Epidemiology and clinical course of Burkholderia cepacia complex infections, particularly those caused by different Burkholderia cenocepacia strains, among patients attending an Italian Cystic Fibrosis Center

In this study, the epidemiology of Burkholderia cepacia complex (Bcc) recovered from the sputum of 75 patients attending the Genoa Cystic Fibrosis (CF) Center at the Gaslini Children's Hospital (Genoa, Italy) was investigated, and the clinical course of the CF patients infected with the different species and genomovars of Bcc was evaluated. All isolates were analyzed for genomovar status by recA gene polymorphism and subsequently random amplified polymorphic DNA fingerprinting. Burkholderia cenocepacia is the predominant species recovered from the CF patients infected with Bcc at the Genoa CF Center. Of the other eight species comprising the Bcc, only a few isolates belonging to B. cepacia genomovar I, Burkholderia stabilis, and Burkholderia pyrrocinia were found. Of the four recA lineages of B. cenocepacia, most patients were infected by epidemic strains belonging to lineages IIIA and IIID, whereas only a few patients harbored IIIB strains. Patient-to-patient spread of Bcc among CF patients was mostly associated with B. cenocepacia, in particular with strains belonging to recA lineages IIIA and IIID. The mortality of CF patients infected with Bcc at the Genoa CF Center was significantly higher than mortality among CF patients not infected with Bcc. All of the deaths were associated with the presence of B. cenocepacia, except the case of a patient infected with B. cepacia genomovar I. Within B. cenocepacia, infection with epidemic strains belonging to lineages IIIA and IIID was associated with higher rates of mortality than was infection with lineage IIIB strains. No significant differences in lung function, body weight, and mortality rate were observed between patients infected with epidemic strains belonging to either B. cenocepacia IIIA or B. cenocepacia IIID.     

PCR-Based Detection and Identification of Burkholderia cepacia Complex Pathogens in Sputum from Cystic Fibrosis Patients

PCR amplification of the recA gene followed by restriction fragment length polymorphism (RFLP) analysis was investigated for the rapid detection and identification of Burkholderia cepacia complex genomovars directly from sputum. Successful amplification of the B. cepacia complex recA gene from cystic fibrosis (CF) patient sputum samples containing B. cepacia genomovar I, Burkholderia multivorans, B. cepacia genomovar III, Burkholderia stabilis, and Burkholderia vietnamiensis was demonstrated. In addition, the genomovar identifications determined directly from sputum were the same as those obtained after selective culturing. Sensitivity experiments revealed that recA-based PCR could reliably detect B. cepacia complex organisms to concentrations of 10(6) CFU g of sputum(-1). To fully assess the diagnostic value of the method, sputum samples from 100 CF patients were screened for B. cepacia complex infection by selective culturing and recA-based PCR. Selective culturing identified 19 samples with presumptive B. cepacia complex infection, which was corroborated by phenotypic analyses. Of the culture-positive sputum samples, 17 were also detected directly by recA-based PCR, while 2 samples were negative. The isolates cultured from both recA-negative sputum samples were subsequently identified as Burkholderia gladioli. RFLP analysis of the recA amplicons revealed 2 patients (12%) infected with B. multivorans, 11 patients (65%) infected with B. cepacia genomovar III-A, and 4 patients (23%) infected with B. cepacia genomovar III-B. These results demonstrate the potential of recA-based PCR-RFLP analysis for the rapid detection and identification of B. cepacia complex genomovars directly from sputum. Where the sensitivity of the assay proves a limitation, sputum samples can be analyzed by selective culturing followed by recA-based analysis of the isolate.     

Differences in invasion and translocation of Burkholderia cepacia complex species in polarised lung epithelial cells in vitro

In order to investigate the mechanisms by which Burkholderia cepacia complex (Bcc) strains cross the epithelial barrier of the lung and cause septicaemia in a subgroup of Cystic Fibrosis (CF) patients, the invasiveness of four Bcc species have been examined in three lung epithelial cells: A549, 16HBE14o- and Calu-3. The latter two cell lines form polarised monolayers when grown on filters. Invasion of both cell lines by B. multivorans strains was reduced when the cells were grown as tight monolayers compared unpolarised cells, suggesting basolateral receptors are required for the process. In contrast, four B. cenocepacia strains showed comparable invasion of both cell lines irrespective of culture model. All four species of Bcc reduced the TER of Calu-3 monolayers. However, while B. cepacia, B. multivorans and B. stabilis strains readily translocated across the epithelial monolayer, B. cenocepacia translocation was slower. Both B. multivorans and B. cenocepacia altered expression of ZO-1 in Calu-3 cells, but not E-cadherin. Overall, the findings that Bcc strains from four species, which differ greatly in their virulence, have the potential to disrupt tight junctions and to translocate across the epithelium, demonstrates this effect is not exclusive to the most virulent species.     

