Draft Genome Sequences of Two Clinical Isolates of Burkholderia mallei Obtained from Nasal Swabs of Glanderous Equines in India.

Burkholderia mallei is a Gram-negative coccobacillus which causes glanders-a fatal disease of equines that may occasionally be transmitted to humans. Several cases of outbreaks have been reported from India since 2006. This paper presents draft genome sequences of two B. mallei strains isolated from equines affected by glanders in India.

Complete Genome Sequence of a Burkholderia pseudomallei Strain Isolated from a Pet Green Iguana in Prague, Czech Republic.

Burkholderia pseudomallei was isolated from pus from an abscess of a pet iguana living in a private household in Prague, Czech Republic. This paper presents the complete genome sequence of B. pseudomallei strain VB976100.

Critical Aspects for Detection of Coxiella burnetii.

Coxiella burnetii is a globally distributed zoonotic γ-proteobacterium with an obligatory intracellular lifestyle. It is the causative agent of Q fever in humans and of coxiellosis among ruminants, although the agent is also detected in ticks, birds, and various other mammalian species. Requirements for intracellular multiplication together with the necessity for biosafety level 3 facilities restrict the cultivation of C. burnetii to specialized laboratories. Development of a novel medium formulation enabling axenic growth of C. burnetii has facilitated fundamental genetic studies. This review provides critical insights into direct diagnostic methods currently available for C. burnetii. It encompasses molecular detection methods, isolation, and propagation of the bacteria and its genetic characterization. Differentiation of C. burnetii from Coxiella-like organisms is an essential diagnostic prerequisite, particularly when handling and analyzing ticks.

Fate of Bacillus anthracis during production of laboratory-scale cream cheese and homemade-style yoghurt.

The viability of Bacillus anthracis during production and storage of cream cheese and yoghurt was evaluated. Experimental cheeses were manufactured from whole milk inoculated with a suspension of B. anthracis vegetative cells and spores at a final concentration of 10(4) cfu/ml. Lactic acid bacteria (LAB) and lab ferment were used to induce milk ripening and milk coagulation. The pH-value of the contaminated milk dropped below 4.5 within the first 6 h and the amount of LAB increased by approximately 2-logs. During cheese production and storage at 5-9 °C for 24 days no growth of B. anthracis was observed. The amount of vegetative cells and spores fluctuated by 1-log. Inoculation of whole milk with heat-treated spores at 10(4) cfu/ml resulted in a slight increase of vegetative cell counts during the first 6 h. This indicated that germination occurred, but replication of vegetative cells was still inhibited in the produced cheese. Incubation of cheeses at room temperature or heating after milk coagulation strongly reduced the amount of LAB but had no effect on the growth behaviour of B. anthracis. The vegetative cell and spore content remained steady at 10(4) cfu/100 mg. During yoghurt production the pH-value decreased within 5 h below 5 and growth of B. anthracis was inhibited throughout storage. A pH-value of 5 or less is likely a critical factor to control the growth of B. anthracis. However, spores remained viable in experimental cream cheeses and yoghurts and are a potential risk of infection.

Isolation of the highly pathogenic and zoonotic agent Burkholderia pseudomallei from a pet green Iguana in Prague, Czech Republic.

BACKGROUND: Melioidosis caused by Burkholderia (B.) pseudomallei is an endemic zoonotic disease mainly reported from northern Australia and Southeast Asia. In Europe, cases of human melioidosis have been reported only from patients travelling to endemic regions. Besides humans, B. pseudomallei has a very broad host range in domestic and wild animals. There are some reports about importation of B. pseudomallei-infected animals from endemic areas into Europe. The present report describes the first case of B. pseudomallei infection of a pet iguana in Europe. CASE PRESENTATION: In a 5-year-old pet Iguana iguana living in a private household in Prague, Czech Republic, B. pseudomallei was isolated from pus of an abscess. The isolate VB976100 was identified by Vitek®2, MALDI-TOF mass spectrometry and polymerase chain reaction as B. pseudomallei. The molecular typing resulted in multi-locus sequence type 436 hitherto, which has been found only once worldwide in a B. pseudomallei strain isolated in the USA and originating from Guatemala. The identification as internal transcribed spacer type G indicates a close relatedness to strains mainly isolated in the Western Hemisphere. These findings support the hypothesis that the iguana became infected in this region or in a breeding facility through contact to other infected animals. CONCLUSIONS: The present case highlights the risk of importation of the highly pathogenic and zoonotic B. pseudomallei into non-endemic regions through animal trade. Therefore, veterinarians treating animals from these areas and physicians examining patients owning such animals should include melioidosis in differential diagnosis whenever specific symptoms appear. Furthermore, veterinary authorities responsible for supervision of traders and pet shops should be aware of this risk of zoonotic transmission.

