Genetic Diversity of Burkholderia contaminans Isolates from Cystic Fibrosis Patients in Argentina

A total of 120 Burkholderia cepacia complex isolates collected during 2004–2010 from 66 patients in two cystic fibrosis reference centers in Argentina were analyzed. Burkholderia contaminans was the species most frequently recovered (57.6%), followed by Burkholderia cenocepacia (15%), a species distribution not reported so far. The recA-PCR-based techniques applied to the B. contaminans isolates revealed that 85% of the population carried the recA-ST-71 allele. Our results showed the utility of BOX-PCR genotyping in analyzing B. contaminans diversity. This approach allowed us to address clonal transmission during an outbreak and the genetic changes occurring in infecting bacteria over the course of chronic infection.     

Development of a species-specific fur gene-based method for identification of the Burkholderia cepacia complex

Burkholderia is an important bacterial genus with a complex taxonomy that contains species of both ecological and pathogenic importance, including nine closely related species collectively termed the Burkholderia cepacia complex (BCC). Unfortunately, 16S rRNA gene analysis has proven to be not sensitive enough to discriminate between species of the BCC. Alternative species identification strategies such as recA-based PCR followed by restriction fragment length polymorphism analysis, although initially useful, have proven to be inaccurate with the increasing species diversity of the BCC. recA gene sequence analysis is more discriminatory and corroborates other biochemical and polyphasic means of taxonomic differentiation. However, it is limited by the fact that certain BCC species are subdivided into discrete recA sequence subgroups that may confuse clinical diagnoses. In this study, an effective approach is described for the rapid differentiation of BCC species from both environmental and clinical sources by means of a single-locus sequencing and PCR assay using fur as a target gene that provides sequence phylogenies that are species specific and, with few exceptions, not divided into subspecies clusters. This assay is specific and can be used to correctly determine the species status of BCC strains tested following sequencing and amplification of the fur gene by both general and species-specific primers. Based on our results, this typing strategy is simpler than and as sensitive as established tests currently in use clinically. This assay is useful for the rapid, definitive identification of all nine current BCC species and potentially novel species groups.     

Genomovar Diversity Amongst<Emphasis Type="Italic"> Burkholderia cepacia</Emphasis> Complex Isolates From an Australian Adult Cystic Fibrosis Unit

In this study, a combination of recA-based PCR assays and 16S rDNA restriction fragment length polymorphism (RFLP) analysis was used to determine the genomovar diversity of clinical Burkholderia cepacia complex isolates. Twenty-eight isolates were prospectively collected from patients attending a large Australian adult cystic fibrosis (CF) unit, 22 isolates were referred from other Australian CF units and a further eight isolates originated from patients without CF. The 28 prospectively collected isolates were distributed amongst the following genomovars: Burkholderia cepacia genomovar I (28.6%), Burkholderia multivorans (21.4%), Burkholderia cepacia genomovar III (39.3%), Burkholderia vietnamiensis (3.6%) and Burkholderia ambifaria (7.1%). The results of this study highlight the usefulness of 16S rDNA RFLP typing for the identification of other Burkholderia spp. and non-fermenting gram-negative bacteria.     

Diagnostic methods and identification challenges experienced in a Burkholderia contaminans outbreak occurred in a tertiary care centre

BackgroundRecently, a novel species contaminans belonging to the family Burkholderia cepacia complex (Bcc) is rising as a hospital pathogen. Detection of Burkholderia contaminans, a member of Bcc can be done only by MALDI TOF and sequencing techniques. We report the diagnostic challenges faced in an outbreak of bacteremia due to B. contaminans grown in diltiazem vials.MethodThe department of microbiology notified the infection control team about a cluster of eleven patients with B. contaminans isolated from blood culture. An outbreak investigation was initiated by performing environmental surveillance and sterility testing of solutions given for the patients. Routine phenotypical methods for identification of species followed by MALDI-TOF and sequencing was performed to identify the pathogen.ResultsAll the patients detected with B. contaminans were having cardiac disease and received diltiazem. Sterility testing of diltiazem vials given for the patient and an unopened vial of same batch has grown B. contaminans. Clonal typing has confirmed the sequence similarities between patient and solution isolates.ConclusionDue to diagnostic challenge in identifying the species of Bcc, MALDI TOF and clonal typing remains the key diagnostic tools available to detect Bcc species at an earliest especially in an outbreak.     

