Microbiology laboratories where MALDI-TOF MS is available can benefit from its capacity to identify most clinically interesting non-tuberculous mycobacteria in a rapid, reliable, and inexpensive manner.
dMycobacterium abscessus, as a species, has been increasingly implicated in respiratory infections, notably in cystic fibrosis patients. The species comprises 3 subspecies, which can be difficult to identify. Since they differ in antibiotic susceptibility and clinical relevance, developing a routine diagnostic tool discriminating Mycobacterium abscessus at the subspecies level is a real challenge. Forty-three Mycobacterium abscessus species isolates, previously identified by multilocus sequence typing, were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A subspecies identification algorithm, based on five discriminating peaks, was drawn up and validated by blind identification of a further 49 strains, 94% of which (n ؍ 46) were correctly identified. Two M. abscessus subsp. massiliense strains were misidentified as M. abscessus subsp. abscessus, and for 1 other strain identification failed. Inter-and intralaboratory reproducibility tests were conclusive. This study presents, for the first time, a classification algorithm for MALDI-TOF MS identification of the 3 M. abscessus subspecies. MALDI-TOF MS proved effective in discriminating within the M. abscessus species and might be easily integrated into the workflow of microbiology labs.
Background
Increasing resistance to antibiotics of Pseudomonas aeruginosa leads to therapeutic deadlock and alternative therapies are needed. We aimed to evaluate the effects of Lactobacillus clinical isolates in vivo, through intranasal administration on a murine model of Pseudomonas aeruginosa pneumonia.
Results
We screened in vitro 50 pulmonary clinical isolates of Lactobacillus for their ability to decrease the synthesis of two QS dependent-virulence factors (elastase and pyocyanin) produced by Pseudomonas aeruginosa strain PAO1.
Two blends of three Lactobacillus isolates were then tested in vivo: one with highly effective anti-PAO1 virulence factors properties (blend named L.rff for L. rhamnosus, two L. fermentum strains), and the second with no properties (blend named L.psb, for L. paracasei, L. salivarius and L. brevis). Each blend was administered intranasally to mice 18 h prior to PAO1 pulmonary infection. Animal survival, bacterial loads, cytological analysis, and cytokines secretion in the lungs were evaluated at 6 or 24 h post infection with PAO1.
Intranasal priming with both lactobacilli blends significantly improved 7-day mice survival from 12% for the control PAO1 group to 71 and 100% for the two groups receiving L.rff and L.psb respectively. No mortality was observed for both control groups receiving either L.rff or L.psb. Additionally, the PAO1 lung clearance was significantly enhanced at 24 h. A 2-log and 4-log reduction was observed in the L.rff + PAO1 and L.psb + PAO1 groups respectively, compared to the control PAO1 group. Significant reductions in neutrophil recruitment and proinflammatory cytokine and chemokine secretion were observed after lactobacilli administration compared to saline solution, whereas IL-10 production was increased.
Conclusions
These results demonstrate that intranasal priming with lactobacilli acts as a prophylaxis, and avoids fatal complications caused by Pseudomonas aeruginosa pneumonia in mice. These results were independent of in vitro anti-Pseudomonas aeruginosa activity on QS-dependent virulence factors. Further experiments are required to identify the immune mechanism before initiating clinical trials.
Eighty-five children were diagnosed with culture-confirmed nontuberculous mycobacterial cervical lymphadenitis within the MYCOMED surveillance network from 2004 to 2013. The mean incidence sharply increased from 0.57 to 3.7 per 100,000 children per year, after the discontinuation of mandatory bacillus Calmette and Guérin immunization in 2007. Cases were documented as Mycobacterium avium (62.3%), Mycobacterium intracellulare (15.3%) and Mycobacterium lentiflavum (12.9%). Outcome was favorable in all, with or without surgery or antimycobacterial treatment.
