Residual risk of Pseudomonas aeruginosa waterborne contamination in an intensive care unit despite the presence of filters at all water points-of-use.

OBJECTIVE: To assess the residual risk of waterborne contamination by Pseudomonas aeruginosa from a water network colonized by a single genotype [sequence type (ST) 299] despite the presence of antimicrobial filters in a medical intensive care unit (ICU). METHODS: During the first 19-month period since the ICU opened, contamination of the water network was assessed monthly by collecting water upstream of the filters. Downstream water was also sampled to assess the efficiency of the filters. P. aeruginosa isolates from patients were collected and compared with the waterborne ST299 P. aeruginosa by multiplex-rep polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. Cross-transmission events by other genotypes of P. aeruginosa were also assessed. RESULTS: Overall, 1.3% of 449 samples of filtered water were positive for P. aeruginosa in inoculum, varying between 1 and 104 colony-forming units/100 mL according to the tap. All P. aeruginosa hydric isolates belonged to ST299 and displayed fewer than two single nucleotide polymorphisms (SNPs). Among 278 clinical isolates from 122 patients, 10 isolates in five patients showed identical profiles to the hydric ST299 clone on both multiplex-rep PCR and PFGE, and differed by an average of fewer than five SNPs, confirming the water network reservoir as the source of contamination by P. aeruginosa for 4.09% of patients. Cross-transmission events by other genotypes of P. aeruginosa were responsible for the contamination of 1.75% of patients. DISCUSSION/CONCLUSION: Antimicrobial filters are not sufficient to protect patients from waterborne pathogens when the water network is highly contaminated. A microbiological survey of filtered water may be needed in units hosting patients at risk of P. aeruginosa infections, even when all water points-of-use are fitted with filters.

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.

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.

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.

Physiological and behavioural responses of Gammarus pulex exposed to acid stress.

Physiological and behavioural responses of the acid-sensitive amphipod Gammarus pulex exposed to a wide range of acid conditions (pH 4.1, 5.1, 6.0) under laboratory conditions were investigated. An exposure of 38 h to acid conditions caused significant decreases in survival rate, osmolality, haemolymph Na+ concentration, ventilatory and locomotor activity compared to organisms exposed to a circumneutral medium (pH 7.9). We highlighted the interest of using individual response distribution, since drastic effects can be detected in organisms exposed to pH 6.0, in particular for osmolality: The response can be divided into two groups, unimpacted and impacted organisms. Moreover this representation permitted to evaluate the health level of individual organisms through the determination of threshold values. Significant correlations between mean pH and mean physiological/behavioural responses were observed. The relationships between individual responses permitted not only to compare endpoints, but also to show that affected organisms were impacted by ionoregulation failure, hypoventilation and low locomotor activity. The energetic reallocation in favour of maintenance functions, such as osmoregulation, is discussed. The results of this study indicate that the values of haemolymph Na+ concentration, osmolality and locomotor activity in G. pulex could be effective ecophysiological/behavioural markers to monitor freshwater ecosystems and to assess the health of organisms or populations.

Ontogeny of the antennal glands in the crayfish Astacus leptodactylus (Crustacea, Decapoda): immunolocalization of Na+,K+-ATPase.

The involvement of the antennal urinary glands in the ontogeny of osmoregulatory functions was investigated during the development of Astacus leptodactylus by measurements of hemolymph and urine osmolality in juvenile and adult crayfish and by the immunodetection of the enzyme Na+,K+-ATPase. In stage II juveniles, 1-year-old juveniles, and adults, all of which were maintained in freshwater, urine was significantly hypotonic to hemolymph. In adults, chloride and sodium concentrations were much lower in urine than in hemolymph. During embryonic development, Na+,K+-ATPase was detected by immunocytochemistry in ionocytes lining the tubule and the bladder, at an eye index (EI) of 220-250 microm, and in the labyrinth, at EI 350 microm. In all regions, immunofluorescence was mainly located at the basolateral side of the cells. No immunofluorescence was detected at any stage in the coelomosac. In late embryonic stages (EI 410-440 microm), in stage I juveniles, and in adults, strong positive immunofluorescence was found from the labyrinth up to and including the bladder. These results show that, as early as hatching, juvenile crayfish are able to produce dilute urine hypotonic to hemolymph. This ability originates from the presence of Na+,K+-ATPase in ion-transporting cells located in the labyrinth, the tubule, and the bladder of the antennal glands and constitutes one of the main adaptations of crayfish to freshwater.