Siderophore Production by Cystic Fibrosis Isolates of Burkholderia cepacia

Sixty-one Burkholderia cepacia isolates from patients with cystic fibrosis (CF) and four plant isolates were screened for production of the siderophores salicylic acid (SA), pyochelin, cepabactin, and ornibactins and fingerprinted by a PCR-based randomly amplified polymorphic DNA (RAPD) method. Of the 24 RAPD types determined, 22 (92%) were associated with isolates that produced SA, 21 (87%) were associated with isolates that produced ornibactins, 15 (60%) were associated with isolates that produced pyochelin, and 3 (12%) were associated with isolates that produced cepabactin. Of the 24 RAPD types plus 2 phenotypic variants of types 1 and 9, 3 were associated with isolates that produced all four siderophores, 8 were associated with isolates that produced three siderophores, 12 were associated with isolates that produced two siderophores, and 3 were associated with isolates that produced only one siderophore. These results suggest that the numbers and types of siderophores produced by CF isolates of B. cepacia correlate with RAPD type and that SA and ornibactins are the most prevalent siderophores produced.     

Five novel acid-tolerant oligotrophic thiosulfate-metabolizing chemolithotrophic acid mine drainage strains affiliated with the genus Burkholderia of Betaproteobacteria and identification of two novel soxB gene homologues

Five acid-tolerant thiosulfate-metabolizing bacteria were isolated from acid mine drainage samples from Garubathan, India. 16S rRNA gene analysis revealed that the strains were affiliated with the genus Burkholderia of the class of Betaproteobacteria. Comparative 16S rRNA gene sequence analyses indicated that the strains designated as GAH1 and GAH2 produced a separate phylogenetic branch having Burkholderia pyrrocinia ATCC 51958^T (96-98%) as the closest relative. Strains GAH4 and Burkholderia tropica Ppe8^T (93%) branched out separately in the phylogenetic tree. Strain GMX2 was most closely related to Burkholderia cepacia ATCC 25417^T (99.6%) and Burkholderia vietnamiensis LMG 10929^T (99%). Strain GAH5 was most closely related to B. pyrrocinia ATCC 51958^T (98%). Oligotrophy has been demonstrated in all AMD strains of Burkholderia spp. All strains showed chemolithoautotrophic and mixotrophic growth in thiosulfate. Furthermore, cell-free extracts of all test strains possessed thiosulfate and sulfite dehydrogenase activities. Phylogenetic analysis of the soxB gene revealed that GAH4 and GAH2 strains formed a novel cluster, Betaproteobacteria II, having highest similarity with Allochromatium vinosum, a member of Gammaproteobacteria II.     

Use of quorum sensing inhibitors to interfere with biofilm formation and development in Burkholderia multivorans and Burkholderia cenocepacia

Burkholderia cepacia complex strains are opportunistic pathogens causing life-threatening infections in cystic fibrosis patients. B. cepacia complex strains are resistant to many antimicrobial agents and commonly produce biofilms in vitro and in vivo. This contributes to their virulence and makes Burkholderia infections difficult to treat. Recently, the quorum sensing (QS) system of Burkholderia spp. has been found to affect their biofilm-forming ability, making it an attractive target for antimicrobial therapy. However, detailed information about the anti-biofilm effect of these compounds is still lacking. In the present study, we evaluated the anti-biofilm effect of several known QS inhibitors. The effect on Burkholderia spp. biofilm formation was examined using crystal violet, resazurin and SYTO9 staining, confocal laser scanning microscopy as well as plating. When used at subinhibitory concentrations, several compounds interfered with biofilm formation by Burkholderia spp. Our results suggest that the QS inhibitors affect later stages of biofilm formation and detachment.     

Outbreak of Burkholderia cepacia bloodstream infections traced to the use of Ringer lactate solution as multiple-dose vial for catheter flushing,Phnom Penh,Cambodia

The Burkholderia cepacia complex is a group of Gram-negative bacteria known as respiratory pathogens in cystic fibrosis patients, but also increasingly reported as a cause of healthcare associated infections. We describe an outbreak of B. cepacia bloodstream infections in a referral hospital in Phnom Penh, Cambodia. Over a 1.5-month period, blood cultures from eight adult patients grew B. cepacia. Bloodstream infection occurred after a median of 2.5 days of hospitalisation. Three patients died: 7, 10 and 17 days after blood cultures were sampled. As part of the outbreak investigation, patient files were reviewed and environmental sampling was performed. All patients had peripheral venous catheters that were flushed with Ringer lactate drawn from a 1 L bag, used as multiple-dose vial at the ward. Cultures of unopened Ringer lactate and disinfectants remained sterile but an in-use bag of Ringer lactate solution and the dispensing pin grew B. cepacia. The isolates from patients and flushing solution were identified as B. cepacia by recA gene sequence analysis, and random amplified polymorphic DNA typing confirmed clonal relatedness. The onset of the outbreak had coincided with the introduction of a dispensing pin with a screw fit that did not allow proper disinfection. Re-enforcement of aseptic procedures with sterile syringe and needle has ended the outbreak. Growth of B. cepacia should alert the possibility of healthcare associated infection also in tropical resource-limited settings. The use of multiple-dose vials should be avoided and newly introduced procedures should be assessed for infection control risks.     

