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2 Oral Carriage & Suffering of Staphylococcus Aureus i

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4 Oral Carriage & Suffering of Staphylococcus Aureus Oral Infection & Staph.Aureus AUTHOR Dr. Biswajit Batabyal EDUCREATION PUBLISHING (Since 2011) iii

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6 Content List S. No. Content Page Abbreviations Abstract 1. Introduction Microbiology Role in disease Atopic dermatitis Toxic shock syndrome and food poisoning 1.5. Mastitis in cows Reproduction Virulence factors Role of pigment in virulence Classical diagnosis Rapid diagnosis and typin Treatment and antibiotic resistance Antibiotics Penicillin Group Cephalosporins Group Macrolide Antibiotics Group Lincosamides Group Rifamycins Linezolid 20 4 v

7 1.19. Quinolones (Fluoroquinolones) Vancomycin Carbapenems Group Mechanisms of antibiotic resistance MRSA in oral cavity Literature Review Aims & Objectives Importance of the Study Aims of the Study Objectives of the Study Research Methodology Study Period & Study Setting Study patients and participants Collection and processing of the samples Identification of isolates Mannitol salt agar Gram Technique Catalase test Coagulase test DNase test Sensitivity testing using Disc Diffusion technique Tryptone soya broth Mueller Hinton agar Methicillin Resistant Staph. aureus detection List of materials used for the study Control strain Maintenance of isolated strains Results vi

8 6. Statistical Analysis Discussion Conclusion Bibliography 108 vii

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10 ABBREVIATIONS AMX : Amoxicillin AMP : Ampicillin AMC : Amoxicillin/clavulanic acid AS : Ampicillin/sulbactam ABST : Antibiotic sensitivity test ATCC : American Type Culture Collection C : Degree centigrade CPS : Coagulase positive Staphylococcus CLSI : Clinical Laboratory Standard Institute CPD : Cefpodoxime CD : Clindamycin CIP : Ciprofloxacin CV : Crystal violet DNA : Deoxyribonucleic acid DNase : Deoxiribonuclease EF : Exfoliative E : Erythromycin EDTA : Ethylenediaminetetraacetic acid Gm : Gram GPC : Gram positive cocci GNB : Gram negative bacilli GDA : Group-D Assistant H 2 O 2 : Hydrogen per oxide HCl : Hydrochloric acid hr : Hour Ig : Immunoglobulin ix

11 IPM : Imipenem IS : Intermediate Sensitive LZ : Linezolid MRSA : Methicillin resistant Staphylococcus aureus MSSA : Methicillin sensitive Staphylococcus aureus MDR : Multi drug resistant mg : Milligram MW : Molecular weight MRP : Meropenem MSA : Mannitol salt agar MHA : Mueller Hinton agar mm : Millimeter ml : Milli litre mcg : Microgram min : Minute NEG : Negative Nacl : Sodium chloride NCCLS : National committee for clinical laboratory standards OX : Oxacillin OF : Ofloxacin OS : Oral Surgery OPD : Outdoor patients department OP & : Oral Pathology & Microbiology OM OT : Operation theater ORSA : Oxacillin resistant Staphylococcus aureus OSSA : Oxacillin sensitive Staphylococcus aureus PI : Piperacillin PIT : Piperacillin/Tazobactam POS : Positive PBP : Penicillin-binding protein PCR : Polymerase chain reaction x

12 PVL : Panton-valentine leukocidin R : Resistant RNA : Ribonucleic acid rrna : Ribosomal Ribonucleic acid RIF : Rifampicin S : Sensitive SA : Staphylococcus aureus SSSS : Staphylococcal scalded skin syndrome Scc mec : Staphylococcal cassette chromosome mec Sec : Second SSIs : Surgical Site infections TSS : Toxic shock syndrome TI : Ticarcillin TCC : Ticarcillin/clavulanic acid TSB : Tryptone soya broth V : Volume VA : Vancomycin VRSA : Vancomycin-resistant Staphylococcus aureus VISA : Vancomycin Intermediate Staphylococcus aureus W : Weight WHO : World Health Oraganisation μg : Microgram μl : Micro liter ***** xi

