Staphylococcus. aureus. In 1983 Dr. Kary Mullis at Cetus Corporation conceives of PCR. PCR is a simple elegant process that enables the production of virtually.
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2.1. Impetigo Impetigo is a common type of skin infection. Bacteria enter skin scratching cause impetigo. Bacteria get below the surface of the skin, usually by some sort of break in the skin and the break in the skin will be noticed (such as a skinned knee or a cut, even an insect bite), but a very small, even microscopic, break is common. Once the bacteria get into the inner layers of the skin, they grow and multiply, creating a blister. Usually, this blister becomes filled with pus and bursts, leaving a crust (Sladden and Johnston, 2004). An acute contagious and superficial infection of the skin caused either by Staph. aureus or, occasionally by β-heamolytic Streptococcus or both, impetigo is common in children, outbreak occurs in institution such as nurseries and boarding schools, sometimes there is a predisposing factor such as insect bites, head lice or trauma, the skin is more vulnerable to secondary bacterial infection in eczema, when the eczema is described as being impetiginized (Vivier and Mckee, 2002). Impetigo can affect healthy skin (primary) and also broken skin, which has been caused by another condition, e.g. eczema (secondary) (Khurana et al., 2002). Two types of bacteria that cause impetigo are group A, βheamolytic Streptococci and Staph. aureus, impetigo is classified as bullous, or non-bullous based on the presence or lack of large blisters (Vivier and Mckee, 2002).
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2.1.1. Bullous Impetigo Bullous impetigo is characterized by bullae, large thin-walled blisters that contain clear or cloudy yellow fluid and measure up to 5 cm in diameter. Bullae are caused by Staphylococcal infections, not by Streptococci, a certain type of Staphylococcal bacteria produces a toxin that causes the large blisters to form. These blisters easily rupture and leave behind a moist area of eroded skin surrounded by a thin ring of the remaining blistered skin. This lesion dries and crusts over, creating a light brown appearance that resembles “varnish”, the lesions are discrete, with little redness or inflammation surrounding them, these large blisters typically occur on the face but may quickly spread to different areas of the skin. A mix of bullous and non-bullous skin lesions may occur (Pickering et al., 2000; Stulberg et al., 2002).
2.1.2. Non-Bullous Impetigo This is the more common type of impetigo and is characterized by reddened sores with honey-yellow crusting on them, the sores may initially appear as small blisters that rupture, ooze, and lead to the layer of crusting, the crust typically appears to be “stuck on”, the infection does not disappear easily with topical cleaning. The lesions are painless, often occur around the mouth and nose or on the arms and legs, and resolve without scarring, these non-bullous lesions are caused by either Streptococcus or Staphylococcus, and in some cases both types of bacteria may be present (Pickering et al., 2000; Stulberg et al., 2002).
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2.2. Furunculosis It is an acute deep abscess of the hair follicles caused by infection with Staph. Aureus, furunculosis differs from folliculitis in that there is a greater degree of inflammation which spreads away from the hair follicle into the surrounding dermis condition which is caused by Staph. aureus which is usually present in the nose axillae or perineum, it is most common in adolescent and young adults and the patient is invariably otherwise healthy (Vivier and Mckee, 2002). A search for diabetes mellitus although always quoted in textbooks is commonly fruitless. Its symptoms may be painful swelling that may discharge pus, furunculosis may present as a boil or carbuncle, a boil is a red, painful nodule containing pus which discharges spontaneously and heals with or without scarring (depending on depth of lesion), this process takes several days, several lesion may be present especially in recurrent cases. A carbuncle is an infection of multiple hair follicles that begins as nodule and enlarges to produce an inflamed mass which discharges pus from multiple follicular orifices, a carbuncle is, therefore a collection of boils, the patient may be quite unwell with a fever. Boils and carbuncles may occur anywhere. A style (hordeolum) is a Staphylococcal infection of the eyelash and represent a small boil, chancriform pyoderma is of uncertain etiology but probably is a reaction to a Staphylococcal infection induced by trauma. It is an indolent welldefined ulcer with a red margin, it occurs around the eye or mouth (Vivier and Mckee, 2002).
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2.3 Pyoderma Brunsting is credited with the initial description of pyoderma gangrenosum (PG) in 1930, classic pG begins as an inflammatory pustule with a surrounding halo that enlarges and begins to ulcerate, a primary lesion-may not always be seen, and a substantial proportion of lesions appear at sites of trauma (pathergy), satellite violaceous papules may appear just peripheral to the border of the ulcer and break-down to fuse with the central ulcer, fully developed lesions are painful ulcers with sharply marginated, undermined, blue to purple borders (James et al., 2011).
2.4 Cellulitis Cellulitis is a suppurative inflammation involving the sub cutaneous tissue, caused most frequently by S. aureus, usually, but not always, this follows some discernible wound. On the leg tinea pedis is the most common portal of entry mild local erythema and tenderness, malaise, and chilly sensations, or a sudden chill and fever may be present at the onset, the erythema rapidly becomes intense and spreads, The area becomes infiltrated and pits on pressure,
sometimes the central part
becomes nodular and surmounted by a vesicle that ruptures and discharges pus and necrotic material, streaks of lymphangitis may spread from the area to the neighboring lymph glands, Gangrene, metastatic abscesses, and grave sepsis may follow, these complications are unusual in immunocompetent adults, but children and compromised adults are at higher risk (James et al., 2011).
