[The controversial Burkholderia cepacia complex, a group of plant growth promoting species and plant, animals and human pathogens]. / El controvertido complejo Burkholderia cepacia, un grupo de especies promotoras del crecimiento vegetal y patógenas de plantas, animales y humanos.

The Burkholderia cepacia complex is a group of 22 species, which are known as opportunistic pathogens in immunocompromised people, especially those suffering from cystic fibrosis. It is also found in nosocomial infections and is difficult to eradicate due to intrinsic resistance to several antibiotics. The species have large genomes (up to 9 Mbp), distributed into 2-5 replicons. These features significantly contribute to genome plasticity, which makes them thrive in different environments like soil, water, plants or even producing nodules in legume plants. Some B. cepacia complex species are beneficial in bioremediation, biocontrol and plant-growth promotion. However, because the B. cepacia complex is involved in human infection, its use in agriculture is restricted. B. cepacia complex is being constantly studied due to the health problems that it causes and because of its agricultural potential. In this review, the history of B. cepacia complex and the most recently published information related to this complex are revised.

Clinical and histopathological features of Burkholderia cepacia complex dermatitis in dogs: a series of four cases.

BACKGROUND: The Burkholderia cepacia complex (Bcc) is an emerging cause of opportunistic infections. Deep pyoderma associated with Bcc infection has been reported previously in dogs receiving ciclosporin. OBJECTIVE: To report the clinical and histopathological features of four additional cases of Bcc dermatitis in dogs, one of which progressed to septicaemia. ANIMALS: Four dogs with a skin culture yielding growth of Bcc and skin biopsies for histopathological investigation. METHODS AND MATERIALS: Retrospective review of medical records and skin biopsies and PCR for Burkholderia on DNA extracted from paraffin-embedded skin and liver to confirm Bcc sepsis. RESULTS: Three different breeds and one mixed breed dog were represented. Two dogs were receiving ciclosporin and one was receiving oclacitinib. One dog had no evidence of immunosuppression. One dog was bathed two days prior to onset of skin lesions. Three dogs presented with dorsally orientated ulcers, crusts and draining tracts; one dog had infection localized to a surgical site. The main histological feature from skin biopsies was severe neutrophilic folliculitis and furunculosis with marked neutrophilic to pyogranulomatous dermatitis. Intracellular Gram-negative and Warthin-Starry positive rods were present in three of four cases. Three dogs were successfully treated with systemic fluoroquinolones or trimethoprim sulfamethoxazole. The Bcc isolate in one dog was resistant to all tested systemic antimicrobials. This dog developed septicaemia and was euthanized. CONCLUSIONS AND CLINICAL IMPORTANCE: Bcc skin infections can occur in immunocompetent and immunocompromised dogs. Bcc isolates may be extensively antimicrobial resistant, presenting a challenge for clinical management. Cutaneous infection may progress to life-threatening sepsis.

Deep pyoderma caused by Burkholderia cepacia complex associated with ciclosporin administration in dogs: a case series.

BACKGROUND: Bacteria of the Burkholderia cepacia complex (Bcc) are ubiquitous Gram-negative bacilli associated with fatal nosocomial infections in humans; multi-antibiotic resistance makes this organism a serious threat in hospital settings. OBJECTIVE: To describe the historical, clinicopathological and treatment characteristics of Bcc-associated deep skin infections in dogs. ANIMALS: Six dogs with skin infections in which skin bacterial cultures resulted in pure growth of Bcc. METHODS: Retrospective study with review of medical records and skin biopsies. RESULTS: All dogs were receiving oral ciclosporin at the time of skin infection development. All dogs were castrated males and four of six were West Highland white terriers. Cutaneous lesions consistent with deep pyoderma were confined mainly to the trunk. In all dogs skin cytology revealed a strong inflammatory response, with moderate to abundant numbers of intracellular (neutrophils and macrophages) and extracellular bacilli. In three dogs histopathology showed a multifocal, nodular to coalescing pyogranulomatous dermatitis associated with multifocal folliculitis and furunculosis. Tissue Giemsa and Gram stains identified numerous Gram-negative rods within macrophages. Antimicrobial susceptibility testing revealed multidrug-resistant Bcc strains with sensitivity to trimethoprim/sulfonamides in all dogs and to marbofloxacin, piperacillin and ceftazidime in three dogs. Successful treatment was achieved in all dogs using trimethoprim/sulfonamides or quinolones (marbofloxacin, ciprofloxacin) or doxycycline in conjunction with ciclosporin withdrawal. CONCLUSIONS AND CLINICAL IMPORTANCE: Clinicians should be aware of the rare potential for Bcc-associated deep skin infections in dogs receiving oral ciclosporin. Owners should be made conscious of the potential transmission risk to humans or other animals.

