Identification of Burkholderia cepacia strains that express a Burkholderia pseudomallei-like capsular polysaccharide.

Burkholderia pseudomallei and Burkholderia cepacia are Gram-negative, soil-dwelling bacteria that are found in a wide variety of environmental niches. While B. pseudomallei is the causative agent of melioidosis in humans and animals, members of the B. cepacia complex typically only cause disease in immunocompromised hosts. In this study, we report the identification of B. cepacia strains isolated from either patients or soil in Laos and Thailand that express a B. pseudomallei-like 6-deoxyheptan capsular polysaccharide (CPS). These B. cepacia strains were initially identified based on their positive reactivity in a latex agglutination assay that uses the CPS-specific monoclonal antibody (mAb) 4B11. Mass spectrometry and recA sequencing confirmed the identity of these isolates as B. cepacia (formerly genomovar I). Total carbohydrates extracted from B. cepacia cell pellets reacted with B. pseudomallei CPS-specific mAbs MCA147, 3C5, and 4C4, but did not react with the B. pseudomallei lipopolysaccharide-specific mAb Pp-PS-W. Whole genome sequencing of the B. cepacia isolates revealed the presence of genes demonstrating significant homology to those comprising the B. pseudomallei CPS biosynthetic gene cluster. Collectively, our results provide compelling evidence that B. cepacia strains expressing the same CPS as B. pseudomallei co-exist in the environment alongside B. pseudomallei. Since CPS is a target that is often used for presumptive identification of B. pseudomallei, it is possible that the occurrence of these unique B. cepacia strains may complicate the diagnosis of melioidosis.IMPORTANCEBurkholderia pseudomallei, the etiologic agent of melioidosis, is an important cause of morbidity and mortality in tropical and subtropical regions worldwide. The 6-deoxyheptan capsular polysaccharide (CPS) expressed by this bacterial pathogen is a promising target antigen that is useful for rapidly diagnosing melioidosis. Using assays incorporating CPS-specific monoclonal antibodies, we identified both clinical and environmental isolates of Burkholderia cepacia that express the same CPS antigen as B. pseudomallei. Because of this, it is important that staff working in melioidosis-endemic areas are aware that these strains co-exist in the same niches as B. pseudomallei and do not solely rely on CPS-based assays such as latex-agglutination, AMD Plus Rapid Tests, or immunofluorescence tests for the definitive identification of B. pseudomallei isolates.

Investigating Pseudomonas aeruginosa population structure and frequency of cross-infection in UK cystic fibrosis clinics - a reference laboratory perspective.

BACKGROUND: We aimed to describe the UK Pseudomonas aeruginosa population structure amongst people with cystic fibrosis (PWCF), and to examine evidence for cross-infection. METHODS: Variable Number Tandem Repeat (VNTR) typing was performed on 4640 isolates from 2619 PWCF received from 55 hospital laboratories between 2017 and 2019. A combination of whole genome sequence (WGS)-based analysis of four clusters from one hospital, and epidemiological analysis of shared strains in twelve hospitals evaluated cross-infection. RESULTS: Of 2619 PWCF, 1324 (51%) harboured common clusters or known transmissible strains, while 1295 carried unique strains/those shared among small numbers of patients. Of the former, 9.5% (250 patients) harboured the Liverpool epidemic strain (LES), followed in prevalence by clone C (7.8%; 205 patients), cluster A (5%;130 patients), and cluster D (3.6%; 94 patients). WGS analysis of 10 LES isolates, 9 of cluster D and 6 isolates each of cluster A and clone C from one hospital revealed LES formed the tightest cluster (between 7 and 205 SNPs), and cluster D the loosest (between 53 and 1531 SNPs). Hospital-specific shared strains were found in some centres, although cross-infection was largely historical, with few new acquisitions. Fifty-nine PWCF (2.3%) harboured "high-risk" clones; one ST235 isolate carried a blaIMP-1 allele. CONCLUSION: Of 2619 PWCF who had P. aeruginosa isolates submitted for VNTR, 51% harboured either common clusters or known transmissible strains, of which LES was the most common. Limited evidence of recent patient-to-patient strain transmission was found, suggesting cross-infection prevention measures and surveillance effectively reduce transmission.

