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We describe the follow-up investigation of an outbreak of endophthalmitis due to Burkholderia contaminans following cataract surgery in a single clinic. Whole-genome sequence analysis of bacteria recovered from affected patients and the clinic identified the clinic’s ventilation system as the source of infection.

We describe the follow-up investigation of an outbreak of endophthalmitis due to

Burkholderia gladioli is a bacterium with a broad ecology spanning disease in humans, animals and plants, but also encompassing multiple beneficial interactions. It is a plant pathogen, a toxin-producing food-poisoning agent, and causes lung infections in people with cystic fibrosis (CF). Contrasting beneficial traits include antifungal production exploited by insects to protect their eggs, plant protective abilities and antibiotic biosynthesis. We explored the genomic diversity and specialized metabolic potential of 206 B. gladioli strains, phylogenomically defining 5 clades. Historical disease pathovars (pv.) B. gladioli pv. allicola and B. gladioli pv. cocovenenans were distinct, while B. gladioli pv. gladioli and B. gladioli pv. agaricicola were indistinguishable; soft-rot disease and CF infection were conserved across all pathovars. Biosynthetic gene clusters (BGCs) for toxoflavin, caryoynencin and enacyloxin were dispersed across B. gladioli , but bongkrekic acid and gladiolin production were clade-specific. Strikingly, 13 % of CF infection strains characterized were bongkrekic acid-positive, uniquely linking this food-poisoning toxin to this aspect of B. gladioli disease. Mapping the population biology and metabolite production of B. gladioli has shed light on its diverse ecology, and by demonstrating that the antibiotic trimethoprim suppresses bongkrekic acid production, a potential therapeutic strategy to minimize poisoning risk in CF has been identified.

Burkholderia gladioli is a bacterium with a broad ecology spanning disease in humans, animals and plants, but also encompassing multiple beneficial interactions. It is a plant pathogen, a toxin-producing food-poisoning agent, and causes lung infections in people with cystic fibrosis (CF). Contrasting beneficial traits include antifungal production exploited by insects to protect their eggs, plant protective abilities and antibiotic biosynthesis. We explored the genomic diversity and specialized metabolic potential of 206 B. gladioli strains, phylogenomically defining 5 clades. Historical disease pathovars (pv.) B. gladioli pv. allicola and B. gladioli pv. cocovenenans were distinct, while B. gladioli pv. gladioli and B. gladioli pv. agaricicola were indistinguishable; soft-rot disease and CF infection were conserved across all pathovars. Biosynthetic gene clusters (BGCs) for toxoflavin, caryoynencin and enacyloxin were dispersed across B. gladioli , but bongkrekic acid and gladiolin production were clade-specific. Strikingly, 13 % of CF infection strains characterized were bongkrekic acid-positive, uniquely linking this food-poisoning toxin to this aspect of B. gladioli disease. Mapping the population biology and metabolite production of B. gladioli has shed light on its diverse ecology, and by demonstrating that the antibiotic trimethoprim suppresses bongkrekic acid production, a potential therapeutic strategy to minimize poisoning risk in CF has been identified.

Burkholderia gladioli is one of few bacteria with a broad ecology spanning disease in humans, animals, and plants, and encompassing beneficial interactions with multiple eukaryotic hosts. It is a plant pathogen, a bongkrekic acid toxin producing food-poisoning agent, and a lung pathogen in people with cystic fibrosis (CF). Contrasting beneficial traits include antifungal production exploited by insects to protect their eggs, plant protective abilities and antibiotic biosynthesis. We explored the ecological diversity and specialized metabolite biosynthesis of 206 B. gladioli strains, phylogenomically defining 5 evolutionary clades. Historical disease pathovars (pv) B. gladioli pv. allicola and B. gladioli pv. cocovenenans were phylogenetically distinct, while B. gladioli pv. gladioli and B. gladioli pv. agaricicola were indistinguishable. Soft-rot disease and CF infection pathogenicity traits were conserved across all pathovars. Biosynthetic gene clusters for toxoflavin, caryoynencin and enacyloxin were dispersed across B. gladioli, but bongkrekic acid and gladiolin production were clade specific. Strikingly, 13% of CF-infection strains characterised (n=194) were bongkrekic acid toxin positive, uniquely linking this food-poisoning risk factor to chronic lung disease. Toxin production was suppressed by exposing strains to the antibiotic trimethoprim, providing a potential therapeutic strategy to minimise poisoning risk in CF.

We tested the in vitro activities of ceftazidime-avibactam, ceftolozane-tazobactam, meropenem-vaborbactam, piperacillin-tazobactam, and 11 other antimicrobial agents against 420 Burkholderia, Achromobacter, Stenotrophomonas, and Pandoraea strains, 89% of which were cultured from respiratory specimens from persons with cystic fibrosis. Among the β-lactam-β-lactamase inhibitor agents, meropenem-vaborbactam had the greatest activity against Burkholderia and Achromobacter, including multidrug-resistant and extensively-drug-resistant strains. None of the newer β-lactam-β-lactamase combination drugs showed increased activity compared to that of the older agents against Stenotrophomonas maltophilia or Pandoraea spp.