A study on the occurrence of Burkholderia cepacia complex in ultrasound gels used in different veterinary clinical settings in India.

Burkholderia cepacia complex (Bcc) organisms are emerging multidrug-resistant pathogens. They are opportunistic and cause severe diseases in humans that may result in fatal outcomes. They are mainly reported as nosocomial pathogens, and transmission often occurs through contaminated pharmaceutical products. From 1993 to 2019, 14 Bcc outbreaks caused by contaminated ultrasound gels (USGs) have been reported in several countries, including India. We screened a total of 63 samples of USGs from various veterinary and human clinical care centers across 17 states of India and isolated 32 Bcc strains of Burkholderia cenocepacia (46.8%), B. cepacia (31.3%), B. pseudomultivorans (18.8%) and B. contaminans (3.1%) species. Some isolates were co-existent in a single ultrasound gel sample. The isolation from unopened gel bottles revealed the intrinsic contamination from manufacturing sites. The MALDI-TOF analysis to identify the Bcc at the species level was supported by the partial sequencing of the recA gene for accurate species identification. The phylogenetic analysis revealed that isolates shared clades with human clinical isolates, which is an important situation because of the possible infections of Bcc by USGs both in humans and animals. The pulsed field gel electrophoresis (PFGE) typing identified the genetic variation among the Bcc isolates present in the USGs. The findings indicated USGs as the potential source of Bcc species.

Burkholderia cenocepacia creates an intramacrophage replication niche in zebrafish embryos, followed by bacterial dissemination and establishment of systemic infection.

Bacteria belonging to the "Burkholderia cepacia complex" (Bcc) often cause fatal pulmonary infections in cystic fibrosis patients, yet little is know about the underlying molecular mechanisms. These Gram-negative bacteria can adopt an intracellular lifestyle, although their ability to replicate intracellularly has been difficult to demonstrate. Here we show that Bcc bacteria survive and multiply in macrophages of zebrafish embryos. Local dissemination by nonlytic release from infected cells was followed by bacteremia and extracellular replication. Burkholderia cenocepacia isolates belonging to the epidemic electrophoretic type 12 (ET12) lineage were highly virulent for the embryos; intravenous injection of <10 bacteria of strain K56-2 killed embryos within 3 days. However, small but significant differences between the clonal ET12 isolates K56-2, J2315, and BC7 were evident. In addition, the innate immune response in young embryos was sufficiently developed to control infection with other less virulent Bcc strains, such as Burkholderia vietnamiensis FC441 and Burkholderia stabilis LMG14294. A K56-2 cepR quorum-sensing regulator mutant was highly attenuated, and its ability to replicate and spread to neighboring cells was greatly reduced. Our data indicate that the zebrafish embryo is an excellent vertebrate model to dissect the molecular basis of intracellular replication and the early innate immune responses in this intricate host-pathogen interaction.

Head Tilt in Immunodeficient Mice Due to Contamination of Drinking Water by Burkholderia gladioli.

Immunodeficient mice in multiple holding rooms presented with head tilt, circling, spinning when picked up by the tail, dehydration, and lethargy. Burkholderia gladioli, a plant pathogen, was identified as the causative agent. Environmental testing revealed the presence of B. gladioli within the automatic watering system, water bottles, and sipper tubes. Here we describe steps taken to reduce the presence of this organism within the automatic watering system and water bottles. Facilities housing immunodeficient mice should take measures to minimize the accumulation of biofilm within their water-supply systems.

Cellulitis caused by the Burkholderia cepacia complex associated with contaminated chlorhexidine 2% scrub in five domestic cats.

