Genetic basis for loss of immuno-reactive O-chain in Salmonella enterica serovar Enteritidis veterinary isolates.

Fifty-two rough Salmonella enterica serovar Enteritidis (S. Enteritidis) isolates from broilers and the environment were characterized for their serological and genotypic properties. Under routine diagnostic serotyping methods such isolates lack the immuno-reactivity of the O-chain of the lipopolysaccharide (LPS), and are referred to as non-typeable. Using a modified slide agglutination method, the isolates could be differentiated into three different serological variants. Twenty-six isolates (50%) were defined as semi-rough, nineteen isolates (37%) as deep-rough, four isolates (8%) as rough and three isolates could not be assigned. Genetically, all semi-rough isolates lacked the wzyB gene encoding the O-antigen polymerase. Two isolates carried a frameshift mutation in wzyB. In 15 of 23 cases deep-rough or rough isolates had a single point mutation, a single - or double-nucleotide insert or deletion in the wbaP gene. The mutational changes lead to expression of truncated (premature) protein, resulting in the loss of the immuno-reactive O-chain. Both rough and smooth S. Enteritidis isolates showed identical or highly similar XbaI-PFGE profiles. Our results indicate that the loss of a functional LPS in S. Enteritidis isolates is caused by a variety of different mutation events within the wzyB (semi-rough) or the wbaP (deep-rough) gene and is not a result of a vertical spread of a specific S. Enteritidis subtype. The defect of the LPS may be a common evolutionary mechanism through which host defence can be escaped.

Molecular epidemiology of Salmonella enterica serovar Kottbus isolated in Germany from humans, food and animals.

Salmonella enterica serovar Kottbus has been continuously isolated from poultry and poultry meat, especially from turkey. We investigated by comparative molecular typing 95 S. Kottbus isolates obtained in Germany between 2000 and 2011 from poultry/poultry meat, pig/pork, cattle, reptiles, the environment as well as from human cases to identify potential infection sources for humans, especially the role of poultry and poultry products as vehicle in transmission of S. Kottbus isolates to humans. Multilocus sequence typing analysis detected three main genetic lineages. Most human isolates belonged to lineage 1 represented by sequence types ST212 and ST808. Part of the isolates isolated from cattle and pork were also linked to this lineage. Nevertheless, human isolates and especially isolates from poultry/poultry meat, and with less extend from other livestock, grouped in lineage 2 represented by ST582. Four additional isolates from reptiles and humans belonging to ST1669 represented the third lineage. The three lineages were also reflected by pulsed-field gel electrophoresis typing data and DNA microarray analysis of 102 pathogenicity genes. Antimicrobial resistance especially to nalidixic acid and ciprofloxacin was predominantly observed in isolates assigned to lineage 2, which contains predominantly resistant isolates compared to lineage 1 and 3. Sequencing of the quinolone resistance-determining region of gyrA revealed a point mutation in codon 83 or 87 responsible for nalidixic acid resistance and MIC values for ciprofloxacin between 0.125 and 0.25mg/l. Overall, this study showed that in Germany a specific S. Kottbus lineage (ST582), which is well-established in poultry, can be transmitted to humans by poultry meat and, consequently, poses a risk for human health.

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.

A quantitative approach towards a better understanding of the dynamics of Salmonella spp. in a pork slaughter-line.

Pork contributes significantly to the public health disease burden caused by Salmonella infections. During the slaughter process pig carcasses can become contaminated with Salmonella. Contamination at the slaughter-line is initiated by pigs carrying Salmonella on their skin or in their faeces. Another contamination route could be resident flora present on the slaughter equipment. To unravel the contribution of these two potential sources of Salmonella a quantitative study was conducted. Process equipment (belly openers and carcass splitters), faeces and carcasses (skin and cutting surfaces) along the slaughter-line were sampled at 11 sampling days spanning a period of 4 months. Most samples taken directly after killing were positive for Salmonella. On 96.6% of the skin samples Salmonella was identified, whereas a lower number of animals tested positive in their rectum (62.5%). The prevalence of Salmonella clearly declined on the carcasses at the re-work station, either on the cut section or on the skin of the carcass or both (35.9%). Throughout the sampling period of the slaughter-line the total number of Salmonella per animal was almost 2 log lower at the re-work station in comparison to directly after slaughter. Seven different serovars were identified during the study with S. Derby (41%) and S. Typhimurium (29%) as the most prominent types. A recurring S. Rissen contamination of one of the carcass splitters indicated the presence of an endemic 'house flora' in the slaughterhouse studied. On many instances several serotypes per individual sample were found. The enumeration of Salmonella and the genotyping data gave unique insight in the dynamics of transmission of this pathogen in a slaughter-line. The data of the presented study support the hypothesis that resident flora on slaughter equipment was a relevant source for contamination of pork.