Development of Galleria mellonella as an alternative infection model for the Burkholderia cepacia complex

Burkholderia is an important bacterial genus with a complex taxonomy that contains species of both ecological and pathogenic importance, including nine closely related species collectively termed the Burkholderia cepacia complex (BCC). In order to more thoroughly investigate the virulence of this bacterial complex of microorganisms, alternative infection models would be useful. To this end, we have adapted and developed the use of the Galleria mellonella wax moth larvae as a host for examining BCC infections. The experimental conditions affecting the BCC killing of the “wax worm” were optimized. BCC virulence levels were determined using 50% lethal doses, and differences were observed between both species and strains of the BCC. The BCC pathogenicity trends obtained compare favorably with results acquired using other published alternative infection models, as well as mammalian infection models. In addition, BCC killing activity was determined by directly measuring relative bacterial loads in three different BCC strains, thus demonstrating innate differences in BCC strain virulence. Finally, genetically mutated BCC strains were compared to a wild-type BCC strain in order to show concomitant reduction of BCC virulence and increased wax worm survival. For experimentation examining the virulent properties of the BCC, the wax worm has proven to be a useful alternative infection model.     

Reliability of Automated Biochemical Identification of Burkholderia pseudomallei Is Regionally Dependent

Misidentifications of Burkholderia pseudomallei as Burkholderia cepacia by Vitek 2 have occurred. Multidimensional scaling ordination of biochemical profiles of 217 Malaysian and Australian B. pseudomallei isolates found clustering of misidentified B. pseudomallei isolates from Malaysian Borneo. Specificity of B. pseudomallei identification in Vitek 2 and potentially other automated identification systems is regionally dependent.     

Molecular epidemiology and antibiotic susceptibility of Burkholderia cepacia-complex isolates from an Italian cystic fibrosis centre

In order to further understanding of how different isolates of Burkholderia cepacia complex persist, spread and cause disease, B. cepacia-complex isolates from 60 patients attending the Cystic Fibrosis Centre of Verona, Italy, between 1997 and 2002 were analyzed. Strains were examined for species, presence of putative epidemic and virulence markers (i.e., cblA and the B. cepacia epidemic-strain marker [BCESM]), genetic relatedness and antibiotic susceptibility. Forty-five percent of patients were infected with B. cenocepacia recA subgroup B, 28% with B. cenocepacia recA subgroup A, 5% with B. multivorans and 5% with B. cepacia. No isolate carried cblA but 35% of B. cenocepacia and one of B. cepacia carried the BCESM transmissibility marker. Pulsed-field gel electrophoresis (PFGE) identified 40 types; 22 of these corresponded to sporadic isolates and 18 to clusters of identical or genetically related strains. Piperacillin, ceftazidime and piperacillin-tazobactam were the most active antibiotics (43.3, 31.1 and 35.5% of resistance, respectively). These results confirm the prevalence of B. cenocepacia in cystic fibrosis patients with rapid clinical deterioration and in those with stable cases of infection. The rates of multiple-source and cross infection were relatively low.     

Mycobacterium abscessus Complex Identification with Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

We determined that the Vitek MS Plus matrix-assisted laser desorption ionization–time of flight mass spectrometry using research-use-only (RUO) v.4.12 and in vitro-diagnostic (IVD) v.3.0 databases accurately identified 41 Mycobacterium abscessus subsp. abscessus and 13 M. abscessus subsp. massiliense isolates identified by whole-genome sequencing to the species but not the subspecies level, from Middlebrook 7H11 and Burkholderia cepacia selective agars. Peak analysis revealed three peaks potentially able to differentiate between subspecies.     

