Antimicrobial resistance, virulence-associated genes, and pulsed-field gel electrophoresis profiles of Salmonella enterica subsp. enterica serovar Typhimurium isolated from piglets with diarrhea in Korea

Salmonella enterica subsp. enterica serovar Typhimurium was isolated from diarrheic piglets in 2 periods, 2000-2001 (n = 25) and 2005-2006 (n = 17). To compare the characteristics of the isolates collected during the 2 periods, all isolates were tested for antimicrobial resistance, the presence of virulence genes, and pulsed-field gel electrophoresis (PFGE) patterns. All 42 isolates were resistant to at least 1 of the 20 antimicrobials tested, and 39 (93%) were resistant to 2 or more antimicrobials. One isolate was resistant to 12 antimicrobials. Profiles of antimicrobial resistance revealed 20 resistance types. Several isolates were also resistant to quinolones and expanded-spectrum cephalosporins. Ten isolates (24%) were resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT); only one isolate had been isolated in 2000-2001, indicating that this type of resistance has rapidly disseminated. Polymerase chain reaction (PCR) assays revealed that all the isolates carried invA. Among the 25 strains isolated in 2000-2001, all carried the sipA, sopA, sopD, sopE2, and ssaR genes, and 96% carried sopB and sifA. Among the 17 strains isolated in 2005-2006, all carried sifA, and approximately 90% carried sipA, sopA, sopB, sopD, sopE2, and ssaR. However, only 6 (14%) of the 42 isolates carried spvC. By PFGE analysis, all 42 strains were classified into 4 major clusters, basically by collection period. The genetic similarity according to PFGE suggests that the strains isolated from diarrheic piglets of this region within the same period may be closely related.     

Antimicrobial resistance in Salmonella enterica serovar Typhimurium from human and animal sources in Italy

Salmonella Typhimurium strains isolated in Italy in the period 2002-2004 from human and animal sources were examined for their antimicrobial susceptibility. Resistance to tetracycline (T, 73.6%), sulfonamides (Su, 73.3%), ampicillin (A, 67.6%), streptomycin (S, 65.4%) and chloramphenicol (C, 32.3%) were frequently observed. Resistance to ciprofloxacin was only observed in a swine strain, but most human strains resistant to nalidixic acid showed reduced susceptibility to that drug (MIC > or = 0.125 mg/l). Overall, 64% of the strains were resistant to four or more drugs. The most common resistance profiles were ACSSuT, prevalent in strains belonging phage type DT104 and ASSuT, prevalently associated with strains unable to be typed.     

Phenotypic and genetic characterization of Salmonella enterica subsp. enterica serovar Typhimurium isolated from pigs in Rio Grande do Sul, Brazil

This study aimed to investigate the relatedness of porcine Salmonella enterica subsp. enterica (S.) serovar Typhimurium strains isolated in Southern Brazil. Sixty-six isolates from pigs belonging to three commercial companies were submitted to phage typing, XbaI-macrorestriction (PFGE), IS200 hybridization, rep-PCR, antimicrobial susceptibility testing, and PCR assay targeting the spvR region. All strains presented a unique rep-PCR pattern and 63 strains had a common IS200 profile. One pulse-type (XA) was the most prevalent (39/66 strains) and included strains of phage types DT177, DT192, DT194 and RDNC. The spvR region was detected in three strains, which harboured plasmids of 90 kb. High rates of tetracycline, sulfonamide and streptomycin resistance were found. Isolates from farms located in different geographic regions but associated to the same commercial companies clustered together and presented a common resistance profile. Results suggested that clonal groups of S. Typhimurium are present in pig commercial companies in Southern Brazil.     