Diversity of Salmonella enterica serovar Derby isolated from pig, pork and humans in Germany.

Salmonella enterica serovar Derby (S. Derby) is one of the most prevalent serovars in pigs in Europe and in the U.S. and ranks among the 10 most frequently isolated serovars in humans. Therefore, a set of 82 epidemiologically unrelated S. Derby strains isolated between 2006 and 2008 from pigs, pork and humans in Germany was selected and investigated in respect to the transmission of clonal groups of the serovar along the food chain. Various phenotypic and genotypic methods were applied and the pathogenicity and resistance gene repertoire was determined. Phenotypically 72% of the strains were susceptible to all 17 antimicrobials tested while the others were monoresistant to tetracycline or multi-resistant with different resistance profiles. Four major clonal groups were identified based on PFGE, sequence data of the virulence genes sopA, sopB and sopD, VNTR-locus STTR5 and MLST revealing also the new sequence type ST774. Thirty different PFGE profiles were detected resulting in four clusters representing the four groups. The pathogenicity gene repertoire of 32 representative S. Derby strains analyzed by microarray showed six types with differences in the Salmonella pathogenicity islands, pathogenicity genes on smaller islets or prophages and fimbriae coding genes. The pathogenicity gene repertoire of the predominant types PAT DE1 and DE2 were most similar to the ones of S. Paratyphi B (dT+, O5-) and to a minor degree to S. Infantis and S. Virchow PATs. Overall this study showed that in Germany currently one major S. Derby clone is frequently isolated from pigs and humans. Contaminated pork was identified as one vehicle and consequently is a risk for human health. To prevent this serovar from entering the food chain, control measurements should be applied at the farm level.

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.

[Characterisation of a phenotypic monophasic variant belonging to Salmonella enterica subsp. enterica serovar Typhimurium from wild birds and its possible transmission to cats and humans]. / Charakterisierung einer phänotypisch monophasischen Variante von Salmonella enterica subsp. enterica Serovar Typhimurium bei Wildvögeln und deren mögliche Ubertragung auf Hauskatzen und Menschen.

In the last two years The National Salmonella Reference Laboratory (NRL-Salm) received an accumulating number of salmonellae with sero-formula 4,12:-:1,2 isolated from perished wild birds, particularly siskins. Within these strains flagellar antigen of the first phase was phenotypically not detectable. By PCR a fragment could be amplified coding specifically for the H:i-flagellar antigen. Consequently, this is a phenotypically monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium var. copenhagen with the sero-formula 4,12:-:1,2. Phage-typing showed most of the isolates belonged to phage type DT40. Some other strains harboured the same lysis pattern but could not assign to a definite phage type. Those strains are designated as RDNC (react with phages but does not conform with definite or provisorial types). Pulsed field gel-electrophoresis (PFGE) confirmed those two lineages of the monophasic variant. Phage type DT40 isolates from humans or cats, able to express both flagellar antigens, did not differ in genotypic properties from those not able to express the flagellar antigen of the first phase. Salmonella strains with identical genotypic characteristics have been isolated from wild birds, human cases particular infants and also cats. This refers to a direct or indirect transmission of the pathogen from wild bird to human. By eating or getting in contact with contaminated birds domestic cats could play an important role as vehicle between bird and human.

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.

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.

Phenotypic and genotypic characterization of antimicrobial resistance in Escherichia coli O111 isolates.

