Clinical Bacteriology - 02 Gram Positive and Negative Cocci

CLINICAL BACTERIOLOGY CATALASE (+) GRAM POSITIVE COCCI STAPHYLOCOCCUS, MICROCOCCUS GRAM POSITIVE & GRAM NEGATIVE COCCI Staphylococcus aureus OUTLINE • Introduction • Catalase (+) Gram Positive Cocci Staphylococcus, Micrococcus o Staphylococcus aureus o Staphylococcus epidermidis o Normal Skin Flora o Staphylococcus schleiferi o Staphylococcus schleiferi o Micrococcus spp. • Catalase (-) Gram Positive Cocci Streptococci And Enterococci o Streptococcus spp. o Enterococcus spp. • Neisseria and Moraxella catarrhalis o Microscopic Characteristics o Tests for Identification o Clinical Diagnosis • • • • • INTRODUCTION • • • • The Staphylococci are catalase positive gram positive cocci that can cause infections in humans. These are organisms can colonize various skin and mucosal surfaces. Staphylococci are transmitted from person to person. Upon transmissions, the organisms may become established as part of the recipient's normal flora and later be introduced to sterile sites by trauma or invasive procedures. Without question, S. aureus is the most virulent species of Staphylococci encountered. The Catalase negative, gram positive cocci such as the Streptococcus and Enterococcus are commonly found as part of normal human flora and are encountered in clinical specimens as contaminants or as components of mixed cultures with minimal or unknown clinical significance. However , when these organisms gain access to normally sterile sites, they can cause life threatening infections. Organisms notably Streptococcus pneumoniae and Streptococcus pyogenes, are notorious pathogens. Although S. pneumoniae can be found as part of the normal upper repiratory flora, this organism is also th leading cause of bacterial pneumonia and meningitis. S. pyogenes which is rarely considered as a normal flora, is deemed clinically important whenever it is encountered. Species of the genus Neisseria and Moraxella catarrhalis are all oxidase positive, gram negative diplococci. Except for N. gonorrhloeae, all are normal inhabitants of the upper respiratory tract of humans. N. gonorrhoeae is primarily a pathogen found in the urogenital tract . It is never considered a normal flora & is always considered clinically significant. Transmission of the two pathogenic species of Neisseria, N. gonorrhoeae and N. meningitidis, is person to person. The former is sexually transmitted while the latter is spread by contaminated respiratory droplets. Infections caused by M. catarrhalis and other Neisseria spp. usually involve a patient's endogenous strain. Infections caused by M. catarrhalis are usually localized to the respiratory tract and rarely disseminate. Most virulent species of staphylocci encountered Normal flora of human anterior nares, nasopharynx, perineal area and skin Various skin, wound and tissue infections commonly caused by S. aureus - this infections can rapidly become life threatening if not treated and manage appropriately S. aureus produce toxin-mediated diseases, such as scalded skin syndrome and toxic shock syndrom Virulence Factors o alpha, beta, gamma and delta - toxins act on host membrane and mediates destruction o Leucocidin (PVL) - mediates destruction of phgocytes o Clumping factor, coagulase and hyalaronidase enhance invasion and survival in tissue o exofoliatins, toxic shock syndrome toxin (TSST-1), enterotoxins - potent exotoxins CHARACTERISTICS OF Staphylococcus aureus • • • • Gram positive cocci usally in clusters S. aureus will grow on 5% sheep blood and chocolate agar but not on macConkey agar. On selective media Mannitol Salt Agar (MSA) is commonly used. On 5% sheep blood agar or chocolate agar the colonies apparance are medium to large; smooth entire, slight raised, translucent; most colonies pigmentedcreamy yellow; most colonies beta hemolytic; 24 hrs of incubation On MSA S. aurues can grow in the presence of salt and ferment manntol produce colonies surrounded by yellow halo; for at least 48 to 72 hours before growth is detected TESTS FOR IDENTIFICATION FOR S. aureus Test Result Catalase Positive Coagulase (slide and tube method) Positive Voges-Proskauer Test Positive PYT Negative Vancomycin Resistant Penicillin and Ampicillin