Investigation of Extended-Spectrum and AmpC ß-Lactamase-Producing Enterobacteriaceae from Retail Seafood in Berlin, Germany.

Retail seafood in Berlin, Germany, was investigated to detect the prevalence and quantitative load of Enterobacteriaceae that produce extended-spectrum ß-lactamase (ESBL) and AmpC ß-lactamase (AmpC). A total of 160 raw seafood samples were screened for the presence of these bacteria using MacConkey agar supplemented with 1 mg/L cefotaxime after nonselective enrichment. Isolated species were subsequently identified using matrix-assisted laser desorption-ionization time-of-flight analysis. ESBL and AmpC production was tested by the disk diffusion method, and ESBL and AmpC genes were characterized using real-time and conventional PCR assays with DNA sequencing. Spread plating was used for quantification of ESBL- and AmpC-producing Enterobacteriaceae. Overall, these bacteria were detected in 21.3% of seafood samples (34 of 160 samples) with prevalences of 22.5 and 20% for shrimp and bivalves, respectively. Of the positive samples, 91.2% contained an ESBL- or AmpC-producing Enterobacteriaceae load of <100 CFU/g (lower detection limit), and 8.8% contained 100 to 1,000 CFU/g. Among the 45 Enterobacteriaceae isolates, Klebsiella pneumoniae (13 isolates) and Escherichia coli (12 isolates) were the predominant species. ESBL and AmpC genes were detected in 33 isolates, with the majority of isolates harboring blaCTX-M (27.3%), blaCMY (21.2%), or blaDHA (21.2%). Our study highlights the hazard associated with seafood containing ESBL- and AmpC-producing Enterobacteriaceae in Germany. Even though the contamination levels were low, the high prevalence of ESBL- and AmpC-producing Enterobacteriaceae in seafood might be of concern to public health because of the potential transmission of these bacteria from seafood to humans through the food chain.

Prevalence of Vibrio spp. in Retail Seafood in Berlin, Germany.

This study was conducted to determine the prevalence of Vibrio spp. in retail seafood in Berlin, Germany. A total of 160 raw seafood samples from supermarkets and seafood shops, consisting of shrimp ( n = 80) and bivalves ( n = 80), were investigated for the presence of Vibrio spp. using the International Organization for Standardization ISO/TS 21872 method and a multiplex PCR. The overall prevalence of Vibrio spp. in retail seafood was 55% (95% CI: 47.2 to 62.8%). The prevalence of Vibrio spp. in shrimp was slightly higher than in bivalves (57.5 versus 52.5%); however, the difference was not statistically significant. Vibrio alginolyticus was the most prevalent species (35.6%), followed by Vibrio parahaemolyticus (27.5%), Vibrio cholerae (6.3%), and Vibrio vulnificus (0.6%). None of the V. parahaemolyticus ( n = 110) isolates encoded tdh/ trh genes, whereas all V. cholerae isolates ( n = 27) were lacking ctxA. Among the chilled samples ( n = 105), the prevalence of Vibrio spp. in unpacked samples was significantly higher than in packed samples ( P = 0.006). Among the packed samples ( n = 55), no significant difference in the prevalence of Vibrio spp. was observed between chilled or frozen products. The results of this study indicated a high prevalence of Vibrio spp. in retail seafood in Germany; positive samples were detected in all types of seafood investigated. The detection of tdh/ trh-negative V. parahaemolyticus isolates should not be neglected because of previous findings on pathogenic strains lacking these virulence markers. Even though thorough cooking might limit the risk of foodborne illness caused by Vibrio, potential cross-contamination during preparation or consumption of raw and undercooked seafood might represent a risk of Vibrio infections.

VIM-1 carbapenemase-producing Escherichia coli isolated from retail seafood, Germany 2016.

Carbapenems belong to the group of last resort antibiotics in human medicine. Therefore, the emergence of growing numbers of carbapenemase-producing bacteria in food-producing animals or the environment is worrying and an important concern for the public health sector. In the present study, a set of 45 Enterobacteriaceae isolated from German retail seafood (clams and shrimps), sampled in 2016, were investigated by real-time PCR for the presence of carbapenemase-producing bacteria. One Escherichia coli (ST10), isolated from a Venus clam (Ruditapes philippinarum) harvested in the Mediterranean Sea (Italy), contained the carbapenemase gene blaVIM-1 as part of the variable region of a class I integron. Whole-genome sequencing indicated that the integron was embedded in a Tn3-like transposon that also contained the fluoroquinolone resistance gene qnrS1. Additional resistance genes such as the extended-spectrum beta-lactamase blaSHV-12 and the AmpC gene blaACC-1 were also present in this isolate. Except blaACC-1, all resistance genes were located on an IncY plasmid. These results confirm previous observations that carbapenemase-producing bacteria have reached the food chain and are of increasing concern for public health.

