Methicillin Resistant Staphylococcus aureus (MRSA): An emerging Veterinary Pathogen

DEPARTMENT OF VETERINARY MICROBIOLOGYCOLLEGE OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY

NARENDRA DEV UNIVERSITY OF AGRICULTURE & TECHNOLOGY NARENDRA NAGAR, (KUMARGANJ), FAIZABAD, U.P. (INDIA)

Credit Seminar For

Master of Veterinary Science(Veterinary Microbiology)

Prerit Kumar SinghI.D. No.- V-8322/2014

Staphylococcus aureus (Staph aureus) bacteria are natural part of flora in most warm blooded animals including humans.

Occasionally animals can become infected by their own normal flora but when an animal normal flora develops resistance to broad-spectrum antibiotics, it becomes a very dangerous health threat.

Methicillin represents the semi-synthetic penicillin-related antibiotic once effective against staphylococci (staph).

Over the years, Staph bacteria have developed resistance to penicillin-related antibiotics, including methicillin.

These resistant bacteria are called Methicillin-Resistant Staphylococcus aureus, or MRSA.

INTRODUCTION

MRSA causes a variety of disseminated, lethal infections in humans as well as animals.

Has the ability to easily transfer resistant genes to other species directly and indirectly.

If these bacteria undergo genetic mutation, making them resistant to even the strongest antibiotic available, including methicillin, it can cause serious illness and even death.

Overuse of antibiotics imposes selective pressures which mediates the acquisition of resistance.

MRSA infections are typically classified as

1. Health care-associated MRSA 2. Community-associated MRSA3. Livestock associated MRSA

The Basic Characteristics Of Staph aureus

Gram positive. Non-motile. Spherical. Grows in bunches (resembling clusters of grapes). Golden color colonies (especially in old culture) Hemolytic on blood agar Produces coagulase, catalase enzymes, Hyaluronidase

(breaks down hyaluronic acid and helps in spreading it), deoxyribonuclease (which breaks down the DNA), lipase (digest lipids), staphylokinase (dissolve fibrin and aid in spread), and beta-lactamase (for drug resistance).

The history of Mithicillin rasistant Staphylococcus aureus (MRSA) The Staph. aureus bacterium commonly known as staph was discovered in the 1880 by Sir Alexander

Ogston. During this era, Staph aureus infections were commonly known to cause painful skin and soft tissue

conditions such as boils, scalded-skin syndrome and impetigo.

Now it is known that more serious forms of Staph aureus infection can progress to bacterial pneumonia and bacteremia —both of which can be fatal.

Staph aureus acquired from improperly prepared or stored food, can also cause a form of food poisoning.

In the 1940s, medical treatment for Staph aureus infections became routine and successful with the discovery and introduction of antibiotic medication, such as penicillin.

From that point on, however, use of antibiotics—including misuse and overuse— has aided natural bacterial evolution by helping the microbes to become resistant to drugs designed to help fight these infections.

In late 1940s and throughout the 1950s, Staph aureus developed resistance to penicillin.

Methicillin, a form of penicillin, was introduced in year 1960 to counter the increasing problem of penicillin-resistant Staph aureus.

Methicillin was one of most common types of antibiotic used to treat Staph aureus infections.

In 1961, British scientists Jevons identified the first strains of Staph aureus bacteria that resisted methicillin. This was the so called birth of MRSA.

Subsequently, new strains of bacteria have developed that can resist previously effective drugs, such as methicillin and most related antibiotics (Palavecino, 2004) .

MRSA is actually resistant to an entire class of penicillin-like antibiotics called beta-lactams. This class of antibiotics includes penicillin, amoxicillin, oxacillin, methicillin, and others (Kuehnert et al., 2005).

The first isolation of the MRSA from animal was from mastitis milk of a cow from Netherland (Deveiese et al., 1972).

The MRSA has since been reported from found domestic species including

Dogs (Van Duijkeren et al., 2004; Walther et al., 2008)

Cats (Bender et al., 2005)

cows (Moon et al., 2007)

Horses (Hartmann et al., 1997; Van den Eede et al., 2009) Sheep (Goni et al., 2004)

Pigs (Voss et al., 2005; Meemken et al., 2008)

Guinea pig

Rabbit Turtle (Walther et al., 2008) Bat

Parrot

Chickens (Lee, 2003; Kwon et al., 2006)

How is MRSA transmitted?The 5 C’s

• Crowding• Contact-frequent skin-to-skin• Compromised skin (i.e. cuts or

abrasions)• Contaminated items and surfaces• Cleanliness, lack of

General structure of Gram positive bacteria

(peptidoglycan) Transpeptidation

Fem

N- acetyl muramic acid

N- acetyl glucosamine

Transglycosylation (extending the glycan chain)

PBPs.catalysed

MecA in suppressed form

It acts by interfering primarily with the synthesis of bacterial cell wall and produce effect by binding to penicillin binding proteins (PBPs). PBPs essentially involved in the maintenance of normal cell morphology and viability of bacteria. PBPs are grouped in to six types on the basis of their molecular weight. Drugs occupy the active site of transpeptidase enzyme (PBP) and inactivate it. Inactivation of transpeptidation in cell wall synthesis leads to blockage of cell wall synthesis.

