Evolution of gentamicin and arsenite resistance acquisition in Ralstonia pickettii water isolates.

Ralstonia pickettii are ubiquitous in water environments. Members of this species are frequently, but not always, resistant to both gentamicin and arsenite. Gentamicin and arsenite co-resistance and the putative molecular mechanisms were investigated. A group of 37 R. pickettii strains isolated from drinking water and hospital wastewater were characterized for gentamicin and arsenite resistance phenotypes, the number and size of plasmids, and screened for genetic elements associated with arsenite tolerance, Integrative and Conjugative Elements (ICEs), among other. The genomes of three representative strains were compared. Most gentamicin resistant (GR) isolates (32/33) were resistant to arsenite, and harbored ICE- and ars operon-related genes. These genetic elements were not detected in any of the five arsenite susceptible strains, regardless of the GR (n = 1) or gentamicin susceptibility (GS) (n = 4) phenotype. The comparison of the genomes of two GR (one resistant and one susceptible to arsenite) and one GS strains suggested that these phenotypes correspond to three phylogroups, distinguished by presence of some genes only in GR isolates, in addition to point mutations in functional genes. The presence of ICEs and ars operon-related genes suggest that arsenite resistance might have been acquired by GR lineages.

Association between gentamicin resistance and stress tolerance in water isolates of Ralstonia pickettii and R. mannitolilytica.

Members of the species Ralstonia pickettii and R. mannitolilytica, although ubiquitous and lacking major virulence factors, have been associated with nosocomial outbreaks. Tolerance to metals, antibiotics, and disinfectants may represent an advantage for their ubiquity and opportunistic pathogenic potential. In this study, we compared five strains that differed on the origin (hospital effluent, tap water, mineral water) and in the susceptibility to aminoglycosides, regarding their tolerance to metals and disinfection. The growth kinetics and biofilm formation capacity were tested in four R. pickettii strains and one R. mannitolilytica at sub-inhibitory concentrations of aminoglycosides or arsenite. The survival to UV radiation, chlorine, or hydrogen peroxide was also compared in aminoglycoside resistant and susceptible strains. Aminoglycoside-resistant strains presented a higher tolerance to arsenite than the susceptible ones and either aminoglycosides or arsenite was observed to stimulate the biofilm formation. Sub-inhibitory concentrations of the aminoglycoside gentamicin or arsenite significantly decreased the growth rate and yield, but only arsenite caused a significant increase of the lag phase. Hydrogen peroxide presented higher disinfection effectiveness against aminoglycoside susceptible than against resistant strains, an effect that was not observed for UV or chlorine. Although this conclusion needs validation based on a larger number of isolates, including clinical, the results suggest that aminoglycoside resistance may be associated with traits that influence Ralstonia spp. fitness in the environment.

Preliminary Results of the Use of a Stabilized Hypochlorous Acid Solution in the Management of Ralstonia Pickettii Biofilm on Silicone Breast Implants.

BACKGROUND: Ralstonia Pickettii biofilms are associated with pocket infections following breast implant surgeries. Biofilm protects bacteria most topically applied antimicrobial irrigations. OBJECTIVES: To evaluate the effectiveness of four antimicrobial solutions on the planktonic form and established biofilm of Ralstonia Pickettii grown on 3 different types of silicone breast implants. METHODS: Time kill assays at clinical concentrations of chlorhexidine gluconate, povidone iodine, triple-antibiotic solution, and a 0.025% hypochlorous acid solution stabilized in amber glass were evaluated. Normal saline was the control. Three types of silicone implants, two with a textured surface and one smooth surface, were selected. Planktonic assays were performed after implants were soaked for one, five, 30, and 120 minute time points. Biofilm assays were performed after 5 and 120 minutes of implant soak time. Both tests evaluated cell-forming units (CFU/mL). RESULTS: Triple antibiotic solution had no effect on R. pickettii and was dropped from the study. Remaining solutions showed total kill of planktonic bacteria at one minute. Saline control showed no significant effect on biofilm as anticipated. Stabilized hypochlorous acid was the only solution tested capable of eradicating R. pickettii biofilm on all implant surfaces tested within the first five minute soak time. CONCLUSIONS: Noncytotoxic, 0.025% hypochlorous acid in normal saline, stabilized in amber glass, successfully eradicated Ralstonia pickettii in planktonic and mature biofilm on three types of silicone implants during initial five minute soak time and may be the preferred antimicrobial solution for pocket lavage. This preliminary study requires further investigation. Leaching and implant compatibility testing is currently in progress.

Emergence of VIM-2 metallo-beta-lactamase producing Ralstonia pickettii clinical isolate in India.

A multidrug-resistant clinical isolate of Ralstonia pickettii from a woman was analysed. Modified Hodge test was positive for carbapenemase production. Conjugation experiment revealed the presence of conjugative plasmid of >140 Kb size typed as IncN type. This is the first report of emergence blaVIM-2 in R. pickettii in India.