Prevalence and antimicrobial resistance of Vibrio spp. in retail shrimps in Vietnam.

The aim of the study was to investigate the prevalence and the antimicrobial resistance patterns of Vibrio (V.) spp. isolated from retail shrimp in Hanoi, Vietnam A total of 202 shrimp samples were collected from retail markets located in ten urban districts of Hanoi. Among those, 201 (99.5%) samples were positive for Vibrio spp. The most common species detected was V parahaemolyticus (96.5%), followed by V. alginolyticus (56.4%), V. cholerae (2%) and V. vulnificus (1.5%). Multiple Vibrio spp. were found in 114 (56.4%) samples. None of the V. parahaemolyticus isolates carried the virulence-associated tdh (thermostable direct haemolysin) and trh (tdh-related haemolysin) genes. In total, 195 V. parahaemolyticus isolates, four V. cholerae isolates and three V. vulnificus isolates were tested for resistance to eight antimicrobial agents. V. parahaemolyticus isolates showed high rates of resistance against ampicillin (87.2%), while a moderate rate was observed for sulfamethoxazole/trimethoprim (18.5%) and intermediate resistance towards tetracycline (24.6%). Low resistance rates (0.5%) were recorded against both ciprofloxacin and cefalothin. Only one V. cholerae isolate with resistance to ampicillin and two V. cholerae isolates with resistance to sulfamethoxazole/trimethoprim were found. All V. vulnificus isolates were susceptible to the eight antimicrobial agents tested. However, the number of V. vulnificus and V. cholerae was small. Multi-resistant isolates were found in V. parahaemolyticus with a low frequency (16.9%). The results of this study revealed the ubiquitous nature of Vibrio spp. in shrimp at retail. To reduce the potential risk of Vibrio infections due to handling or consumption of undercooked seafood, good manufacturing practice as well as safe handling and processing should be encouraged.

Changes in global gene expression of Vibrio parahaemolyticus induced by cold- and heat-stress.

BACKGROUND: Vibrio (V.) parahaemolyticus causes seafood-borne gastro-intestinal bacterial infections in humans worldwide. It is widely found in marine environments and is isolated frequently from seawater, estuarine waters, sediments and raw or insufficiently cooked seafood. Throughout the food chain, V. parahaemolyticus encounters different temperature conditions that might alter metabolism and pathogenicity of the bacterium. In this study, we performed gene expression profiling of V. parahaemolyticus RIMD 2210633 after exposure to 4, 15, 20, 37 and 42 °C to describe the cold and heat shock response. METHODS: Gene expression profiles of V. parahaemolyticus RIMD 2210633 after exposure to 4, 15, 20, 37 and 42 °C were investigated via microarray. Gene expression values and RT-qPCR experiments were compared by plotting the log2 values. Moreover, volcano plots of microarray data were calculated to visualize the distribution of differentially expressed genes at individual temperatures and to assess hybridization qualities and comparability of data. Finally, enriched terms were searched in annotations as well as functional-related gene categories using the Database for Annotation, Visualization and Integrated Discovery. RESULTS: Analysis of 37 °C normalised transcriptomics data resulted in differential expression of 19 genes at 20 °C, 193 genes at 4 °C, 625 genes at 42 °C and 638 genes at 15 °C. Thus, the largest number of significantly expressed genes was observed at 15 and 42 °C with 13.3 and 13%, respectively. Genes of many functional categories were highly regulated even at lower temperatures. Virulence associated genes (tdh1, tdh2, toxR, toxS, vopC, T6SS-1, T6SS-2) remained mostly unaffected by heat or cold stress. CONCLUSION: Along with folding and temperature shock depending systems, an overall temperature-dependent regulation of expression could be shown. Particularly the energy metabolism was affected by changed temperatures. Whole-genome gene expression studies of food related pathogens such as V. parahaemolyticus reveal how these pathogens react to stress impacts to predict its behaviour under conditions like storage and transport.

The emergence of Vibrio pathogens in Europe: ecology, evolution, and pathogenesis (Paris, 11-12th March 2015).

