The Retrospective on Atypical Brucella Species Leads to Novel Definitions.

The genus Brucella currently comprises twelve species of facultative intracellular bacteria with variable zoonotic potential. Six of them have been considered as classical, causing brucellosis in terrestrial mammalian hosts, with two species originated from marine mammals. In the past fifteen years, field research as well as improved pathogen detection and typing have allowed the identification of four new species, namely Brucella microti, Brucella inopinata, Brucella papionis, Brucella vulpis, and of numerous strains, isolated from a wide range of hosts, including for the first time cold-blooded animals. While their genome sequences are still highly similar to those of classical strains, some of them are characterized by atypical phenotypes such as higher growth rate, increased resistance to acid stress, motility, and lethality in the murine infection model. In our review, we provide an overview of state-of-the-art knowledge about these novel Brucella sp., with emphasis on their phylogenetic positions in the genus, their metabolic characteristics, acid stress resistance mechanisms, and their behavior in well-established in cellulo and in vivo infection models. Comparison of phylogenetic classification and phenotypical properties between classical and novel Brucella species and strains finally lead us to propose a more adapted terminology, distinguishing between core and non-core, and typical versus atypical brucellae, respectively.

A systematic review and meta-analysis of the aetiological agents of non-malarial febrile illnesses in Africa.

BACKGROUND: The awareness of non-malarial febrile illnesses (NMFIs) has been on the rise over the last decades. Therefore, we undertook a systematic literature review and meta-analysis of causative agents of non-malarial fevers on the African continent. METHODOLOGY: We searched for literature in African Journals Online, EMBASE, PubMed, Scopus, and Web of Science databases to identify aetiologic agents that had been reported and to determine summary estimates of the proportional morbidity rates (PMr) associated with these pathogens among fever patients. FINDINGS: A total of 133 studies comprising 391,835 patients from 25 of the 54 African countries were eligible. A wide array of aetiologic agents were described with considerable regional differences among the leading agents. Overall, bacterial pathogens tested from blood samples accounted for the largest proportion. The summary estimates from the meta-analysis were low for most of the agents. This may have resulted from a true low prevalence of the agents, the failure to test for many agents or the low sensitivity of the diagnostic methods applied. Our meta-regression analysis of study and population variables showed that diagnostic methods determined the PMr estimates of typhoidal Salmonella and Dengue virus. An increase in the PMr of Klebsiella spp. infections was observed over time. Furthermore, the status of patients as either inpatient or outpatient predicted the PMr of Haemophilus spp. infections. CONCLUSION: The small number of epidemiological studies and the variety of NMFI agents on the African continent emphasizes the need for harmonized studies with larger sample sizes. In particular, diagnostic procedures for NMFIs should be standardized to facilitate comparability of study results and to improve future meta-analyses. Reliable NMFI burden estimates will inform regional public health strategies.

Protein Biomarker Identification for the Discrimination of Brucella melitensis Field Isolates From the Brucella melitensis Rev.1 Vaccine Strain by MALDI-TOF MS.

Brucella melitensis Rev.1 is a live attenuated vaccine strain that is widely used to control brucellosis in small ruminants. For successful surveillance and control programs, rapid identification and characterization of Brucella isolates and reliable differentiation of vaccinated and naturally infected animals are essential prerequisites. Although MALDI-TOF MS is increasingly applied in clinical microbiology laboratories for the diagnosis of brucellosis, species or even strain differentiation by this method remains a challenge. To detect biomarkers, which enable to distinguish the B. melitensis Rev.1 vaccine strain from B. melitensis field isolates, we initially searched for unique marker proteins by in silico comparison of the B. melitensis Rev.1 and 16M proteomes. We found 113 protein sequences of B. melitensis 16M that revealed a homologous sequence in the B. melitensis Rev.1 annotation and 17 of these sequences yielded potential biomarker pairs. MALDI-TOF MS spectra of 18 B. melitensis Rev.1 vaccine and 183 Israeli B. melitensis field isolates were subsequently analyzed to validate the identified marker candidates. This approach detected two genus-wide unique biomarkers with properties most similar to the ribosomal proteins L24 and S12. These two proteins clearly discriminated B. melitensis Rev.1 from the closely related B. melitensis 16M and the Israeli B. melitensis field isolates. In addition, we verified their discriminatory power using a set of B. melitensis strains from various origins and of different MLVA types. Based on our results, we propose MALDI-TOF MS profiling as a rapid, cost-effective alternative to the traditional, time-consuming approach to differentiate certain B. melitensis isolates on strain level.

