Clinical Features of Mycobacterium canettii Infection: A Retrospective Study of 20 Cases Among French Soldiers and Relatives.

BACKGROUND: Mycobacterium canettii forms part of the Mycobacterium tuberculosis complex. Mycobacterium canettii infections are mainly described in the Horn of Africa. The permanent presence of French soldiers in Djibouti raises the question of the risk of being infected with M. canettii. Here, we describe M. canettii infections among French military and their families between 1998 and 2015. METHODS: This retrospective study relied on 3 sources of data: the reference center for mycobacteria in the Biology Department at Percy Military Hospital in Paris, the French Military Center for Epidemiology and Public Health, and the scientific literature. After an exhaustive census of the strains, we studied the epidemiological data on 20 cases among French soldiers and their families. RESULTS: Twenty cases of M. canettii infections are reported, including 5 unpublished cases. Adenitis predominates (n = 15), especially in the cervico facial area and among children; 1 case was observed 1 month after dental care in Djibouti. The pulmonary forms were less frequent (n = 6), and 3 atypical forms are described. All patients had stayed in Djibouti. CONCLUSIONS: Cases of M. canettii infection among the French military consisted mainly of adenitis; disseminated forms were possible with immunodeficiency. Their evolution under specific treatments was comparable to that of tuberculosis. The presumed origin of the infection seemed to be environmental, possibly a water reservoir, and not due to human-to-human contagion.

Clinical Relevance of Type II Fatty Acid Synthesis Bypass in Staphylococcus aureus.

The need for new antimicrobials to treat bacterial infections has led to the use of type II fatty acid synthesis (FASII) enzymes as front-line targets. However, recent studies suggest that FASII inhibitors may not work against the opportunist pathogen Staphylococcus aureus, as environmental fatty acids favor emergence of multi-anti-FASII resistance. As fatty acids are abundant in the host and one FASII inhibitor, triclosan, is widespread, we investigated whether fatty acid pools impact resistance in clinical and veterinary S. aureus isolates. Simple addition of fatty acids to the screening medium led to a 50% increase in triclosan resistance, as tested in 700 isolates. Moreover, nonculturable triclosan-resistant fatty acid auxotrophs, which escape detection under routine conditions, were uncovered in primary patient samples. FASII bypass in selected isolates correlated with polymorphisms in the acc and fabD loci. We conclude that fatty-acid-dependent strategies to escape FASII inhibition are common among S. aureus isolates and correlate with anti-FASII resistance and emergence of nonculturable variants.

Detection of Circulating Mucorales DNA in Critically Ill Burn Patients: Preliminary Report of a Screening Strategy for Early Diagnosis and Treatment.

BACKGROUND: Invasive wound mucormycosis (IWM) is associated with an extremely poor outcome among critically ill burn patients. We describe the detection of circulating Mucorales DNA (cmDNA) for the early diagnosis of IWM in those patients and report the potential value of detecting cmDNA for treatment guidance. METHODS: Severely ill burn patients admitted to our tertiary referral center between October 2013 and February 2016 were included. Retrospective plasma samples were tested for the presence of cmDNA by quantitative real-time polymerase chain reaction (qPCR). Patients were then prospectively screened twice a week, and liposomal amphotericin-B therapy initiated based on a positive qPCR. The primary endpoint was the time between cmDNA detection and standard diagnosis. Secondary endpoints were the time from cmDNA detection and treatment initiation and mortality. RESULTS: Seventy-seven patients (418 samples) were included. The average age was 46 (28-60) years, abbreviated burn severity index was 8 (7-10), and simplified acute physiology score was 33 (23-46). The total body surface area was 33% (22%-52%). cmDNA was detected 11 (4.5-15) days before standard diagnosis. The in-hospital mortality was 62% for patients with IWM and 24% for those without (P = .03). The mortality due to IWM was 80% during period A and 33% during period B (P = .46). CONCLUSIONS: This study suggests that the detection of cmDNA allows earlier diagnosis of IWM in severely ill burn patients and earlier initiation of treatment. Further studies are needed to confirm the impact of earlier treatment initiation on patient outcome.

Progenitor "Mycobacterium canettii" clone responsible for lymph node tuberculosis epidemic, Djibouti.

