RESUMEN

The frequency and genetic bases of antimicrobial drug resistance was determined for 111 Staphylococcus aureus recovered from young healthy carriers in a Spanish region. Resistances to ampicillin (84.7%), kanamycin (27%), erythromycin (25.2%), clindamycin (22.5%), tetracycline (11.7%), amikacin and tobramycin (6.3% each), gentamicin (5.4%), chloramphenicol (2.7%), ciprofloxacin (0.9%; MIC 4 µg/ml), moxifloxacin (0.9%) and mupirocin (0.9%; MIC 60 µg/ml) were found, and all were susceptible to methicillin (MSSA). Nearly 50% of the isolates were resistant to one antibiotic, 30% to two, 15.3% to three and 1.8% to four, while only 6.3% remained fully susceptible. A total of 31 profiles were found. For each phenotypic resistance, at least one gene accounting for it was identified. The detected genes were blaZ; erm(A)-erm(B)-erm(C)-msr(A)-msr(B)-lnu(A), aphA-aadE-sat4-aacA + aphD-aadD, tet(K), cat, and qacA/B, for resistance to ampicillin, macrolides and/or lincosamides, aminoglycosides, tetracycline, chloramphenicol, and quaternary ammonium compounds, respectively. In all isolates carrying cat genes, in all except one of the isolates positive for tet(K), and in most isolates with blaZ, erm(C), msr(A), or msr(B), the gene(s) mapped on resistance plasmids, which were detected in 69.2% of the resistant isolates (65% of the total). The S. aureus from young healthy carriers analysed in the present study do not constitute a reservoir of MRSA, but they represent a repository of multiple determinants conferring resistance to "old" antimicrobials. Some of these have still clinical applications and, considering the increasing resistance to recently introduced antimicrobials, none of them can be disregarded.

Asunto(s)

Antibacterianos/farmacología , Portador Sano/microbiología , Farmacorresistencia Bacteriana , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Genes Bacterianos , Pruebas de Sensibilidad Microbiana , España , Staphylococcus aureus/aislamiento & purificación

RESUMEN

Plasmids encoding resistance and virulence properties in multidrug resistant (MDR) Salmonella enterica (S.) serovar Typhimurium monophasic variant 4,[5],12:i:- isolates recovered from pigs and humans (2006-2008) in Europe were characterised. The isolates were selected based on the detection by PCR-amplification of S. Typhimurium virulence plasmid pSLT genes and were analysed by multi-locus sequence typing (MLST). The resistance genes present in the isolates and the association of these genes with integrons, transposons and insertion sequences were characterised by PCR-sequencing, and their plasmid location was determined by alkaline lysis and by S1-nuclease pulsed-field gel electrophoresis (PFGE) Southern-blot hybridisation. Plasmids were further analysed by replicon typing, plasmid MLST and conjugation experiments. The 10 S. 4,[5],12,i:- selected isolates belonged to ST19. Each isolate carried a large plasmid in which MDR with pSLT-associated virulence genes were located. After analysis, eight different plasmids of three incompatibility groups (IncA/C, IncR and IncF) were detected. Two IncA/C plasmids represented novel variants within the plasmid family of the S. 4,[5],12:i:- Spanish clone, and carried an empty class 1 integron with a conventional qacEΔ1-sul1 3' conserved segment or an In-sul3 type III with estX-psp-aadA2-cmlA1-aadA1-qacH variable region linked to tnpA440-sul3, part of Tn2, Tn21 and Tn1721 transposons, and ISCR2. Four newly described IncR plasmids contained the resistance genes within In-sul3 type I (dfrA12-orfF-aadA2-cmlA1-aadA1-qacH/tnpA440-sul3) and part of Tn10 [tet(B)]. Two pSLT-derivatives with FIIs-ST1+FIB-ST17 replicons carried cmlA1-[aadA1-aadA2]-sul3-dfrA12 and blaTEM-1 genes linked to an In-sul3 type I integron and to Tn2, respectively. In conclusion, three emerging European clones of S. 4,[5],12:i:- harboured MDR plasmids encoding additional virulence functions that could contribute significantly to their evolutionary success.

