**4. MRSA as zoonosis or humanosis**

Several studies have been done to determine the degree to which MRSA plays a role in zoonosis or humanosis. It has been observed that usually the strains originating from companion animals are originally human strains, and that the infection with this MRSA type is considered humanosis. On the other hand, the strains orginating from livestock (livestockassociated - LA) are often divergent from human strains and the infection with this type of LA-MRSA could be considered zoonosis, and in this case MRSA would be an emergent zoonotic agent. Within this context, veterinarians, cattle farmers and pet owners are considered risk groups for acquiring MRSA (Morgan, 2008).

In 2011, a case of empyema was reported in a 79-year-old man in Spain with isolation of MRSA ST398 in purulent exudates from the thorax and trachea and nasal swabs. The ineffective initial therapy with levofloxacin was modified to intravenous linezolid, but the clinical situation of the patient rapidly deteriorated, and he died of multiorgan failure. The

human strains to the animals. In Singapore, ST22-MRSA-IV was isolated from pigs and this strain was previously found increasingly important in the hospital population there. Notably the ST22-MRSA-IV is also a major hospital clone, as is the UK-EMRSA-15 found in the UK, with both strains indicating a contamination of human origin. In Canada, 14% of MRSA isolated from pigs appeared to belong to the human epidemic clone CMRSA-2 (Canadian epidemic MRSA-2, known as USA100), while 74.4% of isolates were LA-MRSA. The remaining strains belonged to rare clones, not related to LA-MRSA or CMRSA-2. Most reports on LA-MRSA in pigs originate from the Netherlands. In Europe, LA-MRSA has also been found in pigs in Germany, Denmark and Belgium, and beyond Europe, in Canada,

Besides the importance of living animals as a source of MRSA, animal origin products also play a role in disseminating these strains to the humans. Lozano et al. (2009) detected MRSA ST398 in food samples in Spain. A total of 318 samples of raw food were evaluated from food-producing animals (148 from chicken, 55 from pork, 46 from veal, 19 from lamb, 10 from turkey, 8 from rabbit and 12 minced-meat samples) and of wild animals (8 *game birds*, 4 wild boar, 4 deer and 4 hare samples). MRSA was detected in 5 of 318 (1.6%) food samples (pork, chicken, rabbit, veal and wild boar). The two strains from pork and veal corresponded to ST398-SCC*mec*V clone (*spa* types t011 and t1197, respectively), the two strains from chicken and rabbit were typed as ST125-SCC*mec*IVa-t067, and the strain from one wild boar was ST217-SCC*mec*IVa-t032, with all the MRSA being PVL negative. The characteristics of these strains suggest that they can be of both animal and human origin, and although the presence of MRSA in food is low, it must be monitored, because it can

Recently, Weese et al. (2011) evaluated the presence of MRSA in feedlot cattle, close to the time of slaughter, in nasal and rectal fecal samples. It was not possible to detect MRSA in these animals, in contrast to recent studies on retail beef (Weese et al., 2010), demonstrating the need for more studies of livestock, as well as farms, processing and retail environments

Several studies have been done to determine the degree to which MRSA plays a role in zoonosis or humanosis. It has been observed that usually the strains originating from companion animals are originally human strains, and that the infection with this MRSA type is considered humanosis. On the other hand, the strains orginating from livestock (livestockassociated - LA) are often divergent from human strains and the infection with this type of LA-MRSA could be considered zoonosis, and in this case MRSA would be an emergent zoonotic agent. Within this context, veterinarians, cattle farmers and pet owners are

In 2011, a case of empyema was reported in a 79-year-old man in Spain with isolation of MRSA ST398 in purulent exudates from the thorax and trachea and nasal swabs. The ineffective initial therapy with levofloxacin was modified to intravenous linezolid, but the clinical situation of the patient rapidly deteriorated, and he died of multiorgan failure. The

to elucidate the epidemiology of contamination with MRSA in meat.

considered risk groups for acquiring MRSA (Morgan, 2008).

Singapore and the USA (Vanderhaeghen et al., 2010).

**3.6 Products of animal origin** 

contribute to its dissemination.

**4. MRSA as zoonosis or humanosis** 

three MRSA strains were typed as ST398, *spa*-type t011, SCC*mec* V and *agr*I and presented the same phenotypic resistance, including β-lactam, tetracycline, clindamycin (but not erythromycin), ciprofloxacin and levofloxacin. It is important to point out that the patient lived with his wife and two sons near a pig farm. Both sons worked on the farm. The patient, but not his wife, sporadically helped on the farm. Nasal samples from three family members indicated MRSA carriage in one son. The characteristics of this isolate were identical to the isolate from the patient. Furthermore, nasal swabs of 18 pigs from the farm were randomly collected, and MRSA isolates were detected in 9 (50%) pigs. One MRSA isolated in each animal was minutely characterized. Eight isolates were typed as ST 398/t011/SCC*mec* V/*agr*I and one remaining isolate as ST398/t1451/SCC*mec* V/*agr*I. All MRSA isolated from the animals had the same phenotypic and genotypic resistance comparing MRSA isolated from the patient and son. These findings indicate potential pighuman zoonosis transmission of MRSA ST398 and that this clone can be associated with severe respiratory pathology in immunocompromised patients, and this microorganism can also be resistant to other first-line antimicrobial agents, such as fluoroquinolones, used to treat these infections. Furthermore, the unusual clindamycin-resistance/erythromycinsusceptibility phenotype can be a key marker (in addition to tetracycline resistance) of the possible presence of livestock-associated MRSA (Lozano et al., 2011).

