M. R. Rodicio

Food Poisoning and Staphylococcus aureus Enterotoxins

Staphylococcus aureus produces a wide variety of toxins including staphylococcal enterotoxins (SEs; SEA to SEE, SEG to SEI, SER to SET) with demonstrated emetic activity, and staphylococcal-like (SEl) proteins, which are not emetic in a primate model (SElL and SElQ) or have yet to be tested (SElJ, SElK, SElM to SElP, SElU, SElU2 and SElV). SEs and SEls have been traditionally subdivided into classical (SEA to SEE) and new (SEG to SElU2) types. All possess superantigenic activity and are encoded by accessory genetic elements, including plasmids, prophages, pathogenicity islands, vSa genomic islands, or by genes located next to the staphylococcal cassette chromosome (SCC) implicated in methicillin resistance. SEs are a major cause of food poisoning, which typically occurs after ingestion of different foods, particularly processed meat and dairy products, contaminated with S. aureus by improper handling and subsequent storage at elevated temperatures. Symptoms are of rapid onset and include nausea and violent vomiting, with or without diarrhea. The illness is usually self-limiting and only occasionally it is severe enough to warrant hospitalization. SEA is the most common cause of staphylococcal food poisoning worldwide, but the involvement of other classical SEs has been also demonstrated. Of the new SE/SEls, only SEH have clearly been associated with food poisoning. However, genes encoding novel SEs as well as SEls with untested emetic activity are widely represented in S. aureus, and their role in pathogenesis may be underestimated.

Virulence and resistance determinants of German Staphylococcus aureus ST398 isolates from nonhuman sources.

ABSTRACT A series of 100 Staphylococcus aureus isolates ascribed to sequence type 398 (ST398) and recovered from different sources (healthy carrier and diseased pigs, dust from pig farms, milk, and meat) in Germany were investigated for their virulence and antimicrobial resistance genetic background. Antimicrobial resistance was determined by the disk diffusion method. Virulence and resistance determinants (37 and 31 genes, respectively) were tested by PCR. Only two virulence profiles, including the accessory gene regulator agrI and three or four hemolysin-encoding genes, were detected. In contrast, 33 resistance profiles were distinguished (only 11 were shown by more than one isolate). Fifty-nine isolates were multiresistant (four or more antimicrobial classes), and 98 were methicillin resistant (mecA positive). All of the ST398 isolates showed resistance to tetracycline [encoded by tet(M) alone or together with tet(K) and/or tet(L)]. In addition, 98% were resistant to other antimicrobials, including macrolide-lincosamine-streptogramin B (70%, encoded by ermA, ermB, and ermC, alone or in combination), trimethoprim (65%, mostly due to dfrK and dfrG), kanamycin and gentamicin [29% and 14%, respectively, mainly related to aac(6′)-Ie-aph(2″)-Ia and/or ant(4′)-Ia but also to aph(3′)-IIIa], chloramphenicol (9%, fexA or cfr), quinupristin-dalfopristin (9%), ciprofloxacin (8%), and trimethoprim-sulfamethoxazole (4%). The heterogeneity of the resistance profiles underlines the ability of the ST398 clone to acquire multiple antimicrobial resistance genes. However, the virulence gene content of the tested isolates was low. Continuous surveillance is needed to clarify whether its pathogenicity potential for animals and humans will increase over time.

Genotypes, exotoxin gene content, and antimicrobial resistance of Staphylococcus aureus strains recovered from foods and food handlers

ABSTRACT Staphylococcal food poisoning, one of the most common food-borne diseases, results from ingestion of one or more staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus in foods. In the present study, 64 S. aureus isolates recovered from foods and food handlers, associated or not associated with food-poisoning outbreaks in Spain, were investigated. They were assigned to 31 strains by spa typing, multilocus sequence typing (MLST), exotoxin gene content, and antimicrobial resistance. The strains belonged to 10 clonal complexes (CCs): CC5 (29.0%), CC30 (25.8%), CC45 (16.1%), CC8, CC15 (two strains each), CC1, CC22, CC25, CC59, and CC121 (one strain each). They contained hemolysin genes (90.3%); lukED (77.4%); exfoliatin genes eta, etd (6.5% each), and etb (3.2%); tst (25.8%); and the following enterotoxin or enterotoxin-like genes or clusters: sea (38.7%), seb (12.9%), sec (16.1%), sed-selj with or without ser (22.9%), selk-selq (6.5%), seh, sell, selp (9.7% each), egc1 (32.3%), and egc2 (48.4%). The number of se and sel genes ranged from zero to 12. All isolates carrying tst, and most isolates with genes encoding classical enterotoxins (SEA, SEB, SEC, and SED), expressed the corresponding toxin(s). Two CC5 isolates from hamburgers (spa type t002, sequence type 5 [ST5]; spa type t2173, ST5) were methicillin resistant and harbored staphylococcal cassette chromosome mec (SCCmec) IVd. Six (19.4%) were mupirocin resistant, and one (spa type t120, ST15) from a food handler carried mupA (MIC, 1,250 μg/ml). Resistance to ampicillin (blaZ) (61.3%), erythromycin (ermA-ermC or ermC) (25.8%), clindamycin (msrA-msrB or msrB) (16.1%), tetracycline (tetK) (3.2%), and amikacin-gentamicin-kanamycin-tobramycin (aphA with aacA plus aphD or aadD) (6.5%) was also observed. The presence of S. aureus strains with an important repertoire of virulence and resistance determinants in the food chain represents a potential health hazard for consumers and merits further observation.