Asunto(s)

Plásmidos/genética , Infecciones por Salmonella/genética , Salmonella typhimurium/genética , Animales , Elementos Transponibles de ADN , Farmacorresistencia Bacteriana Múltiple/genética , Europa (Continente) , Humanos , Integrones/genética , Salmonelosis Animal/genética , Porcinos , Virulencia/genética

RESUMEN

Drug-resistant derivatives of serovar-specific virulence plasmids, such as pSLT, in clinically-relevant Salmonella enterica serovar Typhimurium strains, represent a threat for human health. We have analysed 14 S. Typhimurium isolates recovered in Italy and the United Kingdom from swine and from cases of human infection for the presence of virulence-resistance (VR) plasmids. They were negative for the multidrug resistance (MDR) region of the Salmonella genomic island 1 (SGI1), but expressed resistance to ampicillin, chloramphenicol, streptomycin/spectinomycin, sulfamethoxazole, and tetracyclines. The isolates were characterised by XbaI pulsed-field gel electrophoresis, multilocus sequence typing, and detection of resistance and virulence determinants (PCR/sequencing). Identification of VR plasmids was accomplished by PCR detection of bla genes (encoding ampicillin resistance), class 1 integrons and the pSLT virulence gene spvC. Plasmid analyses were performed by alkaline lysis, S1-nuclease digestion, replicon typing, conjugation, restriction analyses, and Southern blot/hybridization. Two blaOXA-1 positive isolates contained pSLT-derived plasmids related to pUO-StVR2. In nine isolates, eight from swine and one from a patient, MDR-conferring-IncFII-VR plasmids were detected. They contained the blaTEM-1 gene as well as a nonconventional class 1 integron with dfrA12-aadA2 gene cassettes in its variable region, and a sul3 gene in the 3' conserved segment. Restriction analysis suggested a novel pSLT variant. The results obtained underline the role of swine as a potential reservoir for the blaTEM-1-IncFII-plasmids. The occurrence and spread of virulence- and MDR-conferring plasmids should be considered as a potential public health problem.

Asunto(s)

Proteínas Bacterianas/genética , Reservorios de Enfermedades/veterinaria , Genes Bacterianos , Plásmidos , Salmonelosis Animal/microbiología , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , Región de Flanqueo 3' , Animales , Antibacterianos/farmacología , Secuencia de Bases , Secuencia Conservada , Reservorios de Enfermedades/microbiología , Farmacorresistencia Bacteriana Múltiple , Variación Genética , Humanos , Integrones , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Tipificación de Secuencias Multilocus , Salmonelosis Animal/tratamiento farmacológico , Salmonelosis Animal/transmisión , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/aislamiento & purificación , Porcinos , Virulencia

RESUMEN

Salmonella enterica subsp. enterica 4,[5],12:i:- is one of the most prevalent serovars associated with human infections worldwide. Two multidrug-resistant clones, designated Spanish and European clones, are recognized as having importance for public health and are subject to control measures in the European Union. In this study, 23 clinical isolates belonging to the Spanish clone were characterized by multilocus sequence typing, multiple-locus variable number tandem repeat analysis (MLVA), PCR amplification and sequencing, and a DNA microarray targeting 263 genes, in order to provide new insights into their origins and further evolution. The derived data were compared with information available from other studies for S. 4,[5],12:i:- isolates of both the Spanish and the European clones, to identify differential molecular markers which could be potentially used as surveillance tools in the control of dissemination of this serovar. The isolates analyzed were assigned to sequence type 19 and to 17 MLVA patterns, with 3-13-16-NA-311 being the most prevalent. Highly similar virulence, metabolic, and prophage-associated gene profiles were identified, but DNA mobility markers distinguished five genotypes. Two types of deletions, caused by insertion of IS26, presumably donated by pUO-STmR/RV1-like plasmids typically found in the Spanish clone, affected the fljAB operon and surrounding DNA. The Spanish and European clones differ in sequence type, MLVA patterns, gene repertoire, and fljAB deletion type. The observed variability supports an independent evolution of the two successful monophasic clones from different Salmonella enterica serovar Typhimurium ancestors and can be taken into consideration for epidemiological surveillance.

Asunto(s)

Tipificación de Bacteriófagos , Evolución Molecular , Tipificación Molecular , Infecciones por Salmonella/epidemiología , Infecciones por Salmonella/microbiología , Salmonella typhimurium/clasificación , Salmonella typhimurium/genética , Genes Bacterianos , Marcadores Genéticos , Variación Genética , Humanos , Profagos/genética , Salmonella typhimurium/aislamiento & purificación , España/epidemiología , Factores de Virulencia/genética

