DT104 Strains Research Articles

Multidrug-resistantSalmonellaJava

Multidrug-resistant<i>Salmonella</i>Java

Overexpression of the Multidrug Efflux Operon acrEF by Insertional Activation with IS 1 or IS 10 Elements in Salmonella enterica Serovar Typhimurium DT204 acrB Mutants Selected with Fluoroquinolones

High-level fluoroquinolone (FQ) resistance in Salmonella enterica serovar Typhimurium phage type DT204 has been previously shown to be essentially due to both multiple target gene mutations and active efflux by the AcrAB-TolC efflux system. In this study we show that in intermediatly resistant acrB-inactivated serovar Typhimurium DT204 mutants, high-level resistance to FQs can be restored on in vitro selection with FQs. In each FQ- resistant mutant selected from serovar Typhimurium DT204 acrB mutant strains, an insertion sequence (IS1 or IS10) was found integrated upstream of the acrEF operon, coding for AcrEF, an efflux pump highly homologous to AcrAB. In one of the strains, transposition of IS1 caused partial deletion of acrS, the putative local repressor gene of the acrEF operon. Sequence analysis showed that both IS1 and IS10 elements contain putative promoter sequences that might alter the expression of adjacent acrEF genes. Indeed, reverse transcription-PCR experiments showed an 8- to 10-fold increase in expression of acrF in these insertional mutants, relative to their respective parental strain, which correlated well with the resistance levels observed to FQs and other unrelated drugs. It is noteworthy that AcrEF did not contribute to the intrinsic drug resistance of serovar Typhimurium, since acrF deletion in wild-type strains did not result in any increase in drug susceptibility. Moreover, deletion of acrS did not cause any acrF overexpression or any decrease in drug susceptibility, suggesting that acrEF overexpression is mediated solely by the IS1 and IS10 promoter sequences and not by inactivity of AcrS. Southern blot experiments showed that the number of chromosomal IS1 and IS10 elements in the serovar Typhimurium DT204 genome was about 5 and 15 respectively. None were detected in epidemic serovar Typhimurium DT104 strains or in the serovar Typhimurium reference strain LT2. Carrying IS1 and/or IS10 elements in their chromosome may thus be a selective advantage for serovar Typhimurium DT204 strains as opposed to DT104 strains for which no high-level FQ resistance nor insertional mutations were found. Taken together, the results of the present study indicate that the IS1- or IS10- activated AcrEF efflux pump may relay AcrAB in serovar Typhimurium, and underline the importance of transposable elements in the acquisition of FQ and multidrug resistance.

AcrAB-TolC directs efflux-mediated multidrug resistance in Salmonella enterica serovar typhimurium DT104.

Multidrug-resistant Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) strains harbor a genomic island, called Salmonella genomic island 1 (SGI1), which contains an antibiotic resistance gene cluster conferring resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides, and tetracyclines. They may be additionally resistant to quinolones. Among the antibiotic resistance genes there are two, i.e., floR and tet(G), which code for efflux pumps of the major facilitator superfamily with 12 transmembrane segments that confer resistance to chloramphenicol-florfenicol and the tetracyclines, respectively. In the present study we determined, by constructing acrB and tolC mutants, the role of the AcrAB-TolC multidrug efflux system in the multidrug resistance of several DT104 strains displaying additional quinolone resistance or not displaying quinolone resistance. This study shows that the quinolone resistance and the decreased fluoroquinolone susceptibilities of the strains are highly dependent on the AcrAB-TolC efflux system and that single mutations in the quinolone resistance-determining region of gyrA are of little relevance in mediating this resistance. Overproduction of the AcrAB efflux pump, as determined by Western blotting with an anti-AcrA polyclonal antibody, appeared to be the major mechanism of resistance to quinolones. Moreover, chloramphenicol-florfenicol and tetracycline resistance also appeared to be highly dependent on the presence of AcrAB-TolC, since the introduction of mutations in the respective acrB and tolC genes resulted in a susceptible or intermediate resistance phenotype, according to clinical MIC breakpoints, despite the presence of the FloR and Tet(G) efflux pumps. Resistance to other antibiotics, ampicillin, streptomycin, and sulfonamides, was not affected in the acrB and tolC mutants of DT104 strains harboring SGI1. Therefore, AcrAB-TolC appears to direct efflux-mediated resistance to quinolones, chloramphenicol-florfenicol, and tetracyclines in multidrug-resistant S. enterica serovar Typhimurium DT104 strains.