As seen in the reports, animal reservoirs for MRSA are becoming recognized worldwide with increasing awareness of MRSA ST398 colonizing pig and veal farmers, and attending veterinarians, at high rates. In The Netherlands, these groups are now considered high risk, and if admitted to a hospital, they are immediately conducted to isolation, screening for MRSA and decolonization (Loeffler et al., 2009).

MRSA isolates originating from animals have been shown to hold important genes of resistance which could be transferred to less pathogenic human strains, but well adapted, in a nasal co-colonization and resulting in new human lineages, for example (Springer et al., 2009). The gene *czrC,* which confers resistance to cadmium and zinc, was determined in isolates of MRSA CC398 of SCC*mec* type V originating from 23 (74%) pigs and 24 (48%) humans from Denmark. It is suggested that resistance to heavy metals can play a role in coselection of MRSA, because it was strongly related to the clone CC398 (Cavaco et al., 2010).

In lineages MRSA ST398 and MSSA ST9 isolated from pigs in Germany, the major reservoir of these lineages, the gene *cfr* was found, a gene of multi-resistance to the drugs phenicol/lincosamine/oxazolidinone/pleuromutilin/streptogramin A. The risk of its transference to humans with exposure to pig farms is of concern since these lineages can colonize and cause infections in humans (Kehrenberg et al., 2009).

Genes encoding virulence factors can also be carried by animal strains. An example is the PVL gene, encoding an important virulence factor related to MRSA, rarely reported in animals, but can be found in companion animals. PVL-positive CA-MRSA has been reported in cats, dogs, rabbits, birds, bats, turtles, pigs and cattle. The strains associated with pigs that have been rapidly disseminating are currently PVL-negative (Morgan, 2008).

As for animal origin food, meats of several animal species have been evaluated for detection of MRSA. The contamination has been reported in meats of turkey, chicken, veal, pork, beef and lamb. The majority of the isolates were non typeable MRSA. Considering the low

MRSA Epidemiology in Animals 87

performed in accordance with the need of the study and the material and personnel available in each laboratory. Relative speed and the reliability are the desirable characteristics in both methods, because the choice of treatment and infection control measures are determined by the results of such testing (Kaya et al., 2009). Phenotypic methods at first are more accessible and nearly always cheaper; however they depend on the characteristic expression and visualization that cannot occur or be reduced, as for example, by environmental influences and/or regulatory genes (Berger-Bächi, 2002;

Methicillin-resistant *S. aureus* can be identified through different genotypic methods, such as species-specific primers for detection of DNA fragment of *S. aureus*-specific (van Duijkeren et al., 2010, as cited in Martineau et al., 1998) and with gene *mecA* (Murakami et al., 1991) by PCR (polymerase chain reaction), or the detection of DNA fragment of *S. aureus*-specific and

To determine the MRSA clones involved, in the beginning of 1990s the pulsed-field gel electrophoresis (PFGE) of genomic SmaI macrorestriction fragments were introduced and still represents the gold standard with respect to discriminatory power. The clonal groups determined by cluster analysis through PFGE are largely congruent with those defined by MLST. However, with the presence of some lineages of special interest (for example ST398) that are non typeable by the standard restriction enzyme SmaI, other methods have been

As observed in the majority of studies discussed in this chapter, from genotypic methods for classification of predominant strains and determination of evolutionary pathways, MLST and *spa* typing have been the most widely employed: the first because it is an unambiguous discriminatory method for studying MRSA epidemiology and evolution, with results that can be truly portable between laboratories (Enright, 2003) and the second method to indicate genetic microvariation permitting investigation of outbreaks or accomplishment of phylogenetic analysis (Koreen et al., 2004). MLST characterize bacteria isolates unambiguously using the sequences of internal fragments of seven "housekeeping" genes, being a discriminatory method which permits that related strains recovered from different countries be quickly identified (Enright et al., 2002). The *spa* type identified by DNA sequence analysis of the X region of the protein A gene (*spa*) is less expensive, timeconsuming, and error prone than multilocus techniques, such as MLST (Shopsin et al., 1999;

The classification by MLST permits that the genomes of strains deposited in the GenBank database to be compared to establish their evolution and characteristic features. Comparing the genome of CA-MRSA and HA-MRSA from the same clonal lineage as well as their most probable MSSA ancestor, about 78% of the genes are conserved, and the remaining 22% comprise an "accessory genome" including genomic islands, pathogenicity islands, prophages, integrated plasmids, and transposons. However, comparing the *S. aureus*  genome from cattle mastitis (ST151) with human *S. aureus,* it has been demonstrated that this bovine clone probably evolved from a common ancestor by acquiring foreign DNA. Subsequent microarray studies on recent epidemic strains of bovine origin (such as ST97) also revealed the presence of mobile genetic elements absent from *S. aureus* of human origin. Even with these impressive insights into the evolution of *S. aureus*, research has not yet provided many clues on the adaptation of the pathogen to the host, since only limited data

gene *mecA* by multiplex PCR (Huijsdens et al., 2006), for example.

Mohanasoundaram & Lalitha, 2008).

used (Cuny et al., 2010).

Koreen et al., 2004).

number of non typeable MRSA in patients, the role of food products in disseminating MRSA seems to have been overlooked (Morgan, 2008).