Evaluation of species-specific recA-based PCR tests for genomovar level identification within the Burkholderia cepacia complex

The Burkholderia cepacia complex presently comprises nine genomovars: B. cepacia (genomovar I), B. multivorans (genomovar II), B. cepacia genomovar III, B. stabilis (genomovar IV), B. vietnamiensis (genomovar V), B. cepacia genomovar VI, B. ambifaria (genomovar VII), B. anthina (genomovar VIII) and B. pyrrocinia (genomovar IX). Strains of each genomovar can colonise the respiratory tract of cystic fibrosis (CF) patients. However, the majority of infections in CF patients are caused by B. multivorans and B. cepacia genomovar III isolates. Accurate genomovar-level identification is best achieved through a polyphasic approach combining phenotypic and genotypic analyses. In the present study, the sensitivity and specificity of recA-based genomovar specific primer pairs were evaluated with a collection of 508 B. cepacia complex isolates representing all nine genomovars. The assays for the identification of B. multivorans (sensitivity and specificity, 100%), B. cepacia genomovar III (sensitivity, 92%; specificity, 100%), and B. ambifaria (sensitivity and specificity, 100%) were the most efficient. However, the B. cepacia genomovar I assay lacked sensitivity (72%) and cross-reacted with all B. pyrrocinia isolates examined. Several new recA RFLP types were also revealed within the B. cepacia complex. One of these profiles was shared by a clinical and an environmental B. cepacia-like isolate and by the B. ubonensis type strain. The latter organism is a recently described soil bacterium. Its relationship to the various B. cepacia complex genomovars needs further study.     

<Emphasis Type="Italic">Burkholderia cepacia</Emphasis> complex in Serbian patients with cystic fibrosis: prevalence and molecular epidemiology

The Burkholderia cepacia complex (Bcc) organisms remain significant pathogens in patients with cystic fibrosis (CF). This study was performed to evaluate the prevalence, epidemiological characteristics, and presence of molecular markers associated with virulence and transmissibility of the Bcc strains in the National CF Centre in Belgrade, Serbia. The Bcc isolates collected during the four-year study period (2010–2013) were further examined by 16 s rRNA gene, pulsed-field gel electrophoresis of genomic DNA, multilocus sequence typing analysis, and phylogenetic analysis based on concatenated sequence of seven alleles. Fifty out of 184 patients (27.2 %) were colonized with two Bcc species, B. cenocepacia (n?=?49) and B. stabilis (n?=?1). Thirty-four patients (18.5 %) had chronic colonization. Typing methods revealed a high level of similarity among Bcc isolates, indicating a person-to-person transmission or acquisition from a common source. New sequence types (STs) were identified, and none of the STs with an international distribution were found. One centre-specific ST, B. cenocepacia ST856, was highly dominant and shared by 48/50 (96 %) patients colonized by Bcc. This clone was characterized by PCR positivity for both the B. cepacia epidemic strain marker and cable pilin, and showed close genetic relatedness to the epidemic strain CZ1 (ST32). These results indicate that the impact of Bcc on airway colonization in the Serbian CF population is high and virtually exclusively limited to a single clone of B. cenocepacia. The presence of a highly transmissible clone and probable patient-to-patient spread was observed.     