Resistant • Methicillin - Resistance S. aureus (MRSA) resistance to all β-lactams Staphylococcus epidermidis • • • Microscopic Characteristics: o Small to medium; translucent, gray-white colonies o Non-hemolytic o Slime-producing and adhere to the agar surface Clinical Significance: o Normal skin flora and mucous membranes o Causes UTI o Stitch abscess o Prosthetic heart valve infection Tests for identification: o Tube coagulase (-) o 2hr PYR broth hydrolysis (-) o Novobiocin Susceptibility (sensitive) o Alkaline Phosphatase (+) o Polymyxin B (resistant) o Voges-Proskauer (+) Staphylococcus haemolyticus • • • Normal flora of human skin present in fewer numbers Spread of patient’s endogenous strain to normally sterile site, usually as a result of implantation of medical devices. Medium, smooth, butyrous and opaque TESTS FOR IDENTIFICATION Organism NORMAL SKIN FLORA Staphylococcus saprophyticus • • • • • • In humans, S. saprophyticus is found in the normal flora of the female genital tract and perineum. Staphylococcus saprophyticus is a Gram-positive coccus responsible for uncomplicated lower urinary tract infections (UTIs), mainly in young women S. saprophyticus is second only to E. coli as the most frequent causative organism of UTI in women. It was found to contaminate 16·4% of the various food samples with a high prevalence of 34 % in raw beef and pork. It was common in both domestic and imported raw meat products. Introduction of endogenous flora into sterile urinary tract, notably in young, sexually active females. A community acquire infection, not considered agent of nosocomial infections. Gram (+) cocci in clusters PYR Haemolytic Vogues- Mannitol Pattern Proskauer Staphylococcus Slide (+) Narrow (+) lugdunensis coagulase (+) beta hemolytic Staphylococcus Slide (+) (-) intermedium coagulase (+) Staphylococcus Tube Beta haemolyticus coagulase (+) hemolytic • • Culture in Blood Agar Plate (BAP) o Non-pigmented, non-hemolytic colonies Mechanism o Bacterial resistance to methicillin and related penicillin and cephalosporin is mediated by acquisition of gene (mecA) that codes for a novel penicillin-binding protein, PBP2a that has a low affinity for methicillin and related penicillin and cephalosporin. • • • • • • • Normal flora of human skin present in fewer numbers Spread of patient’s endogenous strain to normally sterile site, usually as a result of implantation of medical devices. Medium to large, smooth, glossy, entire edge w/ slightly domed center Unpigmented or cream to yellow-orange S. schleiferi is a coagulase-negative organism which has been described as a pathogen responsible for various nosocomial infections including bacteremia, brain abscess, and infection of intravenous pacemakers. This bacteria have been described to be found typically on skin and mucosal surfaces. Microscopically, it appears medium to large; smooth, glossy, slightly convex with entire edges on 5% Sheep Blood agar. It is susceptible to Novobiocin and produces a heat-stable nuclease. It can be differentiated from S. aureus by production of different nuclease with lack of pigmentation Gram (+) cocci in clusters cocci growth on BAP Biochemical test: Test Urease Coagulase Vogues-Proskauer Results (-) (+) (+) Micrococcus spp. • • • • Staphylococcus lugdunensis • • Staphylococcus lugdunensis - can cause endocarditis & bacteriemia Staphylococcus schleiferi • • Coagulase • Normal flora of human skin, mucosa, and oropharynx Mode of transmission is uncertain Usually considered contaminants of clinical specimens; rarely implicated as cause of infections in humans Test for identification: o Catalase (+) o Modified oxidase (+) o Strict aerobe except Kocuria (Micrococcus) kristinae which is facultatively aerobic o Bacitracin sensitive o Furazolidone resistant o Lysostaphin resistant Small to medium; opaque, convex; nonhemolytic; wide variety of pigments (white, tan, yellow,orange,pink) o CATALASE (-) GRAM POSITIVE COCCI STREPTOCOCCI AND ENTEROCOCCI Streptococcus SPP. 1. 2. 3. 4. 