The S. aureus
strains studied included 44 nasal isolates from healthy pig farmers and 21 from healthy nonfarmer controls (i.e., bank or insurance workers). These participants all had been part of the population included in a previously published epidemiologic study in which the resistance rates in commensal bacteria from healthy pig farmers were compared with the rates in controls matched for age, sex, and county of residence (4
). This population was disseminated over 7 French departments, chosen because they were the leading areas of porcine production. A department is a French administrative territory roughly the size of a British or American county. Each pig farmer worked on a different pig farm. We also studied 14 S. aureus
isolates from the following types of swine infections: cutaneous, for isolates CA-1, CA-2, CA-6, F-9, and F-10; urinary, for isolates CA-3, CA-5, F-8, F-9, IV-11, IV-13, and IV-14; blood, for IV-12; and bone for CA-4. Isolates were collected from 1996 to 2002 in 4 of the 7 departments in which the pig farmers were working and were kindly provided by state veterinary laboratories. All strains had been identified with conventional techniques, and their susceptibility to antimicrobial agents had been determined by the disk-diffusion technique (available from www.sfm.asso.fr
S. aureus strains were lysed with 30 μg/mL lysostaphin, which was incubated for 10 min at 37°C, and DNA was extracted by using MagNA Pure LC automat (Roche, Mannheim, Germany), as recommended by the manufacturer. DNA concentrations were measured by optical density, and extracts were diluted to obtain concentrations of 50 ng/μL DNA for amplification.
The presence of mecA
genes was determined by multiplex polymerase chain reaction (PCR) using mecA1
). Mixes contained 250 μmol/L of each primer, 400 nmol/L of each deoxynucleoside triphosphate (Boehringer GmbH, Mannheim, Germany), 1 × reaction buffer supplied by the manufacturer with 1.5 mmol MgCl2
, 1 U of AmpliTaq DNA polymerase (Applera, Courtaboeuf, France), and 100 ng of DNA extract in a final volume of 50 μL. The PCR was carried out for 1 cycle of 5 min denaturation at 94°C and 20 cycles of 10 s at 94°C, 10 s at 60°C, and 30 s at 72°C. PCR products were visualized under UV irradiation after electrophoresis.
MLST analysis was carried out by sequencing fragments of 7 housekeeping genes (arcC
, and yqiL
), as described (available form www.mlst.net
), except that the primers used for tpi
amplification were tpi2u 5´-GCATTAGCAGATTTAGGCGTTA-3´ and tpi2d 5´-TGCACCTTCTAACAATTGTACGA-3´. All PCR products were purified by using the QIAquick PCR purification kit (Qiagen, Courtaboeuf, France) and sequenced using an ABI Prism sequence (Applera) with Big Dye reaction mixes, using the primers chosen for the initial amplification, and analyzed on the BioEdit biological sequence editor 5.0.6 (7
). Each allele of the 7 housekeeping genes was assigned to a number, and each isolate was characterized by a sequence type (ST), defined by the allelic profile of the housekeeping genes. These profiles were compared to those present in the S. aureus
MLST database (available from www.mlst.net
). The 2 new allele sequences of the yqiL
gene and the 1 new sequence of the aroE
gene were deposited in the MLST database under numbers 72, 73, and 91, respectively. The new STs have also been deposited in the MLST database, under numbers ST432 to ST438, ST440, and ST457.
For each strain, the sequences of all 7 housekeeping genes were concatenated to produce an in-frame sequence of 3,198 bp. A phylogenetic tree () was generated by using the neighbor-joining method, and the robustness of branches was estimated by the bootstrap method. Both are included in Mega version 2.1 software (available from www.megasoftware.net
Figure Unrooted tree showing the phylogenetic relationships among Staphylococcus aureus isolates from pig farmers (PF), bank or insurance workers (BIW), and swine (S). The tree was obtained by the neighbor-joining method, based on the comparison of partial sequences (more ...)
All 79 isolates studied were identified as S. aureus by conventional techniques and harbored the nuc gene. The mecA gene was present in the 5 methicillin-resistant isolates. The Figure shows an unrooted tree in which the aligned sequences of the 79 isolates are compared; it also indicate the ST number and antimicrobial resistance of each isolate.
Nineteen STs were identified among the 65 nasal isolates from pig farmers and nonfarmer controls. Nine (STs 432 to 438, ST440, and ST457) had not been previously described. Twelve of the 19 STs were each found in only 1 isolate, 1 (ST 437) in 2 isolates, and the remaining 6 (ST5, ST8, ST9, ST15, ST34, and ST398) in at least 4 isolates. Only 3 of these 6 STs (ST5, ST15, and ST34) were found in isolates from both pig farmers and nonfarmer controls. ST5 was present in 10 isolates (7 from farmers, 3 from controls), ST15 in 7 (5 from farmers, 2 from controls), and ST34 in 6 (3 from farmers, 3 from controls). Comparison with isolates from the entire MLST database showed that ST5 had previously been reported in 90 isolates from the United Kingdom, Japan, United States, and Poland; ST15 in 33 isolates from the United Kingdom, Australia, and Canada; and ST34 in 15 isolates from the United Kingdom only. The other 3 STs (STs 8, 9, and 398) were only found in isolates from pig farmers. ST8, retrieved from 4 isolates from pig farmers, had previously been reported in 86 isolates from the United Kingdom, Australia, United States, Canada, France, Germany, Netherlands, Denmark, and Greece. ST9 was found in as many as 18 of the 44 pig farmer isolates that we studied but had only been previously described in 5 isolates, all from the United Kingdom. ST398 was retrieved from 6 isolates from pig farmers; previously, it had only been reported in 1 isolate from the Netherlands.
Analysis of the geographic distribution of STs 8, 9, and 398, which were only found in pig farmers, showed that they were dispersed throughout the 7 departments studied. The 18 ST9 isolates were from pig farmers working in 6 of the 7 departments, the 4 ST8 isolates from pig farmers in 3 of 7, and the 6 ST398 isolates from pig farmers in 4 of 7 departments.
Thirteen of the 14 isolates from swine infections had STs that were only found elsewhere in strains from pig farmers. Two of these 13 swine isolates had ST433, which we found in a single pig farmer isolate, 7 had ST9, and 4 ST398 (). STs9, 398, and 433 in the swine isolates originated from 3, 2, and 2 different departments, respectively. The remaining swine isolate had ST97, which was not observed in another isolate. In all, 25 (57%) of the 44 pig farmers isolates had STs identical to those of swine strains. No control isolate was identical to those of the swine.
Four of the 5 methicillin-resistant S. aureus
(MRSA) strains found in pig farmer isolates had STs (ST5 and ST8) previously reported in MRSA (available from www.mlst.net
) or new (ST438). The remaining strain had ST398, which was grouped together with pig farmer isolates that were susceptible to methicillin. Differences in susceptibility to antimicrobial agents other than methicillin were also observed between isolates with identical STs. Although 25 of 25 isolates with ST9 were resistant to penicillin, only 17 were resistant to lincomycin and erythromycin. Of the latter, 5 were coresistant to pristinamycin. One was resistant to kanamycin and pefloxacin. Similar variations in antimicrobial susceptibility were observed among strains with the other STs. Resistance to erythromycin was more frequent in pig farmers than controls (29/44 [66%] vs. 2/21 [10%], as previously reported (4
). Resistance was intermediate in swine strains (5 [38%] of 14).