OBJECTIVES: The aim of this study was to generate baseline data on the prevalence and molecular basis of antimicrobial resistance in Escherichia coli O111 isolates. METHODS: A total of 105 epidemiologically unrelated E. coli O111 isolates from humans and cattle (isolated between 1983 and 2003) were tested for susceptibility to 17 antimicrobial agents by broth microdilution. Resistant isolates were screened by molecular methods for resistance genes, class 1 and 2 integrons and mutations in the quinolone-resistance determining regions. RESULTS: Resistance was found in 76% of the isolates, with a prevalence of 72% for multiresistance. The most prevalent resistances were to streptomycin, sulfamethoxazole and tetracycline (72-68%), followed by spectinomycin, ampicillin and kanamycin/neomycin (39-25%). For each antimicrobial agent, the predominant resistance genes were ampicillin, bla(TEM) (94%); chloramphenicol, catA1 (100%); gentamicin, aac(3)-IV and aac(3)-II (50% each); kanamycin, aphA1 (100%); streptomycin, aadA1- like (66%); sulfamethoxazole, sul1 (59%); tetracycline, tet(A) (86%); and trimethoprim, dfrA1-like (83%). Class 1 integrons were found in 41% of the isolates. They carried aadA1, dfrA1-aadA1 and dfrA15-aadA1. A class 2 integron (dfrA1-sat1-aadA1) was found in one isolate. Only three isolates (3%) were resistant to nalidixic acid (reduced susceptibility to ciprofloxacin), with a single mutation in the gyrA gene. CONCLUSIONS: E. coli O111 strains exhibit a wide repertoire of genetic elements to sustain antimicrobial pressure. Two specific antimicrobial resistance pheno/genotypes, [STR-SPT]-SUL-TET/aadA1-sul1-tet(A) and STR-SUL-TET-AMP-[KAN-NEO]/strA/B-sul2-tet(A)-bla(TEM)-aphA1, are predominant.

Incidence of the recently described sulfonamide resistance gene sul3 among German Salmonella enterica strains isolated from livestock and food.

The sul3 gene recently described in Escherichia coli was found in 22 of 512 (4.3%) German Salmonella isolates from different regions and sources and of different serotypes, antimicrobial resistance phenotypes, and genomic groups. This is the first report on the prevalence of sul3 among Salmonella strains, and the findings support the strong potential of this determinant to spread within bacterial populations.

Phenotypic and genotypic characterization of antimicrobial resistance in German Escherichia coli isolates from cattle, swine and poultry.

OBJECTIVE: Phenotypic and genotypic characterization of the antimicrobial resistance of German Escherichia coli strains isolated during 1999-2001 from cattle, swine and poultry. MATERIALS AND METHODS: Three hundred and seventeen isolates were tested for their resistance to 17 antimicrobial agents by broth microdilution. Resistant strains were screened by molecular methods for resistance genes, integrons and mutations in quinolone-resistance determining regions. RESULTS: Resistance was found in 40% and multiresistance in 32% of the strains. The resistance was significantly higher in isolates from poultry (61%) and swine (60%) than from cattle (25%) (P < 0.01). The most prevalent resistances were to sulfamethoxazole, tetracycline, streptomycin, ampicillin and spectinomycin (30-15%). For each antibiotic, the predominant resistance genes were: ampicillin, blaTEM1-like (92%); chloramphenicol, catA (68%) and cmlA1-like (36%); gentamicin, aac(3)-IV (60%); kanamycin, aphA1 (100%); streptomycin, aadA1-like (61%) and strA/B (59%); sulfamethoxazole, sul2 (66%), sul1 (42%) and sul3 (14%); tetracycline, tet(A) (66%) and tet(B) (42%); and trimethoprim, dfrA1-like (77%), dfrA17 (13%) and dfrA12 (7%). Class 1 integrons were found in 30% of the strains. They carried dfrA1-aadA1a (40%), aadA1a (29%), sat1-aadA1a (16%), dfrA17-aadA5 (11%), oxa1-aadA1a (5%) and dfrA12-aadA2 (3%). Eleven percent of the strains were resistant to nalidixic acid. Of these, 61% presented a reduced susceptibility to ciprofloxacin (MIC = 0.12-2 mg/L) and single mutations in gyrA or gyrA and parC genes, and 39%, full resistance to ciprofloxacin (MIC > or = 4 mg/L) and double and single mutations in gyrA and parC, respectively. CONCLUSION: The study gives baseline information on the magnitude of the resistance problem and its genetic background in contemporary German E. coli from food-producing animals.