Phenotypic and genetic characterization of Salmonella enterica subsp. enterica serovar Typhimurium isolated from pigs in Rio Grande do Sul,Brazil

This study aimed to investigate the relatedness of porcine Salmonella enterica subsp. enterica (S.) serovar Typhimurium strains isolated in Southern Brazil. Sixty-six isolates from pigs belonging to three commercial companies were submitted to phage typing, XbaI-macrorestriction (PFGE), IS200 hybridization, rep-PCR, antimicrobial susceptibility testing, and PCR assay targeting the spvR region. All strains presented a unique rep-PCR pattern and 63 strains had a common IS200 profile. One pulse-type (XA) was the most prevalent (39/66 strains) and included strains of phage types DT177, DT192, DT194 and RDNC. The spvR region was detected in three strains, which harboured plasmids of 90 kb. High rates of tetracycline, sulfonamide and streptomycin resistance were found. Isolates from farms located in different geographic regions but associated to the same commercial companies clustered together and presented a common resistance profile. Results suggested that clonal groups of S. Typhimurium are present in pig commercial companies in Southern Brazil.     

Phage types of Salmonella enterica serovar Enteritidis isolated in South Africa from 1991-1995

A total of 615 strains of Salmonella enterica serovar Enteritidis (SE), received from 1991-1995 at the Onderstepoort Veterinary Institute (OVI), were phage typed. Most SE isolates (54,7%) originated from poultry followed by humans (28,5 %) and poultry eggs (9,6 %). Phage type 34 was the most prevalent (40,5 %) of all isolates, followed by phage type 4 (33,8 %). Other phage types identified were 1, lb, 4a, 7, 7a, 9a, 14, 24, 24var and 35 (in total 2,4% of isolates). Most isolates of SE were received from the Western Cape Province (47,4%) and Gauteng (22,3%). In poultry phage type 4 was dominant, but in humans, eggs, goats, ducks, sheep, pigs and rabbits, phage type 34 was the dominant type. It appeared as if the poultry-associated epidemic of SE in South Africa that occurred from 1991-1995 originated in the Western Cape Province during 1991 amongst poultry and then spread from there to humans and eggs and then to the rest of the country, where it emerged during 1993. Results indicate that phage type 34 was the dominant phage type from 1991-1993, but during 1994-1995 its presence declined. During this latter period the presence of phage type 4 increased. This may suggest that two smaller epidemics consisting of the two different phage types might have been responsible for the epidemic that occurred from 1991-1995.     

Assessment of antibiotic resistance phenotype and integrons in Salmonella enterica serovar Typhimurium isolated from swine

Salmonella enterica serovar Typhimurium (S. Typhimurium) isolated and identified from swine were subjected for the analysis of antibiotic resistance pattern and clinically important class 1 and 2 integrons. In addition, S. Typhimurium isolates exhibiting ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline and florfenicol (ACSSuTF) resistance pattern as described in most Salmonella enterica serotype Typhimurium definitive type 104 (DT104) were characterized by polymerase chain reaction. All the isolates were resistant to more than four antibiotics and showed the highest resistance to streptomycin (94.1%), followed by tetracycline (90.1%), ampicillin (64.7%), chloramphenicol (56.8%) and gentamicin (54.9%). MIC value for the ten isolates ranged between 0.125-2 mug/ml for ciprofloxacin. Among the beta-lactams used, only one of the isolate exhibited resistance to ceftiofur (MIC 8 microg/ml). Sixty eight percent of these multi drug resistance (MDR) S. Typhimurium isolates carried clinically important class 1 integron with 1kb (aadA) and/or 2kb (dhfrXII-orfF-aadA2) resistance gene cassettes. This study reports the increasing trend of multi drug resistance (MDR) S. Typhimurium with clinically important class 1 integron in pigs. In addition, emergence of the ACSSuTF-type resistance in S. Typhimurium PT other than DT104 may limit the use of resistance gene markers in its detection methods by PCR.     

