[Rapid identification of Ralstonia pickettii using PCR-nucleic acid test strips].

OBJECTIVE: To develop a method for rapid detection of Ralstonia pickettii in water for pharmaceutical purpose using PCR-nucleic acid test strips. METHODS: The genomic DNA of Ralstonia pickettii was extracted by boiling method. A pair of specific primers targeting the 16S rDNA with FITC and biotin labeling of the 5' ends was designed and cloned into competent E. coli DH5α cells. The nucleic acid test strips were assembled, and the workload of streptavidin labeled with colloidal gold and antibody concentration in the reaction system was optimized. After verification of the reaction mechanism and assessment of the test sensitivity, specificity and stability, the test strip was used for detecting 7 known strains of Ralstonia pickettii detected in pharmaceutical water, and an evolutionary tree was constructed to analyze the source of contamination. RESULTS: The genomic DNA extracted by boiling method had a purity between 1.8 and 2.0, and the PCR products showed a 100% similarity of with Ralstonia pickettii 16S rDNA registered in GenBank. Using the colloidal gold amplification principle, in every 100 µL colloidal gold solution, 3.5 µL streptavidin was added; the detection line on nitrocellulose membrane was 2.0 mg·mL-1 anti FITC antibody, and the quality control line was 1.2 mg · mL-1 biotinylated BSA, and they generate a red band after binding with positive amplification product. Specificity test of the assembled test strip yielded consistent result with agarose gel electrophoresis without cross reaction with Acinetobacter, Aeromonas, Pseudomonas, or Leclercia adecarboxylata. Sensitivity test of the strip showed a lower detection limit for DNA concentration of 10-5 ng/µL, with a sensitivity 1000 times that of agarose gel electrophoresis. The test strip still had good performance after storage for 3, 6, 9 and 12 months. CONCLUSION: We successfully developed a PCR-nucleic acid test strip for convenient and cost-effective detection of Ralstonia pickettii with good specificity and sensitivity and low cost to facilitate daily monitoring of pharmaceutical water contaminations.

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.

Disrupted tongue microbiota and detection of nonindigenous bacteria on the day of allogeneic hematopoietic stem cell transplantation.

Disruption of the intestinal microbiota caused by intensive chemotherapy, irradiation and antibiotics can result in development of severe gut graft-versus-host disease and infectious complications, leading to poorer outcomes among allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. Although the oral cavity is also densely colonized by indigenous microorganisms, the bacterial composition in allo-HSCT recipients remains unclear. We determined the tongue microbiota composition of 45 patients with hematological disorders on the day of transplantation and compared them to 164 community-dwelling adults. The V1-V2 regions of the 16S rRNA gene sequences demonstrated that the allo-HSCT recipients had less diverse and distinct microbiota from that of community-dwelling adults. The full-length 16S rRNA gene sequences identified 146 bacterial taxa in the microbiota of allo-HSCT recipients, of which 34 bacterial taxa did not correspond to bacteria primarily inhabiting the oral cavity deposited in the expanded Human Oral Microbiome Database. Notably, the detection of Staphylococcus haemolyticus and/or Ralstonia pickettii was significantly associated with a higher risk of mortality during the follow-up period. These results demonstrate that the oral cavity of allo-HSCT recipients is colonized by a disrupted microbiota on the day of transplantation and suggest that detection of specific nonindigenous taxa could be a predictor of transplant outcome.

Evaluating changes to Ralstonia pickettii in high-purity water to guide selection of potential calibration materials for online water bioburden analyzers.

Online water bioburden analyzers (OWBAs) can provide real-time feedback on viable bacteria in high-purity water (HPW) systems for pharmaceutical manufacturers. To calibrate and validate OWBAs, which detect bacteria using scattered light and bacterial autofluorescence, standards are needed that mimic the characteristics of bacteria in HPW. To guide selection of potential standards, e.g., fluorescent microspheres, a relevant bacterial contaminant, Ralstonia pickettii, was characterized for size, count, viability, and autofluorescence after exposure for 24 h to HPW or a nutrient environment. The cells exposed to HPW showed smaller sizes, with lower counts and autofluorescence intensities, but similar spectral features. The cell characteristics are discussed in comparison with a set of fluorescent microspheres, considering factors relevant to OWBAs. These studies suggest that fluorescent microspheres should be relatively small (< 1 µm diameter) and dim, while covering a broad emission range from ≈ (420 to 600) nm to best mimic the representative R. pickettii.

