**5. MRSA detection in animals**

Animal studies of MRSA are limited by certain research bottlenecks compared to human studies, including a lack of standardization regarding culture methodology, susceptibility testing, definition of genetic profile and sampling methods, which ultimately renders comparison difficult. A mass animal screening is logistically difficult, expensive, and impractical in many situations. Moreover, physical challenges exist: for example, carrying out a nasal swab in cats is only possible with appropriate animal restraints (Morgan, 2008).

Despite these drawbacks, several studies have been carried out, as more detailed studies of epidemiological aspects of animal MRSA are indispensable, primarily in food-producing animals of particular concern. These animals are not only reported as the primary source of a recently emergent new type of MRSA, LA-MRSA, but studies also suggest that they are involved in transmission of other strains of MRSA between animals and humans (Vanderhaeghen et al., 2010). MRSA screening must be performed in all diagnostic laboratories, even if done through disk diffusion with oxacillin, which is better than methicillin related to the resistance to degradation and the detection of heteroresistant strains (Weese et al., 2004).

MRSA detection in animals has been performed, generally, with isolation from samples from nasal and oral mucosae and perineum in small animals (Loeffler et al., 2005; Nienhoff et al., 2009), samples from milk in cows (Kwon et al., 2005), samples from nasal, oral and/or perineal swab, in pigs and horses (Baptiste et al., 2005; Voss et al., 2005; Huijsdens et al., 2006), samples from nose and cloacae in poultries (Nemati et al., 2008) and samples from meats (Lozano et al., 2009).

The samples were pre-enriched in different selective mediums with the aim of increasing the sensitivity of the culture technique. Lozano et al. (2009), to detect MRSA in meat, pre-enriched the samples in BHI broth (brain heart infusion) containing 6.5% of sodium chloride at 35°C for 24 h. An aliquot of each growth was seeded on ORSAB plates (oxacillin resistance screening agar base) with oxacillin (2mg/L) and incubated at 35°C for 36 h. Van Duijkeren et al. (2010) compared two methods of culture technique; the first was Müeller Hinton Broth containing 6.5% sodium chloride, which after overnight incubation at 37°C, was transferred to phenol red mannitol broth with 5µg/ml of ceftizoxime and 75µg/ml of aztreonam and after overnight incubation at 37°C was plated onto sheep blood agar and brilliance MRSA agar. In the second method, Tryptone Soy Broth was used containing 4% sodium chloride, 1% mannitol, 16µg/ml of phenol red, 50µg/ml of aztreonam and 5µg/ml ceftizoxime, plating onto sheep blood agar and MRSA brilliance agar after 48 hours of incubation at 37°C. The first method, with the preenrichment containing higher salt concentration, presented better results. Weese et al. (2011) used as pre-enrichment a broth containing tryptone, sodium chloride, mannitol, yeast extract and incubated at 35°C for 24 h to evaluate MRSA presence in feedlot cattle, as well the nose samples as feces. An aliquot of the growth was inoculated onto MRSA Chromogenic agar and incubated at 35°C for 48 hours.

Many methodologies have been employed to identify and characterize strains, ranging from phenotypic to genotypic. Both present advantages and disadvantages and must be

number of non typeable MRSA in patients, the role of food products in disseminating MRSA

Animal studies of MRSA are limited by certain research bottlenecks compared to human studies, including a lack of standardization regarding culture methodology, susceptibility testing, definition of genetic profile and sampling methods, which ultimately renders comparison difficult. A mass animal screening is logistically difficult, expensive, and impractical in many situations. Moreover, physical challenges exist: for example, carrying out a nasal swab in cats is only possible with appropriate animal restraints (Morgan, 2008). Despite these drawbacks, several studies have been carried out, as more detailed studies of epidemiological aspects of animal MRSA are indispensable, primarily in food-producing animals of particular concern. These animals are not only reported as the primary source of a recently emergent new type of MRSA, LA-MRSA, but studies also suggest that they are involved in transmission of other strains of MRSA between animals and humans (Vanderhaeghen et al., 2010). MRSA screening must be performed in all diagnostic laboratories, even if done through disk diffusion with oxacillin, which is better than methicillin related to the resistance to degradation and the detection of heteroresistant

MRSA detection in animals has been performed, generally, with isolation from samples from nasal and oral mucosae and perineum in small animals (Loeffler et al., 2005; Nienhoff et al., 2009), samples from milk in cows (Kwon et al., 2005), samples from nasal, oral and/or perineal swab, in pigs and horses (Baptiste et al., 2005; Voss et al., 2005; Huijsdens et al., 2006), samples from nose and cloacae in poultries (Nemati et al., 2008) and samples from