Polymyxin Resistance and Heteroresistance Are Common in Clinical Isolates of Achromobacter Species and Correlate with Modifications of the Lipid A Moiety of Lipopolysaccharide.

The Achromobacter genus includes opportunistic pathogens that can cause chronic infections in immunocompromised patients, especially in people with cystic fibrosis (CF). Treatment of Achromobacter infections is complicated by antimicrobial resistance. In this study, a collection of Achromobacter clinical isolates, from CF and non-CF sources, was investigated for polymyxin B (PmB) resistance. Additionally, the effect of PmB challenge in a subset of isolates was examined and the presence of PmB-resistant subpopulations within the isolates was described. Further, chemical and mass spectrometry analyses of the lipid A of Achromobacter clinical isolates enabled the determination of the most common structures and showed that PmB challenge was associated with lipid A modifications that included the addition of glucosamine and palmitoylation and the concomitant loss of the free phosphate at the C-1 position. This study demonstrates that lipid A modifications associated with PmB resistance are prevalent in Achromobacter and that subresistant populations displaying the addition of positively charged residues and additional acyl chains to lipid A can be selected for and isolated from PmB-sensitive Achromobacter clinical isolates. IMPORTANCE Achromobacter species can cause chronic and potentially severe infections in immunocompromised patients, especially in those with cystic fibrosis. Bacteria cannot be eradicated due to Achromobacter's intrinsic multidrug resistance. We report that intrinsic resistance to polymyxin B (PmB), a last-resort antimicrobial peptide used to treat infections by multiresistant bacteria, is prevalent in Achromobacter clinical isolates; many isolates also display increased resistance upon PmB challenge. Analysis of the lipopolysaccharide lipid A moiety of several Achromobacter species reveals a penta-acylated lipid A, which in the PmB-resistant isolates was modified by the incorporation of glucosamine residues, an additional acyl chain, loss of phosphates, and hydroxylation of acyl chains, all of which can enhance PmB resistance in other bacteria. We conclude that PmB resistance, particularly in Achromobacter isolates from chronic respiratory infections, is a common phenomenon, and that Achromobacter lipid A displays modifications that may confer increased resistance to polymyxins and potentially other antimicrobial peptides.

Melioidosis Manifesting as Chronic Femoral Osteomyelitis in Patient from Ghana.

A 33-year-old man from Ghana who had diabetes had chronic osteomyelitis of the femoral shaft develop. Tissue samples from surgical debridement grew Burkholderia pseudomallei. He received meropenem, followed by oral trimethoprim/sulfamethoxazole and doxycycline, and fully recovered without complications. Our case report extends the range of countries in Africa as sources of culture-confirmed melioidosis.

Variability in Bacteriophage and Antibiotic Sensitivity in Serial Pseudomonas aeruginosa Isolates from Cystic Fibrosis Airway Cultures over 12 Months.

Antibiotic treatment for Pseudomonas aeruginosa (Pa) in cystic fibrosis is limited in efficacy and may lead to multi-drug resistance (MDR). Alternatives such as bacteriophages are being explored but well designed, and controlled trials are crucial. The rational selection of patients with bacteriophage susceptible infections is required for both safety and efficacy monitoring. We questioned whether bacteriophage susceptibility profiles were constant or variable over time, variability having been reported with antibiotics. Serial Pa isolates (n = 102) from 24 chronically infected cystic fibrosis (CF) patients over one year were investigated with plaque and antibiotic disc diffusion assays. Variable number tandem repeat (VNTR) analysis identified those patients with >1 isolate. A median (range) of 4 (3-6) isolates/patient were studied. Twenty-one (87.5%) individuals had a single VNTR type; three (12.5%) had two VNTR types at different times. Seventy-five percent of isolates were sensitive to bacteriophage at ≥ 1 concentration; 50% of isolates were antibiotic multidrug resistant. Serial isolates, even when representing a single VNTR type, varied in sensitivity to both bacteriophages and antibiotics. The rates of sensitivity to bacteriophage supports the development of this therapy; however, the variability in response has implications for the selection of patients in future trials which must be on the basis of current, not past, isolate testing.