Effect of Fusarium verticillioides on maize-root-associated Burkholderia cenocepacia populations

Burkholderia cepacia complex (Bcc) bacteria are naturally present in the rhizosphere of several crop plants and have been found to antagonize a wide range of important plant pathogens. In this study, we evaluated the effect of the pathogenic fungus Fusarium verticillioides on Bcc populations recovered from the roots of Zea mays plants. Maize plants were cultivated under greenhouse conditions and bacterial colonies were randomly isolated from distinct root portions of Fusarium-treated and control plants. We obtained a total of 120 Bcc isolates which all belonged to the species Burkholderia cenocepacia, a species of the Bcc widely distributed in natural habitats such as the rhizosphere of several crop plants. Results obtained revealed that the presence of the plant pathogen F. verticillioides had an effect at the root colonization level of B. cenocepacia populations, since an increase in indigenous B. cenocepacia bacteria was found in the rhizospheres of maize plants grown in infested soil, compared to the rhizospheres of control plants. The analysis of diversity indices as well as the investigation of genetic polymorphism of B. cenocepacia strains, isolated from Fusarium-treated and control root portions, revealed greater genetic variability in the presence of F. verticillioides, especially in the terminal root system portion. Finally, all B. cenocepacia isolates were also tested for in vitro inhibition of F. verticillioides growth as a functional property. Our results revealed that all B. cenocepacia isolates were able to restrict in vitro fungal growth, suggesting that there was no relationship between genetic polymorphism and biocontrol traits.     

Transmission of Burkholderia cepacia complex: evidence for new epidemic clones infecting cystic fibrosis patients in Italy

To analyze national prevalence, genomovar distribution, and epidemiology of the Burkholderia cepacia complex in Italy, 225 putative B. cepacia complex isolates were obtained from 225 cystic fibrosis (CF) patients attending 18 CF centers. The genomovar status of these isolates was determined by a polyphasic approach, which included whole-cell protein electrophoresis and recA restriction fragment length polymorphism (RFLP) analysis. Two approaches were used to genotype B. cepacia complex isolates: BOX-PCR fingerprinting and pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments. A total of 208 (92%) of 225 isolates belonged to the B. cepacia complex, with Burkholderia cenocepacia as the most prevalent species (61.1%). Clones delineated by PFGE were predominantly linked to a single center; in contrast, BOX-PCR clones were composed of isolates collected either from the same center or from different CF centers and comprised multiple PFGE clusters. Three BOX-PCR clones appeared of special interest. One clone was composed of 17 B. cenocepacia isolates belonging to recA RFLP type H. These isolates were collected from six centers and represented three PFGE clusters. The presence of insertion sequence IS 1363 in all isolates and the comparison with PHDC reference isolates identified this clone as PHDC, an epidemic clone prominent in North American CF patients. The second clone included 22 isolates from eight centers and belonged to recA RFLP type AT. The genomovar status of strains with the latter RFLP type is not known. Most of these isolates belonged to four different PFGE clusters. Finally, a third clone comprised nine B. pyrrocinia isolates belonging to recA RFLP type Se 13. They represented three PFGE clusters and were collected in three CF centers.     

Correlation of wbiI genotype, serotype, and isolate source within species of the Burkholderia cepacia complex

Gram-negative bacteria of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that can infect the lungs of cystic fibrosis (CF) patients and can be transmitted among these patients, causing epidemics in the CF community. Lipopolysaccharide (LPS) is an important virulence factor of many gram-negative bacteria, with the O antigen component of LPS being responsible for serotype specificity. The goal of this work was to develop a genetic method of determining the serotype of Bcc isolates based on the conserved gene wbiI. Homologues of wbiI are found in polysaccharide biosynthesis gene clusters in other bacteria. Primers to a conserved region of the Bcc wbiI gene were able to amplify by PCR a single product in 67 of 80 Bcc isolates tested. Sequencing and restriction enzyme digestion of this wbiI PCR product revealed sufficient DNA polymorphisms to distinguish and group various isolates. In five of nine instances, Bcc isolates of a single serotype had a single wbiI restriction fragment length polymorphism (RFLP) pattern, while isolates of the other four serotypes could have multiple wbiI RFLP types. Species determination of the Bcc isolates revealed no obvious correlation between wbiI RFLP type and species. There was also no apparent correlation between wbiI RFLP type and the ability of a single Bcc isolate to infect an individual with CF. However three of five Bcc outbreaks involved isolates with the same wbiI RFLP type, indicating that wbiI RFLP typing may be a useful tool to help track Bcc outbreaks.     