Biosorption characteristics of a highly Mn(II)-resistant Ralstonia pickettii strain isolated from Mn ore.

Microorganisms play an important role in immobilizing and detoxifying excessive Mn; however, there is so far a lack of sufficient information concerning highly Mn(II)-tolerant bacteria. The present study was conducted to analyze the bio-sorption characteristics of a strain (HM8) isolated from manganese ore wastes. Analytical data from the 16S rDNA sequence determination showed that the species, HM8, had a 99% similarity to Ralstonia pickettii. Results from the designed physiological, biochemical and isothermal adsorption tests indicated that HM8 did not only grow well at a Mn(II) concentration level of 10,000 mg/L but also removed 1,002.83 mg/L of Mn(II) from the bulk solution of the culture, showing that the isolated strain possessed strong capabilities to tolerate and remove Mn(II). In the isothermal bio-sorption experiments performed to investigate the effects of relevant factors on Mn(II) sorption, the highest Mn(II) removal rate was obtained at the contact time 72 h, temperature 40°C, and pH 6.0, while the differences in both strain growth and Mn(II) removal rate between different inoculated HM8 doses were found to be insignificant within the tested range. Scanning electron microscopy showed that, under Mn(II) stress, HM8 cells appeared irregular and cracked, with apparent wrinkles on the surface. The peaks in the Fourier transform infrared spectra showed that hydroxyl and carboxyl groups were the main functional groups for Mn(II) adsorption. The experimental data supported the practical application of HM8 as a biological adsorbent for remediation of heavily Mn contaminated sites.

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.

The clinical and microbiological characteristics of infections in burn patients from the Formosa Fun Coast Dust Explosion.

BACKGROUND/PURPOSE: Bloodstream infection is a leading cause of mortality among burn patients. This study aimed to evaluate the risk factors, causative pathogens, and the relationship between bloodstream infections and other infections among burn patients from the Formosa Fun Coast Dust Explosion. METHODS: This retrospective study evaluated the demographic and clinical characteristics, infection types, causative pathogen(s), and isolates' antibiotic susceptibilities from patients who were hospitalized between June 27 and September 31, 2015. RESULTS: Fifty-eight patients were admitted during the study period (36 males, mean age: 22.6 years). The mean burned total body surface area (TBSA) was 40% for all patients. Eighteen (31%) patients with mean TBSA of 80% had 66 episodes of bloodstream infections caused by 92 isolates. Twelve (18.2%) episodes of bloodstream infections were polymicrobial. Acinetobacter baumannii (19, 20.7%), Ralstonia pickettii (17, 18.5%), and Chryseobacterium meningosepticum (13, 14.1%) were the most common pathogens causing bloodstream infections. A high concordance rate of wound cultures with blood cultures was seen in Staphylococcus aureus (3, 75%) and C. meningosepticum (8, 61.5%) infections. However, no Ralstonia isolate was found in burn wounds of patients with Ralstonia bacteremia. A high concordance rate of central venous catheter cultures with blood cultures was noted in Ralstonia mannitolilytica (5, 62.5%) and Chryseobacterium indologenes (3, 60%) infections. Approximately 21.1% of A. baumannii strains were resistant to carbapenem. All S. aureus isolates were susceptible to methicillin. CONCLUSIONS: Waterborne bacteria should be considered in patients of burns with possible water contact. Empirical broad-spectrum antibiotics should be considered for patients who were hospitalized for severe sepsis, or septic shock with a large burn. Antibiotic treatment should be administered based on the specific pathogens and their detection points.

Sonication contribution to identifying prosthetic joint infection with Ralstonia pickettii: a case report and review of the literature.

