Silvia Herrera-León

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.

Emergence of extended-spectrum β-lactamases and AmpC-type β-lactamases in human Salmonella isolated in Spain from 2001 to 2005

OBJECTIVES To study the resistance to third-generation cephalosporins in Salmonella strains isolated from humans in a 5 year period in Spain, and to identify the responsible genes and their dissemination. METHODS Twenty-seven isolates were analysed by PCR and sequencing to identify the genes responsible for the beta-lactamase resistance phenotypes. The transferability of the phenotypes was tested by conjugation to Escherichia coli K12J53, plasmid detection with S1-PFGE, hybridization and PCRs of the transconjugants. The genetic relationship was determined by PFGE. RESULTS We found bla(CTX-M-9) and bla(CTX-M-10) in Salmonella Virchow PT19. bla(CTX-M-14) was detected in Salmonella (IV) 44:z(4),z(23):-, Salmonella Enteritidis PT6a, Salmonella Typhimurium DT193 and Salmonella Typhimurium DT104B. bla(CTX-M-1) was found in Salmonella Litchfield. bla(CTX-M-15) and bla(CTX-M-32) were found in Salmonella Enteritidis PT1. bla(SHV-12) was found in Salmonella Blockley, Salmonella Hadar PT2, Salmonella Enteritidis PT21, Salmonella Enteritidis PT1 and Salmonella Bredeney. bla(SHV-2) was found in Salmonella Livingstone. bla(CMY-2) was detected in Salmonella Bredeney, Salmonella Newport, Salmonella Enteritidis PT5b and Salmonella Heidelberg. bla(DHA-1) was detected for the first time in Spain in Salmonella Newport. One strain of Salmonella Senftenberg harboured two extended-spectrum beta-lactamases, bla(SHV-12) and bla(CTX-M-9). We have found a large variety of beta-lactamase families as well as several members of major relevance, such as CTX-M-15, CTX-M-32, CMY-2 and DHA-1. XbaI-PFGE, conjugation assays and S1-PFGE hybridization showed that all these beta-lactamases were mediated by plasmids. CONCLUSIONS This study demonstrates the emergence of a public health risk related to resistance to beta-lactams in Salmonella. The resistance trends need to be monitored carefully.

Detection and characterisation of O157:H7 and non-O157 Shiga toxin-producing Escherichia coli in wild boars

The aim of this work was to determine the prevalence and characteristics of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) in free-ranging wild boars killed during the hunting season in southwest Spain. Faecal samples from 212 wild boars (Sus scrofa) were collected and examined for STEC. Characterisation of isolates was performed by PCR, serotyping, phage typing, and pulsed-field gel electrophoresis (PFGE). E. coli O157:H7 and non-O157 STEC were isolated from 7 (3.3%) and 11 (5.2%) animals, respectively, and the resulting 19 isolates were characterised. The PCR procedure indicated that 4 isolates carried the stx(1) gene, 12 carried the stx(2) gene, and 1 contained both of these genes. The ehxA, eae, and saa genes were detected in 13, 8, and 1 of the isolates, respectively. The eae-positive isolates comprised the types eae-gamma 1 and eae-zeta. The isolates belonged to 11 O:H serotypes, including 4 new serotypes not previously reported within STEC strains, and the majority of them were from serotypes previously associated with human infection. E. coli O157:H7 isolates belonged to phage types associated with severe human illness: PT14, PT34, and PT54. Indistinguishable PFGE types were found in E. coli O157:H7 isolates recovered from a wild boar and from a human patient with diarrhoea living in the same geographic area.

Salmonella Derby clonal spread from pork.

The genetic diversity of the Derby serotype of Salmonella enterica in Spain was examined by pulsed-field gel electrophoresis (PFGE). Out of 24 identified PFGE profiles, a major clone was detected in 19% of strains from humans, 52% from food, and 62% from swine. This clone (clone 1) was isolated from pork products, suggesting swine as its source.

Genetic evolution of the Spanish multidrug-resistant Salmonella enterica 4,5,12:i:- monophasic variant.

