Diversity of STs, plasmids and ESBL genes among Escherichia coli from humans, animals and food in Germany, the Netherlands and the UK.

OBJECTIVES: This study aimed to compare ESBL-producing Escherichia coli causing infections in humans with infecting or commensal isolates from animals and isolates from food of animal origin in terms of the strain types, the ESBL gene present and the plasmids that carry the respective ESBL genes. METHODS: A collection of 353 ESBL-positive E. coli isolates from the UK, the Netherlands and Germany were studied by MLST and ESBL genes were identified. Characterization of ESBL gene-carrying plasmids was performed using PCR-based replicon typing. Moreover, IncI1-Iγ and IncN plasmids were characterized by plasmid MLST. RESULTS: The ESBL-producing E. coli represented 158 different STs with ST131, ST10 and ST88 being the most common. Overall, blaCTX-M-1 was the most frequently detected ESBL gene, followed by blaCTX-M-15, which was the most common ESBL gene in the human isolates. The most common plasmid replicon type overall was IncI1-Iγ followed by multiple IncF replicons. CONCLUSIONS: ESBL genes were present in a wide variety of E. coli STs. IncI1-Iγ plasmids that carried the blaCTX-M-1 gene were widely disseminated amongst STs in isolates from animals and humans, whereas other plasmids and STs appeared to be more restricted to isolates from specific hosts.

blaCTX-M-15-carrying Escherichia coli and Salmonella isolates from livestock and food in Germany.

OBJECTIVES: The characterization of CTX-M-15 ß-lactamase-producing Escherichia coli and Salmonella isolates originating mainly from German livestock and food. METHODS: E. coli (526, mainly commensals) and Salmonella (151) non-human isolates resistant to third-generation cephalosporins, originating from routine and monitoring submissions (2003-12) to the Federal Institute for Risk Assessment and different national targeted studies (2011-12), were examined for the presence of blaCTX-M-15 genes by PCR amplification/sequencing. Additional resistance and virulence genes were screened by DNA microarray and PCR amplification. E. coli isolates with blaCTX-M-15 were characterized by phylogenetic grouping, PFGE and multilocus sequence typing (MLST). The blaCTX-M-15 plasmids were analysed by replicon typing, plasmid MLST, S1 nuclease PFGE and Southern blot hybridization experiments. RESULTS: Twenty-one E. coli (livestock, food and a toy; 4.0%) and two Salmonella (horse and swine; 1.3%) isolates were CTX-M-15 producers. E. coli isolates were mainly ascribed to three clonal lineages of sequence types ST678 (German outbreak with enteroaggregative Shiga-toxin-producing E. coli O104:H4; salmon, cucumber and a toy), ST410 (poultry, swine and cattle farms) and ST167/617 (swine farms and turkey meat). The blaCTX-M-15 genes were located on IncI1 and multireplicon IncF plasmids or on the chromosome of E. coli ST410 isolates. CONCLUSIONS: The prevalence of CTX-M-15-producing isolates from non-human sources in Germany is still low. The blaCTX-M-15 gene is, however, present in multidrug-resistant E. coli clones with pathogenic potential in livestock and food. The maintenance of the blaCTX-M-15 gene due to chromosomal carriage is noteworthy. The possibility of an exchange of CTX-M-15-producing isolates or plasmids between livestock and humans (in both directions) deserves continuous surveillance.

Subgrouping of ESBL-producing Escherichia coli from animal and human sources: an approach to quantify the distribution of ESBL types between different reservoirs.