Comparative evaluation of eleven commercial DNA extraction kits for real-time PCR detection of Bacillus anthracis spores in spiked dairy samples.

Spores of Bacillus anthracis are highly resistant and can survive conditions used for food preservation. Sample size and complexity represent the major hurdles for pathogen detection in food-related settings. Eleven commercial DNA extraction kits were evaluated for detection of B. anthracis spores by quantitative real-time PCR (qPCR) in dairy products. DNA was extracted from serial dilutions of B. anthracis spores in milk powder, cream cheese, whole milk and buttermilk. Three kits (QIAamp DNA mini kit, Invisorb Food kit I and II) were determined to produce the lowest limit of detections (LODs) with equally good performance. These kits employed lysozyme and proteinase K treatments or proteinase K in combination with cethyltrimethylamonium bromide-mediated (CTAB) precipitation of cell debris for cell disruption and DNA release. The LODs for these three kits were determined as 10(2) spores/ml of distilled water, 10(3)s pores/20 mg of powdered milk and 10(4) spores/100 mg of cream cheese, respectively. Performance testing of the QIAamp DNA mini kit demonstrated a good reproducibility and appropriate detection limits from 10(3)/ml for butter milk, 10(4)/ml for whole milk and 10(4)/100 mg for low fat cream cheese. However, DNA extraction efficiency was strongly inhibited by cream cheese with higher fat contents with an increased LOD of 10(6)/100 mg spores. This study demonstrated that qPCR detection depends directly on the appropriate DNA extraction method for an individual food matrix and bacterial agent.

Identification of Coxiella burnetii type IV secretion substrates required for intracellular replication and Coxiella-containing vacuole formation.

Coxiella burnetii, the etiological agent of acute and chronic Q fever in humans, is a naturally intracellular pathogen that directs the formation of an acidic Coxiella-containing vacuole (CCV) derived from the host lysosomal network. Central to its pathogenesis is a specialized type IVB secretion system (T4SS) that delivers effectors essential for intracellular replication and CCV formation. Using a bioinformatics-guided approach, 234 T4SS candidate substrates were identified. Expression of each candidate as a TEM-1 ß-lactamase fusion protein led to the identification of 53 substrates that were translocated in a Dot/Icm-dependent manner. Ectopic expression in HeLa cells revealed that these substrates trafficked to distinct subcellular sites, including the endoplasmic reticulum, mitochondrion, and nucleus. Expression in Saccharomyces cerevisiae identified several substrates that were capable of interfering with yeast growth, suggesting that these substrates target crucial host processes. To determine if any of these T4SS substrates are necessary for intracellular replication, we isolated 20 clonal T4SS substrate mutants using the Himar1 transposon and transposase. Among these, 10 mutants exhibited defects in intracellular growth and CCV formation in HeLa and J774A.1 cells but displayed normal growth in bacteriological medium. Collectively, these results indicate that C. burnetii encodes a large repertoire of T4SS substrates that play integral roles in host cell subversion and CCV formation and suggest less redundancy in effector function than has been found in the comparative Legionella Dot/Icm model.

Molecular pathogenesis of the obligate intracellular bacterium Coxiella burnetii.

The agent of Q fever, Coxiella burnetii, is an obligate intracellular bacterium that causes acute and chronic infections. The study of C. burnetii pathogenesis has benefited from two recent fundamental advances: improved genetic tools and the ability to grow the bacterium in extracellular media. In this Review, we describe how these recent advances have improved our understanding of C. burnetii invasion and host cell modulation, including the formation of replication-permissive Coxiella-containing vacuoles. Furthermore, we describe the Dot/Icm (defect in organelle trafficking/intracellular multiplication) system, which is used by C. burnetii to secrete a range of effector proteins into the host cell, and we discuss the role of these effectors in remodelling the host cell.

