Prevalence and variability of siderophore production in the Achromobacter genus.

Achromobacter spp. are opportunistic pathogens of environmental origin increasingly isolated in patients with underlying conditions like cystic fibrosis (CF). Despite recent advances, their virulence factors remain incompletely studied, and siderophore production has not yet been investigated in this genus. The aim of this study was to evaluate the production of siderophores in a large collection of Achromobacter spp. and evaluate the variability according to the origin of the strain and species. A total of 163 strains were studied, including 128 clinical strains (CF and non-CF patients) and 35 strains of environmental origin. Siderophores were quantified by the liquid chrome azurol-sulphonate assay. Species were identified by nrdA gene-based phylogeny. Strains were assigned to 20 species, with Achromobacter xylosoxidans being the most represented (51.5% of strains). Siderophore production was observed in 72.4% of the strains, with amounts ranging from 10.1% to 90% siderophore units. A significantly higher prevalence of siderophore-producing strains and greater production of siderophores were observed for clinical strains compared with strains of environmental origin. Highly variable observations were made according to species: A. xylosoxidans presented unique characteristics (one of the highest prevalence of producing strains and highest amounts produced, particularly by CF strains). Siderophores are important factors for bacterial growth commonly produced by members of the Achromobacter genus. The significance of the observations made during this study must be further investigated. Indeed, the differences observed according to species and the origin of strains suggest that siderophores may represent important determinants of the pathophysiology of Achromobacter spp. infections and also contribute to the particular epidemiological success of A. xylosoxidans in human infections. IMPORTANCE: Achromobacter spp. are recognized as emerging opportunistic pathogens in humans with various underlying diseases, including cystic fibrosis (CF). Although their pathophysiological traits are increasingly studied, their virulence factors remain incompletely described. Particularly, siderophores that represent important factors of bacterial growth have not yet been studied in this genus. A population-based study was performed to explore the ability of members of the Achromobacter genus to produce siderophores, both overall and in relevant subgroups (Achromobacter species; strain origin, either clinical-from CF or non-CF patients-or environmental). This study provides original data showing that siderophore production is a common trait of Achromobacter strains, particularly observed among clinical strains. The major species, Achromobacter xylosoxidans, encompassed both one of the highest prevalence of siderophore-producing strains and strains producing the largest amounts of siderophores, particularly observed for CF strains. These observations may represent additional advantages accounting for the epidemiological success of this species.

Persistent contamination of heater-cooler units for extracorporeal circulation cured by chlorhexidine-alcohol in water tanks.

Recently, surgical site infections due to non-tuberculous mycobacteria (NTM) have been linked to heater-cooler unit contamination. The European Centre for Disease Prevention and Control and manufacturers now recommend the use of hydrogen peroxide in filtered water to fill heater-cooler unit tanks. After implementation of these measures in our hospital, heater-cooler units became heavily contaminated by opportunistic waterborne pathogens such as Pseudomonas aeruginosa and Stenotrophomonas maltophilia. No NTM were detected but fast-growing resistant bacteria could impair their detection. The efficiency of hydrogen peroxide and chlorhexidine-alcohol was compared in situ. Chlorhexidine-alcohol treatment stopped waterborne pathogen contamination and NTM were not cultured whereas their detection efficiency was probably improved.

Tracking the spread routes of opportunistic premise plumbing pathogens in a haematology unit with water points-of-use protected by antimicrobial filters.

