Molecular characterization, spread and evolution of multidrug resistance in Salmonella enterica typhimurium DT104.

Multidrug-resistant Salmonella enterica serovar Typhimurium phage type DT104 has emerged during the last decade as a global health problem because of its involvement in diseases in animals and humans. Multidrug-resistant DT104 strains are mostly resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracyclines (ACSSuT resistance type). The genes coding for such resistances are clustered on the chromosome. This paper reviews new developments in the characterization of S. enterica Typhimurium DT104, its chromosomal antibiotic resistance genes and their spread among other S. enterica Typhimurium phage types and other S. enterica serovars, the development of specific detection methods, virulence characteristics, and the evolution of multidrug-resistance with regard to the emergence of quinolone resistance.     

Prevalence of Escherichia coli O157:H7 prophage-like sequences among German Salmonella enterica serotype Typhimurium phage types and their use in detection of phage type DT104 by the polymerase chain reaction

A 1.6kb DNA fragment identified by random amplifiable polymorphic DNA differentiation (RAPD) from a Salmonella enterica serotype Typhimurium phage type DT104 isolate was used to investigate the prevalence of the region in 160 DT104 isolates, 83 other epidemiological important S. Typhimurium phage types and 20 strains selected from 17 other Salmonella serotypes. PCR screening tests using two different primer-sets derived from the RAPD fragment's nucleotide sequence showed that 76% of the 160 DT104 isolates investigated, including subtypes DT104A, DT104B, DT104B low, DT104H and DT104L, reacted positively. High sensitivity was shown for DT104 strains expressing at least the penta-resistance pattern ACSSuT (97% of 104 strains tested). DT104 susceptible strains showed only a sensitivity of 35% (17 strains tested). In contrast, 83% of the 83 strains from the other S. Typhimurium phage types reacted negatively. Strains from five out of the 17 other serotypes showed a positive signal with one primer-set. The other primer-set exhibited only a positive reaction with one S. Dublin isolate. The analysis of a 2415bp extended sequence revealed homologies to genes encoded by Escherichia coli O157:H7 prophages, suggesting that the described region contains genes of a prophage specific for DT104 and related phage types.     

Epidemiology and characterization of animal Salmonella enterica subspecies Enterica serotype typhimurium DT104 in Hungary

Reports on the internationally emerging significance of multiresistant zoonotic Salmonella in animals and man prompted studies to estimate the significance of multiresistant Salmonella enterica subspecies enterica serotype Typhimurium (S. Typhimurium) phage type DT104 of animal origin in Hungary. A collection of 231 strains (primarily of goose, turkey, poultry and porcine origin from the years 1997-1998) was tested for resistance against 7 selected antibiotics (ampicillin, chloramphenicol, enrofloxacin, nalidixic acid, streptomycin, tetracycline and sulphamethoxazole). Strains with resistance against 3 or more were defined as multiresistant. All strains were phage typed using Felix-Callow's S. Typhimurium phage typing system, and 91 of them (suspect DT104) were also typed according to Anderson's definitive typing (DT) system. In this study, 14% of animal strains from 1997-1998 was classified as DT104, for which turkey, pig and duck seemed to be the main carriers, and the multiresistant non-DT104 strains represented a further 6% of this collection. The prevalence of DT104 was highest among strains of turkey origin (50%), followed by strains of pig (29%), chicken (25%), duck (19%), and goose (3%) origin. The other DT104 related phage types (DT12 and U302) were only detected in the case of 4 strains (2 of porcine, and one each of turkey and of goose origin). The DT104 corresponded to the Felix-Callow types 2/3 or 2c/3 in each case, except in the case of 3 turkey strains where they corresponded to type 35/3. Nalidixic acid resistance was detected in all multiresistant turkey strains and in some of other animal origin but none of these strains were resistant to enrofloxacin. A retrospective analysis (based on the above relationship) indicated that S. Typhimurium strains corresponding to DT104 could be present and increase in the Hungarian farm animal population from about 2% to 20% between 1985 and 1990, in a manner similar to the emergence of human DT104, as reported elsewhere (Pászti et al., 2000). The 91 suspect DT104 strains were also tested for plasmid profile and for spvC gene indicating the presence of the large serotype specific plasmid (Ssp). No characteristic plasmid profile could be attributed to S. Typhimurium DT104. The serovar-specific large plasmid was detected by PCR for spvC in 100% of DT104 strains and in 77% of the non-DT104 strains. The virulence of two DT104 strains was tested in orally infected day-old chicks and compared with virulence of 4 non-DT104 strains. Higher colonizing virulence of DT104 strains could be established as compared to the other strains.     

