Extending the 30-minute rule for red cell units - investigation of the bacterial risk of 60-minute exposures to ambient temperature.

BACKGROUND AND OBJECTIVES: In the UK, a significant proportion of red cell units is discarded due to the 30-min rule governing out of temperature control. Studies have shown that repeated warming to ambient temperature has little impact on red cell quality or bacterial growth. We aimed to validate extension of the rule to 60 minutes by investigation of repeated same, and different, day exposures on bacterial growth. MATERIALS AND METHODS: Red cell units were seeded individually at 100-1000 cfu/ml with Yersinia enterocolitica, Serratia liquefaciens, Pseudomonas putida, Staphylococcus epidermidis, Enterobacter cloacae and Bacillus cereus. Test units were exposed to 30°C for 30 or 60 min on a single occasion at days 15, 17 and 21, or thrice on day 15 of a 35-day storage period. A 10-fold increase in bacterial counts in tests versus controls maintained in cold storage was considered indicative of significant bacterial proliferation. RESULTS: Exposure of units to 30°C for up to 60 min had no substantial impact on the growth of bacteria and all mesophiles declined steadily in tests and controls. Only P. putida showed a near significant elevation in count on exposure for 60 min at day 35. CONCLUSIONS: Extension of the out of temperature rule for red cells to 60 min will potentially not compromise patient safety, although exposures to ambient temperatures should be minimized. Units returned to storage must not be reissued for at least 6 hours and not be exposed to ambient temperatures on more than three occasions.

Bacterial screening of platelet components by National Health Service Blood and Transplant, an effective risk reduction measure.

BACKGROUND: Bacterial contamination of blood components remains a major cause of sepsis in transfusion medicine. Between 2006 and 2010 in the 5 years before the introduction of bacterial screening of platelet (PLT) components by National Health Service Blood and Transplant (NHSBT), seven cases of PLT component-associated transmission of bacterial infection were recorded for 10 patients, three of which were fatal. STUDY DESIGN AND METHODS: Sampling of individual PLT components was undertaken at 36 to 48 hours after donation and tested in the BacT/ALERT system with 8 mL inoculated into each of aerobic and anaerobic culture bottles. Bottles were incubated until the end of the 7-day shelf life and initial reactive bottles were examined for contamination. Bacterial screened time-expired PLTs were tested as in the screen method. RESULTS: From February 2011 to September 2015, a total of 1,239,029 PLT components were screened. Initial-reactive, confirmed-positive, and false-positive rates were 0.37, 0.03, and 0.19%, respectively. False-negative cultures, all with Staphylococcus aureus, occurred on four occasions; three were visually detected before transfusion and one confirmed transmission resulted in patient morbidity. The NHSBT screening protocol effectively reduced the number of clinically adverse transfusion transmissions by 90% in this reporting period, compared to a similar time period before implementation. Delayed testing of 4515 time-expired PLT units after screening revealed no positives. CONCLUSION: The implementation of bacterial screening of PLT components with the NHSBT BacT/ALERT protocol was an effective risk reduction measure and increased the safety of the blood supply.

Evaluation of an ethidium monoazide-enhanced 16S rDNA real-time polymerase chain reaction assay for bacterial screening of platelet concentrates and comparison with automated culture.

BACKGROUND: Culture-based systems are currently the preferred means for bacterial screening of platelet (PLT) concentrates. Alternative bacterial detection techniques based on nucleic acid amplification have also been developed but these have yet to be fully evaluated. In this study we evaluate a novel 16S rDNA polymerase chain reaction (PCR) assay and compare its performance with automated culture. STUDY DESIGN AND METHODS: A total of 2050 time-expired, 176 fresh, and 400 initial-reactive PLT packs were tested by real-time PCR using broadly reactive 16S primers and a "universal" probe (TaqMan, Invitrogen). PLTs were also tested using a microbial detection system (BacT/ALERT, bioMérieux) under aerobic and anaerobic conditions. RESULTS: Seven of 2050 (0.34%) time-expired PLTs were found repeat reactive by PCR on the initial nucleic acid extract but none of these was confirmed positive on testing frozen second aliquots. BacT/ALERT testing also failed to confirm any time-expired PLTs positive on repeat testing, although 0.24% were reactive on the first test. Three of the 400 "initial-reactive" PLT packs were found by both PCR and BacT/ALERT to be contaminated (Escherichia coli, Listeria monocytogenes, and Streptococcus vestibularis identified) and 14 additional packs were confirmed positive by BacT/ALERT only. In 13 of these cases the contaminating organisms were identified as anaerobic skin or oral commensals and the remaining pack was contaminated with Streptococcus pneumoniae. CONCLUSION: These results demonstrate that the 16S PCR assay is less sensitive than BacT/ALERT and inappropriate for early testing of concentrates. However, rapid PCR assays such as this may be suitable for a strategy of late or prerelease testing.