RESUMEN

The population structure of 111 methicillin-susceptible Staphylococcus aureus (MSSA), recovered in Spain from healthy and risk-free carriers was investigated using pulsed-field gel electrophoresis (PFGE), spa (staphylococcal protein A) typing, multi locus sequence typing (MLST) and the accessory gene regulator (agr). Results from the different techniques were highly concordant, and revealed twelve clonal complexes (CCs): CC30 (27%), CC5 (18.9%), CC45 (16.2%), CC15 (11.7%), CC25 (8.1%), CC1, CC9 (3.6% each), CC59, CC97 and CC121 (2.7% each), CC72 (1.8%) and CC8 (0.9%). Isolates with genetic backgrounds of hospital-acquired MSSA were detected and, consistent with the ability of diverse MSSA to act as recipients of the SCCmec cassette, a MSSA isolate from a healthy carrier shared the ST, spa-type and agr-type of a MRSA clone recovered in a hospital of the same region. All except two fragments of the PGFE-profiles of these isolates were identical, and the differential fragment of the MRSA carried mecA. Analyses of the exotoxin gene content of the nasal isolates revealed an increase in the number of exotoxin genes over time. This, together with the detection of lukPV and the high frequency of tst, exfoliatin and enterotoxin genes, is worrisome and requires further surveillance.

Asunto(s)

Portador Sano/microbiología , Exotoxinas/genética , Tipificación Molecular , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/clasificación , Staphylococcus aureus/genética , Portador Sano/epidemiología , Análisis por Conglomerados , Genotipo , Humanos , Epidemiología Molecular , España/epidemiología , Infecciones Estafilocócicas/epidemiología , Staphylococcus aureus/aislamiento & purificación , Factores de Virulencia/genética , Adulto Joven

RESUMEN

The exotoxin gene content was established for 62 Staphylococcus aureus isolates causing bloodstream (n = 31) and wound (n = 31) infections in geriatric patients attending a long-term care Spanish hospital from 1996 to 2006. Content was determined based on PCR screening of genes encoding five haemolysins, three exfoliatins, three leukotoxins and 21 pyrogenic toxin superantigens (PTSAgs), in addition to markers of genomic (νSaß) and pathogenicity (SaPIs) islands. Exotoxin genes were abundant in both bloodstream (11-23 genes) and wound (8-19 genes) isolates, and they were arranged in 55 combinations with only two represented in both groups. All isolates were positive for genes encoding haemolysins (hl; 3-5) and PTSAgs (tst, se and sel; 5-14), whereas exfoliatin (et) and leukotoxin (luk) genes appeared in 98.4 and 51.6 % of isolates, respectively. The hlg, lukPV, tst, sec and selu genes were found significantly more frequently in bloodstream than in wound isolates, whereas hlg-variant, sea, seb, see and selk-selq were more frequent in wound isolates (P<0.05). Distinctive exotoxin gene combinations could potentially be associated with specific mobile genetic elements, including genomic islands [lukED, egc1 (seg, seln, sei, selm, selo) and egc2 (seg, seln, selu, sei, selm, selo)]; pathogenicity islands (etd, seb, sec, sell, selq, selk and tst); bacteriophages (eta, lukPV, sea, selp, selk, selq and see); and plasmids (sed, selj, ser, ses and set). The abundance of exotoxin genes and variety of arrangements shown by S. aureus from geriatric patients could play a role in the adaptation of the pathogen.

Asunto(s)

Exotoxinas/aislamiento & purificación , Sepsis/microbiología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Heridas y Lesiones/microbiología , Anciano , Distribución de Chi-Cuadrado , ADN Bacteriano/química , ADN Bacteriano/genética , Exotoxinas/genética , Humanos , Reacción en Cadena de la Polimerasa , Estudios Retrospectivos , Sepsis/inmunología , España , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Virulencia , Heridas y Lesiones/inmunología

RESUMEN

All Staphylococcus aureus isolates (n=31) that caused bacteraemia in a Spanish geriatric hospital during 1996-2006 were analysed by a simple, rapid and inexpensive PCR technique based on variations in the hsdS1 and hsdS2 genes encoding the sequence recognition subunits of the Sau1 restriction-modification (RM) system. An equal number of isolates collected from surgical wounds over the same time period (control group) were similarly characterized. The RM test allocated 75 % of the isolates to the six major clonal complexes (CC1, CC5, CC8, CC22, CC30 and CC45) for which it was developed. However, recognition of minor CCs and precise identification of the circulating clones required more powerful and comprehensive techniques such as spa typing and multilocus sequence typing (MLST), which are more demanding and expensive. The RM test is not intended to replace spa or MLST typing, but may be of use when time, technical and/or financial resources are limited. Overall, nine and seven CCs were detected in bloodstream and wound isolates, respectively. In both groups, CC5 was the most frequent (35.5 % each), followed by CC45 or CC8 (22.6 and 32.3 % of bloodstream and wound isolates, respectively). The frequency of meticillin resistance was lower in bloodstream (16.1 %) than in wound (51.6 %) isolates (P=0.0025). Among the former, sequence type (ST) 5-staphylococcal cassette chromosome mec (SCCmec) II, ST5-SCCmec IV, ST45-SCCmec IV and ST125-SCCmec IV (now dominant in Spanish hospitals) clones were found. Among the wound isolates, nine meticillin-resistant clones were represented, with three of them (ST125-SCCmec III, ST125-SCCmec V and ST14-SCCmec V) being newly described.

