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Bacteriology Lec Midterms A., Gabrielle, A. Romero Golden yellow pigment STAPHYLOCOCCUS S. citreus — lemon yellow pigment...

Bacteriology Lec Midterms A., Gabrielle, A. Romero Golden yellow pigment STAPHYLOCOCCUS S. citreus — lemon yellow pigment S. albus — porcelain white pigment Spherical, non motile grape like cluster Oil paint appearance Non encapsulated, non spore former MSA: golden yellow pigments Aerobic, facultative anaerobe, some can be obligate anaerobes K tellurite medium: jet black colonies Strongly catalase positive (+) BAP: beta hemolytic Can ferment glucose except S. saprophyticus Coagulase Normal inhabitants of the skin and mucous membranes of humans and other animals. Coagulase test - can differentiate Staphylococcal sp. Enzyme produced clots plasma (staphylocoagulase) General Characteristics (+) = Clot formed (-) = no clot formed Gram positive cocci (purple) Coagulase positive staphylococci ○ Catalase positive (+) ○ S. aureus — human pathogen ○ Singly, in pairs, and in clusters ○ S. delpihini, S. intermedius, S. hycius, S. shleiferi ○ From Greek term ‘staphle’ - “Bunches of grapes” — animal pathogens Morphology (colonial characteristics) Coagulase negative staphylococci ○ Appear creamy, white or rarely light gold, Buttery ○ S. epidermidis — hospital acquired infection looking (if incubated for 18-24hr) ○ S. saprophyticus — UTIs in young sexually active ○ Some species produce β-hemolysis (complete females hemolysis in BAP) Important in urine sample S. aureus’ ○ S. haemolyticus — wounds, septicemia, UTI, native Characteristics valve infection ○ Non motileSy ○ Non spore forming Virulence factors of S. aureus ○ Non encapsulated Enterotoxins ○ Aerobic or facultative anaerobe ○ Heat stable (30-100C) exotoxins that cause One exception S. saccharolyticus (obligate diarrhea and vomiting anaerobe) Toxins A - E and G - I (eight total) Micrococcus ○ Resistant to gastric acid and associated with food poisoning (a, b, d) Micrococcus luteus ○ B, C and sometimes G and I are associated with ○ Gram positive toxic shock ○ Catalase positive (+) TSST - 1 (toxin F) Modified catalase containing a mild ○ Superantigen, have the ability to interact w/ T detergent cells ○ Coagulase negative (-) (coagulate plasma) ○ Majority of cases of menstruating-associated TSS Morphology ○ Associated with tampon use ○ Distinct yellow pigment colony ○ Produced by phage group 1 Generally considered a nonpathogen ○ Cause toxic shock syndrome ○ Toxic shock syndrome toxin - 1 Clinically significant staphylococci TSST - 1 and B, C, G and I are superantigens Staphylococcus aureus Exfoliative toxin (epidermolytic toxin) ○ Habitat: anterior nares (carries) (20% - 30% of humans) ○ Causes sloughing off of the skin and is known to cause scalded skin syndrome or Ritter’s disease ○ Primary pathogen of the genus Also associated with bullous impetigo Produce superficial to systemic infections γ-Hemolysin - Leukocidins (panton - valentine ○ Skin leukocidin) ○ Bacterial sepsis ○ Kills polymorphonuclear leukocytes 3 clinically important species ○ Helps prevent phagocytosis S. aureus Cytolytic toxins of S. aureus S. epidermidis Hemolysins S. saprophyticus ○ Three main type α, β, δ (there is a γ but generally not important) Staphylococcus aureus α hemolysin - destroys platelets, Mode of transmission: traumatic introduction macrophage and tissues ○ Needle stick β hemolysin - shows enhanced activity by ○ Destruction of skin layers (burns, road rash) acting on the sphingomyelin of RBC ○ Medical procedures (iatrogenic) membranes causing lysis. Hot - cold lysin since it works best at 37 and very well Predisposing conditions when stored at 4 degrees celsius ○ Chronic infections δ hemolysin - causes injury to cells and ○ Indwelling devices leukocytes but is less lethal ○ Skin injuries Enzymes (coagulase, protease, hyaluronidase, lipase) ○ Immune response defects ○ Coagulase 1 Bacteriology Lec Midterms A., Gabrielle, A. Romero Very diagnostic but importance in virulence is not completely understood Folliculitis ○ Hyaluronidase Hydrolyzes the hyaluronic acid present in connective tissues helping spread of infection ○ Lipase Breakdown of the fats and oil created by the sebaceous glands on skin surface Protease, lipase, and hyaluronidase are capable of destroying tissue and may facilitate