Lactobacilli intra-tracheal administration protects from Pseudomonas aeruginosa pulmonary infection in mice - a proof of concept.

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.

Evaluation of quantitative PCR for early diagnosis of Pseudomonas aeruginosa infection in cystic fibrosis: a prospective cohort study.

OBJECTIVES: Early detection of Pseudomonas aeruginosa lung positivity is a key element in cystic fibrosis (CF) management. PCR has increased the accuracy of detection of many microorganisms. Clinical relevance of P. aeruginosa quantitative PCR (qPCR) in this context is unclear. Our aim was to determine P. aeruginosa qPCR sensitivity and specificity, and to assess the possible time saved by qPCR in comparison with standard practice (culture). METHODS: A multicentre cohort study was conducted over a 3-year period in 96 patients with CF without chronic P. aeruginosa colonization. Sputum samples were collected at each visit. Conventional culture and two-step qPCR (oprL qPCR and gyrB/ecfX qPCR) were performed for 707 samples. The positivity criteria were based on the qPCR results, defined in a previous study as follow: oprL qPCR positivity alone if bacterial density was <730 CFU/mL or oprL qPCR combined with gyrB/ecfX qPCR if bacterial density was ≥730 CFU/mL. RESULTS: During follow up, 36 of the 96 patients with CF were diagnosed on culture as colonized with P. aeruginosa. This two-step qPCR displayed a sensitivity of 94.3% (95% CI 79.7%-98.6%), and a specificity of 86.3% (95% CI 83.4%-88.7%). It enabled P. aeruginosa acquisition to be diagnosed earlier in 20 patients, providing a median detection time gain of 8 months (interquartile range 3.7-17.6) for them. CONCLUSIONS: Implementing oprL and gyrB/ecfX qPCR in the management of patients with CF allowed earlier detection of first P. aeruginosa lung positivity than culture alone.

Probiotics: a new way to fight bacterial pulmonary infections?

Antibiotics, of which Fleming has identified the first representative, penicillin, in 1928, allowed dramatical improvement of the treatment of patients presenting with infectious diseases. However, once an antibiotic is used, resistance may develop more or less rapidly in some bacteria. It is thus necessary to develop therapeutic alternatives, such as the use of probiotics, defined by the World Health Organization (WHO) as "micro-organisms which, administered live and in adequate amounts, confer a benefit to the health of the host". The scope of these micro-organisms is broad, concerning many areas including that of infectious diseases, especially respiratory infections. We describe the rational use of probiotics in respiratory tract infections and detail the results of various clinical studies describing the use of probiotics in the management of respiratory infections such as nosocomial or community acquired pneumonia, or on specific grounds such as cystic fibrosis. The results are sometimes contradictory, but the therapeutic potential of probiotics seems promising. Implementing research to understand their mechanisms of action is critical to conduct therapeutic tests based on a specific rational for the strains to be used, the dose, as well as the chosen mode and rhythm of administration.

Severity of Escherichia coli bacteraemia is independent of the intrinsic virulence of the strains assessed in a mouse model.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains, a major cause of bacteraemia, typically belong to phylogenetic group B2 and express diverse accessory traits that contribute to virulence in mouse infection models. However, their high genomic diversity obscures the relationship between virulence factors and severity of infection in patients. In this study, we analysed concomitantly the strain's expression of virulence in a mouse model, genomic determinants and the clinical severity of infection in 60 bacteraemic patients. We show that bacterial virulence based on an animal model study and virulence factor determination is not correlated with pejorative outcome of E. coli human blood infections.

Intrafamilial transmission of extended-spectrum-beta-lactamase-producing Escherichia coli and Salmonella enterica Babelsberg among the families of internationally adopted children.

OBJECTIVES: International adoption from developing countries has become an increasing phenomenon in recent years. Given the high prevalence of multidrug-resistant (MDR) bacteria in these countries, the adopted children represent a group at risk for both carriage and infection with MDR bacteria. The dynamics of intrafamilial transmission of MDR bacteria after adoption was studied in a prospective study from January 2002 to January 2005. METHODS: Stool samples, taken at the first visit to the outpatient adoption practice and subsequently every month, from the adopted children of an orphanage of Bamako (Mali) and from all the members of their adoptive families were screened for MDR bacteria and bacterial pathogens. Bacteria were characterized by standard biochemical methods, disc diffusion antibiograms, PFGE and plasmid analysis. beta-Lactamase genes were sought by PCR. RESULTS: Over the study period, 52 ESBL-producing Enterobacteriaceae (E-ESBL), with Escherichia coli (56%) being the most prevalent, were isolated from 24/25 adoptees at arrival in France. During follow-up, the transmission of ESBL-producing E. coli and Salmonella enterica Babelsberg between the adoptees and their adoptive family members has clearly been demonstrated for 5/22 families (23%). The mean duration of the carriage for the adopted children was 9 months (1-15 months). CTX-M-15 was the most prevalent resistance gene among the E-ESBLs (93%), while SHV-12 was found among the S. enterica Babelsberg studied. CONCLUSIONS: International travellers, transfer of patients and now adoption may contribute to the global emergence of MDR bacteria. Thus, in addition to the usual screening of adopted children for infectious diseases, additional screening for MDR bacteria should be recommended, at least for children coming from countries with a high prevalence of MDR bacteria.