Molecular analysis of Salmonella enterica subsp. enterica serovar Agona isolated from slaughter pigs

Salmonella enterica subsp. enterica (S.) serovar Agona plays an important role in Brazil as causative agent of salmonellosis in food-producing animals - in particular, pigs and poultry - as well as in humans. A total of 45 S. Agona isolates collected from slaughter pigs at three different slaughterhouses in Southern Brazil was investigated in this study for their phenotypic and genotypic relatedness. For this, the antimicrobial susceptibility patterns and the phage types were determined. Molecular analysis included the determination of plasmid profiles as well as the analysis of XbaI- and BlnI-generated macro-restriction patterns. Moreover, a novel typing method called subtracted restriction fingerprinting (SRF) was successfully applied to the S. Agona isolates. Based on all properties determined, a dominant clonal group comprising 33 of the 45 isolates was identified. Members of this group were susceptible to all antimicrobials tested, did not carry plasmids, shared the same phage type and were closely related or even indistinguishable by their EcoRI-PauI SRF patterns as well as their XbaI and BlnI macro-restriction patterns. Members of this clonal group were identified at all 3 slaughterhouses at variable frequencies and originated from pig herds raised in 15 different cities in Southern Brazil which were located up to 450 km apart from each other. Since the S. Agona-carrying slaughter pigs were from various integrated production lines, the results of this study suggest that a specific clonal group of S. Agona had entered numerous pig production lines. This observation supports the requirement for the establishment of monitoring and control programmes in Brazil which should also include molecular techniques to better trace the dissemination of S. Agona and other Salmonella serovars in pigs and other food-producing animals.     

Phage Types of Salmonella enterica ssp. enterica serovar Typhimurium Isolated from Production Animals and Humans i Denmark

S. Typhimurium is one of the 2 most common salmonella serotypes causing human salmonellosis in Denmark. In order to illustrate the significance of different production animals as a source of infection, 1461 isolates were characterized by phage typing. The isolates originated from human patients and from cattle, pigs and poultry. By phage typing the isolates could be separated in 35 different phage types. Five types (10, 12, 66, 110 and 135) predominated and comprised 78.8% of the isolates. In humans, 57.3% of the isolates were phage type 12. This phage type was also predominant in pig herds and, to a lesser degree, in cattle. Phage types 110, 120, 135 and 193 constituted 86.5% of the poultry isolates while these phage types only made up 12.9% of the human isolates. The investigation showed that pigs are probably a major source of S. Typhimurium infection in humans in Denmark today.     

Risk factors for clinical Salmonella enterica subsp. enterica serovar Typhimurium infection on Dutch dairy farms

Risk factors for outbreaks in 1999 of clinical Salmonella enterica subsp. enterica serovar Typhimurium infection on dairy farms were studied in a matched case-control study with 47 case farms and 47 control farms. All 47 case farms experienced a clinical outbreak of salmonellosis which was confirmed with a positive bacteriologic culture for serovar Typhimurium in one or more samples. Serovar Typhimurium phage type 401 and 506 (definitive type 104, DT104) were the most frequently isolated phage types (13 isolates). On most farms (66%), clinical signs were seen only among adult cows. The most frequently reported clinical signs were diarrhoea (in 92% of the farms) and depression (in 79% of the farms).Control farms were matched on region and had no history of salmonellosis. A questionnaire was used to collect data on case and control farms. The relationship between serovar Typhimurium status of the farm and possible risk factors was tested using conditional logistic regression. Significant factors in the final model were presence of cats on the farm (OR=0.06), purchase of manure (OR=21.5), feeding colostrum only from own dam (OR=0.08), a non-seasonal calving pattern (OR=25), unrestricted grazing of lactating cows (OR=0.07), and a high mean mowing percentage of pasture (OR=1.02).     

Typing of Salmonella enterica subsp. enterica serovar Mbandaka isolates

Recently, Salmonella enterica subsp. enterica serovar Mbandaka (S. Mbandaka) has gained some importance in the epidemiology of salmonellosis in Poland. Since biotyping, resistance typing, and plasmid profiling were insufficient for strain differentiation, genome macrorestriction by means of pulse-field gel electrophoresis (PFGE) was applied and proved to be the method of choice in S. Mbandaka epidemiological studies. XbaI and BcuI macrorestriction produced 15 and 14 pulse-field profiles (PFP), respectively, but in the case of each enzyme one profile was prevalent. When macrorestriction profiles were combined, a total 24 patterns were found. Based on the similarity of the profiles, four clonal lineages were identified. One clonal lineage contained the majority of poultry, feed and human isolates. Poultry was concluded to be an important source of S. Mbandaka for humans in Poland. Complementary use of various typing techniques improved efficacy of epidemiological studies giving possibility to subdivide S. Mbandaka into 35 types and the index of discrimination reached 0.947.     