ABSTRACT We analyzed a collection of 60 Salmonella enterica 4,5,12:i:- phage type U302 multidrug-resistant monophasic variant strains, isolated in Spain between 2000 and 2007. Most strains showed resistance to ampicillin (A), chloramphenicol (C), sulfamethoxazole (Su), gentamicin (G), streptomycin (S), tetracycline (T), and co-trimoxazole (SxT) (an ACSuGSTSxT resistance pattern). Only one pulsed-field gel electrophoresis (PFGE) type was detected, with 19 subtypes (Simpsons index of diversity [SID] = 0.89). Multiple-locus variable-number tandem-repeat analysis (MLVA) showed more variability, with 32 profiles (SID = 0.97), but only showed diversity at the STTR5 and STTR6 loci. PCR and sequencing demonstrated all strains contained the same allantoin-glyoxylate pathway deletion. Four types of deletions were detected in the fljAB operon, all starting at the same position, at the STM2758 gene, and followed by an IS26 insertion. Furthermore, a representative set of strains of the four deletion types harbored plasmids with IS26. We propose that a Salmonella enterica serotype Typhimurium U302 multidrug-resistant (ACSuGSTSxT) strain, defective for the allantoin-glyoxylate pathway and containing IS26 at plasmid pU302L, could be the ancestor of the variant in Spain.

Epidemiology of subclinical salmonellosis in wild birds from an area of high prevalence of pig salmonellosis: phenotypic and genetic profiles of Salmonella isolates.

The epidemiology of subclinical salmonellosis in wild birds in a region of high Salmonella prevalence in pigs was studied. Three hundred and seventy‐nine faecal samples from 921 birds trapped in 31 locations nearby pig premises, and 431 samples from 581 birds of 10 natural settings far from pig farms were analysed for the presence of Salmonella spp. Positive samples were serotyped and analysed for antimicrobial resistance (AR). Phage typing and pulsed‐field gel electrophoresis (PFGE) on Salmonella Typhimurium isolates were also carried out. The overall proportion of Salmonella‐positive samples was 1.85% (95% CI = 0.93, 2.77). Salmonella isolation was positively associated with samples collected from birds in the proximity of a pig operation (OR = 16.5; 95% CI = 5.17, 52.65), and from non‐migratory (or short‐distance migration) birds (OR = 7.6; 95% CI = 1.20, 48.04) and negatively related to mostly granivorous birds (OR = 0.4; 95% CI = 0.15, 1.13). Salmonella Typhimurium was the most prevalent serotype and four different XbaI PFGE patterns were observed that matched the four phage types identified (U310, U311, DT164 and DT56). Only 20% of the strains showed multi‐AR. In three farms, a high degree of homogeneity among isolates from different birds was observed. These findings suggested that pig farms may act as amplifiers of this infection among wild birds, and the degree of bird density may have much to do on this transmission. Some of the Salmonella serotypes isolated from bird faeces were of potential zoonotic transmission and associated with AR. Monitoring salmonellosis in wild bird is advised.

Class 1 integrons in multidrug-resistant non-typhoidal Salmonella enterica isolated in Spain between 2002 and 2004

In this study, 119 multidrug-resistant isolates of non-typhoidal Salmonella enterica serovars collected in Spain (2002-2004) were screened for integrons. Among the isolates, 73.1% contained class 1 integrons, however classes 2 and 3 were not detected. Integrons containing gene cassettes were found in S. Enteritidis (16/32), S. Typhimurium biphasic (18/32) and monophasic [4,5,12:i:-] (11/19), S. Virchow (17/18) and S. Brandenburg (8/8), but not in S. Hadar (0/10). Ten complete and four incomplete gene cassettes, combined in 10 variable regions, were identified, one of which (2100 bp/dfrA1-597 bp-aadA24) was a new description. Most integrons mapped on plasmids of ca. 40-340 kb. Exceptions were 1000 bp/aadA2 and 1200 bp/bla PSE-1 found on the chromosome of biphasic S. Typhimurium, probably as part of SGI1-like structures.