Clinical and demographic study of non-tuberculous mycobacterial ocular infections in South India

PurposeTo describe demographics, risk factors, antibiotic susceptibility, management and outcomes of ocular infections caused by non-tuberculous mycobacteria (NTM).MethodsA retrospective review of medical case records and microbiology records of patients with ocular infections that were culture positive for non-tuberculous Mycobacteria from January 2014 to December 2018 was done. Antibiotic susceptibility profile was done based on the CLSI guidelines. Laboratory diagnosis for the NTM Species was done by conventional microbiological methods. The species identification was done for stored isolated utilizing polymerase chain reaction targeting 16S rDNA and rpoB gene, followed by DNA sequencing and phylogenetic analysis.ResultsTwenty patients with NTM ocular infections were identified during the study period. A majority of cases presented as 12 infectious keratitis (60%) and three suture-related corneal infiltrates (15%). Common risk factors were history of trauma in 9 (45%) patients and history of ocular surgery in 5 (25%) patients. Patients were treated with combination of amikacin and flouroquinolones/chloramphenicol (70%) and surgical interventions were performed in 25% cases. Only twelve isolates were stored and ten isolates were identified as the M. abscessus subsp. abscessus and two isolates as M. abscessus subsp. massiliense by sequencing and phylogenetic analysis. Majority of the NTM were sensitive to amikacin (75%) followed by moxifloxacin, ciprofloxacin, cephotaxime and tobramycin (35%).ConclusionHigh degree of clinical suspicion, multidrug antibiotic therapy and timely surgical intervention in patients with NTM infections, are advised for better clinical outcomes. Prior ocular trauma, prior ocular surgery and presence of biomaterials were the major predisposing factors. Earlier surgical intervention in cases where abscesses or biomaterials are involved, is necessary for rapid recovery.     

Expanded Multilocus Sequence Typing for Burkholderia Species

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.The multilocus sequence typing (MLST) scheme for the Burkholderia cepacia complex has provided important insights into the population dynamics, diversity, and recombination events in this group of opportunistic pathogens (1-3, 7). It has also been effective in identifying previously misclassified strains and has proved useful in the recent identification of seven novel species in the B. cepacia complex (9, 10). This expansion of the B. cepacia complex and our growing appreciation that other Burkholderia species, particularly B. gladioli, are also involved in human infection, provide an opportunity to expand the capacity of the current MLST scheme. We thus sought to redesign the primers targeting the loci in the current MLST scheme to (i) more reliably amplify these seven loci from all 17 B. cepacia complex species; (ii) amplify these loci from additional Burkholderia species, including B. gladioli and as yet unclassified Burkholderia species; and (iii) enable the use of a single primer set per locus for both amplification and DNA sequencing.We aligned all seven loci (atpD, gltB, gyrB, lepA, phaC, recA, and trpB) in the current MLST scheme from Burkholderia strains for which complete genome sequences were available in the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi) or Joint Genome Institute (http://www.jgi.doe.gov/genome-projects/) database. These strains included Burkholderia ambifaria AMMD, B. cenocepacia J2315, B. cenocepacia AU1054, B. cenocepacia PC184, B. multivorans ATCC 17616, B. vietnamiensis G4, B. dolosa AU0158, B. xenovorans LB400, B. phymatum STM815, B. phytofirmans PsJN, B. mallei ATCC 23344, Burkholderia sp. strain 383, and Burkholderia sp. strain H160. Open reading frames were aligned by using MegAlign (DNASTAR Inc., Madison, Wis.), and Burkholderia genus-level primers were designed to amplify sequences that include the regions amplified by the original MLST primers (3) (Table ​(Table11).

TABLE 1.

Oligonucleotide primer sequences and annealing temperatures for the amplification and sequencing of seven MLST loci for Burkholderia species

Two novel clinical presentations of Burkholderia cepacia infection

We report two cases of multidrug-resistant Burkholderia cepacia (B. cepacia genomovar I) and Burkholderia multivorans causing multiple liver abscesses in a patient with bronchial asthma (case 1) and peritonitis in a patient with cirrhosis and hepatitis C virus disease (case 2), respectively. Both patients were treated successfully.     

Evaluation of PCR pncA-restriction fragment length polymorphism and PCR amplification of genomic regions of difference for the identification of M. bovis strains in lymph nodes cultures

BackgroundA rapid accurate identification of Mycobacterium bovis is essential for surveillance purposes.ObjectivesA PCR pncA-Restriction Fragment Length Polymorphism (RFLP) and a multiplex PCR based on the detection of 3 regions of difference (RD-PCR): RD9, RD4 and RD1 were evaluated for the identification of M. bovis in lymph nodes cultures, in Tunisia, during 2013–2015.MethodsEighty-two M. tuberculosis complex strains were identified using the biochemical tests, GenoType MTBC assay, PCR pncA-RFLP and RD-PCR.ResultsThe PCR pncA-RFLP showed that 54 M. bovis strains, identified by GenoType MTBC, had a mutation at position 169 of pncA gene. Twenty-eight strains did not show any mutation at this position 27 M. tuberculosis isolates and one M. caprae. The PCR pncA-RFLP had a sensitivity of 100.0% (95%CI: 93.3 -100.0) and a specificity of 100.0% (95%CI: 87.9–100.0) for identifying M. bovis. The RD-PCR showed that all M. bovis strains had the RD9 and RD4 deleted but presented RD1. RD-PCR also presented high sensitivity and specificity in detecting M. bovis strains (100.0%).ConclusionsPCR pncA-RFLP and RD-PCR represent very accurate and rapid tools to identify M. bovis. They can be easily implemented in each laboratory due to their low cost and easy use.     