Studies on the involvement of the exopolysaccharide produced by cystic fibrosis-associated isolates of the Burkholderia cepacia complex in biofilm formation and in persistence of respiratory infections

Bacteria belonging to the Burkholderia cepacia complex (BCC) are important opportunistic pathogens that lead to respiratory infections in patients with cystic fibrosis (CF). The clinical outcome following colonization with BCC bacteria is highly variable, and so far, unpredictable. A large percentage (80 to 90%) of BCC isolates from CF patients produce the exopolysaccharide (EPS) cepacian, which has been hypothesized to play a role in the colonization and persistence of these bacteria in the CF lung. In this work, we demonstrate that although it is not required for the initiation of biofilm formation, cepacian plays a role in the establishment of thick biofilms. This conclusion was based on a comparison of the abilities of EPS-defective mutants derived from a B. cepacia mucoid CF isolate by random plasposon insertion mutagenesis and the ability of the parental strain to form biofilms. However, the systematic characterization of 108 CF isolates, corresponding to 15 distinct strains, indicated that other strain-dependent factors are also involved in the development of thick, mature biofilms. The isolates examined belonged to the species B. cepacia, B. multivorans, B. cenocepacia, and B. stabilis and were obtained during a 7-year period of surveillance from 21 CF patients receiving care at the major Portuguese CF center. Most of them (90%) were serial isolates from 12 persistently infected patients. In spite of the concept that bacteria growing in biofilms display more resistance to antibiotics and to host phagocyte killing than do planktonically growing cells, no clear correlation could be established between the ability of the various strains examined to produce EPS and/or to form biofilms in vitro and the persistence or virulence of the respiratory infections they caused in different patients.     

Molecular analysis of Burkholderia cepacia complex isolates from a Portuguese cystic fibrosis center: a 7-year study

This work reports results of a systematic molecular analysis involving 113 Burkholderia cepacia complex isolates obtained from 23 cystic fibrosis (CF) patients under surveillance over a 7-year period at the major Portuguese CF center, the Santa Maria Hospital in Lisbon. The majority of the isolates were serial isolates from persistently infected patients (more than one-half of the population examined). In agreement with previous studies, B. cenocepacia (formerly genomovar III) was the most prevalent species; it was isolated from 52% of the patients infected with B. cepacia complex isolates. Contrasting with previous studies, a very significant percentage of the Portuguese CF subpopulation examined was infected with B. cepacia genomovar I (36%) and B. stabilis (18%). B. multivorans was recovered from two of the infected patients. All four of the species or genomovars were associated with poor clinical outcome, including the cepacia syndrome, and gave rise to chronic and transient infections, with the clinical condition depending on the patient and other still-unidentified factors. The B. cepacia epidemic strain marker region was found exclusively in genomovar III strains, while cblA was detected in genomovars I and III, only. There was no clear relation between the presence of these markers and transmissibility. Altogether, our results indicate that the use of these markers or the genomovar status in identifying patients at higher risk for infection is uncertain.     

Epidemiology of Burkholderia cepacia infection in patients with cystic fibrosis: analysis by randomly amplified polymorphic DNA fingerprinting.

We fingerprinted a collection of 627 Burkholderia cepacia isolates from 255 patients with cystic fibrosis (CF) and 43 patients without CF and from the environment, by a PCR-based randomly amplified polymorphic DNA (RAPD) method with primers selected for their ability to produce discriminatory polymorphisms. The RAPD typing method was found to be reproducible and discriminatory, more sensitive than PCR ribotyping, and able to group epidemiologically related B. cepacia strains previously typed by both pulsed-field gel electrophoresis and conventional ribotyping. Seven strain types infecting multiple CF patients were found at several different CF treatment centers in Canada, the United States, the United Kingdom, France, and Australia, indicating the presence of epidemic strain types. Most CF patients were each colonized with a single strain type, and several patients harbored the same strain type for 5 or more years. B. cepacia isolates recovered from other clinical sources (44 isolates examined) and from the environment (58 isolates examined) possessed RAPD fingerprints that were generally distinct from CF-associated strain types (525 isolates examined). RAPD is a versatile fingerprinting method for studying the epidemiology of B. cepacia.     