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2.5 Ecthyma Ecthyma is an ulcerative Staphylococcal or Streptococcal pyoderma, nearly always of the shins or dorsal feet, the disease begins with a vesicle or vesicopustule, which enlarges and in a few days becomes thickly crusted. When the crust is removed there is a superficial saucer-shaped ulcer with a raw base and elevated edges, the lesions tend to heal after a few weeks, leaving scars but rarely may proceed to gangrene when resistance is low, in fact in a debilitated patient a focus of pyogenic infection elsewhere often precedes the onset of ecthyma in many cases local adenopathy may be present, uncleanliness, malnutrition, and trauma are predisposing causes (James et al., 2011).
2.6 Folliculitis Folliculitis, thin walled pustules at the follicle orifices. Favorite locations are the extremities and scalp, although it is also seen on the face, especially periorally. These frigile, yellowish white, domed pustules develop in crops and heal in a few days, S. aureus is the most frequent cause. The infection may secondarily arise in scratches, insect bites, or other skin injuries, Staphylococcal folliculitis may affect other areas, such as the eyelashes, axillae, pubis, and thighs, on the pubis it may be transmitted among sexual partners, and mini epidemics of folliculitis and furunculosis of the genital and gluteal areas may be considered a sexually transmitted disease (STD), Staphylococcal folliculitis has also been reported frequently among patients with acquired immunodeficiency syndrome and may be a cause of pruritus, an atypical, plaque-like form has been reported (James et al., 2011).
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2.7. Paronychia Paronychia is an inflammatory reaction involving the folds of the skin surrounding the fingernail, the causative bacteria are usually Staph. aureus, it is characterized by acute or chronic purulent, tender, and painful swellings of the tissues around the nai1, caused by an abscess in the nailfold, when the infection becomes chronic, horizontal ridges appear at the base of the nail, with recurrent bouts new ridges appear, the primary predisposing factor that is identifiable is separation of the eponychium from the nail plate, the separation is usually caused by trauma as a result of moisture-induced maceration of the nailfolds from frequent wetting of the hands, the relationship is close enough to justify treating chronic paronychia as work related in bartenders, food servers, nurses (James et al., 2011).
2.8. Antibiotics 2.8.1. Methicillin group Antibiotics are specific chemical substances derived from or produced by living organisms that are capable of inhibiting the life processes of other organisms, the first antibiotics were isolated from microorganisms but some are now obtained from higher plants ex. flowers of Crepis taraxacifolia and animals, over 3,000 antibiotics have been identified but only a few dozens are used in medicine. Antibiotics are the most widely prescribed class of drugs comprising 12% of the prescriptions in the United States, the penicillin was the first antibiotics discovered as natural products from the mold Penicillium. In 1928, Sir Alexander Fleming, Professor of bacteriology at St. Mary's Hospital in London, cultured Staph. Aureus, he noticed zones of inhibition where mold spores were growing, he named the mold Penicillium rubrum. It 9
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was determined that a secretion of the mold was effective against Grampositive bacteria (Bud, 2009). Penicillin is a group of antibiotics derived from Penicillium fungi. They include penicillin G, procaine penicillin, benzathine penicillin, and penicillin V. etc, Penicillin antibiotics are historically significant because they are the first drugs that were effective against many previously serious diseases such as syphilis and infections caused by Staphylococci and Streptococci, penicillin is still widely used today, though many types of bacteria are now resistant. All penicillin is β-lactam antibiotics and is used in the treatment of bacterial infections caused by susceptible, usually Gram-positive, organisms, the chemical structure of penicillin was determined by Dorothy Crowfoot Hodgkin in 1945, penicillin has become the most widely used antibiotic to date, and is still used for many Grampositive bacterial infections (Garrod, 1960).
2.8.2. Mode of Action - Enzyme Inhibition All penicillin derivatives produce their bactericidal effects by inhibition of bacterial cell wall synthesis. Specifically, the cross linking of peptides on the mucosaccharide chains is prevented, if cell walls are improperly made cell walls allow water to flow into the cell causing it to burst. Resemblances between a segment of penicillin structure and the backbone of a peptide chain have been used to explain the mechanism of action of β-lactam antibiotics (Bud, 2009).
2.8.3. Mechanisms of bacterial resistance to methicillin Staph. aureus is ubiquitous. It readily grows on human skin and mucous membranes. MRSA is a strain of Staph. aureus which by definition is resistant to the semi-synthetic penicillin (i.e. methicillin, 10
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nafcillin, and oxacillin). As such, it is resistant to most other β-lactam antibiotics (including other penicillin, cephalosporin, and cephamycin). Additionally, MRSA is often resistant to other classes of antibiotics (Topal, 2001). Such infections are difficult to treat as the bacteria are resistant to the most useful antimicrobial agents (Harbarth et al., 2008).
