Staphylococcal bovine mastitis in France: enterotoxins, resistance and the human Geraldine methicillin-resistant Staphylococcus aureus clone

Abstract

Sir, Staphylococcus aureus, which potentially produces toxins that are common sources of food poisoning worldwide, represents about one-third of the bacteria isolated from bovine mastitis in France. Recently, in this country, a food poisoning outbreak due to the presence of the staphylococcal enterotoxin E in cheese made from unpasteurized cow’s milk was reported. This prompted us to retrospectively characterize a collection of S. aureus associated with bovine mastitis in order to detect the presence of enterotoxin genes and resistance phenotypes that might be of public health concern. Between February 2007 and June 2008, 139 non-duplicate S. aureus isolates from bovine mastitis were collected from 15 French veterinary laboratories covering all main cattle-rearing areas. Isolates were sent to the national reference laboratory of the Anses (French agency for food, environmental and occupational health safety) in Lyon, France, and identified using a triplex PCR targeting the 16S rRNA, mecA and S. aureus-specific nuc genes. Antimicrobial susceptibility was tested by the disc diffusion method on Mueller–Hinton agar and interpreted according to the breakpoints recommended by the Antibiogram Committee of the French Society of Microbiology (http://www.sfm.asso.fr). Sixteen antibiotics of veterinary and/or human interest were tested, using S. aureus ATCC 25923 as the quality control strain. Finally, the genes sea to see and seg to sej (enterotoxins), eta and etb (exfoliative toxins), tst (toxic shock syndrome toxin) and lukS-PV-lukF-PV (Panton–Valentine toxin) were screened by PCR. Enterotoxin genes were detected in 68.3% (95/139) of the isolates, and 54% (75/139) harboured a combination of two to four genes, with one strain even presenting a combination of six genes (Table 1). The genes seb, see, eta, etb and lukS-PV-lukF-PV were not detected, whereas sea, encoding the most frequent enterotoxin associated with food poisoning, was detected in 5 isolates (3.6%). In contrast, seg and sei were found in 70 and 71 isolates, respectively, and co-occurred in 61 isolates (associated or not with other toxins). Both genes are part of the enterotoxin gene cluster (selo, selm, sei, seln and seg), which has often been associated with S. aureus from bovine mastitis. The combination of sed and sej, usually localized on a plasmid, was also detected in 10 isolates (7.2%). Finally, tst and sec, which are part of the bovine S. aureus pathogenicity island SAPIbov, were detected in 12 isolates (while tst was most probably not associated with SAPIbov in the strain presenting six virulence genes). The proportion of penicillin-resistant isolates reached 41.0%, but antimicrobial resistance was otherwise weak, with only 10 isolates (7.2%) resistant to tetracycline and 9 to macrolides/lincosamides/streptogramin B (MLSB phenotype; 6.5%). One isolate was resistant to penicillin, cefoxitin, kanamycin and tobramycin, but was susceptible to all other antibiotics, including gentamicin. Since methicillin-resistant S. aureus (MRSA) is very rare in cases of bovine mastitis in France, this isolate was further characterized using a microarray-based assay (S. aureus Genotyping, Identibac– Alere) that simultaneously allows (i) detection of clinically relevant virulence factors, resistance determinants and typing markers of S. aureus and (ii) assignment of isolates to strains, clones or clonal complexes. Bovine MRSA isolates have been described only rarely, –7 but there are recent reports of mastitis associated with MRSA ST398 in Germany, which presented only very few virulence genes as characterized by the same microarray technique, and in Belgium. Here, surprisingly, the MRSA was assigned to the human epidemic Geraldine clone, which has emerged in France since 2007. Indeed, sequencing revealed a ST5 strain belonging to the spa-type t002, which perfectly matched all the characteristics of the Geraldine clone, including the accessory gene regulator allele (agr2) and the SCCmec cassette type I (ccrA1, ccrB1), as well as the complete patterns of resistance [mecA, blaR, blaI, blaZ, aadD, tet(L) and fosB] and virulence (tst, sec, sed, sel, sem, seo, clfA-B, epbS, eno and efb) genes. The immune evasion cluster, which is rarely found in animals and is typical of human adaptation, and which is thought to be involved in the success of this particular clone, was also detected. Thus, the presence of the Geraldine clone in a case of bovine mastitis, isolated in August 2007 in the Orne district, strongly argues for on-farm human transmission to the cow. This is of great concern since S. aureus is known to easily transmit within a farm and classically causes contagious mastitis. Moreover, resistance to methicillin (and multiresistance) makes it so difficult to treat that