Direct identification and molecular characterization of zoonotic hazards in raw milk by metagenomics using Brucella as a model pathogen.

Metagenomics is a valuable diagnostic tool for enhancing microbial food safety because (i) it enables the untargeted detection of pathogens, (ii) it is fast since primary isolation of micro-organisms is not required, and (iii) it has high discriminatory power allowing for a detailed molecular characterization of pathogens. For shotgun metagenomics, total nucleic acids (NAs) are isolated from complex samples such as foodstuff. Along with microbial NAs, high amounts of matrix NAs are extracted that might outcompete microbial NAs during next-generation sequencing and compromise sensitivity for the detection of low abundance micro-organisms. Sensitive laboratory methods are indispensable for detecting highly pathogenic foodborne bacteria like Brucella spp., because a low infectious dose is sufficient to cause human disease through the consumption of contaminated dairy or meat products. In our study, we applied shotgun metagenomic sequencing for the identification and characterization of Brucella spp. in artificially and naturally contaminated raw milk from various ruminant species. With the depletion of eukaryotic cells prior to DNA extraction, Brucella was detectable at 10 bacterial cells ml-1, while at the same time microbiological culture and isolation of the fastidious bacteria commonly failed. Moreover, we were able to retrieve the genotype of a Brucella isolate from a metagenomic dataset, indicating the potential of metagenomics for outbreak investigations using SNPs and core-genome multilocus sequence typing (cgMLST). To improve diagnostic applications, we developed a new bioinformatics approach for strain prediction based on SNPs to identify the correct species and define a certain strain with only low numbers of genus-specific reads per sample. This pipeline turned out to be more sensitive and specific than Mash Screen. In raw milk samples, we simultaneously detected numerous other zoonotic pathogens, antimicrobial resistance genes and virulence factors. Our study showed that metagenomics is a highly sensitive tool for biological risk assessment of foodstuffs, particularly when pathogen isolation is hazardous or challenging.

Antimicrobial resistance of Helicobacter pylori in an eastern German region.

BACKGROUND: Antimicrobial therapy is recommended to eradicate Helicobacter (H.) pylori in infected individuals. As first-line treatments are empiric, knowledge of antimicrobial resistance is key to successful eradication. AIMS: We investigated primary resistance in an eastern German region to derive recommendations for eradication treatment. METHODS: We used molecular genetic methods to examine Helicobacter rapid urease test (RUT) positive gastric specimens of 533 patients from Berlin and the federal state of Brandenburg with allegedly no prior eradication treatment. Tissue samples were removed from RUT and screened by real-time PCR for mutations conferring resistance to clarithromycin. In addition, 182 samples out of 533 were tested for resistance to levofloxacin and tetracycline. RESULTS: Primary resistances were 10.9% (58 out of 533) to clarithromycin; 13.7% (25/182) to levofloxacin; and 2.2% to tetracycline (4/182). Combined resistance to clarithromycin/levofloxacin was low (2.2%, 4/182). Female sex was significantly associated with clarithromycin resistance. CONCLUSION: Clarithromycin may be a suitable first-line antibiotic for about 90% of outpatients. A simple molecular test may help physicians avoid prescription of an ineffective first-line regimen.

MALDI-TOF MS and genomic analysis can make the difference in the clarification of canine brucellosis outbreaks.