5. Beta-Hemolytic Streptococci o S. pyogenes (Group A) o S. agalactiae (Group B) Group C, F and G beta-hemolytic streptococci Streptococcus pneumoniae Viridans/ Alpha-hemolytic Streptococci o S. mutans, S. salivarius, S. mitis, S. bovis, o S.urinalis, S.anginosus/ S. milleri Nutritionally Varient Streptococci • STREPTOCOCCI • • • • • Gram positive cocci occurring in pairs or in chains Non-motile Non-spore forming Facultative anaerobe Catalase negative Therapeutic option: ▪ Penicilin is drug of Choice. o Streptococcus agalactiae (Group B beta-hemolytic streptococci) Streptococcus agalactiae o Group B beta-hemolytic streptococci o Infections associated with neonates and are acquired during the birthing process. o Translucent to opaque; flat, glossy; narrow zone of o beta hemolysis; some strains non-hemolytic o Capsular material interferes with phagocytic activity and complement cascade activation o CAMP test positive – S. agalactiae able to hydrolyze hippurate. Positive test shows appearance of an arrowhead shape at the juncture of the S. agalactiae and S. aureus streaks o Serologic agglutination - identifying non-betahemolytic S. agalactiae BETA-HEMOLYTIC STREPTOCOCCI • Streptococcus pyogenes o Group A Beta-Hemolytic Streptococci o “Flesh- eating bacteria” o Mode of Transmission ▪ Person to person or Direct contact w/ mucosa/ secretion ▪ Inhalation of Contaminated droplets from Sneeze and cough o Habitat (reservoir) ▪ Clinically Significant when present in specimen ▪ Inhabits Skin and Upper Respiratory tract ▪ Not a normal flora but may be lugged on nasal, pharyngeal and Mucosa o Virulence Factor: ▪ Streptolysin O ▪ Streptolysin ▪ Cross-reaction of Ab against Streptococcal Antigen. o Can cause : ▪ Scarlet fever ▪ Streptococcal toxic shock syndrome ▪ Acute pharyngitis o Appearance on 5% Sheep Blood Agar ▪ Grayish white, transparent to trancelucent, matte or glossy ▪ With large zone of Beta hemolysis o Detection of S. pyogenes antigen (throat specimen) ▪ Latex agglutination/ enzyme linked immune assay (ELISA) ▪ Very specific/ sensitivity approximately 60% ▪ May result false positive if specimen lack of S.pyogenes. o Gram Stain: ▪ Gram positive cocci , round or oval-shaped. ▪ Hydrolysis of Hippurate (-) ▪ PYR (+) ▪ Voges Proskauer (-) ▪ CAMP TEST (-) • Streptococcus pneumoniae o Group C, F and G beta-hemolytic streptococci o Primary cause of bacterial pneumonia, meningitis, and otitis Media o Colonizes nasopharynx of humans o Lancet-shaped & occurs singly, in pairs or in short chains o Small, gray, glistening, colonies, maybe mucoid if has a polysaccharide capsule, alpha-hemolytic o Antiphagocytic properties of the polysaccharide capsule is the key to the organism’s virulence o enhancing the growth= blood agar plate are incubated in a candle jar or CO2 chamber (5%-10% CO2) for 24hours, and it will produced small round glistening dome-shaped colonies that are transparent with an entire edge. o Bile solubility test positive – ability of bile salts to lyse S. pneumoniae o Optochin susceptible – producing zone of inhibition • Viridans/ Alpha-hemolytic Streptococci o Opportunistic pathogens of low virulence o Slowly evolving endocarditis, bacteremia and infections in immunocompromised and meningitis in patients suffering trauma o Minute to small, gray, domed, smooth or matte; alpha hemolytic or non hemolytic Moraxella Catarrhalis • • Enterococcus SPP. 1. 2. • Enterococcus fecalis Enterococcus faecium Other Enterococcus spp. Isolated from humans o E. durans o E. mundtii o E. dispar o E. gallinarum o E. avium o E. hirae o E. raffinosus o E. casseliflavus • o Enterococci • • • • • • • • Also called the Group D Streptoccocus (according to the Lancefield classification) Colonizes gastrointestinal system of humans and animals Gram (+), non-spore forming, facultative anaerobes Catalase negative Can tolerate conditions of: High sodium chloride High pH High temperature (60 degrees – up to 30 mins) Enterococcus • • • • 2 species that causes infections in humans: o Enterococcus faecalis o Enterococcus faecium Opportunistic pathogens (mostly causes HAIs) BAP: small, cream or white, smooth, Alpha/Beta/Non-hemolytic Clinical laboratories identify Enterococcus spp. By demonstrating that the isolate is PYR (+) and LAP (+) and that it grows at 45° C and in 6.5% NaCl. Moraxella Catarrhalis colonies on Chocolate Agar: o Large, gray, opaque o Nonpigmented o Smooth; friable “hockey puck” consistency o Colony may be moved intact over surface of