Journal of Scientific Research ISSN 0555-7674 Vol. XXXX No. 2, December, 2010

The continuing threat of methicillin-resistant Staphylococcus aureus- Past Present Future

Zaeema Khan1, Saba Faisal2,3, Shahida Hasnain3 1School of Biological Sciences, University of the Punjab, Lahore, Punjab, Pakistan. 2Citi Lab and Research Centre, Faisal Town Lahore, Punjab, Pakistan. 3Department of Microbiology & Molecular Genetics, , University of the Punjab, Lahore, Punjab, Pakistan.

Abstract: Methicillin-resistant Staphylococcus aureus has been a major problem in the worldwide health care centres and institutions since the last 60 years . It has been a major pathogen responsible for nosocomial infections and recently has created life-threatening situations. resistance was seen a few years after the drug was introduced. To solve this problem another drug of the penicillin family was introduced : the semi- synthetic methicillin or oxacillin. This solved the resistance for a while until in 1960 in the UK, strains of Staphylococcus aureus were isolated that showed resistance to methicillin thus termed as Methicillin-resistant Staphylococcus aureus MRSA or Superbug. In the previous few decades MRSA has shown increased resistance not only to methicillin but also to a wide range of other such as gentamycin, erythromycin, and tetracycline .After the emergence of multidrug resistance in MRSA, the only drug of choice for treating MRSA infections was the glycopeptide . It was considered the drug of “last resort” until the unfortunate emergence of another alarming mutated strains of MRSA-Vancomycin-intermediate Staphylococcus aureus (VISA), and now recently the biggest threat of all is vancomycin-resistant Staphylococcus aureus (VRSA) which has acquired Vancomycin vanA gene from Enterococcus faecalis. The emergence of VISA or VRSA isolates alone are the huge threat mankind faces. Thus it has become imperative that analysis of MRSA strains take place and their genetic characteristics may be well understood to fight against the evolution of MRSA into VISA or VRSA.

Keywords: Methicillin-resistant Staphylococcus aureus, antibiotic resistance, penicillin, nosocomial infection, vancomycin.

1-For Proof and Correspondence: Zaeema Khan Zaeema Khan

History S. aureus first showed resistance to Alexander Fleming in 1928 observed the penicillin by the presence of a â- zone of inhibition that Penicillium lactamase enzyme penicillinase which notatum had produced around hydrolysed the tetrahedral ring ( by staphylococci [1]. Fleming discovered acquisition of a chromosomal encoded the antibiotic penicillin and in 1940 BlaZ gene which encoded the Florey and Chain isolated and purified it lactamase). [2]. Penicillin was introduced in 1943, a time when antibiotic resistance in Penicillin showed narrow spectrum staphylococci was completely unknown activity and proved to be ineffective and was used extensively in World War II against evolving strains. Derivatives of for the treatment of wounded soldiers of penicillin were essential which led to the the Allied Forces [3]. It was hardly development of ampicillin now suspected that several years later classified as a moderate-spectrum â- penicillin resistance would evolve in lactam antibiotic. It showed better staphylococci. Resistance to penicillin activity than the original and may have developed as early as 1946 [3]. has been used extensively since 1961. But both ampicillin and amonicillin were Mode of Action of the ß-Lactam ineffective against ß-lactamase Penicillin penicillinase producing bacteria. ß- Penicillin belongs to the ß-lactam lactamase resistant penicillins were then antibiotics which have a bactericidal further developed. Methicillin was mode of action and act by inhibiting the developed by Beecham in 1959. peptidoglycan synthesis in bacteria. Vancomycin was introduced in 1956, Gram-positive bacteria have a peptido- Methicillin in 1960, and Ampicillin in glycan layer covering 90% of the cell 1961. Resistance against methicillin was wall [4]. Because these antibiotics are observed the earliest in 1961 i.e. one year variants of active site “serine” hydrolases after its introduction, resistance to they can bind to the active site of ampicillin was observed in 1973 and penicillin binding proteins [5]. First an resistance to vancomycin was observed attack on the amide bond on D-Ala-D- in 1988 [3]. Modifications in the original Ala on the serine active site takes place. penicillin molecules Pen G and V gave Then acylation occurs after opening of rise to synthetic antibiotics. In the â-lactam ring and irreversible methicillin the unsubstituted aryl groups binding of the open ring to D-Ala-D-Ala were replaced with 2, 6 dimethoxy on the serine active site. This is the substituents, in oxacillin were replaced completion of the catalytic reaction by the phenyloxazolyl group. This which hinders the final peptidoglycan blocked the ß-lactamase active site, synthesis cross- linking [4]. making it resistant to hydrolysis. Methicillin shows similar activity like Emergence of Resistance that of all other penicillins. It acts on the In 1961 in the United Kingdom the very peptidoglycan synthesis by acylation and first emergence of penicillin-resistant S. cross linking of the peptidoglycan layer. aureus was observed[6]. It showed Methicillin proved to be the answer to resistance against dimethoxyphenecillin, penicillin resistant staphylococcus. large doses of which were required to treat patients due to its rapid absorption The second mode of ß-lactam resistance and secretion. [6]. is due to possession of altered penicillin- binding proteins. MRSA possesses a