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14 ABSTRACT The oral cavity carriage and antibiotic susceptibility patterns of Staphylococcus aureus in Dental hospital staff and healthy general population were determined. Oral cavity swabs were taken from 113 healthy general population and 90 health care workers. Antibiotic disc susceptibility testing was conducted following the CLSI method. Staphylococcus aureus carriage was noted in 28.3% of healthy general population and 38.9% of health care workers. Resistance to commonly used oral antibiotics of healthy general population & health care workers, ampicillin 93.8% & 97.2%, amoxicillin/clavulanic acid 84.4% & 77.2%, amoxicillin 43.8% & 57.2%, ciprofloxacin 53.2% & 57.2% and ofloxacin 37.5% & 42.9%, respectively. 5.7% methicillin resistant Staphylococcus aureus was detected among the hospital personnel from isolated strain. The MRSA isolates showed multiple drug resistance (MDR), except imipenem. Hospitals should assess the advantages and disadvantages of routinely culturing personnel, however, in outbreak situation hospital personnel especially young persons may be sources of nosocomial infection. Staphylococcus aureus is a well recognized pathogen associated with a variety of clinical syndrome. The role of Staph. aureus in some types of oral disease may be more important than previously recognized. The present study was designed to investigate the prevalence of Staphylococcus aureus, MRSA and their rate of resistance to different anti staphylococcal antibiotics. For this study, Gurunanak Institute of Dental Science & Research (Kolkata), selected patients who were suffering from Staphylococcus aureus oral infection. Isolated Staphylococcus aureus was tested for Oxacillin (1 mcg) sensitivity and their antibiotic susceptibility was investigated by using eighteen antibiotics followed by Disk diffusion technique following CLSI method. Out of the 223 specimens collected, 109 (48.8%) were xiii

15 isolated. All the 109 (48.8%) specimens were studied in detail. 5.5% of the isolates were shown to be methicillin resistant Staph. aureus (MRSA). Percentage (%) of resistance in commonly used oral antibiotics are ampicillin 98.1%, amoxycillin/clavulanic acid 73.3%, amoxycillin 44.9%, ofloxacin 48.6% and ciprofloxacin 41.2%. The MRSA isolates showed multiple drug resistance (MDR), except linezolid and imipenem. In line with more recent surveys, this retrospective study suggests that Staph. aureus may be more frequent isolate from the oral cavity than hitherto suspected. The role of Staph. aureus in several diseases of the oral mucosa merits further investigation. The problem of infection has been persistent in the surgical world even after the introduction of antibiotics. Pathogens that infect surgical site can be acquired from the hospital environment or other infected patients. A total of 66 pus samples from postoperative oral & maxillofacial surgical infections were received in the Department of Microbiology, Gurunanak Institute of Dental Science & Research, Panihati, Kolkata, over a period of one year. The isolates were identified using standard laboratory procedures. All the isolates were tested for susceptibility to various commonly used antibiotics and screened for oxacillin susceptibility according to CLSI guidelines. Out of 66 pus samples received, 34(51.5%) were culture positive for Staph. aureus. Methicillin resistance was documented in 14 (41.2%) of the Staph. aureus isolates. Highest efficacy was observed with linezolid (97.0%). All MRSA isolates were 100% sensitive to linezolid. The hospital acquired surgical site infection is alarming. Hospital disinfection and treatment protocols should be practiced. ***** xiv

16 Oral Carriage & Suffering of Staphylococcus Aureus 1 INTRODUCTION Staphylococcus aureus literally the golden cluster seed or the seed gold and also known as golden staph and Oro staphira is a facultative anaerobic, Gram-positive coccus and is the most common cause of staph infections. It is frequently part of the skin flora found in the nose and on skin. About 20% of the human populations are long term carriers of Staph. aureus [1]. The carotenoid pigment staphyloxanthin is responsible for Staph. aureus characteristic golden color, which may be seen in colonies of the organism. This pigment acts as a virulence factor with an antioxidant action that helps the microbe evade death by reactive oxygen species used by the host immune system. Staph organisms which lack the pigment are more easily killed by host defenses. Staph. aureus can cause a range of illnesses from minor skin infection, such as pimples, impetigo, boils (furuncles), cellulitis folliculitis, carbuncles, scalded skin syndrome, and abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome (TSS), chest pain, bacteremia, and sepsis. Its incidence is from skin, soft tissue, respiratory, bone, joint, endovascular to wound infections. It is still one of the five most common causes of nosocomial infections, often causing postsurgical wound infections. Abbreviated to Staph. aureus or Staph. aureus in medical literature, Staph. aureus should not be confused with the similarly named and similarly dangerous (and also medically relevant) species of the genus Streptococcus. Staph. aureus was discovered in Aberdeen, Scotland in 1880 by the surgeon Sir Alexander Ogston in pus from surgical 1