Brucellosis is one of the most common bacterial zoonoses worldwide affecting not only livestock and wildlife but also pets. Canine brucellosis is characterized by reproductive failure in dogs. Human Brucella canis infections are rarely reported but probably underestimated due to insufficient diagnostic surveillance. To improve diagnostics, we investigated dogs in a breeding kennel that showed clinical manifestations of brucellosis and revealed positive blood cultures. As an alternative to the time-consuming and hazardous classical identification procedures, a newly developed species-specific intact-cell matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis was applied, which allowed for rapid identification of B. canis and differentiation from closely related B. suis biovar 1. High-throughput sequencing and comparative genomics using single nucleotide polymorphism analysis clustered our isolates together with canine and human strains from various Central and South American countries in a distinct sub-lineage. Hence, molecular epidemiology clearly defined the outbreak cluster and demonstrated the endemic situation in South America. Our study illustrates that MALDI-TOF MS analysis using a validated in-house reference database facilitates rapid B. canis identification at species level. Additional whole genome sequencing provides more detailed outbreak information and leads to a deeper understanding of the epidemiology of canine brucellosis.

Peptidase PepP is a novel virulence factor of Campylobacter jejuni contributing to murine campylobacteriosis.

Mechanisms of host-pathogen interactions resulting in immunopathological responses upon human Campylobacter jejuni infection are not completely understood, but the recent availability of murine infection models mimicking key features of campylobacteriosis helps solving this dilemma. During a screen for proteases expressed by C. jejuni, we identified a peptidase of the M24 family as a potential novel virulence factor, which was named PepP. The gene is strongly conserved in various Campylobacter species. A constructed deletion mutant ΔpepP of C. jejuni strain 81-176 grew as efficiently compared to isogenic wild-type (WT) or pepP complemented bacteria. To shed light on the potential role of this protease in mediating immunopathological responses in the mammalian host, we perorally challenged microbiota-depleted IL-10-/- mice with these strains. All strains stably colonized the murine gastrointestinal tract with comparably high loads. Remarkably, pepP deficiency was associated with less severe induced malaise, with less distinct apoptotic and innate immune cell responses, but also with more pronounced proliferative/regenerative epithelial cell responses in the large intestine at d6post-infection. Furthermore, pro-inflammatory mediators were lower in the colon, ileum, and mesenteric lymph nodes of mice that had been challenged with the ΔpepP mutant compared to the WT or pepP complemented strains. This also held true for extra-intestinal organs including liver, kidneys, and lungs, and, strikingly, to systemic compartments. Taken together, protease PepP is a novel virulence determinant involved in mediating campylobacteriosis. The finding that apoptosis in the colon is significantly diminished in mice infected with the pepP mutant highlights the epithelial layer as the first and main target of PepP in the intestine.

Metabolic and fitness determinants for in vitro growth and intestinal colonization of the bacterial pathogen Campylobacter jejuni.

Campylobacter jejuni is one of the leading infectious causes of food-borne illness around the world. Its ability to persistently colonize the intestinal tract of a broad range of hosts, including food-producing animals, is central to its epidemiology since most infections are due to the consumption of contaminated food products. Using a highly saturated transposon insertion library combined with next-generation sequencing and a mouse model of infection, we have carried out a comprehensive genome-wide analysis of the fitness determinants for growth in vitro and in vivo of a highly pathogenic strain of C. jejuni. A comparison of the C. jejuni requirements to colonize the mouse intestine with those necessary to grow in different culture media in vitro, combined with isotopologue profiling and metabolic flow analysis, allowed us to identify its metabolic requirements to establish infection, including the ability to acquire certain nutrients, metabolize specific substrates, or maintain intracellular ion homeostasis. This comprehensive analysis has identified metabolic pathways that could provide the basis for the development of novel strategies to prevent C. jejuni colonization of food-producing animals or to treat human infections.

A transferable plasticity region in Campylobacter coli allows isolates of an otherwise non-glycolytic food-borne pathogen to catabolize glucose.

Thermophilic Campylobacter species colonize the intestine of agricultural and domestic animals commensally but cause severe gastroenteritis in humans. In contrast to other enteropathogenic bacteria, Campylobacter has been considered to be non-glycolytic, a metabolic property originally used for their taxonomic classification. Contrary to this dogma, we demonstrate that several Campylobacter coli strains are able to utilize glucose as a growth substrate. Isotopologue profiling experiments with (13) C-labeled glucose suggested that these strains catabolize glucose via the pentose phosphate and Entner-Doudoroff (ED) pathways and use glucose efficiently for de novo synthesis of amino acids and cell surface carbohydrates. Whole genome sequencing of glycolytic C. coli isolates identified a genomic island located within a ribosomal RNA gene cluster that encodes for all ED pathway enzymes and a glucose permease. We could show in vitro that a non-glycolytic C. coli strain could acquire glycolytic activity through natural transformation with chromosomal DNA of C. coli and C. jejuni subsp. doylei strains possessing the ED pathway encoding plasticity region. These results reveal for the first time the ability of a Campylobacter species to catabolize glucose and provide new insights into how genetic macrodiversity through intra- and interspecies gene transfer expand the metabolic capacity of this food-borne pathogen.