Asunto(s)

Bacteriemia/microbiología , Tipificación Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/clasificación , Staphylococcus aureus/genética , Anciano , Anciano de 80 o más Años , Proteínas Bacterianas/genética , Análisis por Conglomerados , Enzimas de Restricción-Modificación del ADN/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Femenino , Genotipo , Servicios de Salud para Ancianos , Hospitales , Humanos , Cuidados a Largo Plazo , Masculino , España , Staphylococcus aureus/aislamiento & purificación , Infección de Heridas/microbiología

RESUMEN

OBJECTIVES: To broaden knowledge of the molecular bases and genetics of multidrug resistance in clinical isolates of Salmonella enterica serotype 4,5,12:i:- belonging to the Spanish clone. METHODS: The relatedness of the isolates was determined by phage typing and XbaI-PFGE. Resistance genes, integrons and transposable elements were identified by PCR amplification and sequencing. Plasmids were characterized by alkaline lysis, S1-PFGE, conjugation, replicon typing and Southern blot hybridization. RESULTS: The isolates were closely related and resistant to five to seven antimicrobials (ampicillin, chloramphenicol, gentamicin, streptomycin/spectinomycin, sulphonamides, trimethoprim and tetracycline, arranged in different combinations). Most of the responsible genes were provided by a conventional class 1 integron with the dfrA12-orfF-aadA2 variable region, an atypical class 1 integron containing sul3 next to the estX-psp-aadA2-cmlA1-aadA1 variable region and a truncated Tn1721 transposon carrying tet(A). A defective Tn21 with the mer operon and ISVsa3 associated with sul2 were also detected. All resistance genes and mobile genetic elements were located on large, non-conjugative and highly variable plasmids carrying one (A/C) or two (A/C and N) replicons, as well as virulence genes of pSLT. CONCLUSIONS: IncA/C plasmids are responsible for multidrug resistance in an increasing number of relevant human and animal bacterial pathogens, and hence are regarded as an important threat to public health. Those found in the Spanish clone of Salmonella 4,5,12:i:- constitute a relevant example of short-term evolution, and could have been involved in the successful adaptation of this pathogen.

Asunto(s)

Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple , Plásmidos , Salmonelosis Animal/epidemiología , Infecciones por Salmonella/epidemiología , Salmonella enterica/genética , Factores de Virulencia/genética , Animales , Técnicas de Tipificación Bacteriana , Tipificación de Bacteriófagos , Southern Blotting , Conjugación Genética , Elementos Transponibles de ADN , Electroforesis en Gel de Campo Pulsado , Genes Bacterianos , Humanos , Integrones , Epidemiología Molecular , Tipificación Molecular , Reacción en Cadena de la Polimerasa , Infecciones por Salmonella/microbiología , Salmonelosis Animal/microbiología , Salmonella enterica/clasificación , Salmonella enterica/efectos de los fármacos , Salmonella enterica/aislamiento & purificación , Análisis de Secuencia de ADN , Serotipificación , España/epidemiología , Virulencia

RESUMEN

One hundred and twenty pathogenic isolates of Pseudomonas syringae pv. phaseolicola recovered in Spain were subjected to biochemical and genomic typing, and investigated for virulence gene complement. Fifty-six were recovered from common beans (Phaseolus vulgaris) of the type Granja Asturiana, grown in a northern Spanish region (Asturias), and 64 from other common beans cultured in the neighbouring region of Castilla y León. Typing by PmeI digestion followed by pulsed-field gel electrophoresis revealed 27 profiles, with only three being common to both regions. Relationships between profiles distributed the isolates into two clusters: A (subdivided into subclusters A1 and A2) and B. Cluster A included all isolates from Granja Asturiana and about a quarter of the isolates from Castilla y León. Isolates from cluster A were negative for mannitol utilization and hybridized to probes for the argK-tox region responsible for phaseolotoxin production. Isolates that grouped in cluster B, which were only found in Castilla y León, were able to utilize mannitol but did not hybridize to probes for the argK-tox region. Separation of the isolates into three genomic groups, subsequently termed PphA1, PphA2 and PphB, was also supported by effector gene complement and location. In PphB, all effector genes tested (hopX1, hopF1, avrB2 and avrD1) mapped on chromosomal fragments, but faint hybridization of avrB2 with plasmids of about 40 kb was also observed. In PphA hopX1 mapped on the chromosome; in PphA1 avrB2 and avrD1 were carried on virulence plasmids (most of approx. 125 kb) and hopF1 was not detected, while in PphA2 the three genes were located on plasmids (approx. 75-160 kb). These results can be used as a framework to investigate the basis of regional variation in population structure, and for further epidemiological surveillance of P. syringae pv. phaseolicola.