Prevalence of Shiga toxin-producing Escherichia coli, Salmonella spp. and Campylobacter spp. in large game animals intended for consumption: Relationship with management practices and livestock influence

Although wild ruminants have been identified as reservoirs of Shiga-toxin producing Escherichia coli (STEC), little information is available concerning the role of Salmonella spp. and Campylobacter spp. in large game species. We evaluated the presence of these pathogens in faeces (N=574) and carcasses (N=585) sampled from red deer (N=295), wild boar (N=333) and other ungulates (fallow deer, mouflon) (N=9). Animal sampling was done in situ from 33 hunting estates during two hunting seasons. Salmonella spp. and Campylobacter spp. strains associated with human campylobacteriosis were infrequently detected indicating that both pathogens had a limited zoonotic risk in our study area. The overall STEC prevalence in animals was 21% (134/637), being significantly higher in faeces from red deer (90 out of 264). A total of 58 isolates were serotyped. Serotypes O146:H- and O27:H30 were the most frequent in red deer and the majority of isolates from red deer and wild boar were from serotypes previously found in STEC strains associated with human infection, including the serotype O157:H7. The STEC prevalence in red deer faeces was significantly higher with the presence of livestock (p<0, 01) where high densities of red deer (p<0.001) were present. To the best of our knowledge, this is the first study reporting the occurrence of Salmonella spp. and STEC in carcasses of large game animals. Furthermore, this study confirmed by pulsed-field gel electrophoresis (PFGE) that cross contamination of STEC during carcass dressing occurred, implying the likelihood of these pathogens entering into the food chain.

Pheno-genotypic characterisation of Escherichia coli O157:H7 isolates from domestic and wild ruminants.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 represents a major public health concern worldwide, with ruminants recognised as their main natural reservoir. The aim of this work was to determine the phenotypic features and genetic relationships of 46 E. coli O157:H7 isolates obtained from sheep, cattle and deer faeces and from unpasteurised goat milk in Spain over a period of 11 years. Characterisation was performed by polymerase chain reaction (PCR), phage typing and pulsed-field gel electrophoresis (PFGE). An atypical E. coli O157:H7 strain (sorbitol-fermenting and beta-glucuronidase positive) originating from deer faeces was detected. Genes encoding Shiga toxins were detected in 69.6% of isolates, all of them carrying only the stx(2) gene. The isolates were from nine different phage types, although 67.4% were restricted to only three: PT14, PT34 and PT54. PT54 was the most prevalent phage type and contained isolates from cattle, sheep and deer. Majority of the isolates were from phage types previously found in strains associated with human infection. XbaI-PFGE identified 33 different types and 11 groups of closely related types (more than 85% similarity), one of which included 21 (45.7%) isolates originating from different animal species, including deer. These results indicate common origin or inter-species spread of genetically similar E. coli O157:H7 isolates and contribute to earlier investigations identifying deer as a natural source of E. coli O157:H7. The study also highlights the emergence of phenotypic variants of E. coli O157:H7, which may not be identified by routine culture methods or by biochemical tests used to characterise serotype O157:H7.

Surveillance of antibiotic resistance evolution and detection of class 1 and 2 integrons in human isolates of multi-resistant Salmonella Typhimurium obtained in Uruguay between 1976 and 2000

OBJECTIVES To study the evolution of antibiotic resistance in isolates of Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium) obtained in Uruguay between the years 1976 and 2000, and to determine the incidence of class 1 and 2 integrons in the multi-resistant isolates. METHODS We studied 258 strains of Salmonella Typhimurium from various sources, isolated between 1976 and 2000. We determined the evolution of antibiotic resistance and the distribution of class 1 and 2 integrons in all isolates by means of disk diffusion assays and PCR. RESULTS During the period 1989-2000 resistance to streptomycin was 56.8%, tetracycline 13.6%, sulfonamides 11.2%, and ampicillin 7.2%. Resistance to gentamicin, kanamycin, chloramphenicol, and nalidixic acid were lower than 5%; no resistance was detected to fluoroquinolones, oxyiminocephalosporins, and amikacin. These results show a dramatic decrease with respect to values found in the period 1976-1988. In this period, resistance to streptomycin was 63.2%, tetracycline 36.8%, sulfonamides 32.3%, and ampicillin 27.8%. Throughout the two periods, 29 multi-resistant Salmonella Typhimurium strains were isolated harboring some class of integron: 15 strains had only intI2, 11 strains presented both intI1 and intI2, and three isolates only intI1. CONCLUSIONS Our results show a marked decrease in resistance throughout these years, along with a correlation between resistance to different antibiotics and the presence of integrons.