Anne-Laure Michon

Intragenomic and intraspecific heterogeneity in rrs may surpass interspecific variability in a natural population of Veillonella.

As well as intraspecific heterogeneity, intragenomic heterogeneity between 16S rRNA gene copies has been described for a range of bacteria. Due to the wide use of 16S rRNA gene sequence analysis for taxonomy, identification and metagenomics, evaluating the extent of these heterogeneities in natural populations is an essential prerequisite. We investigated inter- and intragenomic 16S rRNA gene heterogeneity of the variable region V3 in a population of 149 clinical isolates of Veillonella spp. of human origin and in 13 type or reference Veillonella strains using PCR-temporal temperature gel electrophoresis (TTGE). 16S rRNA gene diversity was high in the studied population, as 45 different banding patterns were observed. Intragenomic heterogeneity was demonstrated for 110 (74 %) isolates and 8 (61.5 %) type or reference strains displaying two or three different gene copies. Polymorphic nucleotide positions accounted for 0.5-2.5 % of the sequence and were scattered in helices H16 and H17 of the rRNA molecule. Some of them changed the secondary structure of H17. Phylotaxonomic structure of the population based on the single-copy housekeeping gene rpoB was compared with TTGE patterns. The intragenomic V3 heterogeneity, as well as recombination events between strains or isolates of different rpoB clades, impaired the 16S rRNA-based identification for some Veillonella species. Such approaches should be conducted in other bacterial populations to optimize the interpretation of 16S rRNA gene sequences in taxonomy and/or diversity studies.

Intrapatient diversity of Achromobacter spp. involved in chronic colonization of Cystic Fibrosis airways.

Achromobacter spp. are increasingly identified in Cystic Fibrosis (CF) patients and their ability to persistently colonize the CF respiratory tract (CFRT) suggests that Achromobacter species possess adaptive characteristics. We studied genome dynamics in 118 isolates recovered from 13 patients with Achromobacter chronic colonization (5-26 isolates per patient recovered over 13-61 months). Isolates were identified to species level by nrdA gene sequencing, subjected to Pulsed-Field Gel Electrophoresis (PFGE) and multiplex rep-PCR (MR-PCR), and rrs intragenomic diversity was studied by PCR-Temporal Temperature Gel Electrophoresis (TTGE). Intrapatient diversity was assessed: (i) from dynamics of XbaI and/or SpeI-based pulsotypes, (ii) from comparison of MR-PCR profiles, and (iii) by longitudinal analysis of rrs intragenomic diversity. Patients were chronically colonized by Achromobacter xylosoxidans (n=10), Achromobacter dolens (n=1) or Achromobacter insuavis (n=2). All strains displayed genomic diversification over time but A. insuavis showed higher pulsotype diversity compared to other species. Intragenomic rrs heterogeneity was found in strains from 6 of 13 patients and may be persistently observed. Achromobacter genome evolution observed during chronic colonization of the CFRT warrants further investigation of the adaptation features of the different species, as well as of the selective forces driving this adaptation in the CFRT.

Current insights in invasive group A streptococcal infections in pediatrics

A rising incidence of invasive group A Streptococcus infections (IGASI) has been noted in children in the past three decades. The relative frequency of the infection types showed marked differences to IGASI in adults, and severity of the disease resulted in a mortality rate usually comprising between 3.6% and 8.3%. The emm1-type group A Streptococcus (GAS) subclone displaying a particular pattern of virulence factors was widely disseminated and prevalent in children with IGASI while the emm3-type GAS subclone appeared as a recent emerging genotype. However, the implication of these hypervirulent clones in the increase of IGASI in children is still controversial. Recent advances in our knowledge on pathogenesis of IGASI underlined that deregulation of virulence factor production, individual susceptibility, as well as exuberant cytokine response are important factors that may account for the severity of the disease in children. Future changes in IGASI epidemiology are awaited from current prospects for a safe and effective vaccine against GAS. IGASI are complex infections associating septic, toxic, and immunological disorders. Treatment has to be effective on both the etiologic agent and its toxins, due to the severity of the disease associated to the spread of highly virulent bacterial clones. More generally, emergence of virulent clones responsible for septic and toxic disease is a matter of concern in pediatric infectiology in the absence of vaccination strategy.