Global change has caused a worldwide increase in reports of Vibrio-associated diseases with ecosystem-wide impacts on humans and marine animals. In Europe, higher prevalence of human infections followed regional climatic trends with outbreaks occurring during episodes of unusually warm weather. Similar patterns were also observed in Vibrio-associated diseases affecting marine organisms such as fish, bivalves and corals. Basic knowledge is still lacking on the ecology and evolutionary biology of these bacteria as well as on their virulence mechanisms. Current limitations in experimental systems to study infection and the lack of diagnostic tools still prevent a better understanding of Vibrio emergence. A major challenge is to foster cooperation between fundamental and applied research in order to investigate the consequences of pathogen emergence in natural Vibrio populations and answer federative questions that meet societal needs. Here we report the proceedings of the first European workshop dedicated to these specific goals of the Vibrio research community by connecting current knowledge to societal issues related to ocean health and food security.

Gene expression profiles of Vibrio parahaemolyticus in viable but non-culturable state.

Viable but non-culturable (VBNC) state is referred to as a dormant state of non-sporulating bacteria enhancing the survival in adverse environments. To our knowledge, only few studies have been conducted on whole genomic expression of Vibrio parahaemolyticus VBNC state. Since a degradation of nucleic acids in V. vulnificus non-culturable state has been detected, we hypothesize that gene regulation of VBNC cells is highly reduced, downregulation of gene expression is dominant and only metabolic functions crucial for survival are kept on a sustained basis. Hence, we performed the whole transcriptomic profiles of V. parahaemolyticus in three phases (exponential, early stationary phase and VBNC state). Compared with exponential and early stationary phase, in V. parahaemolyticus VBNC cells we found 509 induced genes and 309 repressed by more than 4-fold among 4820 investigated genes. Upregulation was dominant in most of non-metabolism functional categories, while five metabolism-related functional categories revealed downregulation in VBNC state. To our knowledge, this is the first study of comprehensive transcriptomic analyses of three phases of V. parahaemolyticus RIMD2210633. Although the mechanism of VBNC state is not yet clear, massive regulation of gene expression occurs in VBNC state compared with expression in other two phases, indicating VBNC cells are active.

Pathogenic vibrios in environmental, seafood and clinical sources in Germany.

Bacteria of the family Vibrionaceae naturally occur in marine and estuarine environments. Only few species of Vibrionaceae are associated with human cases of gastroenteritis, ear and wound infections, caused by ingestion of seafood or contact with Vibrio containing water. Increasing consumption of seafood (fish, fishery products and shellfish) poses a possible source of Vibrio infections in Germany. Additionally, there is a growing concern that abundances of pathogenic vibrios may increase in German coastal waters as a result of e.g. climate change resulting in probably rising surface water temperatures. According to the One Health concept the VibrioNet consortium started in 2010 to investigate the occurrence and relevance of non-cholera vibrios of human concern in Germany. Vibrios from environmental, seafood and clinical sources were analyzed with the aim to find connections between different reservoirs or sources and to identify potential ways of transmission of these pathogens to assess the risk of infections associated with them. Potentially pathogenic strains mostly belong to the species Vibrio parahaemolyticus, Vibrio vulnificus and non-O1/non-O139 Vibrio cholerae. Investigations on imported seafood and mussels from primary production areas confirmed the frequent occurrence of these species. Moreover, studies of German coastal waters and sediments showed the presence and seasonality of these marine bacteria. So far the incidence of clinical cases of vibriosis in Germany is low. Between 1994 and 2013 thirteen cases of Vibrio spp. associated wound infections and/or septicaemia have been reported. However, the high prevalence of vibrios in aquatic environments and aquatic organisms is of concern and demands continued control of food and surveillance for clinical infections with pathogenic vibrios.

Population analysis of Vibrio parahaemolyticus originating from different geographical regions demonstrates a high genetic diversity.