BACKGROUND: Water networks in hospitals are frequently contaminated by opportunistic premise plumbing pathogens (OPPPs) leading to installation of antimicrobial filters on water points-of-use (POU) in order to limit patients' exposure. AIM: To assess the spread of OPPPs through secondary water routes (outside the plumbing system) in an adult haematology unit in which 52 out of 73 water POU were high risk for patients and protected by antimicrobial filters. METHODS: An observational audit identified six secondary water routes for which bacteria tracking and typing were performed in 315 surface samplings. Bacterial isolates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and compared to the infra-species level by multiplex repetitive element sequence-based polymerase chain reaction and/or by restriction fragment length polymorphism in pulse-field gel electrophoresis. FINDINGS: Pseudomonas aeruginosa and Stenotrophomonas maltophilia, as well as non-pathogenic OPPP indicators, were detected in water collected upstream of antimicrobial filters. P. aeruginosa was the sole OPPP retrieved from tested surfaces (5.1%). The same clone of P. aeruginosa spread from water source to dry surfaces in the same room and cross-contaminated two sinks in different rooms. Three clones of non-pathogenic OPPP indicators spread more widely in different rooms. CONCLUSION: A strategy based on filtration of most (but not all) water POU in a haematology unit could be sufficient to limit the spread of OPPPs to the environment, provided a functional mapping of 'high-risk' POU has been undertaken. The residual spread of OPPPs and OPPP indicators linked to non-filtered water POU argues for careful monitoring of non-filtered water use.

Intraclonal variations of resistance and phenotype in Pseudomonas aeruginosa epidemic high-risk clone ST308: A key to success within a hospital?

Most multidrug-resistant (MDR) and extensively drug-resistant (XDR) P. aeruginosa strains belonged to epidemic high-risk (EHR) clones that succeeded worldwide in the context of hospital outbreaks. In order to study the intraclonal diversity in EHR P. aeruginosa, we selected clinical and environmental strains of the EHR clone ST308 that caused outbreak clusters over five years in a hospital and then persisted in the hospital environment during four additional years, causing sporadic infections. Unexpectedly, resistance phenotype was very diverse within the population, independently of the origin (environmental or human) and the period of isolation (during or after outbreaks). Most MDR/XDR strains belonged to clusters in pulsed-field gel electrophoresis (PFGE) while singleton strains instead displayed susceptible or moderately resistant phenotypes. High diversity was observed for motility and biofilm formation without correlation with the origin and the period. Resistance to biocides was not linked to epidemic success or to environmental persistence. Finally, the EHR clone ST308 did not display common adaptive traits, nor traits related to an origin or a period of isolation in the hospital. The major character of this EHR clone ST308 is its intraclonal diversity that probably warrants its adaptation and persistence in hospital whatever the conditions and therefore its epidemic behaviour. This diversity could result from adaptive radiation with the evolution of multiple lineages that fill available niches within a complex ecosystem such as a hospital.

Skin microbiota is the main reservoir of Roseomonas mucosa, an emerging opportunistic pathogen so far assumed to be environmental.

Roseomonas spp. are increasingly involved in human infectious diseases. The environmental source for infection is generally admitted in published cases owing to the origin of most Roseomonas species and to their affiliation to the family Acetobacteraceae in Rhodospirillales, which mainly groups environmental bacteria. For a better delineation of Roseomonas habitat and infectious reservoir, we related phenotype, phylotype (16S rRNA gene), genomotype (pulsed-field gel electrophoresis) and origin of 33 strains isolated from humans, hospital environment and natural environment. Genetic and metagenomic databases were also surveyed. The population structure of the genus showed clades associated with humans, whereas others grouped environmental strains only. Roseomonas mucosa is the main human-associated species and the study supported the idea that opportunistic infections due to this species are related to the patient skin microbiota rather than to the environment. In contrast, some strains belonging to other species isolated from patients with cystic fibrosis were related to environmental clades, suggesting an exogenous source for patient colonization. Accurate knowledge about the reservoirs of opportunistic pathogens that have long been considered of environmental origin is still needed and would be helpful to improve infection control and epidemiological survey of emerging human pathogens.

Source-case investigation of Mycobacterium wolinskyi cardiac surgical site infection.