High Heterogeneity within Methicillin-Resistant Staphylococcus aureus ST398 Isolates, Defined by Cfr9I Macrorestriction-Pulsed-Field Gel Electrophoresis Profiles and spa and SCCmec Types

ABSTRACT During recent years, the animal-associated methicillin-resistant Staphylococcus aureus clone ST398 has extensively been studied. The DNA of these isolates turned out to be refractory to SmaI restriction, and consequently, SmaI is unsuitable for subtyping this clone by standard pulsed-field gel electrophoresis (PFGE). Very recently, ST398 DNA was shown to be digested by Cfr9I, a neoschizomer of SmaI. In the present study, we employed Cfr9I PFGE on 100 German and 5 Dutch ST398 isolates and compared their PFGE profiles, protein A gene variable repeat regions (spa types), and types of the staphylococcal cassette chromosome mec (SCCmec). The isolates (from healthy carrier pigs, clinical samples from pigs, dust from farms, milk, and meat) were assigned to 35 profiles, which were correlated to the SCCmec type. A dendrogram with the Cfr9I patterns assigned all profiles to two clusters. Cluster A grouped nearly all isolates with SCCmec type V, and cluster B comprised all SCCmec type IVa and V* (a type V variant first identified as III) carriers plus one isolate with SCCmec type V. Both clusters also grouped methicillin-susceptible S. aureus isolates. The association of the majority of isolates with SCCmec type V in one large cluster indicated the presence of a successful subclone within the clonal complex CC398 from pigs, which has diversified. In general, the combination of Cfr9I PFGE with spa and SCCmec typing demonstrated the heterogeneity of the series analyzed and can be further used for outbreak investigations and traceability studies of the MRSA ST398 emerging clone.

Antimicrobial Resistance and Virulence Determinants in European Salmonella Genomic Island 1-Positive Salmonella enterica Isolates from Different Origins

ABSTRACT Salmonella genomic island 1 (SGI1) contains a multidrug resistance region conferring the ampicillin-chloramphenicol-streptomycin-sulfamethoxazole-tetracycline resistance phenotype encoded by bla PSE-1, floR, aadA2, sul1, and tet(G). Its increasing spread via interbacterial transfer and the emergence of new variants are important public health concerns. We investigated the molecular properties of SGI1-carrying Salmonella enterica serovars selected from a European strain collection. A total of 38 strains belonging to S. enterica serovar Agona, S. enterica serovar Albany, S. enterica serovar Derby, S. enterica serovar Kentucky, S. enterica serovar Newport, S. enterica serovar Paratyphi B dT+, and S. enterica serovar Typhimurium, isolated between 2002 and 2006 in eight European countries from humans, animals, and food, were subjected to antimicrobial susceptibility testing, molecular typing methods (XbaI pulsed-field gel electrophoresis [PFGE], plasmid analysis, and multilocus variable-number tandem-repeat analysis [MLVA]), as well as detection of resistance and virulence determinants (PCR/sequencing and DNA microarray analysis). Typing experiments revealed wide heterogeneity inside the strain collection and even within serovars. PFGE analysis distinguished a total of 26 different patterns. In contrast, the characterization of the phenotypic and genotypic antimicrobial resistance revealed serovar-specific features. Apart from the classical SGI1 organization found in 61% of the strains, seven different variants were identified with antimicrobial resistance properties associated with SGI1-A (S. Derby), SGI1-C (S. Derby), SGI1-F (S. Albany), SGI1-L (S. Newport), SGI1-K (S. Kentucky), SGI1-M (S. Typhimurium), and, eventually, a novel variant similar to SGI1-C with additional gentamicin resistance encoded by aadB. Only minor serovar-specific differences among virulence patterns were detected. In conclusion, the SGI1 carriers exhibited pathogenetic backgrounds comparable to the ones published for susceptible isolates. However, because of their multidrug resistance, they may be more relevant in clinical settings.