agar. Moraxella catarrhalis colonies on Blood Agar, are nonhemolytic. Members of the genus Neisseria and Moraxella catarrhalis appear as gram-negative diplococci with adjacent sides flattened. Moraxella catarrhalis on chocolate agar Neisseria spp. • • • • Neisseria gonorrhea colonies on Chocolate Agar small, grayish white, convex, translucent, shiny colonies with either smooth or irregular margins; may be up to five different colony types on primary plates. Neisseria meningitidis colonies on Chocolate Agar Medium, smooth, round, moist, gray to white; encapsulated strains are mucoid; may be greenish cast in agar underneath colonies Neiserria spp. resembles tiny coffee or kidney beans in the Gram Stain. New York City Agar Neisseria meningitidis Neisseria gonorrhea TESTS FOR IDENTIFICATION Neisseria and Moraxella catarrhalis MICROSCOPIC CHARACTERISTICS • • • • These organisms are either coccobacilli or short to medium sized, gram negative rods. Sub inhibitory concentrations of penicillin cause the coccoid forms of these bacteria to elongate to abacillus morphology. True cocci, such as most Neisseria spp. and Moraxella (Branhamella) catarrhalis, maintain their original coccus shape in the presence of penicillin. Members of genus Neisseria and Moraxella catarrhalis appear as gram-negative diplococci w/ adjacent sides flattened. • • • • Elongation in the presence of penicillin done by streaking a blood agar plate and adding a 10-unit penicillin disk to the first quadrant before overnight incubation at 35° C. A Gram stain of the growth taken from around the edge of the zone of inhibition readily demonstrates whether the isolate in question is a true coccus or has elongated. Determination of carbohydrate utilization patterns historically has been performed in cysteine trypticase soy agar (CTA) w/ 1% dextrose, maltose, lactose, and sucrose Chromogenic substrate enzyme tests for betagalactosidase, gamma-glutamyl aminopeptidase, and prolyl-hydroxylprolyl aminopeptidase. M. catarrhalis lacks all three of these enzymes. Neisseria spp. • • • • • • • The pathogenic Neisseria spp. are very sensitive to drying and temperature extremes. The presence of prolyl-hydroxylprolylaminopeptidase alone identifies an organism as N. gonorrhoeae. The direct Gram stain of body fluids for either N. gonorrhoeae or N. meningitidis is best accomplished using a cytocentrifuge. Direct examination of endocervical secretions in symptomatic women is still only presumptive evidence of gonorrhea and the diagnosis must be confirmed by culture. Swabs are acceptable for N. gonorrhoeae if the specimen will be plated within 6 hours. Particle agglutination methods are available for immunoserologic identification of N. gonorrhoeae. The ONPG test. N. lactamica is ONPG positive, and N. gonorrhoeae is ONPG negative. Moraxella catarrhalis • • • • M. catarrhalis can be differentiated from the gonococci and meningococci based on its growth on blood agar at 22°C and on nutrient agar at 35°C The reduction of nitrate to nitrite. Inability to utilize carbohydrates. DNase production. M. catarrhalis is the only member of this group of organisms that hydrolyzes DNA. BIOLOGICAL AND PHYSIOLOGIC CHARACTERISTICS OF Moraxella catarrhalis and Coccoid Neisseria spp. BIOCHEMICAL TESTS CLINICAL DIAGNOSIS • • • Neiserria gonorrhea o Leading cause of sexually Transmitted diseases. Epididymitis in males and acute cervicitis in females. o Other localized infections include pharyngitis, anorectal infections, opthalmia of newborns from infected mothers and conjunctivitis o Pelvic inflammatory disease. Gonorrhea from N. gonorrhea Moraxella catarrhalis o Most infections are localized to sites associated with the respiratory tract and include otitis media, sinusitis, and pneumonia. o Lower respiratory tract infections target elderly people and those with chronic obstructive pulmonary disease. Otitis media Acquired pneumonia from M. catarrhalis Neiserria meningitidis o Life-threatening, acute, purulent meningitis. o Development of Petechiae may be associated with meningococcal bacteria. o Disseminated intravascular coagulation which can be fatal and shock. o Common infections include conjunctivitis, pneumonia, and sinusitis. Meningococcemia by N. meningitidis •