17 Dr. Biswajit Batabyal abscesses [2]. Each year, some 500,000 patients in American hospitals contract a staphylococcal infection [3] Microbiology Staph. aureus is a facultative anaerobic, Gram-positive coccus, which appears as grape-like clusters when viewed through a microscope and has large, round, golden-yellow colonies, often with hemolysis, when grown on blood agar plates [4]. The golden appearance is the etymological root of the bacteria s name; golden in Latin. Staph. aureus is catalase-positive (meaning that it can produce the enzyme, catalase ) and able to convert hydrogen peroxide (H 2 O 2 ) to water and oxygen, which makes the catalase test useful to distinguish staphylococci from enterococci and streptococci. A small percentage of Staph. aureus can be differentiated from most other staphylococci by the coagulase test: Staph. aureus is primarily coagulase positive (meaning that it can produce the enzyme coagulase ) that causes clot formation, whereas most other Staphylococcus species are coagulase-negative [4]. However, while the majority of Staph. aureus are coagulasepositive, some may be atypical in that they do not produce coagulase(the most common organism in patients with nosocomial bacteremia is coagulase-negative staphylococcus) [5]. Incorrect identification of an isolate can impact implementation of effective treatment and /or control measures [6]. Fig.1.1.Grampositive cocci in cluster 2

18 Oral Carriage & Suffering of Staphylococcus Aureus Fig.1.2. Staph. aureus Electron micrograph 1.2. Role In Disease Staph. aureus are responsible for food poisoning through the production of an enterotoxin and pathogenicity is also associated with coagulase positivity. Staph. aureus may occur as a commensal on skin; it also occurs in the nose frequently (in about a third of the population) [7] and throat less commonly. The occurrence of Staph. aureus under these circumstances does not always indicate infection and, therefore, does not always require treatment (indeed, treatment may be ineffective and re-colonization may occur). It can survive on domesticated animals such as dogs, cats, and horses, and can cause bumble foot in chickens. It can survive for hours to days, weeks, or even months on dry environmental surfaces depending on strain [8]. It can host phages, such as Panton-Valentine leukocidin, that increase its virulence. Staph. aureus can infect other tissues when barriers have been breached (e.g. skin or mucosal lining). This leads to furuncles (boils) and carbuncles (a collection of furuncles). In infants Staph. aureus infection can cause a severe disease staphylococcal scalded skin syndrome (SSSS) [9]. Staph. aureus alv infections can be spread through contact with pus from an infected wound, skin-to-skin contact with an infected person by producing hyaluronidase that destroys tissues, and contact with objects such as towels, sheets, clothing, or athletic equipment used by an infected person. Deeply penetrating Staph. aureus infections can be severe. Prosthetic joints put a person at particular risk for septic arthritis, and staphylococcal endocarditis 3

19 Dr. Biswajit Batabyal (infection of the heart valves) and pneumonia, which may be rapidly spread Atopic Dermatitis Staph. aureus is extremely prevalent in atopic dermatitis patients, who are less resistant to it than other people. It often causes complications. The disease is most likely found in fertile active places including, the armpits, hair, and scalp. The large pimples that appear in those areas may cause the worst of the infection if popped. This can lead to scaled skin syndrome. A severe form of this is Ritter s disease seen in neonates Toxic Shock Syndrome And Food Poisoning Some strains of Staph. aureus, which produce the exotoxin TSST- 1, are the causative agents of toxic shock syndrome. Some strains of Staph. aureus also produce an enterotoxin that is the causative agent of Staph. aureus gastroenteritis. The gastroenteritis is selflimiting, with the person recovering in 8-24 hours. Symptoms include nausea, vomiting, diarrhea, and abdominal pain Mastitis In Cows Staph. aureus is one of the causal agents of mastitis in dairy cows. Its large polysaccharide capsule protects the organism from recognition by the cow s immune defenses [10] Reproduction Staph. aureus reproduces asexually. It starts this process by reproducing its DNA. The membrane stretches out and separates the DNA molecules. The cells from a hollow space that eventually divides out into two new cells. The new cell wall does not fully separate from the existing cell wall, which is why the cells are 4