Isolates of the Burkholderia cepacia complex (BCC) are known as plant and human pathogens. We describe herein BCC infections as the cause of subcutaneous abscesses and purulent cellulitis in 5 cats. All cats were presented with an open wound, and 4 received standard wound care and empiric antibiotic therapy. Despite treatment, clinical signs worsened in 4 cats. Isolates of the BCC were obtained from all 5 cases. Two cats were submitted for postmortem examination. Subcutaneous abscesses with draining fistulas were observed. Histopathology revealed severe, pyogranulomatous cellulitis with intralesional gram-negative bacilli. Based on susceptibility results, the other 3 cats were administered effective antibiotics and recovered without complications. The BCC was cultured from the 2% chlorhexidine surgical scrub solution used in the clinic, suggesting the source of infection for 4 of 5 cats. Given the ability to grow in antiseptic solutions, the extra steps required to culture from antiseptics, and innate multidrug resistance, the BCC poses a challenge to both detect and treat. Although the BCC causes disease almost exclusively in humans with cystic fibrosis or immunodeficiency, the bacteria should also be a differential for nosocomial infections in veterinary patients.

Procedurally similar competitive immunoassay systems for the serodiagnosis of Babesia equi, Babesia caballi, Trypanosoma equiperdum, and Burkholderia mallei infection in horses.

Procedurally similar competitive enzyme-linked immunoassay (cELISA) methods were developed for the serodiagnosis of Babesia equi and Babesia caballi (piroplasmosis), Trypanosoma equiperdum (dourine), and Burkholderia mallei (glanders) infections in horses. Apparent test specificities for the B. equi, B. caballi, T. equiperdum, and B. mallei cELISAs were 99.2%, 99.5%, 98.9%, and 98.9%, respectively. Concordances and kappa values between the complement fixation (CF) and the cELISA procedures for the serodiagnosis of B. equi, B. caballi, T. equiperdum, and B. mallei infections in experimentally exposed horses were 76% and 0.55, 89% and 0.78, 97% and 0.95, and 70% and 0.44, respectively. The cELISA method may be a technically more reproducible, objective, and convenient approach for piroplasmosis, dourine, and glanders serodiagnosis in qualifying animals for international movement and disease eradication programs than the CF systems currently in use. Use of the cELISA method also obviated the problems associated with testing hemolyzed or anticomplementary sera.

Outbreak of otitis media caused by Burkholderia gladioli infection in immunocompromised mice.

An athymic nude mouse with severe head tilt due to otitis media was identified. Within weeks of identification of this first case, immune-deficient mice of various genotypes from the same facility were similarly affected, and cases from other facilities were found within two months. Culture of ear exudate specimens from affected mice yielded bacteria that were initially identified as Burkholderia cepacia, a plant pathogen considered an important opportunistic pathogen in persons with cystic fibrosis or chronic granulomatous disease. Several of these isolates, however, were subsequently identified as B. gladioli on the basis of results of biochemical analysis and a species-specific polymerase chain reaction (PCR) assay. Genotyping analysis revealed clonality among the isolates, indicating a shared strain among affected mice. A 16S rDNA-based PCR assay specific for the genera Burkholderia and Ralstonia, and a selective culture medium were used in efforts to characterize the epidemiology of this outbreak. In addition to culture of specimens from the oropharyngeal cavity of affected mice, samples were obtained from the environment, feces, sipper tubes, drinking water, and soiled bedding from cages of affected individuals. Burkholderia gladioli was most consistently detected in oropharyngeal swab specimens from affected mice. The PCR assay was equivalent to selective culture in identifying mice in the carrier state that did not have clinical signs of infection. However, neither detection method had sufficient sensitivity to reliably identify all carrier mice, causing the organism to persist at low levels unless entire colonies of immune-deficient mice were removed. The organism was highly resistant to antibiotic therapy. The source and epidemiology of this organism remain unknown. This epizootic serves as an important reminder that immunocompromised rodent colonies may harbor important human opportunistic pathogens.

First cases of animal diseases published since 2000. 5. Sheep.