Antimicrobial resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from animals in Korea: comparison of phenotypic and genotypic resistance characterization

Fourteen and 22 each of Salmonella Enteritidis and Salmonella Typhimurium (S. Typhimurium) were isolated from animals from 1983 to 1999 in Korea and tested for their antibiotic resistance patterns, phage types and resistance gene patterns. S. Typhimurium isolates were highly resistant to streptomycin, sulfisoxazole and tetracycline, 95, 95 and 86%, respectively. The incidence of multiple antibiotic resistance (resistant to more than two drugs tested) of S. Typhimurium isolates was extremely high (100%) comparing to S. Enteritidis isolates (21%). Two of the five ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfisoxazole and tetracycline) resistant type S. Typhimurium isolates were phage type definitive type 104 (DT104).For the detection of resistance related genes in S. Enteritidis and S. Typhimurium isolates, particularly ACSSuT type S. Typhimurium, antibiotic resistance genes, cmlA/tetR, bla(PSE-1) and bla(TEM), and genus Salmonella specific gene, sipB/C, were amplified using four pairs of primers in a hot-start multiplex polymerase chain reaction (PCR). Two Korean isolates of S. Typhimurium DT104 showed bla(TEM) amplicons instead of bla(PSE-1) for the ampicillin resistance and they were susceptible to florfenicol. The multiplex PCR used in this study was useful in characterization of multiple drug resistant Salmonella isolates, especially ACSSuT type S. Typhimurium, and identification of beta-lactamase gene distribution among Salmonella isolates.     

Comparison of the in vitro pathogenicity of two Salmonella Typhimurium phage types

The in vitro pathogenicity of Salmonella enterica serovar Typhimurium phage type (pt) 90 and pt 506 (also known as DT 104) isolates from human and porcine origin was studied in adhesion and invasion assays to the human cell line Caco-2 and the porcine cell line IPI-2. Interleukin-8 (IL-8) production by these two cell lines in response to stimulation by the two Salmonella phage types was also measured. Generally, Salmonella Typhimurium pt 506 and pt 90 adhered to and invaded Caco-2 cells and IPI-2 cells equally well. The release of IL-8 by Caco-2 cells or by IPI-2 cells was similar, independent of the Salmonella phage type used for stimulation of the cells. These data suggest that Salmonella Typhimurium pt 90 has a similar ability to cause Salmonella infections as Salmonella Typhimurium DT 104.     

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.     

Salmonella typhimurium DT104 in farmed rabbits

A total of 1,000 rectal samples were collected from rabbits coming from 25 rabbit farms in southern Italy. All samples were processed for isolation of Salmonella spp. by standard culture method based on the ISO 6579:2002 method. Salmonella spp. was isolated from 1/25 rabbit farms analyzed. In particular, four out of 1,000 rectal swab samples, taken from young rabbits, were serotyped as S. Typhimurium and phage typed as S. Typhimurium DT104. All the isolates were resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (ACSSuT pentaresistance type). The findings of the present study suggest the rabbit as potential carrier of S. Typhimurium DT104.     

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.     

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.     

