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.     

Development of a multilocus sequence typing scheme for the opportunistic pathogen Pseudomonas aeruginosa

A multilocus sequence typing (MLST) scheme has been developed for Pseudomonas aeruginosa which provides molecular typing data that are highly discriminatory and electronically portable between laboratories. MLST data confirm the data from previous studies that suggest that P. aeruginosa is best described as nonclonal but as having an epidemic population. The index of association was 0.17, indicating a freely recombining population; however, there was evidence of clusters of closely related strains or clonal complexes among the members of this population. It is apparent that the sequence types (STs) from single isolates, representing each of the present epidemic clones in the United Kingdom from Liverpool, Manchester, and the West Midlands, are not closely related to each other. This suggests distinct evolutionary origins for each of these epidemic clones in the United Kingdom. Furthermore, these clones are distinct from European clone C. Comparison of the results of MLST with those of toxA typing and serotyping revealed that strains with identical STs may possess different toxA types and diverse serotypes. Given that recombination is important in the population of P. aeruginosa, the lack of a linkage between toxA type and serotype is not surprising and reveals the strength of the MLST approach for obtaining a better understanding of the epidemiology of P. aeruginosa.     

Identification of DNA markers for a transmissible Pseudomonas aeruginosa cystic fibrosis strain

A number of transmissible Pseudomonas aeruginosa strains have been identified which potentially constitute an emerging threat to patients with cystic fibrosis (CF). We sought to identify DNA markers that were specific to a transmissible P. aeruginosa CF clone and evaluate these probes on a large collection of genotypically distinct P. aeruginosa strains. Using subtractive DNA hybridization, in combination with analysis using the P. aeruginosa PAO1 genome chip, DNA markers specific for or absent from the Manchester transmissible CF strain (MA) were identified. Five subtractive DNA hybridization markers (MA15, MA18, MA21, MA22, and MA30) were found to be specific to strain MA and were located within a novel 13,318-bp genomic island, designated the MA island. The MA island encoded 18 genes and consisted of two bacteriophage-like regions; one region encoded the MA-specific subtractive hybridization markers, while the other bacteriophage-like region contained a Vibrio cholera-like toxin gene. Probes MA15, MA18, MA21, MA22, and MA30 were all found to be specific to strain MA when a collection of 141 P. aeruginosa strains was examined by hybridization with each DNA marker. In contrast, a previously isolated DNA marker for the Liverpool transmissible CF strain, PS21, was not found to be specific, detecting two additional strain types in the collection screened. Both the Manchester and Liverpool strain types were not encountered in CF populations outside the United Kingdom. The MA genomic island and Vibrio cholera-like toxin gene within it constitute novel genetic factors associated with a transmissible P. aeruginosa strain and their role in pathogenesis remains to be determined.     

Identification of IS1356, a new insertion sequence, and its association with IS402 in epidemic strains of Burkholderia cepacia infecting cystic fibrosis patients.

Burkholderia cepacia is now recognized as an important opportunistic pathogen in cystic fibrosis (CF) and other compromised patients. Epidemicity among CF patients has been attributed to at least one particularly infectious strain (strain ET12), and both genetic evidence and anecdotal evidence suggest that this strain, currently endemic in Ontario, and those causing an epidemic in the United Kingdom, are indeed the same. Our study was conducted to determine whether there was any association between the presence of various insertion sequence (IS) elements, the cable pilin subunit gene (cblA), electrophoretic type (ET), and ribotype (RT) in a collection of 97 clinical and 2 environmental isolates of B. cepacia. No apparent linkage was found for IS elements IS401, IS402, IS406, IS407, and IS408 with ET or RT. The cblA target, said to be a marker for high infectivity, was detected in 100% (38 of 38) of strains of B. cepacia ET12 and in a single strain of ET13 that differed in a single enzyme allele. A new IS, IS1356, identified during the investigation, was present in 71.7% of all isolates, and 50.7% of these isolates harbored IS1356 as a hybrid IS element inserted into IS402. IS1356 is 1,353 bp in length, and when it is inserted into IS402 it results in a 10-bp duplication at the site of insertion. IS1356 contains one major open reading frame of 1,260 bp coding for a putative transposase which has significant homology to ISRm3 in Rhizobium meliloti (59%) and to an undesignated IS element in Corynebacterium diphtheriae (49%). The IS402-IS1356 element was found exclusively in the epidemic strains from Ontario and the United Kingdom, being detected in 94.7% (36 of 38 isolates) of B. cepacia ET12 isolates. Of the two ET12 isolates found to be devoid of the IS402-IS1356 element, both contained IS1356 unassociated with IS402, one was temporally unrelated to the epidemic, and the other was from a CF patient in a geographic area remote from Ontario and the United Kingdom. It is evident that the IS402-IS1356 hybrid element, the cblA pilin subunit gene, and the allelic suite represented by multilocus enzyme electrophoretic type ET12 may provide useful markers for the epidemic, highly transmissible transatlantic strain isolated in Ontario and the United Kingdom.     