BACKGROUND: Vibrio parahaemolyticus is frequently isolated from environmental and seafood samples and associated with gastroenteritis outbreakes in American, European, Asian and African countries. To distinguish between different lineages of V. parahaemolyticus various genotyping techniques have been used, incl. multilocus sequence typing (MLST). Even though some studies have already applied MLST analysis to characterize V. parahaemolyticus strain sets, these studies have been restricted to specific geographical areas (e.g. U.S. coast, Thailand and Peru), have focused exclusively on pandemic or non-pandemic pathogenic isolates or have been based on a limited strain number. RESULTS: To generate a global picture of V. parahaemolyticus genotype distribution, a collection of 130 environmental and seafood related V. parahaemolyticus isolates of different geographical origins (Sri Lanka, Ecuador, North Sea and Baltic Sea as well as German retail) was subjected to MLST analysis after modification of gyrB and recA PCRs. The V. parahaemolyticus population was composed of 82 unique Sequence Types (STs), of which 68 (82.9%) were new to the pubMLST database. After translating the in-frame nucleotide sequences into amino acid sequences, less diversity was detectable: a total of 31 different peptide Sequence Types (pSTs) with 19 (61.3%) new pSTs were generated from the analyzed isolates. Most STs did not show a global dissemination, but some were supra-regionally distributed and clusters of STs were dependent on geographical origin. On peptide level no general clustering of strains from specific geographical regions was observed, thereby the most common pSTs were found on all continents (Asia, South America and Europe) and rare pSTs were restricted to distinct countries or even geographical regions. One lineage of pSTs associated only with strains from North and Baltic Sea strains was identified. CONCLUSIONS: Our study reveals a high genetic diversity in the analyzed V. parahaemolyticus strain set as well as for geographical strain subsets, with a high proportion of newly discovered alleles and STs. Differences between the subsets were identified. Our data support the postulated population structure of V. parahaemolyticus which follows the 'epidemic' model of clonal expansion. Application of peptide based AA-MLST allowed the identification of reliable relationships between strains.

Heat stress adaptation induces cross-protection against lethal acid stress conditions in Arcobacter butzleri but not in Campylobacter jejuni.

The ability of many bacteria to adapt to stressful conditions may later protect them against the same type of stress (specific adaptive response) or different types of stresses (multiple adaptive response, also termed cross-protection). Arcobacter butzleri and Campylobacter jejuni are close phylogenetic relatives that occur in many foods of animal origin and have been linked with human illness (mainly diarrhoea). In the present study, sublethal stress adaptation temperatures (48 °C and 10 °C) and mild and lethal acid conditions (pH 5.0 and pH 4.0) were determined for A. butzleri and C. jejuni. In addition, it was evaluated whether these sublethal stress adaptations cause specific adaptive responses or cross-protection against subsequent mild or lethal acid stresses in these bacteria. The studies were conducted in broth adjusted to the different conditions and the results were determined by the dilution series plating method. It was shown that heat stress adapted A. butzleri (incubated for 2 h at 48 °C) were significantly more resistant to subsequent lethal acid stress (pH 4.0) than non-adapted cells at the 1 h time-point (p < 0.01 in Wilcoxon rank sum test). No specific adaptive responses against the stresses in A. butzleri or C. jejuni and no cross-protection in C. jejuni were found. The ability of heat stressed A. butzleri to tolerate later lethal acid conditions should be taken into account when designing new food decontamination and processing strategies.

Prevalence and molecular characteristics of Vibrio spp. isolated from preharvest shrimp of the North Western Province of Sri Lanka.

This study investigated the prevalence and molecular characteristics of Vibrio spp. in farmed shrimp (Penaeus monodon) in Sri Lanka. A total of 170 shrimp samples (100 g of whole shrimp each) taken from individual ponds from 54 farms were collected 1 week prior to harvest from the North Western Province of Sri Lanka. Overall, 98.1% of the farms and 95.1% of the ponds were positive for Vibrio spp. in shrimp; at the pond level, V. parahaemolyticus (91.2%) was most common, followed by V. alginolyticus (18.8%), V. cholerae non-O1/non-O139 (4.1%), and V. vulnificus (2.4%). Multiple Vibrio spp. were detected in 20.6% of the ponds. None of the V. parahaemolyticus isolates (n = 419) were positive for the virulence-associated tdh (thermostable direct hemolysin) and trh (TDH-related hemolysin) genes. V. cholerae was confirmed by the presence of ompW, and all isolates (n = 8) were negative for the cholera toxin (ctxA) gene. V. cholerae isolates were serogrouped by PCR and identified as V. cholerae non-O1/non-O139. All four V. vulnificus strains, isolated from different ponds of two geographical regions, showed pathogenic potential; they belonged to vcgC sequence type, type B 16S rRNA genotype and contained a pilF polymorphism associated with human pathogenicity. The results of this study revealed the ubiquitous nature of vibrios in farmed shrimp. To minimize the potential risk of Vibrio infections due to handling or consumption of raw or undercooked seafood products, good manufacturing practices as well as proper handling and processing should be addressed.