In this fifth article of a series of papers listing first case reports of animal diseases published since 2000, the following five cases of sheep diseases are discussed: Ependymoma. Mastitis caused by Burkholderia cepacia infection. Meningoencephalitis associated with Globicatella sanguinis infection. Neospora caninum infection in an adult sheep and her twin fetuses. Plasma cell tumor. After a short introduction, the bibliographical data, the abstract of the author(s), and some additional information derived from the article are given. The article will be regularly updated adding overlooked as well as new first reports.

Stress conditions triggering mucoid morphotype variation in Burkholderia species and effect on virulence in Galleria mellonella and biofilm formation in vitro.

Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens causing chronic respiratory infections particularly among cystic fibrosis patients. During these chronic infections, mucoid-to-nonmucoid morphotype variation occurs, with the two morphotypes exhibiting different phenotypic properties. Here we show that in vitro, the mucoid clinical isolate Burkholderia multivorans D2095 gives rise to stable nonmucoid variants in response to prolonged stationary phase, presence of antibiotics, and osmotic and oxidative stresses. Furthermore, in vitro colony morphotype variation within other members of the Burkholderia genus occurred in Bcc and non-Bcc strains, irrespectively of their clinical or environmental origin. Survival to starvation and iron limitation was comparable for the mucoid parental isolate and the respective nonmucoid variant, while susceptibility to antibiotics and to oxidative stress was increased in the nonmucoid variants. Acute infection of Galleria mellonella larvae showed that, in general, the nonmucoid variants were less virulent than the respective parental mucoid isolate, suggesting a role for the exopolysaccharide in virulence. In addition, most of the tested nonmucoid variants produced more biofilm biomass than their respective mucoid parental isolate. As biofilms are often associated with increased persistence of pathogens in the CF lungs and are an indicative of different cell-to-cell interactions, it is possible that the nonmucoid variants are better adapted to persist in this host environment.

Taxon K, a complex within the Burkholderia cepacia complex, comprises at least two novel species, Burkholderia contaminans sp. nov. and Burkholderia lata sp. nov.

The aim of the present study was to re-examine the taxonomic position and structure of taxon K (also known as group K) within the Burkholderia cepacia complex (Bcc). For this purpose, a representative set of strains was examined by a traditional polyphasic taxonomic approach, by multilocus sequence typing (MLST) analysis and by analysis of available whole-genome sequences. Analysis of the recA gene sequence revealed three different lineages, designated recA-I, recA-II and recA-III. DNA-DNA hybridization experiments demonstrated that recA-I and recA-II isolates each represented a single novel species. However, DNA-DNA hybridization values of recA-II strains towards recA-III strains and among recA-III strains were at the threshold level for species delineation. By MLST, recA-I isolates were clearly distinguished from the others and represented a distinct lineage referred to as MLST-I, whereas recA-II and recA-III isolates formed a second MLST lineage referred to as MLST-II. A divergence value of 3.5 % was obtained when MLST-I was compared with MLST-II. The internal level of concatenated sequence divergence within MLST-I and MLST-II was 1.4 and 2.7 %, respectively; by comparison with the level of concatenated sequence divergence in established Bcc species, these data demonstrate that the MLST-I and MLST-II lineages represent two distinct species within the Bcc. The latter conclusion was supported by comparison of the whole-genome average nucleotide identity (ANI) level of MLST-I and MLST-II strains with strains of established Bcc species and by a whole-genome-based phylogenetic analysis. We formally propose to classify taxon K bacteria from the MLST-I and MLST-II lineages as Burkholderia contaminans sp. nov. (with strain J2956T =LMG 23361T =CCUG 55526T as the type strain) and Burkholderia lata sp. nov. (with strain 383T =ATCC 17760T =LMG 22485T =CCUG 55525T as the type strain), respectively. The MLST approach was confirmed as a valuable instrument in polyphasic taxonomic studies; more importantly, the cumulative data for about 1000 Bcc isolates analysed demonstrate that the 3 % concatenated sequence divergence level correlates with the 70 % DNA-DNA hybridization or 95 % whole-genome ANI threshold levels for species delineation.

Detection of Burkholderia cepacia DNA from artificially infected EDTA-blood and lung tissue comparing different DNA isolation methods.