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.     

International typing study of toxin A-negative,toxin B-positive Clostridium difficile variants

Clinically important strains of Clostridium difficile that do not produce toxin A but produce toxin B and are cytotoxic (A(-)/B(+)) have been reported from multiple countries. In order to compare the relatedness of these strains, we typed 23 A(-)/B(+) C. difficile isolates from the United Kingdom (6 isolates), Belgium (11 isolates), and the United States (6 isolates) by three well-described typing methods. Restriction endonuclease analysis (REA), PCR ribotyping, and serogrouping differentiated 11, 4, and 3 different strain types, respectively. Twenty-one of the 23 A(-)/B(+) variants had a 1.8-kb truncation of the toxin A gene characteristic of toxinotype VIII strains; 20 of the 21 toxinotype VIII-like strains were PCR type 17. PCR type 17 isolates could be differentiated into two separate strain groups by serogrouping and by REA. REA further discriminated these isolates into eight subgroups (REA types). PCR type 17-serogroup F-REA group CF isolates were recovered from all three countries, and one specific REA type, CF4, was recovered from patients with C. difficile disease in the United Kingdom and the United States. C. difficile A(-)/B(+) variants of apparent clonal origin are widely distributed in Europe and North America.     

Characteristics of a new epidemic MRSA in Germany ancestral to United Kingdom EMRSA 15

In 1996 a new epidemic MRSA emerged in three hospitals North of Berlin. This strain, Barnim epidemic MRSA, was isolated in 15 hospitals in Northern Germany in 1997 and 29 hospitals throughout Germany in 1998. Isolates of this clone are non-typeable by phages, its resistance phenotype is PEN, OXA, ERY, CLI, CIP (genotype: mecA, ermC, mutations in grlA and gyrA). The Sma I macrorestriction pattern corresponds to particular phage group II strains which is confirmed by the 16S-23S rRNA gene spacer pattern. Isolates of this clone differ by less than three Sma I macrorestriction fragments from isolates of the EMRSA15 clone from the United Kingdom, the most common epidemic MRSA isolates in the United Kingdom in recent years. Both epidemic strains produce enterotoxin C and possess the sec determinant for this toxin, the configuration of the mec regulon is mecI-, mecRB+, mecRC+. Both share the same Alu I pattern of PCR amplimers of the 3' end region of the coagulase gene. EMRSA 15 and Barnim EMRSA share a common multilocus sequence type indicating a recent, shared evolutionary origin.     

Genotypic analysis of Burkholderia cepacia isolates from 13 French cystic fibrosis centers.

Burkholderia cepacia has been involved in outbreaks of pulmonary infection among patients with cystic fibrosis (CF), and the spread of a highly transmissible clone has been reported throughout the United Kingdom and Canada. These data prompted a DNA-based typing study of the strains recovered in French CF centers. Ninety-five isolates recovered from 71 patients attending 13 CF centers in 9 regions of France were characterized by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Twenty-one genotypes were identified among the 95 isolates, and the results of RAPD and PFGE were concordant for 89 isolates (94%). Cross-colonization was demonstrated in 7 of the 13 CF centers. The investigation of serial isolates showed that most chronically colonized patients harbored a single B. cepacia strain. A geographically clustered distribution of B. cepacia genotypes was observed, except for one genotype, which was detected in four regions but was proven to be different from the genotype of the British-Canadian highly transmissible strain. The present study confirms the ability of B. cepacia to spread among CF communities in France and the importance of epidemiological surveys in the institution of prevention policies.     