Defining the metabolic requirements for the growth and colonization capacity of Campylobacter jejuni.

During the last decade Campylobacter jejuni has been recognized as the leading cause of bacterial gastroenteritis worldwide. This facultative intracellular pathogen is a member of the Epsilonproteobacteria and requires microaerobic atmosphere and nutrient rich media for efficient proliferation in vitro. Its catabolic capacity is highly restricted in contrast to Salmonella Typhimurium and other enteropathogenic bacteria because several common pathways for carbohydrate utilization are either missing or incomplete. Despite these metabolic limitations, C. jejuni efficiently colonizes various animal hosts as a commensal intestinal inhabitant. Moreover, C. jejuni is tremendously successful in competing with the human intestinal microbiota; an infectious dose of few hundreds bacteria is sufficient to overcome the colonization resistance of humans and can lead to campylobacteriosis. Besides the importance and clear clinical manifestation of this disease, the pathogenesis mechanisms of C. jejuni infections are still poorly understood. In recent years comparative genome sequence, transcriptome and metabolome analyses as well as mutagenesis studies combined with animal infection models have provided a new understanding of how the specific metabolic capacity of C. jejuni drives its persistence in the intestinal habitat of various hosts. Furthermore, new insights into the metabolic requirements that support the intracellular survival of C. jejuni were obtained. Because C. jejuni harbors distinct properties in establishing an infection in comparison to pathogenic Enterobacteriaceae, it represents an excellent organism for elucidating new aspects of the dynamic interaction and metabolic cross talk between a bacterial pathogen, the microbiota and the host.

Relevance of Campylobacter to public health--the need for a One Health approach.

Campylobacter species belong to the most important foodborne bacteria which cause gastroenteritis in humans in both developed and developing countries. With increasing reporting rates, the public awareness towards Campylobacter infections is growing continuously. This strengthens the necessity to establish intervention measures for prevention and control of thermophilic Campylobacter spp. along the food chain, as in particular poultry and poultry meat represent a major source of human infections. An interdisciplinary One Health approach and a combined effort of all stakeholders are necessary to ultimately reduce the burden of campylobacteriosis cases in humans. Numerous studies point out, however, that at present a complete elimination of Campylobacter in the food chain is not feasible. The present aim should therefore be to establish control measures and intervention strategies to minimize the occurrence of Campylobacter spp. in livestock (e.g. poultry flocks) and to reduce the quantitative Campylobacter burden in animals and foods. To this end, a combination of intervention methods at different stages of the food chain appears most promising. That has to be accompanied by targeted consumer advice and education campaigns to raise the awareness towards Campylobacter infections.

Utilization of host-derived cysteine-containing peptides overcomes the restricted sulphur metabolism of Campylobacter jejuni.

The non-glycolytic food-borne pathogen Campylobacter jejuni successfully colonizes the intestine of various hosts in spite of its restricted metabolic properties. While several amino acids are known to be used by C. jejuni as energy sources, none of these have been found to be essential for growth. Here we demonstrated through phenotype microarray analysis that cysteine utilization increases the metabolic activity of C. jejuni. Furthermore, cysteine was crucial for its growth as C. jejuni was unable to synthesize it from sulphate or methionine. Our study showed that C. jejuni compensates this limited anabolic capacity by utilizing sulphide, thiosulphate, glutathione and the dipeptides γGlu-Cys, Cys-Gly and Gly-Cys as sulphur sources and cysteine precursors. A panel of C. jejuni mutants in putative peptidases and peptide transporters were generated and tested for their participation in the catabolism of the cysteine-containing peptides, and the predicted transporter protein CJJ81176_0236 was discovered to facilitate the growth with the dipeptide Cys-Gly, Ile-Arg and Ile-Trp. It was named Campylobacter peptide transporter A (CptA) and is the first representative of the oligopeptide transporter OPT family demonstrated to participate in the glutathione-derivative Cys-Gly catabolism in prokaryotes. Our study provides new insights into how host- and microbiota-derived substrates like sulphide, thiosulphate and short peptides are used by C. jejuni to compensate its restricted metabolic capacities.