Integron content of Salmonella enterica serotype Typhimurium strains isolated in Hungary in the years 1997-1999

The integron content of 52 DT104/U302 phage type strains and 53 non-DT104/U302 strains of Salmonella enterica serotype Typhimurium (S. Typhimurium) was studied in PCR experiments using a 5'-CS/3'-CS primer pair (Lévesque et al., 1995). Forty-three out of 44 streptomycin- and/or ampicillin-resistant DT104 and related phage type strains were found to carry a 1 kb and/or 1.2 kb long integron. The other resistance markers did not affect the number and size of integrons; no integron-free multidrug-resistant (MDR) DT104 strains were found. The two large groups of DT104 strains (Felix-Callow's phage types 2 and 2c) proved to be identical in respect of integron patterns (IPs), supporting the views of those authors who consider DT104 a single clone. Strains of human and animal origin did not differ from each other in their IPs. Within the non-DT104 phage types, ampicillin- and/or streptomycin-resistant, integron-free MDR strains were also found. Based on amplicons varying between 290 and 3500 bp an IP system was suggested. The commonest amplicon sizes in non-DT104 strains were 1450 and 2050 bp. The IPs of DT104 strains and of non-DT104 strains containing an integron of 1 and 1.2 kb size were stable. In contrast, the IPs of other non-DT104 strains showed a varying degree of instability. Integron loss was frequently associated with spontaneous plasmid elimination and changes of R-type among the descendants of a given strain.     

Antimicrobial resistance in Salmonella isolated from animals and their environment in England and Wales from 1988 to 1999

Resistance to 16 antimicrobial agents was monitored in 109,125 Salmonella cultures isolated from animals, their environment and feedstuffs between 1988 and 1999. The sensitivity of the 6512 isolates of Salmonella enterica enterica serotype Dublin to all the antimicrobial agents tested varied from 98.2 per cent in 1997 to 99.7 per cent in 1990 and 1996. In contrast, among 28,053 isolates of Salmonella enterica enterica serotype Typhimurium, there was a marked decrease in their sensitivity to all the antimicrobial agents tested, from 57.4 per cent in 1992 to 7.6 per cent in 1995, owing to the widespread occurrence in farm animals of S Typhimurium isolates of the definitive type DT104, resistant to ampicillin, sulphonamides, streptomycin, chloramphenicol and tetracyclines, although the percentage of sensitive isolates increased to 18.4 per cent in 1999, when the incidence of DT104 had decreased. Some isolates of DT104 also showed an increase in resistance to potentiated sulphonamides (2.4 per cent in 1989 to 19.2 per cent in 1999) and nalidixic acid (0 per cent in 1992, 3.8 per cent in 1995 to a peak of 16.9 per cent in 1998). In 1996, 5.1 per cent of 1086 isolates of S Typhimurium from cattle and 35.9 per cent of 192 isolates of S Typhimurium from poultry showed resistance to nalidixic acid. Of the other 74,528 Salmonella isolates, the percentage of strains sensitive to all the antimicrobials tested decreased slightly from 88.2 per cent in 1988 to 70.6 per cent in 1996 and then increased slightly to 73.7 per cent in 1999. The commonest of these other Salmonella serotypes was Salmonella Enteritidis (20,982), which remained predominantly susceptible (ranging from 81.4 to 97.4 per cent) during the study period. Few isolates were resistant to commonly used veterinary antimicrobials, for example, furazolidone, the use of which was banned in 1990, and the aminoglycoside, apramycin.     

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).     

High prevalence of multiple resistance to antibiotics in Salmonella serovars isolated from a poultry slaughterhouse in Spain

Salmonellosis is a major foodborne infection in Spain, and strains that are resistant to a great variety of antibiotics have become a major public health concern. The aim of this study was to determine the level of antibiotic resistance in 133 Salmonella isolates obtained from a poultry slaughterhouse in Zaragoza (NE Spain). Antimicrobial resistance testing was performed by disk diffusion method using 19 antibiotics. Results were interpreted following the NCCLS criteria. Overall, the highest percentage of resistance was found to the following antimicrobial agents: sulfadiazine (96.2%), neomycin (53.4%), tetracycline (21.8%), and streptomycin (11.3%). All isolates were found to be resistant to one or more of the antibiotics tested. Multiple resistance was observed in 87 strains (65.4%). We found 23 different patterns of resistance in Salmonella Enteritidis. Resistance to sulfadiazine was the most common single resistance. The most frequent patterns of multiresistant strains were neomycin+sulfadiazine and neomycin+tetracycline+sulfadiazine. S. 4,5,12:b: showed the highest percentages of resistance to the tested drugs, with five different resistance patterns found. Ampicillin+chloramphenicol+streptomycin+sulphonamides+tetracycline (ACSSuT) resistance pattern, commonly associated with S. Typhimurium DT 104, was not detected in strains of the same phage type from broilers. The appearance of substantial multiresistance in foodborne Salmonella isolates suggests the need for more prudent use of antibiotics by farmers, veterinarians, and physicians.     

