Species distribution and antimicrobial susceptibility of Burkholderia cepacia complex isolates in clinical infections: Experience from a tertiary care hospital, Southern India.

PURPOSE: Burkholderia cepacia complex (Bcc) is a diverse group of environmental bacteria associated with opportunistic infections. The identification of Bcc using conventional methods poses challenges. Bcc infections are difficult to treat due to intrinsic antibiotic resistance. The study aimed to investigate the species distribution and antimicrobial susceptibility of clinical Bcc isolates. METHODS: A total of 153 Bcc isolates obtained from clinical samples were analysed. Species identification was carried out using automated methods, including MALDI-TOF MS and VITEK2. Antimicrobial susceptibility testing was performed using the disc diffusion method. RESULTS: Burkholderia cenocepacia (70.5%) emerged as the most prevalent species, followed by Burkholderia contaminans (9.8%) and Burkholderia cepacia (7.2%). Ventilator-associated pneumonia (38.6%) was the most common infection, followed by sepsis (28.1%). Co-existence of Bcc with other pathogens in many cases suggested potential co-infection scenarios. Antimicrobial susceptibility revealed that ceftazidime, co-trimoxazole and meropenem were the most effective drugs, while levofloxacin proved to be the least effective. Moderate susceptibility was noted to minocycline, with 4.6% of isolates exhibiting multi-drug resistance. CONCLUSION: This study provides valuable insights into the prevalence, clinical associations, and antibiotic susceptibility of Bcc in India. It highlights the importance of Bcc as a nosocomial pathogen, especially in vulnerable patient populations. The findings contribute to understanding Bcc infections, their distribution, and emphasize the necessity for accurate identification methods in clinical settings.

Identification of the Optimal Cultivation Period Required to Isolate Representatives of Mycobacterium abscessus Complex Isolated from Patients with Cystic Fibrosis.

BACKGROUND: In patients with cystic fibrosis (CF), representatives of the fast-growing Mycobacterium abscessus complex (MABSc) are often distinguished, but the culture of the material taken from such patients increases the growth time. We analyzed the terms of cultivation of MABSc representatives on dense nutrient media and also evaluated the productivity of a modified nutrient medium based on agar for the isolation of Burkholderia cepacia complex (BCC). METHODS: Sixty-four strains of MABSc isolated from patients with CF and suspected tuberculosis were analyzed. The material from the patients was cultured on a universal chromogenic medium, 5% blood agar, yolk-salt agar, selective medium for isolation of BCC, and Löwenstein-Jensen medium. The cultures were incubated for 5 days (37°C, aerobic conditions), after for 23 days (28°C, aerobic conditions). The productivity of the developed nutrient medium was evaluated by the number of cells that gave visible growth after culturing 0.1 mL of a bacterial suspension of 103 CFU/mL. RESULTS: 76.8% of the strains grew in a 2-week period, and 23.2% of the strains were obtained at a later date from 18 to 28 days (average: 21.23 days). The modified medium with a concentration of 240 mg of iron (III) polymaltose hydroxide proved to be the most optimal for the isolation of MABSc. CONCLUSION: When using a chromogenic medium for culture material from patients with CF, it is necessary to extend incubation up to 28 days to increase the probability of MABSc isolation. The modified BCC medium showed a good selectivity result but required further investigation.

A culture-independent nucleic acid diagnostics method for use in the detection and quantification of Burkholderia cepacia complex contamination in aqueous finished pharmaceutical products.