"Mycobacterium canettii," an opportunistic human pathogen living in an unknown environmental reservoir, is the progenitor species from which Mycobacterium tuberculosis emerged. Since its discovery in 1969, most of the ≈70 known M. canettii strains were isolated in the Republic of Djibouti, frequently from expatriate children and adults. We show here, by whole-genome sequencing, that most strains collected from February 2010 through March 2013, and associated with 2 outbreaks of lymph node tuberculosis in children, belong to a unique epidemic clone within M. canettii. Evolution of this clone, which has been recovered regularly since 1983, may mimic the birth of M. tuberculosis. Thus, recognizing this organism and identifying its reservoir are clinically important.

Rapid identification of international multidrug-resistant Pseudomonas aeruginosa clones by multiple-locus variable number of tandem repeats analysis and investigation of their susceptibility to lytic bacteriophages.

The objective of this study was to determine the genetic diversity of multidrug-resistant (MDR) Pseudomonas aeruginosa strains isolated over a period of 12 months in two French hospitals and to test their susceptibility to bacteriophages. A total of 47 MDR isolates recovered from hospitalized patients were genotyped using multiple-locus variable number of tandem repeats analysis. The genotypes were distributed into five clones (including 19, 5, 5, 3, and 3 isolates, respectively) and 12 singletons. Comparison to 77 MDR strains from three other countries, and MLST analysis of selected isolates showed the predominance of international MDR clones. The larger clone, CC235, contained 59 isolates displaying different antibiotic resistance mechanisms, including the presence of the GES1, VIM-2, VIM-4, and IMP-1 ß-lactamases. Three newly isolated P. aeruginosa bacteriophages were found to lyse 42 of the 44 analyzed strains, distributed into the different clonal complexes. This pilot study suggests that systematic genotyping of P. aeruginosa MDR strains could improve our epidemiological understanding of transmission at both the local (hospital) and the national level and that phage therapy could be an alternative or a complementary treatment to antibiotics for treating MDR-infected patients.

Acinetobacter parvus bacteraemia community-acquired.

Clinicians are generally familiar with Acinetobacter baumannii as an aetiological agent of serious nosocomial infections in intensive care units. Other Acinetobacter species can also be responsible for life-threatening sepsis. Here, we report about a bacteraemia caused by Acinetobacter parvus, community-acquired, identified with a 16S rRNA gene sequence analysis and the MALDI-TOF mass spectrometry system.

[Update on Mycobacterium bovis infections in France: 4 cases reports]. / Actualités de l'infection à Mycobacterium bovis en France : à propos de 4 cas.

Mycobacterium bovis (M. bovis) is a cause of zoonosis. It is rare in developed countries since cattle control. We report four cases of M. bovis infection in people aged more 60 years. They were probably infected during infancy, consuming unpasteurized milk. It is the main transmission mode in developing countries where veterinary controls aren't made. M. bovis infections clinical aspects are varied and treatment is complicated by natural pyrazinamide resistance. Recent diagnostic methods using molecular biology are quick and specific and facilitate identification.

Isolation of Nocardia beijingensis from a pulmonary abscess reveals human immunodeficiency virus infection.

A strain of Nocardia was isolated from a pulmonary abscess of a human immunodeficiency virus-infected patient in France. Comparative 16S rRNA gene sequence analysis revealed that the isolate represented a strain of Nocardia beijingensis. Antimicrobial susceptibility testing was essential to guide the clinicians to successfully treat this infection.

Evaluation of prognostic scores for respiratory syncytial virus infection in a French multicentre cohort of allogeneic haematopoietic stem cell transplantation recipients.

Haematopoietic stem cell transplantation (HSCT) recipients are at risk for severe respiratory syncytial virus (RSV) infection. Two prognostic scores have been proposed to predict the risk of progression from upper respiratory tract infection (URTI) to lower respiratory tract infection (LRTI) and death. This was a multicentre study of allogeneic HSCT recipients diagnosed with an RSV infection between 2010 and 2019 who were retrospectively stratified by the immunodeficiency scoring index (ISI) and the severe immunodeficiency (SID) score. Endpoints were overall survival, RSV-attributable mortality and progression to LRTI after URTI. Prognostic analyses were performed using Cox regression models. We included 147 consecutive patients, including 94 (63.9%) initially diagnosed with URTI and 53 (36.1%) with LRTI. At 90 days, 14 patients had died (survival rate, 90.5%; 95% CI: 85.9-95.3), and nine deaths were attributable to RSV (attributable mortality rate, 5.4%; 95% CI: 2.5-10.0). The cumulative 90-day incidence of LRTI after URTI was 13.8% (95% CI: 7.8-21.6). Neither score showed prognostic value for mortality, while the ISI allowed the prediction of progression to LRTI (p = 0.0008). Our results do not fully replicate the results previously reported in cohorts of HSCT recipients. This may reflect the recent epidemiology of RSV infections in this HSCT cohort.