20 Oral Carriage & Suffering of Staphylococcus Aureus observed in clusters. This cell will eventually reproduce and cells will attach onto it [11] Virulence Factors Toxins Depending on the strains, Staph. aureus is capable of secreting several toxins, which can be categorized into three groups. Many of these toxins are associated with specific diseases. Super antigens PTSAgs have super antigen activities that induce toxic shock syndrome (TSS). This group includes the toxin TSST-1, which causes TSS associated with tampon use. The staphylococcal enterotoxins, which cause a form of food poisoning, are included in this group. Exfoliative toxins EF toxins are implicated in the disease staphylococcal scalded-skin syndrome (SSSS), which occurs most commonly in infants and young children. It also may occur as epidemics in hospital nurseries. The protease activity of the exofoliative toxins causes peeling of the skin observed with SSSS. Other toxins Staphylococcal toxins that act on cell membranes include alphatoxin, beta-toxin, delta-toxin, and several bicomponent toxins. The bicomponent toxin Panton-Valentine leukocidin (PVL) is associated with severe necrotizing pneumonia in children. The genes encoding the components of PVL are encoded on a bacteriophage found in community-associated methicillin-resistant Staph. aureus (MRSA) strains. Protein A Protein A is a protein that is anchored to staphylococcal peptidoglycan pentaglycine bridges (chains of five glycine residues) by the transpeptidase Sortase A [12]. Protein A is an IgGbinding protein that binds to the Fc region of an antibody. In fact, 5

21 Dr. Biswajit Batabyal studies involving mutation of genes coding for protein A resulted in a lowered virulence of S. aureus as measured by survival in blood, which has led to speculation that Protein A contributed virulence requires binding of antibody Fc regions[13]. Protein A in various recombinant forms has been used for decades to bind and purify a wide range of antibodies by immune affinity chromatography. Transpeptidases such as the sortases that are responsible foranchorning factors like Protein A to the staphylococcal peptidoglycan are being studied in hopes of developing new antibiotics to target MRSA infections [14] Role Of Pigment In Virulence Some strains of Staph. aureus are capable of producing staphyloxanthin-a carotenoid pigment that acts as a virulence factor. It has an antioxidant action that helps the microbe evade death by reactive oxygen species used by the host immune system. Staphyloxanthin is responsible for Staph. aureus characteristic golden color [15]. When comparing a normal strain of Staph. aureus with a strain modified to lack staphyloxanthin, the wild type pigmented strain was more likely to survive incubation with an oxidizing chemical such as hydrogen peroxide than the mutant strain was. Colonies of the two strains were also exposed to human neutrophils. The mutant colonies quickly succumbed while many of the pigmented colonies survived. Wounds on mice were inoculated with the two strains. The pigmented strains created lingering abscesses. Wounds with the unpigmented strains healed quickly. These tests suggest that the staphyloxanthin may be key to the ability of Staph. aureus to survive immune system stacks. Drugs designed to inhibit the bacterium s production of the staphyloxanthin may weaken it and renew its susceptibility to antibiotics [16]. In fact, because of similarities in the pathways for biosynthesis of staphyloxanthin and human cholesterol, a drug developed in the context of cholesterol-lowering therapy was shown to block Staph. aureus pigmentation and disease progression in a mouse infection model [17]. 6

22 Oral Carriage & Suffering of Staphylococcus Aureus 1.9. Classical Diagnosis Depending upon the type of infection present, an appropriate specimen is obtained accordingly and sent to the laboratory for definitive identification by using biochemical or enzyme-based tests. A Gram stain is first performed to guide the way, which should show typical gram-positive bacteria, cocci, in clusters. Second, the isolate is culture on mannitol salt agar, which is a selective medium with 7-9% NaCl that allows Staph. aureus to grow, producing yellow-colored colonies as a result of mannitol fermentation and subsequent drop in the medium s ph. Furthermore, for differentiation on the species level, catalase (positive for all Staphylococcus species), coagulase (fibrin clot formation, positive for Staph. aureus), DNAse (zone of clearance on nutrient agar), lipase (a yellow color and rancid odor smell), and phosphatase (a pink color) tests are all done. For staphylococcal food poisoning, phage typing can be performed to determine if the staphylococci recovered from the food to determine the source of infection Rapid Diagnosis And Typing Diagnostic microbiology laboratories and reference laboratories are key for identifying outbreaks and new strains of Staph. aureus. Recent genetic advances have enabled reliable and rapid techniques for the identification and characterization of clinical isolates of Staph. aureus in real-time. These tools support infection control strategies to limit bacterial spread and ensure the appropriate use of antibiotics. These techniques include real-time PCR and quantitative PCR and are increasingly being employed in clinical laboratories [18, 19]. 7