Defense mechanisms against oxidative stress in Coxiella burnetii: adaptation to a unique intracellular niche.

Survival of intracellular pathogenic bacteria depends on the ability to resist host-mediated degradation and to generate a replicative niche within the host. Usually, after internalization by professional phagocytic cells, the bacteria containing vacuole or phagosome traffics through the endocytic pathway, progressively acidifies and develops into a degradative mature phagolysosome. In this environment bacteria are exposed to a wide variety of anti-microbial agents, such as defensins, proteases, and reactive oxygen species (ROS) and reactive nitrogen species (RNS). Most parasitizing bacteria have evolved strategies to interfere with this maturation process and to direct the development of an environment that supports survival and replication. C. burnetii also follows this paradigm, but directs the biogenesis of a unique parasitophorous vacuole (PV), which resembles, yet is distinct from a terminal phagolysosome. Within the environment of the PV, C. burnetii is exposed to varying levels of ROS and RNS, which represent the primary defense mechanism of the host cell against this invading microorganism. Major mediators for ROS and RNS are superoxide (O (2) (-) ) and nitric oxide (NO(*)), generated by the cellular NADPH oxidase (phox) and inducible nitric oxide synthase (iNOS), respectively. C. burnetii employs several strategies to evade oxidative stress; on the host side (i) delaying phagolysosome fusion and (ii) inhibiting cellular NADPH oxidase. On the bacterial side, maintaining genome stability by (iii) evolving a preference for a low iron environment, (iv) expressing a minimal and likely crucial set of DNA repair genes and (v) detoxifying the PV by ROS and RNS degrading enzymes. Overall defense mechanisms in C. burnetii against oxidative and nitrosative stress and the regulation thereof are not fully defined and our knowledge is mainly based on genome sequence information. Comparison with E. coli as a model bacterium reveals that defense strategies of C. burnetii differ significantly and emphasize a highly adaptive evolution to this harsh and unique niche.

Characterization of the GDP-D-mannose biosynthesis pathway in Coxiella burnetii: the initial steps for GDP-ß-D-virenose biosynthesis.

Coxiella burnetii, the etiologic agent of human Q fever, is a gram-negative and naturally obligate intracellular bacterium. The O-specific polysaccharide chain (O-PS) of the lipopolysaccharide (LPS) of C. burnetii is considered a heteropolymer of the two unusual sugars ß-D-virenose and dihydrohydroxystreptose and mannose. We hypothesize that GDP-D-mannose is a metabolic intermediate to GDP-ß-D-virenose. GDP-D-mannose is synthesized from fructose-6-phosphate in 3 successive reactions; Isomerization to mannose-6-phosphate catalyzed by a phosphomannose isomerase (PMI), followed by conversion to mannose-1-phosphate mediated by a phosphomannomutase (PMM) and addition of GDP by a GDP-mannose pyrophosphorylase (GMP). GDP-D-mannose is then likely converted to GDP-6-deoxy-D-lyxo-hex-4-ulopyranose (GDP-Sug), a virenose intermediate, by a GDP-mannose-4,6-dehydratase (GMD). To test the validity of this pathway in C. burnetii, three open reading frames (CBU0671, CBU0294 and CBU0689) annotated as bifunctional type II PMI, as PMM or GMD were functionally characterized by complementation of corresponding E. coli mutant strains and in enzymatic assays. CBU0671, failed to complement an Escherichia coli manA (PMM) mutant strain. However, complementation of an E. coli manC (GMP) mutant strain restored capsular polysaccharide biosynthesis. CBU0294 complemented a Pseudomonas aeruginosa algC (GMP) mutant strain and showed phosphoglucomutase activity (PGM) in a pgm E. coli mutant strain. Despite the inability to complement a manA mutant, recombinant C. burnetii PMI protein showed PMM enzymatic activity in biochemical assays. CBU0689 showed dehydratase activity and determined kinetic parameters were consistent with previously reported data from other organisms. These results show the biological function of three C. burnetii LPS biosynthesis enzymes required for the formation of GDP-D-mannose and GDP-Sug. A fundamental understanding of C. burnetii genes that encode PMI, PMM and GMP is critical to fully understand the biosynthesic pathway of GDP-ß-D-virenose and LPS structure in C. burnetii.

Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii.