The non-tuberculous mycobacteria (NTM) Mycobacterium wolinskyi caused bacteraemia and massive colonization of an aortic prosthesis in a patient 16 days after cardiac surgery, necessitating repeat surgery and targeted antimicrobial chemotherapy. The infection control team investigated the source and conditions of infection. Peri-operative management of the patient complied with recommendations. The environmental investigation showed that although M. wolinskyi was not recovered, diverse NTM species were present in water from point-of-use taps and heater-cooler units for extracorporeal circulation. This case and increasing evidence of emerging NTM infections in cardiac surgery led to the implementation of infection control procedures in cardiac surgery wards.

Carbapenemase-producing Enterobacteriaceae: use of a dynamic registry of cases and contacts for outbreak management.

BACKGROUND: The emergence and spread of carbapenemase-producing Enterobacteriaceae (CPE) have become a major public health problem. Control and prevention of CPE infections hinge on isolation precautions for carriers and active screening and follow-up of contacts. AIM: To implement an open registry of cases and contacts for acute outbreak management, long-term data collection and epidemiological investigation. METHODS: All cases, defined as patients (infected or colonized) with a CPE-positive culture during their hospitalization, and contacts (e.g. patients cared for by the same healthcare team as a case) were registered in an ongoing database. Hospital stays were cross-referenced for every new entry and epidemiological links (e.g. shared contacts) investigated. All cases and contacts not cleared by complete screening were registered on an active list. FINDINGS: Between October 2012 and November 2014, we registered 30 cases and 1268 contacts, among which 24 were linked to two or three separate cases. Only 6.5% of contacts fulfilled complete screening with three rectal swabs, and 1145 contacts are still registered on the active surveillance list. Two outbreaks (12 and nine cases) occurred nine months apart. Cross-referencing of hospital stays using the registry revealed epidemiological links between seemingly unrelated cases of CPE-positive patients and suggested an environmental source of transmission, which was demonstrated thereafter. CONCLUSION: We implemented a simple and multi-purpose tool to manage CPE episodes and investigate epidemiological links. Efforts are necessary to improve screening of contact patients who may be occult sources of transmission. A regional registry could be helpful.

Proteobacteria from the human skin microbiota: Species-level diversity and hypotheses.

The human skin microbiota is quantitatively dominated by Gram-positive bacteria, detected by both culture and metagenomics. However, metagenomics revealed a huge variety of Gram-negative taxa generally considered from environmental origin. For species affiliation of bacteria in skin microbiota, clones of 16S rRNA gene and colonies growing on diverse culture media were analyzed. Species-level identification was achieved for 81% of both clones and colonies. Fifty species distributed in 26 genera were identified by culture, mostly belonging to Actinobacteria and Firmicutes, while 45 species-level operational taxonomic units distributed in 30 genera were detected by sequencing, with a high diversity of Proteobacteria. This mixed approach allowed the detection of 100% of the genera forming the known core skin Gram-negative microbiota and 43% of the known diversity of Gram-negative genera in human skin. The orphan genera represented 50% of the current skin pan-microbiota. Improved culture conditions allowed the isolation of Roseomonas mucosa, Aurantimonas altamirensis and Agrobacterium tumefaciens strains from healthy skin. For proteobacterial species previously described in the environment, we proposed the existence of skin-specific ecotypes, which might play a role in the fine-tuning of skin homeostasis and opportunistic infections but also act as a shuttle between environmental and human microbial communities. Therefore, skin-associated proteobacteria deserve to be considered in the One-Health concept connecting human health to the health of animals and the environment.

Rhizobium pusense is the main human pathogen in the genus Agrobacterium/Rhizobium.

Rhizobium pusense was recently described after isolation from the rhizosphere of chickpea. Multilocus sequence-based analysis of clinical isolates identified as Agrobacterium (Rhizobium) radiobacter demonstrated that R. pusense is the main human pathogen within Agrobacterium (Rhizobium) spp. Clinical microbiology of Agrobacterium (Rhizobium) should be considered in the light of recent taxonomic changes.