Asunto(s)

Electroforesis en Gel de Campo Pulsado/métodos , Phaseolus/microbiología , Plásmidos/análisis , Pseudomonas syringae/genética , Pseudomonas syringae/patogenicidad , Factores de Virulencia/genética , Genes Bacterianos , Ornitina/análogos & derivados , Ornitina/genética , Pseudomonas syringae/clasificación , España

RESUMEN

Fifty-five bovine, 50 equine, 60 ovine, and 50 porcine carcasses were sampled in a slaughterhouse in eastern Spain. Two samples were taken from each carcass, one using the excision method and the other using the swabbing method. Four different materials were used for swabbing: cellulose, polyurethane, or viscose sponges, and medical gauze. Samples were collected at the end of the process by four different people before the carcasses were taken to the cooler. The samples were examined for total viable bacteria counts (TVCs) and Enterobacteriaceae counts (ECs). The mean TVC for all species sampled by excision was 4.50 log CFU/cm(2), which was significantly higher than the 3.53 log CFU/cm(2) obtained by swabbing. The TVCs obtained using gauze and the cellulose and polyurethane sponges were significantly higher (P < 0.05) than the corresponding TVCs obtained using viscose sponges. Animal species, the person who collected the samples, and microbiological load also had a significant effect on TVC. ECs were obtained from 82.8% of excision samples, from larger percentages of samples obtained using cellulose or polyurethane sponges or gauze swabs, but from smaller percentages of samples obtained using viscose sponges. The Enterobacteriaceae load significantly influenced the EC. In contrast, animal species and the person who collected the samples had no significant effect. The cellulose sponge, polyurethane sponge, and gauze gave high mean log counts of aerobic bacteria and Enterobacteriaceae, which makes these swab types suitable for use in slaughterhouses for the purpose of assessing production process hygiene.

Asunto(s)

Mataderos , Técnicas Bacteriológicas/métodos , Recuento de Colonia Microbiana/métodos , Enterobacteriaceae/aislamiento & purificación , Contaminación de Alimentos/análisis , Carne/microbiología , Mataderos/instrumentación , Mataderos/normas , Animales , Bovinos/microbiología , Seguridad de Productos para el Consumidor , Microbiología de Alimentos , Caballos/microbiología , Humanos , Higiene , Ovinos/microbiología , Piel/microbiología , España , Especificidad de la Especie , Porcinos/microbiología

RESUMEN

OBJECTIVES: Detection and characterization of extended-spectrum beta-lactamases (ESBLs) and AmpC-encoding genes was conducted in German Salmonella isolated from different sources from 2003 to 2007. METHODS: Non-duplicate German isolates from the National Salmonella Reference Laboratory Collection (2003-07) with ceftiofur MICs of > or =4 mg/L were tested for beta-lactam/beta-lactamase inhibitor susceptibility, presence of ESBLs or AmpC-encoding genes, class 1 and 2 integrons, other resistance genes, and IS26 and ISEcp1 sequences by PCR/sequencing. The isoelectric point of the beta-lactamase was determined. Strains were analysed by PFGE and plasmid profiling. The bla genes were mapped by Southern-blot hybridization. Plasmids were characterized by rep-PCR typing. RESULTS: Sixteen isolates (10 Salmonella Typhimurium, 2 Salmonella Anatum, 2 Salmonella Paratyphi B dT + , 1 Salmonella Infantis and 1 Salmonella London) carried bla(CTX-M) (15 bla(CTX-M-1) and one bla(CTX-M-15)) genes located on self-transferable IncB/O, IncI1 and/or IncN plasmids. Seven of the Salmonella Typhimurium isolates carried the SGI1-M variant. Six isolates (five Salmonella Agona and one Salmonella Kentucky) carried the bla(CMY-2) gene on IncI1 conjugative plasmids. bla(TEM-20) genes were detected in two Salmonella Paratyphi B dT+ isolates, and bla(TEM-52) in one Salmonella Paratyphi B dT+ and one Salmonella Virchow, located on IncI1 plasmids. All Salmonella Paratyphi isolates harboured a 2300 bp/dfrA1-sat2-aadA1 class 2 integron. CONCLUSIONS: Among the 22 679 German Salmonella isolates investigated, the ESBL and AmpC beta-lactamase prevalence was still low; however, it is slowly increasing. Various beta-lactamase genes are linked to a variety of genetic elements capable of horizontal DNA transfer. Consequently, their dissemination is likely and demands adequate risk management strategies.