The Burkholderia cepacia complex (Bcc) is the number one bacterial complex associated with contaminated Finished Pharmaceutical Products (FPPs). This has resulted in multiple healthcare related infection morbidity and mortality events in conjunction with significant FPP recalls globally. Current microbiological quality control of FPPs before release for distribution depends on lengthy, laborious, non-specific, traditional culture-dependent methods which lack sensitivity. Here, we present the development of a culture-independent Bcc Nucleic Acid Diagnostic (NAD) method for detecting Bcc contaminants associated with Over-The-Counter aqueous FPPs. The culture-independent Bcc NAD method was validated to be specific for detecting Bcc at different contamination levels from spiked aqueous FPPs. The accuracy in Bcc quantitative measurements was achieved by the high degree of Bcc recovery from aqueous FPPs. The low variation observed between several repeated Bcc quantitative measurements further demonstrated the precision of Bcc quantification in FPPs. The robustness of the culture-independent Bcc NAD method was determined when its accuracy and precision were not significantly affected during testing of numerous aqueous FPP types with different ingredient matrices, antimicrobial preservative components and routes of administration. The culture-independent Bcc NAD method showed an ability to detect Bcc in spiked aqueous FPPs at a concentration of 20 Bcc CFU/mL. The rapid (≤ 4 hours from sample in to result out), robust, culture-independent Bcc NAD method presented provides rigorous test specificity, accuracy, precision, and sensitivity. This method, validated with equivalence to ISO standard ISO/TS 12869:2019, can be a valuable diagnostic tool in supporting microbiological quality control procedures to aid the pharmaceutical industry in preventing Bcc contamination of aqueous FPPs for consumer safety.

Presence of the Hmq System and Production of 4-Hydroxy-3-Methyl-2-Alkylquinolines Are Heterogeneously Distributed between Burkholderia cepacia Complex Species and More Prevalent among Environmental than Clinical Isolates.

The Burkholderia cepacia complex (Bcc) comprises several species of closely related, versatile bacteria. Some Bcc strains produce 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs), analogous to the 4-hydroxy-2-alkylquinolines of Pseudomonas aeruginosa. Using in silico analyses, we previously estimated that the hmqABCDEFG operon, which encodes enzymes involved in the biosynthesis of HMAQs, is carried by about one-third of Bcc strains, with considerable inter- and intraspecies variability. In the present study, we investigated by PCR, using consensus primers, the distribution of hmqABCDEFG in a collection of 312 Bcc strains (222 of clinical and 90 of environmental origins) belonging to 18 Bcc species. We confirmed that this operon is not distributed evenly among Bcc species. Among the 30% of strains bearing the hmqABCDEFG operon, we found that 92% of environmental isolates and 82% of clinically isolated Bcc strains produce levels of HMAQs detectable by liquid chromatography-mass spectrometry in at least one of the tested culture conditions. Among the hmqABCDEFG-positive but HMAQ-negative strains, none expressed the hmqA gene under the specified culture conditions. Interestingly, the hmqABCDEFG operon is more prevalent among plant root environment species (e.g., Burkholderia ambifaria and Burkholderia cepacia) and absent in species commonly found in chronically colonized individuals with cystic fibrosis (e.g., Burkholderia cenocepacia and Burkholderia multivorans), suggesting a role for the Hmq system in niche adaptation. We investigated the impact of the Hmq system on plant growth promotion and found that Pisum sativum root development by B. ambifaria required a functional HMAQ system. IMPORTANCE Environmental bacteria belonging to the various closely related species forming the Burkholderia cepacia complex (Bcc) can infect plants and animals, including humans. Their pathogenicity is regulated by intercellular communication, or quorum sensing, allowing them to collaborate instead of acting individually. Bcc organisms generally exploit interacting quorum sensing systems based on N-acyl-homoserine lactones as signaling molecules. Several Bcc strains also carry an hmqABCDEFG operon responsible for the biosynthesis of 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs), molecules analogous to the Pseudomonas quinolone signal (PQS) system of P. aeruginosa. Our finding that the prevalences of the Hmq system and HMAQ production are very different between various Bcc species suggests a key role in niche adaptation or pathogenicity. This is supported by a significant reduction in plant growth promotion in the absence of HMAQ production for a beneficial Bcc strain.

Implementation of microbiota analysis in clinical trials for cystic fibrosis lung infection: Experience from the OligoG phase 2b clinical trials.