Differentiation of Burkholderia Species by PCR-Restriction Fragment Length Polymorphism Analysis of the 16S rRNA Gene and Application to Cystic Fibrosis Isolates

Burkholderia cepacia, which is an important pathogen in cystic fibrosis (CF) owing to the potential severity of the infections and the high transmissibility of some clones, has been recently shown to be a complex of five genomic groups, i.e., genomovars I, II (B. multivorans), III, and IV and B. vietnamiensis. B. gladioli is also involved, though rarely, in CF. Since standard laboratory procedures fail to provide an accurate identification of these organisms, we assessed the ability of restriction fragment length polymorphism (RFLP) analysis of amplified 16S ribosomal DNA (rDNA), with the combination of the patterns obtained with six endonucleases, to differentiate Burkholderia species. This method was applied to 16 type and reference strains of the genus Burkholderia and to 51 presumed B. cepacia clinical isolates, each representative of one clone previously determined by PCR ribotyping. The 12 Burkholderia type strains tested were differentiated, including B. cepacia, B. multivorans, B. vietnamiensis, and B. gladioli, but neither the genomovar I and III reference strains nor the genomovar IV reference strain and B. pyrrociniaT were distinguishable. CF clinical isolates were mainly distributed in RFLP group 2 (which includes B. multivoransT) and RFLP group 1 (which includes B. cepacia genomovar I and III reference strains, as well as nosocomial clinical isolates). Two of the five highly transmissible clones in French CF centers belonged to RFLP group 2, and three belonged to RFLP group 1. The remaining isolates either clustered with other Burkholderia species (B. cepacia genomovar IV or B. pyrrocinia, B. vietnamiensis, and B. gladioli) or harbored unique combinations of patterns. Thus, if further validated by hybridization studies, PCR-RFLP of 16S rDNA could be an interesting identification tool and contribute to a better evaluation of the respective clinical risks associated with each Burkholderia species or genomovar in patients with CF.     

Hypermutation in Burkholderia cepacia complex is mediated by DNA mismatch repair inactivation and is highly prevalent in cystic fibrosis chronic respiratory infection

The Burkholderia cepacia complex (Bcc) represents an important group of pathogens involved in long-term lung infection in cystic fibrosis (CF) patients. A positive selection of hypermutators, linked to antimicrobial resistance development, has been previously reported for Pseudomonas aeruginosa in this chronic infection setting. Hypermutability, however, has not yet been systematically evaluated in Bcc species. A total of 125 well characterized Bcc isolates recovered from 48 CF patients, 10 non-CF patients and 15 environmental samples were analyzed. In order to determine the prevalence of mutators their spontaneous mutation rates to rifampicin resistance were determined. In addition, the genetic basis of the mutator phenotypes was investigated by sequencing the mutS and mutL genes, the main components of the mismatch repair system (MRS). The overall prevalence of hypermutators in the collection analyzed was 13.6%, with highest occurrence (40.7%) among the chronically infected CF patients, belonging mainly to B. cenocepacia, B. multivorans, B. cepacia, and B. contaminans –the most frequently recovered Bcc species from CF patients worldwide. Thirteen (76.5%) of the hypermutators were defective in mutS and/or mutL. Finally, searching for a possible association between antimicrobial resistance and hypermutability, the resistance-profiles to 17 antimicrobial agents was evaluated. High antimicrobial resistance rates were documented for all the Bcc species recovered from CF patients, but, except for ciprofloxacin, a significant association with hypermutation was not detected. In conclusion, in the present study we demonstrate for the first time that, MRS-deficient Bcc species mutators are highly prevalent and positively selected in CF chronic lung infections. Hypermutation therefore, might be playing a key role in increasing bacterial adaptability to the CF-airway environment, facilitating the persistence of chronic lung infections.     

Six-year molecular analysis of Burkholderia cepacia complex isolates among cystic fibrosis patients at a referral center for lung transplantation

Over a 6-year period, Burkholderia cepacia complex species were isolated from cystic fibrosis (CF) patients receiving care at The University of North Carolina Hospitals (clinic CF patients) and from those referred from other treatment centers. Fifty-six isolates collected from 30 referred patients and 26 clinic CF patients were characterized by pulsed-field gel electrophoresis (PFGE) and were assayed by PCR to detect the cable pilin gene, cblA. PFGE results indicated that six separate clusters (clusters A to F) were present among the 56 isolates and that three clusters (clusters A, B, and E) consisted only of isolates from referred patients infected with B. cepacia complex isolates prior to referral. However, one cluster (cluster C) consisted of isolates from four CF patients, and hospital records indicate that this cluster began with an isolate that came from a referred patient and that spread to three clinic CF patients. Cluster D consisted of two isolates from clinic CF patients, and hospitalization records are consistent with nosocomial, patient-to-patient spread. cblA was present in only 4 of the 56 isolates and included isolates in cluster E from the referred patients. Our results indicate a lack of spread of a previously characterized, transmissible clone from referred patients to our clinic CF population. Only two instances of nosocomial, patient-to-patient spread could be documented over the 6-year period. An additional spread of an isolate (cluster F) from a referred patient to a clinic patient could not be documented as nosocomial and may have been the result of spread in a nonhospitalized setting. The majority (36 of 56) of our B. cepacia complex-infected CF patients harbor isolates with unique genotypes, indicating that a diversity of sources account for infection. These data suggest that CF patients infected with B. cepacia complex and referred for lung transplantation evaluation were not a major source of B. cepacia complex strains that infected our resident CF clinic population.     