Hypermutable Pseudomonas aeruginosa in Cystic fibrosis patients from two Brazilian cities.

Hypermutable (HPM) strains of Pseudomonas aeruginosa have been found at high frequencies in cystic fibrosis (CF) patients in Europe. We report the results of testing for HPM frequencies, mutator genotype, and antimicrobial resistance of P. aeruginosa strains from Brazilian CF patients. A modified disk diffusion technique was used to quantify antibiotic-resistant subpopulations of an isolate, and estimations of the frequency of mutation to rifampin resistance were determined for 705 isolates from 149 patients attending clinics in two Brazilian cities. Mutations in the mutS gene were detected by sequencing assays. We found 194 (27.5%) HPM isolates in samples from 99 (66.4%) patients. Thirty-five HPM isolates (18.0%) from 31 (31.3%) patients exhibited a high increased spontaneous mutation rate compared with controls, and eight isolates from six patients displayed a defective mutS gene. The dominant HPM population was associated with very low antibiotic resistance levels, while HPM subpopulations were generally more resistant to antimicrobials. A relatively high prevalence of HPM P. aeruginosa in CF patients was associated with surprisingly low antibiotic resistance levels, in contrast to some earlier studies.

Development of an ethidium monoazide-enhanced internally controlled universal 16S rDNA real-time polymerase chain reaction assay for detection of bacterial contamination in platelet concentrates.

BACKGROUND: Bacterial contamination of platelet (PLT) concentrates remains a problem for blood transfusion services. Culture-based bacterial screening techniques are available but offer inadequate speed and sensitivity. Alternative techniques based on polymerase chain reaction (PCR) amplification have been described but their performance is often compromised by traces of bacterial DNA in reagents. STUDY DESIGN AND METHODS: Universal 16S rDNA primers were used to develop a real-time PCR assay (TaqMan, Applied Biosystems) and various reagent decontamination strategies were explored. Detection sensitivity was assessed by spiking PLT concentrates with known concentrations of 13 different organisms. RESULTS: Restriction enzyme digestion, master mix ultrafiltration, and use of alternative Taq polymerases all reduced the level of reagent DNA contamination to some extent but all proved unreliable. In contrast, ethidium monoazide (EMA) treatment of the PCR master mix followed by photoactivation was reliable and effective, permitting a full 40 amplification cycles, and totally eliminated contamination without compromising assay sensitivity. All 13 organisms were efficiently detected and the limit of detection for Escherichia coli-spiked PLTs was approximately 1 colony-forming unit/mL. Coamplification of human mitochondrial DNA served to confirm efficient nucleic acid extraction and the absence of PCR inhibition in each sample. One of five automated extraction platforms evaluated was found to be contamination free and capable of high-throughput processing. CONCLUSION: Cross-linking of EMA to DNA via photoactivation solved the previously intractable problem of reagent contamination and permitted the development of a high-sensitivity universal bacterial detection system. Trials are ongoing to assess the suitability of the system for high-throughput screening of PLT concentrates.

Comparison of the worldwide transmissible Pseudomonas aeruginosa with isolates from brazilian cystic fibrosis patients.

Cross-infection with Pseudomonas aeruginosa among cystic fibrosis (CF) patients is a rare occurrence. However, the emergence of transmissible strains has been reported between unrelated individuals. We analyzed the genetic relationship among P. aeruginosa isolates from Brazilian CF patients and transmissible clones which are worldwide spread. The data does not indicate the presence of closely related variant clones.

Evaluation of a multiplex PCR for detection of serotypes K1, K2 and K5 in Klebsiella sp. and comparison of isolates within these serotypes.

A multiplex PCR using targets within the serotype-specific region of the capsular polysaccharide synthesis gene cluster of serotypes K1, K2 and K5 was evaluated using the 77 reference serotype strains of Klebsiella, and a panel of clinical isolates subjected previously to conventional serotyping. The PCR was highly specific for these serotypes, which are those most associated with virulence in humans and horses. PCR confirmed that isolates of the K5 serotype had cross-reacted with antiserum for other serotypes, particularly for K7. K5 isolates received by our laboratory were almost exclusively from thoroughbred horses, and were submitted for screening prior to breeding programmes. Most, including a reference strain isolated in 1955, belonged to a cluster of genetically similar isolates of sequence type (ST) 60. K1 isolates, all from humans, belonged to a previously identified cluster of ST 23.