38 The continuing threat of methicillin-resistant Staphylococcus chwomosomal mecA gene which erythromycin, tetK tetM encode produce PBP2A or the penicillin binding resistance to tetracycline[14]. protein. ß-lactams cannot bind as effectively to these altered PBPs, and, as Community Acquired MRSA (CA- a result, the ß-lactams are less effective at MRSA) disrupting cell wall synthesis [11]. S. aureus is commonly associated with Notable examples of this mode of skin infections [15] and causes infections resistance include 2A which is a cell wall in various parts of the body including biosynthetic enzyme MRSA. Other endocardium, long, soft tissues [16]. S. methicillin group of antibiotics were then aureus is an important hospital pathogen used to treat MRSA. Notably causing various infections and toxinoses flucloxacillin and dicloxacillin. Altered including cutaneous abscesses with PBPs do not necessarily rule out all varying severity and lethal necrotizing treatment options with ß-lactam fasciitis and life threatening necrotizing antibiotics [12]. pneumonia. [17,18]. With treatment with various ß lactams and other modern Multiple drugs this dangerous pathogen has MRSA shows evolved into MRSA making the against a variety of different antibiotics infections even more lethal leaving very erythromycin, tetracycline, and limited therapy options [19,20]. S. such as gentamycin aureus is also responsible for impetigo [13]. Resistance genes on plasmids have and staphylococcal scalded skin shown resistance to heavy metals such as syndrome causing blisters in children cadmium acetate, mercuric chloride, and neonates. Recently it has been resistance to chemicals such as observed that from outpatients CA- propamidine isoethionate, ethidium MRSA has been isolated and found bromide [14], and resistance to responsible for impetigo which is of antibiotics such as , serious concern since the CA-MRSA neomycin, erythromycin, mupirocin, carries resistance genes not only for ß- gentamycin, kanamycin, streptomycin, lactams but also for multidrugs [21]. S. tetracycline, tobramycin, clindamycin, aureus is a commensal bacterium fusidic acid, trimethoprim, ciprofloxacin commonly found in the anterior nares of [14]. Vancomycin was the ideal drug the human body. It has now been used to treat MRSA infections by 1986. established that the presence of S. aureus The threat of VISA or even VRSA led to in the nose has important epidemio- the development of the oxazolidnone logical significance especially when , introduced in 1999 used to treat there is a widespread multidrug resistant multidrug resistant staphylococci [3]. As in S. aureus. Administration of a wide discussed in detail before resistance to range of antibiotics to hospitalized antibiotics in S. aureus has been patients has been a major risk factor for mediated through two ways. The first is MRSA infection [22]. the presence of the BlaZ lactamase on the chromosome. In MRSA the expression of The commensal bacteria in the nose penicillin –insensitive PBP2a, encoded undergo modification with the by the chromosomal mecA gene is administration of systemic antibiotic responsible for resistance. Resistance drugs [23]. Misuse of penicillin resulting genes for other antibiotics include aac6'- in environmental widespread has been aph2”, aph (3')-III for gentamycin shown to be a significant factor for the resistance, ermA, ermB, ermC for colonization and transmission of