the spread of infection to Furuncle adjoining tissues. Protein A ○ Bind the Fc portion of antibodies to avoid phagocytosis Masking of its immunogenic protein with host proteins to look like “self” Assists in blocking phagocytosis Types of hemolysis in blood agar Alpha - grayish brown Beta - colorless Gamma - no hemolysis Staph and Streptococcus species Epidemiology of S. aureus Primary reservoir ○ Nares Scalded skin syndrome Other reservoir Extensive exfoliative dermatitis ○ Axillae, vagina, pharynx and other skin surfaces ○ Staphylococcal (SSSS) Hospital outbreaks More likely to occur in renal failure patients ○ Nurseries Immunocompromised ○ Burn units Severity ranges from mild to severe ○ Surgical patients ○ Localized lesion to large generalized area with Infections/Disease of S. aureus profuse peeling of the epidermal layer Lasts about 2 - 4 days Most clinically significant species ○ Spontaneous recovery in children various cutaneous infections, purulent abscesses ○ Adult cases can lead to mortality Impetigo or cellulitis Skin and wound infections Toxic epidermal necrolysis ○ Pus formers Multiple causes ○ Furuncle (boil) ○ Drug induced A painful inflammation of the skin and ○ Infections subcutaneous tissue. Extension of folliculitis, large, raised, deep abscesses. ○ Vaccines ○ Carbuncles Cause officially not known Boils that have invasive lesions develop Similar to SSSS from multiple furuncles and may progress ○ Treatment with steroids helpful into deeper tissues Unlike SSSS ○ Folliculitis High mortality rate Infection of the hair follicle or oil gland ○ Bullous impetigo Toxic shock syndrome also known as impetigo contagiosa Association with super absorbent tampons Large pustules surrounded by small zone Clinical presentation of erythema (redness) ○ High fever Sometimes occur due to blocked follicles, sebaceous ○ Rash of trunk spreading to extremities glands and sweat glands. ○ Watery diarrhea S. aureus is a common cause of infective endocarditis ○ Vomiting and toxin-induced diseases, such as food poisoning, and is associated with scalded skin syndrome (SSS) Dehydration and toxic shock syndrome (TSS). Leads to hypotension DIC - disseminated intravascular coagulation Increase in BUN and creatinine 2 Bacteriology Lec Midterms A., Gabrielle, A. Romero Fatal in 2% - 5% of cases due to multiorgan system failure Glycocalyx - skin flora Staphylococcus epidermidis, a normal skin flora organism, is often associated with slime layers on venous catheters This bacterium moves in from the skin surface (where it does not produce slime) to the catheter where it turns on slime production to firmly attach to the catheter Once the slime is produced, it is impossible to get rid of it and the catheter usually has to be removed Food poisoning Staphylococcus saprophyticus Toxin not bacterial growth causes disease UTIs in young sexually active women Enterotoxin A - D (A and D most common) ○ Due in part to increased adherence to epithelial ○ From enterotoxin producing strains contaminating cells lining the urogenital tract the rich foods (mayonnaise) Rarely present in other skin areas or mucous ○ Inadequate refrigeration membranes ○ Include rich foods potato salad or mayonnaise Urine cultures Symptoms ○ If present in low amounts, it is still considered ○ Appear rapidly about 2 - 8 hours after ingesting significant food Second most common cause of UTI ○ Usually resolve in 24 - 48 hours sometimes less Coagulase negative (-), novobiocin resistant ○ nausea and vomiting, abdominal pain and cramping Other coagulase negative staphylococci S. haemolyticus Staphylococcal food poisoning ○ Second most common coagulase negative (-) staph Enterotoxin ○ Can be found in wounds, UTIs, bacteremia, Heat stable endocarditis symptoms include: Recently noted resistance to vancomycin ○ Nausea, vomiting, abdominal cramps Opportunistic pathogens Caused by unhygienic preparations of food ○ S. lugdunensis - both community-associated and hospital acquired infections, more virulent than Other infections other is known to contain the gene mecA, which Secondary pneumonia encodes oxacillin resistance ○ After influenza A infection but relatively rare ○ S. schleiferi But it has high mortality rate ○ S. pseudintermedius - infection in dogs and cats. Vet staff and pet owners at risk. Bacteremia and endocarditis ○ IV - drug addicts