2.8.4. Mechanisms of resistance The four main mechanisms for resistance to antimicrobial agents. 1. Drug inactivation or modification for example, enzymatic deactivation of Penicillin G in some penicillin-resistant bacteria through the production of β-lactamases. 2. Alteration of target site: for example, alteration of PBP the binding target site of penicillin in MRSA and other penicillin-resistant bacteria. 3. Alteration of metabolic pathway. 4. Reduced drug accumulation by decreasing drug permeability and/or increasing active efflux (pumping out) of the drugs across the cell surface (Li and Nikadio, 2009).
2.9. Methicillin Resistance Staphylococcus aureus 2.9.1. History MRSA is a bacterial strain that is highly resistant to some antibiotics (Al-Talib et al., 2009; David, 2009). MRSA was first reported decades ago in the United States and was related to exposures in the healthcare system. These bacteria are resistant to β-lactam antibiotics, including methicillin, oxacillin, penicillin, and amoxicillin (Stroube, 2008). It is well-known that the Staph. aureus, was discovered in the 11
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1880, during this era, Staph. aureus infection commonly caused painful skin and soft tissue conditions such as boils, scalded-skin syndrome and impetigo. More serious forms of Staph. aureus infection can progress to bacterial pneumonia and bacteremia in the bloodstream both of which can be fatal. Staph. aureus acquired from improperly prepared or stored food can also cause a form of food poisoning (Simon and Buka, 2007). In the 1940, medical treatment for Staph. aureus infections became routine and successful with the discovery and introduction of antibiotic in medicine, such as penicillin, the use of antibiotics including misuse and overuse has aided natural bacterial evolution by helping the microbes become resistant to drugs designed to help fight these infections, in the late 1940 and throughout the 1950, Staph. aureus developed resistance to penicillin. Methicillin, a form of penicillin, was introduced to counter the increasing problem of penicillin-resistant Staph. aureus. Methicillin was one of most common types of antibiotics used to treat Staph. aureus infections; Once antibiotics were made openly available, they were commonly used to treat numerous types of medical conditions, including those that were later proven unfazed by the drug, it only took four years for the Staphylococcus bacteria to develop a resistance to penicillin, with the first strains of an evolved Staph. bacteria were discovered in 1947 (Simon and Buka, 2007). In 1959 methicillin, the first β-lactamaseresistant penicillin, was licensed in England. In 1960 the first MRSA isolates were identified in England (Daum, 2010). In 1961, British scientists identified the first strains of Staph. aureus bacteria that resisted methicillin, this was the so-called birth of MRSA. The first reported human case of MRSA in the United States came in 1968 (Simon and Buka, 2007). The first group of people to become infected with MRSA to any significant extent were intravenous 12
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drug users in the early 1980 in the USA, It is now found worldwide and it is a huge problem in the hospitals of the US, Canada, Europe and Australia. More recently, MRSA has become a serious threat to patients in China, the Middle East and in Asia. It is currently a lesser problem in African states. Subsequently, new strains of bacteria have developed that can now resist previously effective drugs, such as methicillin and most related antibiotics (Simon and Buka, 2007; So and Farrington, 2008). In 1982, large outbreak of MRSA infections among intravenous drug users in Detroit, Michigan, In late 1980–1990, outbreaks of MRSA noted in Australia among Aboriginal population with no exposure to hospitals, In Mid-1990 scattered reports of CA-MRSA infections in children in the United States, when Comparing two periods-1993-1995 and 1995-1997 among children with no risk factors for health care exposure, there was a 25-fold increase in the rate of hospitalizations due to MRSA, In 1998–2008, the community acquired-MRSA while rates of Hospital Acquired-MRSA infection remained stable, rates of CA-MRSA increased, According to the first reports of healthy, young children dying of severe MRSA infections was reported in 1999. In 2001, there was a shift from USA400 to USA300 as predominant strain of CA-MRSA in the United States. In 2003 defined SCCmec IV was recognized. CAMRSA risk factors were identified in 2005 to date include: athletes, military recruits, incarcerated people, emergency room patients, urban children, HIV patients, men who have sex with men, indigenous populations (Moreillon, 2008; Daum, 2010).