Microarray-based genotyping of Salmonella: inter-laboratory evaluation of reproducibility and standardization potential.

Bacterial food-borne infections in humans caused by Salmonella spp. are considered a crucial food safety issue. Therefore, it is important for the risk assessments of Salmonella to consider the genomic variation among different isolates in order to control pathogen-induced infections. Microarray technology is a promising diagnostic tool that provides genomic information on many genes simultaneously. However, standardization of DNA microarray analysis is needed before it can be used as a routine method for characterizing Salmonella isolates across borders and laboratories. A comparative study was designed in which the agreement of data from a DNA microarray assay used for typing Salmonella spp. between two different labs was assessed. The study was expected to reveal the possibility of obtaining the same results in different labs using different equipment in order to evaluate the reproducibility of the microarray technique as a first step towards standardization. The low-density array contains 281 57-60-mer oligonucleotide probes for detecting a wide range of specific genomic marker genes associated with antibiotic resistance, cell envelope structures, mobile genetic elements and pathogenicity. Several critical methodology parameters that differed between the two labs were identified. These related to printing facilities, choice of hybridization buffer, wash buffers used following the hybridization and choice of procedure for purifying genomic DNA. Critical parameters were randomized in a four-factorial experiment and statistical measures of inter-lab consistency and agreement were performed based on the kappa coefficient. A high level of agreement (kappa=0.7-1.0) in microarray results was obtained even when employing different printing and hybridization facilities, different procedures for purifying genomic DNA and different wash buffers. However, less agreement (Kappa=0.2-0.6) between microarray results were observed when using different hybridization buffers, indicating this parameter as being highly critical when transferring a standard microarray assay between laboratories. In conclusion, this study indicates that DNA microarray assays can be reproduced in at least two different facilities, which is a pre-requisite for the development of standard guidelines.

A multiplex ligation detection assay for the characterization of Salmonella enterica strains.

A proof of principle of a multi-target assay for genotyping Salmonella has been developed targeting 62 genomic marker sequences of Salmonella related to pathogenicity. The assay is based on multiplex ligation detection reaction (LDR) followed by customized ArrayTube® microarray detection. The feasibility of the developed assay was verified in a method comparison study with conventional PCR using 16 Salmonella 'test' strains comprising eight serovars. Subsequently, the feasibility of the LDR microarray assay was also tested by analyzing 41 strains belonging to 23 serovars. With the exception of four serovars each serovar was characterized by a unique virulence associated gene repertoire. The LDR microarray platform proved to be a convenient, rapid and easy to use tool with potential in tracing a Salmonella contamination in the food chain, for outbreak studies, and to provide data for risk assessors that support bio-traceability models.

Association between phylogeny, virulence potential and serovars of Salmonella enterica.

Salmonella enterica subsp. enterica is one of the leading causes of zoonotic food-borne disease worldwide. The consequence of these infections is a serious impact on economics of the society in the form of lost productivity and expenses for medical care. The objective of this study was to analyze the difference in genomic content between selected serovars, especially the content of pathogenicity genes and this was done with a DNA microarray. Furthermore, we investigated the phylogenetic relationship between serovars using multilocus sequence typing (MLST). We chose serovars Typhimurium and Enteritidis as they are responsible for 75% of human infections in Europe. Additionally, we included serovars Derby, Dublin, Saintpaul, 4,5,12:i:-, Java and 4,5,12:b:- which are suspected to have different degrees of virulence to humans. MLST analysis clustered strains according to serovar with the exception of Java and Derby. DNA microarray clustered strains according to serovar and serogroup except for serovar 4,5,12:b:-. Differences in content of pathogenicity related genes between serovars with various host preferences and virulence towards humans were not observed. However, our strains from the supposedly less virulent serovar Derby lacked a combination of genes important for virulence. It might be speculated that other serovars can sustain their pathogenicity lacking one or two of these genes, whereas lack of many virulence genes will result in reduced virulence. A partial lack of concordance between MLST and microarray was found and this can be explained by the underlying data. On one hand, microarray data include highly variable regions which are known to be involved in horizontal gene transfer. On the other hand, MLST data is restricted to seven sequences and disregards contribution of horizontally acquired genes when evaluating evolution. The DNA microarray and MLST analysis complement each other giving a clearer image of evolution of these serovars and, furthermore, a visualization of the horizontally acquired genes.