Diagnostic multiplex PCR assay for the identification of the Liverpool, Midlands 1 and Manchester CF epidemic strains of Pseudomonas aeruginosa.

Individual PCR amplification tests have been developed for three UK CF epidemic strains, the Liverpool epidemic strain (LES), Midlands 1 and the Manchester epidemic strain (MES). We report a simple diagnostic multiplex PCR test that can be used to screen for all three of these strains. To evaluate the test, we screened collections of LES, MES and Midlands 1 isolates, along with various CF and non-CF non-epidemic Pseudomonas aeruginosa strains. The test was 100% sensitive and 100% specific in the identification of these UK CF epidemic strains.

Widespread pyocyanin over-production among isolates of a cystic fibrosis epidemic strain.

BACKGROUND: Some isolates of the Liverpool cystic fibrosis epidemic strain of Pseudomonas aeruginosa exhibit an unusual virulence-related phenotype, characterized by over-production of quorum sensing-regulated exoproducts such as pyocyanin and LasA protease. Our aim was to determine the prevalence of this unusual phenotype amongst isolates of the epidemic strain, and to study other intraclonal phenotypic and genotypic variations. RESULTS: The unusual phenotype was detected in at least one epidemic strain isolate from the majority of cystic fibrosis patients tested, and can be retained for up to seven years during chronic infection. Multiple sequential isolates of the epidemic strain taken from six patients over a period of up to nine years exhibited a wide range of phenotypes, including different antimicrobial susceptibilities. Our data suggest that each sputum sample contains a mixture of phenotypes and genotypes within the epidemic strain population, including within colony morphotypes. Many isolates exhibit premature (during early rather than late exponential growth) and over-production of pyocyanin, which has a number of toxic effects directly relevant to cystic fibrosis. CONCLUSION: The widespread occurrence of this unusual phenotype suggests that it may play an important role in the success of the epidemic strain.

Detection of antibodies to Pseudomonas aeruginosa in serum and oral fluid from patients with cystic fibrosis.

Cystic fibrosis (CF) patients who are chronically infected with Pseudomonas aeruginosa make serum antibodies to bacterial surface LPS as well as other pseudomonas antigens. This study investigated the feasibility of using oral fluid samples for the detection of pseudomonas antibodies in CF patients and compared these results with corresponding serum antibodies. Most strains of P. aeruginosa produce two forms of LPS molecule, termed A-band (described as a common antigen) and B-band (O-serotype-specific antigen), apparently bound to a common core oligosaccharide moiety. A-band LPS was demonstrated in 45 out of 49 clinical isolates of P. aeruginosa by SDS-PAGE and immunoblotting with a specific antibody. Oral fluids were collected from 17 adult CF patients, all of whom were sputum culture positive for P. aeruginosa (13 also provided serum samples), 11 primary ciliary dyskinesia (PCD) patients and 37 healthy volunteers. Antibodies to A-band LPS were detected by immunoblotting in all of the CF patients' oral fluids but 10 of the volunteer samples gave weak reactions with immunoblotting. Six of the PCD patients gave a weak reaction with A-band antibodies and only one demonstrated antibodies to core LPS. In a quantitative ELISA, 15 of the 17 CF patients' oral fluids were shown to contain antibodies to A-band LPS, whilst none of the volunteer samples contained antibodies to A-band LPS. All serum samples from the CF patients were positive by both methods. Thus this is a sensitive procedure for the detection of antibodies to A-band LPS of P. aeruginosa in oral fluid and serum from patients with CF.

Genetically similar isolates of Klebsiella pneumoniae serotype K1 causing liver abscesses in three continents.