Escherichia (E.) coli producing extended-spectrum beta-lactamases (ESBLs) are an increasing problem for public health. The success of ESBLs may be due to spread of ESBL-producing bacterial clones, transfer of ESBL gene-carrying plasmids or exchange of ESBL encoding genes on mobile elements. This makes it difficult to identify transmission routes and sources for ESBL-producing bacteria. The objectives of this study were to compare the distribution of genotypic and phenotypic properties of E. coli isolates from different animal and human sources collected in studies in the scope of the national research project RESET. ESBL-producing E. coli from two longitudinal and four cross-sectional studies in broiler, swine and cattle farms, a cross-sectional and a case-control study in humans and diagnostic isolates from humans and animals were used. In the RESET consortium, all laboratories followed harmonized methodologies for antimicrobial susceptibility testing, confirmation of the ESBL phenotype, specific PCR assays for the detection of bla(TEM), bla(CTX), and bla(SHV) genes and sequence analysis of the complete ESBL gene as well as a multiplex PCR for the detection of the four major phylogenetic groups of E. coli. Most ESBL genes were found in both, human and non-human populations but quantitative differences for distinct ESBL-types were detectable. The enzymes CTX-M-1 (63.3% of all animal isolates, 29.3% of all human isolates), CTX-M-15 (17.7% vs. 48.0%) and CTX-M-14 (5.3% vs. 8.7%) were the most common ones. More than 70% of the animal isolates and more than 50% of the human isolates contained the broadly distributed ESBL genes bla(CTX-M-1), bla(CTX-M-15), or the combinations bla(SHV-12)+bla(TEM) or bla(CTX-M-1)+bla(TEM). While the majority of animal isolates carried bla(CTX-M-1) (37.5%) or the combination bla(CTX-M-1)+bla(TEM) (25.8%), this was the case for only 16.7% and 12.6%, respectively, of the human isolates. In contrast, 28.2% of the human isolates carried bla(CTX-M-15) compared to 10.8% of the animal isolates. When grouping data by ESBL types and phylogroups bla(CTX-M-1) genes, mostly combined with phylogroup A or B1, were detected frequently in all settings. In contrast, bla(CTX-M-15) genes common in human and animal populations were mainly combined with phylogroup A, but not with the more virulent phylogroup B2 with the exception of companion animals, where a few isolates were detectable. When E. coli subtype definition included ESBL types, phylogenetic grouping and antimicrobial susceptibility data, the proportion of isolates allocated to common clusters was markedly reduced. Nevertheless, relevant proportions of same subtypes were detected in isolates from the human and livestock and companion animal populations included in this study, suggesting exchange of bacteria or bacterial genes between these populations or a common reservoir. In addition, these results clearly showed that there is some similarity between ESBL genes, and bacterial properties in isolates from the different populations. Finally, our current approach provides good insight into common and population-specific clusters, which can be used as a basis for the selection of ESBL-producing isolates from interesting clusters for further detailed characterizations, e.g. by whole genome sequencing.

pMdT1, a small ColE1-like plasmid mobilizing a new variant of the aac(6')-Ib-cr gene in Salmonella enterica serovar Typhimurium.

OBJECTIVES: To characterize a 5.9 kb aac(6')-Ib-cr-harbouring plasmid that was detected in a clinical Salmonella Typhimurium DT104B strain. METHODS: Extraction and purification of plasmid DNA and electrotransformation assays were carried out in order to obtain kanamycin-resistant transformants. MICs of several fluoroquinolones and aminoglycosides were determined. DNA sequencing was performed by primer walking on purified plasmid preparations. The new plasmid nucleotide sequence was analysed and compared with available sequences using bioinformatic tools. RESULTS: pMdT1 is a 5.9 kb mobilizable ColE1-like plasmid that harbours aac(6')-Ib-cr4, a gene encoding a new variant of the AAC(6')-Ib-cr protein (225 amino acids). This active protein conferred resistance to tobramycin and kanamycin, and also decreased susceptibility to ciprofloxacin and norfloxacin in the transformant strain, as MICs demonstrated. The mobilization region, necessary for horizontal transfer and composed of the mobA, mobB, mobC and mobD genes, displayed a high degree of identity with those from representative ColE1-like plasmids. The basis of mobility (bom), oriT and origin of replication regions were also detected. Apart from the acetylase-encoding gene, three other open reading frames (ORFs) were determined. No similarities were found when the ORF1 sequence was compared with the sequences included in GenBank. The deduced ORF2 protein predicted a CopG-like structure characteristic of transcriptional regulators, and the deduced ORF3 protein was identical to macrophage stimulating factors. CONCLUSIONS: The pMdT1 is the smallest mobilizable ColE1-like plasmid containing an aac(6')-Ib-cr gene that has been described so far.

Population structure of Salmonella enterica serovar 4,[5],12:b:- strains and likely sources of human infection.