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2.9.2. Definition of Methicillin Resistance Staphylococcus. aureus MRSA is the name given to a strain of the Staph. aureus bacteria that is resistant to a number of antibiotics. MRSA bacteria are relatively harmless unless they gain entry into the body, where they can cause serious damage. MRSA infections are easily spread. Anyone can contract the condition. Some individuals, however, are at greater risk. Bacteria are single-celled microorganisms too small to be seen without a microscope. They are found everywhere, including in and on the human body. Some forms of bacteria are harmful, but other types are harmless (Albrich and Harbarth, 2008). In fact, some types of harmless bacteria are used to make vaccines and medicines, other types help the body to function by taking up space in the body that would otherwise serve as colonization sites for more dangerous bacteria, these bacteria outnumber human body cells ten to one, other forms of bacteria are not useful, they produce harmful chemicals or toxins that attack the body, Staph. aureus falls into this group, about one out of three individuals is colonized with or carries small quantities of Staph. aureus on their skin, groin, armpits, and/or in their noses and throats, one out of ten carries methicillin-resistant Staph. aureus. In most cases, these bacterial colonies are harmless, many of these colonies are too small to cause any damage, and, because Staph. aureus is not a good competitor, it is unable to displace helpful bacteria in order to gain a stronghold, Staph. colonies become dangerous when an initiating event, such as a break in the skin, occurs; this then provides the bacteria a portal of entry into the body (Albrich and Harbarth, 2008). Once inside the body, Staph. aureus secretes powerful toxins, which attack and destroy host cells, converting them into nutrients for 14
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bacterial growth. As the bacteria multiply, more and more toxins are released. Some of these toxins break down internal tissue, which allows the bacteria to enter the bloodstream, once in the bloodstream, the toxins can destroy disease-fighting white blood cells, break apart red blood cells, cause the formation of blood clots, attack the top layer of skin, and, eventually, harm every organ in the body, because of these factors, scientists and health care professionals often refer to Staph. aureus as a “superbug.” According to Michael Otto of the National Institute of Allergy and Infectious Diseases, “Different bacteria have different strategies to attack the human immune system. Staph. aureus seems to have a lot of strategies (Sheen, 2010).
2.9.3.
Molecular
detection
of
Methicillin
Resistance
Staphylococcus. aureus In 1983 Dr. Kary Mullis at Cetus Corporation conceives of PCR. PCR is a simple elegant process that enables the production of virtually unlimited copies of genetic material in the laboratory, although the Nobel Prize for the conception of PCR was given to Kary Mullis, Ph.D., in 1993, the technology was developed and applied by the labs of Henry Erlich, Ph.D., David Gelfand, Ph.D., and other teams of scientists at Cetus. The full history of the technology development and application is marked by an extraordinary collaboration of scientists working in a corporate setting, working together to identify and overcome the obstacles to the practical use of PCR for DNA analysis in research and medicine (Saiki et al., 1988; Murakami et al., 1991; Hawkey; 1998; Bartlett and stirling, 2003). PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications, these 15
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include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes, the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing) and the detection and diagnosis of infectious diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR (Weiss, 2009). The method relies on Thermal Cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA, primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification, as PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations (figure 2.3) (Bartlett and stirling, 2003). And the detection of this gene by polymerase chain reaction (PCR) is considered as "gold standard" for detection of Methicillin Resistance in Staphylococci (Maes et al., 2002; Ercis et al., 2008). Molecular genetics is the field of biology and genetics that studies the structure and function of genes at a molecular level, the field studies how the genes are transferred from generation to generation. Molecular genetics employs the methods of genetics and molecular biology, it is so called to differentiate it from other sub fields of genetics such as ecological genetics and population genetics, an important area within molecular genetics is the use of molecular information to determine the patterns of descent, and therefore the correct scientific classification of 16
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organisms: this is called molecular systematic (Hardison, 2005; Schwartz and Maresca, 2006).
2.10. Staphylococcal Cassette Chromosome SCCmec elements, unique genomic islands that are found in Staphylococcal species, have two essential components, the ccr gene complex (ccr) and the mec gene complex (mec). The ccr gene complex is composed of ccr genes and surrounding open reading frames (ORFs), and the mec gene complex is composed of the mecA gene, regulatory genes, and insertion sequences upstream or downstream of mecA. Several mec and ccr allotypes have been found among SCCmec elements (Chongtrakool et al., 2006). Five types of SCCmec element (SCCmec I, II, III, IV, and V) and a small number of variants have been characterized. Each SCCmec element integrates at the same site (attBscc) at the 3_ end of an open reading frame (ORF) of unknown function, designated orfX. SCCmec consists of the mec gene and cassette chromosome recombinase (ccr) gene complexes. Five classes of mec gene complex (A to E), which vary in their genetic structure, have been described. Each mec complex consists of an intact copy of mecA, a copy of IS431mec and, when present, complete or truncated mec regulatory genes mecI and mecR1, the ccr complex consists of the ccr genes ccrA and ccrB in combination (ccrAB) or ccrC alone, as well as adjacent ORFs (Shore et al., 2005).