Culture-independent microbiota analysis is widely used in research and being increasingly used in translational studies. However, methods for standardisation and application of these analyses in clinical trials are limited. Here we report the microbiota analysis that accompanied two phase 2b clinical trials of the novel, non-antibiotic therapy OligoG CF-5/20 for cystic fibrosis (CF) lung infection. Standardised protocols (DNA extraction, PCR, qPCR and 16S rRNA gene sequencing analysis) were developed for application to the Pseudomonas aeruginosa (NCT02157922) and Burkholderia cepacia complex (NCT02453789) clinical trials involving 45 and 13 adult trial participants, respectively. Microbiota analysis identified that paired sputum samples from an individual participant, taken within 2 h of each other, had reproducible bacterial diversity profiles. Although culture microbiology had identified patients as either colonised by P. aeruginosa or B. cepacia complex species at recruitment, microbiota analysis revealed patient lung infection communities were not always dominated by these key CF pathogens. Microbiota profiles were patient-specific and remained stable over the course of both clinical trials (6 sampling points over the course of 140 days). Within the Burkholderia trial, participants were infected with B. cenocepacia (n = 4), B. multivorans (n = 6), or an undetermined species (n = 3). Colonisation with either B. cenocepacia or B. multivorans influenced the overall bacterial community structure in sputum. Overall, we have shown that sputum microbiota in adults with CF is stable over a 2 h time-frame, suggesting collection of a single sample on a collection day is sufficient to capture the microbiota diversity. Despite the uniform pathogen culture-positivity status at recruitment, trial participants were highly heterogeneous in their lung microbiota. Understanding the microbiota profiles of individuals with CF ahead of future clinical trials would be beneficial in the context of patient stratification and trial design.

Distribution of Burkholderia cepacia complex species isolated from industrial processes and contaminated products in Argentina.

Burkholderia cepacia complex (Bcc) members have clinical relevance as opportunistic pathogens in patients with cystic fibrosis and are responsible of numerous nosocomial infections. These closely related bacteria are also reported as frequent contaminants of industrial products. In this retrospective study, we use PCR and recA gene sequence analysis to identify at species level Bcc isolates recovered from massive consumption products and industrial processes in Argentina during the last 25 years. The sequences obtained were also compared with recA sequences from clinical Bcc isolates deposited in GenBank database. We detected Bcc in purified water and preserved products from pharmaceutics, cosmetics, household cleaning articles, and beverages industries. B. contaminans (which is prevalent among people with cystic fibrosis in Argentina) was the most frequent Bcc species identified (42% of the Bcc isolates studied). B. cepacia (10%), B. cenocepacia (5%), B. vietnamiensis (16%), B. arboris (3%), and the recently defined B. aenigmatica (24%) were also detected. Rec A sequences from all B. cepacia and most B. contaminans industrial isolates obtained in this study displayed 100% identity with recA sequences from isolates infecting Argentinean patients. This information brings evidence for considering industrial massive consumption products as a potential source of Bcc infections. In addition, identification at species level in industrial microbiological laboratories is necessary for a better epidemiological surveillance. Particularly in Argentina, more studies are required in order to reveal the role of these products in the acquisition of B. contaminans infections.

Molecular characterization of clinical isolates from vascular access infection: A single-institution study.

Hemodialysis requires repeated, reliable access to the systemic circulation; therefore, a well-functioning vascular access (VA) procedure is crucial for stable hemodialysis. VA infections (VAIs) constitute the most challenging complication and cause considerable morbidity, loss of access, and even death. In this study, we investigated the molecular profiles of different bacterial isolates retrieved from various types of VA grafts. We collected clinical isolates from hemodialysis patients with VAIs in our institution for the period between 2013 and 2018. We identified the bacterial isolates using standard biochemical procedures; we used a polymerase chain reaction for coagulase-negative staphylococci (CoNS) and Burkholderia cepacia complex (BCC) species identification. The antibiotic resistance and molecular profile were analyzed using the disk diffusion method and multilocus sequence typing, respectively. We studied 150 isolates retrieved from patients with VAI and observed that Staphylococcus aureus was the predominant bacterial species, followed by S. argenteus, BCC, and CoNS. According to multilocus sequence typing data, we identified a wide variety of sequence types (STs) in S. aureus isolates, with ST59, ST45, and ST239 being the predominant types. Burkholderia cepacia with two new ST types, namely ST1723 and ST1724, accounted for most of the BCC infections, along with ST102 B. contaminans, which were mainly isolated from infected tunneled-cuffed catheters. In summary, the increased incidence of S. argenteus and BCC infections provides insights into their potential clinical effects in VAIs. The various STs identified in different bacterial species indicate the high genetic diversity of bacterial species isolated from VAIs in our institution.

How new molecular tools can help bugbusters: a Burkholderia cepacia complex outbreak investigation.