Salmonella enterica serovar 4,[5],12:b:- is a monophasic serovar not able to express the second-phase flagellar antigen (H2 antigen). In Germany, the serovar is occasionally isolated from poultry, reptiles, fish, food, and humans. In this study, a selection of 67 epidemiologically unrelated Salmonella enterica serovar 4,[5],12:b:- strains isolated in Germany between 2000 and 2011 from the environment, animal, food, and humans was investigated by phenotypic and genotypic methods to better understand the population structure and to identify potential sources of human infections. Strains of this monophasic serovar were highly diverse. Within the 67 strains analyzed, we identified 52 different pulsed-field gel electrophoresis XbaI profiles, 12 different multilocus sequence types (STs), and 18 different pathogenicity array types. The relatedness of strains based on the pathogenicity gene repertoire (102 markers tested) was in good agreement with grouping by MLST. S. enterica serovar 4,[5],12:b:- is distributed across multiple unrelated eBurst groups and consequently is highly polyphyletic. Two sequence types (ST88 and ST127) were linked to S. enterica serovar Paratyphi B (d-tartrate positive), two single-locus variants of ST1583 were linked to S. enterica serovar Abony, and one sequence type (ST1484) was associated with S. enterica serovar Mygdal, a recently defined, new serovar. From the characterization of clinical isolates and those of nonhuman origin, it can be concluded that the potential sources of sporadic human infections with S. enterica serovar 4,[5],12:b:- most likely are mushrooms, shellfish/fish, and poultry.

Human infections attributable to the D-tartrate-fermenting variant of Salmonella enterica serovar Paratyphi B in Germany originate in reptiles and, on rare occasions, poultry.

In this study, the population structure, incidence, and potential sources of human infection caused by the d-tartrate-fermenting variant of Salmonella enterica serovar Paratyphi B [S. Paratyphi B (dT+)] was investigated. In Germany, the serovar is frequently isolated from broilers. Therefore, a selection of 108 epidemiologically unrelated S. enterica serovar Paratyphi B (dT+) strains isolated in Germany between 2002 and 2010 especially from humans, poultry/poultry meat, and reptiles was investigated by phenotypic and genotypic methods. Strains isolated from poultry and products thereof were strongly associated with multilocus sequence type ST28 and showed antimicrobial multiresistance profiles. Pulsed-field gel electrophoresis XbaI profiles were highly homogeneous, with only a few minor XbaI profile variants. All strains isolated from reptiles, except one, were strongly associated with ST88, another distantly related type. Most of the strains were susceptible to antimicrobial agents, and XbaI profiles were heterogeneous. Strains isolated from humans yielded seven sequence types (STs) clustering in three distantly related lineages. The first lineage, comprising five STs, represented mainly strains belonging to ST43 and ST149. The other two lineages were represented only by one ST each, ST28 and ST88. The relatedness of strains based on the pathogenicity gene repertoire (102 markers tested) was mostly in agreement with the multilocus sequence type. Because ST28 was frequently isolated from poultry but rarely in humans over the 9-year period investigated, overall, this study indicates that in Germany S. enterica serovar Paratyphi B (dT+) poses a health risk preferentially by contact with reptiles and, to a less extent, by exposure to poultry or poultry meat.

Clonal dissemination of Salmonella enterica serovar Infantis in Germany.

Salmonella enterica serovar Infantis (Salmonella Infantis) is consistently isolated from broiler chickens, pigs, and humans worldwide. This study investigated 93 epidemiologically unrelated Salmonella Infantis strains isolated in Germany between 2005 and 2008 in respect to their transmission along the food chain. Various phenotypic and genotypic methods were applied, and the pathogenicity and resistance gene repertoire was determined. Phenotypically, 66% of the strains were susceptible to all 17 antimicrobials tested, while the others were almost all multidrug-resistant (two or more antimicrobial resistances), with different resistance profiles and preferentially isolated from broiler chickens. A number of phage types (PTs) were shared by strains from pigs, broiler chickens, and humans (predominated by PT 29). One, PT 1, was only detected in strains from pigs/pork and humans. Pulsed-field gel electrophoresis (PFGE) subdivided strains in seven different clusters, named A-G, consisting of 35 various XbaI profiles with coefficient of similarity values of 0.73-0.97. The majority of XbaI profiles were assigned to clusters A and C, and two predominant XbaI profiles were common in strains isolated from all sources investigated. Multi-locus sequence typing (MLST) analysis of selected strains representing the seven PFGE clusters revealed that they all belonged to ST32. The pathogenicity gene repertoire of 37 representative Salmonella Infantis strains analyzed by microarray was also identical. The resistance gene repertoire correlated perfectly with the phenotypic antimicrobial resistance profiles, and multidrug-resistant strains were associated with class 1 integrons. Overall, this study showed that two major closely related genotypes of Salmonella Infantis can transmit in Germany to humans through contaminated broiler meat or pork, and consequently presents a hazard for human health.