Detection and typing of integrons in epidemic strains of Acinetobacter baumannii found in the United Kingdom

Integrons were sought in Acinetobacter isolates from hospitals in the United Kingdom by integrase gene PCR. Isolates were compared by pulsed-field gel electrophoresis, and most belonged to a small number of outbreak strains or clones of A. baumannii, which are highly successful in the United Kingdom. Class 1 integrons were found in all of the outbreak isolates but in none of the sporadic isolates. No class 2 integrons were found. Three integrons were identified among the main outbreak strains and clones. While a particular integron was usually associated with a strain or clone, some members carried a different integron. Some integrons were associated with more than one strain. The cassette arrays of two of the integrons were very similar, both containing gene aacC1, which confers resistance to gentamicin, two open reading frames coding for unknown products (orfX, orfX'), and gene aadA1a, which confers resistance to spectinomycin and streptomycin. The larger of these integrons had two copies of the first (orfX) of the gene cassettes coding for unknown products. The third integron, with a cassette array containing gene aacA4, which codes for amikacin, netilmicin, and tobramycin resistance; a chloramphenicol acetyltransferase, catB8; and gene aadA1, conferring resistance to spectinomycin and streptomycin, was associated with an OXA-23 carbapenemase-producing clone, which has spread rapidly in hospitals in the United Kingdom during 2003 and 2004. These integron cassette arrays have been found in other outbreak strains of A. baumannii from other countries. We conclude that integrons are useful markers for epidemic strains of A. baumannii and that integron typing provides valuable information for epidemiological studies.     

Involvement of SHV-12 and SHV-2a encoding plasmids in outbreaks of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a Tunisian neonatal ward

Previous genotypic investigations of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae recovered in a Tunisian neonatal ward revealed the spread of two epidemic strains and a high number of genetically unrelated isolates. The aim of the present study was to determine the role of the dissemination of self-transferrable plasmids harboring bla genes in the outbreaks experienced by the ward. The 49 previously identified clinical isolates of ESBL-producing K. pneumoniae were examined for relationships between their enzymes and plasmids. Analysis of crude extracts by isoelectric focusing showed four beta-lactamase-activities at pI 8.2, 7.6, 6, and 5.4. Clinical isolates contained large plasmids that could be transferred by conjugation and transformation conferring resistance to expanded-spectrum cephalosporins. DNA amplification and sequencing were performed to confirm the identities of transferred beta-lactamases. Nucleotide sequence analysis of SHV-specific PCR products from six isolates identified two bla(SHV) genes corresponding to SHV derived ESBLs, SHV-12 and SHV-2a. PstI digestion of plasmid DNA from transformants revealed six restriction patterns. The occurrence of the prevalent plasmid pattern in both epidemic strains and unrelated isolates indicated that diffusion and endemic persistence of the bla(SHV-ESBL) genes in the ward were due to concomitant spread of epidemic strains and plasmid dissemination among unrelated strains.     

Linkage analysis of geographic and clinical clusters in Pseudomonas cepacia infections by multilocus enzyme electrophoresis and ribotyping.

Multilocus enzyme electrophoresis and ribotyping were used to characterize 83 strains of Pseudomonas cepacia, mostly isolated from cystic fibrosis (CF) patients, although a number of isolates from non-CF nosocomial infections and reference environmental strains were represented. Twenty enzyme electrophoretic types (ETs) were determined; of these, one clone (ET12) was associated with six of nine ribotypes (RTs) said to be geographically representative of the United Kingdom and all of the Ontario (Canada) isolates from CF patients. This clone was not associated with nosocomial infections or environmental strains and was never found in CF isolates from British Columbia or Nova Scotia, Canada, or a center in the eastern United States. Individual isolate EcoRI RT signatures did not cluster geographically as did the ET signatures by clonal analysis. Frequently RTs occurred in more than a single ET. Known point source focal nosocomial outbreaks were typified by single ETs and stable RTs. Dendrographic analysis of the strains grouped those strains from CF patients, nosocomial outbreaks, and environmental sources into separate ET families, and diversity analysis indicated that, with the exception of ET17, CF isolates clustered in unique and closely related ETs different from those from nosocomial and environmental sources. This study has also shown the potential of multilocus enzyme electrophoresis to monitor the intercontinental spread of P. cepacia strains in CF patients, and this may have a significant impact on plans for CF patient summer camps and design of infection control practices. Whether the intercontinental ET12 clone, which predominates in the United Kingdom and the province of Ontario, linked by summer camp acquisition, has increased virulence for CF patients remains to be established.     