Detection of integrons and antibiotic-resistance genes in Salmonella enterica serovar Typhimurium isolates with resistance to ampicillin and variable susceptibility to amoxicillin-clavulanate

We characterized 29 antimicrobial-resistant Salmonella enterica serovar Typhimurium strains, including four belonging to the monophasic variant 4,5,12:i:–, mostly isolated from infants. They were selected from 3230 strains isolated in the years 1990–2001 on the basis of resistance to ampicillin and variable susceptibility to the amoxicillin-clavulanate combination. Twenty-three strains were resistant to more than four antibiotics. All the strains carried the bla TEM gene and most were able to transfer this gene by conjugation. Sequencing of the gene from one of the amoxicillin-clavulanate-resistant strains allowed identification of the encoded β-lactamase as TEM-1; all of these strains carried a second gene encoding β-lactamase production, either pse-1 or oxa1. However, the association of bla TEM plus pse-1 genes did not always confer resistance to amoxicillin-clavulanate. The pse-1 gene, found in 17 strains, was located in the Salmonella Genomic Island-1 (SGI1), which carries two integrons and encodes multiple drug-resistance. None of the oxa1-bearing strains had the SGI1, yet this gene was found as part of an integron that also carried the aadA1 gene and was not plasmid-associated. Thirteen of the strains harbouring SGI1 belonged to the definitive phage type (DT) 104, and most of those remaining to DT104b and U302; particularly, strains carrying the oxa1-aadA1 integron belonged to the last two phage types. Pulsed field electrophoresis confirmed the clonal organization of DT104 strains, whereas U302 strains fell into different groups, depending on their resistance determinants.

Analysis of integrons in human isolates of Salmonella enterica serovar typhimurium isolated in the Slovak Republic

About 110 sporadic, epidemiologically unrelated Salmonella enterica serovar typhimurium strains isolated in the Slovak Republic were analyzed for the presence of integrons. Of these 110 examined strains, 47 were of definitive phage type DT104 and 63 were strains of various phage type, RDNC and untypeable, designated here as non-DT104 strains. All isolates were also tested for antimicrobial resistance to 10 antibiotics as well as for the presence of virulence plasmid. Of 63 non-DT104 strains, 15 isolates were multiple-resistant, independently from phage type, other strains were resistant to one, two or three drugs. Resistance to ampicillin, streptomycin, tetracycline and sulfisoxazole was most frequently observed. Among the DT104 isolates up 65.9% exhibited characteristic pentaresistance – ACSSuT phenotype. The integron content was studied in PCR experiments using a 5′-CS/3′-CS primer pair. Fourteen non-DT104 strains, independently from phage type, were found to carry integrons with amplicons 650–1900 bp in size. Thirty-six DT104 strains contained integrons of 1000 and 1200 bp and 31 of they exhibited the ACSSuT phenotype. No integron was found in 10 DT104 strains, which included strains mostly resistant only to streptomycin, tetracycline and sulfisoxazole. The majority of non-DT104 strains did not possess any integrons. Our findings show the widespread existence of both resistant and multiple-resistant epidemiologically unrelated Salmonella typhimurium strains and suggest that integrons contribute to this antimicrobial resistance. The presence of 90-kb virulence plasmid in the 54 non-DT104 and in the all DT104 strains was found.