An outbreak of bloodstream infection (BSI) caused by members of the Burkholderia cepacia complex (Bcc) took place from March 2012 until April 2014 involving thirteen patients. AIM: To describe an outbreak investigation of BSI Bcc and showing how genetic sequencing tools contributed to confirm the hypothesis of extrinsic contamination proposed by an observational study. METHODS: The Infection Control Department revised and reinforced good practices of infusion therapy and catheter care, visits to affected wards, a case control study, and environmental screening based on the case-control findings. RESULTS: Data from the case-control study found an association of cases with central venous catheter (OR 1.36; CI 1.15-1.67) and intravenous cisatracurium use (OR 10.75; CI 1.67-68.89). Visits to the operatory block revealed problems related to the cold chain used for the preservation of thermolabile cisatracurium. We could not retrieve Bcc from environmental samples using classic microbiology. New samples from the same surfaces were obtained for genetic sequencing. Bcc was identified in the cooler box, refrigerator and reusable ice packages. CONCLUSION: Environmental screening using genetic sequencing proved to be a useful tool for confirming our hypothesis of extrinsic contamination raised by the case-control study.

Of onions and men: Report of cavitary community acquired pneumonia due to Burkholderia cepacia complex in an immunocompetent patient and review of the literature.

Burkholderia cepacia complex consists of highly antibiotic resistant gram negative bacilli that are plant symbionts and also potential agents of human infection.  This bacterial family's claim to fame in clinical medicine is as the scourge of cystic fibrosis patients, in whom it is a notorious respiratory pathogen.  Outside of cystic fibrosis, it rarely comes to mind as an etiology of community acquired pneumonia with or without lung cavitation in immunocompetent hosts.  We describe a case of an otherwise healthy, community-dwelling man who presented with subacute cavitary lung disease, the causative organism of which turned out to be Burkholderia cepacia complex.  Our report is accompanied by a review of the literature, which identified an additional eleven cases in the same category.  We analyze all of the available cases for the emergence of any identifiable patterns or peculiarities.

Host Adaptation Predisposes Pseudomonas aeruginosa to Type VI Secretion System-Mediated Predation by the Burkholderia cepacia Complex.

Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) species are opportunistic lung pathogens of cystic fibrosis (CF) patients. While P. aeruginosa can initiate long-term infections in younger CF patients, Bcc infections only arise in teenagers and adults. Both P. aeruginosa and Bcc use type VI secretion systems (T6SSs) to mediate interbacterial competition. Here, we show P. aeruginosa isolates from teenage and adult CF patients, but not those from young CF patients, are outcompeted by the epidemic Bcc isolate Burkholderia cenocepacia strain AU1054 in a T6SS-dependent manner. The genomes of susceptible P. aeruginosa isolates harbor T6SS-abrogating mutations, the repair of which, in some cases, rendered the isolates resistant. Moreover, seven of eight Bcc strains outcompeted P. aeruginosa strains isolated from the same patients. Our findings suggest certain mutations that arise as P. aeruginosa adapts to the CF lung abrogate T6SS activity, making P. aeruginosa and its human host susceptible to potentially fatal Bcc superinfection.

Management and outcomes of Burkholderia cepacia complex bacteremia in patients without cystic fibrosis: a retrospective observational study.

Burkholderia cepacia complex (BCC) is an emerging pathogen of nosocomial infection in chronic or critically ill patients without cystic fibrosis (CF). The objective was to evaluate the management and outcomes of BCC bacteremia in patients without CF. We conducted a retrospective study of non-CF adult patients with BCC bacteremia between January 1997 and December 2016 at 4 tertiary hospitals in South Korea. A total of 216 non-CF patients with BCC bacteremia were identified. Most cases were hospital-acquired (79.2%), and the most common source was a central venous catheter (CVC) (42.1%). The rates of susceptibility to trimethoprim-sulfamethoxazole and piperacillin-tazobactam of BCC isolates were high as 92.8% and 90.3%, respectively. The rates of susceptibility to ceftazidime, meropenem, and levofloxacin were 75.5%, 72.3%, and 64.1%, respectively. The 14-day, 30-day, and in-hospital mortality rate was 19.4%, 23.1%, and 31.0%, respectively. Female (OR = 3.1; 95% CI, 1.4-6.8), liver cirrhosis (OR = 6.2; 95% CI, 1.6-16.6), septic shock (OR = 11.2; 95% CI, 5.1-24.8), and catheter-related infection (OR = 2.6, 95% CI, 1.2-5.8) were the independent risk factors for 30-day mortality. The outcome did not differ according to type of antibiotics used. Among 91 patients with CVC-related BCC bacteremia, delayed CVC removal (> 3 days) had a higher rate of persistent bacteremia (54.5 vs. 26.1%; P = 0.03) and lower rate of clinical response (49.0 vs. 71.9%; P = 0.04), compared with early CVC removal (within 3 days). BCC bacteremia occurring in non-CF patients was mostly hospital-acquired and CVC-related. Early removal of the catheter is crucial in treatment of CVC-related BCC bacteremia.

