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Due to health concerns associated with the increase of antimicrobial resistance in foodborne pathogens such as Salmonella, plant extracts have become important natural alternatives to known antimicrobials. The eradication effect of the water-soluble stinging nettle extracts with 2 mg/mL, 4 mg/mL, 6 mg/mL, 8 mg/mL, 12 mg/mL, 16 mg/mL, and 20 mg/mL concentrations, on pre-formed biofilms and swimming motility of Salmonella enterica subspecies enterica serovars, including Newport, Typhimurium, Enteritidis, Virchow, Othmarschen and Mikawasima, was investigated in vitro. Degradation of biofilm formation on spinach inoculated with each serovar was ascertained within different exposure time of 40 mg/mL concentrated extract as well. Moreover, major genes responsible for biofilm formation (i.e., rpoS, mlrA, ycfR, fimA, spiA and csgA) were screened in these isolates. The extract significantly decreased swimming motilities of Mikawasima and Virchow serovars. The highest reductions were found as 0.88 Log CFU/mL and 2.00 Log CFU/cm2 in vitro and on spinach, respectively.

WOS: 000390071600012 PubMed ID: 27768932 192 Food samples (commonly consumed 8 food types), 355 animal samples (animal feces of bovine, ovine, goat and chicken) and 50 samples from clinical human cases in Sanliurfa city, Turkey in a year were collected to determine the Salmonella enterica subsp. enterica mosaic in Turkey. 161 Salmonella isolates represented 17 serotypes, 20 sequence types (STs) and 44 PFGE patterns (PTs). 3 serotypes, S. Enteritidis, S. Typhimurium and S. Kentucky, were recovered from three different hosts. The highest discriminatory power was obtained by PFGE (SID = 0.945), followed by MLST (SID = 0.902) and serotyping (SID = 0.885) for all isolates. The prevalence of antimicrobial resistance genes (aadA1, aadA2, strA, strB, aphA(1-lab) bla(TEM-1), bla(PSE-1), tetA) was highly correlated with phenotypic profiles of aminoglycoside, beta-lactam and tetracycline groups (kappa >0.85). From our knowledge, this is the first study reporting spatial and temporal distribution of Salmonella species through phenotypic and genetic approaches over farm to fork chain in Turkey. Thus, our data provided further information for evolution, ecology and transmission of Salmonella in Turkey. (C) 2016 Published by Elsevier B.V. Scientific and Technical Council of TurkeyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [TUBITAK 3501(1120192)] We acknowledge Dr. Martin Wiedmann from Cornell University for using BioNumerics, Dr. Nihat Dilsiz from Harran University Medicine Faculty, Harran University Veterinary Faculty for the valuable discussion, and Dr. Tolga Can from Middle East Technical University, Computer Engineering for building a web-based database. This work is partially supported by The Scientific and Technical Council of Turkey Grant TUBITAK 3501(1120192).

Salmonella represents an important zoonotic pathogen worldwide, but the transmission dynamics between humans and animals as well as within animal populations are incompletely understood. We characterized Salmonella isolates from cattle and humans in two geographic regions of the United States, the Pacific Northwest and the Northeast, using three common subtyping methods (pulsed-field gel electrophoresis [PFGE], multilocus variable number of tandem repeat analysis [MLVA], and multilocus sequence typing [MLST]). In addition, we analyzed the distribution of antimicrobial resistance among human and cattle Salmonella isolates from the two study areas and characterized Salmonella persistence on individual dairy farms. For both Salmonella enterica subsp. enterica serotypes Newport and Typhimurium, we found multidrug resistance to be significantly associated with bovine origin of isolates, with the odds of multidrug resistance for Newport isolates from cattle approximately 18 times higher than for Newport isolates from humans. Isolates from the Northwest were significantly more likely to be multidrug resistant than those from the Northeast, and susceptible and resistant isolates appeared to represent distinct Salmonella subtypes. We detected evidence for strain diversification during Salmonella persistence on farms, which included changes in antimicrobial resistance as well as genetic changes manifested in PFGE and MLVA pattern shifts. While discriminatory power was serotype dependent, the combination of PFGE data with either MLVA or resistance typing data consistently allowed for improved subtype discrimination. Our results are consistent with the idea that cattle are an important reservoir of multidrug-resistant Salmonella infections in humans. In addition, the study provides evidence for the value of including antimicrobial resistance data in epidemiological investigations and highlights the benefits and potential problems of combining subtyping methods.