Fourier transform infrared spectroscopy for rapid identification of nonfermenting gram-negative bacteria isolated from sputum samples from cystic fibrosis patients

The accurate and rapid identification of bacteria isolated from the respiratory tract of patients with cystic fibrosis (CF) is critical in epidemiological studies, during intrahospital outbreaks, for patient treatment, and for determination of therapeutic options. While the most common organisms isolated from sputum samples are Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, in recent decades an increasing fraction of CF patients has been colonized by other nonfermenting (NF) gram-negative rods, such as Burkholderia cepacia complex (BCC) bacteria, Stenotrophomonas maltophilia, Ralstonia pickettii, Acinetobacter spp., and Achromobacter spp. In the present study, we developed a novel strategy for the rapid identification of NF rods based on Fourier transform infrared spectroscopy (FTIR) in combination with artificial neural networks (ANNs). A total of 15 reference strains and 169 clinical isolates of NF gram-negative bacteria recovered from sputum samples from 150 CF patients were used in this study. The clinical isolates were identified according to the guidelines for clinical microbiology practices for respiratory tract specimens from CF patients; and particularly, BCC bacteria were further identified by recA-based PCR followed by restriction fragment length polymorphism analysis with HaeIII, and their identities were confirmed by recA species-specific PCR. In addition, some strains belonging to genera different from BCC were identified by 16S rRNA gene sequencing. A standardized experimental protocol was established, and an FTIR spectral database containing more than 2,000 infrared spectra was created. The ANN identification system consisted of two hierarchical levels. The top-level network allowed the identification of P. aeruginosa, S. maltophilia, Achromobacter xylosoxidans, Acinetobacter spp., R. pickettii, and BCC bacteria with an identification success rate of 98.1%. The second-level network was developed to differentiate the four most clinically relevant species of BCC, B. cepacia, B. multivorans, B. cenocepacia, and B. stabilis (genomovars I to IV, respectively), with a correct identification rate of 93.8%. Our results demonstrate the high degree of reliability and strong potential of ANN-based FTIR spectrum analysis for the rapid identification of NF rods suitable for use in routine clinical microbiology laboratories.     

Lack of evidence of nosocomial cross-infection byBurkholderia cepacia among danish cystic fibrosis patients

Burkholderia cepacia isolates from nine of the ten Danish cystic fibrosis (CF) patients known between 1975 and the present day to carry this organism were investigated. Eight distinct genotypes were found with polymerase chain reaction ribotyping and pulsed-field gel electrophoresis. The results indicate that there is little patient-to-patient cross-infection withBurkholderia cepacia within the Danish CF population, even though the majority of patients attend the same CF clinic on a regular basis.     

Microbiologic characteristics of pathogenic bacteria from hospitalized trauma patients who survived Wenchuan earthquake

The purpose of this study is to investigate the microbiological characterization of pathogenic bacteria isolated from trauma patients after Wenchuan earthquake in 2008. Most infections were identified in the patients over 60?years of age, with an incidence rate of 78.5%, and more infections in wound (43.3%) and respiratory tract (37.1%) sites were identified. A total of 97 non-duplicated clinical pathogens were isolated from 91 trauma patients. Of those pathogens, 62 (63.9%) were Gram-negative bacilli, 23 (23.7%) were Gram-positive cocci, 9 (9.3%) were fungi, and 3 (3.1%) were anaerobes, such as Clostridium perfringens. The distribution spectrum of pathogens isolated from trauma patients after earthquake was different to that from non-earthquake trauma patients in our hospital at the same time. The most prevalent pathogenic isolates were Escherichia coli (15.4%), Acinetobacter baumannii (14.4%), Staphylococcus aureus (12.3%), Burkholderia cepacia (11.3%), and Enterococcus spp. (9.3%). The drug susceptibility results showed that most of the Gram-negative bacilli, except for Pseudomonas aeruginosa and Burkholderia cepacia, were susceptible to imipenem, but resistant to the first- and the second-generation cephalosporins. Most of the Gram-positive cocci were susceptible to vancomycin, linezolid, and Synercid/dalfopristin. Characteristics of pathogenic bacterium isolated from trauma patients after earthquake have been demonstrated which play an important role in the appropriate treatment of infections.