Is prolonged incubation required for optimal recovery of Burkholderia cepacia complex in sputum from cystic fibrosis patients? Data versus dogma.

Cystic fibrosis (CF) expert groups globally recommend using selective medium for isolation of Burkholderia cepacia complex (BCC) from respiratory specimens of CF patients. However, there is no consensus available for optimal duration of incubation and recommendations are variable. The purpose of our study was to compare the difference in recovery of BCC in CF samples at 48 hours versus 7 days when inoculated on Burkholderia cepacia selective agar. A total of 307 consecutive clinical respiratory specimens from our local CF unit were studied prospectively (August 2017 to December 2017). All specimens were inoculated on Burkholderia cepacia medium, containing polymyxin B, gentamicin and ticarcillin. In our laboratory, these plates are routinely incubated for 48 hours as per the manufacturer's recommendation. However, for this study all plates with no growth at 48 hours were further incubated for total of 7 days at 35°C in O2. Plates were read daily to look for any growth. Microbial identification was performed using MALDI-TOF Vitek MS (database V3.0). Of the 307 CF respiratory specimens cultured, 177 (58%) were from paediatric and 130 (42%) were from adult patients; 155 (50%) specimens were sputum, 148 (48%) were cough swabs and four (1%) were bronchoalveolar lavage (BAL). All specimens from adults were sputum except one BAL. Thirteen (4%) cultures from eight adult and five paediatric specimens grew BCC. The majority (294, 96%) of specimens had no growth when incubated for 7 days. All 13 positive isolates recovered within 48 hours and there were no additional positive isolates found beyond 48 hours of incubation. We conclude from our analysis that prolonged incubation is not warranted for recovery of BCC in CF specimens if selective medium containing gentamicin and polymyxin is used. By adopting this approach of non-extended incubation, the burden of work on laboratory personnel can be significantly reduced and much faster turnaround time for CF cultures achieved. Our study confirms the results of recently published data on this point and challenges the prevailing dogma of utility of extended incubation for BCC isolation. For devising consensus statements for microbiology laboratories on this issue, CF societies and expert groups should consider reviewing data from the recent studies.

Aetiological agents for pulmonary exacerbations in children with cystic fibrosis: An observational study from a tertiary care centre in northern India.

Background & objectives: Pulmonary disease is the main cause of morbidity and mortality in cystic fibrosis (CF). The infection occurs with a unique spectrum of bacterial pathogens that are usually acquired in an age-dependent fashion. The objective of this study was to find out the aetiological agents in respiratory specimens from children with CF during pulmonary exacerbation and relate with demographic variables. Methods: In this observational study, airway secretions from children (n=104) with CF presenting with pulmonary exacerbations were collected and tested for bacteria, fungi, mycobacteria and viral pathogens using appropriate laboratory techniques. The frequencies of isolation of various organisms were calculated and associated with various demographic profiles. Results: Bacteria were isolated in 37 (35.5%) and viral RNA in 27 (29.3%) children. Pseudomonas was the most common bacteria grown in 31 (29.8%) followed by Burkholderia cepacia complex (Bcc) in three (2.8%) patients. Among viruses, Rhinovirus was the most common, identified in 16 (17.4%) samples followed by coronavirus in four (4.3%). Fungi and mycobacteria were isolated from 23 (22.1%) and four (3.8%) children, respectively. Aspergillus flavus was the most common fungus isolated in 13 (12.5%) children. Interpretation & conclusions: Pseudomonas was the most common organism isolated during exacerbation. Non-tuberculous mycobacteria were not isolated, whereas infection with Bcc and Mycobacterium tuberculosis was observed, which could probably have a role in CF morbidity. Polymicrobial infections were associated with severe exacerbations.