BACKGROUND: In the context of an increase number of primary and revision total hip and total knee arthroplasty performed yearly, an increased risk of complication is expected. Prosthetic joint infection (PJI) remains the most common and feared arthroplasty complication. Ralstonia pickettii is a Gram-negative bacterium, that has also been identified in biofilms. It remains an extremely rare cause of PJI. There is no report of an identification of R. pickettii on an extracted spacer loaded with antibiotic. CASE PRESENTATION: We present the case of an 83-years-old Caucasian male patient, that underwent a right cemented total hip replacement surgery. The patient is diagnosed with an early PJI with no isolated microorganism. A debridement and change of mobile parts is performed. At the beginning of 2016, the patient in readmitted into the Orthopedic Department for sever, right abdominal and groin pain and elevated serum erythrocyte sedimentation rate and C-reactive protein. A joint aspiration is performed with a negative microbiological examination. A two-stage exchange with long interval management is adopted, and a preformed spacer loaded with gentamicin was implanted. In July 2016, based on the proinflammatory markers evolution, a shift a three-stage exchange strategy is decided. In September 2016, a debridement, and changing of the preformed spacer loaded with gentamicin with another was carried out. Bacteriological examination of the tissues sampled intraoperatively was positive for Pseudomonas aeruginosa. From the sonication fluid, no bacteria were isolated on culture or identified using the bbFISH assay. During the hospitalization period, the patient received i.v. ceftazidime 3x2g/day and p.o. ciprofloxacin 2x750mg/day, antibiotic therapy that was continued after discharge with p.o. ciprofloxacin 2x750mg/day for 6 weeks. In February 2017, a reimplantation of a revision prosthesis is performed. The retrieved spacer is sonicated, and after 4 days of incubation of the sonication fluid, R. pickettii is isolated. A long term antibiotic therapy with cotrimoxazole being prescribed. CONCLUSIONS: Bacteria culture of sonication fluid remains the gold standard in diagnosing prosthetic joint infections. R. pickettii remains an extremely rare cause of prosthetic joint infection. Optimal management of R. pickettii prosthetic joint infections of has not been established.

Neumonía bilateral por Ralstonia pickettii en una paciente inmunocompetente / Bilateral pneumonia due to Ralstonia picketti in immunocompetent patient

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Sepsis fulminante por Ralstonia pickettii / Fulminant sepsis due to Ralstonia pickettii

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Ralstonia pickettii bacteremia in hemodialysis patients: a report of two cases. / Bacteriemia por Ralstonia pickettii en pacientes en hemodiálisis: reporte de dos casos.

Ralstonia pickettii is a low-virulence gram-negative bacillus that may be associated with infections related to health care and may cause bacteremia. Ralstonia pickettii bacteremia is uncommon but is related to the contamination of medical products, mainly in immunodepressed patients. We present two cases of patients on chronic hemodialysis with Ralstonia pickettii bacteremia linked to contamination of the dialysis water. Similar cases have been published with links to intravenous fluid administration, medication ampules, and the use of extracorporeal oxygenation membranes, among other factors. The detection of Ralstonia pickettii bacteremia should provoke suspicion and a search for contaminated medical products, fluids, and/or medications.

Bacteriemia por Ralstonia pickettii en pacientes en hemodiálisis: reporte de dos casos / Ralstonia pickettii bacteremia in hemodialysis patients: a report of two cases

RESUMEN Ralstonia pickettii es un bacilo gram negativo de baja virulencia que puede asociarse a infecciones relacionadas a los cuidados de la salud y provocar bacteriemias. La bacteriemia por Ralstonia pickettii es poco frecuente pero se relaciona con la contaminación de productos de uso médico principalmente en pacientes inmunodeprimidos. Presentamos dos casos en pacientes en hemodiálisis crónica vinculados a contaminación del agua de diálisis. Se han publicado casos similares vinculados a la administración de fluídos intravenosos, ampollas de medicación, asociado a membranas de circulación extracorpórea, entre otros. La detección de una bacteriemia por Ralstonia pickettii, debe sospechar e iniciar la búsqueda de productos de uso médico contaminados, fluídos y/o medicación.

