Aurora Echeita

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.

Serotypes, virulence genes and intimin types of Shiga toxin (verocytotoxin)-producing Escherichia coli isolates from minced beef in Lugo (Spain) from 1995 through 2003

BackgroundShiga toxin-producing Escherichia coli (STEC) have emerged as pathogens that can cause food-borne infections and severe and potentially fatal illnesses in humans, such as haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). In Spain, like in many other countries, STEC strains have been frequently isolated from ruminants, and represent a significant cause of sporadic cases of human infection. In view of the lack of data on STEC isolated from food in Spain, the objectives of this study were to determine the level of microbiological contamination and the prevalence of STEC O157:H7 and non-O157 in a large sampling of minced beef collected from 30 local stores in Lugo city between 1995 and 2003. Also to establish if those STEC isolated from food possessed the same virulence profiles as STEC strains causing human infections.ResultsSTEC were detected in 95 (12%) of the 785 minced beef samples tested. STEC O157:H7 was isolated from eight (1.0%) samples and non-O157 STEC from 90 (11%) samples. Ninety-six STEC isolates were further characterized by PCR and serotyping. PCR showed that 28 (29%) isolates carried stx1 genes, 49 (51%) possessed stx2 genes, and 19 (20%) both stx1 and stx2. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 43 (45%) and in 25 (26%) of the isolates, respectively. Typing of the eae variants detected four types: γ1 (nine isolates), β1 (eight isolates), ε1 (three isolates), and θ (two isolates). The majority (68%) of STEC isolates belonged to serotypes previously detected in human STEC and 38% to serotypes associated with STEC isolated from patients with HUS. Ten new serotypes not previously described in raw beef products were also detected. The highly virulent seropathotypes O26:H11 stx1eae-β1, O157:H7 stx1stx2eae-γ1 and O157:H7 stx2eae-γ1, which are the most frequently observed among STEC causing human infections in Spain, were detected in 10 of the 96 STEC isolates. Furthermore, phage typing of STEC O157:H7 isolates showed that the majority (seven of eight isolates) belonged to the main phage types previously detected in STEC O157:H7 strains associated with severe human illnesses.ConclusionThe results of this study do not differ greatly from those reported in other countries with regard to prevalence of O157 and non-O157 STEC in minced beef. As we suspected, serotypes different from O157:H7 also play an important role in food contamination in Spain, including the highly virulent seropathotype O26:H11 stx1eae-β1. Thus, our data confirm minced beef in the city of Lugo as vehicles of highly pathogenic STEC. This requires that control measures to be introduced and implemented to increase the safety of minced beef.

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.

Relationship of pulsed-field profiles with key phage types of Salmonella enterica serotype Enteritidis in Europe: results of an international multi-centre study

Salmonella is one of the most common causes of foodborne infection in Europe with Salmonella enterica serovar Enteritidis (S. Enteritidis) being the most commonly identified serovar. The predominant phage type for S. Enteritidis is phage type (PT) 4, although PT 8 has increased in incidence. Within these phage types, pulsed-field gel electrophoresis (PFGE) provides a method of further subdivision. The international project, Salm-gene, was established in 2001 to develop a database of PFGE profiles within nine European countries and to establish criteria for real-time pattern recognition. It uses DNA fingerprints of salmonellas to investigate outbreaks and to evaluate trends and emerging issues of foodborne infection within Europe. The Salm-gene database contains details of about 11 700 S. Enteritidis isolates, demonstrating more than 65 unique PFGE profiles. The clonal nature of S. Enteritidis is evidenced by the high similarity and distribution of PFGE profiles. Over 56% (6603/11 716) of the submitted isolates of several different phage types were profile SENTXB.0001, although this profile is most closely associated with PT 4. The next most common profiles, SENTXB.0002 and SENTXB.0005, were closely associated with PT 8 and PT 21 respectively. Studies to investigate the relationship of profile types with outbreaks and possible vehicles of infection suggest that the incidence of PFGE profile SENTXB.0002, and thus PT 8, in some countries may be due to importation of foods or food production animals from Eastern Europe, where PT 8 is amongst the most frequently identified phage types. Collation of subtyping data, especially in the commonly recognized phage types, is necessary in order to evaluate trends and emerging issues in salmonella infection.

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.

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.

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.

Prevalence and antimicrobial susceptibility of Salmonella infections in free-range pigs

The prevalence of Salmonella spp. infection was determined in 67 free-range pig herds in southern Spain. Microbiological assessment was performed on ileocolic lymph nodes collected at slaughter according to ISO 6579:2002 procedures. Overall, 33% of herds were infected and the prevalence of infection was 5.3%. Salmonella spp. serovars most frequently isolated were Anatum and Typhimurium, although uncommon serovars such as Hessarek and Mikawasima were also detected. Isolates were tested against 16 antimicrobial agents and exhibited resistance to streptomycin (46%), tetracycline (30%), sulphonamides (25%) and ampicillin (23%) by the break-point method. Multi-drug resistance, defined as resistance to ≥ 4 antimicrobials, was 36%.

Salmonella Indiana as a cause of abortion in ewes: Genetic diversity and resistance patterns

Salmonella enterica subspecies enterica Indiana, a food-borne serovar uncommon in most countries, was responsible for an outbreak of abortion in a flock of Lacaune dairy ewes in southern Spain. Drinking water and feedstuff samples were analysed in an attempt to determine the source of the infection. Pigeons (Columba livia) and turtledoves (Streptopelia turtur) in close contact with the ewes were captured and examined for the bacterium. Seventeen S. Indiana strains were isolated from the ewes and wild birds and the genetic similarity among them analysed by Pulsed Field Gel Electrophoresis (PFGE) after the digestion of their genomic DNA with the restriction enzyme XbaI. The results suggest the wild birds might be responsible for the outbreak in the ewes. The strains recovered were fully susceptible to 15 out of the 16 antimicrobial agents tested: ampicillin, amoxycillin clavulanate, cephalothin, ceftriaxone, gentamicin, neomycin, streptomycin, tetracycline, ciprofloxacin, enrofloxacin, sulphonamides, trimethoprim-sulphamethoxazole, apramycin, colistin and chloramphenicol. Differences in the resistance pattern to nalidixic acid were observed; 11 strains (64.7%) were nalidixic acid resistant (R-Nx) and 6 (35.3%) sensitive (S-Nx). Among the R-Nx strains, a substitution of Gly to Cys at position 81 (Gly81àCys) of the gyrA gene in 10 strains isolated from wild birds and ovine foetuses, and of Asp to Tyr at position 87 (Asp87àTyr) in one strain isolated from ewe faeces, were revealed by sequencing the gene. To control the outbreak, enrofloxacin treatment was administered for 5 days. The same therapy was used to prevent infection during following gestation cycles, administering the antimicrobial agent at presentation and over 4 weeks before birth. Anti-bird meshes and closed drinking and feeding troughs were also installed to prevent further contact of the ewes with wild birds.