Oral vaccination of pigs with an invasive gyrA- cpxA-rpoB Salmonella Typhimurium mutant

The potency to protect pigs against colonization and against clinical salmonellosis was evaluated after oral immunization with a live gyrA-cpxA-rpoB Salmonella ( S.) Typhimurium mutant ( S. Tm. Nal2/Rif9/Rtt). Twenty 4-week-old male hybrid piglets were immunized orally, a control group received a placebo. Three weeks postimmunization, all pigs were challenged orally with a highly virulent S. Typhimurium DT104 strain. Clinical investigation revealed that immunization prevented the vaccinated pigs from clinical symptoms of salmonellosis. While all placebo-treated animals showed a 2–4-day episode of moderate to severe clinical symptoms, 90% of immunized pigs did not show any clinical signs at all. The bacteriological results showed a marked beneficial effect of the oral immunization. Vaccinated pigs showed a significantly decreased rate of colonization of the inner organs (42.5% versus 87.5%) when compared to the placebo-treated animals. Furthermore, in comparison to the non-immunized pigs, the vaccines developed a higher specific immunoglobulin (Ig)A antibody activity, but a significant lower IgM antibody activity in serum. The findings underline the ability of an attenuated oral live S. Typhimurium mutant to prevent clinical symptoms of salmonellosis in pigs and to significantly reduce the colonization of tissues and inner organs, as well as the shedding of S. Typhimurium.

Molecular Weight of Chitosan Influences Antimicrobial Activity in Oil-in-Water Emulsions

The objective of this study was to evaluate the antimicrobial efficiency of chitosans in oil-in-water emulsions. Model emulsions were prepared with 20% corn oil, 1.5% Tween 20, 1.5% Trypticase soy broth, 0.58% acetic acid, and chitosan polysaccharide or chitosan oligosaccharide in concentrations of 0, 0.1, 0.2, 0.5, and 0.7%. A control containing HCl was included to determine the role of acetic acid in the overall antibacterial activity. The pH of samples and controls was adjusted to 4.5. Emulsions were inoculated with Listeria monocytogenes (strains Scott A and 310) or Salmonella Typhimurium DT104 (strains 2486 and 2576) at a level of 107 CFU/ml. Inoculated emulsions were incubated at 10 and 25° C for 4 days and analyzed for bacterial count every 24 h. Both tested Salmonella strains were more susceptible to acetic acid than Listeria. However, L. monocytogenes was more affected by chitosan than either Salmonella strain. During the storage at 25° C, initial inoculum in the emulsions with 0.58% acetic acid and 0.1% chitosan polysaccharide was reduced to below the detection limits after 24, 48, 72, or 96 h for L. monocytogenes 310, Salmonella Typhimurium DT104 2576, Salmonella Typhimurium DT104 2486, or L. monocytogenes Scott A, respectively. Chitosan oligosaccharide was less effective against all tested bacteria and showed a concentration-dependent effect. The antimicrobial efficacy of chitosan was reduced at 10° C, and reduction of microbial loads was delayed for approximately 24 h compared with 25° C. Results suggest that addition of 0.1% chitosan polysaccharidewould be sufficient to ensure the microbial safety of oil-in-water emulsions regardless of storage temperature.

Six modern plagues and how we are causing them

Every time we sneeze, there seems to be a new form of flu: bird flu, swine flu, Spanish flu, Hong Kong flu, H5N1, and most recently, H5N7. While these diseases appear to emerge from thin air, in fact, human activity is driving them. And the problem is not just flu, but a series of rapidly evolving and dangerous modern plagues. According to veterinarian and journalist Mark Walters, we are contributing to, if not overtly causing, some of the scariest epidemics of our time. Through human stories and cutting-edge science, Walters explores the origins of seven diseases: Mad Cow Disease, HIV/AIDS, Salmonella DT104, Lyme Disease, Hantavirus, West Nile, and new strains of flu. He shows that they originate from manipulation of the environment, from emitting carbon and clear-cutting forests to feeding naturally herbivorous cows recycled animal protein. Readers will both learn how today's plagues first developed and discover patterns that could help prevent the diseases of tomorrow.

Role of TolC and parC mutation in high-level fluoroquinolone resistance in Salmonella enterica serotype Typhimurium DT204.