Multi-locus sequence typing of Salmonella enterica subsp. enterica serovar Enteritidis strains in Japan between 1973 and 2004

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) was responsible for a worldwide pandemic during the 1980s and 1990s; however, changes in the dominant lineage before and after this event remain unknown. This study determined S. Enteritidis lineages before and after this pandemic event in Japan using multilocus sequence typing (MLST). Thirty S. Enteritidis strains were collected in Japan between 1973 and 2004, consisting of 27 human strains from individual episodes, a bovine strain, a liquid egg strain and an eggshell strain. Strains showed nine phage types and 17 pulsed-field profiles with pulsed-field gel electrophoresis. All strains had homologous type 11 sequences without any nucleotide differences in seven housekeeping genes. These MLST results suggest that S. Enteritidis with the diversities revealed by phage typing and pulsed-field profiling has a highly clonal population. Although type 11 S. Enteritidis may exhibit both pleiotropic surface structure and pulsed-field type variation, it is likely to be a stable lineage derived from an ancestor before the 1980s and/or 1990s pandemic in Japan.     

Insertional mutation of marA vitiates inducible multiple antimicrobial resistance in Salmonella enterica subsp. enterica serovar Choleraesuis

marA has been shown to mediate a multiple antimicrobial resistance (MAR) phenotype following induction in some members of the Enterobacteriaceae. When Salmonella Choleraesuis was exposed to inducing agents they displayed higher minimal inhibitory concentrations (MIC) to multiple antimicrobial agents and an increase in marA expression as determined by northern hybridization analysis. The objective of the present study was to determine if mutation of marA vitiated multiple antimicrobial resistance inducibility in S. Choleraesuis. A loss-of-function mutation of marA in a single S. Choleraesuis isolate was created by insertion of the dihydrofolate reductase (DHFR) gene cassette within marA using double homologous recombination. This mutation was complemented with an expression plasmid possessing marA under the control of an IPTG-inducible promoter. Mutation and complementation of marA was verified using polymerase chain reaction, Northern hybridization, and Western blotting assays. Minimum inhibitory concentrations (MICs) of tetracycline, chloramphenicol, nalidixic acid, and rifampin were determined against induced and uninduced wildtype, marA-disrupted and marA-complemented strains using a microbroth dilution assay. Minimum inhibitory concentrations against induced wildtype and marA-complemented strains increased four- to eight-fold for all antimicrobials tested when compared to the uninduced strains while the MICs of the induced marA-disrupted mutant remained the same. However, this increase was abrogated when the cells were grown in the presence of the efflux pump inhibitor compound EPI phe-arg-naphthylamide. The results indicate that a functional marA is solely required for an inducible multiple antimicrobial resistance phenotype in S. Choleraesuis.     

Genomic diversity and resistome profiles of Salmonella enterica subsp. enterica serovar Kentucky isolated from food and animal sources in Ireland

Salmonella enterica subsp. enterica serovar Kentucky is frequently isolated from poultry, dairy and beef cattle, the environment and people with clinical salmonellosis globally. However, the sources of this serovar and its diversity and antimicrobial resistance capacities remain poorly described in many regions. To further understand the genetic diversity and antimicrobial sensitivity patterns among S. Kentucky strains isolated from non-human sources in Ireland, we sequenced and analysed the genomes of 61 isolates collected from avian, bovine, canine, ovine, piscine, porcine, environmental and vegetation sources between 2000 and 2016. The majority of isolates (n = 57, 93%) were sequence type (ST) 314, while only three isolates were ST198 and one was ST152. Several isolates were multidrug-resistant (MDR) and 14 carried at least one acquired antimicrobial resistance gene. When compared to a database of publicly available ST314, four distinct clades were identified (clades I–IV), with the majority of isolates from Ireland clustering together in Clade I. Two of the three ST198 isolates were characteristic of those originating outside of the Americas (Clade ST198.2), while one was distantly clustered with isolates from South and North America (Clade ST198.1). The genomes of the two clade ST198.2 isolates encoded Salmonella Genomic Island 1 (SGI1), were multidrug-resistant and encoded polymorphisms in the DNA gyrase (gyrA) and DNA topoisomerase (parC) known to confer resistance to fluoroquinolones. The single ST152 isolate was from raw beef, clustered with isolates from food and bovine sources in North America and was pan-susceptible. Results of this study indicate that most S. Kentucky isolates from non-human sources in Ireland are closely related ST314 and only a few isolates are antimicrobial-resistant. This study also demonstrates the presence of multidrug-resistant ST198 in food sources in Ireland.     