Propionibacterium acnes populations involved in deep pathological samples and their dynamics along the cardiac surgical pathway.

Propionibacterium acnes belongs to the normal skin microbiota, but it is also responsible for acne vulgaris and causes serious infections such as endocarditis and surgical site infections (SSI). The P. acnes population is structured into phylogenetic groups, with phylotype I being associated with acne. Herein, we explore the link between phylotypes and clinical origins in a collection of P. acnes isolated from different body sites, involved in deep infections or healthcare-associated infections (HAI), with particular emphasis on strains from cardiac SSI. Cardiac SSI have been further studied in terms of P. acnes population dynamics during the care pathway. The recA and tly genes phylotypes were compared to hemolytic behavior, susceptibility to antimicrobial agents, and clinical origins. An original approach of recA polymerase chain reaction temporal temperature gel electrophoresis (PCR-TTGE) was developed and applied for the direct identification of P. acnes phylotypes in surgical samples, in order to assess their temporal dynamics during the surgical course. Our results underlined the preferential involvement of IA-2/IB and II phylogroups in HAI and SSI. Unlike IA and II, type IA-2/IB presented a gradual increase with the depth of sampling in the peroperative phase of cardiac surgery. Phylotypes IA and IA-2/IB were both predominant in scar tissues and on postoperative skin, suggesting a specific predisposition to recolonize skin. Particular association of the phylotype IA-2/IB with SSI and its propensity to colonize wounds in cardiac surgery was observed. We assumed that the follow-up of P. acnes phylotypes during pathological processes could give new clues for P. acnes pathogenicity.

Group B streptococci in milk and late neonatal infections: an analysis of cases in the literature.

BACKGROUND: The source for late-onset neonatal infections (LONI) due to group B Streptococcus (GBS) has not been fully explored. We reviewed GBS LONI cases associated with contaminated breast milk to determine whether breast milk was a possible route for neonatal infection. DATA SOURCES: A PubMed search from January 1977 to March 2013 was performed with MeSH words "Streptococcus agalactiae", "group B Streptococcus", "infection", "milk", "human", "late-onset infection" and/or "neonate"; relevant cross references were also reviewed. RESULTS: Forty-eight documented cases of GBS LONI matched our search criteria and were retrieved from the literature. When performed, molecular typing identified clonal isolates in the neonate and milk samples taken after LONI in all cases, with the hypervirulent sequence type 17 (ST-17) clone identified in two of these cases. Caesarean delivery combined with the absence of GBS recovery from maternal samples other than milk was noted for four cases. The rate of recurrent infections was high (35%) and, together with the data reviewed, points to a potential role of breast milk in GBS LONI. CONCLUSIONS: The cases reviewed here, together with the evidence of breast milk transmission for other pathogens, suggest that breast milk, which would account for repeated GBS transmission to the neonate, may favour gut translocation and subsequent LONI. Further investigations are nevertheless needed to study the relative importance of this contamination route compared with persistent postnatal gut colonisation and the dynamics of milk and neonatal gut colonisation.

Which antibiotics and breakpoints should be used for Aeromonas susceptibility testing? Considerations from a comparison of agar dilution and disk diffusion methods using Enterobacteriaceae breakpoints.