This report describes transmission of a Burkholderia cenocepacia ET12 strain (ET12-Bc) at the Toronto Adult Cystic Fibrosis (CF) Centre occurring from 2008 to 2017. Epidemiological and genomic data from 11 patients with CF were evaluated. Isolates were analysed using whole genome sequencing (WGS). Epidemiological investigation and WGS analysis suggested nosocomial transmission, despite enhanced infection control precautions. This was associated with subsequent deaths in 10 patients. ET12-Bc positive patients are no longer cared for on the same unit as ET12-Bc negative patients.

Aim: To develop NB-201, a nanoemulsion compound, as a novel microbicidal agent against methicillin-resistant Staphylococcus aureus (MRSA) infection, which is a common threat to public health but with limited therapeutic options. Materials & methods: NB-201 was tested in in vitro and in vivo murine and porcine models infected with MRSA. Results: Topical treatment of MRSA-infected wounds with NB-201 significantly decreased bacterial load and had no toxic effects on healthy skin tissues. NB-201 attenuated neutrophil sequestration in MRSA-infected wounds and inhibited epidermal and deep dermal inflammation. The levels of proinflammatory cytokines were reduced in NB-201-treated MRSA-infected wounds. Conclusion: NB-201 can greatly reduce inflammation characteristic of infected wounds and has antimicrobial activity that effectively kills MRSA regardless of the genetic basis of antibiotic resistance.

A repeat multi-locus sequence analysis (MLSA) of concatenated nusA, eno, rpoB, gltB, lepA, nuoL and nrdA sequences of strains classified as Achromobacter marplatensis was performed. The results revealed that earlier reported sequence data of the proposed type strain were erroneous, and that the corrected concatenated sequence divergence between the A. marplatensis LMG 26219T (=CCUG 56371T) sequence type and that of strains of Achromobacter spiritinus was well below the 2.1% threshold value that delineates species of the genus Achromobacter. These results therefore demonstrated that strains which were classified as A. spiritinus should be reclassified as A. marplatensis and that the name Achromobacter spiritinus should no longer be used. An emendation of the description of Achromobacter marplatensis is warranted.

We tested the activities of ceftolozane-tazobactam and 13 other antimicrobial agents against 221 strains of Burkholderia cepacia complex and Burkholderia gladioli . Most strains (82%) were cultured from persons with cystic fibrosis, and most (85%) were recovered since 2011. The ceftolozane-tazobactam MIC was ≤8 μg/ml for 77% of the strains. However, the MIC range was broad (≤0.5 to >64 μg/ml; MIC 50/90 , 2/32 μg/ml). Significant differences in susceptibility to some antimicrobial agents were observed between species.

The differentiation of Bordetella species, particularly those causing human infection, is problematic. We found that sequence analysis of an internal fragment of nrdA allowed differentiation of the currently named Bordetella species. Analysis of 107 “ Bordetella ” isolates recovered almost exclusively from human respiratory tract specimens identified several putative novel species.

The phenotypic and genotypic characteristics of seventeen Achromobacter strains representing MLST genogroups 2, 5, 7 and 14 were examined. Although genogroup 2 and 14 strains shared a DNA-DNA hybridization level of about 70%, the type strains of both genogroups differed in numerous biochemical characteristics and all genogroup 2 and 14 strains could by distinguished by nitrite reduction, denitrification and growth on acetamide. Given the MLST sequence divergence which identified genogroups 2 and 14 as clearly distinct populations, the availability of nrdA sequence analysis as a single locus identification tool for all Achromobacter species and genogroups, and the differential phenotypic characteristics, we propose to formally classify Achromobacter genogroups 2, 5, 7 and 14 as four novel Achromobacter species for which we propose the names Achromobacter insuavis sp. nov. (with strain LMG 26845(T) [=CCUG 62426(T)] as the type strain), Achromobacter aegrifaciens sp. nov. (with strain LMG 26852(T) [=CCUG 62438(T)] as the type strain), Achromobacter anxifer sp. nov. (with strain LMG 26857(T) [=CCUG 62444(T)] as the type strain), and Achromobacter dolens sp. nov. (with strain LMG 26840(T) [=CCUG 62421(T)] as the type strain).

Background We recently described a multilocus sequence typing scheme for Achromobacter that identified several novel species in this genus. Methods We assessed the ability of nrdA sequence analysis to differentiate Achromobacter species, including the seven previously named species and 14 recently described genogroups. Confirmation of distinctness between groups was confirmed using the k parameter. Using this single locus sequence to differentiate species, we analyzed Achromobacter isolates obtained from 341 CF patients in the U.S. Results We found that Achromobacter xylosoxidans accounts for 42% of Achromobacter infections, while Achromobacter ruhlandii accounted for 23.5% of infections. Isolates from 17% of patients were members of the novel genogroup 14. The remaining 17.5% of strains belonged to 11 other species/genogroups. Conclusion The use of nrdA sequence analysis allows differentiation of the several Achromobacter species that can infect persons with CF. Achromobacter species other than A. xylosoxidans account for the majority of Achromobacter infection in CF patients in the U.S.