To study the role of TolC and of parC mutation in high-level fluoroquinolone resistance in clonal clinical strains of Salmonella enterica serotype Typhimurium phage type DT204 (S. Typhimurium DT204). Deletion of the tolC gene (DeltatolC) was first performed in a susceptible S. Typhimurium DT104 strain lacking target gene mutations involved in fluoroquinolone resistance. P22 transduction was further used to transduce DeltatolC from this strain to a high-level fluoroquinolone-resistant S. Typhimurium DT204 strain carrying several target gene mutations, including one in parC (ciprofloxacin MIC of 32 mg/L). Deletion of tolC in the high-level fluoroquinolone-resistant S. Typhimurium DT204 strain resulted in the same decrease in resistance levels (16- to 32-fold) as shown previously for an acrB mutant of the same strain, suggesting that AcrAB-TolC is the main efflux system involved in high-level fluoroquinolone resistance of S. Typhimurium DT204 strains. In some S. Typhimurium DT204 DeltatolC transductants, concomitant loss of the parC (Ser-80-->Ile) mutation, located approximately 9.3 kb upstream of tolC, resulted in a further 16- to 32-fold decrease in resistance levels to fluoroquinolones and thus a hypersusceptible phenotype (ciprofloxacin MIC of 0.063 mg/L). The AcrAB-TolC efflux system, together with multiple target gene mutations, including the parC mutation, appear essential to confer high-level fluoroquinolone resistance in S. Typhimurium DT204.

Effect of a 5 day enrofloxacin treatment on Salmonella enterica serotype Typhimurium DT104 in the pig.

There are concerns that the use of enrofloxacin in livestock production may contribute to the development of fluoroquinolone resistance in zoonotic bacteria. The objective of our study was to investigate the effect of a single 5 day enrofloxacin treatment on Salmonella enterica serotype Typhimurium DT104 in a pig model. Our results showed that a single treatment failed to eradicate S. Typhimurium DT104, which continued to be isolated up to 35 days after treatment. We also provide evidence that treatment positively selects for S. Typhimurium DT104 strains that are already nalidixic acid resistant (gyrA Asn-87) or cyclohexane resistant, the latter being indicative of an up-regulated efflux pump. Emergence of fluoroquinolone resistance was not detected during treatment or post-treatment in any of the Salmonella strains monitored. However, the effect of enrofloxacin on the nalidixic acid-resistant and cyclohexane-resistant S. Typhimurium DT104 outlasted the current withdrawal time of 10 days for Baytril (commercial veterinary formulation of enrofloxacin). In conclusion, our study has provided direct evidence that enrofloxacin-treated pigs could be entering abattoirs with higher numbers of quinolone-resistant zoonotic bacteria than untreated pigs, increasing the risk of these entering the food chain.

Antimicrobial Drug–resistant Salmonella Typhimurium (Reply to Dahl)