Burkholderia cenocepacia, B. multivorans, B. ambifaria and B. vietnamiensis isolates from cystic fibrosis patients have different profiles of exoenzyme production

Knowledge about the virulence mechanisms of species from the Burkholderia cepacia complex (BCC) is still limited. The genomovar heterogeneity and production of different virulence factors are likely to contribute to the variation in the clinical outcome observed in BCC-infected cystic fibrosis (CF) patients. Therefore, in this study we investigated the genetic polimorphism, the presence of genetic makers associated with virulence and transmissibility in BCC, and the profile of exoenzyme production of 59 BCC isolates obtained from 59 CF patients attending the reference CF centre in Rio de Janeiro, Brazil. The DNA sequence analyses of the recA gene allowed us to identify 40 of these 59 BCC species as being B. cenocepacia, 9 as B. vietnamiensis, 6 as B. multivorans and 4 as B. ambifaria. The assessment of the bacterial genetic polymorphism by PFGE revealed that B. cenocepacia and the B. multivorans isolates belonged to four and two different PFGE profiles with prevalence of two clones, A and B, respectively. All B. vietnamiensis and B. ambifaria belonged to only one PFGE profile (J and E, respectively). None of the isolates exhibited the genetic markers cblA and BCESM, assessed by polymerase chain reaction. In contrast, the profile of enzymatic activity, assessed by phenotypic methods, differed among the BCC species: protease activity was detected only in B. cenocepacia and B. ambifaria isolates, whereas only B. vietnamiensis isolates produced hemolysin. Although the phospholipase C activity was similar among the different species, the level of lipase activity produced by B. multivorans was higher than in the other species. We speculate that the differential characteristics of exoenzyme production may account for the differences in the pathogenic potentials of each BCC species.     

Revised approach for identification of isolates within the Burkholderia cepacia complex and description of clinical isolates not assigned to any of the known genomovars

One hundred thirty-eight clinical isolates of the Burkholderia cepacia complex (Bcc) were identified using a modified strategy that involved PCR detection of the cblA gene for the ET12 lineage simultaneously with detection of the Bcc recA PCR product; recA sequence cluster analysis also was part of the strategy. Four strains could not be assigned to any of the known genomovars.     

Distribution of genes encoding putative transmissibility factors among epidemic and nonepidemic strains of Burkholderia cepacia from cystic fibrosis patients in the United Kingdom

In the last 15 years, Burkholderia cepacia has emerged as a significant pathogen in cystic fibrosis (CF) patients, mainly due to the severity of infection observed in a subset of patients and the fear of transmission of the organism to noncolonized patients. Although patients who deteriorate rapidly cannot be predicted by microbiological characteristics, three genetic markers have been described for strains that spread between patients. These are the cblA gene, encoding giant cable pili; a hybrid of two insertion sequences, IS1356 and IS402; and a 1.4-kb open reading frame known as the B. cepacia epidemic strain marker (BCESM). The latter two are of unknown function. An epidemic strain lineage was previously identified among CF patients in the United Kingdom that apparently had spread from North America and that was characterized by a specific random amplified polymorphic DNA (RAPD) pattern. We searched for the described genetic markers using specific PCR assays with 117 patient isolates of B. cepacia from 40 United Kingdom hospitals. Isolates were grouped according to genomovar and epidemic strain lineage RAPD pattern with a 10-base primer, P272. A total of 41 isolates from patients in 12 hospitals were classified as the epidemic strain, and 40 of these were distributed in genomovars IIIa (11 isolates), IIIb (1 isolate), and IIIc (28 isolates). All isolates of the epidemic strain were positive for the cblA gene and BCESM, but two lacked the insertion sequence hybrid. None of the 76 sporadic isolates contained cblA or the insertion sequence hybrid, but 11 of them were positive for BCESM. Nonepidemic isolates were distributed among genomovars I or IV (9), II (49), IIIa (11), IIIb (3), and IIIc (4). There were three clusters of cross-infection (one involving two patients and two involving three patients) with isolates of genomovar II. We conclude that in the United Kingdom, a single clonal lineage has spread between and within some hospitals providing care for CF patients. The presence of the cblA gene is the most specific marker for the epidemic strain. We recommend that all isolates of B. cepacia from CF patients should be screened by PCR to influence segregation and infection control strategies.     

