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.

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.Methicillin-resistant Staphylococcus aureus (MRSA) of sequence type 398 (ST398) has gained particular attention during recent years because of its association with pigs and its ability to colonize pig farmers and other people in close contact with pigs (7,12,47). The MRSA isolates of ST398 usually lack important virulence determinants that are typical in other community and hospital MRSA isolates. The majority of the ST398 isolates analyzed so far carry only hemolysin-encoding genes (13,21,31,32), although a small number of cases in which the isolates carried the bicomponent leukotoxin PantonValentine (lukPV genes) (43, 49) or staphylococcal enterotoxins (SEs, se genes) (21, 26) have also been reported. Genes for other toxins, like exfoliatins (ET, et genes), leukotoxins, and toxic shock syndrome toxin (TSST-1, tst gene) have not been found yet in ST398 isolates (13,21,31,32,44). The regulation of the expression of most extracellular virulence factors in S. aureus is under the control of a two-component signaling system called the accessory gene regulator (agr), which is polymorphic and divided into four distinct genetic groups (I to IV). A correlation exists between some agr groups and certain pathotypes and clonal complexes (CCs) (48), and CC398 seem to be associated with agr group I (ag...

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.

Non-typhoidal serovars of Salmonella enterica (NTS) are a leading cause of food-borne disease in animals and humans worldwide. Like other zoonotic bacteria, NTS have the potential to act as reservoirs and vehicles for the transmission of antimicrobial drug resistance in different settings. Of particular concern is the resistance to critical “last resort” antimicrobials, such as carbapenems. In contrast to other Enterobacteriaceae (e.g., Klebsiella pneumoniae, Escherichia coli, and Enterobacter, which are major nosocomial pathogens affecting debilitated and immunocompromised patients), carbapenem resistance is still very rare in NTS. Nevertheless, it has already been detected in isolates recovered from humans, companion animals, livestock, wild animals, and food. Five carbapenemases with major clinical importance—namely KPC (Klebsiella pneumoniae carbapenemase) (class A), IMP (imipenemase), NDM (New Delhi metallo-β-lactamase), VIM (Verona integron-encoded metallo-β-lactamase) (class B), and OXA-48 (oxacillinase, class D)—have been reported in NTS. Carbapenem resistance due to the production of extended spectrum- or AmpC β-lactamases combined with porin loss has also been detected in NTS. Horizontal gene transfer of carbapenemase-encoding genes (which are frequently located on self-transferable plasmids), together with co- and cross-selective adaptations, could have been involved in the development of carbapenem resistance by NTS. Once acquired by a zoonotic bacterium, resistance can be transmitted from humans to animals and from animals to humans through the food chain. Continuous surveillance of resistance to these “last resort” antibiotics is required to establish possible links between reservoirs and to limit the bidirectional transfer of the encoding genes between S. enterica and other commensal or pathogenic bacteria.

Salmonella enterica serovar 4,[5],12:i:- is a monophasic variant of S. Typhimurium incapable of expressing the second-phase flagellar antigen (fljAB operon), and it is recognized to be one of the most prevalent serovars causing human infections. A clonal lineage characterized by phage type DT193, PulseNet PFGE profile STYMXB.0131 and multidrug resistance to ampicillin, streptomycin, sulphonamides and tetracycline (R-type ASSuT) is commonly circulating in Europe. In this study we determined the deletions affecting the fljAB operon and the resistance region responsible for the R-type ASSuT in a strain of Salmonella enterica serovar 4,5,12:i:- DT193/STYMXB.0131, through an approach based on PCRs and Southern blot hybridization of genomic DNA. Using a set of nine specific PCRs, the prevalence of the resistance region was assessed in a collection of 144 S. enterica serovar 4,[5],12:i:-/ASSuT/STYMXB.0131 strains isolated from Germany, Switzerland and Italy. A 28 kb-region is embedded between the loci STM2759 and iroB, replacing the DNA located in between, including the fljAB operon. It encompasses the genes blaTEM−1, strA-strB, sul2 and tet(B) responsible for the R-type ASSuT together with genes involved in plasmid replication and orfs of unknown function characteristically located on IncH1 plasmids. Its location and internal structure is fairly conserved in S. enterica serovar 4,[5],12:i:-/ASSuT/STYMXB.0131 strains regardless of the isolation source or country. Hence, in the S. enterica serovar 4,[5],12:i:-/ASSuT/STYMXB.0131 clonal lineage widespread in Germany, Switzerland and Italy, a resistance region derived from IncH1 plasmids has replaced the chromosomal region encoding the second flagellar phase and is an example of the stabilization of new plasmid-derived genetic material due to integration into the bacterial chromosome.