Changes of multi-drug resistance pattern in Salmonella enterica subspecies enterica serovar typhimurium isolates from food-producing animals in Japan

A total of 153 isolates of Salmonella Typhimurium derived from food-producing animals in Japan between 2002 and 2005 were investigated for antimicrobial resistance and phage types related to definitive phage type 104 (DT104). The predominant resistance type was resistance to ampicillin, dihydrostreptomycin, kanamycin, and oxytetracycline in bovine (45.2%, 48/104) and resistance to dihydrostreptomycin and oxytetracycline in porcine isolates (58.7%, 27/48). DT104-related phage type was found in 32 of 104 bovine isolates, two of 48 porcine isolates, and one of eight isolates from poultry, showing that the proportion of the phage type in S. Typhimurium isolates from cattle and pigs significantly (P<0.01) decreased from 71.9% and 31.4% in 1999-2001 to 30.8% and 4.1% in 2002-2005, respectively.     

Salmonella Genomic Island 1 (SGI1) and genetic characteristics of animal and food isolates of Salmonella typhimurium DT104 in Hungary

To study the genetic characteristics of DT104 strains of Salmonella Typhimurium and the prevalence of Salmonella Genomic Island (SGI1) in Hungary, 140 recent Salmonella strains of food and animal origin were examined. For the first time in Hungary, the SGI1 was found in 17 out of 59 S. Typhimurium isolates (all proven to be DT104 phage type). These 17 strains were then subtyped by pulsed-field gel electrophoresis (PFGE) into 6 pulsotypes which were less correlated with the geographic origin than with the animal species of origin.     

Salmonella enterica infections in Spanish swine fattening units

The present study is the first conducted in Spain to estimate the bacteriological herd prevalence of Salmonella enterica in fattening units and to describe the Salmonella serovar diversity on these farms using a sample representative of the entire swine population. For this purpose, 10 faecal samples were collected from 10 different pens containing pigs close to market weight in a total of 232 fattening units. Total sample size was proportionally distributed according to the fattener census in each of the regions of the country and all the samples were examined by culture of 25 g of faecal material. One hundred (43.1%) farms had at least one Salmonella-positive sample (95% CI: 37-49.1%). Salmonella enterica was detected in 290 (12.5%) pooled faecal floor samples (95% CI: 11.2-13.8%). The apparent herd prevalence of salmonellosis was similar among multi-site, finishing and farrow to finish farms. Overall, 24 different serovars were identified, with S. Typhimurium, S. Rissen and S. Derby being the most common both at herd and sample level. Results of phage typing were available for the 91 isolates of S. Typhimurium. A total number of 10 different phage types were identified, with DT 193 being the most frequent. Phage types DT 104, DT 104b and DT U302, which have been associated with several multi-resistant patterns, accounted for 23% and 29% of the Typhimurium total isolates or Typhimurium infected farms respectively.     

The dual role of wild phages for horizontal gene transfer among Salmonella strains

Salmonella bacteriophages seem to mediate horizontal transfer of virulence functions among Salmonella strains in two different ways: by general transduction and also by lysogenic conversion. The majority of wild phages isolated from Salmonella strains belong to the P22 like phages and were able to transduce. Our data show that the lysogenic conversion is generally accompanied by changes in the susceptibility to the typing phages used for epidemiological purposes. Similar phage type conversions to S. Typhimurium DT104 could be detected upon lysogenization with two other S. Typhimurium strains. For some S. Typhimurium strains the typical phage pattern is actually associated with alterations of virulence characteristics. For example, all tested wild type isolates of phage types DT49 and DT204 were found to be SopE phi-lysogens. The Anderson typing phages interfere with the prophages and/or cryptic phages and so the complex genetic short-term evolution can be demonstrated in the lab. This is one reason for the successful application of phage typing in Salmonella epidemiology since the 50s.     

