M. A. Usera

Serotypes, phage types and virulence genes of Shiga-producing Escherichia coli isolated from sheep in Spain

PROBLEM ADDRESSED Shiga toxin-producing Escherichia coli (STEC), have emerged as food poisoning pathogens which can cause severe diseases in humans. OBJECTIVE The aim of this study was to determinate the serotypes and virulence genes of STEC strains isolated from sheep in Spain, with the purpose of determining whether sheep represent a potential source of STEC pathogenic for humans. METHODS AND APPROACH Faecal swabs obtained from 697 healthy lambs on 35 flocks in Spain during the years 2000 and 2001 were examined for STEC using phenotypic (Vero cells) and genotypic (PCR) methods. RESULTS STEC O157:H7 strains were isolated from seven (1%) animals in six flocks, whereas non-O157 STEC strains were isolated from 246 (35%) lambs in 33 flocks. A total of 253 ovine STEC strains were identified in this study. PCR showed that 110 (43%) strains carried stx(1) genes, 10 (4%) possessed stx(2) genes and 133 (53%) both stx(1) and stx(2). Enterohaemolysin (ehxA) and intimin (eae) virulence genes were detected in 120 (47%) and in 9 (4%) of the STEC strains. STEC strains belonged to 22 O serogroups and 44 O:H serotypes. However, 70% were of one of these six serogroups (O6, O91, O117, O128, O146, O166) and 71% belonged to only nine serotypes (O6:H10, O76:H19, O91:H-, O117:H-, O128:H-, O128:H2, O146:H21, O157:H7, O166:H28). A total of 10 new O:H serotypes not previously reported in STEC strains were found in this study. Seven strains of serotype O157:H7 possessed intimin type gamma1, and two strains of serotype O156:H- had the new intimin zeta. STEC O157:H7 strains were phage types 54 (four strains), 34 (two strains) and 14 (one strain). CONCLUSIONS This study confirms that healthy sheep are a major reservoir of STEC pathogenic for humans. However, because the eae gene is present only in a very small proportion of ovine non-O157 STEC, most ovine strains may be less pathogenic.

Serotypes and virulence genes of bovine Shigatoxigenic Escherichia coli (STEC) isolated from a feedlot in Argentina

Grazing-fed cattle were previously demonstrated to be reservoir of non-O157 Shigatoxigenic Escherichia coli (STEC) serotypes in Argentina. The acid-resistance of some STEC strains makes it reasonable to assume the presence in feedlot of particular STEC serotypes. Fifty-nine animals were sampled every 2 weeks during 6 months by rectal swabs. Twenty-seven of 59 animals (45.8%) were shown to be Stx2(+); 3/59 (5.1%) carried Stx1(+) and 7/59 (11.9%) were Stx1(+) Stx2(+). Among 44 STEC isolates, 31 isolates were associated to 10 O serogroups (O2, O15, O25, O103, O145, O146, O157, O171, O174, O175) and 13 were considered non-typable (NT). Six H antigens (H2, H7, H8, H19, H21, H25) were distributed in 21 isolates whereas 23 were non-mobile (H-). Seventeen of 44 strains (38.6%) were eaeA(+) and 14 (31.8%) harbored the 60MDa plasmid. The megaplasmid (Mp) and eaeA gene were simultaneously found in a limited number of serotypes belonging to the enterohaemorrhagic E. coli (EHEC). E. coli O157:H7 strains, isolated from four (6.8%) animals, corresponded to the Stx2(+), eaeA(+), Mp(+) pattern. Three O157:H7 strains belonged to phage type 4 and the other strain was atypical. Many serotypes isolated from grain-fed cattle (O2:H25, O15:H21, O25:H19, O145:H-, O146:H-, O146:H21, O157:H7, O175:H8) also differed from those isolated by us previously from grazing animals. The serotypes O15:H21, O25:H19 and O175:H8 had not been identified at present as belonging to STEC. This work provides new data for the understanding of the ecology of STEC in grain-fed cattle and confirms that cattle are an important reservoir of STEC.

Atypical, fljB-negative Salmonella enterica subsp. enterica strain of serovar 4,5,12:i:- appears to be a monophasic variant of serovar Typhimurium.