[Tuberculous meningitis: diagnosis and therapeutic difficulties]. / Méningites tuberculeuses: difficultés diagnostiques et thérapeutiques.

Tuberculosis remains a public-health problem in 2010 with 9 millions cases and 1,7 million deaths worldwide each year. Tuberculosis meningitis is rare (0.5 to 1%) but is associated with high mortality and disability among survivors. An early starting of treatment is crucial. Despite molecular biology methods, microbiological diagnosis remains a challenge for the biologist. We report here 2 cases of tuberculous meningitis with different clinical and biological presentations, which underline diagnosis and therapeutic difficulties encountered in the management of this disease. The first one occurred in an HIV infected patient and the second one was caused by a multidrug-resistant strain. Clinical issues were severe with important neurological residual disability and death. Biological methods available for tuberculous meningitis diagnosis are exposed.

Comparison of two commercial assays for the characterization of rpoB mutations in Mycobacterium tuberculosis and description of new mutations conferring weak resistance to rifampicin.

OBJECTIVES: The aim of this study was to compare the efficiency of two commercial assays, INNO-LiPA Rif.TB and MTBDRplus, for the identification of mutations in the rpoB hot-spot region and to assess the efficiency of these mutations in conferring resistance to rifampicin. METHODS: A collection of 126 rifampicin-resistant Mycobacterium tuberculosis and Mycobacterium africanum isolates and 18 rifampicin-susceptible isolates from different countries were analysed using the two hybridization assays. RESULTS: For 60 strains the hot-spot region of the rpoB gene was sequenced, confirming the results of the hybridization assays and allowing the identification of new mutations. In total, 17 mutations involving 10 codons were observed, two of which are newly described (D516Y and E562G/P564L). Mutations L533P, H526L, D516Y and L511P and the double mutation E562G/P564L conferred a low level of resistance. CONCLUSIONS: The assays INNO-LiPA Rif.TB and MTBDRplus identified rpoB mutations in 98.4% of cases. MTBDRplus provided additional information due to the overlapping hybridization probes and in addition allowed the investigation of katG mutations for isoniazid resistance.

Efficacy and tolerability of a cocktail of bacteriophages to treat burn wounds infected by Pseudomonas aeruginosa (PhagoBurn): a randomised, controlled, double-blind phase 1/2 trial.

BACKGROUND: Wound infections are the main cause of sepsis in patients with burns and increase burn-related morbidity and mortality. Bacteriophages, natural bacterial viruses, are being considered as an alternative therapy to treat infections caused by multidrug-resistant bacteria. We aimed to compare the efficacy and tolerability of a cocktail of lytic anti-Pseudomonas aeruginosa bacteriophages with standard of care for patients with burns. METHODS: In this randomised phase 1/2 trial, patients with a confirmed burn wound infection were recruited from nine burn centres in hospitals in France and Belgium. Patients were eligible if they were aged 18 years or older and had a burn wound clinically infected with P aeruginosa. Eligible participants were randomly assigned (1:1) by use of an interactive web response system to a cocktail of 12 natural lytic anti-P aeruginosa bacteriophages (PP1131; 1 × 106 plaque-forming units [PFU] per mL) or standard of care (1% sulfadiazine silver emulsion cream), both given as a daily topical treatment for 7 days, with 14 days of follow-up. Masking of treatment from clinicians was not possible because of the appearance of the two treatments (standard of care a thick cream, PP1131 a clear liquid applied via a dressing), but assignments were masked from microbiologists who analysed the samples and patients (treatment applied while patients were under general anaesthetic). The primary endpoint was median time to sustained reduction in bacterial burden by at least two quadrants via a four-quadrant method, assessed by use of daily swabs in all participants with a microbiologically documented infection at day 0 who were given at least one sulfadiazine silver or phage dressing (modified intention-to-treat population). Safety was assessed in all participants who received at least one dressing according to protocol. Ancillary studies were done in the per-protocol population (all PP1131 participants who completed 7 days of treatment) to assess the reasons for success or failure of phage therapy. This trial is registered with the European Clinical Trials database, number 2014-000714-65, and ClinicalTrials.gov, number NCT02116010, and is now closed. FINDINGS: Between July 22, 2015, and Jan 2, 2017, across two recruitment periods spanning 13 months, 27 patients were recruited and randomly assigned to receive phage therapy (n=13) or standard of care (n=14). One patient in the standard of care group was not exposed to treatment, giving a safety population of 26 patients (PP1131 n=13, standard of care n=13), and one patient in the PP1131 group did not have an infection at day 0, giving an efficacy population of 25 patients (PP1131 n=12, standard of care n=13). The trial was stopped on Jan 2, 2017, because of the insufficient efficacy of PP1131. The primary endpoint was reached in a median of 144 h (95% CI 48-not reached) in the PP1131 group versus a median of 47 h (23-122) in the standard of care group (hazard ratio 0·29, 95% CI 0·10-0·79; p=0·018). In the PP1131 group, six (50%) of 12 analysable participants had a maximal bacterial burden versus two (15%) of 13 in the standard of care group. PP1131 titre decreased after manufacturing and participants were given a lower concentration of phages than expected (1 × 102 PFU/mL per daily dose). In the PP1131 group, three (23%) of 13 analysable participants had adverse events versus seven (54%) of 13 in the standard of care group. One participant in each group died after follow-up and the deaths were determined to not be related to treatment. The ancillary study showed that the bacteria isolated from patients with failed PP1131 treatment were resistant to low phage doses. INTERPRETATION: At very low concentrations, PP1131 decreased bacterial burden in burn wounds at a slower pace than standard of care. Further studies using increased phage concentrations and phagograms in a larger sample of participants are warranted. FUNDING: European Commission: Framework Programme 7.