We report here the complete genome sequences of Bordetella flabilis and Bordetella bronchialis recovered from cultures of individuals with cystic fibrosis (CF), and “ Bordetella pseudohinzii ” recovered from a CF mouse model.

We report the complete genome sequences of 17 rapidly growing nontuberculous mycobacterial (NTM) strains, including 16 Mycobacterium abscessus complex strains and one M. immunogenum strain. These sequences add value to studies of the genetic diversity of rapidly growing NTM strains recovered from human specimens.

The phenotypic and genotypic characteristics of a historical collection of strains identified as Achromobacter denitrificanswere examined. Sequence analysis of a 765 bp nrdA gene fragment revealed that eight of these strains belonged to the recently described Achromobacter aegrifaciens, Achromobacter mucicolens, and Achromobacter insolitus, and that one strain belonged to Achromobacter xylosoxidans. The analysis also suggested the presence of four novel species of the genus Achromobacter among the remaining strains. The latter was confirmed by multilocus sequence analysis of concatenated nusA, eno, rpoB, gltB, lepA, nuoL andnrdA gene fragments and extensive genotypic and phenotypic characterization. We propose to name these novel species as Achromobacter agilis sp. nov., nom. rev. (type strain LMG 3411T=CCUG 62454T), Achromobacter pestifer sp. nov., nom. rev. (type strain LMG 3431T=CCUG 61959T) , Achromobacter kerstersii sp. nov. (type strain LMG 3441T=CCUG 62449T) and Achromobacter deleyi sp. nov. (type strain LMG 3458T=CCUG 62433T).

We report the complete genome sequence of a Mycobacterium abscessus subsp. bolletii isolate recovered from a sputum culture from an individual with cystic fibrosis. This sequence is the first completed whole-genome sequence of M. abscessus subsp. bolletii and adds value to studies of M. abscessus complex genomics.

Here, we report the draft genome sequences of 63 Pseudomonas aeruginosa isolates, recovered in culture of sputum from 15 individuals with cystic fibrosis (CF) receiving care in a single CF care center over a 13-year period. These sequences add value to studies of within-host evolution of bacterial pathogens during chronic infection.

We report the complete genome assembly of the Streptococcus pyogenes type emm 4 strain MEW427 (also referred to as strain UM001 in the Pediatric Acute-Onset Neuropsychiatric Syndrome [PANS] Research Consortium), a throat isolate from a child with acute-onset neuropsychiatric symptoms meeting clinical criteria for PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus). The genome length is 1,814,455 bp with 38.51% G+C%.

We present here the complete genome sequence of Streptococcus pyogenes type emm 28 strain MEW123, a streptomycin-resistant derivative of a pediatric throat isolate. The genome length is 1,878,699 bp, with 38.29% G+C% content. The genome sequence adds value to this virulent emm 28 representative strain and will aid in the investigation of streptococcal pathogenesis.

The genus Achromobacter currently is comprised of seven species, including Achromobacter xylosoxidans , an opportunistic and nosocomial pathogen that displays broad-spectrum antimicrobial resistance and is recognized as causing chronic respiratory tract infection in persons with cystic fibrosis (CF). To enable strain typing for global epidemiologic investigations, to clarify the taxonomy of “ Achromobacter -like” strains, and to elucidate the population structure of this genus, we developed a genus-level multilocus sequence typing (MLST) scheme. We employed in silico analyses of whole-genome sequences of several phylogenetically related genera, including Bordetella , Burkholderia , Cupriavidus , Herminiimonas , Janthinobacterium , Methylibium , and Ralstonia , for selecting loci and designing PCR primers. Using this MLST scheme, we analyzed 107 genetically diverse Achromobacter isolates cultured from biologic specimens from CF and non-CF patients, 1 isolate recovered from sludge, and an additional 39 strains obtained from culture collections. Sequence data from these 147 strains, plus three recently genome-sequenced Achromobacter strains, were assigned to 129 sequence types based on seven loci. Calculation of the nucleotide divergence of concatenated locus sequences within and between MLST clusters confirmed the seven previously named Achromobacter species and revealed 14 additional genogroups. Indices of association showed significant linkage disequilibrium in all of the species/genogroups able to be tested, indicating that each group has a clonal population structure. No clear segregation of species/genogroups between CF and non-CF sources was found.