Molecular methods to investigate adhesion, transmigration, invasion and intracellular survival of the foodborne pathogen Campylobacter jejuni.

Campylobacter jejuni is a spiral-shaped Gram-negative pathogen and major agent of gastrointestinal foodborne illness in humans worldwide. This pathogen encodes numerous described pathogenicity-associated factors involved in important processes including bacterial adhesion to, transmigration across, invasion into and intracellular survival within intestinal epithelial cells. This review article highlights various molecular techniques applied in the studies of each of these individual steps of C. jejuni host cell interactions in vitro including gentamicin protection assay, chemotaxis and motility assays, transwell and intracellular survival assays, G-Lisa, siRNA knockdown, immunohistochemistry, immunofluorescence, electron microscopy and luciferase reporter assays. We discuss the strengths and limitations of the methods as well as the different cell model systems applied. Future work should employ new technologies including modern microscopic, proteomics-based and cell signaling approaches to identify and characterise novel virulence mechanisms, which are crucial to provide fresh insights into the diversity of strategies employed by this important pathogen to cause disease.

Contribution of amino acid catabolism to the tissue specific persistence of Campylobacter jejuni in a murine colonization model.

Campylobacter jejuni is a major cause of food-borne disease in industrialized countries. Carbohydrate utilization by C. jejuni is severely restricted, and knowledge about which substrates fuel C. jejuni infection and growth is limited. Some amino acids have been shown to serve as carbon sources both in vitro and in vivo. In the present study we investigated the contribution of serine and proline catabolism to the invitro and invivo growth of C. jejuni 81-176. We confirmed that the serine transporter SdaC and the serine ammonia-lyase SdaA are required for serine utilization, and demonstrated that a predicted proline permease PutP and a bifunctional proline/delta-1-pyrroline-5-carboxylate dehydrogenase PutA are required for proline utilization by C. jejuni 81-176. C. jejuni 81-176 mutants unable to utilize serine were shown to be severely defective for colonization of the intestine and systemic tissues in a mouse model of infection. In contrast, C. jejuni 81-176 mutants unable to utilize proline were only defective for intestinal colonization. These results further emphasize the importance of amino acid utilization in C. jejuni colonization of various tissues.

Closely related Campylobacter jejuni strains from different sources reveal a generalist rather than a specialist lifestyle.

BACKGROUND: Campylobacter jejuni and Campylobacter coli are human intestinal pathogens of global importance. Zoonotic transmission from livestock animals or animal-derived food is the likely cause for most of these infections. However, little is known about their general and host-specific mechanisms of colonization, or virulence and pathogenicity factors. In certain hosts, Campylobacter species colonize persistently and do not cause disease, while they cause acute intestinal disease in humans. RESULTS: Here, we investigate putative host-specificity using phenotypic characterization and genome-wide analysis of genetically closely related C. jejuni strains from different sources. A collection of 473 fresh Campylobacter isolates from Germany was assembled between 2006 and 2010 and characterized using MLST. A subset of closely related C. jejuni strains of the highly prevalent sequence type ST-21 was selected from different hosts and isolation sources. PCR typing of strain-variable genes provided evidence that some genes differed between these strains. Furthermore, phenotypic variation of these strains was tested using the following criteria: metabolic variation, protein expression patterns, and eukaryotic cell interaction. The results demonstrated remarkable phenotypic diversity within the ST-21 group, which however did not correlate with isolation source. Whole genome sequencing was performed for five ST-21 strains from chicken, human, bovine, and food sources, in order to gain insight into ST-21 genome diversity. The comparisons showed extensive genomic diversity, primarily due to recombination and gain of phage-related genes. By contrast, no genomic features associated with isolation source or host were identified. CONCLUSIONS: The genome information and phenotypic data obtained in vitro and in a chicken infection model provided little evidence of fixed adaptation to a specific host. Instead, the dominant C. jejuni ST-21 appeared to be characterized by phenotypic flexibility and high genetic microdiversity, revealing properties of a generalist. High genetic flexibility might allow generalist variants of C. jejuni to reversibly express diverse fitness factors in changing environments.