Transmission, adaptation and geographical spread of the Pseudomonas aeruginosa Liverpool epidemic strain.

The Liverpool epidemic strain (LES) is an important transmissible clonal lineage of Pseudomonas aeruginosa that chronically infects the lungs of people with cystic fibrosis (CF). Previous studies have focused on the genomics of the LES in a limited number of isolates, mostly from one CF centre in the UK, and from studies highlighting identification of the LES in Canada. Here we significantly extend the current LES genome database by genome sequencing 91 isolates from multiple CF centres across the UK, and we describe the comparative genomics of this large collection of LES isolates from the UK and Canada. Phylogenetic analysis revealed that the 145 LES genomes analysed formed a distinct clonal lineage when compared with the wider P. aeruginosa population. Notably, the isolates formed two clades: one associated with isolates from Canada, and the other associated with UK isolates. Further analysis of the UK LES isolates revealed clustering by clinic geography. Where isolates clustered closely together, the association was often supported by clinical data linking isolates or patients. When compared with the earliest known isolate, LESB58 (from 1988), many UK LES isolates shared common loss-of-function mutations, such as in genes gltR and fleR. Other loss-of-function mutations identified in previous studies as common adaptations during CF chronic lung infections were also identified in multiple LES isolates. Analysis of the LES accessory genome (including genomic islands and prophages) revealed variations in the carriage of large genomic regions, with some evidence for shared genomic island/prophage complement according to clinic location. Our study reveals divergence and adaptation during the spread of the LES, within the UK and between continents.

Structured surveillance of Achromobacter, Pandoraea and Ralstonia species from patients in England with cystic fibrosis.

A structured survey of the cystic fibrosis pathogens Achromobacter, Pandoraea and Ralstonia species from thirteen sentinel hospitals throughout England was undertaken by Public Health England. One isolate per patient of these genera collected from CF patients during the seven-month survey period in 2015 was requested from participating hospitals. Species-level identification was performed using nrdA/gyrB sequence cluster analysis, and genotyping by pulsed-field gel electrophoresis. In total, 176 isolates were included in the survey; 138 Achromobacter spp. (78.4%), 29 Pandoraea spp. (16.5%) and 9 Ralstonia spp. (5.1%). Novel Achromobacter and Pandoraea clusters were identified. High levels of antimicrobial resistance were found, particularly among Pandoraea isolates. Genotyping analysis revealed considerable diversity, however one geographically-widespread cluster of A. xylosoxidans isolates from six hospitals was found, in addition to two other clusters, both comprising isolates from two hospitals, either derived from the same region (A. xylosoxidans), or from hospitals within the same city (P. apista).

Investigation of a Pandoraea apista cluster common to adult and paediatric cystic fibrosis patients attending two hospitals in the same city.

PURPOSE: We examined evidence for transmission of Pandorea apista among cystic fibrosis (CF) patients attending paediatric and adult services in one city who had previously been found to harbour related isolates by pulsed-field gel electrophoresis (PFGE). METHODOLOGY: The whole-genome sequences of 18 isolates from this cluster from 15 CF patients were examined, along with 2 cluster isolates from 2 other centres. The annotated sequence of one of these, Pa14367, was examined for virulence factors and antibiotic resistance-associated genes in comparison with data from a 'non-cluster' isolate, Pa16226. RESULTS: Single-nucleotide polymorphism (SNP) analysis suggested that cluster isolates from the same city differed from one another by a minimum of 1 and a maximum of 383 SNPs (an average of 213 SNPs; standard deviation: 18.5), while isolates from the 2 other hospitals differed from these by a minimum of 34 and 61 SNPs, respectively. Pa16226 differed from all cluster isolates by a minimum of 22 706 SNPs. Evidence for patient-to-patient transmission among isolates from the same city was relatively limited, although transmission from a common source could not be excluded. The annotated genomes of Pa14367 and Pa16226 carried putative integrative and conjugative elements (ICEs), coding for type IV secretion systems, and genes associated with heavy metal degradation and carbon dioxide fixation, and a wide selection of genes coding for efflux pumps, beta-lactamases and penicillin-binding proteins. CONCLUSION: Epidemiological analysis suggested that this cluster could not always be attributed to patient-to-patient transmission. The acquisition of ICE-related virulence factors may have had an impact on its prevalence.