Antimicrobial resistance and virulence determinants in European Salmonella genomic island 1-positive Salmonella enterica isolates from different origins.

Salmonella genomic island 1 (SGI1) contains a multidrug resistance region conferring the ampicillin-chloramphenicol-streptomycin-sulfamethoxazole-tetracycline resistance phenotype encoded by bla(PSE-1), floR, aadA2, sul1, and tet(G). Its increasing spread via interbacterial transfer and the emergence of new variants are important public health concerns. We investigated the molecular properties of SGI1-carrying Salmonella enterica serovars selected from a European strain collection. A total of 38 strains belonging to S. enterica serovar Agona, S. enterica serovar Albany, S. enterica serovar Derby, S. enterica serovar Kentucky, S. enterica serovar Newport, S. enterica serovar Paratyphi B dT+, and S. enterica serovar Typhimurium, isolated between 2002 and 2006 in eight European countries from humans, animals, and food, were subjected to antimicrobial susceptibility testing, molecular typing methods (XbaI pulsed-field gel electrophoresis [PFGE], plasmid analysis, and multilocus variable-number tandem-repeat analysis [MLVA]), as well as detection of resistance and virulence determinants (PCR/sequencing and DNA microarray analysis). Typing experiments revealed wide heterogeneity inside the strain collection and even within serovars. PFGE analysis distinguished a total of 26 different patterns. In contrast, the characterization of the phenotypic and genotypic antimicrobial resistance revealed serovar-specific features. Apart from the classical SGI1 organization found in 61% of the strains, seven different variants were identified with antimicrobial resistance properties associated with SGI1-A (S. Derby), SGI1-C (S. Derby), SGI1-F (S. Albany), SGI1-L (S. Newport), SGI1-K (S. Kentucky), SGI1-M (S. Typhimurium), and, eventually, a novel variant similar to SGI1-C with additional gentamicin resistance encoded by aadB. Only minor serovar-specific differences among virulence patterns were detected. In conclusion, the SGI1 carriers exhibited pathogenetic backgrounds comparable to the ones published for susceptible isolates. However, because of their multidrug resistance, they may be more relevant in clinical settings.

Evolution and population structure of Salmonella enterica serovar Newport.

Salmonellosis caused by Salmonella enterica serovar Newport is a major global public health concern, particularly because S. Newport isolates that are resistant to multiple drugs (MDR), including third-generation cephalosporins (MDR-AmpC phenotype), have been commonly isolated from food animals. We analyzed 384 S. Newport isolates from various sources by a multilocus sequence typing (MLST) scheme to study the evolution and population structure of the serovar. These were compared to the population structure of S. enterica serovars Enteritidis, Kentucky, Paratyphi B, and Typhimurium. Our S. Newport collection fell into three lineages, Newport-I, Newport-II, and Newport-III, each of which contained multiple sequence types (STs). Newport-I has only a few STs, unlike Newport-II or Newport-III, and has possibly emerged recently. Newport-I is more prevalent among humans in Europe than in North America, whereas Newport-II is preferentially associated with animals. Two STs of Newport-II encompassed all MDR-AmpC isolates, suggesting recent global spread after the acquisition of the bla(CMY-2) gene. In contrast, most Newport-III isolates were from humans in North America and were pansusceptible to antibiotics. Newport was intermediate in population structure to the other serovars, which varied from a single monophyletic lineage in S. Enteritidis or S. Typhimurium to four discrete lineages within S. Paratyphi B. Both mutation and homologous recombination are responsible for diversification within each of these lineages, but the relative frequencies differed with the lineage. We conclude that serovars of S. enterica provide a variety of different population structures.

A predominant multidrug-resistant Salmonella enterica serovar Saintpaul clonal line in German turkey and related food products.