39 Zaeema Khan penicillin-resistant S. aureus in the nose Aborigines of Western Australia [30]. and to other patients. Frequent dosages of Following that it appeared in apparently tetracycline given to patients with healthy individuals having no risk factors tetracycline-resistant S. aureus also in 1997 and 1999 in the USA and facilitated the widespread of tetracycline Europe[31]. In children CA-MRSA resistant strains thus antibiotic pressure infection spread through infected skin by has been a major factor contributing to contact can be the causative agent of fatal the emergence of MRSA [22] which has necrotizing pneumonia. An important become a significant threat as MSSA and characteristic of CA-MRSA is the MRSA strains colonize the human body presence of the mec IV the methicillin- persistently [15]. In a study conducted in resistance locus located on the 1998 in Bangladesh it was shown that staphylococcal cassette chromosome MRSA was prevalent among diabetic SCCmec and a virulent Panton-Valentine patients; 37.2% among infected leucocidin (PVL) gene. SCCmec hospitalized diabetics and 21.6% among elements have been categorized into 5 non-hospitalized diabetics in the allelic types out of which throughout the community [24]. world HA-MRSA carries SCCmec type I, type II, type III. The other two SCCmec Epidemic MRSA elements type IV and type V have been Methicillin-resistant Staphylococcus dispersed greatly among CA-MRSA aureus has been mainly associated with strains. [38]. CA-MRSA has prevalence the hospital set up and various other in the community since the 1990's. healthcare institutions [28]. There are high there are high risk factors of CA- MRSA has emerged in various parts acquiring MRSA infection during of the world Australia, New Zealand, surgery, renal dialysis and by Canada, the USA, Europe and in Hong contaminated medical equipment. In Kong. In Hong Kong the strain isolated hospital environments where MRSA from a 50 year old man with a good readily causes infection it is often health, showed resistance to 1ìg considered as epidemic MRSA or oxacillin disk but it was non-multidrug EMRSA. Nursing homes in various parts resistant [32]. A crucial point about CA- of the world have been associated with MRSA is that there are no specific risk MRSA infections and may be considered factors which the people in the as a reservoir. Healthcare institutions community are exposed to. In the south MRSA infection has been a major Asian region in Bangladesh from the problem causing mortality [27]. In recent outpatients department of a hospital years MRSA has been isolated from located in Dhaka community acquired people in healthy communities who are MRSA has been isolated [33]. In Los not at risk of MRSA infection. It is still Angeles it was noted that CA-MRSA was unclear why some MRSA strains are responsible for a surprisingly higher associated with hospitals and health care number of infections of necrotizing centers whereas some strains are present fasciitis[17] . Studies and data from a in healthy individuals in the community MRSA infection in a Lebanese man [29]. showed the presence of many toxins and the etA and etD exfoliative toxin genes Increasing Prevalence of MRSA were described. The resistant genes were around the world the mecA gene and resistance genes for First case of Community Acquired- neomycin and streptothricin. Various MRSA was reported 1993 in the pathogenicity islands have been