presenting with fever LABORATORY DIAGNOSIS OF STAPHYLOCOCCUS ○ Enter through injection sites Osteomyelitis Microscopic examination ○ Occurs secondary to bacteremia and results in bacteria invading the bone Numerous gram - positive cocci ○ Fever, chills, swelling and pain around the injection PMNs area Purulent exudates joint fluids, aspirated secretions Arthritis if bacteria in the joint Aspirate is best sample Staphylococcus epidermidis Isolation of staphylococci Predominantly nosocomial infections Grow easily on blood agar plates and thioglycolate ○ Skin flora gets introduced in catheters, heart If heavily contaminated, they can be selected with the valves, CSF shunts following: ○ Produces a slime layer (biofilm) that helps ○ Mannitol salt agar adherence to prosthetics and avoidance of ○ Columbia colistin - nalidixic acid agar (CNA) phagocytosis ○ Phenylethyl alcohol agar ○ UTIs Selective media Resident flora of the skin Coagulase negative (-) novobiocin sensitive Mannitol salt agar (7.5%) NaCl Slime factor Halophiles - salt loving bacteria Stitch abscess, mild UTI, endocarditis, bacteremia, Ability of S. aureus to ferment mannitol (non halophilic meningitis organism) and tolerate 10% NaCl a common source of hospital-acquired infections and ○ Ferment mannitol often a contaminant in improperly collected blood culture specimens. MSA - acidic → phenol red → yellow indwelling catheters and prosthetic devices, are often ○ Non fermentation of mannitol - non acidic → caused by isolates shown to produce a biofilm - key phenol red remains red / pink component in bacterial pathogenesis and is a complex Positive result golden yellow colonies interaction between host, indwelling device, and pH indicator: phenol red bacteria poly(γ-DL-glutamic acid) - protective advantage against host defenses 3 Bacteriology Lec Midterms A., Gabrielle, A. Romero Positive results: coagulation of plasma Uses citrated plasma Tube method - demonstrate free coagulase Slide method - demonstrate bound coagulase Only S. aureus will be positive Read after 4 hrs of incubation at 37C Reading within 4 hrs: prevent false negative result Reading within 20 hrs: prevent false positive result Group of coagulase positive staphylococci Identification test: catalase Principle: tests for enzyme catalase ○ 2H2O2→ 2H2O + O2 Drop H2O2 onto smear Bubbling = POS (most bacteria, O2 generated) No bubbling = NEG (streptococci and other lactic acid bacteria, no O2 generated) Catalase test Uses of hydrogen peroxide Positive for S. aureus; (-) streptococcus species Coagulase - negative staphylococci Positive result rapid effervescence of gas Growth and metabolism - catalase Staphylococci contain enzyme catalase which converts H2O2 into water and oxygen The test is performed by emulsifying a colony of staphylococcus in hydrogen peroxide and observing a bubbling reaction as oxygen is liberated This test is simple to perform and quickly differentiates the staphylococci from the streptococci, the other major group of Gram positive cocci Coagulase Cell bound coagulase (clumping factor) ○ Clots human, rabbit, or pig plasma Slide test method ○ Mix suspension of organism with a small amount of rabbit plasma ○ Check for clumping (if clumping then positive) If clumping is negative, a tube test should be performed ○ Why? 5% don’t produce cell bound coagulase Extracellular free coagulase ○ Extracellular enzyme secreted that clots plasma Separating micrococcus ○ Tube test Bacitracin disk test Check for coagulation 4 hours after ○ Micrococcus inoculation and 24 hours after Susceptible (micrococcus luteus) Detects staphylocoagulase, or free Lemon yellow color of colony doesn’t hurt coagulase. Staphylocoagulase is an extracellular molecule that causes a clot to Do not produce acid under anaerobic conditions in form when bacterial cells are incubated glucose O/F media with plasma ○ Coagulase negative staphylococci Prevent autolysis and false negative result Resistant (S. saprophyticus, S. Hallmark test for staphylococcus aureus epidermidis, or others) Other staph can produce positive coagulation Differentiating coagulase negative staphylococci ○ Usually do not exhibit the same colony morphology If it’s a urine sample ○ S. saprophyticus Coagulase test Presumptive ID can be done using novobiocin Best single criterion for pathogenicity 4 Bacteriology Lec Midterms