rpoB gene sequencing highlights the prevalence of an E. miricola cluster over other Elizabethkingia species among UK cystic fibrosis patients.

Difficulties in distinguishing species of the Elizabethkingia genus by MALDI-TOF prompted use of rpoB sequencing to investigate species distribution among 44 isolates from cystic fibrosis (CF) patients. Forty-three isolates from 38 patients formed a cluster comprising E. miricola and proposed novel species E. bruuniana sp. nov., the exception clustering with proposed species E. ursingii sp. nov., also part of this wider cluster. All 44 isolates were PCR-positive for urease gene ureG, whereas only one of 23 E. anophelis isolates from non-CF patients was positive, suggesting that this gene is largely associated with the E. miricola cluster. Antibiotic susceptibilities of 12 CF isolates revealed all were resistant to beta-lactams with the exception of piperacillin-tazobactam, and were only susceptible to minocycline and co-trimoxazole. Pulsed-field gel electrophoresis analysis revealed 4 shared strains among 17 CF patients in one pediatric clinic, but epidemiological investigations did not support patient-to-patient transmission except between one sibling pair.

Prevalence of Burkholderia species, including members of Burkholderia cepacia complex, among UK cystic and non-cystic fibrosis patients.

PURPOSE: We aimed to establish the prevalence of different Burkholderia species among UK cystic fibrosis (CF) and non-CF patients over a 2 year period. METHODOLOGY: Matrix-assisted laser desorption/ionization-time of flight mass spectrometry was used to identify isolates to genus level, followed by recA/gyrB sequence clustering or species-specific PCR. In all, 1047 Burkholderia isolates were submitted for identification from 361 CF patients and 112 non-CF patients, 25 from the hospital environment and three from a commercial company. Potential cross-infection was assessed by pulsed-field gel electrophoresis (PFGE) and multi- locus-sequence typing (MLST). MICs were determined for 161 Burkholderia cepacia complex (Bcc) isolates. CF Trust registry data were sought to examine clinical parameters relating to Bcc infection. RESULTS: Burkholderia multivorans was the most prevalent species among CF patients affecting 56 % (192) patients, followed by Burkholderia cenocepacia IIIA (15 %; 52 patients). Five novel recA clusters were found. Among non-CF patients, Burkholderia cepacia was the most prevalent species (37/112; 34 %), with 18 of 40 isolates part of a UK-wide B. cepacia 'cluster'. This and three other clusters were investigated by PFGE and MLST. Cable-pili positive isolates included two novel sequence types and representatives of ET12. Antibiotic susceptibility varied between and within species and CF/non- CF isolates. CF Trust registry data suggested no significant difference in lung function between patients harbouring B. cenocepacia, B. multivorans and other Bcc species (P=0.81). CONCLUSION: The dominance of B. multivorans in CF, the presence of a B. cepacia cluster among non-CF patients and the existence of putative novel species all highlighted the continuing role of Burkholderia species as opportunistic pathogens.

Use of nrdA gene sequence clustering to estimate the prevalence of different Achromobacter species among Cystic Fibrosis patients in the UK.