Aeromonas species are environmental organisms that are responsible for numerous infections in humans and animals. Their antimicrobial susceptibility is usually evaluated using Enterobacteriaceae breakpoints. Although disk diffusion and minimum inhibitory concentration (MIC)-based methods are important for infectious disease management and epidemiological surveys of resistance, comparisons between these two methods have not been extensively studied for Aeromonas isolates. We propose the first extensive comparison of agar dilution and disk diffusion susceptibility testing methods, performed for 20 antimicrobial agents, including unevaluated or incompletely evaluated antibiotics (ticarcillin with or without clavulanic acid, ertapenem, tigecycline), on 146 Aeromonas isolates affiliated with six Aeromonas species via molecular means. We evaluated the level of agreement between Enterobacteriaceae breakpoints-based methods. Reliable agreement (>95%) was observed for piperacillin, cefotaxime, cefepime, nalidixic acid, ofloxacin, ciprofloxacin, gentamicin, amikacin, tetracycline and cotrimoxazole, whereas marked inconsistencies between the methods were noted for carbapenems, amoxicillin-clavulanic acid, ticarcillin, ticarcillin-clavulanic acid, tobramycin and tigecycline. The results indicate that beta-lactam and aminoglycoside susceptibility testing should be limited to piperacillin, cephems, gentamicin and amikacin. Co-amoxiclav should be avoided given the lack of agreement between the two methods. Adjusting the zone diameter breakpoints for tigecycline and cefoxitin could also improve the agreement to >95% and reduce the error rates to acceptable levels.

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.

Utility of the Etest GRD for detecting Staphylococcus aureus with reduced susceptibility to glycopeptides in cystic fibrosis patients.

Glycopeptide-intermediate S. aureus (GISA), particularly heterogeneous GISA (hGISA), remain difficult to detect in the routine practice of medical microbiology. Novel tools have been evaluated comparatively to the population analysis profile-area under the curve (PAP-AUC) reference method for detecting GISA/hGISA. Among them, the Etest GRD showed relatively high specificity (85.8-97%) and negative predictive value (97%) but lower sensibility (57-95%) and positive predictive value (30.8%). We investigated the utility of the Etest GRD for detecting GISA/hGISA among 180 strains isolated from 106 cystic fibrosis (CF) patients. Etest GRD was performed on all isolates, and those exhibiting a GISA/hGISA phenotype were further tested by PAP-AUC and other agar routine assays for GISA/hGISA detection. The Etest GRD allowed the detection of 15 GISA/hGISA strains, of which eight were confirmed by the reference method. Despite the 3.9% level of false positive results, the Etest GRD constitutes a useful routine tool for detecting GISA/hGISA overlooked by other routine assays, two strains being detected by the Etest GRD only. GISA/hGISA represented 7.7% of MRSA and 2.1% of MSSA, and were found in 4.7% of CF patients colonized/infected by S. aureus, which is the highest rate reported to date in this population.

[Molecular fingerprint of bacterial communities and 16S rDNA intra-species heterogeneity: a pitfall that should be considered]. / Empreinte moléculaire des communautés bactériennes et hétérogénéité intraspécifique de l'ADNr 16S: est-il raisonnable d'occulter le problème?

UNLABELLED: Molecular fingerprinting methods are currently used to study microbial communities by culture independent approaches. They are proposed as identification tool owing to the availability of rapid automated methods. The 16S rRNA gene (16S rDNA) is an efficient marker for bacterial identification and microbial communities analysis. However, the 16S rDNA polymorphism among strains of the same species is an underestimated pitfall of the fingerprinting approaches. AIM OF THE STUDY: We studied the 16S rDNA variability among strains of three bacterial species of medical interest. MATERIAL AND METHODS: Total DNA was extracted from clinical isolates of Pseudomonas aeruginosa (N=20), Clostridium difficile (N=20) and Enterobacter cloacae (N=14). The Polymerase Chain Reaction (PCR) products obtained with consensus primers flanking the 16S rDNA variable regions V3 and V6-V7-V8 were separated by Temporal Temperature Gradient gel Electrophoresis (TTGE). DNA extracted from TTGE bands were sequenced and analysed. RESULTS: All the isolates of P. aeruginosa and of C. difficile displayed one single TTGE band with constant migration distances suggesting that there was no 16S rDNA polymorphism among strains in these two species. Oppositely, the isolates of E. cloacae gave complex TTGE patterns formed by multiple bands with variable migration distances. These patterns corresponded to 16S rRNA genes variable in a single genome as well as among strains of the species. CONCLUSION: Intra-species and/or intragenomic variability of 16S rDNA should be taken into account for pertinent interpretation of molecular fingerprint. For this purpose, a comprehensive description of the polymorphism of this marker is necessary.