The spreading of antibiotic resistance is a major public health issue, which requires alternative treatments to antibiotics. Lactobacilli have shown abilities to prevent pneumonia in clinical studies when given by oral route, certainly through the gut-lung axis involvement. Rationally, respiratory administration of lactobacilli has been developed and studied in murine model, to prevent from respiratory pathogens. It allows a direct effect of probiotics into the respiratory system. To our knowledge, no study has ever focused on the effect of probiotic intra-respiratory administration to prevent from Pseudomonas aeruginosa (PA) pneumonia, a major respiratory pathogen associated with high morbidity rates. In this study, we evaluated the beneficial activity of three Lactobacillus strains (Lactobacillus fermentum K.C6.3.1E, Lactobacillus zeae Od.76, Lactobacillus paracasei ES.D.88) previously screened by ourselves and known to be particularly efficient in vitro in inhibiting PAO1 virulence factors. Cytotoxic assays in alveolar epithelial cell line A549 were performed, followed by the comparison of two lactobacilli prophylactic protocols (one or two administrations) by intra-tracheal administration in a C57BL/6 murine model of PA pneumonia. A549 cells viability was improved from 23 to 75% when lactobacilli were administered before PAO1 incubation, demonstrating a protective effect (P<0.001). A significant decrease of 2 log of PAO1 was observed 4 h after PAO1 instillation (3×106 cfu/mouse) in both groups receiving lactobacilli (9×106 cfu/mouse) compared to PAO1 group (P<0.05). One single prophylactic administration of lactobacilli significantly decreased the secretion by 50% in bronchoalveolar lavages of interleukin (IL)-6 and tumour necrosis factor-α compared to PAO1. No difference of secretion was observed for the IL-10 secretion, whatever the prophylactic study design. This is the first study highlighting that direct lung administration of Lactobacillus strains protect against PA pneumonia. Next step will be to decipher the mechanisms involved before developing this novel approach for human applications.
The first objective of this study was to determine the GenoType NTM-DR assay performance for subspecies identification in Mycobacterium abscessus complex isolates. The second objective was to evaluate the GenoType NTM-DR assay ability to detect clarithromycin and amikacin resistance in M. abscessus complex isolates compared with drug susceptibility testing (DST) and PCR sequencing of the erm(41), rrl and rrs genes. The concordance between the GenoType NTM-DR and MLST results concerning subspecies identification was 100%. The wild type and mutated alleles of the rrl and rrs genes were detected by the GenoType NTM-DR assay and PCR sequencing with 100% (115/115) agreement. Similarly, 100% concordance between GenoType NTM-DR and DST was observed for clarithromycin and amikacin testing. Sensitivity for the detection of clarithromycin and amikacin resistance was 100%. The GenoType NTM-DR assay provides a robust and complementary tool to the gold standard methods (MLST and broth microdilution) for subspecies identification and drug resistance detection.
IntroductionPseudomonas aeruginosa pulmonary infections are the primary cause of morbi-mortality in patients with cystic fibrosis (CF). In this cohort study, the objective was to identify candidate biomarkers of P. aeruginosa infection within the airway microbiota.MethodsA 3-year prospective multicentre study (PYOMUCO study) was conducted in Western France and included patients initially P. aeruginosa free for at least 1 year. A 16S-targeted metagenomics approach was applied on iterative sputum samples of a first set of patients (n=33). The composition of airway microbiota was compared according to their P. aeruginosa status at the end of the follow-up (colonised vs non-colonised), and biomarkers associated with P. aeruginosa were screened. In a second step, the distribution of a candidate biomarker according to the two groups of patients was verified by qPCR on a second set of patients (n=52) coming from the same cohort and its load quantified throughout the follow-up.ResultsPorphyromonas (mainly P. catoniae) was found to be an enriched phylotype in patients uninfected by P. aeruginosa (p<0.001). This result was confirmed by quantitative PCR. Conversely, in patients who became P. aeruginosa-positive, P. catoniae significantly decreased before P. aeruginosa acquisition (p=0.014).DiscussionFurther studies on replication cohorts are needed to validate this potential predictive biomarker, which may be relevant for the follow-up in the early years of patients with CF. The identification of infection candidate biomarkers may offer new strategies for CF precision medicine.
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