Advances toward the Elucidation of Hypertonic Saline Effects on Pseudomonas aeruginosa from Cystic Fibrosis Patients

Objectives Nebulized hypertonic saline (HTS) has beneficial effects including reducing pulmonary exacerbations in Cystic Fibrosis (CF) patients. Several mechanisms may explain these effects but antimicrobial activity of NaCl remains largely unexplored. We aimed to measure the antimicrobial effect of NaCl on Pseudomonas aeruginosa isolated from the respiratory tract in CF patients. Methods NaCl minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined for strains characterized for mucoidy, antimicrobial resistance, and ability to form biofilm using 0,9% to 15% NaCl solutions. NaCl effects on biofilm formation, preformed biofilm, and mobility were evaluated. Kinetics of antimicrobial effects was studied. Results The growth of all isolates (n = 85) from 34 patients was inhibited by 6% NaCl solution. A 10% concentration had a bactericidal activity on 90% of the isolates. Mucoid and multidrug resistant (MDR) isolates displayed lower MICs compared to non-mucoid and to non-MDR isolates, respectively. Time-kill kinetics showed that NaCl exhibited a rapid, dose and growth phase dependent bactericidal effect. Three percent or more of NaCl inhibited biofilm formation for 69% of strongly adherent isolates. A dose-dependent decrease of preformed biofilm viability and an inhibitory activity on bacterial motility were observed. Conclusions NaCl inhibited the growth of all isolates and killed 38% of tested isolates within concentration range currently used in therapeutics. Our results suggest that anti-pseudomonal activity is another mechanism of action of HTS to add to those already established. Clinical trials are needed to compare diverse HTS conditions of use (rhythm, dose and mode of delivery) to obtain efficient and optimized anti-P. aeruginosa effects. More generally, NaCl effect on other opportunistic pathogens as well as on global microbiotae recovered during polymicrobial diseases warrants further investigations.

Intragenomic and intraspecific heterogeneity of the 16S rRNA gene in seven bacterial species from the respiratory tract of cystic fibrosis patients assessed by PCR-Temporal Temperature Gel Electrophoresis

16S rRNA gene-based cultivation-independent methods are increasingly used to study the diversity of microbiota during health and disease. One bias of these methods is the variability of 16S rRNA gene that may exist among strains of a same species (intraspecific heterogeneity) or between rrs copies in a genome (intragenomic heterogeneity). We evaluated the level of intraspecific and intragenomic 16S rDNA variability in seven species frequently encountered in respiratory tract samples in cystic fibrosis (CF). A total of 179 strains were subjected to V3 region 16S rDNA PCR-TTGE. Using this easy-to-perform and rapid method, different levels of V3 region rrs heterogeneity were demonstrated. No intraspecific and intragenomic rrs heterogeneity was demonstrated for Moraxella catarrhalis (n=16), Pseudomonas aeruginosa (n=31) and Streptococcus pneumoniae (n=14) showing a single PCR-TTGE band characteristic of the species. Low level of intraspecific heterogeneity was observed for Staphylococcus aureus (n=30), Stenotrophomonas maltophilia (n=29) and Achromobacter xylosoxidans (n=28), and 17%, 38% and 96% of these strains showed intragenomic heterogeneity (two to four different rrs copies), respectively. Haemophilus influenzae (n=31) displayed the higher level of intraspecific variability with 23 different PCR-TTGE patterns and 61% of the strains showed intragenomic rrs heterogeneity (two to four different rrs copies). Although only one hypervariable region of the 16S rRNA gene was explored, intraspecific and intragenomic rrs heterogeneity was frequently observed in this study and should be taken into consideration for a better interpretation of 16S rRNA gene-based diversity profiles in denaturing gels and to avoid any overestimation of the respiratory microbiota diversity in CF.

Impact of High Diversity of Achromobacter Populations within Cystic Fibrosis Sputum Samples on Antimicrobial Susceptibility Testing

ABSTRACT Chronic colonization by opportunistic environmental bacteria is frequent in the airways of cystic fibrosis (CF) patients. Studies of Pseudomonas aeruginosa evolution during persistence have highlighted the emergence of pathoadaptive genotypes and phenotypes, leading to complex and diversified inpatient colonizing populations also observed at the intraspecimen level. Such diversity, including heterogeneity in resistance profiles, has been considered an adaptive strategy devoted to host persistence. Longitudinal genomic diversity has been shown for the emergent opportunistic pathogen Achromobacter, but phenotypic and genomic diversity has not yet been studied within a simple CF sputum sample. Here, we studied the genomic diversity and antimicrobial resistance heterogeneity of 132 Achromobacter species strains (8 to 27 strains of identical or distinct colonial morphotypes per specimen) recovered from the sputum samples of 9 chronically colonized CF patients. We highlighted the high within-sample and within-morphotype diversity of antimicrobial resistance (disk diffusion) and genomic (pulsed-field gel electrophoresis) profiles. No sputum sample included strains with identical pulsotypes or antibiotic susceptibility patterns. Differences in clinical categorization were observed for the 9 patients and concerned 3 to 11 antibiotics, including antibiotics recommended for use against Achromobacter. Within-sample antimicrobial resistance heterogeneity, not predictable from colonial morphology, suggested that it may represent a selective advantage against antibiotics in an Achromobacter persisting population and potentially compromise the antibiotic management of CF airway infections.