BACKGROUND: We aimed to estimate the prevalence of different Achromobacter species among UK Cystic Fibrosis (CF) patients. METHODS: nrdA sequence clustering was used to identify 147 Achromobacter isolates from 96 patients from 27 hospitals to species level. Potential cross-infection was investigated by MLST, pulsed-field gel electrophoresis and whole genome sequencing (WGS). RESULTS: Achromobacter xylosoxidans was the most prevalent species affecting 59 of 96 (61%) patients, followed by Achromobacter insuavis and Achromobacter dolens (12.4% and 8%, respectively). Three novel nrdA clusters were identified. One was further characterised by sequencing the intrinsic blaOXA gene, revealing novel variants. WGS of A. insuavis 2a isolates from four patients attending the same paediatric unit revealed that three were ST144, but differed from one another by a minimum of 385 SNPs, suggesting cross-infection was unlikely. CONCLUSIONS: nrdA sequence clustering permitted an estimation of UK Achromobacter species prevalence, highlighted additional novel species, and aided cross-infection investigations.

A novel culture medium for isolation of rapidly-growing mycobacteria from the sputum of patients with cystic fibrosis.

BACKGROUND: Isolation of mycobacteria from the sputum of patients with cystic fibrosis (CF) is challenging due to the overgrowth of cultures by other bacteria and fungi. In this setting, Burkholderia cepacia selective agar (BCSA) has been recommended as a convenient and effective culture medium for the isolation of rapidly-growing, non-tuberculous mycobacteria (NTM). A novel selective culture medium (RGM medium) was evaluated for the isolation of rapidly-growing NTM from the sputum of children and adults with CF. METHODS: A total of 118 isolates of rapidly-growing mycobacteria and 98 other bacteria and fungi were inoculated onto RGM medium. These were assessed for growth at 30°C over a seven day period. A total of 502 consecutive sputum samples were collected from 210 patients with CF. Each sample was homogenized and cultured onto RGM medium and also onto BCSA. Cultures were incubated for 10days at 30°C. RESULTS: Of 118 isolates of mycobacteria all but one grew well on RGM medium, whereas 94% of other bacteria and fungi were inhibited. A total of 55 sputum samples (from 33 distinct patients) yielded NTM using a combination of both RGM and BCSA (prevalence: 15.7%). NTM were recovered from 54 sputum samples using RGM medium compared with only 17 samples using BCSA (sensitivity 98% vs. 31%; P≤0.0001). A total of 419 isolates of non-mycobacteria were recovered from sputum samples on BCSA compared with 46 on RGM medium. CONCLUSIONS: RGM medium offers a simple and effective culture method for the isolation of rapidly-growing mycobacteria from sputum samples from patients with CF without decontamination of samples. RGM medium allows for the systematic screening of all sputum samples routinely referred for culture from patients with CF.

Clinical utilization of genomics data produced by the international Pseudomonas aeruginosa consortium.

The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consortium Database (http://ipcd.ibis.ulaval.ca/). Here, we present our strategy and the results that emerged from the analysis of the first 389 genomes. With as yet unmatched resolution, our results confirm that P. aeruginosa strains can be divided into three major groups that are further divided into subgroups, some not previously reported in the literature. We also provide the first snapshot of P. aeruginosa strain diversity with respect to antibiotic resistance. Our approach will allow us to draw potential links between environmental strains and those implicated in human and animal infections, understand how patients become infected and how the infection evolves over time as well as identify prognostic markers for better evidence-based decisions on patient care.

Whole-genome sequencing and epidemiological analysis do not provide evidence for cross-transmission of mycobacterium abscessus in a cohort of pediatric cystic fibrosis patients.