The samples were pre-enriched in different selective mediums with the aim of increasing the sensitivity of the culture technique. Lozano et al. (2009), to detect MRSA in meat, pre-enriched the samples in BHI broth (brain heart infusion) containing 6.5% of sodium chloride at 35°C for 24 h. An aliquot of each growth was seeded on ORSAB plates (oxacillin resistance screening agar base) with oxacillin (2mg/L) and incubated at 35°C for 36 h. Van Duijkeren et al. (2010) compared two methods of culture technique; the first was Müeller Hinton Broth containing 6.5% sodium chloride, which after overnight incubation at 37°C, was transferred to phenol red mannitol broth with 5µg/ml of ceftizoxime and 75µg/ml of aztreonam and after overnight incubation at 37°C was plated onto sheep blood agar and brilliance MRSA agar. In the second method, Tryptone Soy Broth was used containing 4% sodium chloride, 1% mannitol, 16µg/ml of phenol red, 50µg/ml of aztreonam and 5µg/ml ceftizoxime, plating onto sheep blood agar and MRSA brilliance agar after 48 hours of incubation at 37°C. The first method, with the preenrichment containing higher salt concentration, presented better results. Weese et al. (2011) used as pre-enrichment a broth containing tryptone, sodium chloride, mannitol, yeast extract and incubated at 35°C for 24 h to evaluate MRSA presence in feedlot cattle, as well the nose samples as feces. An aliquot of the growth was inoculated onto MRSA Chromogenic agar and

Many methodologies have been employed to identify and characterize strains, ranging from phenotypic to genotypic. Both present advantages and disadvantages and must be

seems to have been overlooked (Morgan, 2008).

**5. MRSA detection in animals** 

strains (Weese et al., 2004).

meats (Lozano et al., 2009).

incubated at 35°C for 48 hours.

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; Mohanasoundaram & Lalitha, 2008).

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

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 used (Cuny et al., 2010).

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; Koreen et al., 2004).

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

MRSA Epidemiology in Animals 89

Proper cleaning and disinfection of contaminated environments, including transport

Control and/or treatment of colonized and infected animals with or without antimicrobials

Th1e affected animals need to be immediately separated from healthy animals. In extreme cases culling of infected animals is a further option. Milk of animals with mastitis by MRSA must be destroyed, and in some cases the infected quarter must be prematurely dried-off.

If the antimicrobial treatment is chosen, it is necessary to evaluate its risk-benefit compared with other alternatives. The choice of antimicrobials should always be based on a susceptibility test, and all precautions should be taken that the drug reaches the infected site

The options of control for colonized horses, as well as livestock animals, include the nonantimicrobial management and the antimicrobial treatment of colonized or infected horses. In a Canadian study, two farms with horses colonized by MRSA drastically reduced the number of affected animals with active screening and strict implementation of control protocols of infection, without the use of antimicrobial therapy. Antimicrobial treatment must be applied only if the colonization is persistent or in cases in which control measures

Preventive measures for the infected animals are the same for the previously mentioned livestock animals. In equine hospitals, MRSA management for veterinary practices, guidelines stipulated by the British Small Animal Veterinary Association (BSAVA), for example, can also be applicable. In the confirmed or suspected cases of infection by MRSA, horses must be isolated and treated as if disseminating a nosocomial and zoonotic agent. It is necessary to take precautions with staff hygiene as well, using protective barriers, such as gloves, aprons, and boots. Moreover, the entire animal must be evaluated before being

In colonized animals, it not recommended to decolonize animals having mucosae colonization with MRSA. And it is observed that apparently some pets eliminate the carriage of MRSA spontaneously if the re-colonization is prevented. Antimicrobial therapy is only indicated in exceptionally persistent cases or when control measures are impossible., If the infection is local in infected animals, meticulous management of the wound can avoid the necessity of antimicrobial use. The risk of resistance development, the susceptibility profile of the isolate, the severity of the infection and the presence of systemic disease (fever, leukocytosis), and the patient's underlying disease or any comorbidity must be taken into account when choosing the antimicrobial treatment. In deciding to treat or eventually

euthanize, veterinarians must consider the national veterinary guidelines available.

admitted to the hospital to ensure prevention of MRSA dissemination.

**6.1.3 Specific measures for companion animals** 

Animal owners should be informed about the risks and necessary precautions.

vehicles. Special attention should be paid to dust in stables

**6.1.1.1 Reducing carriers on MRSA-positive livestock farms** 

is necessary for the reduction of carriers.

with appropriate concentrations.

are impossible.

**6.1.2 Specific measures for horses** 

are available (Cuny et al., 2010). In this respect, further research is needed to address these gaps, as well as to better understand the evolution of these strains in humans and animals.