Pulsed-field gel electrophoresis (PFGE) characterization of 335 temporally and spatially matched clinical, bovine, and human Salmonella enterica subsp. enterica isolates revealed 167 XbaI PFGE patterns. These isolates were previously classified into 51 serotypes and 73 sequence types, as determined by multilocus sequence typing. Discriminatory power of PFGE (Simpson's index, D = 0.991) was considerably higher than that of multilocus sequence typing (D = 0.920) or serotyping (D = 0.913). Although 128 PFGE types each only represented a single isolate, 8 PFGE types represented >4 isolates, including (i) three serotype Enteritidis and Heidelberg patterns that were only identified among human isolates, (ii) two PFGE patterns (each representing serotypes Bardo and Newport) that were significantly more common among bovine isolates as compared with human isolates; (iii) two PFGE types that each includes two serotypes (4,5,12:i:- and Typhimurium; Thompson and 1,7:-:1,5); and (iv) one PFGE type that includes eight Typhimurium isolates from humans and cattle. Characterization of isolates collected over multiple farm visits indicated that given specific PFGE types persisted over time on 11 farms. On an additional seven farms, isolates with a given sequence type represented multiple PFGE type, which typically only differed by

Salmonella spp. are one of the most common causes of bacterial food-borne diseases worldwide (34). In the United States nontyphoidal Salmonella serotypes cause an estimated 1.4 million human salmonellosis cases, including approximately 550 deaths annually (27). Serotyping with the Kaufmann-White scheme is used commonly as a first step to differentiate Salmonella isolates. Serotyping of Salmonella isolates is based on lipopolysaccharide moieties on the cell surface (O antigens) and the flagellar proteins (H antigens), as well as capsular protein antigens (Vi antigen), which are only found in a few Salmonella serotypes (e.g., Salmonella enterica serotype Typhi). According to the Kaufmann-White scheme, Salmonella includes over 2,500 recognized serotypes (20). Many Salmonella bacteria are motile due to peritrichous flagella (28), which include a basal body, a propeller, and a hook. The motility of Salmonella depends on the rotation of the flagellar propeller (i.e., the filament), which includes either FliC (phase 1 antigen) or FljB (phase 2 antigen) flagellin (11). Most Salmonella serotypes, including Salmonella enterica serotype Typhimurium, are biphasic, meaning that they can express two distinct flagellar antigens (i.e., phase 1 and phase 2 antigens). Regulation of phase 1 and 2 antigen expression is under the control of the recombinase Hin. This recombinase facilitates inversion of a promoter element so that it either (i) transcribes fljB (which encodes the phase 2 antigen FljB) and fljA (which encodes a repressor of fliC, the gene encoding the phase 1 antigen FliC) (4, 37) or (ii) does not transcribe either of these genes. If this promoter is located in an orientation that does not allow for transcription of fljB and fljA, the lack of a repression of fliC transcription leads to expression of phase 1 flagellar antigens. Salmonella enterica serotype 4,5,12:i:− is a serotype that appears to be antigenically similar and genetically closely related to Salmonella serotype Typhimurium (which has the antigenic formula 4,5,12:i:1,2) but lacks expression of the second-phase flagellar antigen, which is 1,2 in Salmonella serotype Typhimurium (28). Salmonella serotype 4,5,12:i:− was the sixth most common Salmonella serotype among cases of human disease in the United States in 2006 (10) and the fourth most common serotype among human isolates in Spain in 1998 (18). Overall, the prevalence of Salmonella serotype 4,5,12:i:− among human cases has increased considerably in many countries in the world over the last 10 years (9, 10, 18, 29, 36). This Salmonella serotype has also been responsible for a number of human salmonellosis outbreaks over the last decades, including in Spain (1998), the United States (2004 and 2007), and Luxemburg (2006). Salmonella serotype 4,5,12:i:− has been isolated, particularly over the last decade, from a number of different foods and animals (1, 6, 13, 29, 38). While a number of separate studies, using molecular subtyping and characterization tools (e.g., genomic microarrays and PCR assays to test for gene presence/absence), have shown that Salmonella serotype 4,5,12:i:− isolates from Spain (15, 18) and the United States (1, 2, 38) are genetically closely related to Salmonella serotype Typhimurium, we are not aware of any comparative studies of Salmonella serotype 4,5,12:i:− isolates from Europe and the United States that have been published to date. In order to provide a better understanding of the transmission, ecology, and evolution of Salmonella serotype 4,5,12:i:−, we have assembled a collection of 190 Salmonella serotype 4,5,12:i:− and Typhimurium isolates from various sources and from two countries, the United States and Spain. These isolates were characterized by different molecular subtyping methods (i.e., multilocus sequence typing [MLST] and pulsed-field gel electrophoresis [PFGE]), followed by characterization of selected isolates for genomic deletions that may be responsible for the lack of phase 2 flagellum expression.