Mode of action

Mechanism Of Resistance mecA gene is responsible for development of

methicillin resistance. mecA gene is chromosomally located on a mobile genetic element called the staphylococcal cassette chromosome (SCC) which encodes for penicillin-binding protein 2a (PBP-2a) and responsible for synthesis of penicillin-binding protein 2a (PBP2a; also called PBP2) a 78-kDa protein. Expression of PBP-2a is controlled by mecR1 and mecI regulator genes located upstream of mecA gene. A mutation in the mec regulators leads to expression of mecA gene (PBP-2a). A PBP2a substitutes for the other PBPs and because of its low affinity for all β-lactam antibiotics, this enables staphylococci to survive exposure to high concentrations of these agents.

MRSA Symptoms

MRSA symptoms in animals.

The MRSA skin infection usually starts as a small red bump or boil which can develop into a deep painful abscess.

Common locations in the body where an infection occurs are the skin,

ears, and at wound sites, especially after surgery.

Initially, MRSA can look like any other infection, but it doesn’t respond to antibiotics.

When the skin lesions refuse to heal, that’s often when people start to realize that they’re dealing with a potentially life-threatening infection.

The infection can progress to necrotizing fasciitis. It can also move to the lungs as necrotizing pneumonia. About a third of MRSA infections in the lungs cause death.

A septic infection of the entire body can also develop.

Because MRSA is so difficult to treat, it can progress from a mild skin rash into a life-threatening infection that invades your pet’s bones, joints and major organs, as well as the bloodstream. About half of MRSA infections in the bloodstream are fatal.

MRSA Has Become a Problem for animals due to-

• We are overusing antibiotics in human and animal medicine.

• Animal exposed to even more antibiotics when they eat factory-farmed animal feed and animal products.

• The decision to use antibiotics should be taken lightly on animals.

• They should be prescribed unless absolutely not necessary.

Many of the health problems for which antibiotics are routinely overprescribed respond just as well and often better to safer alternatives like herbs, common sense approaches like disinfecting wounds, as well as nutritional supplements.

Unless your animal has a life-threatening illness or injury that can only be treated with antibiotics, let your veterinarian know that you prefer to at least try and treat, if possible, without antibiotics.

MRSA Diagnosis

Agar dilution test

Agar dilution is a method used by researchers to determine the resistance of pathogens to antibiotics. It is the dilution method most frequently used to test the effectiveness of new antibiotics.

NOTE :- Minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation.

• A minimum of four to five colonies isolated from an overnight growth are transferred to sterile saline. The suspension is adjusted to a 0.5 McFarland standard (108 cfu/ml) and spot inoculated on Mueller–Hinton agar plates supplemented with 2% NaCl and containing 256–0.125 μg oxacillin/ml in serial doubling dilutions. The oxacillin Mueller–Hinton plates are incubated at 35oC for 24 hours. MIC of ≥4 μg/ml is considered resistant and MIC of ≤2 is considered susceptible.

Broth micro-dilution

Broth micro-dilution is a method used to test the susceptibility of bacteria to antibiotics. It is the most commonly used method to perform this test in the United States.

Disc diffusion test

The zone of inhibition must be read with transmitted light and not reflected light. Zone diameter of ≤10 mm is considered as resistant, ≥13 mm as susceptible whereas 11-12 mm is considered as intermediate. If intermediate results are obtained for S. aureus, testing for mecA, PBP2a. If there is a definite zone of inhibition around the disk, the organism is susceptible no zone occurs ,the organism is rasistant.

Etest oxacillin MIC test

Chromogenic media for MRSACurrently available chromogenic media for MRSA detection –

ChromID MRSA- ChromID targets the α-glucosidase enzyme of S. aureus, resulting in green-colored colonies of MRSA.

MRSA Select - MRSA Select incorporates a cephamycin derivative and characterizes MRSA colonies by a pink color.

MRSA Ident agar

CHROMagar MRSA-

Denim Blue agar-

Results in rose to mauve MRSA colonies

Produces denim blue colonies of MRSA

Dusky pink or ruby-colored MRSA colonies

OXOID PENICILLIN BINDING PROTEIN (PBP2 ) LATEX AGGLUTINATION TEST KIT ′

The method involves extraction of PBP2a from suspensions of colonies and detection by latex agglutination. The kit contains latex particles sensitized with a monoclonal antibody against PBP2a. Visible agglutination indicates a positive result and the presence of PBP2a, the mecA gene product.

Molecular methods:

Detection of mecA gene by PCR is considered as the gold standard. DNA extraction is performed on the isolate and mecA gene is amplified using specific primers. The master mix containing PCR buffer, dNTP mix, primer, Taq DNA polymerase, and MgCl2 and template DNA is subjected to hot start PCR. This is followed by 30 cycles of denaturation at 94oC for 45 seconds, annealing at 50oC for 45 seconds, and extension at 72oC for 1 minute and final extension step at 72oC for 3 minutes.

PCR products are visualized on 2% agarose gel with ethidium bromide dye under UV transilluminator.

Conclusion

The reports last few years strongly suggested the role of animals in perpetuation of MRSA.

Scientific data shows that the lineage of animal and human strains differs.

The human infection caused by animal strains are more deadly.

So the detailed studies on occurrence and genetic variation among MRSA strain of animal origin is essential.