Bacterial DNA (Burkholderia cepacia) was prepared from artificially infected equine ethylenediaminetetraacetic acid (EDTA)-blood and lung tissue by using four standard methods (lysis buffer containing proteinase K, phenol/chloroform/isoamylalcohol-extraction, microwave-treatment, heat treatment) and six commercially available kits (Puregene, High Pure PCR Template Preparation Kit, InstaGene, QiaAmp Tissue Kit, DNAzol and Elu-Quik). After a subsequent polymerase chain reaction (PCR), their efficacy and sensitivity were compared. Concerning the detection limits, the simple lysis with a proteinase K-containing buffer led to the best results for EDTA-blood as well as for artificially infected lung tissue.

Serodiagnosis of Burkholderia mallei infections in horses: state-of-the-art and perspectives.

Burkholderia mallei causes glanders or farcy in solipeds, a disease that must be reported to the OIE (Office International des Epizooties, Paris, France). The number of reported outbreaks has increased steadily during the last decade. Serodiagnosis is hampered by the considerable number of false-positives and -negatives of the internationally prescribed tests. The major problem leading to low sensitivity and specificity of complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e. crude preparations of whole cells. Future perspectives for the development and evaluation of serological test kits using well-characterized single antigens are discussed in the light of recent molecular research on B. mallei and the closely related saprozoonotic agent B. pseudomallei.

Mouse model of sublethal and lethal intraperitoneal glanders (Burkholderia mallei).

Sixty male BALB/c mice were inoculated intraperitoneally with either a sublethal or a lethal dose of Burkholderia mallei China 7 strain, then killed at multiple time points postinoculation. Histopathologic changes were qualitatively similar in both groups and consisted of pyogranulomatous inflammation. In sublethal study mice, changes were first seen at 6 hours in mediastinal lymph nodes, then in spleen, liver, peripheral lymph nodes, and bone marrow at day 3. These changes generally reached maximal incidence and severity by day 4 but decreased by comparison in all tissues except the liver. Changes were first seen in lethal study mice also at 6 hours in mediastinal lymph nodes and in spleens. At day 1, changes were present in liver, peripheral lymph nodes, and bone marrow. The incidence and severity of these changes were maximal at day 2. In contrast to sublethal study mice, the incidence and severity of the changes did not decrease through the remainder of the study. The most significant difference between the two groups was the rapid involvement of the spleen in the lethal study mice. Changes indicative of impaired vascular perfusion were more frequently seen in the sublethal study mice. Our findings indicate that mice are susceptible to B. mallei infection and may serve as an appropriate model for glanders infection in a resistant host such as human beings. Additionally, by immunoelectron microscopy, we showed the presence of type I O-antigenic polysaccharide (capsular) antigen surrounding B. mallei.

Outbreak of subclinical mastitis in a flock of dairy sheep associated with Burkholderia cepacia complex infection.

An outbreak of subclinical mastitis in a flock of 620 milking sheep was investigated. Microbiological and epidemiological analyses identified the causative agent as belonging to the Burkholderia cepacia complex (formerly Pseudomonas cepacia). Every ewe in the milking flock was individually tested for subclinical mastitis on two separate occasions, 6 weeks apart, by the California (rapid) mastitis test (CMT). The proportion of CMT-positive ewes was 69 of 393 (17.6%) on the first sampling and 27 of 490 (5.5%) on the second sampling. Pure B. cepacia cultures identified with the API 20 NE system were grown from 64 of 96 (66.7%) CMT-positive ewes and from 1 of 33 (3.0%) CMT-negative ewes. Statistical analysis confirmed the significant association between a positive CMT result and a positive culture result for B. cepacia complex. Additional polyphasic taxonomic analyses of eight isolates showed that seven belonged to B. cepacia genomovar III; the remaining isolate was identified as Burkholderia vietnamiensis (formerly B. cepacia genomovar V). Bacteriological investigation of samples from milking equipment and other environmental sites failed to identify "B. cepacia" in any of the samples taken. To our knowledge, this is the first report of an outbreak of natural infection in animals caused by B. cepacia complex and the first description of B. cepacia complex infection in sheep.