A set of 84 Staphylococcus aureus isolates collected from the milk of cows with subclinical mastitis in Asturias (a cattle region of Spain) and six control strains were tested for sequences of genes encoding hemolysins (hla, hlb, hld, hlg, and hlg-2), leukotoxins (lukPV, lukM, and lukED), toxic shock syndrome toxin (tst), and enterotoxins (sea to see, seg to ser, and seu) by conventional and multiplex PCR. It was found that 84, 83, 11, and 39 isolates carried some type of hl, luk, tst, or se gene, respectively, which were arranged in 14 exotoxin genotypes. All of the isolates were negative for lukPV, hlg, sea, sed, see, sej, sek, sep, seq, and ser. Two gene groupings could be related with pathogenicity islands-[lukED, seg, sei, sem, sen, seo ؎ seu] with Sa␤-1 and [tst, sec, sel] with SaPIbov, present in 45 and 13.1% of the isolates, respectively-while 11.9% of them carried both islands. Only one contained seb (together with Sa␤-1), and another contained seh (together with lukED). The isolates were also analyzed by pulsed-field gel electrophoresis performed with SmaI. Thirty-nine SmaI profiles (similarity coefficient [S] ‫؍‬ 0.94 to 0.21) were differentiated; 12, 1, and 10 of these, respectively, were generated by isolates presumptively carrying Sa␤-1, SaPIbov, or both. Five SmaI profiles (S > 0.8) formed a cluster, which contained 20 and 10 isolates carrying one (Sa␤-1) or both islands. These data show the high frequency of genes encoding cytotoxins and pyrogenic toxin superantigens, their relationship with pathogenicity islands, and their distribution among a diversity of genetic types of S. aureus related to subclinical mastitis.

ABSTRACTStaphylococcal food poisoning, one of the most common food-borne diseases, results from ingestion of one or more staphylococcal enterotoxins (SEs) produced byStaphylococcus aureusin foods. In the present study, 64S. aureusisolates recovered from foods and food handlers, associated or not associated with food-poisoning outbreaks in Spain, were investigated. They were assigned to 31 strains byspatyping, 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 geneseta,etd(6.5% each), andetb(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-seljwith or withoutser(22.9%),selk-selq(6.5%),seh,sell,selp(9.7% each),egc1(32.3%), andegc2(48.4%). The number ofseandselgenes ranged from zero to 12. All isolates carryingtst, and most isolates with genes encoding classical enterotoxins (SEA, SEB, SEC, and SED), expressed the corresponding toxin(s). Two CC5 isolates from hamburgers (spatype t002, sequence type 5 [ST5];spatype t2173, ST5) were methicillin resistant and harbored staphylococcal cassette chromosomemec(SCCmec) IVd. Six (19.4%) were mupirocin resistant, and one (spatype t120, ST15) from a food handler carriedmupA(MIC, 1,250 μg/ml). Resistance to ampicillin (blaZ) (61.3%), erythromycin (ermA-ermCorermC) (25.8%), clindamycin (msrA-msrB or msrB) (16.1%), tetracycline (tetK) (3.2%), and amikacin-gentamicin-kanamycin-tobramycin (aphAwithaacAplusaphDoraadD) (6.5%) was also observed. The presence ofS. aureusstrains with an important repertoire of virulence and resistance determinants in the food chain represents a potential health hazard for consumers and merits further observation.

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.