Asunto(s)

Antibacterianos/farmacología , Cefalosporinas/farmacología , Microbiología de Alimentos , Salmonelosis Animal/microbiología , Salmonella enterica/efectos de los fármacos , Resistencia betalactámica , beta-Lactamasas/biosíntesis , Animales , Animales Domésticos/microbiología , ADN Bacteriano/química , ADN Bacteriano/genética , Electroforesis en Gel de Campo Pulsado , Alemania , Humanos , Punto Isoeléctrico , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Plásmidos/análisis , Salmonella enterica/genética , Salmonella enterica/aislamiento & purificación , Análisis de Secuencia de ADN , beta-Lactamasas/química , beta-Lactamasas/genética

RESUMEN

pUO-StVR2 is a virulence-resistance plasmid which originated from pSLT of Salmonella enterica serovar Typhimurium through acquisition of a complex resistance island, flanked by regions that provide a toxin-antitoxin system and an iron uptake system. The presence of resistance and virulence determinants on the same plasmid allows coselection of both properties, potentially increasing health risks.

Asunto(s)

Farmacorresistencia Bacteriana/genética , Evolución Molecular , Plásmidos/genética , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/patogenicidad , Proteínas Bacterianas/genética , Elementos Transponibles de ADN , ADN Bacteriano/genética , Humanos , Datos de Secuencia Molecular , Salmonella typhimurium/genética , Análisis de Secuencia de ADN , Virulencia/genética

RESUMEN

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.

Asunto(s)

Farmacorresistencia Bacteriana Múltiple/genética , Integrones/genética , Salmonella enterica/clasificación , Salmonella enterica/efectos de los fármacos , Salmonella enteritidis/efectos de los fármacos , Salmonella typhimurium/efectos de los fármacos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Humanos , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Mapeo Restrictivo , Salmonella enterica/genética , Salmonella enterica/aislamiento & purificación , Salmonella enteritidis/genética , Salmonella enteritidis/aislamiento & purificación , Salmonella typhimurium/genética , Salmonella typhimurium/aislamiento & purificación , Serotipificación , España

RESUMEN

The epidemiological impact in Spain of an emerging group of multidrug-resistant Salmonella enterica serotype Typhimurium, characterized by the presence of virulence-resistance hybrid plasmids (termed pUO-StVR) that are related to the S. Typhimurium virulence plasmid pSLT, was evaluated. Adscription to the group was based on detection of the bla(OXA-1) gene (encoding ampicillin resistance) by PCR, and identification of a pUO-StVR plasmid through hybridization with specific probes for virulence (spvC) and resistance (bla(OXA-1)) genes. In this way, 57 out of 134 ampicillin-resistant clinical isolates of S. Typhimurium, collected over 2002-2004 in 21 Spanish cities, were assigned to the group, which can be already regarded as endemic. Most isolates (>89%) shared the following features: (i) resistance to ampicillin, chloramphenicol, streptomycin/spectinomycin, sulfonamides, and tetracycline, encoded by bla(OXA-1)-catA1-aadA1-sul1-tet(B); (ii) a class 1 integron (InH) with the bla(OXA-1)-aadA1 gene cassettes within its variable region of ca. 2000bp; (iii) the spvC, rck, samA, oriT, traT, traX, repA (RepFIIA), and parA/B genes (but not rsk and pefABCD) of pSLT; (iv) a hybrid plasmid of ca. 125kb, termed pUO-StVR2, where the resistance and virulence genes are located. However, intra-group diversity was also detected, since a total of four resistance phenotypes, five resistance genotypes, two integron profiles, five plasmid variants (pUO-StVR2, 4-7, differing in size, restriction profile and/or resistance pattern), 15 XbaI-BlnI combined macrorestriction profiles, and five phage types were identified. Each hybrid plasmid was revealed as a distinctive BlnI band, through hybridization with pUO-StVR2. The genetic markers used, together with the knowledge generated in the present study, could be applied to epidemiological surveillance of S. Typhimurium pUO-StVR worldwide.