Molecular epidemiology of human enterovirus 71 in the United Kingdom from 1998 to 2006

The last decade witnessed a significant increase in epidemic activity of human enterovirus 71 (EV71) in the Western Pacific Region (WPR). In most European countries, this risk is unrecognized despite occasional cases of severe disease and two severe outbreaks in Eastern Europe 30 years ago. In this study we report the first examination of the molecular epidemiology of EV71 in the United Kingdom from 1998 to 2006. Genomic regions encoding the 1D coat protein (VP1) and 3D polymerase (Pol) from 32 EV71 isolates associated with neurological or cutaneous manifestations were sequenced. Phylogenetic analyses of VP1 and 3D Pol sequences identified genotype C as the dominant strain. Several United Kingdom isolates had genetic linkages with predated C1 or C2 strains from Europe and the WPR. Recombination events were not detected between United Kingdom strains. However, a previously published Taiwanese strain was identified as an intergenotypic recombinant. EV71 genotype C appears to have continuous circulation in the United Kingdom from 1998 to 2006 with repeated introductions of new strains replacing previous strains. It is necessary to continuously monitor the molecular evolution and recombination events of EV71.     

Numerical analysis of electrophoretic protein patterns of methicillin-resistant strains of Staphylococcus aureus.

A total of 50 strains of Staphylococcus aureus, including 41 methicillin-resistant S. aureus (MRSA) strains, were characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins. The protein patterns contained 40-50 discrete bands and were highly reproducible. Partial patterns were used as the basis of a computer-assisted numerical analysis. The MRSA strains clustered into four phenons at the 83% similarity level; and further division of phenon 1, at the 86% similarity level, resulted in a total of six clusters. All of the MRSA isolates from an MRSA epidemic in the United Kingdom were found to cluster in phenon 1 together with 9 of the 12 MRSA isolates from eastern Australia and 3 other MRSA isolates from the United Kingdom. The remaining three eastern Australian isolates clustered separately in phenon 2. Phenon 3 appeared to be exclusive to strains that were both susceptible and resistant to methicillin and that reacted with group V phages, and phenon 4 comprised 11 isolates, all of which were other MRSA isolates from the United Kingdom. We conclude that computer-assisted numerical analysis by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins provides additional criteria for the study of the epidemiology and the evolution of MRSA.     

Wide dispersion of ST175 clone despite high genetic diversity of carbapenem-nonsusceptible Pseudomonas aeruginosa clinical strains in 16 Spanish hospitals

During the COMParative Activity of Carbapenems Testing (COMPACT) surveillance study, 448 Pseudomonas aeruginosa clinical isolates were obtained from 16 Spanish hospitals. Nonsusceptibility (EUCAST breakpoints) to imipenem (35%), meropenem (33%), and/or doripenem (33%) was observed with 175 isolates (39%). Simultaneous resistance to these three drugs was observed with 126 of the 175 isolates (72%). Except for colistin, high resistance rates were observed among noncarbapenem antibiotics. Clonal relatedness was investigated by pulsed-field gel electrophoresis (PFGE) with SpeI, discriminating 68 patterns. Multilocus sequence typing (MLST) was performed on 84 isolates representing different PFGE types and all participating hospitals. Thirty-nine sequence types (STs) could be distinguished, and of these, ST175 (48 isolates, 10 hospitals), ST646 (16 isolates, 4 hospitals), ST532 (13 isolates, 3 hospitals), and ST111 (13 isolates, 7 hospitals) were the most frequently encountered. Minimum-spanning tree analysis confirmed a wide dissemination of different clones among participant hospitals, particularly ST175. PFGE pattern comparison within the four most frequent STs revealed that ST175 isolates were relatively uniform, while ST646, ST532, and ST111 isolates were highly diverse, with almost every isolate belonging to a unique pulsotype, even when originating from the same center. The population of carbapenem-nonsusceptible P. aeruginosa isolates from 16 hospitals is highly diverse, with one ST (ST175) representing a highly conserved clone disseminated in 10 of the 16 participant hospitals. This ST175 clone should be added to the list of P. aeruginosa clones at high risk for epidemic spread, such as the Liverpool, Manchester, and Melbourne clones previously found in cystic fibrosis patients and ST235 in the nosocomial setting.     

