) of an M. abscessus transposon mutant library revealed the important role of MAB_0855, a yet uncharacterized Mycobacterial membrane protein Large (MmpL). Large-scale comparisons with SGM and RGM genomes uncovered MmpL12 proteins as putative orthologs of MAB_0855 and a locus-scale synteny between the MAB_0855 and Mycobacterium chelonae mmpL8 loci. A KO mutant of the MAB_0855 gene, designated herein as mmpL8MAB , had impaired adhesion to MΦ and displayed a decreased intracellular viability. Despite retaining the ability to block phagosomal acidification, like the WT strain, the mmpL8MAB mutant was delayed in damaging the phagosomal membrane and in making contact with the cytosol. Virulence attenuation of the mutant was confirmed in vivo by impaired zebrafish killing and a diminished propensity to induce granuloma formation. The previously shown role of MmpL in lipid transport prompted us to investigate the potential lipid substrates of MmpL8MAB Systematic lipid analysis revealed that MmpL8MAB was required for the proper expression of a glycolipid entity, a glycosyl diacylated nonadecyl diol (GDND) alcohol comprising different combinations of oleic and stearic acids. This study shows the importance of MmpL8MAB in modifying interactions between the bacteria and phagocytic cells and in the production of a previously unknown glycolipid family.

Factors associated with bacteraemia due to multidrug-resistant organisms among bacteraemic patients with multidrug-resistant organism carriage: a case control study.

Background: Infections caused by multidrug-resistant organisms (MDRO) are emerging worldwide. Physicians are increasingly faced with the question of whether patients need empiric antibiotic treatment covering these pathogens. This question is especially essential among MDRO carriers. We aim to determine the occurrence of MDRO bacteraemia among bacteraemic patients colonized with MDRO, and the associated factors with MDRO bacteraemia among this population. Methods: We performed a retrospective monocentric study among MDRO carriers hospitalized with bacteraemia between January 2013 and August 2016 in a French hospital. We compared characteristics of patients with MDRO and non-MDRO bacteraemia. Results: Overall, 368 episodes of bacteraemia were reviewed; 98/368 (26.6%) occurred among MDRO carriers.Main colonizing bacteria were extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (40/98; 40.8%), ESBL-producing Klebsiella pneumoniae (35/98; 35.7%); methicillin-resistant Staphylococcus aureus (26/98; 26.5%) and multidrug-resistant Pseudomonas aeruginosa (PA) (12/98; 12.2%).There was no significant difference considering population with MDRO bacteraemia vs. non-MDRO bacteraemia, except for immunosuppression [OR 2.86; p = 0.0207], severity of the episode [OR 3.13; p = 0.0232], carriage of PA [OR 5.24; p = 0.0395], and hospital-acquired infection [OR 2.49; p = 0.034].In the multivariate analysis, factors significantly associated with MDRO bacteraemia among colonized patient were only immunosuppression [OR = 2.96; p = 0.0354] and the hospital-acquired origin of bacteraemia [OR = 2.62; p = 0.0427]. Conclusions: According to our study, occurrence of bacteraemia due to MDRO among MDRO carriers was high. Factors associated with MDRO bacteraemia were severity of the episode and hospital-acquired origin of the bacteraemia. Thus, during bacteraemia among patients colonized with MDRO, if such characteristics are present, broad-spectrum antimicrobial treatment is recommended.

Identification of genes required for Mycobacterium abscessus growth in vivo with a prominent role of the ESX-4 locus.

Mycobacterium abscessus, a rapidly growing mycobacterium (RGM) and an opportunistic human pathogen, is responsible for a wide spectrum of clinical manifestations ranging from pulmonary to skin and soft tissue infections. This intracellular organism can resist the bactericidal defense mechanisms of amoebae and macrophages, an ability that has not been observed in other RGM. M. abscessus can up-regulate several virulence factors during transient infection of amoebae, thereby becoming more virulent in subsequent respiratory infections in mice. Here, we sought to identify the M. abscessus genes required for replication within amoebae. To this end, we constructed and screened a transposon (Tn) insertion library of an M. abscessus subspecies massiliense clinical isolate for attenuated clones. This approach identified five genes within the ESX-4 locus, which in M. abscessus encodes an ESX-4 type VII secretion system that exceptionally also includes the ESX conserved EccE component. To confirm the screening results and to get further insight into the contribution of ESX-4 to M. abscessus growth and survival in amoebae and macrophages, we generated a deletion mutant of eccB4 that encodes a core structural element of ESX-4. This mutant was less efficient at blocking phagosomal acidification than its parental strain. Importantly, and in contrast to the wild-type strain, it also failed to damage phagosomes and showed reduced signs of phagosome-to-cytosol contact, as demonstrated by a combination of cellular and immunological assays. This study attributes an unexpected and genuine biological role to the underexplored mycobacterial ESX-4 system and its substrates.