Background Transmission of Burkholderia cenocepacia ET-12 strain (ET-12Bc) can cause epidemics in the cystic fibrosis (CF) population. The Toronto Adult CF center currently follows 500 patients; 20% have infection with B. cepacia complex (BCC), including 48 patients infected with ET-12Bc. The center adheres to the 2013 infection prevention and control guidelines and patients are also segregated by clinics. Despite this, there have been 11 new acquisitions of ET-12Bc since 2008. The objective of this study was to describe the investigation of an ET-12Bc outbreak in CF patients, using whole-genome sequencing (WGS). Methods Investigations included multilocus sequence typing (MLST) and WGS of 34 isolates (11 new ET-12Bc acquisitions, 18 isolates of known ET-12Bc patients (including all patients with hospital admissions that overlapped with new acquisitions), four isolates from CF patients in the USA and the J2315 reference strain). Each of the seven MLST alleles from ET12Bc strain J2315 was downloaded from PubMLST and used to “Blast” each of the 16 WGS databases. WGS was done using 150 bp paired-end runs on an Illumina HiSeq4000. Single nucleotide polymorphisms (SNPs) between the newly sequenced strains and J2315 were profiled. Results Ten patients had a hospital admission within the 2 months preceding their first ET-12Bc positive sputum culture, except for one in whom ET-12Bc was detected 12 months following hospital admission. In all isolates, the seven alleles (atpD, gltB, gyrB, recA, lepA, phaC and trpB) matched 100% to sequence type 28 and clonal complex 31, and were identical to J2315. WGS SNP analysis confirmed that transmission occurred from known cases on the unit in 10/11 (90.9%) patients. To date, 8/11 patients with new acquisitions have died (median survival of 95 days). Conclusion Our investigations found epidemiological evidence suggestive of ET-12Bc transmission on the CF unit, which was confirmed by MLST and WGS SNP analysis. Compared with MLST, WGS SNP analysis had better discriminatory power and was well correlated with the identified epidemiological links between patients. Recognition of ET-12Bc transmission with associated high mortality rates has led to a change in our hospital policy. ET-12Bc positive patients will no longer be cared for on the same unit as ET-12Bc negative patients with CF. Disclosures All authors: No reported disclosures.

Nine Burkholderia cepacia complex (Bcc) bacteria were isolated during environmental surveys for the ecological niche of Burkholderia pseudomallei, the aetiological agent of melioidosis, in the Northern Territory of Australia. They represented two multi-locus sequence analysis-based clusters, referred to as Bcc B and Bcc L. Three additional environmental and clinical Bcc B isolates were identified upon deposition of the sequences in the PubMLST database. Analysis of the concatenated nucleotide sequence divergence levels within both groups (1.4 and 1.9 %, respectively) and towards established Bcc species (4.0 and 3.9 %, respectively) demonstrated that the two taxa represented novel Bcc species. All 12 isolates were further characterized using 16S rRNA and recA gene sequence analysis, RAPD analysis, DNA base content determination, fatty acid methyl ester analysis and biochemical profiling. Analysis of recA gene sequences revealed a remarkable diversity within each of these taxa, but, together, the results supported the affiliation of the two taxa to the Bcc. Bcc B strains can be differentiated from most other Bcc members by the assimilation of maltose. Bcc L strains can be differentiated from other Bcc members by the absence of assimilation of N-acetylglucosamine. The names Burkholderia stagnalis sp. nov. with type strain LMG 28156T ( = CCUG 65686T) and Burkholderia territorii sp. nov. with type strain LMG 28158T ( = CCUG 65687T) are proposed for Bcc B and Bcc L bacteria, respectively.

The phenotypic and genotypic characteristics of four Bordetella hinzii-like strains from human respiratory specimens and representing nrdA gene sequence based genogroups 3, 14 and 15 were examined. In a 16S rRNA gene sequence based phylogenetic tree, the four strains consistently formed a single coherent lineage but their assignment to the genus Bordetella was equivocal. The respiratory quinone, polar lipid and fatty acid profiles generally conformed to those of species of the genus Bordetella and were characterized by the presence of ubiquinone 8, of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several aminolipids, and of high percentages of C16 : 0, cyclo-C17 : 0 and summed feature 2, as major chemotaxonomic marker molecules, respectively. The DNA G+C content was about 66 mol%, which corresponded with that of the high-percentage DNA G+C content genera of the family Alcaligenaceae including the genus Bordetella. DNA–DNA hybridization experiments revealed the presence of three distinct genomospecies and thus confirmed phenotypic differences as revealed by means of extensive biochemical characterization. We therefore propose to formally classify Bordetella genogroups 3, 14 and 15 as Bordetella bronchialis sp. nov. (type strain LMG 28640T = AU3182T = CCUG 56828T), Bordetella sputigena sp. nov. (type strain LMG 28641T = CCUG 56478T) and Bordetella flabilis sp. nov. (type strain LMG 28642T = AU10664T = CCUG 56827T). In addition, we propose to reclassify Achromobacter sediminum into the novel genus Verticia, as Verticia sediminum, gen. nov., comb. nov., on the basis of its unique phylogenetic position, its marine origin and its distinctive phenotypic, fatty acid and polar lipid profile.

Two new PCR assays (for Ralstonia species and Ralstonia respiraculi ), together with previously published PCR assays, were used to assess Ralstonia isolates recovered from 111 cystic fibrosis patients. Ralstonia mannitolilytica accounted for 46% of isolates, while R. respiraculi and Ralstonia pickettii accounted for 19% and 18%, respectively. Ralstonia basilensis and Ralstonia metallidurans , species not previously recovered from human samples, were also identified.

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.