Characterization of Salmonella enterica serovar Typhimurium strains of veterinary origin by molecular typing methods

Twenty-eight strains of Salmonella enterica serovar Typhimurium were characterized by three PCR-based methods. Ten strains harbored type I integrons and two different integron profiles were detected. Typing by amplified fragment length polymorphism (AFLP) resulted in observation of 10 profiles that differed by one to six bands. Salmonella strains were screened for presence of phage genes using a PCR-phage typing; five genes from P22 phage and genes encoding putative virulence factors from phages Gifsy-1, Gifsy-2 and Fels-1 were selected for testing. This set of genes was sufficient for dividing the strains into eight different PCR-phage profiles. Similar grouping of strains was observed in case of all the employed DNA techniques and they corresponded well with the phage type and antimicrobial resistance of the strains. The highest discriminating power was achieved with use of the AFLP, yet the detection of integrons and PCR-phage typing also proved to be valuable in typing the S. Typhimurium strains.     

Phenotypic and genotypic differentiation of porcine Salmonella enterica subsp. enterica serovar Derby isolates

Sixty-two Salmonella enterica subsp. enterica serovar Derby isolates from slaughter pigs and meat products isolated in Southern Brazil were analyzed for their genomic relationships and for the presence of antimicrobial resistance genes. Twenty-four S. Derby isolates were indistinguishable by their subtracted restriction fingerprinting (SRF) pattern, XbaI- and BlnI-macrorestriction patterns, phage type, plasmid profile, and resistance pattern. In contrast to the BlnI-macrorestriction patterns, the XbaI-macrorestriction patterns were in good agreement with the results of SRF analysis and phage typing. Among the four phage types detected, PT10 and PT21 were the most common. The combination of all typing methods revealed a great diversity among the S. Derby isolates. All strains carried plasmids and the 60 resistant isolates showed at least tetracycline resistance. The resistance genes found were sul1 and/or sul2 (sulfonamide resistance), aadA2 (streptomycin/spectinomycin resistance), tet(A) (tetracycline resistance), tet(B) (tetracycline/minocycline resistance), bla(TEM) (ampicillin resistance), and dfrA14 (trimethoprim resistance). A correlation of the geno- and phenotypic characteristics with the origin of the isolates revealed a substantial temporal variation in the occurrence of specific S. Derby isolates in different independent pig production lines in Southern Brazil. The large number of resistant isolates underlined the potential risk that S. Derby isolates can pose to human health when they enter the food chain.     

Presence of arsenic resistance in Salmonella enterica serovar Kentucky and other serovars isolated from poultry

A collection of 125 Salmonella enterica poultry isolates (71 serovar Kentucky isolates, and the remainder belonging to serovars Alachua, Enteritidis, Hadar, Heidelberg, Montevideo, Mbandaka, Senftenberg, Typhimurium, and Worthington) were tested for the ability to grow on tryptic soy agar containing sodium arsenite [As(III)] or arsenate [As(V)]. All serovar Kentucky isolates and 18 of the non-Kentucky isolates were able to grow in the presence of 0.1 mM As(III), and 69 grew in the presence of 1 mM As(V). Thirty of the non-Kentucky isolates did not grow at these As(III) and As(V) concentrations, but seven grew at 1 mM As(III) and 10 mM As(V). PCR-based analysis demonstrated the presence of arsB and arsD sequences in all Kentucky isolates, whereas one or both of these sequences were present in only 30 of the other isolates. It remains to be determined if these arsenic-resistance determinants benefit Salmonella exposed to man-made arsenic-containing compounds in poultry environments.     