In Reply to Dahl: The emergence and spread of multidrug-resistant Salmonella enterica serovar Typhimurium DT104 (MDR DT104) contributed to an international increase in antimicrobial drug resistance in S. Typhimurium in the late 1990s (1,2). This type of Salmonella is usually resistant to five drugs: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (R-type ACSSuT) and easily acquires resistance to other drugs, including quinolones, trimethoprim, and aminoglycosides (1,3–5). To determine death rates after infection with MDR DT104 or closely related strains, we identified patients who were infected with strains at least resistant to ACSSuT (6). Analysis limited to strains that were only R-type ACSSuT would have given a misleading result since MDR DT104 often, as mentioned, develops additional resistance to other classes of antimicrobial drugs in addition to the ACSSuT-complex. This fact needs to be taken into account in any attempt to quantify the overall public health impact of MDR DT104 and related strains. We found, in our matched cohort study (6), that 283 patients infected with strains resistant to at least ACSSuT were 4.8 times more likely to die than the general Danish population, compared with 2.3 for 953 patients infected with pansusceptible strains. This difference in death rates ocurred mainly because 40 of the 283 strains had R-type ACSSuTNx (i.e., additional resistance to nalidixic acid), and infection with this strain in particular is associated with a high death rate (relative mortality 13.1). As Dahl suggests, infection with R-type ACSSuT (Nx susceptible) was not associated with excess mortality in the 243 patients included in the analysis, and the measured effect of ACSSuT was achieved by the inclusion of the nalidixic acid–resistant strains in this group. However, all deaths associated with nalidixic acid–resistant strains occurred in the 40 patients with R-type ACSSuTNx (being DT104s), whereas none of the 43 patients infected with non-ACSSut strains resistant to nalidixic acid died. This finding may be related to small numbers in these subanalyses. However, because 25 of the patients with R-type ACSSuTNx were part of an outbreak, they may have had an average higher exposure dose, which may have contributed to some deaths (3). In addition, an interaction between different resistance traits in Salmonella may exist, which may lead to more deaths and disease, or DT104 may be somewhat more virulent than most other S. Typhimurium subtypes. The database that we used for our analysis was updated in May 2002. We have now identified 13 deaths in 342 patients infected with strains resistant to ACSSuT (but Nx susceptible), which corresponds to a relative mortality rate of 4.18 (95% confidence interval [CI]) 2.18 to 8.02) compared with a matched sample of the general population. Of 1,432 patients infected with pansusceptible strains, 43 patients died (relative mortality rate 2.64; 95% CI 1.88 to 3.70). In other words, the mortality rate in patients infected with strains resistant to ACSSuT (Nx susceptible) was 1.6 times higher than in patients with pansusceptible strains (p value for homogeneity 0.22). These estimates were not adjusted for coexisting conditions as were the estimates in the paper (6). We agree with Dahl that particular problems are associated with quinolone resistance in zoonotic salmonellae and that fluoroquinolones may have reduced efficacy to treat patients infected with Salmonella strains that are nalidixic acid (quinolone) resistant (7). We therefore encourage initiatives to preserve the efficacy of fluoroquinolones, including a limitation of their use in agriculture. Whether infection with S. Typhimurium R-type ACSSuT, with no additional resistance, is associated with higher disease or death rates than pansusceptible S. Typhimurium remains unclear. Although the difference was not significant (p=0.22), our recent estimates suggest that the death rate is approximately 60% higher in patients infected with such strains. This view is corroborated by recent studies from the United States, which suggest that S. Typhimurium R-type ACSSuT is associated with an increased risk for blood stream infection (8) and that resistance in nontyphoid Salmonella is associated with an increased risk for admission to hospital (9).

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.

Analysis of gene cassettes of streptomycin-spectinomycin resistance of Hungarian Salmonella enterica serotype Typhimurium strains.

By PCR using the ant(3")-Ia primer pair the aadA gene was detected in 34 streptomycin- and spectinomycin-resistant Salmonella enterica serotype Typhimurium strains. Out of them 12 belonged to DT104 and 22 to non-DT104 phage type. Using different primer combinations it was demonstrated that this gene was integron-associated in all cases: in the DT104 strains it was generally contained by a 1 kb integron while in the majority of the non-DT104 strains by a 2.05 kb (less often by a 1.9 or 1 kb) integron. In the case of integrons carrying multiple cassettes the cassette containing the aadA gene was located closer to the 3' end of the integron. The aadA genes of DT104 and non-DT104 strains were different: in the former group the aadA2 gene, while in the latter group (constituted by strains of five different phages types as well as unclassifiable and untypable strains) the aadA1 gene could be identified. The RH50/RH51 primer pair described by Collis and Hall (1992) proved to be suitable for rapid discrimination between the aadA1 and aadA2 genes on the basis that the RH51 primer bound exclusively to the aadA2 gene.

Antibiotic resistance genes and Salmonella genomic island 1 in Salmonella enterica serovar Typhimurium isolated in Italy.

Fifty-four epidemiologically unrelated multidrug-resistant Salmonella enterica serovar Typhimurium isolates, collected between 1992 and 2000 in Italy, were analyzed for the presence of integrons. Strains were also tested for Salmonella genomic island 1 (SGI1), carrying antibiotic resistance genes in DT104 strains. A complete SGI1 was found in the majority of the DT104 strains. Two DT104 strains, showing resistance to streptomycin-spectinomycin and sulfonamides, carried a partially deleted SGI1 lacking the flo(st), tetR, and tetA genes, conferring chloramphenicol-florfenicol and tetracycline resistance, and the integron harboring the pse-1 gene cassette, conferring ampicillin resistance. The presence of SGI1 was also observed in serovar Typhimurium strains belonging to other phage types, suggesting either the potential mobility of this genomic island or changes in the phage-related phenotype of DT104 strains.