ABSTRACT An fljB-negative, multidrug-resistantSalmonella enterica serovar 4,5,12:i:− phage type DT U302 strain (resistant to ampicillin, chloramphenicol, sulfonamide, gentamicin, streptomycin, tetracycline, and sulfamethoxazole-trimethoprim) emerged and spread in Spain in 1997. Sequences specific for Salmonella serovar Typhimurium and phage type DT 104 and U302 were present in this atypicalSalmonella strain, suggesting that it is a monophasicSalmonella serovar Typhimurium variant.

Verotoxin-producing Escherichia coli in Spain: prevalence, serotypes, and virulence genes of O157:H7 and non-O157 VTEC in ruminants, raw beef products, and humans.

In Spain, as in many other countries, verotoxin-producing Escherichia coli (VTEC) strains have been frequently isolated from cattle, sheep, and foods. VTEC strains have caused seven outbreaks in Spain (six caused by E. coli O157:H7 and one by E. coli O111:H– [nonmotile]) in recent years. An analysis of the serotypes indicated serological diversity. Among the strains isolated from humans, serotypes O26:H11, O111:H–, and O157:H7 were found to be more prevalent. The most frequently detected serotypes in cattle were O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and OUT (O untypeable):H19. Different VTEC serotypes (e.g., O5:H–, O6:H10, O91:H–, O117:H–, O128:H–, O128:H2, O146:H8, O146:H21, O156:H–, and OUT:H21) were found more frequently in sheep. These observations suggest a host serotype specificity for some VTEC. Numerous bovine and ovine VTEC serotypes detected in Spain were associated with human illnesses, confirming that ruminants are important reservoirs of pathogenic VTEC. VTEC can produce one or two toxins (VT1 and VT2) that cause human illnesses. These toxins are different proteins encoded by different genes. Another virulence factor expressed by VTEC is the protein intimin that is responsible for intimate attachment of VTEC and effacing lesions in the intestinal mucosa. This virulence factor is encoded by the chromosomal gene eae. The eae gene was found at a much less frequency in bovine (17%) and ovine (5%) than in human (45%) non-O157 VTEC strains. This may support the evidence that the eae gene contributes significantly to the virulence of human VTEC strains and that many animal non-O157 VTEC strains are less pathogenic to humans.

DNA Microarray-Based Typing of an Atypical Monophasic Salmonella enterica Serovar

ABSTRACT A multidrug-resistant fljB-lacking Salmonella enterica serovar [4,5,12:i:−] emerged in Spain in 1997. We analyzed the genome from four strains of this serovar using a microarray containing almost all the predicted protein coding regions of serovar Typhimurium strain LT2, including the pSLT plasmid. Only a few differences from serovar Typhimurium LT2 were observed, suggesting the serovar to be Typhimurium as well. Six regions of interest were identified from the microarray data. Cluster I was a deletion of 13 genes, corresponding to part of the regulon responsible for the anaerobic assimilation of allantoin. Clusters II and IV were associated with the absence of the Fels-1 and Fels-2 prophage. Cluster III was a small group of Gifsy-1 prophage-related genes that appeared to be deleted or replaced. Cluster V was a deletion of 16 genes, including iroB and the operon fljAB, which is reflected in the serovar designation. Region VI was the gene STM2240, which appears to have an additional homologue in these strains. The regions spanning the deletions involving the allantoin operon and the fljAB operon were PCR amplified and sequenced. PCR across these regions may be an effective marker for this particular emergent serovar. While the microarray data for all isolates of the new serovar were essentially identical for all LT2 chromosomal genes, the isolates differed in their similarity to pSLT, consistent with the heterogeneity in plasmid content among isolates of the new serovar. Recent isolates have acquired a more-complete subset of homologues to this virulence plasmid. In general, microarrays can provide useful complementary data to other typing methods.

Multiplex PCR for Distinguishing the Most Common Phase-1 Flagellar Antigens of Salmonella spp.