A., Gabrielle, A. Romero ○ Streak organism onto a blood plate and add novobiocin disk to heavy growth quadrant Methicillin - resistant staphylococci If resistant MRSA - isolates resistant to nafcillin/oxacillin ○ Zone size below or at resistant level Methicillin - resistant S. epidermidis ○ S. saprophyticus ○ MRSE If susceptible Infection control ○ Likely S. epidermidis but can be others, would use ○ Barrier protection tests in table14 - 5z ○ Contact isolation Novobiocin susceptibility test ○ Handwashing Treat with vancomycin ○ Test for susceptibility with cefoxitin disk mecA gene ○ Encodes penicillin binding proteins Gold standard ○ mecA gene detected by PCR Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) - Common in athletes, correctional facility inmates, military recruits in close contact environments, pediatric patients, and novobiocin susceptibility using a 5-µg novobiocin disk. tattoo recipients. S. saprophyticus is resistant to novobiocin, whereas most other CoNS are susceptible. Vancomycin - resistant staphylococci Rapid identification Vancomycin - drug of choice, sometimes only drug BBL staphyloslide available for serious staphylococcal infections Seradyn color slide VISA (Vancomysin Intermediate SA) - first recovered in Staphaurex Japan Bacti staph VRSA (Vancomycin-resistant SA) Plasma coated carrier particles detect both clumping ○ Isolated in US 2002 - from patients with factor and protein A underlying conditions Resistant and intermediate forms Staphylococcal schematic Macrolide resistance Resistance to clindamycin may not be obvious ○ Erythromycin and clindamycin should have same resistance patterns D test ○ Use erythromycin and clindamycin disks Growth between disks but not on side of clindamycin Inducible resistance - grow if bacteria also exposed to erythromycin. Flowchart for the identification of gram positive bacteria Key testing for ID of clinically significant staphylococci Antimicrobial susceptibility Non beta - lactamase producing staph ○ Use penicillin ○ However up to 85% - 90% of S. aureus are resistant Thus do beta - lactamase test Always perform susceptibility testing ○ Especially in serious infections 5 Bacteriology Lec Midterms A., Gabrielle, A. Romero Gamma hemolytic - do not hemolyze - no hemolytic STREPTOCOCCUS AND ENTEROCOCCUS SPP. reaction surrounding the colonies Family: Streptococcaceae Based on the antigenic nature of cell wall carbohydrates: Gram positive cocci in chain or pairs C polysaccharide Non-motile, non-sporeformer, non-encapsulated Rebecca Lancefield: Found out that the C carbohydrate Facultatively Anaerobic, may be considered Aerotolerant can be extracted from the streptococcal cell wall by Anaerobes placing the organisms in dilute acid and heating for 10 Peptostreptococcus: minutes. Obligate Anaerobe Requires Carbon Dioxide - Stimulates growth Growth enhanced by blood, serum, glucose incorporated into the agar plate, colonies are small and transparent BAP (Blood Agar Plate): Pinpoint Hemolysis - Require enriched media with blood or serum for growth Have a group or common C carbohydrate (polysaccharide) - used to serologically classify an isolate Microscopy: Gram-positive spherical cells, some exhibit β-hemolytic streptococci or pyogenic (pus-forming) lancet-shaped cells, arranged in chains or pairs streptococci and species that are non–β-hemolytic Culture (BAP): Grayish, pinpoint, translucent to slightly (nonpyogenic) opaque; Some species are mucoid Pyogenic streptococci isolated frequently from humans Biochemical Tests: (-) Catalase, Oxidase, and Gas production; Non-motile, Ferment Carbohydrates include Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. equisimilis, and Streptococcus anginosus group Classification of streptococci Academic or Bergey’s Classification Based on temperature requirements Enterecoccus - former Group D, has D antigen present on the cell wall. Transfer in other family “enterococcae”. Can also see Alpha and Beta. SUMMARY OF CLASSIFICATION Group A streptococci (GAS) Microscopy: Cocci in long chains Facultative Anaerobe, grows best in the presence of 10% CO2 Grows on enriched media only Hemolytic Pattern: Beta Resistant to drying, can be recovered from swabs after several hours of collection Antigenic Structure: M Protein is attached to Peptidoglycan cell wall ❖ Essential for virulence: Evasion of