Characterization of VariantSalmonellaGenomic Island 1 Multidrug Resistance Regions from Serovars Typhimurium DT104 and Agona

Strains of multidrug-resistant Salmonella enterica serovar Typhimurium DT104 (DT104) and S. enterica serovar Agona (Agona) have been found to harbor Salmonella genomic island 1 (SGI1), a 43-kb genomic region that contains many of the drug resistance genes. Such strains are resistant to ampicillin (pse-1), chloramphenicol/florfenicol (floR), streptomycin/spectinomycin (aadA2), sulfonamides (sul1), and tetracycline [tet(G)] (commonly called the ACSSuT phenotype). All five resistance genes are found in a 13-kb multidrug resistance (MDR) region consisting of an unusual class I integron structure related to In4. We examined DT104 and Agona strains that exhibited other resistance phenotypes to determine if the resistance genes were associated with variant SGI1 MDR regions. All strains were found to harbor variant SGI1-like elements by using a combination of Southern hybridization, PCR mapping, and sequencing. Variant SGI1-like elements were found with MDR regions consisting of (i) an integron consisting of the SGI1 MDR region with the addition of a region containing a putative transposase gene (orf513) and dfrA10 located between duplicated qacEDelta1/sulI genes (SGI1-A; ACSSuTTm); (ii) an integron with either an aadA2 (SSu) or a pse-1 (ASu) cassette (SGI1-C and SGI1-B, respectively); (iii) an integron consisting of the SGI1-C MDR region plus an orf513/dfrA10 region as in SGI1-A (SGI1-D; ASSuTm; ampicillin resistance due to a TEM beta-lactamase); and (iv) an integron related to that in SGI1 but which contains a 10-kb inversion between two copies of IS6100, one which is inserted in floR (SGI1-E; ASSuT). We hypothesize that the MDR of SGI1 is subject to recombinational events that lead to the various resistance phenotypes in the Salmonella strains in which it is found.

Diversity in Antimicrobial Resistance and Other Characteristics AmongSalmonellaTyphimurium DT104 Isolates

Multiresistant Salmonella enterica subspecies enterica serovar Typhimurium definitive type 104 (S. Typhimurium DT104 or DT104) bacteria are important pathogens in animals and humans. DT104 isolates are often called pentaresistant strains that spread clonally. The purpose of this study was to determine phenotypic, genotypic, and epidemiologic characteristics of 175 S. Typhimurium DT104 strains isolated from food-producing animals in Canada. More than 90% of the isolates were resistant to ampicillin (Amp), chloramphenicol (Chl), florfenicol (Flo), sulfisoxazole (Sul), and tetracycline (Tet), 53% of the isolates were additionally resistant to spectinomycin (Spc) and streptomycin (Str), and 28% to kanamycin (Kan) and neomycin (Neo). Sixty-one percent of the strains harbored a single 60-MDa plasmid, 21% contained 60- and 2.0-MDa plasmids, and 4% had 60, 4.6- and 2.0-MDa plasmids. Resistance to Kan and Neo was encoded by the aminoglycoside aphA-1 gene on 2.0-MDa plasmids, whereas resistance to trimethoprim (Tmp) and Sul was encoded by the dhfrIb gene on 4.6-MDa plasmids. Polymerase chain reactions (PCR) showed the presence of integrons with the ant (3")-Ia aminoglycoside adenyltransferase and the bla(PSE-1) beta-lactamase gene cassettes, and the presence of the flost gene in all but one strain resistant to Spc and Str, Amp, and Chl and Flo, respectively. DT104 isolates from cattle at six feedlots represented a separate clone; they were sensitive to Str and Spc and lacked the ant (3")-Ia gene. Pulsed-field gel electrophoresis (PFGE) using Bln I, Spe I, and Xba I resulted in 15, 12, and 8 PFGE patterns, respectively. In summary, we observed considerable diversity in phenotypic, genotypic, and epidemiological characteristics among the DT104 isolates.

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 1.6 kb 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 2415 bp 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.