2.11. MecA gene Strains of MRSA have become the most important causative agents of Hospital Acquired diseases worldwide, the genetic determinant of resistance, mecA, is not a gene native to Staph. aureus but was acquired
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from an extra species source by an unknown mechanism (Wu et al., 2001). MecA doesn’t express phenotype, it is genotype character one in 10
4
or 10
6
(Ercis et al., 2008), and his expression of resistance is
variable and commonly heterogeneous within strains that lead to erratic performance (Alepopoulou et al., 1991). Penicillin binding protein (PBP) 2' is the most important mechanism of the resistance to β-lactams in MRSA, and the mecA gene is the coding gene of PBP2', and located in the SmaI fragment G of the chromosome map by Pattle P. a part of the structure of mecA is similar to that of the penicillinase gene (Kanno, 1992). PBP-2a functions as a transpeptidase in cell wall synthesis in MRSA at high concentrations of β-lactam antibiotics that inhibit the growth of methicillin-susceptible strains with normal PBPs. This additional PBP is encoded by the structural gene mecA on the chromosome (Song et al., 1987), which has also been detected in methicillin resistant strains of other Staphylococcal species (Ubukata et al., 1990; Kobayashi et al., 1994). The mecA gene is a component of a large DNA fragment designated mec DNA, which is located at the specific site of the Staph. aureus chromosome and has been suggested to be transmitted from other bacterial species (Hiramatsu et al., 1996; Wu et al., 1996). The acquisition of mec DNA is considered to be the first genetic requisite for methicillin resistance of Staphylococci (Kuwahara-Arai et al., 1996). Expression of PBP-2a is controlled by two regulator genes on mec DNA, mecI and mecR1, located upstream of mecA, which encode mecA repressor protein and signal transducer protein, respectively (Hiramatsu et al., 1992) An MRSA carrying intact mecI and mecR1 together with mecA 18
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has been called pre-MRSA, which is represented by prototype Staph. aureus, Since intact mecI product strongly represses the expression of PBP-2a, the pre-MRSA is apparently methicillin susceptible (Hiramatsu, 1995). Hence, it is hypothesized that removal of the repressor function for mecA is a prerequisite for constitutive expression of methicillin resistance in Staph. aureus with mec DNA. Indeed, the deletion of mecI or point mutations in the mecI gene has been found in a number of methicillinresistant Staphylococcal isolates, in some strains, point mutations were detected in the mecA promoter region corresponding to a presumptive operator of mecA, the binding site of the repressor protein. Furthermore, genetic alteration on the chromosome which causes high methicillin resistance was presented as another mechanism of evolution of MRSA, although the details are not known (Kobayashi et al., 1998).
Figure 2.1: MecA gene structure (Stapleton and Taylor, 2002). Two methicillin resistance phenotypes are discernible by efficiency of plating (EOP) studies: homotypic and heterotypic (De-Lencastre and Tomasz, 1993). Homotypic (homogeneous) strains uniformly express high level resistance, whereas heterotypic (heterogeneous) organisms exhibit a strain-unique variation in resistance expression. In heterotypic isolates, highly resistant subpopulations are present but they are a small proportion of the largely susceptible majority. However, under the 19
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antibiotics,
the
more
resistant
minority
subpopulation predominates, providing clinical resistance. The genes that regulate or affect heterotypic resistance expression are not known, and it is not clear what role the regulation of mecA transcription plays in this process. Several sequences that regulate mecA transcription have been described. These include the β-lactamase regulatory sequences, blaR1blaI, and the analogous and partially homologous chromosomal sequences, mecR1-mecI. mecR1 and mecI are located immediately to mecA and are divergently transcribed from it (Hiramatsu, 1995). MecR1 is predicted to be a transmembrane signal transducer protein and MecI is predicted to be a repressor, on the basis of amino acid sequence similarities of gene products that regulate β-lactamase production in both Staph. aureus and Bacillus licheniformis (Niemeyer et al., 1996; Loeffler and Lloyd, 2010).
2.11.1. Structure of mecA gene The most prevalent and widely disseminated mec complex had the structure mecI-mecR1-mecA-IS431R (or IS431mec), designated the class A mecA gene complex, in contrast, mecA was bracketed by two copies of IS431, forming the structure IS431L-mecA-IS431R which had low-level methicillin resistance and characteristic heterogeneous methicillin resistance as judged by population analysis. In these strains, IS431L was located to the left of an intact mecI gene, forming the structure IS431Lclass A mecA gene complex. In other Staph. strains, IS431L was associated with the deletion of mecI and mecR1, forming the structure IS431L-mecR1-mecA-IS431mec, designated the class C mecA gene complex containing the IS431L-class A mecA gene complex with low concentrations of methicillin, the mutants had intermediate level of 20
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methicillin resistance. The mecA gene transcription was shown to be derepressed in a representative mutant strain, The indicated that the mecIencoded repressor function is responsible for the low-level methicillin resistance of some Staph. clinical strains and that the IS431-mediated mecI gene deletion causes the expression of methicillin resistance through the derepression of mecA gene transcription (Katayama et al., 2001). Adjacent to mecA on the Staphylococcal chromosome are two genes, mecR1 and mecI, that are co-transcribed divergently from mecA. The mecR1 gene encodes a membrane bound signal transduction protein (MecR1) while mecI encodes a transcriptional regulator (MecI). Between mecA and mecR1 are the promoters for these genes and an operator region that encompasses the 10 sequence of mecA and the 35 sequence of mecR1 (Figure 2.1) (Hiramatsu et al., 1992; Kobayashi et al., 1998; Stapleton and Taylor, 2007).