40 The continuing threat of methicillin-resistant Staphylococcus observed in various MRSA strains all In a study conducted in several Asian around the world. In Japan there are countries it was found that out of a total of strains carrying etA gene but are rarely 1357 MRSA isolates 347 (25.6%) were found outside of Japan. In the European found to be resistant to 4mg/Litre of CA-MRSA etD and edinB containing vancomycin. Heterointermediate pathogenicity island is observed [34]. In resistance to vancomycin hVISA Michigan in the United States MRSA analysis showed the presence of hVISA shows plasmid encoded resistance to in Japan (8.2%), India (6.3%), South aminoglycosides, trimethoprim, Korea (6.1%), the Philippines (3.6%), disinfectants and ß-lactams carrying the Vietnam (2.4%), Singapore (2.3%), and resistance genes aacA-aphD, dfrA, qacC Thailand (2.1%). This incidence of and blaZ respectively [35]. hVISA is attributed to the extensive use of glycopeptides in Asian countries [39]. In hospitals the criteria for defining a In Pakistan up till now, there has been no Hospital Acquired-MRSA case includes case of vancomycin intermediate or the above mentioned risk factors resistant S. aureus. In a study conducted showing a history of prolonged stay in a in Rawalpindi, the MIC's of vancomycin health care facility within a year of the were calculated for MRSA and it was culture date of the MRSA strain, the concluded that up till now there is no patient admitted to a hospital and presence of VISA or VRSA in Pakistan. acquiring a MRSA infection after 48 [41] hours and the indwelling percutaneous medical devices (gastrostomy tube) and Role of the Glycopeptide Vancomycin finally a positive culture for MRSA [36]. and Vancomycin Intermediate and Recent surgery and hospitalization, Vancomycin Resistant Staphylococcus living in health-care institutions e.g. aureus (VISA and VRSA) elderly nursing homes, medical devices The treatment of MRSA infections has including catheters, and percutaneous become problematic due to the devices and dialysis are all now acquisition of multiple drug resistance prominent risk factors leading to MRSA genes. Due to widespread MRSA infection [37]. infections there has been great pressure to use glycopeptides for treatment. The In a multicentre study in Bangladesh glycopeptide vancomycin has been used there were 510 MRSA isolates out of to treat patients with MRSA infections 3611 samples (14.1%). Incidence of [40]. Now through plasmid acquired MRSA in four Bangladesh hospitals vancomycin resistance has been acquired revealed that the rates of MRSA range by some MRSA strains. Vancomycin from 32.0% and 63.00%. The study resistance was first observed in a strain of conducted in 2005 showed that the Enterococcus faecalis in 1988. This high incidence of MRSA has greatly increased resistance is mediated by a mobile in Bangladesh over the past 4-5 years in genetic element. The resistance gene was different hospitals of various regions of VanA located in a gene cluster. Bangladesh.[33]. In Japan MRSA has Transference of the mobile genetic already been found in healthy children. element to MRSA creates high level In the analysis of 818 children, 231 teicoplanin and vancomycin resistance in children (28.2%) carried methicillin- MRSA. The emergence of vancomycin r e s i s t a n t c o a g u l a s e - n e g a t i v e intermediate and vancomycin resistant staphylococci (MRC-NS), 35 children Staphylococcus aureus has become a (4.3%) carried MRSA. major threat [35].

41 Zaeema Khan

MRSA infections have been treated with having intermediate resistance to vancomycin since there have been few vancomycin MICs of 1-4mg/litre. drugs it is susceptible to. Most MRSA Although this is an intermediate strains showed susceptibility to susceptibility there is controversy vancomycin near the value of 1 µg/ml till whether hVISA is of medical the late 1990s. In Pakistan [41] as well as significance[45]. Bangladesh [33] MRSA strains show susceptibility to vancomycin MIC < 4 Future Prospects of Treatment of µg/ml. In 1997 in a study in Kuwait MRSA infection MRSA showed MIC values of 0-5-2 In vitro studies showed that copper mg/L for vancomycin and teicoplanin but sulfate has antimicrobial activity against from 1999 the strains showed reduced MRSA. Soluble copper silicate has been susceptibility to both drugs MIC 3–4 studied in detail as a possible future mg/L.[14]. Since the end of 1999 several treatment for dermatological MRSA countries have reported stains with infections and nasal decolonization reduced susceptibility to vancomycin therapy. In phase I studies a topical cream and such vancomycin intermediate containing 0.22% Cu wt/wt strains showed MIC values of 8-16 was shown to be tolerated by 350 people. µg/ml. The very first reports of It has been shown that MRSA showed an vancomycin resistant S. aureus strains MIC of 175mg Cu/liter. At 2X MIC and was reported in two hospitals in the 4X MIC bactericidal and post antibiotic United States in 2002 showing high effects (in more than 1 hour) were resistance MIC of 32 µg/ml [42]. This observed. [46]. was a very alarming discovery raising serious concern that the spread of VRSA References would make MRSA infection drug 1. Fleming A. On the Antibacterial therapy very difficult and could even Action of Cultures of a Penicillium, give rise to staphylococcal infections with Special Reference to Their Use with no or treatment in the Isolation of B. influenzae. Br J available. [43,44]. Exp Pathol, 1929; 10:226-236.

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