Antimicrobial susceptibilities and clinical sources of Dialister species.

Seventy-four strains representing the four species of the genus Dialister were isolated from various clinical samples. Dialister pneumosintes and Dialister micraerophilus were the two mainly encountered species. Fifty-five isolates were tested against 14 antimicrobial agents. Decreased susceptibilities to piperacillin, metronidazole, macrolides, fluoroquinolones, and rifampin were demonstrated. The clinical impact of these decreased susceptibilities remains to be investigated but should prompt microbiologists to perform antimicrobial susceptibility testing for clinically important Dialister spp.

Prevotella nanceiensis sp. nov., isolated from human clinical samples.

Three strains of anaerobic, non-pigmented, Gram-negative bacilli isolated from various human clinical samples were characterized in terms of phenotypic and genotypic tests, including sequence analysis of 16S rRNA and rpoB genes. The strains were most closely related to the type strains of Prevotella marshii and Prevotella shahii on the basis of both 16S rRNA (89.8 and 89.0 % identity, respectively) and rpoB gene sequences (83.1 and 82.8 % identity, respectively). Phylogenetic analysis showed that the isolates constituted a robust homogeneous group distinct from known species in the genus Prevotella. The rrn skeleton (as determined by PFGE) and the DNA G+C content, determined to be 39.4 mol% for strain LBN 293(T), distinguished the novel isolates from the type strains of P. marshii and P. shahii. The three strains were saccharolytic and produced acetic, lactic and succinic acids as major metabolic end products. Polyphasic investigations supported the proposal of a novel species, Prevotella nanceiensis sp. nov., with LBN 293(T) (=AIP 261.03(T) =CIP 108993(T) =CCUG 54409(T)) as the type strain.

Three years of multi-laboratory external quality control for the molecular detection of Toxoplasma gondii in amniotic fluid in France.

Between 2002 and 2004, panels of amniotic fluid containing varying concentrations of Toxoplasma gondii were sent to up to 23 laboratories in France for molecular (PCR-based) detection as part of a national quality assurance initiative in the molecular prenatal diagnosis of toxoplasmosis. Participants were free to enroll and no fees were required. The general level of sensitivity was high, and the rate of false-positive reactions was relatively low. Considerable diversity among PCR methods and primers was revealed. This external quality assurance scheme provided the opportunity to improve laboratory practice and performance, and to increase communication among laboratories involved in making this diagnosis.

Unusual implication of biopsy forceps in outbreaks of Pseudomonas aeruginosa infections and pseudo-infections related to bronchoscopy.

Between January and April 2003, a sudden increase in positive respiratory tract specimens for Pseudomonas aeruginosa was observed in an intensive care unit of the University Teaching Hospital of Montpellier, France. Most of the strains were cultured from bronchoalveolar lavage fluid samples, suggesting that bronchoscopic procedures could be implicated. The relationships between isolates were investigated by antibiotyping and pulsed-field gel electrophoresis. Both phenotypic and molecular markers allowed identification of two consecutive nosocomial outbreaks of respiratory infections related to two different bronchoscopes. These two outbreaks implicated nine and seven patients, respectively. Four of these 16 patients had true infections and recovered with antibiotic therapy. Inspection of both bronchoscopes revealed a damaged internal channel caused by defective biopsy forceps. These defects led to improper cleaning and disinfection of the bronchoscopes despite adherence to all current reprocessing procedures. The two outbreaks were controlled after replacing the inner channels of the bronchoscopes and switching from use of re-usable to disposable biopsy forceps. These outbreaks emphasize the need to establish surveillance procedures for detecting contamination of bronchoscopes, and the importance of recording each endoscopic procedure to facilitate further investigations if needed.