Phagocytosis, Anti-Phagocytose ❖ 80 different types: E.g., M5, M10 ❖ Resistance to infection is related to M Protein Antibody Production Streptococcus pyogenes ”Fever-producing” and “Flesh-eating” bacterium, affecting deeper tissues and organs Primary reservoir: Humans (throat and skin) Principal Virulence Factor: M Protein (Attached to the Peptidoglycan, Antiphagocytic, for Alpha hemolysis- Partial hemolysis - Incomplete hemolysis adherence to mucosal cells) - - Greenish or brownish zone colonies encoded by gene emm, has 200 diff. Beta hemolysis - Full - Complete hemolysis - Bacteria can Serotypes and subtypes. produce “hemolysin”- can produce toxin 6 Bacteriology Lec Midterms A., Gabrielle, A. Romero - M1 serotype- most common in pharyngitis Additional virulence factor -. Lipoteichoic acid and protein F - adhesion molecules, affixed to proteins on the bacterial surface. - Fibronectin-binding protein - , secures the attachment of streptococci to the oral mucosal cells - Hyaluronic acid capsules- Weakly immunogenic. Prevents phagocytosis by neutrophils or macrophages, mask it antigens and remained unrecognized by host’s immune system. Culture (BAP): Grayish-white, small, translucent, smooth with a well-defined B-hemolysis EPIDEMIOLOGY Not considered part of the normal flora Source: Human upper respiratory tract (throat, pharynx, nose) Most common site of disease: Pharynx (Strep Throat) Transmission: Direct contact, Fomites CULTURE: ○ Small, translucent, smooth; well-defined β-hemolysis CLINICAL SYNDROMES Necrotizing Fasciitis (NF/Galloping Gangrene/Flesh Eating Bacteria Syndrome) Rare, life-threatening infection of the skin and tissues under the skin that supports the muscles, blood vessels, and nerves. Imputation required. Acquired even in the absence of skin trauma/break in the skin Types: Type 1 NF (Aerobic and Anaerobic bacteria) - saltwater with Vibrio spp. Streptolysin O - Unstable in the presence of oxygen. We Type 2 NF (Streptococcus pyogenes) can use it as a means of determining the presence of Type 3 NF (Clostridium perfringens) Strepyogenis, by serologic testing “Anti streptolysin O” - Early Symptoms: Fever with erythematous, painful, more than 1:4 positive with streptococcus pyegenes. swollen area of the skin Causes hemolysis when it is anaerobic. Highly Late Symptoms: Black spots on the skin or ulcers, pus immunogenic. dripping from the infected area, and diarrhea Streptolysin S - non-antigenic. In the surface hemolysis. Aerboic incubation DAMAGING SEQUELAE OF STREPTOCOCCI STREPTOCOCCAL PYOGENIC EXOTOXINS Rheumatic fever- damage in the heart, after strep throat Acute glomerulonephritis - damage in the kidney, release TYPE B TOXIN - Dermal (skin) of albumin, lessen albumin in blood - fluids leak out the Effect of these toxins tissue - forming edema - Fever: primary effect (not the rashes) - Rashes: dermal activity à ̀ hypersensitivity reaction - Heat-labile, produced by lysogenic strains Specimen Collection and Recovery for Streptococcus pyogenes GROUP A streptococci Specimen Collection Always swab 2 sample, 1 for gram staining and for culture Swab brushed over the posterior pharynx and each tonsillar area - avoid other areas (teeth, tongue, hard palate, soft palate, lips) If exudate is present, it should also be touched with the swab Avoid contamination by tongue and uvula 7 Bacteriology Lec Midterms A., Gabrielle, A. Romero Recovery: Acid-stable polysaccharide in cell wall Sheep Blood Agar (SBA) plate is inoculated and Also known as Group B streptococci (GBS) streaked for isolation Nine recognized capsular polysaccharide serotypes SBA containing Sulfamethoxazole (SMZ) Three major serotypes: Ia, Ib, II Improves recovery of β-hemolytic streptococci from Contain a terminal residue of Sialic Acid throat cultures Weakly immunogenic, can inhibit activation of the Inhibit the normal flora, which can isolate more alternative complement pathway pyogenes, resistant to SXT Virulence Factors Observe after 24 hours for the presence of β-hemolytic Capsule: Prevents Phagocytosis but is ineffective after colonies Opsonization If none: Incubate for an additional 24 hours, check Sialic Acid: The most significant every 24 hrs component of the capsule and critical virulence determinant LABORATORY TESTS FOR GROUP A STREPTOCOCCI With mutant strains, loss of capsular sialic acid was associated with loss