Figure 2.2: Structures of Staphylococcal cassette chromosome types based on multiple-locus sequence typing (Okuma et al., 2002). 21
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Some clinical strains are found to remain susceptible to methicillin (pre-methicillin-resistant Staph. aureus status), in those strains, mecA gene transcription is strongly repressed by the function of the accompanying regulator genes, mecI and mecR1 (Hiramatsu, 1995; Hiramatsu et al., 1991) transcription of mecA, encoding PBP2' (or PBP2a), was essential for the phenotypic expression of methicillin resistance (Song et al., 1987). Since mecI encodes a strong repressor of mecA gene transcription, pre-MRSA strains have only a marginal level of resistance to methicillin (Hiramatsu, 1995). Experimental inactivation of mecI causes derepressed production of PBP2' and makes the cell express methicillin resistance (Sharma et al., 1998). This requirement of mecI inactivation for the phenotypic expression of methicillin resistance coincides well with the observation with clinical MRSA isolates in which the mecI gene is either mutated or completely deleted in the tested strains (Weller, 1999). In pre-MRSA in some strain, mecA is flanked by the intact set of mec regulator genes on the left-hand side, and by a DNA region of about 3 kb, called the hyper variable region on the right-hand side (Ryffel et al., 1991). The right boundary of the latter region is demarcated by a copy of insertion sequence IS431mec (or IS431R) (Barberis-Maino et al., 1987). The mecA locus of pre-MRSA strains thus forms a prototypic structure of mecI-mecR1-mecA-IS431R that is designated the class A mecA gene complex (mec complex). In other clinical MRSA strains, the entire mecI gene and the 3 part of mecR1 are deleted from the class A mec complex and a fragment of the insertion sequence IS1272 (IS1272) has been association with the deletion point (Archer et al., 1994). The 22
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designate this structure, IS1272-mecR1-mecA-IS431R, the class B mec complex. Recently, it has been recognized that some methicillin-resistant Staphylococcus (MRS) strains express a low-level methicillin resistance in spite of their carriage of mecA. They have previously shown that mecI is widely disseminated in Staph. species (Suzuki et al., 1993). Dickinson and Archer have demonstrated that intact mecI is found in some Staph. epidermidis clinical strains with low-level oxacillin resistance (Dickinson and Archer, 2000). They have shown that allelic replacement inactivation of mecI raises the oxacillin resistance of these strains by several fold, indicating a role for mecI-mediated repression in the low-level β-lactam resistance in certain Staph. epidermidis strains, on the other hand, Kobayashi et al., have found some Staph. epidermidis clinical strains in a Japanese Hospital that carry intact mecI in spite of their overt β-lactam resistance expression (Katayama et al., 2001).
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Figure 2.3: This data model defines major classes of attributes for an MRSA profile (for example, genotyping methods, virulence factors and clinical outcomes) and relationships between them. blaZ, β-lactamase gene; drfA, trimethoprim resistance gene; Ent, enterotoxin; erm, macrolide resistance gene; Et, exfoliative toxin; femA, gene encoding a cytoplasmic protein necessary for the expression of methicillin resistance; Luk-PV, Panton-Valentine leukocidin; mecA, gene encoding PBP2a, the low-binding-affinity penicillin-binding protein that mediates methicillinresistance; MRSA, methicillin resistant Staph. aureus; SCCmec, Staphylococcus cassette chromosome; spa, Staphylococcal protein A gene type; ST, sequence type; tetK, tetracycline resistance gene; tst, Staphylococcal toxic shock toxin gene; vanA, vanB, vanC, vancomycin resistance genes (Sintchenko et al., 2007).
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2.11.2. Mode of action of mecA gene The most commonly known carrier of the mecA gene is the bacterium known as MRSA, the mecA gene allows a bacterium to be resistant to antibiotics such as Methicillin, Penicillin, Oxacillin, Tetracyclin and other penicillin-like antibiotics. The mecA gene does not allow the ringlike structure of penicillin-like antibiotics to attack the enzymes that help form the cell wall of the bacterium (transpeptidases), and hence the bacteria is allowed to replicate as normal. The mecA gene encodes the protein PBP2A (Penicillin binding protein 2A). PBP2a has a low affinity for β-lactams such as Methicillin, Penicillin, and this enables transpeptidase activity in the presence of β-lactams to allow cell wall synthesis (Stapleton and Taylor, 2002).
2.12. FemA gene The concept of fem factors was first developed in 1983 by Brigitte Berger-Bächi, who defined fem genes via a reduction of methicillin resistance obtained by Transposon mediated inactivation of a gene even though the expression of mecA, the primary resistance gene encoding the extra penicillin-binding protein 2a (PBP2a) found in all MRSA, was not influenced. Exploitation of this experimental approach, mainly by the groups of Berger-Bächi (Reitz et al., 1967; Crusberg et al., 1970) led to the detection of a large number of fem factors, which have also sometimes been called aux (auxiliary) genes. Today at least six fem genes have been described and it has been estimated that there are at least ten equivalent but distinctly named (e.g. aux) genes (Crusberg et al., 1970; Labischinski et al., 1998). Two additional genes (femB and femC) were subsequently identified on the large SmaI-A fragment, and a fourth gene (femD) was 25
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identified on the SmaI fragment I of the chromosome of Staph. aureus (De-Lencastre et al., 1994). The femA gene encoding the femA protein, which is involved in the synthesis of the Staphylococcal cell wall (Mehrotra et al., 2000; Hwang et al., 2007). The femA and femB operon, that act as regulator genes are essential for the expression of methicillin resistance in Staph. aureus (Suzuki et al., 1993). The cooperation of both femA and mecA determinants seems to be required, but the mechanism is not well understood. Interestingly, femA appears to be a unique feature of Staph. aureus; it is not found in other Staphylococcus species (Vannuffel et al., 1995). It is well-known that femA, one of several genes involved in the synthesis
of
the
branched-peptide
structure
of
Staph.