Characterization of the survival ability of Cupriavidus metallidurans and Ralstonia pickettii from space-related environments.

Four Cupriavidus metallidurans and eight Ralstonia pickettii isolates from the space industry and the International Space Station (ISS) were characterized in detail. Nine of the 12 isolates were able to form a biofilm on plastics and all were resistant to several antibiotics. R. pickettii isolates from the surface of the Mars Orbiter prior to flight were 2.5 times more resistant to UV-C(254nm) radiation compared to the R. pickettii type strain. All isolates showed moderate to high tolerance against at least seven different metal ions. They were tolerant to medium to high silver concentrations (0.5-4 µM), which are higher than the ionic silver disinfectant concentrations measured regularly in the drinking water aboard the ISS. Furthermore, all isolates survived a 23-month exposure to 2 µM AgNO(3) in drinking water. These resistance properties are putatively encoded by their endogenous megaplasmids. This study demonstrated that extreme resistance is not required to withstand the disinfection and sterilization procedures implemented in the ISS and space industry. All isolates acquired moderate to high tolerance against several stressors and can grow in oligotrophic conditions, enabling them to persist in these environments.

Efficacy of various chemical disinfectants on biofilms formed in spacecraft potable water system components.

As the provision of potable water is critical for successful habitation of the International Space Station (ISS), life support systems were installed in December 2008 to recycle both humidity from the atmosphere and urine to conserve available water in the Station. In-flight pre-consumption testing from the dispensing needle at the Potable Water Dispenser (PWD) indicated that bacterial concentrations exceeded the current ISS specifications of 50 colony-forming units (CFU) ml(-1). Subsequent investigations revealed that a corrugated stainless steel flex hose upstream of the dispensing needle in the PWD was filled with nonsterile water and left at room temperature for more than 1 month before launch. To simulate biofilm formation that was suspected in the flight system, sterile flex hoses were seeded with a consortium of bacterial isolates previously recovered from other ISS water systems, including Ralstonia pickettii, Burkholderia multivorans, Caulobacter vibrioides, and Cupriavidus pauculus. After incubation for 5 days, the hoses were challenged with various chemical disinfectants including hydrogen peroxide (H2O2), colloidal silver, and buffered pH solutions to determine the ability of the disinfectants to decrease and maintain bacterial concentrations below ISS specifications. The disinfection efficacy over time was measured by collecting daily heterotrophic plate counts after exposure to the disinfectants. A single flush with either 6% H2O2 solution or a mixture of 3% H2O2 and 400 ppb colloidal silver effectively reduced the bacterial concentrations to <1 CFU ml(-1) for a period of up to 3 months.

Characterization of regulatory element Rp3 of regulation of beta-lactamases from Ralstonia pickettii.

The two chromosomally encoded beta-lactamases, OXA-22 and OXA-60, from Ralstonia pickettii are inducible by beta-lactam molecules. Disruption of RP3 abolished induction of both beta-lactamases and the resistance to pH, osmolarity and survival in the stationary phase, suggesting that RP3 might be a global regulator. Interactions between RP3, OXA-22 and OXA-60 were investigated at a transcript and protein level using 5'-rapid amplification of cDNA ends experiments, real-time reverse transcription (RT)-PCR and footprinting assays. The rp3 gene was actively transcribed and the promoter sequences corresponded to a nontypical sigma(70)-type promoter. RT-PCR analysis showed that rp3 expression as well as that of the bla(OXA) genes was positively regulated: the level of transcripts of rp3, bla(OXA-22) and bla(OXA-60) genes were, respectively, increased 20-, 100- and 2000-fold upon imipenem induction. DNAse I footprinting showed that RP3 specifically bound to tandem repeats centered at positions -55.5 and -73.5 upstream from the bla(OXA-22) and bla(OXA-60) transcriptional start sites. Interestingly, the binding site at bla(OXA-60) overlapped the -35 region of the rp3 promoter, although the region essential for induction lies at the beginning of the orf-rp3. This result indicates that RP3 is most probably only one component of a novel regulatory system involved in the expression of beta-lactamases in R. pickettii.

Streptomyces griseus enhances denitrification by Ralstonia pickettii K50, which is possibly mediated by histidine produced during co-culture.

Ralstonia pickettii K50 (strain K50) is a denitrifying bacterium that produces low levels of N(2)O under aerobic conditions. In this study, we found that co-culturing of strain K50 with Streptomyces griseus significantly enhanced the denitrification activity of strain K50 in an artificial wastewater (AWW) system. Most factors that enhance denitrification activity were in the high molecular weight fraction of the cell-free broth of S. griseus, and were suggested to be extracellular proteases. Further investigation revealed that the cultivation of strain K50 in protease-treated AWW medium fully enhanced denitrification, and that a shortage of amino acids in the medium limited it. Among the 20 standard amino acids tested, only histidine had a significant effect in inducing denitrification by strain K50. Our results indicate that histidine is a novel inducer of bacterial denitrification.