The magA gene was sought in hypermucoviscous isolates of Klebsiella spp., the Klebsiella K serotype reference strains and in isolates of the K1 serotype of Klebsiella pneumoniae from the UK, Hong Kong, Israel, Taiwan and Australia. Only K1 isolates were PCR positive for magA; this gene was found in all such isolates tested. Hypermucoviscosity was not confined to magA positive isolates, nor was it found in all magA positive isolates. Comparison of XbaI PFGE profiles revealed that most (19/23) of the magA positive isolates clustered within 72 % similarity, with a further subcluster of isolates, from three different continents, clustering within >80 %. All of the 16 isolates tested within the main cluster had the same sequence type (ST 23) by multilocus sequence typing, with the exception of one isolate, which had a single nucleotide difference at one of the seven loci. This study indicates that a genotype strongly associated with highly invasive disease in Taiwan, where large numbers of cases have been reported, is geographically very widespread.

The role of ISAba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii.

ISAba1 was found in all widespread clones of Acinetobacter baumannii in the United Kingdom. All isolates studied had a blaOXA-51-like carbapenemase gene; some also had blaOXA-23-like and/or blaOXA-58-like. Among isolates with blaOXA-51-like as sole carbapenemase gene, only those with ISAba1 adjacent to blaOXA-51-like were carbapenem resistant. Minor differences in blaOXA-51-like sequence were observed in resistant and susceptible isolates. Isolates with blaOXA-23-like in addition were consistently resistant to carbapenems; in all of these ISAba1 lay upstream of blaOXA-23-like, but was not associated with blaOXA-51-like. These results suggest that ISAba1 is providing the promoter for blaOXA-51-like and, probably, for blaOXA-23-like.

Development of a diagnostic test for the Midlands 1 cystic fibrosis epidemic strain of Pseudomonas aeruginosa.

In a previous study of isolates from cystic fibrosis (CF) patients in England and Wales, the Midlands 1 strain of Pseudomonas aeruginosa was identified as the second most common clone, representing 10% of isolates and found in nearly one-third of all CF centres [Scott, F. W. & Pitt, T. L. (2004). J Med Microbiol 53, 609-615]. Using suppression subtractive hybridization, 54 sequences were identified as present in a Midlands 1 strain but were absent from strain PAO1. The distribution of 14 of these sequences amongst representatives of Midlands 1, other CF epidemic strains and unrelated P. aeruginosa CF isolates was determined using PCR assays. Using these data, a PCR-based test was developed that was specific for the Midlands 1 clone, which was confirmed using dot-blot hybridization. By applying the test to CF isolates from a CF centre in Liverpool, a Midlands 1 clone was identified. The identity was confirmed using typing by PFGE. The PCR test should facilitate a greater understanding of the distribution of the Midlands 1 strain in the UK and elsewhere.

A proof-of-concept model for the identification of the key events in the infection process with specific reference to Pseudomonas aeruginosa in corneal infections.

BACKGROUND: It is a common medical practice to characterise an infection based on the causative agent and to adopt therapeutic and prevention strategies targeting the agent itself. However, from an epidemiological perspective, exposure to a microbe can be harmless to a host as a result of low-level exposure or due to host immune response, with opportunistic infection only occurring as a result of changes in the host, pathogen, or surrounding environment. METHODS: We have attempted to review systematically the key host, pathogen, and environmental factors that may significantly impact clinical outcomes of exposure to a pathogen, using Pseudomonas aeruginosa eye infection as a case study. RESULTS AND DISCUSSION: Extended contact lens wearing and compromised hygiene may predispose users to microbial keratitis, which can be a severe and vision-threatening infection. P. aeruginosa has a wide array of virulence-associated genes and sensing systems to initiate and maintain cell populations at the corneal surface and beyond. We have adapted the well-known concept of the epidemiological triangle in combination with the classic risk assessment framework (hazard identification, characterisation, and exposure) to develop a conceptual pathway-based model that demonstrates the overlapping relationships between the host, the pathogen, and the environment; and to illustrate the key events in P. aeruginosa eye infection. CONCLUSION: This strategy differs from traditional approaches that consider potential risk factors in isolation, and hopefully will aid the identification of data and models to inform preventive and therapeutic measures in addition to risk assessment. Furthermore, this may facilitate the identification of knowledge gaps to direct research in areas of greatest impact to avert or mitigate adverse outcomes of infection.

Development of 5' nuclease real-time PCR assays for the rapid identification of the burkholderia mallei//burkholderia pseudomallei complex.