Identification of Campylobacter jejuni genes involved in its interaction with epithelial cells.

Campylobacter jejuni is the leading cause of infectious gastroenteritis in industrialized nations. Its ability to enter and survive within nonphagocytic cells is thought to be very important for pathogenesis. However, little is known about the C. jejuni determinants that mediate these processes. Through an extensive transposon mutagenesis screen, we have identified several loci that are required for C. jejuni efficient entry and survival within epithelial cells. Among these loci, insertional mutations in aspA, aspB, and sodB resulted in drastic reduction in C. jejuni entry and/or survival within host cells and a severe defect in colonization in an animal model. The implications of these findings for the understanding of C. jejuni-host cell interactions are discussed.

Characterization of a Campylobacter jejuni VirK protein homolog as a novel virulence determinant.

Campylobacter jejuni is a leading cause of food-borne illness in the United States. Despite significant recent advances, its mechanisms of pathogenesis are poorly understood. A unique feature of this pathogen is that, with some exceptions, it lacks homologs of known virulence factors from other pathogens. Through a genetic screen, we have identified a C. jejuni homolog of the VirK family of virulence factors, which is essential for antimicrobial peptide resistance and mouse virulence.

Metabolic diversity in Campylobacter jejuni enhances specific tissue colonization.

Campylobacter jejuni is a leading cause of foodborne illness in industrialized countries. This pathogen exhibits significant strain-to-strain variability, which results in differences in virulence potential and clinical presentations. Here, we report that acquisition of the capacity to utilize specific nutrients enhanced the ability of a highly pathogenic strain of C. jejuni to colonize specific tissues. The acquisition of a gene encoding a gamma-glutamyltranspeptidase enabled this strain to utilize glutamine and glutathione and enhanced its ability to colonize the intestine. Furthermore, the acquisition of a DNA segment, which added a sec-dependent secretion signal to an otherwise cytoplasmic asparaginase, allowed this pathogen to utilize asparagine and to more efficiently colonize the liver. Our results reveal that subtle genetic changes in a bacterial pathogen result in significant changes in its ability to colonize specific tissues. In addition, these studies revealed remarkably specific nutritional requirements for a pathogen to effectively colonize different tissues.

A MyD88-deficient mouse model reveals a role for Nramp1 in Campylobacter jejuni infection.

Campylobacter jejuni is a major worldwide cause of enteric illnesses. Adult immunocompetent mice are not susceptible to C. jejuni infection. However, we show here that mice deficient in the adaptor protein myeloid differentiation factor 88 (MyD88), which is required for signaling through most Toll-like receptors, can be stably colonized by C. jejuni but not by isogenic derivatives carrying mutations in known virulence genes. We also found that Nramp1 deficiency increases the mouse susceptibility to C. jejuni infection when administered systemically. These results indicate that MyD88-deficient mice could be a useful model to study C. jejuni colonization and reveal a potential role for Nramp1 in the control of this bacterial pathogen.

Unique features of a highly pathogenic Campylobacter jejuni strain.

Campylobacter jejuni, a major human enteric pathogen, exhibits significant strain-to-strain differences which result in differences in pathogenic potential. C. jejuni 81-176 is a highly virulent strain that exhibits unique pathogenic features and is used by many research laboratories. We have determined the nucleotide sequence of its genome and compared it to the genomes of other sequenced C. jejuni strains. We identified a number of unique genetic features which may confer specific metabolic and pathogenic properties on this strain. We have also identified regions of the C. jejuni genome that are hot spots for the integration of horizontally acquired genetic material. This information should help the understanding of the pathogenesis of C. jejuni and, in particular, the unique features of this highly pathogenic strain.