ABSTRACT Most Salmonella serotypes alternatively express either phase-1 or phase-2 flagellar antigens, encoded by the fliC and fljB genes, respectively. Flagellar phase reversal for the identification of both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fliC genes encoding the H:i, H:r, H:l,v, H:e,h, H:z10, H:b, and H:d antigens have been sequenced; and the specific sites for each antigen in selected Salmonella serotypes have been determined. These results, together with flagellar G-complex variable internal sequences obtained by the Foodborne and Diarrheal Diseases Branch at the Centers for Disease Control and Prevention in Atlanta, Ga., have been used to design a multiplex PCR to identify the G-complex antigens as well as the H:i, H:r, H:l,v, H:e,h, Hz10, H:b, and H:d first-phase antigens. These antigens are part of the most common Salmonella serotypes possessing first-phase flagellar antigens. Salmonella enterica serotype Enteritidis is identified by adding a specific primer pair published previously (P. G. Agron, R. L. Walker, H. Kinde, S. J. Sawyer, D. C. Hayes, J. Wollard, and G. L. Andersen, Appl. Environ. Microbiol. 67:4984-4991, 2001). This multiplex PCR includes 13 primers. A total of 161 Salmonella strains associated with 72 different serotypes were tested. Each strain generated one first-phase-specific antigen fragment ranging from 100 to 500 bp; Salmonella serotype Enteritidis, however, generated two amplicons of 500 bp that corresponded to the G complex and a 333-bp serotype-specific amplicon, respectively. Twenty-three strains representing 19 serotypes with flagellar genes different from those targeted in this work did not generate any fragments. The method is quick, specific, and reproducible and is independent of the phase expressed by the bacteria when they are tested.

Multiplex PCR-based detection and identification of the most common Salmonella second-phase flagellar antigens.

Most Salmonella serotypes alternatively express phase 1 or phase 2 flagellar antigens encoded by fliC and fljB genes respectively. Flagellar phase reversal to identify both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fljB genes encoding H:1,w, H:e,n,x and H:e,n,z15 antigens have been sequenced and the specific sites for each antigen determined in selected Salmonella serotypes. These results, together with flagellar H1 complex variable internal sequences previously published, have been used to design a multiplex-PCR to identify H:1,2, H:1,5, H:1,6, H:1,7, H:1,w, H:e,n,x and H:e,n,z15 second-phase antigens. These antigens are part of the most common Salmonella serotypes possessing second-phase flagellar antigens. This multiplex-PCR includes 10 primers. A total of 140 Salmonella strains associated with 49 different serotypes were tested. Each strain generated one second-phase-specific antigen fragment, ranging between 50 and 400 bps. Twenty-five strains associated with 17 serotypes, with no second-phase antigen or with an antigen different from those tested in this work, did not generate any fragments. The method is quick, specific and reproducible and is independent of the phase expressed by the bacteria when tested.

Mechanism of Resistance to Several Antimicrobial Agents in Salmonella Clinical Isolates Causing Traveler's Diarrhea

ABSTRACT The evolution of antimicrobial resistance in Salmonella isolates causing travelers diarrhea (TD) and their mechanisms of resistance to several antimicrobial agents were analyzed. From 1995 to 2002, a total of 62 Salmonella strains were isolated from stools of patients with TD. The antimicrobial susceptibility to 12 antibiotics was determined, and the molecular mechanisms of resistance to several of them were detected as well. The highest levels of resistance were found against tetracycline and ampicillin (21 and 19%, respectively), followed by resistance to nalidixic acid (16%), which was mainly detected from 2000 onward. Molecular mechanisms of resistance were analyzed in 16 isolates. In these isolates, which were resistant to ampicillin, two genes encoding β-lactamases were detected: oxa-1 (one isolate) and tem-like (seven isolates [in one strain concomitantly with a carb-2]). Resistance to tetracycline was mainly related to tetA (five cases) and to tetB and tetG (one case each). Resistance to chloramphenicol was related to the presence of the floR and cmlA genes and to chloramphenicol acetyltransferase activity in one case each. Different genes encoding dihydrofolate-reductases (dfrA1, dfrA12, dfrA14, and dfrA17) were detected in trimethoprim-resistant isolates. Resistance to nalidixic acid was related to the presence of mutations in the amino acid codons 83 or 87 of the gyrA gene. Further surveillance of the Salmonella spp. causing TD is needed to detect trends in their resistance to antimicrobial agents, as we have shown in our study with nalidixic acid. Moreover, such studies will lead to better treatment and strategies to prevent and limit their spread.