Changing epidemiology of the respiratory bacteriology of patients with cystic fibrosis-data from the European cystic fibrosis society patient registry.

BACKGROUND: Monitoring changes in the epidemiology of cystic fibrosis (CF) pathogens is essential for clinical research, quality improvement, and clinical management. METHODS: We analyzed data reported to the European Cystic Fibrosis Society Patient Registry (ECFSPR) from 2011 to 2016 to determine the overall and the age-specific annual prevalence and incidence of selected CF pathogens and their trends during these years. The ECFSPR collects data on three chronic infections: Pseudomonas aeruginosa (PsA), Burkholderia cepacia complex Species (BCC) and Staphylococcus aureus (SA), as well as on the occurrence of non-tuberculous mycobacteria (NTM) and Stenotrophomonas maltophilia (SM). The same analyses were performed for different country groups, according to their gross national income (GNI). RESULTS: The pathogens with the highest prevalence were SA and PsA, with prevalence, in 2016, equal to 38.3% and 29.8% respectively, followed by SM (8.1%). The pathogens with the lowest prevalence were NTM (3.3%) and BCC (3.1%). The overall prevalence and incidence significantly decreased for PsA; they also decreased for BCC, while they increased significantly for SA. The overall prevalence of NTM and SM increased significantly. The most considerable prevalence changes were observed for PsA, which decreased across all income country groups and all age strata (with the exception of 0-1 years) The prevalence and incidence of pathogens differed significantly according to GNI. CONCLUSIONS: The epidemiology of CF pathogens in Europe has changed; epidemiologic data differ significantly among countries with different socio-economic status. The causes of these observations are multifactorial and include improvements in clinical care and infection control.

Burkholderia Cepacia Complex Causing Pneumonia in an Immunocompetent Non-Cystic Fibrosis Patient: Case Report and Review of Literature.

BACKGROUND: Burkholderia cepacia complex is widespread in the environment and has been recognized as a cause of opportunistic pulmonary infections, particularly in patients with Cystic Fibrosis (CF). The natural ecology of the bacteria as part of plant growth-promoting rhizosphere provides stark contrast to its infectious potential. Its preponderance as a nosocomial pathogen may be due to its ability to survive in antiseptic solutions, contaminate equipments and intrinsic antimicrobial resistance. CASE: An elderly, diabetic male was evaluated for hemoptysis, fever and cough. Chest computed tomography showed a thick walled cavity in the left lung and hilar lymphadenopathy. Sputum examination showed Gram negative bacilli and no acid fast bacilli. Sputum culture yielded growth of non-fermentative Gram negative bacilli on two occasions, but blood culture was sterile. The isolate was identified as B. cepacia by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). The patient's general condition remained poor and in spite of initiation of antibiotics, the patient expired after an episode of massive hemoptysis. CONCLUSION: This report raises concerns regarding the spread and severity of B. cepacia infection in non-compromised patients in the community and the need to suspect and identify it. Since the organism is inherently resistant to antipseudomonal penicillins, aminoglycosides and polymyxin B, differentiation from Pseudomonas spp. and determining antimicrobial susceptibility is paramount for treatment.

Oligotrophic Media Compared with a Tryptic Soy Agar or Broth for the Recovery of Burkholderia cepacia Complex from Different Storage Temperatures and Culture Conditions.