DNA microarray analysis of Salmonella serotype Typhimurium strains causing different symptoms of disease.

BACKGROUND: Salmonella enterica subsp. enterica is one of the leading food-borne pathogens in the USA and European countries. Outcome of human Salmonella serotype Typhimurium infections ranges from mild self-limiting diarrhoea to severe diarrhoea that requires hospitalization. Increased knowledge of the mechanisms that are responsible for causing infection and especially the severity of infection is of high interest. RESULTS: Strains were selected from patients with mild infections (n = 9) and patients with severe infections (n = 9) and clinical data allowed us to correct for known underlying diseases. Additionally, outbreak isolates (n = 3) were selected. Strains were analyzed on a DNA-DNA microarray for presence or absence of 281 genes covering marker groups of genes related to pathogenicity, phages, antimicrobial resistance, fimbriae, mobility, serotype and metabolism. Strains showed highly similar profiles when comparing virulence associated genes, but differences between strains were detected in the prophage marker group. The Salmonella virulence plasmid was present in 72% of the strains, but presence or absence of the virulence plasmid did not correspond to disease symptoms. A dendrogram clustered strains into four groups. Clustering confirmed DT104 as being a clonal phagetype. Clustering of the remaining strains was mainly correlated to presence or absence of the virulence plasmid and mobile elements such as transposons. Each of the four clusters in the tree represented an almost equal amount of strains causing severe or mild symptoms of infection. CONCLUSIONS: We investigated clinical significance of known virulence factors of Salmonella serotype Typhimurium strains causing different disease symptoms, and conclude that the few detected differences in Salmonella serotype Typhimurium do not affect outcome of human disease.

Virulotyping and antimicrobial resistance typing of Salmonella enterica serovars relevant to human health in Europe.

The combination of virulence gene and antimicrobial resistance gene typing using DNA arrays is a recently developed genomics-based approach to bacterial molecular epidemiology. We have now applied this technology to 523 Salmonella enterica subsp. enterica strains collected from various host sources and public health and veterinary institutes across nine European countries. The strain set included the five predominant Salmonella serovars isolated in Europe (Enteritidis, Typhimurium, Infantis, Virchow, and Hadar). Initially, these strains were screened for 10 potential virulence factors (avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) by polymerase chain reaction. The results indicated that only 14 profiles comprising these genes (virulotypes) were observed throughout Europe. Moreover, most of these virulotypes were restricted to only one (n = 9) or two (n = 4) serovars. The data also indicated that the virulotype did not vary significantly with host source or geographical location. Subsequently, a representative subset of 77 strains was investigated using a microarray designed to detect 102 virulence and 49 resistance determinants. The results confirmed and extended the previous observations using the virulo-polymerase chain reaction screen. Strains belonging to the same serovar grouped together, indicating that the broader virulence-associated gene complement corresponded with the serovar. There were, however, some differences in the virulence gene profiles between strains belonging to an individual serovar. This variation occurred primarily within those virulence genes that were prophage encoded, in fimbrial clusters or in the virulence plasmid. It seems likely that such changes enable Salmonella to adapt to different environmental conditions, which might be reflected in serovar-specific ecology. In this strain subset a number of resistance genes were detected and were serovar restricted to a varying degree. Once again the profiles of those genes encoding resistance were similar or the same for each serovar in all hosts and countries investigated.

DNA microarray for molecular epidemiology of Salmonella.

Salmonellosis is a common infection estimated to affect 3 billion people and to cause 200,000 deaths every year. Infections can appear as enteric fever, gastroenteritis, bacteremia, or extraintestinal focal infection. The course of the disease depends on a variety of factors, including infective dose, immune status of the host, and the genetic background of both the host and the pathogen. It has been recognized that certain Salmonella types play a major role in the epidemiology of Salmonella. Here we describe a DNA microarray comprised of 282 sixty-mer oligonucleotide probes to study the epidemiology of Salmonella enterica subsp. enterica isolates at the genotypic level. The probes detect targets encoding genes associated with pathogenicity, antibiotic resistance, fimbriae, prophages, flagella (H antigens), lipopolysaccharides (O antigens), plasmids, insertion sequence elements, and metabolism. The probes are printed on glass slides, and whole-genomic fluorescence-labeled Salmonella DNA is hybridized to the substrate. For quality assurance, a number of controls are included on the microarray.