Burkholderia pseudomallei is the causative agent of melioidosis and was classified as a biologic agent by the Centers for Disease Control and Prevention (Atlanta, GA). Acute melioidosis has a case fatality rate of >40%, and septicemia is fatal in up to 90%. The aim of the study was to design 5'-nuclease real-time PCR assays for the rapid and reliable identification of the B. mallei/B. pseudomallei complex. Real-time PCR assays using TaqMan probes targeting the 16S rDNA and fliC were developed on an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). Specificity was assessed with 64 B. pseudomallei, nine B. mallei, 126 other Burkholderia strains of 29 species, and 45 clinically relevant non-Burkholderia organisms. Sensitivity, specificity, and positive and negative predictive value of the assays were 100%. Discrimination between B. pseudomallei and B. mallei, an organism which can be regarded as a clone of B. pseudomallei, could not be achieved. A probit analysis revealed that 7.5 and 52 genome equivalents (GE) of B. pseudomallei could be detected using the fliC and the 16S rDNA assays (P = .05), respectively. In spiked blood samples, the detection limit was approximately 300 and 3.000 GE for fliC and the 16S rDNA, respectively. In conclusion, we recommend the simultaneous use of the 16S rDNA and fliC real-time PCR assays for the rapid and specific identification of the B. mallei/B. pseudomallei complex in positive blood cultures or from suspicious bacterial colonies allowing the early onset of appropriate antibiotic therapy.

Identification and characterization of transmissible Pseudomonas aeruginosa strains in cystic fibrosis patients in England and Wales.

Most past studies of cross-infection with Pseudomonas aeruginosa among cystic fibrosis (CF) patients in the UK suggest that it is a rare occurrence. However, two recent reports of highly transmissible strains in patients in regional centres in England (Liverpool and Manchester) have raised questions as to the extent of the problem and prompted a nationwide survey to establish the distribution of P. aeruginosa strain genotypes among these patients. Isolates of P. aeruginosa were requested from over 120 hospitals in England and Wales and a sample size of approximately 20% of the CF patient population in each centre was recommended. In total, 1225 isolates were received from 31 centres (range 1 to 330). Single patient isolates were typed by SpeI macrorestriction and PFGE. A panel of strains of the common genotypes including representatives of reported transmissible strains was assembled and further characterized by fluorescent amplified fragment length polymorphism (FAFLP) genotyping. At least 72% of all patients harboured strains with unique genotypes. Small clusters of related strains were evident in some centres, presumably indicating limited transmission of local strains. The most prevalent strain was indistinguishable from that previously described as the 'Liverpool' genotype, and accounted for approximately 11% of patient isolates from 15 centres in England and Wales. The second most common genotype (termed Midlands 1) was recovered from 86 patients in nine centres and the third genotype, which matched closely the PFGE profile of Clone C, a genotype originally described in Germany, was found in eight centres and was isolated from 15 patients. A fourth genotype, identical to the published Manchester strain, was found in three centres. FAFLP analysis revealed some microheterogeneity among strains of the Liverpool genotype but all isolates of this genotype were positive by PCR for a strain-specific marker. These data suggest that cross-infection with P. aeruginosa has occurred both within and widely between CF centres in England and Wales. The two most common genotypes accounted for more than one-fifth of patients' isolates examined and transmissible genotypes were found in all but three centres studied. These results emphasize the need for continued surveillance of P. aeruginosa genotypes in CF patients to provide informed infection control policy in treatment centres.

Fecal transplantation: passing fashion or here to stay?

Several trillions of bacteria, distributed among more than 1,000 species, are natural inhabitants of the human intestinal tract and constitute what is now known as the gut microbiota. Although its composition varies within and between individuals with age, diet, and health status, it is becoming increasingly recognized that imbalances in the bacterial microbiota (dysbiosis) are linked to a number of conditions such as antibiotic-associated diarrhea, inflammatory bowel disease, and obesity, among others. Fecal transplantation where a preparation of stool from a microbiologically screened donor is administered into the colon of an affected recipient has been shown to be highly effective for the treatment of recurrent Clostridium difficile infection. Several trials of this therapy are now underway for gut dysbiosis in a number of patient disease groups raising concerns on the risk of transmission of infectious agents from donor to recipient, possible long-term adverse consequences of treatment, and effective regulation of the stool material used for the procedure. A worrying aspect is the emergence of private stool banks providing samples to the general public for self-administration.

Strains of Burkholderia cenocepacia genomovar IIIA possessing the cblA gene that are distinct from ET12.

Three strains of Burkholderia cenocepacia genomovar IIIA that were polymerase chain reaction positive for cblA, bcrA, and the epidemic strain marker, but were distinct from representatives of ET12 by pulsed-field gel electrophoresis, are described. One of these strains was shown to express cable pili by electron microscopy.