Outbreak of Invasive Wound Mucormycosis in a Burn Unit Due to Multiple Strains of Mucor circinelloides f. circinelloides Resolved by Whole-Genome Sequencing.

Mucorales are ubiquitous environmental molds responsible for mucormycosis in diabetic, immunocompromised, and severely burned patients. Small outbreaks of invasive wound mucormycosis (IWM) have already been reported in burn units without extensive microbiological investigations. We faced an outbreak of IWM in our center and investigated the clinical isolates with whole-genome sequencing (WGS) analysis. We analyzed M. circinelloides isolates from patients in our burn unit (BU1, Hôpital Saint-Louis, Paris, France) together with nonoutbreak isolates from Burn Unit 2 (BU2, Paris area) and from France over a 2-year period (2013 to 2015). A total of 21 isolates, including 14 isolates from six BU1 patients, were analyzed by whole-genome sequencing (WGS). Phylogenetic classification based on de novo assembly and assembly free approaches showed that the clinical isolates clustered in four highly divergent clades. Clade 1 contained at least one of the strains from the six epidemiologically linked BU1 patients. The clinical isolates were specific to each patient. Two patients were infected with more than two strains from different clades, suggesting that an environmental reservoir of clonally unrelated isolates was the source of contamination. Only two patients from BU1 shared one strain, which could correspond to direct transmission or contamination with the same environmental source. In conclusion, WGS of several isolates per patients coupled with precise epidemiological data revealed a complex situation combining potential cross-transmission between patients and multiple contaminations with a heterogeneous pool of strains from a cryptic environmental reservoir.IMPORTANCE Invasive wound mucormycosis (IWM) is a severe infection due to environmental molds belonging to the order Mucorales. Severely burned patients are particularly at risk for IWM. Here, we used whole-genome sequencing (WGS) analysis to resolve an outbreak of IWM due to Mucor circinelloides that occurred in our hospital (BU1). We sequenced 21 clinical isolates, including 14 from BU1 and 7 unrelated isolates, and compared them to the reference genome (1006PhL). This analysis revealed that the outbreak was mainly due to multiple strains that seemed patient specific, suggesting that the patients were more likely infected from a pool of diverse strains from the environment rather than from direct transmission among them. This study revealed the complexity of a Mucorales outbreak in the settings of IWM in burn patients, which has been highlighted based on WGS combined with careful sampling.

A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes.