Characterization of pathogenic and resistant genome repertoire reveals two clonal lines in Salmonella enterica subsp. enterica serovar Paratyphi B (+)-tartrate positive.

A total of 36 contemporary human, animal, and environmental (+)-tartrate-fermenting (dT+) Salmonella enterica serovar Paratyphi B isolates, formerly called Salmonella serovar Java, and five related monophasic S. enterica serovar 4,5,12:b:- isolates from Belgium, Germany, the Netherlands, and the United Kingdom were investigated for clonality and antimicrobial resistance profiles, as well as their virulence and resistance gene repertoire. Two major clonal lines, which could be phenotypically differentiated by the expression of the O:5 antigen, were identified. All O:5 antigen negative strains were multidrug resistant and originated (with two exceptions) from Belgian, Dutch, or German poultry. Strains exhibiting the O:5 antigen encoded by the oafA gene revealed a more heterogeneous group including multidrug-resistant and susceptible strains. Compared to O:5 antigen negative isolates, Salmonella Paratyphi B dT+ O:5 positive strains possessed additional virulence determinants. The Salmonella genomic island 1 was only found in O:5 positive strains. Five monophasic Salmonella 4,5,12:b:- lacking the phase-2 flagellar antigen were assigned to Salmonella Paratyphi B dT+ isolates of the O:5 positive group. The conclusion of the analysis is that Salmonella Paratyphi B dT+ O:5 negative and O:5 positive isolates evolved from a different lineage. Salmonella Paratyphi B dT+ O:5 positive strains possess additional fimbrial and virulence genes that probably enable this clone to interact with a broader range of hosts and the environment. Salmonella Paratyphi B dT+ O:5 negative continuously persists in poultry across Western Europe, especially Belgium, the Netherlands, and Germany.

Poultry-associated Salmonella enterica subsp. enterica serovar 4,12:d:- reveals high clonality and a distinct pathogenicity gene repertoire.

A European baseline survey during the years 2005 and 2006 has revealed that the monophasic Salmonella enterica subsp. enterica serovar 4,12:d:- was, with a prevalence of 23.6%, the most frequently isolated serovar in German broiler flocks. In Denmark and the United Kingdom, its serovar prevalences were 15.15% and 2.8%, respectively. Although poultry is a major source of human salmonellosis, serovar 4,12:d:- is rarely isolated in humans (approximately 0.09% per year). Molecular typing studies using pulsed-field gel electrophoresis and DNA microarray analysis show that the serovar is highly clonal and lacks genes with known contributions to pathogenicity. In contrast to other poultry-associated serovars, all strains were susceptible to 17 antimicrobial agents tested and did not encode any resistance determinant. Furthermore, serovar 4,12:d:- lacked the genes involved in galactonate metabolism and in the glycolysis and glyconeogenesis important for energy production in the cells. The conclusion of the study is that serovar 4,12:d:- seems to be primarily adapted to broilers and therefore causes only rare infections in humans.

Regulation of CXCR3 and CXCR4 expression during terminal differentiation of memory B cells into plasma cells.

C-X-C motif chemokine receptor 3 (CXCR3) and CXCR4 expressed on immunoglobulin G (IgG)-plasma-cell precursors formed in memory immune responses are crucial modulators of the homing of these cells. Here, we studied the regulation of the expression of these chemokine receptors during the differentiation of human memory B cells into plasma cells. We show that CXCR3 is absent on CD27- naive B cells but is expressed on a fraction of memory B cells, preferentially on those coexpressing IgG1. On differentiation into plasma-cell precursors, CXCR3+ memory B cells maintain the expression of this chemokine receptor. CXCR3- memory B cells up-regulate CXCR3 and migrate toward concentration gradients of its ligands only when costimulated with interferon gamma (IFN-gamma), but not interleukin 4 (IL-4), IL-1beta, IL-6, IFN-alpha, IFN-beta, or tumor necrosis factor alpha (TNF-alpha). In contrast, the differentiation of CXCR4- B cells into plasma cells is generally accompanied by the induction of CXCR4 expression. These results show that lack of CXCR4 expression on plasma-cell precursors is not a limiting factor for plasma-cell homing and that the expression of CXCR3 on memory B cells and plasma-cell precursors is induced by IFN-gamma, provided in human T helper type 1 (Th1)-biased immune responses. Once induced in memory B cells, CXCR3 expression remains part of the individual cellular memory.