Identificación bacteriana mediante secuenciación del ARNr 16S: fundamento, metodología y aplicaciones en microbiología clínica

other types of techniques turns out impossible, difficult, or requires a lot of time. Amplification of the gene to be sequenced uses preferably DNA extracted from a bacterial pure culture, but can be achieved also directly from a clinical sample. The latter has led to the discovery of new pathogens. Bearing in mind its potential, as the technical resources improve and the prize becomes more competitive, the identification based on 16S rDNA sequencing will certainly find a wider application in the clinical microbiology laboratory.

The emerging methicillin‐resistant Staphylococcus aureus ST398 clone can easily be typed using the Cfr9I SmaI‐neoschizomer

Aim:  To establish a PFGE protocol using Cfr9I, neoschizomer of SmaI, for typing of Staphylococcus aureus isolates belonging to the emerging MRSA ST398 clone.

Molecular typing of Staphylococcus aureus bloodstream isolates from geriatric patients attending a long-term care Spanish hospital.

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.

Spread of a multiresistant CTX-M-9-producing Salmonella enterica serotype Virchow phage type 19 in Spain.

The purpose of this study was to survey Salmonella enterica serotype Virchow phage type 19 (S. Virchow PT19) strains submitted to the Spanish National Reference Laboratory for Salmonella (SNRLS) from 2002 to 2006 in order to determine the rate type and genetic background of β-lactam resistance and to further identify the associated resistances. Ninety-nine S. Virchow PT19 strains were analysed. Antimicrobial susceptibility was determined by the disk diffusion method using Mueller–Hinton agar medium. Polymerase chain reaction (PCR) assays and, later, sequencing of the obtained fragments were performed for the molecular characterisation of the resistances. Pulsed-field gel electrophoresis (PFGE) and plasmid analysis (using conjugation, Southern blot hybridisation and replicon typing) were used for characterisation. The characterisation of S. Virchow PT19 strains allowed the identification of a clonal multiresistant S. Virchow PT19 harbouring an IncH12 plasmid with the blaCTX-M-9 gene within the complex integron In60 distributed across Spain. An IncH12 plasmid widely reported and studied in Enterobacteria is described in a clonal multiresistant S. Virchow PT19 which has successfully spread throughout Spain

Brote de gastroenteritis en una guardería causado por una cepa de Salmonella enterica serovar Typhimurium portadora del plásmido híbrido de resistencia-virulencia pUO-StVR2

Objetivo El estudio epidemiologico y microbiologico de un brote de salmonelosis ocurrido en 2004, que afecto a 22 ninos de una guarderia en Oviedo. Metodos Se determinaron las tasas de ataque y curves epidemicas y se aplicaron tecnicas de tipificacion bacteriana. Resultados El brote pudo atribuirse a una cepa de Salmonella enterica serovar Typhimurium perteneciente a un grupo emergente que alberga un plasmido hibrido de virulencia-resistencia de 125-130 kb, denominado pUO-StVR2. Las tasas de ataque para los casos confirmados frente a los posibles fue 27,2% frente a 23,5% para los ninos y 0 frente a 26,5% para el personal del centro. La fuente de infeccion no pudo ser identificada, aunque segun la evolucion temporal de los casos se podria considerar que el modo de transmision fue el contacto cuidadoras-ninos, facilitando la diseminacion fecal-oral. Todos menos uno de los 27 aislamientos analizados (de 22 pacientes) presentaban caracteristicas identicas: perfil-R, perfil de plasmidos, RAPD-tipo, PFGE-tipo y eran no fagotipificables (la excepcion fue un aislamiento con fagotipo DT104b). pUO-StVR2 es un derivado del plasmido de virulencia, pSLT, de la cepa tipo LT2 que ha ganado una region-R compleja (ACSSuT/blaOXA-catA1-strA/B-aadA1-sul1-sul2-tet[B]) en la que los genes blaOXA-aadA1 forman parte de la region variable de un integron de clase 1. Conclusion Este brote constituye un ejemplo de como una cepa de S. enterica serovar Typhimurium, perteneciente a un tipo probablemente ya endemico en la peninsula Iberica, puede transmitirse a la comunidad y afectar a un colectivo susceptible.