Propionibacterium acnes plays a central role in the pathogenesis of acne and is responsible for severe opportunistic infections. Numerous typing schemes have been developed that allow the identification of phylotypes, but they are often insufficient to differentiate subtypes. To better understand the genetic diversity of this species and to perform epidemiological analyses, high throughput discriminant genotyping techniques are needed. Here we describe the development of a multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) method. Thirteen VNTRs were identified in the genome of P. acnes and were used to genotype a collection of clinical isolates. In addition, publically available sequencing data for 102 genomes were analyzed in silico, providing an MLVA genotype. The clustering of MLVA data was in perfect congruence with whole genome based clustering. Analysis of the clustered regularly interspaced short palindromic repeat (CRISPR) element uncovered new spacers, a supplementary source of genotypic information. The present MLVA13 scheme and associated internet database represents a first line genotyping assay to investigate large number of isolates. Particular strains may then be submitted to full genome sequencing in order to better analyze their pathogenic potential.

High prevalence of multidrug resistant tuberculosis in Djibouti: a retrospective study.

INTRODUCTION: The Republic of Djibouti is an African country that exhibits one of the highest incidence rate of tuberculosis in the world. The aim of this study was to evaluate the prevalence of multidrug-resistant tuberculosis among new cases. METHODOLOGY: We studied retrospectively every tuberculosis case diagnosed over a 12-month period in patients hospitalized at the French Military Hospital of Bouffard. During this period, 1,274 samples from 675 patients were tested. RESULTS: We isolated 266 mycobacteria corresponding to 180 cases of tuberculosis. Thirty-three were fully susceptible and 57% met the tuberculosis criteria, with 46% primary resistance. No extensively-drug-resistant tuberculosis was found. CONCLUSION: Our results highlight a major concern about the situation in this part of the world.

Significance of the identification in the Horn of Africa of an exceptionally deep branching Mycobacterium tuberculosis clade.

Molecular and phylogeographic studies have led to the definition within the Mycobacterium tuberculosis complex (MTBC) of a number of geotypes and ecotypes showing a preferential geographic location or host preference. The MTBC is thought to have emerged in Africa, most likely the Horn of Africa, and to have spread worldwide with human migrations. Under this assumption, there is a possibility that unknown deep branching lineages are present in this region. We genotyped by spoligotyping and multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) 435 MTBC isolates recovered from patients. Four hundred and eleven isolates were collected in the Republic of Djibouti over a 12 year period, with the other 24 isolates originating from neighbouring countries. All major M. tuberculosis lineages were identified, with only two M. africanum and one M. bovis isolates. Upon comparison with typing data of worldwide origin we observed that several isolates showed clustering characteristics compatible with new deep branching. Whole genome sequencing (WGS) of seven isolates and comparison with available WGS data from 38 genomes distributed in the different lineages confirms the identification of ancestral nodes for several clades and most importantly of one new lineage, here referred to as lineage 7. Investigation of specific deletions confirms the novelty of this lineage, and analysis of its precise phylogenetic position indicates that the other three superlineages constituting the MTBC emerged independently but within a relatively short timeframe from the Horn of Africa. The availability of such strains compared to the predominant lineages and sharing very ancient ancestry will open new avenues for identifying some of the genetic factors responsible for the success of the modern lineages. Additional deep branching lineages may be readily and efficiently identified by large-scale MLVA screening of isolates from sub-Saharan African countries followed by WGS analysis of a few selected isolates.

Diversity of Acinetobacter baumannii in four French military hospitals, as assessed by multiple locus variable number of tandem repeats analysis.

BACKGROUND: Infections by A. calcoaceticus-A. baumannii (ACB) complex isolates represent a serious threat for wounded and burn patients. Three international multidrug-resistant (MDR) clones (EU clone I-III) are responsible for a large proportion of nosocomial infections with A. baumannii but other emerging strains with high epidemic potential also occur. METHODOLOGY/PRINCIPAL FINDINGS: We automatized a Multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) protocol and used it to investigate the genetic diversity of 136 ACB isolates from four military hospitals and one childrens hospital. Acinetobacter sp other than baumannii isolates represented 22.6% (31/137) with a majority being A. pittii. The genotyping protocol designed for A.baumannii was also efficient to cluster A. pittii isolates. Fifty-five percent of A. baumannii isolates belonged to the two international clones I and II, and we identified new clones which members were found in the different hospitals. Analysis of two CRISPR-cas systems helped define two clonal complexes and provided phylogenetic information to help trace back their emergence. CONCLUSIONS/SIGNIFICANCE: The increasing occurrence of A. baumannii infections in the hospital calls for measures to rapidly characterize the isolates and identify emerging clones. The automatized MLVA protocol can be the instrument for such surveys. In addition, the investigation of CRISPR/cas systems may give important keys to understand the evolution of some highly successful clonal complexes.