Impact of Pseudomonas aeruginosa Genomic Instability on the Application of Typing Methods for Chronic Cystic Fibrosis Infections

The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa is widespread among cystic fibrosis (CF) patients in the United Kingdom and has emerged recently in North America. In this study, we report the analysis of 24 “anomalous” CF isolates of P. aeruginosa that produced inconsistent results with regard to either pulsed-field gel electrophoresis (PFGE) or PCR tests for the LES. We used a new typing method, the ArrayTube genotyping system, to determine that of the 24 anomalous isolates tested, 13 were confirmed as the LES. LES isolates could not be clearly distinguished from non-LES isolates by two other commonly used genetic fingerprinting tests, randomly amplified polymorphic DNA (RAPD) analysis and BOX-PCR, and varied considerably in their carriage of LES genomic islands and prophages. The genomic instability of the LES suggests that identification of this emerging transmissible strain could be a challenging task, and it questions whether discrimination is always a desirable feature of bacterial typing methods in the context of chronic CF infections.Chronic Pseudomonas aeruginosa infections in cystic fibrosis (CF) patients are associated with a decline in lung function and increased mortality (11). Infection usually occurs during adolescence and, once established, the pathogen is impossible to eradicate (17). In recent years, the view of P. aeruginosa infection in CF patients has been altered because of the emergence of transmissible strains. Originally, it was thought that patients acquired their own unique strains from the environment and only in specific circumstances, such as siblings with CF, were patients found to share the same strain (35). However, in 1996 Cheng et al. (6) described the use of molecular typing to demonstrate the spread of a drug-resistant strain of P. aeruginosa (named the Liverpool epidemic strain [LES]) among patients in a children''s CF center in Liverpool, United Kingdom (6). Seven years later, an analysis of patient samples taken after the year 2000 identified the LES in 79% of 80 P. aeruginosa-colonized CF patients in the Liverpool adult CF center, confirming the spread and longevity of LES infections (29). In a survey of 31 CF centers in England and Wales in 2004, involving more than 1,200 isolates of P. aeruginosa, the LES was identified as the most common clone (33). Furthermore, the LES has also been identified in Scotland (10). Recently, cases of CF patients infected with the LES have also emerged in Canada (1).In addition to its transmissibility, the LES appears to be more aggressive than other P. aeruginosa strains. It has been shown to replace previously established strains of P. aeruginosa (superinfection) (24), has infected both non-CF parents of a CF patient (25), and has infected a pet cat, causing significant morbidity (26). Furthermore, the LES is associated with greater morbidity (2) and increased renal failure (3) and appears to have enhanced survival in air (30).Patient segregation based on LES status requires simple but effective strain typing methods with adequate discriminatory powers to achieve both the initial identification of the LES and subsequent epidemiological surveillance. The genome of P. aeruginosa is a mosaic structure consisting of a core genome and a variable accessory genome (23). The accessory genome includes large insertions, such as prophages and genomic islands, contributing to genome size variations between 5.2 and 7 Mb (38). The earliest archived isolate of the LES (LESB58, from 1988) has recently been genome sequenced (40), revealing the presence of six prophages and five genomic islands.Specific PCR assays have been developed for detection of the LES (31, 34). Following reports of false positives for the original marker PS21 (20), a second marker (LES-F9) was identified (34). Neither PS21 nor LES-F9, which map to separate genomic islands (LESGI-3 and LESGI-1, respectively), is 100% specific to the LES, but the combination of the two has not been reported previously in any non-LES strain. The use of PCR assays in routine diagnostic tests in Liverpool and elsewhere in the United Kingdom has led to the implementation of successful segregation measures. However, we have a number of putative LES CF isolates that do not give concordant results with respect to the presence of PS21, the presence of LES-F9, and the molecular typing method pulsed-field gel electrophoresis (PFGE). The Clondiag ArrayTube (AT) system is a portable method for interrogating both the conserved and accessory genomes of P. aeruginosa isolates. The process is rapid, relatively inexpensive, and robust and has been used to type P. aeruginosa isolates from diverse habitats (39).Here, we report the use of the AT system to resolve the strain status of a collection of suspected LES isolates of P. aeruginosa from CF patients. We provide evidence that the genomic instability exhibited by the LES could impact the validity of routine typing schemes, causing both false-positive and false-negative identification, and suggest that normal paradigms for bacterial typing may be limited in the context of chronic infections in CF.     