Previous studies have identified specific Burkholderia cepacia complex strains that are common to multiple persons with cystic fibrosis (CF). Such so-called epidemic strains have an apparent enhanced capacity for inter-patient spread and reside primarily in Burkholderia cenocepacia (formerly B. cepacia complex genomovar III). We sought to identify strains from B. cepacia complex species other than B. cenocepacia that are similarly shared by multiple CF patients. We performed genotype analysis of 360 recent sputum culture isolates from 360 persons residing in 29 cities by using repetitive extragenic palendromic polymerase chain reaction (rep-PCR) and pulsed field gel electrophoresis. The results indicate that sharing of a common Burkholderia multivorans strain occurs relatively infrequently; however, several small clusters of patients infected with the same strain were identified. A cluster of seven patients infected with the same B. cepacia (genomovar I) strain was found. We also identified a large group of 28 patients receiving care in the same treatment center and infected with the same Burkholderia dolosa strain. These observations suggest that B. cepacia complex strains in species other than B. cenocepacia may be spread among CF patients.

Persons with cystic fibrosis (CF) are susceptible to chronic pulmonary infection due to certain Burkholderia species, but it is not clear whether this typically involves persistent infection with the same strain or sequential infection with distinct strains. We analyzed 1095 Burkholderia isolates recovered from serial sputum cultures from 379 patients with CF receiving care in 112 CF treatment centers in the United States. Genotyping was performed by random amplified polymorphic DNA typing or pulsed-field gel electrophoresis. Overall, a change in infecting strain was found in 24 (6.9%) of 347 patients infected with Burkholderia cepacia complex and in 3 (9%) of 32 patients infected with Burkholderia gladioli. Several patients were likely coinfected, at least transiently, with >1 B. cepacia complex strain. The potential for strain replacement during chronic infection may confound studies of the relationship between strain and clinical outcome and must be considered in designing effective infection-control practices.

The Burkholderia cepacia complex consists of several closely related bacterial species (or genomovars) which although generally not pathogenic for healthy individuals, contribute significantly to morbidity and mortality among persons with cystic fibrosis (CF). Certain B. cepacia complex strains are more frequently recovered from CF sputum cultures than are others, and these typically reside in genomovar III. The ET12 clone is a genomovar III strain that predominates among CF patients in Canada and the United Kingdom and is characterized by distinctive cblA-encoded pili that have a cable-like morphology. In a previous survey of B. cepacia complex isolates recovered from 606 CF patients in the US, a single genomovar III ET12 isolate (isolate AU0007) was identified; several cblA-containing genomovar I isolates, however, were also detected. In the study reported here, analysis by PFGE revealed several distinct strain types among these genomovar I isolates, and sequence analysis of their cblA genes demonstrated 87.8-88.4% identity to the ET12 cblA sequence. Southern analysis indicated that the cblA variant from each genomovar I isolate resides on a 4 kbp EcoRI fragment, in contrast to ET12 isolates, in which cblA localizes to a 5 kbp EcoRI fragment. Western blot assay indicated expression of the 16 kDa major pilin subunit by ET12 isolates, including AU0007, but neither whole-cell nor surface-protein extracts of the genomovar I reacted. Electron microscopy revealed the complete absence of pili expression by the genomovar I isolates. In contrast to typical ET12 isolates, AU0007 appeared to be hyperpiliated with rigid pili that lacked the cable morphology and did not bind cytokeratin 13, which has been previously identified as the epithelial cell receptor for the ET12 cable-pili-associated adhesin.

Using a polyphasic approach (including cellular protein and fatty acid analysis, biochemical characterization, 16S ribosomal DNA sequencing, and DNA-DNA hybridizations), we characterized 51 bacterial isolates recovered from respiratory secretions of cystic fibrosis (CF) patients. Our analyses showed that 24 isolates belong to taxa that have so far not (or only rarely) been reported from CF patients. These taxa include Acinetobacter sp., Bordetella hinzii , Burkholderia fungorum , Comamonas testosteroni , Chryseobacterium sp., Herbaspirillum sp., Moraxella osloensis , Pandoraea genomospecies 4, Ralstonia gilardii , Ralstonia mannitolilytica , Rhizobium radiobacter , and Xanthomonas sp. In addition, one isolate most likely represents a novel Ralstonia species, whereas nine isolates belong to novel taxa within the α- Proteobacteria . Eight of these latter isolates are classified into the novel genus Inquilinus gen. nov. as Inquilinus limosus gen. nov., sp. nov., or as Inquilinus sp. The remaining 17 isolates are characterized as members of the family Enterobacteriaceae . The recovery of these species suggests that the CF lung is an ecological niche capable of supporting the growth of a wide variety of bacteria rarely seen in clinical samples. Elucidation of the factors that account for the association between these unusual species and the respiratory tract of CF patients may provide important insights into the pathophysiology of CF infection. Because accurate identification of these organisms in the clinical microbiology laboratory may be problematic, the present study highlights the utility of reference laboratories capable of identifying unusual species recovered from CF sputum.

Standard microbiology references describe Stenotrophomonas maltophilia as oxidase negative and variable with respect to utilization of lactose and sucrose. Analysis of a collection of 766 S. maltophilia isolates indicated that approximately 20% are oxidase positive and that this species should be reevaluated for other phenotypes, including oxidative fermentation of lactose and sucrose.