Recently, Salmonella enterica subsp. enterica serovar Saintpaul has increasingly been observed in several countries, including Germany. However, the pathogenic potential and epidemiology of this serovar are not very well known. This study describes biological attributes of S. Saintpaul isolates obtained from turkeys in Germany based on characterization of their pheno- and genotypic properties. Fifty-five S. Saintpaul isolates from German turkeys and turkey-derived food products isolated from 2000 to 2007 were analyzed by using antimicrobial agent, organic solvent, and disinfectant susceptibility tests, isoelectric focusing, detection of resistance determinants, plasmid profiling, pulsed-field gel electrophoresis (PFGE), and hybridization experiments. These isolates were compared to an outgroup consisting of 24 S. Saintpaul isolates obtained from humans and chickens in Germany and from poultry and poultry products (including turkeys) in Netherlands. A common core resistance pattern was detected for 27 German turkey and turkey product isolates. This pattern included resistance (full or intermediate) to ampicillin, amoxicillin-clavulanic acid, gentamicin, kanamycin, nalidixic acid, streptomycin, spectinomycin, and sulfamethoxazole and intermediate resistance or decreased susceptibility to ciprofloxacin (MIC, 2 or 1 mug/ml, respectively) and several third-generation cephalosporins (including ceftiofur and cefoxitin [MIC, 4 to 2 and 16 to 2 mug/ml, respectively]). These isolates had the same core resistance genotype, with bla(TEM-1), aadB, aadA2, sul1, a Ser83-->Glu83 mutation in the gyrA gene, and a chromosomal class 1 integron carrying the aadB-aadA2 gene cassette. Their XbaI, BlnI, and combined XbaI-BlnI PFGE patterns revealed levels of genetic similarity of 93, 75, and 90%, respectively. This study revealed that a multiresistant S. Saintpaul clonal line is widespread in turkeys and turkey products in Germany and was also detected among German human fecal and Dutch poultry isolates.

Pork contaminated with Salmonella enterica serovar 4,[5],12:i:-, an emerging health risk for humans.

Salmonella enterica subsp. enterica serovar 4,[5],12:i:- is a monophasic variant of S. enterica serovar Typhimurium (antigenic formula 4,[5],12:i:1,2). Worldwide, especially in several European countries and the United States, it has been reported among the 10 most frequently isolated serovars in pigs and humans. In the study reported here, 148 strains of the monophasic serovar isolated from pigs, pork, and humans in 2006 and 2007 in Germany were characterized by various phenotypic and genotypic methods. This characterization was done in order to investigate their clonality, the prevalence of identical subtypes in pigs, pork, and humans, and the genetic relatedness to other S. enterica serovar Typhimurium subtypes in respect to the pathogenic and resistance gene repertoire. Two major clonal lineages of the monophasic serovar were detected which can be differentiated by their phage types and pulsed-field gel electrophoresis (PFGE) profiles. Seventy percent of the strains tested belonged to definite phage type DT193, and those strains were mainly assigned to PFGE cluster B. Nineteen percent of the strains were typed to phage type DT120 and of these 86% belonged to PFGE cluster A. Sixty-five percent of the isolates of both lineages carried core multiresistance to ampicillin, streptomycin, tetracycline, and sulfamethoxazole encoded by the genes bla(TEM1-like), strA-strB, tet(B), and sul2. No correlation to the source of isolation was observed in either lineage. Microarray analysis of 61 S. enterica serovar 4,[5],12:i:- and 20 S. enterica serovar Typhimurium isolates tested determining the presence or absence of 102 representative pathogenicity genes in Salmonella revealed no differences except minor variations in single strains within and between the serovars, e.g., by presence of the virulence plasmid in four strains. Overall the study indicates that in Germany S. enterica serovar 4,[5],12:i:- strains isolated from pig, pork, and human are highly related, showing their transmission along the food chain. Since the pathogenicity gene repertoire is highly similar to that of S. enterica serovar Typhimurium, it is essential that interventions are introduced at the farm level in order to limit human infection.

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.

Extended-spectrum {beta}-lactamases and AmpC {beta}-lactamases in ceftiofur-resistant Salmonella enterica isolates from food and livestock obtained in Germany during 2003-07.