Identification by subtractive hybridization of a novel insertion element specific for two widespread Burkholderia cepacia genomovar III strains

Species of the Burkholderia cepacia complex cause chronic and life-threatening infections in persons with cystic fibrosis. Epidemic strains infect multiple patients, reside primarily in genomovar III, and have an apparent enhanced capacity for human infection and/or interpatient transmission. By using subtractive hybridization, a novel insertion element, designated IS1363, was identified in epidemic strain PHDC, known to infect many cystic fibrosis patients in the mid-Atlantic region of the United States. IS1363 was also found in most isolates of the ET12 lineage, responsible for infecting large numbers of patients in Ontario, Canada, and the United Kingdom. Southern blot analysis demonstrated that whereas multiple copies of IS1363 were present in strain PHDC, only one copy was present in ET12 isolates. IS1363 was used to probe a collection of 943 B. cepacia complex isolates, representing all nine genomovars, recovered from 761 cystic fibrosis patients or the natural environment. IS1363 was not found in other genomovar III strains and, with the exception of B. ambifaria, was absent from other B. cepacia complex species. Genotyping analyses of all IS1363-positive isolates demonstrated that strain PHDC was more widely distributed in the United States than previously appreciated; 212 cystic fibrosis patients in 24 states were identified as being infected with PHDC.     

Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database

Oxacillin-resistant Staphylococcus aureus (ORSA) is a virulent pathogen responsible for both health care-associated and community onset disease. We used SmaI-digested genomic DNA separated by pulsed-field gel electrophoresis (PFGE) to characterize 957 S. aureus isolates and establish a database of PFGE patterns. In addition to PFGE patterns of U.S. strains, the database contains patterns of representative epidemic-type strains from the United Kingdom, Canada, and Australia; previously described ORSA clonal-type isolates; 13 vancomycin-intermediate S. aureus (VISA) isolates, and two high-level vancomycin-resistant, vanA-positive strains (VRSA). Among the isolates from the United States, we identified eight lineages, designated as pulsed-field types (PFTs) USA100 through USA800, seven of which included both ORSA and oxacillin-susceptible S. aureus isolates. With the exception of the PFT pairs USA100 and USA800, and USA300 and USA500, each of the PFTs had a unique multilocus sequence type and spa type motif. The USA100 PFT, previously designated as the New York/Tokyo clone, was the most common PFT in the database, representing 44% of the ORSA isolates. USA100 isolates were typically multiresistant and included all but one of the U.S. VISA strains and both VRSA isolates. Multiresistant ORSA isolates from the USA200, -500, and -600 PFTs have PFGE patterns similar to those of previously described epidemic strains from Europe and Australia. The USA300 and -400 PFTs contained community isolates resistant only to beta-lactam drugs and erythromycin. Noticeably absent from the U.S. database were isolates with the previously described Brazilian and EMRSA15 PFGE patterns. These data suggest that there are a limited number of ORSA genotypes present in the United States.     