BACKGROUND: Mycobacterium abscessus has emerged as a major pathogen in cystic fibrosis (CF) patients and has been associated with poor clinical outcomes, particularly following lung transplant. We investigated the acquisition of this bacterium in a cohort of pediatric CF patients. METHODS: Demographic and patient location data were used to uncover epidemiological links between patients with genetically related strains of M. abscessus that had been previously typed by variable-number tandem repeat profiling. Whole-genome sequencing was applied to 27 M. abscessus isolates from the 20 patients in this cohort to provide definitive data on the genetic relatedness of strains. RESULTS: Whole-genome sequencing data demonstrated that M. abscessus isolates from 16 patients were unrelated, differing by at least 34 single-nucleotide polymorphisms (SNPs) from any other isolate, suggesting that independent acquisition events have occurred. Only 2 clusters of very closely related (<25 SNPs) isolates from different patients were seen. The first cluster contained 8 isolates, differing by a maximum of 17 SNPs, from a sibling pair who had intense exposure to each other both inside and outside the hospital. The second cluster contained 3 isolates, differing by a maximum of 24 SNPs, from 2 individuals with no apparent epidemiological links. CONCLUSIONS: We have not demonstrated cross-transmission of M. abscessus within our hospital, except between 1 sibling pair. Alternative routes of acquisition of M. abscessus infection, in particular the environment, require further investigation.

Mycobacterium abscessus infection in cystic fibrosis: molecular typing and clinical outcomes.

Mycobacterium abscessus is a significant pathogen in the cystic fibrosis patient population. PCR amplification and sequencing can provide accurate subspecies identification, and can predict macrolide susceptibility, which is becoming increasingly important for patient management. Molecular techniques for further typing of isolates provide tools for the ongoing investigations into the clinical impact of particular M. abscessus strains. Whole-genome sequencing is likely to be the only technique that provides sufficient resolution for investigating transmission events between patients.

Clusters of genetically similar isolates of Pseudomonas aeruginosa from multiple hospitals in the UK.

Variable number tandem repeat (VNTR) analysis at nine loci of isolates of Pseudomonas aeruginosa submitted to the national reference laboratory from UK hospitals, from over 2000 patients, between June 2010 and June 2012 revealed four widely found types that collectively were received from approximately a fifth of patients, including from those with cystic fibrosis. These types were also prevalent among related submissions from the clinical environment and were received from up to 54 (out of 143) hospitals. Multi-locus sequence typing and blaOXA-50-like sequencing confirmed the clonal relationship within each cluster, and representatives from multiple centres clustered within about 70 % by pulsed-field gel electrophoresis. Illumina sequencing of 12 isolates of cluster A of VNTR profile 8, 3, 4, 5, 2, 3, 5, 2, x (where the repeat number at the last, most discriminatory locus is variable) revealed a large number of variably present targets in the accessory genome and seven of these were sought by PCR among a larger set of isolates. Representatives from patients within a single centre mostly had distinct accessory gene profiles, suggesting that these patients acquired the strain independently, while those with clear epidemiological links shared the same profile. Profiles also varied between representatives from different centres. Epidemiological investigations of widely found types such as these require the use of finer-typing methods, which increasingly will be informed by next generation sequencing.

Molecular fingerprinting of Mycobacterium abscessus strains in a cohort of pediatric cystic fibrosis patients.

Forty-one Mycobacterium abscessus complex isolates from 17 pediatric cystic fibrosis (CF) patients were typed using a novel variable-number tandem repeat (VNTR) scheme and an automated repetitive-PCR (rep-PCR) system. Both VNTR and rep-PCR typing methods differentiate between members of the M. abscessus complex. The isolates from individual patients are indistinguishable, and the data strongly suggest that individual CF patients are persistently infected with one strain and also suggests that different CF patients can harbor the same strain.

Melioidosis acquired by traveler to Nigeria.

We describe melioidosis associated with travel to Nigeria in a woman with diabetes, a major predisposing factor for this infection. With the prevalence of diabetes projected to increase dramatically in many developing countries, the global reach of melioidosis may expand.

Identification of Achromobacter xylosoxidans by detection of the bla(OXA-114-like) gene intrinsic in this species.

Achromobacter xylosoxidans is an emerging pathogen among patients with cystic fibrosis. Here we describe a specific PCR for identification of this organism, based on detection of bla(OXA-114-like). Comparison of isolates by pulsed-field gel electrophoresis revealed evidence of cross-infection in some cases, but most patients harbored their own strain.