Objective—To determine the duration of fecal shedding of and serologic response to Salmonella spp after natural infection in dairy calves and characterize Salmonella organisms recovered from these herds. Design—Longitudinal study. Animals—Calves from 2 dairy herds (A and B) in the northeast United States that were identified at the beginning of a Salmonella outbreak. Procedures—Fecal samples were collected twice per week (herd A) or once per week (herd B); blood samples were collected for serologic testing once per week in both herds. Bacteriologic culture of fecal samples was performed, and Salmonella isolates were characterized by serotype, pulsed-field gel electrophoresis (PFGE) pattern, and antimicrobial resistance profile. Results—All Salmonella isolates from herd A were serovar Typhimurium var Copenhagen, had the same PFGE pattern, and were resistant to at least 9 antimicrobials. All isolates from herd B were Salmonella Typhimurium, represented 2 PFGE patterns, and were susceptible to all antimicrobials evaluated. The estimated duration of fecal shedding was 14 days in herd A and 9 days in herd B. Few calves were seropositive for antibody against Salmonella lipopolysaccharide within the first week after birth (0 of 20 in herd A and 13 of 79 in herd B) or seroconverted (6 in herd A and 4 in herd B). Fecal shedding was more common in calves that seroconverted, but overall, there was not a strong association between seropositivity and fecal shedding of Salmonella organisms. Conclusions and Clinical Relevance—Although the herds differed in serologic response and Salmonella subtype, the duration of fecal shedding among calves was similar between herds.

In recent years, the proportion of Salmonella enterica infections represented by S. enterica serovar Newport has increased markedly among humans and animals. Multilocus variable-number tandem-repeat analysis (MLVA) has proven to be useful in discriminating other highly clonal Salmonella serovars. Here, we report on the development of a highly discriminatory MLVA for Salmonella serovar Newport.

Salmonella spp. represent one of the most common causes of bacterial foodborne illnesses around the world. The species Salmonella enterica contains more than 2500 serotypes, and emergence of new human pathogenic Salmonella strains and serotypes represents a major public health issue. Salmonella enterica subsp. enterica serotype 4,5,12:i:– represents a monophasic variant of Salmonella Typhimurium, which has rarely been identified before the mid-1990s. The prevalence of this serotype among human salmonellosis cases has increased considerably since the mid-1990s and Salmonella 4,5,12:i:– currently (i.e., the first decade of the 2000s) represents one of the most common serotypes among human cases in many countries around the world. This paper discusses our current knowledge of the global ecology, epidemiology, transmission, and evolution of this emerging Salmonella serotype.

Nontyphoidal salmonellosis has been characterized by a pattern of dissemination of clonal Salmonella enterica. The most well-known example of this was S. enterica serovar Typhimurium definitive phage type 104 (DT104), a clone that emerged and disseminated globally in the 1990s (1). Other examples of epidemic clones include Salmonella serovar Wien, which spread from North Africa through Europe in the 1970s; S. Typhimurium phage type 10 (DT10), which disseminated across Canada in the 1970s; and other phage types of S. Typhimurium associated with cattle that disseminated widely in Europe during the 1980s (2). More recently, multidrug-resistant (MDR) S. Agona was disseminated in poultry in Belgium; MDR S. Paratyphi B dT+ in Germany, Belgium, and the Netherlands; and MDR S. Newport that harbors the plasmid-mediated AmpC resistance in the United States (3). Epidemic clones may remain largely restricted to the animal reservoir for years before their incidence rises among human infections: S. Typhimurium phage type 204c disseminated throughout the United Kingdom in cattle from 1980 through 1984, but its proportion among human S. Typhimurium infections was not high during that period (4). Some epidemic clones, for example DT10 and DT104, expanded in the absence of a specific resistance advantage (1). The mechanisms underlying emergence, dissemination, and subsequent decline of novel Salmonella clones are unknown, although some researchers have hypothesized that fitness-associated genetic factors may allow more efficient dissemination in specific hosts and environments (5) and that acquisition of these fitness genes may be phage mediated (6). One approach to understanding clonal replacement events is to conduct prospective surveillance of Salmonella infections in animal and human hosts. We initiated such a study to compare human- and animal-origin Salmonella strains by serovar, antimicrobial drug resistance pattern, and pulsed-field gel electrophoresis (PFGE).