Phage Types and Genotypes of Shiga Toxin-Producing Escherichia coli O157:H7 Isolates from Humans and Animals in Spain: Identification and Characterization of Two Predominating Phage Types (PT2 and PT8)

ABSTRACT Phage typing and DNA macrorestriction fragment analysis by pulsed-field electrophoresis (PFGE) were used for the epidemiological subtyping of a collection of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated in Spain between 1980 and 1999. Phage typing distinguished a total of 18 phage types among 171 strains isolated from different sources (67 humans, 82 bovines, 12 ovines, and 10 beef products). However, five phage types, phage type 2 (PT2; 42 strains), PT8 (33 strains), PT14 (14 strains), PT21/28 (11 strains), and PT54 (16 strains), accounted for 68% of the study isolates. PT2 and PT8 were the most frequently found among strains from both humans (51%) and bovines (46%). Interestingly, we detected a significant association between PT2 and PT14 and the presence of acute pathologies. A group of 108 of the 171 strains were analyzed by PFGE, and 53 distinct XbaI macrorestriction patterns were identified, with 38 strains exhibiting unique PFGE patterns. In contrast, phage typing identified 15 different phage types. A total of 66 phage type-PFGE subtype combinations were identified among the 108 strains. PFGE subtyping differentiated between unrelated strains that exhibited the same phage type. The most common phage type-PFGE pattern combinations were PT2-PFGE type 1 (1 human and 11 bovine strains), PT8-PFGE type 8 (2 human, 6 bovine, and 1 beef product strains), PT2-PFGE subtype 4A (1 human, 3 bovine, and 1 beef product strains). Nine (29%) of 31 human strains showed phage type-PFGE pattern combinations that were detected among the bovine strains included in this study, and 26 (38%) of 68 bovine strains produced phage type-PFGE pattern combinations observed among human strains included in this study, confirming that cattle are a major reservoir of strains pathogenic for humans. PT2 and PT8 strains formed two groups which differed from each other in their motilities, stx genotypes, PFGE patterns, and the severity of the illnesses that they caused.

Antibiotic Resistance of Salmonella spp. from Animal Sources in Spain in 1996 and 2000

Emergence of resistant and multiresistant bacteria has become an important worldwide sanitary problem. International agencies recommend improving resistance surveillance studies in not only human but also animal origin strains. Because of its ubiquitous characteristics and zoonotic agent consideration, Salmonella spp. can be used as a good indicator microorganism for resistance surveillance studies. Salmonella spp. strains from animal sources isolated in 1996 (107) and 2000 (474) in Spain were tested against 12 different antimicrobials agents, using the disc diffusion method. Results were interpreted following the NCCLS criteria. Data showed that Salmonella spp. strains (61.7% in 1996 and 81.5% in 2000) were resistant to at least one antibiotic. Pig-related strains were considerably more resistant than strains from other sources. Enteritidis serotype was less resistant than other serotypes, except for ampicillin in 1996 (50% resistant) and nalidixic acid in 2000 (65.1% resistant). An emergent monophasic serotype, 4,5,12:i:-, first detected in 1997 in Spain was 100% resistant and 90% multiresistant. Typhimurium serotype was the most common Salmonella serotype from animal sources in both years. It was widely distributed among animals and was among the serotypes with a higher degree of resistance. The ampicillin, chloramphenicol, sulfonamides, streptomycin, and tetracycline resistance pattern, commonly associated with Salmonella serotype Typhimurium DT 104, had spread among other Typhimurium phage types and other Salmonella serotypes. Salmonella spp. strains isolated from feeding stuffs were considerably more susceptible than animal source strains, suggesting that the high Salmonella spp. resistance percentage was probably due to the use of antibiotics in animal farms rather than the consumption of contaminated feeding stuffs.