aureus
peptidoglycan (Berger-Bachi and Rohrer, 2002; Cai et al., 2007). Mutations in femA, the gene required for incorporation of the second and third glycines into the cross bridge, were found following PCR amplification and nucleotide sequence analysis. Complementation of antibiotic-resistant mutants with pBBB31, which encodes femA, restored the phenotype of oxacillin resistance, resistance to antibiotic was associated with mutations in femA, but resistance could be suppressed by the coadministration of β-lactams antibiotics (Climo et al., 2001). FemA genes encode proteins which influence the level of methicillin resistance of Staph. aureus (Kobayashi et al., 1994). The tertiary structure of fem proteins was predicted based on protein structure homology modeling, using web-based automated comparative protein modeling (Giannouli et al., 2010; Giannouli et al., 2011).
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The femA gene may be used in gene disruption studies in Staph. epidermidis, although it is believed that the femA protein is involved in the formation of a pentaglycine bridge in the cell wall of the bacterium, gene disruption will allow one to ascertain the precise effect of the loss of the femA gene, gene disruption experiments in Staph. aureus have revealed that a loss of femA results in an 40% reduction in cell wall glycine content, a similar result might be anticipated for Staph. epidermidis, once determined, this information can be used to generate an assay for agents which inhibit the femA protein, and are therefore useful in combination with antibiotics to treat
methicillin-resistant-bacteria
(Alborn et al., 1997).
2.12.1. Mode of action of femA gene The femA gene encodes a protein precursor which plays a role in pre-peptidoglycan biosynthesis in Staph. aureus and is also considered as a factor influencing the level of methicillin resistance, a femA homolo-gous gene was recently characterized in Staph. epidermidis, entailing the possibility of femA phylogenetic (Vannuffel et al., 1999). Resistance to antibiotic has previously been described among MRSA strains with alterations in the formation of the pentaglycine cross bridge. Current evidence suggests that the pentaglycine cross bridge is formed in Staph. aureus under the control of three separate genes. The femA and femB encode factors that catalyze the successive addition of the second through fifth glycines (Kopp et al., 1996; Climo et al., 2001). Expressing femA during the exponential growth phase is varying amounts, in the stationary phase, the femA content was diminished. Strains in which femA was inactivated by insertion of Tn55I into the control region of the femAB operon are still expressed about 10% of the
27
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protein compared to their parent strains. Tn55I insertion in the middle of the femB gene did not affect the femA expression (Johnson et al., 1995). Resistance to antibiotic has been documented and this resistance generally results from mutations in the femA gene, which encodes the factor responsible for addition of the second and third glycines to the pentaglycine bridge of the cell wall (Ehlert et al., 1997). Inactivation of the femA function results in monoglycine cross bridges, thus eliminating the target for antibiotic cleavage , In addition to the reduction of the glycine content in the cell wall of femA mutants, these mutants have been characterized as having reduced cross-linkage and cell wall turnover, aberrant cell septum formation, and retardation of cell separation (Stranden et al., 1997). It is also well documented that MRSA strains that become resistant to lysostaphin lose their methicillin resistance phenotype (Rohrer and Berger-Bachi, 2003). The development of resistance to antibiotics often brings with it a fitness cost to the bacteria, These fitness costs are likely, however, ameliorated by subsequent evolution in the form of compensatory mutations (MaisnierPatin and Andersson, 2004). As each new class of antibiotics is introduced, evolution selects for the mutants which can survive the antibiotic challenge. This mutant is often less fit than their ancestral lineage, and thus, there is a second round of evolution which can occur to select for mutants which have compensated for the fitness deficit (Kusuma et al., 2007). A characteristic property of the Staph. aureus peptidoglycan is the long and flexible pentaglycine interpeptide bridge that allows a high cross linking of the peptidoglycan strands (Labischinski, 1992). It also acts as an anchor for cell wall directed surface proteins (Schneewind et al., 1995). The interpeptide bridge is synthesized by sequential addition of 28
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glycine residues by cell wall specific glycyl-tRNA (Matsuhashi et al., 1967), and requires the action of at least two proteins, femA and femB, that are encoded by the femAB operon. femA directs the incorporation of the glycines Gly2-Gly3, femB that of the glycines Gly4-Gly5 (Strandén et al., 1997). femAB-like sequences could be detected by Southern blots in all Staphylococcal type strains (unpublished results) and a femAB-like operon was characterized in Staph. epidermidis (Alborn et al., 1996) suggesting that femA and femB analogues belong to staphylococcal housekeeping genes needed in the biosynthesis of the interpeptide bridge (Tschierske et al., 1997). Since lysostaphin is under development as therapy for staphylococcal infections and resistance to lysostaphin could possibly occur during lysostaphin therapy (Kusuma et al., 2007).