of virulence Other products produced by GBS Hemolysin, CAMP factor, DNases, Hyaluronidase, Protease, Neuraminidase No evidence that these products play a role in virulence Diseases Nosocomial transmission: Unwashed hands of the mother or healthcare worker Infection of the fetus during passage (Neonatal Sepsis, Neonatal Meningitis, Post- Puerperal fever) Most common cause of Neonatal Meningitis Significant cause of invasive disease in newborns, leading cause of death in infants. Colonization of vagina and rectal area w/ GBS - found in 10-30% of pregnant women. Adult Infections: Group A: Resistant to SXT, Susceptible to Bacitracin Mother after childbirth or abortion Group B: Resistant to SXT and Bacitracin Endometritis or wound infections Group A & B: Susceptible to Penicillin Endocarditis Older Adults Immunodeficiency Skin and Soft tissue infections, Intraabdominal abscess Bacteremia, Pneumonia endocarditis, UTI Early Onset Infection Less than 7 days old Presence of GBS in the vagina of the mother 80% of cases: Vertical Transmission (from mother) LABORATORY DIAGNOSIS FOR GROUP A STREPTOCOCCI Premature Birth, Membrane rupture, develops into Positive culture: β-Hemolytic, Pinpoint colonies Pneumonia or Meningitis with bacteremia Microscopy and Staining: Gram Positive Cocci in chains Result: Very high mortality if not treated quickly (Examination of pus/ throat swabs) Late Onset Infection Rapid Detection Tests (RADTs) for GAS Pharyngitis 7 days old and up Enzyme Immunoassays (EIAs) Usually, 1 week to 3 months Optical Immunoassays (OIAs) Primarily Meningitis Rapid PCR Serotype III GBS clone ST-17 - responsible for the Serological Testing majority of the early- and late-onset infections worldwide Positive Streptococcal Antigen Test Elevated or Rising Streptococcal Antibody Titer (ASO) Treatment Elevated Acute Phase Reactants (ESR, CRP) Penicillin and Ampicillin (Most strains sensitive > Drug of choice) Group A streptococci Treatment Combination of Penicillin/Ampicillin with Treatment: Penicillin (Drug of choice), Erythromycin Aminoglycosides (Life-threatening cases, Pregnant Prophylaxis: Can prevent RF but not GN carriers) Intrapartum prophylaxis and selective administration of antibiotics to infants (90% reduction of sepsis) Streptococcus agalactiae (Group B streptococci) Ceftriaxone or Cefotaxime Normal flora of the female genital tract, lower GIT Vancomycin Pregnancy: Screened for Group B strep at 35-37 weeks of gestation Specimen Collection and Handling Virulence factor: In Pregnant women: Capsule, Sialic acid Collect vaginal and rectal material (swab) between Avirulent factors: 35-37 weeks gestation Hemolysin, CAMP Factor, Screen pregnant women with asymptomatic Neuraminidase bacteriuria Culture: Grayish white, Inoculate to select broth, such as Todd Hewitt broth with mucoid colonies, small Antimicrobials (Lim broth), TransVag broth with 8 ug/mL zone of β-Hemolysis after Gentamicin and 15 ug/mL Nalidixic Acid, or Strep B Carrot 24-48 hours of incubation Broth at 35 ̊C Incubate for 18-24 hours at 35C Granada Agar: Yellow Lim Broth: Improved Todd-Hewitt Broth to Orange colonies Possess the Group B specific antigen 8 Bacteriology Lec Midterms A., Gabrielle, A. Romero Contains all components of THB and with added antimicrobials (10 ug/mL of Colistin and 15 ug/mL of Nalidixic Acid) Carrot Broth: Chromogenic medium in which the presence of Group B streptococcus gives the clear medium an orange or red color after 6-24 hours of incubation Subculture to SBA LABORATORY DIAGNOSIS FOR GROUP B STREPTOCOCCI Viridans streptococci Viridans: Greening, referring to the α hemolysis exhibited (Alpha prime) Lack of Lancefield group antigens (Lack of M Protein) α-hemolytic or may be non-hemolytic Alpha Prime: Small inner zone of intact RBCs and a wider outer zone of Beta hemolysis Normal Flora: Oral cavity, Nasopharynx (Oropharyngeal commensals), GIT, Female genital tract Considered as opportunistic pathogens of low virulence (Immunocompromised) (+) LAP Test Oropharyngeal commensal Prophylactic Antimicrobial therapy for women whose May gain entry through dental procedures samples will test positive results on: Fastidious Organisms: Some requiring CO2 for growth Vaginal and Rectal Screenings Urine culture Women with previous history An unknown status of GBS at the time of delivery GROUP C, G, AND F STREPTOCOCCI