Is hepatitis C virus NS3 protease quasispecies heterogeneity predictive of progression from cirrhosis to hepatocellular carcinoma?

We investigated whether an HCV NS3 protease quasispecies heterogeneity was associated with progression from viral cirrhosis to hepatocellular carcinoma (HCC). The NS3 protease quasispecies structure of 10 HCV-1b cirrhotic patients (controls) was compared with that of 10 paired HCV-1b cirrhotic patients who displayed progression to HCC (cases). NS3 protease genetic complexity and diversity did not differ significantly between cases and controls. Amino acid substitutions were detected at 20 (11%) and 25 (14%) sites in at least two variants of the NS3 protease in cases and controls, respectively. Significant differences in the percentage of substituted clones were observed for 10 NS3 sites. Mutations Y56F, I71V, T72I, Q86P, P89S, S101G/D, R117H, S122G/T/N, V132I and V170I were more frequently observed in the NS3 protease sequences of controls than in those of cases. Residue V107 was substituted in NS3 cases but not in controls. However, these differences did not allow the definition of a specific NS3 profile related to HCC occurrence. The NS3 secondary structure B1-1 previously identified as potentially predictive of HCC was identified with a higher frequency in cases quasispecies (84.2%) than in controls (55.9%; P < 0.05). Our results suggest that there may be a relationship to fibrosis progression when diversity parameters are considered together with secondary structure profiles. Further investigations are required to determine the cellular interactions of HCV NS3 protease in the context of carcinogenesis.

Mutator natural Escherichia coli isolates have an unusual virulence phenotype.

A small percentage of natural Escherichia coli isolates (both commensal and pathogenic) have a mutator phenotype related to defects in methyl-directed mismatch repair (MR) genes. We investigated whether there was a direct link between the mutator phenotype and virulence by (i) studying the relationships between mutation rate and virulence in a mouse model of extraintestinal virulence for 88 commensal and extraintestinal pathogenic E. coli isolates and (ii) comparing the virulence in mice of MR-deficient and MR-proficient strains that were otherwise isogenic. The results provide no support for the hypothesis that the mutator phenotype has a direct role in virulence or is associated with increased virulence. Most of the natural mutator strains studied displayed an unusual virulence phenotype with (i) a lack of correspondence between the number of virulence determinants and pathogenicity in mice and (ii) an intermediate level of virulence. On a large evolutionary scale, the mutator phenotype may help parasites to achieve an intermediate rate of virulence which mathematical models predict to be selected for during long-term parasite-host interactions.

The link between phylogeny and virulence in Escherichia coli extraintestinal infection.

Previous studies suggesting a link between Escherichia coli phylogenetic groups and extraintestinal virulence have been hampered by the difficulty in establishing the intrinsic virulence of a bacterial strain. Indeed, unidentified virulence factors do exist, and the susceptibility of the host to infection is highly variable. To overcome these difficulties, we have developed a mouse model of extraintestinal virulence to test the virulence of the strains under normalized conditions. We then assessed the phylogenetic relationships compared to the E. coli reference (ECOR) collection, the presence of several known virulence determinants, and the lethality to mice of 82 human adult E. coli strains isolated from normal feces and during the course of extraintestinal infections. Commensal strains belong mainly to phylogenetic groups A and B1, are devoid of virulence determinants, and do not kill the mice. Strains exhibiting the same characteristics as the commensal strains can be isolated under pathogenic conditions, thus indicating the role of host-dependent factors, such as susceptibility linked to underlying disease, in the development of infection. Some strains of phylogenetic groups A, B1, and D are able to kill the mice, their virulence being most often correlated with the presence of virulence determinants. Lastly, strains of the B2 phylogenetic group represent a divergent lineage of highly virulent strains which kill the mice at high frequency and possess the highest level of virulence determinants. The observed link between virulence and phylogeny could correspond to the necessity of virulence determinants in a genetic background that is adequate for the emergence of a virulent clone, an expression of the interdependency of pathogenicity and metabolic activities in pathogenic bacteria.