Thermal Inactivation of Salmonella spp., Salmonella typhimurium DT104, and Escherichia coli 0157:H7 in Ground Beef

ABSTRACT: In 19.1% fat ground beef, Escherichia coli 0157:H7 was less heat‐ resistant at ≥58°C than the Salmonella typhimurium DT104 and Salmonella senftenberg, but at 55°C the D value was similar to DT104 strains and higher than an eight‐strain Salmonella cocktail. Inactivation of E. coli 0157:H7 was more temperature‐dependent than the cocktail and DT104 strains. E. coli and DT104 strains were more heat‐resistant in beef containing 19% fat than 4.8% fat. The cocktail was more thermally stable in stationary as compared to log phase. Freezing of inoculated raw meat decreased heat resistance of the cocktail. The pathogenic strain, growth phase of the organism, state of the meat (fresh or frozen) and meat composition must be considered when designing protocols to verify thermal processes.

Sample preparation methods for PCR detection of Escherichia coli O157:H7, Salmonella typhimurium, andListeria monocytogenes on beef chuck shoulder using a single enrichment medium

To improve the utility of the polymerase chain reaction (PCR) for food samples, methods for preparing template DNA were developed to remove PCR inhibitors. Beef chuck shoulder medallions, artificially contaminated, individually or in combination, with Escherichia coli serotype O157:H7 strain FSIS 45753-35, Salmonella typhimurium DT104 strain 13HP, or Listeria monocytogenes strain Scott A at concentrations of 10, 1 and 0·5 cfu/cm2were swabbed with a sponge, and the sponges were enriched for 18 h at 37°C in universal pre-enrichment broth (UPB). Enriched broth cultures (EBC), cell pellets (CP), or phosphate-buffered saline–washed cell pellets (PBSCP) from enriched sponge samples were compared for detection of E. coli O157:H7, S. typhimurium DT104, or L. monocytogenes by the PCR using the BAXTMsystem. Recovery of the three organisms was effective for detection of each pathogen at initial levels of 10, 1 and 0·5 cfu/cm2when inoculated separately, or in combination, onto the beef samples. Use of EBC, CP, or PBSCP of sponge-swabbed samples eliminated problems associated with inhibition of the PCR by food components, time-consuming extraction of DNA, and inhibition due to large amounts of non-target DNA derived from the food. The procedure involving enrichment of sponge-swabbed beef samples in UPB followed by PCR amplification using EBC with the BAXTMsystem is the most efficient and simple method for detection of E. coli O157:H7, S. typhimurium DT104, and L. monocytogenes.

Effect of challenge temperature and solute type on heat tolerance of Salmonella serovars at low water activity.

Salmonella spp. are reported to have an increased heat tolerance at low water activity (a(w); measured by relative vapor pressure [rvp]), achieved either by drying or by incorporating solutes. Much of the published data, however, cover only a narrow treatment range and have been analyzed by assuming first-order death kinetics. In this study, the death of Salmonella enterica serovar Typhimurium DT104 when exposed to 54 combinations of temperature (55 to 80 degrees C) and a(w) (rvp 0.65 to 0.90, reduced using glucose-fructose) was investigated. The Weibull model (LogS = -bt(n)) was used to describe microbial inactivation, and surface response models were developed to predict death rates for serovar Typhimurium at all points within the design surface. The models were evaluated with data generated by using six different Salmonella strains in place of serovar Typhimurium DT104 strain 30, two different solutes in place of glucose-fructose to reduce a(w), or six low-a(w) foods artificially contaminated with Salmonella in place of the sugar broths. The data demonstrate that, at temperatures of > or =70 degrees C, Salmonella cells at low a(w) were more heat tolerant than those at a higher a(w) but below 65 degrees C the reverse was true. The same patterns were generated when sucrose (rvp 0.80 compared with 0.90) or NaCl (0.75 compared with 0.90) was used to reduce a(w), but the extent of the protection afforded varied with solute type. The predictions of thermal death rates in the low-a(w) foods were usually fail-safe, but the few exceptions highlight the importance of validating models with specific foods that may have additional factors affecting survival.