Prevalence, serovars, phage types, and antibiotic susceptibilities of Salmonella strains isolated from animals in the United Arab Emirates from 1996 to 2009

The aim of this study was to give some insights into the prevalence, serovars, phage types, and antibiotic resistances of Salmonella from animal origin in the United Arab Emirates. Data on diagnostic samples from animals (n?=?20,871) examined for Salmonella between 1996 and 2009 were extracted from the databases of the Central Veterinary Research Laboratory in Dubai and from typed strains (n?=?1052) from the Robert Koch Institute, Wernigerode Branch in Germany and analyzed for general and animal-specific trends. Salmonella was isolated from 1,928 (9 %) of the 20,871 samples examined. Among the 1,052 typed strains, most were from camels (n?=?232), falcons (n?=?166), bustards (n?=?101), antelopes (n?=?66), and horses (n?=?63). The predominant serovars were Salmonella Typhimurium (25 %), Salmonella Kentucky (8 %), followed by Salmonella Frintrop (7 %), and Salmonella Hindmarsh (5 %). When analyzed by animal species, the most frequent serovars in camels were Salmonella Frintrop (28 %) and Salmonella Hindmarsh (21 %), in falcons Salmonella Typhimurium (32 %), in bustards Salmonella Kentucky (19 %), in antelopes Salmonella Typhimurium (9 %), and in horses Salmonella Typhimurium (17 %) and S. Kentucky (16 %). Resistance of all typed Salmonella strains (n?=?1052) was most often seen to tetracycline (23 %), streptomycin (22 %), nalidixic acid (18 %), and ampicillin (15 %). These data show trends in the epidemiology of Salmonella in different animal species which can be used as a base for future prevention, control, and therapy strategies.     

Salmonella spp. in Bavarian liquid pig manure: occurrence and relevance for the distribution of antibiotic resistance

In a representative study, 380 manure samples of pig farms distributed all over Bavaria (Germany) were screened for the presence of Salmonella spp. at the time of manure application to soil using methods adapted to DIN EN ISO 6579. The isolates were tested according to DIN 58940 for their susceptibility towards 26 antimicrobial substances - such substances partly administered in animals, but mainly used in human therapy. Six out of 380 manure samples and, in addition, the only separated liquid manure sample examined, contained Salmonella isolates. Serotypes represented S. Typhimurium (n = 5), S. Derby (n = 1) and S. Infantis/S. Serogroup C1-(6,7:r:-)-form (each n = 1). Within the serovar S. Typhimurium, the most common phage type was DT104 (n = 3). All Salmonella isolates originated from 'big' farms (>30/45 sows on combined/breeding farms or >220 fattening pigs). Four out of six manure samples contained resistant isolates; all resistant Salmonellae were multi-resistant. Two out of three DT104-isolates showed the typical penta-resistance pattern often found in S. Typhimurium DT104, which was, furthermore, supplemented by other resistances. One Escherichia coli isolated together with a phage type DT104 S. Typhimurium also expressed the same penta-resistance pattern.     

Epidemiology of Salmonella enterica serovars enteritidis and Typhimurium in animals and people in Scotland between 1990 and 2001

Two serovars of salmonella which are currently of particular importance in both human and animal infections are Salmonella enterica serovars Enteritidis phage type 4 (PT4) and Typhimurium definitive type 104 (DT104). This paper describes the trends in the relationships between the levels of infection of people and a range of farm animal species with these two serovars and explores some of the reasons behind them. In 1996, there was a peak of 520 reports of S Typhimurium DT104 infection in people in Scotland, but the number has decreased every year since, to 96 in 2001. In cattle the incidence of S Typhimurium DT104 also peaked in 1996, with 138 incidents, and it has similarly decreased every year to 2001 when there were 10 reported incidents. Similar declines have been observed in its incidence in sheep and pigs. In people the number of reports of S Enteritidis PT4 peaked in 1997 at 1684 and then declined to 457 in 2001. In chickens, the number of reports of S Enteritidis PT4 peaked in 1998 at 34 incidents, but no incidents were reported in the following three years.