Recovered from URT, Vagina, and Skin of humans Possess M Protein (like Group A streptococci) Isolates of this group with beta hemolysis are detected by serotyping with latex agglutination reagents Virulence Factors Associated with Pharyngitis and Skin infections Not well established (Impetigo) A polysaccharide capsule and cytolysin have been Mode of Acquisition: Person-to-Person contact identified in some members of the anginosus group Group C streptococci: Main source of streptokinase Other factors with possible virulence tendencies Mostly animal pathogens Extracellular dextran, cell surface-associated proteins, In humans: S. dysgalactiae subsp. equisimilis extracellular enzymes In horses: S. equi subsp. zooepidemicus Beta-hemolytic except S. dysgalactiae VIRIDANS STREPTOCOCCI DISEASES Express protein G (surface of bacteria) Species: S. dysgalactiae subsp. equisimilis, S. equi DISEASE DESCRIPTION subsp. zooepidemicus Culture: Small, flat, grayish-white with regular to Subacute Production of extracellular complex narrow zone of alpha or beta hemolysis Bacterial polysaccharides: glucans, dextrans, levans > Antimicrobial Susceptibility Testing: Endocarditis enhance attachments to host cell surfaces Group C streptococci – Susceptible to Bacitracin and such as cardiac endothelial cells SXT Group G streptococci - Bacitracin variable Transient Associated with endocarditis 2 Forms: Bacteremia More common in children with hematologic malignancies (Viridans Streptococcal Bacteremia) Other Diseases Fulminant Cardiovascular Collapse Meningitis Oral Infections: Gingivitis and Dental 9  Bacteriology Lec Midterms A., Gabrielle, A. Romero Carries Abscesses, Osteomyelitis, Empyema Subacute Bacterial Endocarditis: Most common cause is the Viridans streptococci; More common in children than in adults with hematologic malignancies S. gallolyticus subsp. gallolyticus and S. anginosus group: Often isolated in blood cultures of individuals with Gastrointestinal carcinoma Viridans streptococci: Diagnosis Laboratory Diagnosis Gram Staining LAP (+), PYR (-) Culture GROUP D Streptococci Colonies: Minute to small Laboratory Diagnosis gray, domed, smooth or matte, alpha (some non) hemolytic Diagnostic Tests for S. bovis group (Group D Optochin Test: Optochin streptococci) resistant Growth in Bile Esculin Medium: Bile Esculin Medium – (+) Reagents: Esculin and 1-4% Bile salt Black color complex in the agar (+): Black color complex exhibited in the agar within 48 Growth 6.5% NaCl: No growth at high alkalinity hours Penicillin test: Susceptible 6.5% NaCl (Nutrient both base) test: No growth Bile Insoluble (Negative reaction/Absence of turbidity) PYR Test: Negative (No color change) Penicillin Test: S. bovis susceptible to Penicillin ENTEROCOCCUS SPP. Belong to the family Enterococcaceae Former: Group D Streptococci All species produce D antigen, belongs to the Lancefield D group Microbiota: GIT of humans and animals, Female gut Newer Identification Methods Pseudocatalase reaction: Weak bubbling in catalase test Development of molecular methods (E.g., DNA (Due to release of Peroxidase) hybridization and PCR) for the identification over viridans Species: E. faecalis, E. faecium, E. avium, E. allinarum, streptococci has been challenging E. durans, E. raffinosus Multilocus sequence analysis may be used to ID unknown streptococci species E. faecium, E. faecalis: Commonly identified species MALDI-TOF systems are in the evaluation stage Can grow under extreme conditions like Bile, 6.5% Treatment NaCl Salt, or Alkaline pH or at 45°C Penicillin with or without Aminoglycosides Specimens: Cultured ASAP Ceftriaxone Grows well at 35 ̊C in the presence of CO 2 Vancomycin (Resistant strains and Allergic to Penicillin) Differential Tests: B-D-Glucuronidase Test (BGUR) (+) Tellurite Test: E. faecalis Differentiates the B-Hemolytic streptococci in larger (+) Arabinose Formation: E. faecium colony group C and G from the small colony group C and Hemolytic Pattern: Alpha, Beta, Gamma G of the S. anginosus group or milleri complex BGUR: Enzyme that is found in isolates of large, colony-forming, B-hemolytic group C and G streptococci Substrate: Methylumbelliferyl-B-D-Glucuronide (+): Fluorescence of Large colony B-Hemolytic Groups C and G Quality Control: Positive: Escherichia coli ATCC 11775 Negative: Raoultella planticola NCTC 9528 