2.13.
Clinical
importance
of
Methicillin
Resistance
Staphylococcus. aureus In clinical practice, MRSA poses an increasing threat, as not only can the organism survive for long periods in the environment, but also it can colonize the skin, nose, and throat of patients and healthcare staff, and is readily spread by direct contact. Risk factors for acquisition of MRSA include previous hospitalization, pressure sores, and recent treatment with antibiotics. Intensive care patients have a higher risk of developing MRSA infection compared to medical patients (Coello et al., 1997). Some outbreaks of MRSA in neonatal units come from the community rather than the hospital, others are apparently due to maternal colonization acquired in hospital (Brooks, 2005).
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2.14. The epidemiology of Methicillin Resistance Staph. aureus As surveillance of MRSA infections in the UK has primarily involved ascertainment of cases, the majority of the data available relate to infections in hospitalized patients, it is clear from these data, that hospitalized patients vary in terms of their risk of developing MRSA bacteremia, as discussed, analysis of data from the voluntary reporting scheme stratified by patient age showed that MRSA is more common in adults (Johnson et al., 2005). It is a commonly recognized organism that usually manifests itself in the form of various surface conditions, such as skin and soft tissue infections. Bones and heart valves also can be infected with Staph. Aureus, more invasive forms of Staphylococcal infection cause significant tissue damage. Necrotizing fasciitis, toxic shock syndrome, and purpura fulminans have all been caused by Staph. aureus. Postinfluenza pneumonia caused by Staph. aureus was responsible for many deaths in the flu pandemics of 1918, 1957, and 1968. The discovery and implementation of penicillin-based antibiotics in the 1940s briefly improved the outlook for patients infected with Staph. Aureus, resistance to penicillins in the form of β-lactamases quickly followed, however, and methicillin, a semisynthetic penicillin, was designed in 1959 to resist βlactamase degradation, MRSA was first noted in the United Kingdom in 1961 and in the United States in 1968, MRSA is currently a nosocomial pathogen with a high prevalence in acute-care and long-term-care settings and is considered the most commonly identified antibiotic-resistant pathogen in hospitals, until about 5 years ago, outbreaks of MRSA in the community were rare and usually linked to outbreaks at a local hospital or skilled nursing facility (Hirschmann, 2007). 30
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The important point for clinicians is that most invasive MRSA infections now occur in the community, rather than in the hospital, about 10% of the MRSA infections in this study were cellulitis, a reminder that skin and soft tissue are the sites of a significant number of MRSA infections, several studies indicate that most invasive infections from Staph. aureus both methicillin susceptible and methicillin resistantoriginate from organisms that patients carry in their own nares or on their skin (Hirschmann, 2007).
2.15. Prevention of Methicillin Resistance Staphylococcus aureus There is no way to completely prevent MRSA infections. There are, however, a number of measures that individuals can take to lessen the spread of the bacteria and protect themselves from becoming infected like hand washing, it is most important way to stop the spread of infection and protect oneself, and covering wounds important hygienic practices include cleaning and covering open sores, which serves two purposes. First, if the sore is not infected, it prevents the bacteria from entering the body the soap also can be beneficial (Sheen, 2010). Because MRSA is so antibiotic resistant, it is termed a "superbug" by some investigators. This MRSA is a variation of an already recognized human pathogen, the bacteria are usually found in the human armpit, groin, nose (most frequently), and throat. Fortunately, only about 1%-2% of people are colonized by MRSA, usually in the nose, according to the U.S. Centers for Disease Control and Prevention (CDC). In the majority of cases, the colonizing bacteria do not cause disease. However, damage to the skin or other injury may allow the bacteria to overcome the natural protective mechanisms of the body leading to infection; because of its
31
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ability to destroy skin, it is also one of the types of bacteria that has been termed a "flesh-eating bacterium (Nathwani et al., 2008). Alongside the development of sequenced-based typing methods, attention has become focused on the comparison of different MRSA genomes, in particular, the number of studies on the distribution of genes that are directly involved in causing disease has increased (Peacock et al., 2002; Nashev et al., 2004; Kuhn et al., 2006; Soliman et al,. 2009). At least seven types plus subtypes of SCCmec have been identified and their identification can be used to epidemiologically characterize isolates and investigate their relatedness (Deurenberg and Stobberingh, 2009). MRSA strains emerged soon after the introduction of methicillin into clinical practice, in addition to being a nosocomial pathogen, MRSA has become a community pathogen, strains that possess mecA gene are either heterogeneous or homogeneous in their expression of resistance. The heterogeneous expression occasionally results in minimal inhibitory concentrations that appear to be borderline and consequently the isolates may be interpreted as susceptible (Swenson et al., 2007).
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