Performances of several commercial test systems were reviewed to determine their relative levels of accuracy in identifying Burkholderia cepacia complex isolates recovered from cystic fibrosis sputum culture. Positive predictive values ranged from 71 to 98%; negative predictive values ranged from 50 to 82%. All systems misidentified B. cepacia complex. The species most frequently misidentified as B. cepacia was Burkholderia gladioli . These data support the results of previous studies that recommend confirmatory testing, including the use of DNA-based methods, for sputum culture isolates presumptively identified as B. cepacia .

Bordetella spp. are not normally included when considering the opportunistic bacterial species that are typically involved in respiratory tract infections in individuals with cystic fibrosis (CF). By using a combination of bacterial genotyping and 16S rDNA sequencing, Bordetella spp. were identified in cultures obtained from 43 individuals with CF. Most (n = 23) patients were infected with Bordetella bronchiseptica/parapertussis; five were infected with Bordetella hinzii, four with Bordetella petrii, three with Bordetella avium, and eight with unidentified Bordetella spp. Consideration should be given to the presence of these organisms in the evaluation of CF sputum cultures.

We analyzed Burkholderia cepacia complex isolates recovered from 1,218 cystic fibrosis (CF) patients and 90 patients without CF. Although all B. cepacia complex species were found, some were rarely identified. The distribution of species differed between the CF and non-CF populations and appears to be changing over time among CF patients.

Analysis of partial gyrB gene sequences revealed six taxa in a group of 17 Burkholderia glathei -like isolates which were further examined by (GTG)5-PCR fingerprinting, 16S rRNA gene sequence analysis, DNA–DNA hybridizations, determination of the DNA G+C content, whole-cell fatty acid analysis and an analysis of cell and colony morphology and more than 180 biochemical characteristics. The results demonstrated that one taxon consisting of three human clinical isolates represented Burkholderia zhejiangensis , a recently described methyl-parathion-degrading bacterium isolated from a wastewater-treatment system in China. The remaining taxa represented five novel species isolated from soil or rhizosphere soil samples, and could be distinguished by both genotypic and phenotypic characteristics. We therefore propose to formally classify these bacteria as Burkholderia humi sp. nov. (type strain, LMG 22934T = CCUG 63059T), Burkholderia choica sp. nov. (type strain, LMG 22940T = CCUG 63063T), Burkholderia telluris sp. nov. (type strain, LMG 22936T = CCUG 63060T), Burkholderia udeis sp. nov. (type strain, LMG 27134T = CCUG 63061T) and Burkholderia terrestris sp. nov. (type strain, LMG 22937T = CCUG 63062T).

Eleven Burkholderia cepacia-like isolates of human clinical and environmental origin were examined by a polyphasic approach including recA and 16S rRNA sequence analysis, multilocus sequence analysis (MLSA), DNA base content determination, fatty acid methyl ester analysis, and biochemical characterization. The results of this study demonstrate that these isolates represent a novel species within the B. cepacia complex (Bcc) for which we propose the name Burkholderia pseudomultivorans. The type strain is strain LMG 26883(T) (=CCUG 62895(T)). B. pseudomultivorans can be differentiated from other Bcc species by recA gene sequence analysis, MLSA, and several biochemical tests including growth at 42°C, acidification of sucrose and adonitol, lysine decarboxylase and β-galactosidase activity, and esculin hydrolysis.

The phenotypic and genotypic characteristics of fourteen human clinical Achromobacter strains representing four genogroups which were delineated by sequence analysis of nusA, eno, rpoB, gltB, lepA, nuoL and nrdA loci, demonstrated that they represent four novel Achromobacter species. The present study also characterized and provided two additional reference strains for Achromobacter ruhlandii and Achromobacter marplatensis, species for which, thus far, only single strains are publicly available, and further validated the use of 2.1% concatenated nusA, eno, rpoB, gltB, lepA, nuoL and nrdA sequence divergence as a threshold value for species delineation in this genus. Finally, although most Achromobacter species can be distinguished by biochemical characteristics, the present study also highlighted considerable phenotypic intraspecies variability and demonstrated that the type strains may be phenotypically poor representatives of the species. We propose to classify the fourteen human clinical strains as Achromobacter mucicolens sp. nov. (with strain LMG 26685(T) [=CCUG 61961(T)] as the type strain), Achromobacter animicus sp. nov. (with strain LMG 26690(T) [=CCUG 61966(T)] as the type strain), Achromobacter spiritinus sp. nov. (with strain LMG 26692(T) [=CCUG 61968(T)] as the type strain), and Achromobacter pulmonis sp. nov. (with strain LMG 26696(T) [=CCUG 61972(T)] as the type strain).

PCR primers targeting loci in the current Burkholderia cepacia complex multilocus sequence typing scheme were redesigned to (i) more reliably amplify these loci from B . cepacia complex species, (ii) amplify these same loci from additional Burkholderia species, and (iii) enable the use of a single primer set per locus for both amplification and DNA sequencing.