Asunto(s)

Farmacorresistencia Bacteriana Múltiple/genética , Plásmidos/genética , Salmonella typhimurium/genética , Resistencia a la Ampicilina/genética , Tipificación de Bacteriófagos , Enfermedades Endémicas/estadística & datos numéricos , Humanos , Integrones/genética , Infecciones por Salmonella/epidemiología , Salmonella typhimurium/aislamiento & purificación , España/epidemiología , Virulencia

RESUMEN

OBJECTIVES: To evaluate the incidence, molecular basis and distribution among genomic types of antimicrobial drug resistance in Salmonella enterica (S.) serovar Brandenburg isolates recorded in the Principality of Asturias, Spain. METHODS: Thirty-seven S. Brandenburg isolates were tested for susceptibility to antimicrobial agents and typed by random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE). PCR amplifications, together with DNA cloning and sequencing, were used to identify resistance genes, integrons and transposons and to establish the structure and physical associations between them. Conjugation experiments were applied to establish the location of the identified elements. RESULTS: Twenty-one isolates were resistant to one or more unrelated drugs. Resistances to streptomycin, tetracycline, kanamycin, chloramphenicol, ampicillin and trimethoprim-sulfamethoxazole, encoded by aadA1, tet(A) or tet(B), aphA1, catA1, bla(TEM) and dfrA1-sul1-sul3, respectively, were most frequently observed. Multidrug resistance (32.4%) was mainly mediated by mobile genetic elements. These included: (i) class 1 integrons (with dfrA1-aadA1 gene cassettes in their variable region), which were part of Tn21-related transposons associated with Tn9; (ii) a Tn1721-derivative containing tet(A); (iii) a defective Tn10 that carried tet(B), and was linked to an integron; and (iv) large conjugative plasmids carrying a class 1 integron-Tn21-Tn9-like structure, together with the Tn1721- or the Tn10-related element. Two-way-RAPD and XbaI-PFGE discriminated the isolates into 15 and 12 profiles, respectively. CONCLUSIONS: Complex genetic elements have apparently been responsible for the recruitment, assembly and dispersion of resistance genes among the highly diverse genomic types of S. Brandenburg, identified as causal agents of human salmonellosis in the Principality of Asturias, over recent years.

Asunto(s)

Conjugación Genética/genética , Elementos Transponibles de ADN/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Integrones/genética , Salmonella enterica/clasificación , Antibacterianos/farmacología , Clonación Molecular , ADN Bacteriano/análisis , Electroforesis en Gel de Campo Pulsado , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa/métodos , Técnica del ADN Polimorfo Amplificado Aleatorio , Infecciones por Salmonella/epidemiología , Infecciones por Salmonella/microbiología , Salmonella enterica/efectos de los fármacos , Salmonella enterica/genética , Salmonella enterica/aislamiento & purificación , Análisis de Secuencia de ADN , Serotipificación

RESUMEN

OBJECTIVES: To characterize class 1 and class 2 integrons which were simultaneously detected in non-typhoid Salmonella enterica strains of non-prevalent serovars, and to investigate their possible association with transposons and/or plasmids. METHODS: Eight multidrug-resistant S. enterica strains belonging to serovars Virchow (4), Panama (2), Grumpensis (1) and Worthington (1), each containing a class 1 and a class 2 integron, were analysed. Nested PCR amplification was used to determine the gene-cassette configuration of the integrons. Overlapping PCR amplifications were applied in integron-transposon linkage experiments. Conjugation and hybridization experiments were used to localize integrons and transposons in the bacterial genome (plasmid and chromosome associated). RESULTS: One of two different class 1 integrons (with variable regions of 1000 bp/aadA1 and 2300 bp/sat-smr-aadA1) inserted into Tn21-like transposons, were found to coexist with the class 2 integron (2300 bp/dfrA1-sat1-aadA1) of Tn7 in the analysed strains. Class 1 integrons were always found in large conjugative plasmids whereas apparently intact or defective copies of the Tn7 integron could be located on the same plasmid and/or the bacterial chromosome. CONCLUSIONS: This report describes different associations between mobile genetic elements that play a crucial role in the capture and spread of antimicrobial drug resistance. As far as we are aware, this is the first description of class 2 integrons in serovars Panama, Grumpensis and Worthington.

Asunto(s)

Elementos Transponibles de ADN/genética , ADN Bacteriano/genética , Integrones/genética , Plásmidos/genética , Salmonella enterica/genética , Cromosomas Bacterianos/genética , Conjugación Genética , Farmacorresistencia Bacteriana Múltiple , Genoma Bacteriano , Secuencias Repetitivas Esparcidas , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa

RESUMEN

Two large conjugative resistance (R) plasmids from clinical strains of Salmonella enterica serovar Virchow carried a class 2 integron with the 5' conserved sequence (5'CS)-dfrA1-sat1-aadA1-3'CS gene array, which is associated with defective Tn7 transposons. In addition, each contained a different class 1 integron (with 5'CS-aadA1-3'CS or 5'CS-sat-smr-aadA1-3'CS gene arrays) linked to Tn21-Tn9 sequences, and several non-integron-associated R determinants. An intact copy of Tn7 (including the class 2 integron) was present in the chromosome of each strain.