23 Dr. Biswajit Batabyal Treatment And Antibiotic Resistance The treatment of choice for Staph. aureus infection is penicillin; but, in most countries, penicillin-resistance is extremely common and first-line therapy is most commonly a penicillinase-resistant beta-lactam antibiotic (for example, oxacillin or flucloxacillin). Combination therapy with gentamicin may be used to treat serious infections like endocarditis [20, 21], but its use is controversial because of the high risk of damage to the kidneys [22]. The duration of treatment depends on the site of infection and on severity. Antibiotic resistance in Staph. aureus was uncommon when penicillin was first introduced in Indeed, the original petri dish on which Alexander Fleming of Imperial College London observed the antibacterial activity of the penicillium fungus was growing a culture of Staph. aureus. By 1950, 40% of hospital Staph. aureus isolates were penicillin-resistant; and, by 1960,this had risen to 80% [23]. Researchers from Italy have identified a bacteriophage active against Staphylococcus aureus, including methicillin-resistant strains (MRSA), in mice and possibly humans [24]. Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium responsible for several difficult-to-treat infections in humans. It is also called multidrug-resistant Staphylococcus aureus and oxacillin-resistant Staphylococcus aureus (ORSA). MRSA is any strain of Staphylococcus aureus that has developed resistance to beta-lactam antibiotics, which include the penicillins (methicillin, dicloxacillin, nafcillin, oxacillin, etc.) and the cephalosporins. Strains unable to resist these antibiotics are classified as methicillin-sensitive Staphylococcus aureus, or MSSA. The development of such resistance does not cause the organism to be more intrinsically virulent than strains of Staphylococcus aureus that have no antibiotic resistance, but resistance does make MRSA infection more difficult to treat with standard types of antibiotics and thus more dangerous. MRSA is especially troublesome in hospitals and nursing homes, where patients with open wounds, invasive devices, and weakened 8

24 Oral Carriage & Suffering of Staphylococcus Aureus immune systems are at greater risk of infection than the general public Antibiotics The word antibiotic [25] is derived from Greek stems that mean against life. In 1889, the French researcher Paul Vuillemin coined the term to describe a substance he isolated some years earlier from Pseudomonas aeruginosa. The substance, called pyocyanin, inhibits the growth of other bacteria in test tubes, but it was too toxic to be useful in disease therapy. Vuillemin s term has survived to the current era. Antibiotics are now considered to be chemical products or derivatives of microorganisms that are inhibitory to other microorganisms. Scientists are uncertain as to how the ability to produce antibiotics arose in living things, but it is conceivable that random genetic mutations were responsible. Clearly, the ability to produce an antibiotic conferred an extraordinary evolutionary advantage on the processor in the struggle for survival. In this section we shall discuss the sources of antibiotics-which are used in this study, their modes of action, and side effects, and how they are used by physicians to control infections disease Penicillin Since the 1940s, Penicillin [25] has remained the most widely used antibiotic because of its low cost and thousands of derivatives. Penicillin G, or Benzyl penicillin, is currently the most popular penicillin antibiotic and is usually the one intended when doctor prescribe Penicillin. Other types are Penicillin F and Penicillin V, all with the same basic structure of a beta-lactam nucleus and several attached groups. The Penicillins are active against a variety of Gram-positive bacteria, including Staphylococci, Streptococci, Clostridia, and Pneumococci. In higher concentrations, they are also inhibitory to the Gram-negative diplococci that cause gonorrhea and meningitis, and they are useful against syphilis spirochetes. 9

25 Dr. Biswajit Batabyal Penicillin functions during the synthesis of the bacterial cell wall. It blocks the cross-linking of carbohydrates in the peptidoglycan layer during wall formation. Antimicrobial Activity [26]: The initial step in Penicillin action is binding of the drug to cell receptors. These receptors are PBPs, at least some of which are enzymes involved in transpeptidation reactions. From three to six (or more) PBPs per cell can be present. After penicillin molecules have attached to the receptors, peptidoglycan synthesis is inhibited as final transpeptidation is blocked. A final bacterialcidal event is the removal or inactivation of an inhibitor of autolytic enzymes in the cell wall. This activates the autolytic enzymes and results in cell lysis. Organisms with defective autolysin function are inhibited but not killed by Beta-lactam drugs, and they are said to be tolerant. Since active cell wall synthesis is required for penicillin action, metabolically inactive microorganisms are not susceptible. Penicillin G and Penicillin V are often measured in unit (1 million Units=0.6 gm), but the semi synthetic penicillins are measured in grams. Whereas microgm/ml of Penicillin G is lethal for a majority of susceptible gram positive organisms, times more is required to kill gram negative bacteria(except Neisseriae). Resistance Resistance to penicillins falls into several categories: 1. Production of Beta-lactamases by Staphylococci, gram negative bacteria, haemophili, gonococci, and others. More than 50 different Beta-lactamases are known, most of them produced under the control of bacterial plasmids. Some Betalactamases are inducible by the new cephalosporins. 2. Lack of penicillin receptors (PBPs) or altered PBPs (e.g., Pneumococci, enterococci) or inaccessibility of receptors because of permeability barriers of bacterial outer membranes. These are often under chromosomal control. 10

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