Although Turkey is one of the major producers of fruits and vegetables in the world, there has been no information available on the prevalence of pathogens in fresh produce. To fill this gap, we collected 503 fresh produce samples including tomato, parsley, iceberg lettuce, green-leaf lettuce and five different fresh pepper varieties (i.e., green, kapya, bell, mazamort and Charleston) from 3 major districts within 9 supermarkets and 3 bazaars in Ankara, Turkey to investigate the presence of Salmonella. Salmonella was detected in 0.8% (4/503) of samples by conventional culturing method with molecular confirmation conducted through polymerase chain reaction (PCR). For further characterization of isolates, serotyping, antimicrobial susceptibility testing, multi-locus sequence typing (MLST; aroC, thrA, purE, sucA, hisD, hemD and dnaN) and pulsed-field gel electrophoresis (PFGE) were performed. Salmonella enterica subsp. enterica serotypes Anatum, Charity, Enteritidis and Mikawasima were isolated from two parsley, one pepper and one lettuce samples, respectively. MLST resulted in 4 sequence types (STs) for each serotype, including one novel ST for serotype Mikawasima. Similarly, PFGE revealed four different XbaI PFGE patterns. The results of this survey, obtained by the most common subtyping methods (i.e. serotyping, MLST and PFGE) worldwide, contributes to the development of a national database in Turkey, which is essential for investigating the evolutionary pathways, geographical distribution and genetic diversity of Salmonella strains.

A collection of 179 human and 156 bovine clinicalSalmonellaisolates obtained from across New York state over the course of 1 year was characterized using serotyping and a multilocus sequence typing (MLST) scheme based on the sequencing of three genes (fimA,manB, andmdh). The 335 isolates were differentiated into 52 serotypes and 72 sequence types (STs). Analyses of bovine isolates collected on different farms over time indicated that specific subtypes can persist over time on a given farm; in particular, a number of farms showed evidence for the persistence of a specificSalmonella entericaserotype Newport sequence type. Serotypes and STs were not randomly distributed among human and bovine isolates, and selected serotypes and STs were associated exclusively with either human or bovine sources. A number of common STs were geographically widespread. For example, ST6, which includes isolates representing serotype Typhimurium as well as the emerging serotype 4,5,12:i:-, was found among human and bovine isolates in a number of counties in New York state. Phylogenetic analyses supported the possibility that serotype 4,5,12:i:- is closely related toSalmonellaserotype Typhimurium.Salmonellaserotype Newport was found to represent two distinct evolutionary lineages that differ in their frequencies among human and bovine isolates. A number ofSalmonellaisolates carried two copies ofmanB(33 isolates) or showed small deletion events infimA(nine isolates); these duplication and deletion events may provide mechanisms for the rapid diversification ofSalmonellasurface molecules. We conclude that the combined use of an economical three-gene MLST scheme and serotyping can provide considerable new insights into the evolution and transmission ofSalmonella.

Background The bacterium Salmonella enterica includes a diversity of serotypes that cause disease in humans and different animal species. Some Salmonella serotypes show a broad host range, some are host restricted and exclusively associated with one particular host, and some are associated with one particular host species, but able to cause disease in other host species and are thus considered "host adapted". Five Salmonella genome sequences, representing a broad host range serotype (Typhimurium), two host restricted serotypes (Typhi [two genomes] and Paratyphi) and one host adapted serotype (Choleraesuis) were used to identify core genome genes that show evidence for recombination and positive selection. Results Overall, 3323 orthologous genes were identified in all 5 Salmonella genomes analyzed. Use of four different methods to assess homologous recombination identified 270 genes that showed evidence for recombination with at least one of these methods (false discovery rate [FDR] ompC, a gene encoding an outer membrane protein, which has also been found to be under positive selection in other bacteria. A total of 8, 16, 7, and 5 genes showed evidence for positive selection in Choleraesuis, Typhi, Typhimurium, and Paratyphi branch analyses, respectively. Sequencing and evolutionary analyses of four genes in an additional 42 isolates representing 23 serotypes confirmed branch specific positive selection and recombination patterns. Conclusion Our data show that, among the four serotypes analyzed, (i) less than 10% of Salmonella genes in the core genome show evidence for homologous recombination, (ii) a number of Salmonella genes are under positive selection, including genes that appear to contribute to virulence, and (iii) branch specific positive selection contributes to the evolution of host restricted Salmonella serotypes.