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http://origin.cdc.gov/ncidod/eid/vol11no05/04-0866.htm
Clonal Comparison of Staphylococcus aureus Isolates from Healthy Pig
Farmers, Human Controls, and Pigs
Laurence Armand-Lefevre,* Raymond Ruimy,* and Antoine Andremont*
*Groupe Hospitalier Bichat-Claude Bernard, Paris, France

Pig farming is a risk factor for increased nasal Staphylococcus aureus
colonization. Using sequence typing and phylogenetic comparisons, we
showed that overcolonization of farmers was caused by a few bacterial
strains that were not present in nonfarmers but often caused swine
infections. This finding suggests a high rate of strain exchange
between pigs and farmers.

Pig farmers work in close contact with animals that are given heavy
loads of antimicrobial agents and therefore are highly colonized by
resistant bacteria (1). The transfer of resistant bacteria from farm
animals to farmers has been demonstrated in several instances (2,3).
In a recent comparison of pig farmers and nonfarming controls, farmers
were at a significantly greater risk for colonization by resistant
commensal bacteria, including fecal enterobacteria and enterococci,
nongroupable throat streptococci, and nasal Staphylococcus aureus (4).
The rate of nasal S. aureus colonization was also significantly higher
in farmers, in whom it reached 44.6%, compared to 24.1% in controls
(4). The latter rate was similar to that observed in published
cross-sectional prevalence studies conducted among study participants
living in healthy communities (5). However in the previous study, we
did not investigate the sources and origin of nasal S. aureus
colonization and resistance in farmers. Here, we used the gene-based,
recently developed technique of multilocus sequence typing (MLST)
(available from www.mlst.net) to describe the characteristics of these
nasal S. aureus strains from farmers and controls and the
relationships between strains, and investigated their possible animal
origin by comparing them with strains isolated from infected pigs from
the same geographic area.

The Study
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 and nuc genes was determined by multiplex
polymerase chain reaction (PCR) using mecA1, mecA2, nuc1, and nuc2
primers (6). 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, aroE, glpF, gmk, pta, tpi, 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.

 Figure  
   
 Click to view enlarged image

Figure. Unrooted tree showing the phylogenetic relationships among
Staphylococcus aureus isolates from pig farmers...
  
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 (Figure) 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).

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 (Figure). 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).

Conclusions
Our results strongly suggest that the high risk for nasal S. aureus
colonization that we previously reported in pig farmers (4) was due to
strains exchanged with swine: 25 (57%) of the 44 pig farmer isolates
grouped together with the swine isolates and had 3 STs (9, 398, and
433) that were not found in control isolates. If these pig farmers had
not been taken into account, the rate of nasal carriage in pig farmers
would have been close to that found in controls (5). The number of pig
strains tested was small because swine mastitis, unlike bovine
mastitis, is rare and because no other collection of S. aureus from
swine was available for testing (we did not sample pigs when we
performed the previous (4) study of pig farmers and controls).

The hypothesis that pig farmers exchanged strains bearing these
specific STs with swine was not formally demonstrated in our study
because we did not test strains isolated from swine and from farmers
on the same farms at the same time. However, MLST is a powerful tool
for comparing strains and determining their phylogenetic and
epidemiologic relatedness (8). This tool, together with the similarity
of the pig farmer and swine strains (which both had STs 9, 398, and
433) and the absence of these STs in strains from epidemiologically
matched controls, argues strongly in support of an exchange of
specific strains between pig farmers and pigs.

The widespread geographic distribution of strains with similar STs in
strains in pig farmers and swine only was puzzling because pig farmers
were working in different farms scattered among the 7 major French
pig-raising departments and separated by tens or sometimes hundreds of
kilometers. The potential sources of contamination of pigs and farmers
by strains such as those with STs 9, 398, or 433, which were common to
both, were not investigated. One possibility might be that the sows
used for pig production are the vectors of transmission because young
sows are transferred from 1 farm to another when production needs to
be increased. A specific investigation is needed to explore this
possibility. Contamination of 9 sheep farms and their dairy products
by a single S. aureus strain was previously demonstrated in France,
but the routes of dissemination were not investigated (9). In
California, the widespread distribution of a multidrug-resistant clone
of Escherichia coli that caused community-acquired urinary tract
infections was suspected to be of animal origin, but again, the routes
of dissemination were not investigated (10).

We observed differences in susceptibility to antimicrobial agents
between strains with the same ST, which suggests that the final
phenotypes were selected locally, depending on the use of
antimicrobial agents. This finding was particularly striking with
susceptibility to macrolides and related drugs, a class of
antimicrobial agent widely used in pig farming (11), although
individual farms may use it differently. Unfortunately, data on the
use of antimicrobial agents by each farm were not available.

Animal-to-human transmission during farming has been demonstrated for
enterobacteria and enterococci in several instances (1,2) but only
once before for S. aureus (9). Although they are rare (12), animal
MRSA have been suggested as a source of infection for humans (13). Our
results suggest that such transmission may be frequent, particularly
since virtually no barrier precautions were used by the pig farmers
studied in our previous investigation (4).

In the few published studies on the molecular epidemiology of nasal
strains from carriers, genetic backgrounds of the strains were very
diverse (14), just as in our controls. This finding further underlines
the particular way in which most pig farmer strains are grouped
together. Whatever the exact route of transmission, single S. aureus
strains, probably acquired from pigs, colonized the nostrils of pig
farmers throughout large geographic areas and that this colonization
probably caused their overall increase in nasal S. aureus
colonization. Since nasal carriage is a recognized source of S. aureus
bacteremia with severe consequences (15), our findings suggest that
pig farming could be a staphylococcal hazard for farmers, under the
conditions in which it is practiced today. Several points could not be
addressed in the study, including whether colonization of the farmers
by pig S. aureus isolates was permanent or temporary, whether the pig
isolates were also disseminated in the farmer's families and to other
persons living in the area, whether skin and soft tissues of pig
farmers were infected and, if so, whether or not it was due to S.
aureus isolates identical to those from pigs. These questions will be
addressed in further, specifically designed, epidemiologic studies.

Acknowledgments
We thank Alain Lacourt, Eric Laporte, and Hervé Morvan for providing
bacterial strains from swine infections; Marie-Jeanne Julliard and
Sabine Couriol for secretarial assistance; and Mathilde Dreyfus for
English revision.

This work was supported by the Agence Française pour le Sécurité
Sanitaire de l'Environnement (AFSSE), contract number RD-2003-001.The
original population study during which the S. aureus strains studied
here were isolated had been performed by the authors in collaboration
with the following institutions: Fédération Nationale des Coopératives
Bovines et Viandes, Institut de Veille Sanitaire, and Mutualité
Sociale Agricole.

Dr. Armand-Lefevre is a clinical bacteriologist in the bacteriology
laboratory of Bichat-Claude Bernard Hospital. Her current interest is
the evolution of bacterial resistance in the natural ecosystems of
humans.

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Suggested citation for this article:
Armand-Lefevre L, Ruimy R, Andremont A. Clonal comparison of
Staphylococcus aureus isolates from healthy pig farmers, human
controls, and pigs. Emerg Infect Dis [serial on the Internet]. 2005
May [date cited]. Available from
http://www.cdc.gov/ncidod/EID/vol11no05/04-0866.htm