Burkholderia cepacia complex infection in Italian patients with cystic fibrosis: prevalence, epidemiology, and genomovar status

The prevalence, epidemiology, and genomovar status of Burkholderia cepacia complex strains recovered from Italian cystic fibrosis (CF) patients were investigated using genetic typing and species identification methods. Four CF treatment centers were examined: two in Sicily, one in central Italy, and one in northern Italy. B. cepacia complex bacteria were isolated from 59 out of 683 CF patients attending these centers (8.6%). For the two geographically related treatment centers in Sicily, there was a high incidence of infection caused by a single epidemic clone possessing the cblA gene and belonging to B. cepacia genomovar III, recA group III-A, closely related to the major North America-United Kingdom clone, ET12; instability of the cblA sequence was also demonstrated for clonal isolates. In summary, of all the strains of B. cepacia encountered in the Italian CF population, the genomovar III, recA group III-A strains were the most prevalent and transmissible. However, patient-to-patient spread was also observed with several other genomovars, including strains of novel taxonomic status within the B. cepacia complex. A combination of genetic identification and molecular typing analysis is recommended to fully define specific risks posed by the genomovar status of strains within the B. cepacia complex.     

Rapid single-nucleotide polymorphism-based identification of clonal Pseudomonas aeruginosa isolates from patients with cystic fibrosis by the use of real-time PCR and high-resolution melting curve analysis

Pseudomonas aeruginosa genotyping relies mainly upon DNA fingerprinting methods, which can be subjective, expensive and time-consuming. The detection of at least three different clonal P. aeruginosa strains in patients attending two cystic fibrosis (CF) centres in a single Australian city prompted the design of a non-gel-based PCR method to enable clinical microbiology laboratories to readily identify these clonal strains. We designed a detection method utilizing heat-denatured P. aeruginosa isolates and a ten-single-nucleotide polymorphism (SNP) profile. Strain differences were detected by SYBR Green-based real-time PCR and high-resolution melting curve analysis (HRM10SNP assay). Overall, 106 P. aeruginosa sputum isolates collected from 74 patients with CF, as well as five reference strains, were analysed with the HRM10SNP assay, and the results were compared with those obtained by pulsed-field gel electrophoresis (PFGE). The HRM10SNP assay accurately identified all 45 isolates as members of one of the three major clonal strains characterized by PFGE in two Brisbane CF centres (Australian epidemic strain-1, Australian epidemic strain-2 and P42) from 61 other P. aeruginosa strains from Australian CF patients and two representative overseas epidemic strain isolates. The HRM10SNP method is simple, is relatively inexpensive and can be completed in <3 h. In our setting, it could be made easily available for clinical microbiology laboratories to screen for local P. aeruginosa strains and to guide infection control policies. Further studies are needed to determine whether the HRM10SNP assay can also be modified to detect additional clonal strains that are prevalent in other CF centres.     

High genotypic diversity of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis in the Czech Republic

Over the last decade, epidemic and frequently multidrug-resistant Pseudomonas aeruginosa has increasingly been found among European cystic fibrosis (CF) patients. In the Czech Republic, more than half of the registered CF patients attend the Prague CF centre. At this centre, a Burkholderia cenocepacia strain was recently found to have spread among the patients. The aim of the present study was to assess whether P. aeruginosa isolates from patients at this centre were also genetically related and, if so, whether they were multidrug-resistant. We investigated a collection of 69 isolates from as many patients who represented 80% of the total number of P. aeruginosa-positive patients in 2004. The organisms were typed by AFLP and SpeI macrorestriction analyses (PFGE). Using these methods, 44 unique strains and nine groups of two to five isolates each were distinguished. Among these groups, two each had a prevalence of 7% in the patient population, while others had a prevalence of < or =3%. The diversity observed with PFGE was largely in agreement with the diversity found by AFLP analysis. All isolates were susceptible to colistin; 94-96% were susceptible to piperacillin, ceftazidime, cefepime, meropenem, amikacin or tobramycin; and 84-87% were susceptible to ciprofloxacin, gentamicin or netilmicin. In conclusion, the organisms recovered from Czech CF patients showed high genotypic diversity and good susceptibility to antipseudomonal agents. The absence of highly epidemic P. aeruginosa strains may result from infection control measures taken upon recognition of the epidemic B. cenocepacia.