The Burkholderia cepacia complex (BCC) is capable of remaining viable in low-nutrient environments and harsh conditions, posing a contamination risk in non-sterile pharmaceutical products as well as a challenge for detection. To develop optimal recovery methods to detect BCC, three oligotrophic media were evaluated and compared with nutrient media for the recovery of BCC from autoclaved distilled water or antiseptic solutions. Serial dilutions (10-1 to 10-12 CFU/ml) of 20 BCC strains were inoculated into autoclaved distilled water and stored at 6°C, 23°C and 42°C for 42 days. Six suspensions of Burkholderia cenocepacia were used to inoculate aqueous solutions containing 5 µg/ml and 50 µg/ml chlorhexidine gluconate (CHX) and 10 µg/ml benzalkonium chloride (BZK), and stored at 23°C for a further 199 days. Nutrient media such as Tryptic Soy Agar (TSA) or Tryptic Soy Broth (TSB), oligotrophic media (1/10 strength TSA or TSB, Reasoner's 2nd Agar [R2A] or Reasoner's 2nd Broth [R2AB], and 1/3 strength R2A or R2AB) were compared by inoculating these media with BCC from autoclaved distilled water and from antiseptic samples. The recovery of BCC in water or antiseptics was higher in culture broth than on solid media. Oligotrophic medium showed a higher recovery efficiency than TSA or TSB for the detection of 20 BCC samples. Results from multiple comparisons allowed us to directly identify significant differences between TSA or TSB and oligotrophic media. An oligotrophic medium pre-enrichment resuscitation step is offered for the United States Pharmacopeia (USP) proposed compendial test method for BCC detection.

Impact of clonally-related Burkholderia contaminans strains in two patients attending an Italian cystic fibrosis centre: a case report.

BACKGROUND: Burkholderia contaminans is one of the 20 closely related bacterial of the Burkholderia cepacia complex, a group of bacteria that are ubiquitous in the environment and capable of infecting people with cystic fibrosis (CF). This species is an emerging pathogen and it has been widely isolated from CF patients in Argentina, Spain, Portugal, Australia, Canada, USA with a low prevalence in Ireland, France, Russia, Switzerland, Czech Republic, and Italy. This is the first report of B. contaminans affecting two Italian CF patients attending the same CF Centre. We correlate B. contaminans colonisation with lung function decline and co-infection with other clinically relevant CF pathogens. CASE PRESENTATION: B. contaminans was identified by Multi Locus Sequence Typing in routine sputum analysis of two Caucasian CF women homozygous for Phe508del CFTR mutation. Sequence Type 102 was detected in both strains. It is known that B. contaminans ST102 was isolated both from CF and non-CF patients, with an intercontinental spread across the world. Random Amplified Polymorphic DNA analysis revealed the genetic relatedness between the two strains. We examined their susceptibility to antimicrobial agents, comparing the latter with that recorded for other B. contaminans isolated from different countries. We also described key virulence factors possibly linked with a clinical outcome. Specifically, we attempted to correlate colonization with the incidence of acute exacerbation of symptoms and lung function decline. CONCLUSIONS: This case presentation suggests that acquisition of B. contaminans ST102 is not directly associated with a lung function decline. We retain that the presence of other CF pathogens (i.e. MRSA and Trichosporon) along with B. contaminans ST102 might have contributed to the worsening of clinical conditions in our CF patients. The circumstances leading to the establishment of B. contaminans ST102 infections are still unknown. We highlight the importance to proper detect and typing bacteria implicated in CF infection by using molecular techniques.

Evaluation of different molecular and phenotypic methods for identification of environmental Burkholderia cepacia complex.

The correct identification of different genera and bacterial species is essential, especially when these bacteria cause infections and appropriate therapies need to be chosen. Bacteria belonging to the Burkholderia cepacia complex are considered important opportunistic pathogens, causing different types of infections in immunocompromised, principally in patients with cystic fibrosis. Twenty-one isolates were obtained from different soil samples and identified by sequencing of 16S rRNA, 23S rRNA, recA gene, MLST and by VITEK 2 and MALDI-TOF MS systems. Then, statistical analyses were performed. VITEK 2 and MALDI-TOF MS systems showed different bacterial genera. Sequencing of the 16S rRNA, 23S rRNA gene and amplification of recA gene showed that all the isolates belong to the B. cepacia complex. Sequencing of the recA gene showed a predominance of B. cenocepacia. The PCR of the recA gene showed a high specificity when it is necessary to identify the bacteria belonging to the B. cepacia complex in comparison with 16S and 23S rRNA genes sequencing. MLST analyzes showed a diversity of STs, which have not yet been correlated to the species. Phenotypic identification was not suitable for the identification of these pathogens since in many cases different genera have been reported, including identification by using MALDI-TOF MS.