Asunto(s)

Conjugación Genética , Integrones , Plásmidos , Salmonella enterica/genética , Secuencia de Bases , Elementos Transponibles de ADN , Farmacorresistencia Bacteriana/genética , Humanos , Datos de Secuencia Molecular

RESUMEN

A total of 80 strains of Salmonella enterica serovar Enteritidis, causing gastroenteritis (G) or bacteraemia (B), and three control strains (C), were subjected to: (i) detection of 14 chromosomally and 1 plasmid-located virulence genes by PCR, (ii) detection of DNA polymorphisms by XbaI and BlnI PFGE, and cluster analysis, (iii) mapping of the 15 screened sequences on macrorestriction profiles and (iv) comparison of the screening and mapping results with data available for other Salmonella strains. Identical virulence genotypes and very similar macrorestriction profiles were shown by most S. Enteritidis strains. However, a number of B strains belonged to genomic types with polymorphisms affecting fragments carrying (SPI2-slyA), (SPI2-slyA-phoP/Q-agfA), (SPI4 and/or stn) and spvC. The information obtained provides the basis for further studies on the genetic background of virulence and the molecular epidemiology of S. Enteritidis.

Asunto(s)

Mapeo Cromosómico , Cromosomas Bacterianos/genética , Genoma Bacteriano , Mapeo Restrictivo , Salmonella enteritidis/clasificación , Salmonella enteritidis/patogenicidad , Reacción en Cadena de la Polimerasa , Salmonella enteritidis/genética , Virulencia/genética

RESUMEN

Staphylococcus aureus recovered from nasal carriers, producers and non-producers (43 isolates each) of classical pyrogenic toxin superantigens (PTSAgs), were screened for 17 additional PTSAg-genes by PCR. Percentages of 88.4 and 65.1 were positive for some new enterotoxin-gene, and 76.7 and 55.8 for enterotoxin-gene-clusters (egc-like), respectively. The 86 isolates belonged to 17 toxin-genotypes (all eta-, etb-, etd-, see- and sep-negative), and generated 40 SmaI-genomic profiles that in a dendrogram of similarity (S0.7) clustered into nine lineages and 11 non-clustered branches. Correlations between classical PTSAgs and SmaI-lineages were established and egc-like groupings appeared dispersed in six lineages.

Asunto(s)

Toxinas Bacterianas/genética , Portador Sano/microbiología , Exotoxinas/genética , Nariz/microbiología , Staphylococcus aureus/aislamiento & purificación , Superantígenos/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Electroforesis en Gel de Campo Pulsado , Humanos , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , España , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo

RESUMEN

A set of 269 Staphylococcus aureus isolates recovered from nasal carriers and manually handled foods in a region of Spain was analyzed for pyrogenic toxin production and toxin genes. Fifty-seven isolates producing at least one of four enterotoxins (SEA, SEB, SEC, SED), 10 isolates producing only toxic shock syndrome toxin (TSST-1), and 10 isolates producing both toxin types were found. The 77 toxigenic isolates were discriminated into 36 SmaI genomic and 13 EcoRI plasmid profiles. A strong relationship between toxin profiles with both SmaI genomic and EcoRI plasmid profiles was revealed. SmaI genomic profiles showing six or less mismatching fragments and similarity coefficient > or =0.7 were included in a lineage. Eight lineages were differentiated; six of them grouped both human and food isolates and two of these also included outbreak-implicated isolates. Two lineages, represented by TSST-SEA and TSST-1, on the one hand, and SEC and SEC-SED isolates, on the other hand, were the most frequent, but only the second was outbreak-related. When SmaI genomic and EcoRI plasmid profiles were hybridized with tst, sea, seb, and sec toxin probes, it was observed that each probe mapped on a different SmaI fragment from isolates falling into the same lineage. All of the probes only mapped on genomic fragments, but sed also mapped on three plasmid fragments. When sej and ser probes were included, they mapped together with sed on the chromosome and on the plasmids. Two plasmids (ca. 33 and 36 kb) carried the expected sed-sej-ser genes, while the other (ca. 53.5 kb) carried sed-sej and ser-like genes. The latter plasmid and the chromosomal location of sed-sej-ser are new findings from this study.

Asunto(s)

Toxinas Bacterianas/aislamiento & purificación , Enterotoxinas/aislamiento & purificación , Manipulación de Alimentos , Intoxicación Alimentaria Estafilocócica , Staphylococcus aureus/química , Superantígenos/aislamiento & purificación , Toxinas Bacterianas/genética , Cromosomas Bacterianos , ADN Bacteriano/análisis , Enterotoxinas/genética , Microbiología de Alimentos , Humanos , Mucosa Nasal/microbiología , Plásmidos , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/microbiología