What to expect from molecular tools for non-documented pediatric infectious diseases

Objective: Evaluation of the contribution of molecular tools to the overall diagnosis of infectious diseases in children. Methods: Results of 16S rDNA analysis (179 children; 228 specimens), combined to specific amplification of Kingella kingae (126 children; 166 osteoarticular specimens), were retrospectively analyzed for samples with inconclusive cultures. Result: The overall positive yield in diagnosis was 12.8% of the patients for 16S rDNA PCR, 40.5% for K. kingae PCR and 45.2% for combined use of both methods. Results were related to clinical and biological data (direct examination, certainty/uncertainty of clinical diagnosis, fever, biological markers, previous antibiotics), and to the number of samples analyzed per patient, allowing the identification of specific situations with significant contribution of PCR methods. Conclusion: Molecular techniques constitute valuable tools to improve the bacterial infection diagnosis in children; however, specific indications, dedicated samples, and number of analyzed samples per patient are key points to optimize their contribution.

Bacteremia due to imipenem-resistant Roseomonas mucosa in a child with acute lymphoblastic leukemia.

Roseomonas are described as opportunistic pathogens rarely involved in human infections. Their identification requires molecular methods and their antimicrobial susceptibility pattern varies according to the species. We report the first case of bacteremia due to Roseomonas mucosa in a child with leukemia and reviewed pediatric cases of Roseomonas infection, for which undoubted strain identification was available. Favorable outcome was observed despite resistance to numerous β-lactams that may account for delayed effective treatment, suggesting the low virulence of Roseomonas in children. Here, the strain also displayed unusual resistance to imipenem, highlighting the possible acquisition of additional resistance by this pathogen.

Tigécycline : CMI 50/90 vis-à-vis de 1766 bacilles à Gram-négatif (entérobactéries résistantes aux céphalosporines de troisième génération), Acinetobacter baumannii et Bacteroides du groupe fragilis , CHU – Montpellier, 2008–2011

Tigecycline is a new glycylcyclin with a wide spectre including multi-resistant bacteria. Our laboratory tests in routine the in vitro activity of the TGC towards clinically significant isolates of 3rd generation cephalosporins resistant enterobacteriaceae (EC3R), Acinetobacter baumannii and Bacteroides fragilis group (BFG). The objective of this study is to describe the in vitro activity of TGC against these strains isolated between 2008 and 2011 in the university hospital of Montpellier. In this study period, 1070 isolates EC3R including 541 extended spectrum β-lactamase-producers (ESBL) strains, 47 isolates of A. baumannii including 40 multi-resistant isolates and 645 isolates of BFG were tested. Minimum inhibitory concentrations (MIC) were determined using the E-test method. TGC was active against 86.2% of EC3R with a MIC 90 less or equal to 1mg/L (Escherichia coli being the most sensitive species). A. baumannii and BFG were also inhibited at low concentrations of TGC with a MIC 90 less or equal to 2mg/L respectively for 47% and 84.2% of the isolates. Our study confirms the activity of TGC against the EC3R including ESBL-producers strains. The relevance of the therapeutic use of TGC on the BFG isolates with a MIC greater than 2mg/L should be better documented. Often prescribed in therapeutic impasse, the proper use of TGC would require: clarifying the threshold of sensitivity for some species (i.e., A. baumannii, Bacteroides fragilis group); a better understanding of correlation between in vitro and in vivo activity.

Rapid bench identification of methicillin-sensitive and methicillin-resistant Staphylococcus aureus: A multicenter comparative evaluation of Alere PBP2a Culture Colony Test (Alere) Versus Slidex MRSA detection (bioMérieux)

Using 30 clinical isolates of Staphylococcus aureus representative of the most prevalent clones circulating in France, the performance of the Alere™ PBP2a Culture Colony Test (CCT) and the Slidex(®) MRSA detection kit (SMD) were compared in 5 different labs. CCT demonstrated better performance and was easier to conduct in routine.