Coxiella burnetii is an obligate intracellular bacterial pathogen responsible for acute and chronic Q fever. This bacterium harbors a type IV secretion system (T4SS) highly similar to the Dot/Icm of Legionella pneumophila that is believed to be essential for its infectivity. Protein substrates of the Coxiella T4SS are predicted to facilitate the biogenesis of a phagosome permissive for its intracellular growth. However, due to the lack of genetic systems, protein transfer by the C. burnetii Dot/Icm has not been demonstrated. In this study, we report the identification of 32 substrates of the C. burnetii Dot/Icm system using a fluorescence-based ß-lactamase (TEM1) translocation assay as well as the calmodulin-dependent adenylate cyclase (CyaA) assay in the surrogate host L. pneumophila. Notably, 26 identified T4SS substrates are hypothetical proteins without predicted function. Candidate secretion substrates were obtained by using (i) a genetic screen to identify C. burnetii proteins interacting with DotF, a component of the T4SS, and (ii) bioinformatic approaches to retrieve candidate genes that harbor characteristics associated with previously reported substrates of the Dot/Icm system from both C. burnetii and L. pneumophila. Moreover, we have developed a shuttle plasmid that allows the expression of recombinant proteins in C. burnetii as TEM fusion products. Using this system, we demonstrated that a Dot/Icm substrate identified with L. pneumophila was also translocated by C. burnetii in a process that requires its C terminus, providing direct genetic evidence of a functional T4SS in C. burnetii.

Antiserum against Raoultella terrigena ATCC 33257 identifies a large number of Raoultella and Klebsiella clinical isolates as serotype O12.

Raoultella terrigena ATCC 33257, recently reclassified from the genus Klebsiella, is a drinking water isolate and belongs to a large group of non-typeable Klebsiella and Raoultella strains. Using an O-antiserum against a capsule-deficient mutant of this strain, we could show a high prevalence (10.5%) of the R. terrigena O-serotype among non-typeable, clinical Klebsiella and Raoultella isolates. We observed a strong serological cross-reaction with the K. pneumoniae O12 reference strain, indicating that a large percentage of these non-typeable strains may belong to the O12 serotype, although these are currently not detectable by the K. pneumoniae O12 reference antiserum in use. Therefore, we analyzed the O-polysaccharide (O-PS) structure and genetic organization of the wb gene cluster of R. terrigena ATCC 33257, and both confirmed a close relation of R. terrigena and K. pneumoniae O12. The two strains possess an identical O-PS, lipopolysaccharide core structure, and genetic organization of the wb gene cluster. Heterologous expression of the R. terrigena wb gene cluster in Escherichia coli K-12 resulted in the WecA-dependent synthesis of an O-PS reactive with the K. pneumoniae O12 antiserum. The serological data presented here suggest a higher prevalence of the O12-serotype among Klebsiella and Raoultella isolates than generally assumed.

Constitutive SOS expression and damage-inducible AddAB-mediated recombinational repair systems for Coxiella burnetii as potential adaptations for survival within macrophages.

SUMMARY: Coxiella burnetii, a Gram-negative obligate intracellular pathogen, replicates within an parasitophorous vacuole with lysosomal characteristics. To understand how C. burnetii maintains genomic integrity in this environment, a database search for genes involved in DNA repair was performed. Major components of repair, SOS response and recombination were identified, including recA and ruvABC, but lexA and recBCD were absent. Instead, C. burnetii possesses addAB orthologous genes, functional equivalents to recBCD. Survival after treatment with UV, mitomycin C (MC) or methyl methanesulfonate (MMS), as well as homologous recombination in Hfr mating was restored in Escherichia coli deletion strains by C. burnetii recA or addAB. Despite the absence of LexA, co-protease activity for C. burnetii RecA was demonstrated. Dominant-negative inhibition of C. burnetii RecA by recA mutant alleles, modelled after E. coli recA1 and recA56, was observed and more apparent with expression of C. burnetii RecAG159D mutant protein. Expression of a subset of repair genes in C. burnetii was monitored and, in contrast to the non-inducible E. coli recBCD, addAB expression was strongly upregulated under oxidative stress. Constitutive SOS gene expression due to the lack of LexA and induction of AddAB likely reflect a unique repair adaptation of C. burnetii to its hostile niche.