Burkholderia xenovorans LB400 (LB400), a well studied, effective polychlorinated biphenyl-degrader, has one of the two largest known bacterial genomes and is the first nonpathogenic Burkholderia isolate sequenced. From an evolutionary perspective, we find significant differences in functional specialization between the three replicons of LB400, as well as a more relaxed selective pressure for genes located on the two smaller vs. the largest replicon. High genomic plasticity, diversity, and specialization within the Burkholderia genus are exemplified by the conservation of only 44% of the genes between LB400 and Burkholderia cepacia complex strain 383. Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp. The latter is largely explained by our findings that >20% of the LB400 sequence was recently acquired by means of lateral gene transfer. Although a range of genetic factors associated with in vivo survival and intercellular interactions are present, these genetic factors are likely related to niche breadth rather than determinants of pathogenicity. The presence of at least eleven “central aromatic” and twenty “peripheral aromatic” pathways in LB400, among the highest in any sequenced bacterial genome, supports this hypothesis. Finally, in addition to the experimentally observed redundancy in benzoate degradation and formaldehyde oxidation pathways, the fact that 17.6% of proteins have a better LB400 paralog than an ortholog in a different genome highlights the importance of gene duplication and repeated acquirement, which, coupled with their divergence, raises questions regarding the role of paralogs and potential functional redundancies in large-genome microbes.

Background: Burkholderia cenocepacia can cause life threatening respiratory tract infections in patients with cystic fibrosis (CF) and has a significant impact on survival. There is extensive evidence for patient to patient spread and nosocomial transmission of this organism, and several widespread B cenocepacia strains have been described including the transatlantic ET12 clone. A study was performed to compare B cenocepacia isolates recovered from CF patients receiving care in several European countries and strains isolated from other clinical samples and the environment, with reference isolates from the epidemic B cenocepacia strain PHDC which has so far only been recovered from CF patients and soil in the USA. Methods: A large collection of B cenocepacia isolates, including a large number recovered from CF patients receiving care in several European countries, Canada and the USA, were genotyped by means of randomly amplified polymorphic DNA typing (RAPD) and rep-PCR using the BOX-A1R primer (BOX-PCR). Results: Nineteen Burkholderia cenocepacia isolates cultured from clinical samples in Europe (18 recently recovered from CF patients in France and Italy and one recovered in 1964 from urine in the UK) showed RAPD fingerprinting patterns that were similar to patterns obtained from isolates of B cenocepacia strain PHDC. Subsequent analysis of these isolates using BOX-PCR confirmed that the European isolates and strain PHDC represent the same clone. Conclusion: Strain PHDC represents a second transatlantic B cenocepacia clone capable of colonising patients with CF.

Pseudomonas aeruginosa is the major opportunistic bacterial pathogen in persons with cystic fibrosis (CF); pulmonary infection occurs in approximately 80% of adult CF patients. Much of CF patient management depends on accurate identification of P. aeruginosa from sputum culture. However, identification of this species may be problematic due to the marked phenotypic variability demonstrated by CF sputum isolates and the presence of other closely related species. To facilitate species identification, we used 16S ribosomal DNA (rDNA) sequence data to design PCR assays intended to provide genus- or species-level identification. Both assays yielded DNA fragments of the predicted size. We tested 42 culture collection strains (including 14 P. aeruginosa strains and 28 strains representing 16 other closely related Pseudomonas species) and 43 strains that had been previously identified as belonging to 28 nonpseudomonal species also recovered from CF patient sputum. Based on these 85 strains, the specificity and sensitivity of both assays were 100%. To further assess the utility of the PCR assays, we tested 66 recent CF sputum isolates. The results indicated that preliminary phenotypic testing had misidentified several isolates. The 16S rDNA sequence was determined for 38 isolates, and in all cases it confirmed the results of the PCR assays. Thus, we have designed two PCR assays: one is specific for the genus Pseudomonas , while the other is specific for P. aeruginosa . Both assays show 100% sensitivity and specificity.

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 IS 1363 , was identified in epidemic strain PHDC, known to infect many cystic fibrosis patients in the mid-Atlantic region of the United States. IS 1363 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 IS 1363 were present in strain PHDC, only one copy was present in ET12 isolates. IS 1363 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. IS 1363 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 IS 1363 -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.

The Burkholderia cepacia complex consists of several closely related bacterial species (or genomovars) which although generally not pathogenic for healthy individuals, contribute significantly to morbidity and mortality among persons with cystic fibrosis (CF). Certain B. cepacia complex strains are more frequently recovered from CF sputum cultures than are others, and these typically reside in genomovar III. The ET12 clone is a genomovar III strain that predominates among CF patients in Canada and the United Kingdom and is characterized by distinctive cblA-encoded pili that have a cable-like morphology. In a previous survey of B. cepacia complex isolates recovered from 606 CF patients in the US, a single genomovar III ET12 isolate (isolate AU0007) was identified; several cblA-containing genomovar I isolates, however, were also detected. In the study reported here, analysis by PFGE revealed several distinct strain types among these genomovar I isolates, and sequence analysis of their cblA genes demonstrated 87.8-88.4% identity to the ET12 cblA sequence. Southern analysis indicated that the cblA variant from each genomovar I isolate resides on a 4 kbp EcoRI fragment, in contrast to ET12 isolates, in which cblA localizes to a 5 kbp EcoRI fragment. Western blot assay indicated expression of the 16 kDa major pilin subunit by ET12 isolates, including AU0007, but neither whole-cell nor surface-protein extracts of the genomovar I reacted. Electron microscopy revealed the complete absence of pili expression by the genomovar I isolates. In contrast to typical ET12 isolates, AU0007 appeared to be hyperpiliated with rigid pili that lacked the cable morphology and did not bind cytokeratin 13, which has been previously identified as the epithelial cell receptor for the ET12 cable-pili-associated adhesin.