ABSTRACT Ralstonia pickettii is a low-virulence gram-negative bacillus that may be associated with infections related to health care and may cause bacteremia. Ralstonia pickettii bacteremia is uncommon but is related to the contamination of medical products, mainly in immunodepressed patients. We present two cases of patients on chronic hemodialysis with Ralstonia pickettii bacteremia linked to contamination of the dialysis water. Similar cases have been published with links to intravenous fluid administration, medication ampules, and the use of extracorporeal oxygenation membranes, among other factors. The detection of Ralstonia pickettii bacteremia should provoke suspicion and a search for contaminated medical products, fluids, and/or medications.

[Outbreak with Ralstonia pickettii caused by contaminated magnesium vials]. / Ausbruch mit Ralstonia pickettii durch kontaminierte Magnesium-Ampullen.

HISTORY: In February 2013, 5 patients in an intensive care unit (ICU) were found to have positive blood cultures with Ralstonia pickettii within one week. Because all patients got intravenous therapy, improper work of a staff member was suspected. Some days later, a 6th patient was found with a positive blood culture of Ralstonia pickettii in another department of the hospital. INVESTIGATIONS: Hygienic investigations showed no evidence of failures in preparation of intravenous therapy. All patients were on different intravenous drugs, but every patient had received glucose 5 % and magnesium. We examined samples of glucose and magnesia as well as samples from environment. RESULTS AND COURSE: Glucose and magnesium samples were examined by membrane filter method. Ralstonia pitteckii was detected in some Magnesium vials. We concluded, that contamination of Magnesium vials might have been the reason for blood stream infection of patients. Pharmacists and authorities were informed and all vials were collected and replaced by vials from another company. Later a nationwide recall of Magnesium vials was performed by the producing company. No further Ralstonia pickettii was found in blood cultures in our hospital. CONCLUSION: Unusual pathogens in blood cultures should lead to reflection of rarer causes such as contamination of medicines.

Déjà vu: Ralstonia mannitolilytica infection associated with a humidifying respiratory therapy device, Israel, June to July 2011.

Following a bloodstream infection in June 2011 with Ralstonia mannitolilytica in a premature infant treated with a humidifying respiratory therapy device, an investigation was initiated at the Hadassah Medical Centres in Jerusalem. The device delivers a warmed and humidified mixture of air and oxygen to patients by nasal cannula. The investigation revealed colonisation with R. mannitolilytica of two of 15 patients and contamination of components of five of six devices deployed in the premature units of the Hadassah hospitals. Ten isolates from the investigation were highly related and indistinguishable from isolates described in an outbreak in 2005 in the United States (US). Measures successful in containing the US outbreak were not included in user instructions provided to our hospitals by the distributor of the device.

The fate of elbows with unexpected positive intraoperative cultures during revision elbow arthroplasty.

BACKGROUND: An intraoperative culture sample obtained during revision elbow arthroplasty that is unexpectedly positive poses a dilemma for the surgeon. The purpose of our study was to determine the prevalence of positive cultures during revision elbow arthroplasty when infection is not suspected preoperatively, and the long-term implications of these positive cultures. METHODS: Two hundred and thirteen consecutive revision elbow arthroplasties were performed at our institution between 2000 and 2007. Of these, sixteen patients had unexpected positive intraoperative cultures. RESULTS: The majority of cultures grew either Staphylococcus epidermidis or Propionibacterium acnes. Twelve patients had more than two years of follow-up. One of the twelve patients was treated as for an infection because of unexplained early implant loosening and the isolation of Staphylococcus epidermidis. Ten of the twelve elbows were treated as "contaminants" and did not receive long-term antibiotic treatment. Nine of these ten remained infection-free at the time of the final follow-up, while the remaining one developed an infection with a different organism. CONCLUSIONS: In our series, there was a 7.5% chance of encountering an unexpected positive result on intraoperative culture at the time of revision elbow arthroplasty. The majority of patients were successfully treated without antibiotics with a low rate of failure. A minority were considered as infections, typically presenting with unexplained early loosening and isolation of an organism on solid culture medium.

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.