Trends in phage types and antimicrobial resistance of Salmonella enterica serovar Enteritidis isolated from animals in Great Britain from 1990 to 2005

Surveillance data for Salmonella enterica serovar Enteritidis incidents and isolations from food animals in Great Britain from 1990 to 2005 were analysed to detect any trends and provide the basis for a comparison between phage types (pt) and antimicrobial sensitivity patterns in human beings and animals. During 2001 to 2005 there was a decrease in incidents involving most species except ducks. Only the numbers of incidents involving pts 6, 6a, 9b and 14b (in ducks) and pts 6a and 13a (in mammals) increased significantly during this period, whereas there were 93 per cent fewer incidents involving pt 4 than in 1990 to 2000. After adjustment for pt, the isolates from ducks were more resistant to nalidixic acid, tetracyclines and sulfonamides, and were more likely to be multiresistant than isolates from chickens. Isolates from turkeys tended to be more resistant to sulfonamides than isolates from chickens. pts 1, 5a, 6, 6a and 35 had the highest level of resistance after adjusting for species. During 2001 to 2005 there was an increase in resistance among pts 1, 6 and 7, in most cases involving nalidixic acid.     

Evaluation of molecular typing methods for Salmonella enterica serovar Typhimurium DT104 isolated in Germany from healthy pigs

The discriminatory power of four different DNA based typing methods was tested for the molecular subtyping of Salmonella Typhimurium phage type DT104 isolates. German DT104 strains (n = 133) originating from slaughter pigs were analysed by plasmid profiling, and 32 of them by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes XbaI, SpeI or BlnI, random amplification of polymorphic DNA (RAPD) using 13 different primers and IS200 typing. A resulting subtyping scheme was obtained which is based on the most discriminatory power of the individual methods i.e. plasmid profiling and PFGE with all three enzymes. The index of discrimination obtained by the subtyping scheme was 0.909 closely approaching the maximum value of one. Although minor differences occurred in the molecular DNA pattern of single DT104 strains, a dominating subtyping pattern was observed confirming other studies which showed, that S. Typhimurium DT104 isolates are highly clonal.     

Herd-level diagnosis for Salmonella enterica subsp. enterica serovar Dublin infection in bovine dairy herds

Herd-level sensitivities of bacteriological and serological methods were compared in 79 bovine dairy herds, recently infected with Salmonella enterica subsp. enterica serovar Dublin. All farms experienced clinical signs of salmonellosis for the first time and had no history of vaccination against salmonellosis. At the start of the study, infection with serovar Dublin was confirmed with at least one positive bacteriologic culture for serovar Dublin from a clinical case (gold standard for herd infection).Bacteriological culture was done on samples of dung-pits, drinking water, bulk-milk filters, and faeces of animals with current or earlier clinical signs of salmonellosis. Blood samples of all animals and bulk-milk samples were tested using an ELISA.Herd-level sensitivity (HSe) of culture of dung-pits, drinking water, bulk-milk filters, and faeces of animals with current or earlier signs of salmonellosis was 45, 5, 7, and 38%, respectively. HSe for serology of all animals was 100%. If blood samples of all calves 4-6 months old were examined, at least one calf was seropositive on 91% of the infected farms. If serology was performed on samples of animals with current or earlier signs of salmonellosis, at least one animal was seropositive on 80% of the infected farms. HSe for bulk-milk samples was 54%. However, if clinical signs of salmonellosis were observed only in lactating animals, sensitivity of bulk-milk serology was 79%.Interesting combinations of methods were the combination of serology of bulk milk with either serology of animals with current or earlier signs of salmonellosis (HSe=91%), or serology of all calves of 4-6 months old (HSe=99%).