Molecular typing of,and distribution of genetic markers among,Burkholderia cepacia complex isolates from Brazil

PCR tests were used to assign genomovar status to 39 non-cystic fibrosis (non-CF) and 11 CF Burkholderia cepacia complex isolates from patients in hospitals in Recife, Brazil. Non-CF isolates were assigned to genomovar IIIA (71.8%), genomovar I (15.4%), B. vietnamiensis (7.7%), and B. multivorans (5.1%). CF isolates were assigned to genomovar IIIA (18.2%), B. vietnamiensis (18.2%), and genomovar I (9.1%). Six CF isolates sharing recA PCR-restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) patterns could not be assigned to a genomovar. 16S rDNA sequence obtained from these isolates indicated a closest relationship to B. anthina, but the recA sequence was equally divergent from several genomovars. PCR screening indicated the presence of cblA in only two isolates, whereas the B. cepacia epidemic strain marker was found in 22 of 28 genomovar IIIA isolates. A type III secretion gene was detected in all but genomovar I isolates. RAPD and PCR-RFLP assays, targeting both recA and fliC, indicated a large amount of genetic variability among the isolates, with many novel patterns being observed. Nine genomovar IIIA isolates from different non-CF patients and clinical sources had identical genotypes, indicating the presence of a common clone.     

Burkholderia cenocepacia sp. nov.--a new twist to an old story

DNA-DNA hybridisation experiments between isolates representing Burkholderia cepacia genomovar III recA lineages IIIA and IIIB reinforced the classification of both phylogenetic subgroups as a single genospecies, distinct from B. cepacia (genomovar I). A formal classification of B. cepacia genomovar III encompassing the recA lineages IIIA and IIIB, and the new recA lineages IIIC and IIID, as B. cenocepacia sp. nov., with LMG 16656 as the type strain, is proposed.     

Are all community methicillin-resistant Staphylococcus aureus related? A comparison of their mec regions

AIMS: To compare the relationship of community-acquired, methicillin-resistant Staphylococcus aureus (CMRSA) from five Australian States and New Zealand. METHODS: Contour-clamped homogeneous electric field (CHEF) electrophoresis and analysis of the mec complex and ccr gene complex by PCR were used to compare 22 CMRSA isolates from Western Australia (WA), South Australia (SA), Victoria (VIC), New South Wales (NSW) and New Zealand (NZ) and three hospital-acquired epidemic MRSA (EMRSA). RESULTS: Sixteen community isolates were found to carry Class B mec complex and Type 2 ccr gene complex. Two WA isolates carried the Class B1 mec complex and three VIC and one SA isolate carried a previously unreported mec complex, which has been labelled E. The ccr gene type of the Class B1 and Class E isolates could not be determined. These isolates may carry previously unreported ccr gene complexes. The relatedness of the CHEF patterns of the CMRSA was dependent on their geographical origin. A similar CHEF pattern was found in some WA MRSA, VIC and SA isolates. NSW and NZ CMRSA had the same CHEF patterns and were similar to three VIC isolates and EMRSA-16. Two SA CMRSA isolates had CHEF patterns similar to the English EMRSA-15 strain. A multiply resistant, nosocomial EMRSA from Australia had a class A mec complex, and a CHEF pattern, which was unrelated to any of the CMRSA. CONCLUSION: Most of the CMRSA isolated from five Australian states and New Zealand had unrelated CHEF patterns. However, the majority of them carried the Type IV SCCmec cassette (Class B mec and Type 2 ccr gene complexes), which indicates that they may have acquired their mec complex from the same source or that they have evolved from the same progenitor. Some of the CMRSA had a previously undescribed SCCmec cassette.