We analyzed a collection of 97 well-characterized Burkholderia cepacia genomovar III isolates to evaluate multiple genomic typing systems, including pulsed-field gel electrophoresis (PFGE), BOX-PCR fingerprinting and random amplified polymorphic DNA (RAPD) typing. The typeability, reproducibility, and discriminatory power of these techniques were evaluated, and the results were compared to each other and to data obtained in previous studies by using multilocus restriction typing (MLRT). All methods showed excellent typeability. PFGE with Spe I was more reproducible than RAPD and BOX-PCR fingerprinting. The discriminatory power of the methods was variable, with PFGE and RAPD typing having a higher index of discrimination than BOX-PCR fingerprinting. In general, the results obtained by PFGE, BOX-PCR fingerprinting, and MLRT were in good agreement. Our data indicate that different genomic-based methods can be used to type B. cepacia genomovar III isolates depending on the situation and the epidemiologic question being addressed. PFGE and RAPD fingerprinting are best suited to addressing small-scale studies (i.e., local epidemiology), whereas BOX-PCR fingerprinting is more appropriate for large-scale studies (i.e., global epidemiology). In this regard, BOX-PCR fingerprinting can be considered a rapid and easy alternative to MLRT.

Objectives: We sought to determine whether the same Burkholderia cepacia complex strain has persisted as the dominant clonal lineage among patients in a large cystic fibrosis (CF) treatment center during the past 2 decades. Study design: The inter-city spread of B cepacia through transfer of a colonized patient and the impact of infection control measures in containing inter-patient transmission were investigated. We analyzed all available B cepacia complex isolates recovered from 1981 to 1987 and from 1996 to 2000 at one large CF treatment center (Center A) and from 1997 to 2000 at another center (Center B). Incidence of B cepacia complex infection and infection control measures in both centers were assessed. Results: Seventeen (81%) of 21 Center A patients from whom B cepacia complex bacteria were recovered between 1981 and 1987 and 40 (97%) of 41 patients culture-positive between 1996 and 2000 were infected with the same genomovar III strain. Transfer of a colonized patient from Center A to Center B was associated with an increase in B cepacia complex infection in Center B, all of which was with the Center A dominant strain. This strain, designated PHDC , lacks both B cepacia epidemic strain and cblA markers. Conclusions: B cepacia complex strains may remain endemic in CF treatment centers for many years. Responsible bacterial and host factors and optimal infection control measures to prevent inter-patient spread remain to be identified. (J Pediatr 2001;139:643–9)

Several distinct species (genomovars) comprise bacteria previously identified merely as Burkholderia cepacia. Understanding how these species, collectively referred to as the B. cepacia complex, differ in their epidemiology and pathogenic potential in cystic fibrosis (CF) is important in efforts to refine management strategies. B. cepacia isolates recovered from 606 CF patients receiving care at 132 treatment centers in 105 cities in the United States were assessed to determine species within the B. cepacia complex and examined for the presence of putative transmissibility markers (B. cepacia epidemic strain marker [BCESM] and cable pilin subunit gene [cblA]). Fifty percent of patients were infected with B. cepacia complex genomovar III, 38% with B. multivorans (formerly genomovar II), and 5% with B. vietnamiensis (formerly genomovar V); fewer than 5% of patients were infected with either genomovar I, B. stabilis (formerly genomovar IV), genomovar VI, or genomovar VII. BCESM was found in 46% of genomovar III isolates and not in any other species. Only one isolate, from a patient infected with the ET12 epidemic lineage, contained the complete cblA pilin subunit gene. Our data indicate a differential capacity for human infection among the phylogenetically closely related species of the B. cepacia complex. The low frequency of BCESM and cblA suggests that they are not sufficient markers of B. cepacia virulence or transmissibility.

Summary Life threatening infection with species of the Burkholderia cepacia complex frequently occurs as a result of cross infection among individuals with cystic fibrosis. Stringent infection control measures have decreased but not eliminated such infection in this vulnerable population, implying that non-patient reservoirs contribute to ongoing acquisition. However, strains common to both the natural environment and patients with cystic fibrosis have not yet been described. By use of various genotyping methods, we have identified from agricultural soil the B cepacia genomovar III strain that is most frequently recovered from cystic fibrosis patients in the mid-Atlantic region of the USA. This finding indicates that human pathogenic strains are not necessarily distinct from environmental strains, and might help explain ongoing human acquisition despite strict infection control measures.