OBJECTIVES: Detection and characterization of extended-spectrum beta-lactamases (ESBLs) and AmpC-encoding genes was conducted in German Salmonella isolated from different sources from 2003 to 2007. METHODS: Non-duplicate German isolates from the National Salmonella Reference Laboratory Collection (2003-07) with ceftiofur MICs of > or =4 mg/L were tested for beta-lactam/beta-lactamase inhibitor susceptibility, presence of ESBLs or AmpC-encoding genes, class 1 and 2 integrons, other resistance genes, and IS26 and ISEcp1 sequences by PCR/sequencing. The isoelectric point of the beta-lactamase was determined. Strains were analysed by PFGE and plasmid profiling. The bla genes were mapped by Southern-blot hybridization. Plasmids were characterized by rep-PCR typing. RESULTS: Sixteen isolates (10 Salmonella Typhimurium, 2 Salmonella Anatum, 2 Salmonella Paratyphi B dT + , 1 Salmonella Infantis and 1 Salmonella London) carried bla(CTX-M) (15 bla(CTX-M-1) and one bla(CTX-M-15)) genes located on self-transferable IncB/O, IncI1 and/or IncN plasmids. Seven of the Salmonella Typhimurium isolates carried the SGI1-M variant. Six isolates (five Salmonella Agona and one Salmonella Kentucky) carried the bla(CMY-2) gene on IncI1 conjugative plasmids. bla(TEM-20) genes were detected in two Salmonella Paratyphi B dT+ isolates, and bla(TEM-52) in one Salmonella Paratyphi B dT+ and one Salmonella Virchow, located on IncI1 plasmids. All Salmonella Paratyphi isolates harboured a 2300 bp/dfrA1-sat2-aadA1 class 2 integron. CONCLUSIONS: Among the 22 679 German Salmonella isolates investigated, the ESBL and AmpC beta-lactamase prevalence was still low; however, it is slowly increasing. Various beta-lactamase genes are linked to a variety of genetic elements capable of horizontal DNA transfer. Consequently, their dissemination is likely and demands adequate risk management strategies.

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.

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.

Rapid classification and identification of salmonellae at the species and subspecies levels by whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry .

Variations in the mass spectral profiles of multiple housekeeping proteins of 126 strains representing Salmonella enterica subsp. enterica (subspecies I), S. enterica subsp. salamae (subspecies II), S. enterica subsp. arizonae (subspecies IIIa), S. enterica subsp. diarizonae (subspecies IIIb), S. enterica subsp. houtenae (subspecies IV), and S. enterica subsp. indica (subspecies VI), and Salmonella bongori were analyzed to obtain a phylogenetic classification of salmonellae based on whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometric bacterial typing. Sinapinic acid produced highly informative spectra containing a large number of biomarkers and covering a wide molecular mass range (2,000 to 40,000 Da). Genus-, species-, and subspecies-identifying biomarker ions were assigned on the basis of available genome sequence data for Salmonella, and more than 200 biomarker peaks, which corresponded mainly to abundant and highly basic ribosomal or nucleic acid binding proteins, were selected. A detailed comparative analysis of the biomarker profiles of Salmonella strains revealed sequence variations corresponding to single or multiple amino acid changes in multiple housekeeping proteins. The resulting mass spectrometry-based bacterial classification was very comparable to the results of DNA sequence-based methods. A rapid protocol that allowed identification of Salmonella subspecies in minutes was established.

Multi-locus variable-number tandem repeat analysis for outbreak studies of Salmonella enterica serotype Enteritidis.

BACKGROUND: Salmonella enterica subsp. enterica serotype Enteritidis is known as an important and pathogenic clonal group which continues to cause worldwide sporadic cases and outbreaks in humans. Here a new multiple-locus variable-number tandem repeat analysis (MLVA) method is reported for highly-discriminative subtyping of Salmonella Enteritidis. Emphasis was given on the most predominant phage types PT4 and PT8. The method comprises multiplex PCR specifically amplifying repeated sequences from nine different loci followed by an automatic fragment size analysis using a multicolor capillary electrophoresis instrument. A total of 240 human, animal, food and environmental isolates of S. Enteritidis including 23 definite phage types were used for development and validation. Furthermore, the MLVA types were compared to the phage types of several isolates from two recent outbreaks to determine the concordance between both methods and to estimate their in vivo stability. The in vitro stability of the two MLVA types specifically for PT4 and PT8 strains were determined by multiple freeze-thaw cycles. RESULTS: Seventy-nine different MLVA types were identified in 240 S. Enteritidis strains. The Simpson's diversity index for the MLVA method was 0.919 and Nei diversity values for the nine VNTR loci ranged from 0.07 to 0.65. Twenty-four MLVA types could be assigned to 62 PT4 strains and 21 types to 81 PT8 strains. All outbreak isolates had an indistinguishable outbreak specific MLVA type. The in vitro stability experiments showed no changes of the MLVA type compared to the original isolate. CONCLUSION: This MLVA method is useful to discriminate S. Enteritidis strains even within a single phage type. It is easy in use, fast, and cheap compared to other high-resolution molecular methods and therefore an important tool for surveillance and outbreak studies for S. Enteritidis.