NCTC: National Collection of Type Cultures GROUP D STREPTOCOCCI Enterococcal Grows at 6.5% NaCl inhibited but not killed by Penicillin Non-enterococcal group remained part of the Group D UTI, Cardiovascular infection, and Meningitis streptococci. E. faecalis, E. faecium, E. durans Species are indigenous microbiota of human and animal Non-Enterococcal (Group D streptococci) intestinal tracts and female genitourinary tract Inhibited by 6.5% NaCl and killed by Penicillin Opportunistic pathogens, frequently causing UTI, Endocarditis nosocomial infections S. bovis, S. equinus Resistant to multiple antimicrobial agents with S. bovis no longer a valid species name (S. bovis and temperature-dependent growth properties S. equinus were the same species) Infection and disease development attributed to person-to-person transmission and handling of fomites and contaminated medical instruments S. gallolyticus subsp. gallolyticus in blood cultures: Gastrointestinal carcinoma One of the most feared nosocomial pathogens Nosocomial UTI: Most common (Urinary catheterization or other Urologic manipulations) 10 Bacteriology Lec Midterms A., Gabrielle, A. Romero Prolonged hospitalization, Bacteremia, Wound vanA: Highly resistant to Vancomycin, located in the Infections Plasmids and Transposons Endocarditis (5-10% of bacterial endocarditis) Vancomycin-containing agar and Chromogenic VRE media: Used for screening PCR-based assays have been used for identification Virulence Factors Grow in extreme conditions Resistant to multiple antimicrobial agents Surface adhesion proteins Extracellular serine protease Gelatinase Two subunit toxins called Cytolysin in Enterococcus faecalis - quorum-sensing mechanism Laboratory Diagnosis: Microscopy: Gram Staining (G+ cocci in pairs and long chains) Weak bubbling in catalase test (Pseudocatalase reaction) Culture: TSB or BHI with 5% Sheep Blood (35 ̊C with low levels of CO2) Recommended Media for Contaminated Specimens: Bile Esculin Azide Agar, CNA, PEA, Cephalexin-Aztreonam- Arabinose agar Colonies are creamy white, glistening, and smooth with varied hemolysis E. faecalis: Identified by its ability to grow in the presence of Tellurite Bile Esculin Test: Bile Resistant > Black color 6.5% NaCl > Turbidity (Pink > Yellow) Penicillin Test: Resistant Streptococcus pneumoniae Pneumococcus or Diplococcus pneumoniae Microscopy: Gram Positive, in pairs, short chains, lancet-shaped (pointed ends) or oval, non-motile Cell wall contains an antigen known as the C Substance which is not related to the C Carbohydrate of the Lancefield Groups Facultative Anaerobe; Capnophilic Alpha Hemolytic; Fastidious Encapsulated strains (>90%; Virulent); Non- Encapsulated strains (Avirulent) C-reactive protein (CRP) - reacts with C substance - increases during inflammation and infection Antimicrobial Resistance Causative agent of Lobar Pneumonia Resistance to several agents Most common cause of bacterial pneumonia in the Acquired resistance to Aminoglycosides, β-lactams, and elderly and immunocompromised individuals Glycopeptides Has been identified as an etiology of the Community Resistant to Cephalosporins and some Aminoglycosides Acquired Pneumonia (CAP) in adults and a major (Gentamicin, Erythromycin) cause of Healthcare-Associated Pneumonia (HCAP) Enterococci are resistant to both Penicillin test and Most common organism associated with Efromycin Acid Disk (100 ug) test bacterial meningitis in adults Vancomycin-resistant enterococci (VRE) Colonizer of Nasopharynx of healthy individuals Predominantly E. faecium due to the resistant genes (5-75%) known as the van genes (vanA, vanB, vanC) It is an asymptomatic members of a normal Eight phenotypes have been described respiratory tract such as the nasopharynx Capsule vanA and vanB most frequently encountered Principal virulence factor 11 Bacteriology Lec Midterms A., Gabrielle, A. Romero Made up of complex polysaccharides >90 different serotypes Immunogenic and can be identified with the appropriate antiserum Basis of Quellung Reaction (Neufield test) Capsule is antigenic - Antibody directed against the the capsular antigen is protective. Quellung reaction – the capsule swells in the presence of specific anti-capsular serum; also serves to serotype the isolate specifically (serotypes 1-3). COMMUNITY-ACQUIRED PNEUMONIA (CAP) IN ADULTS Opsonization of the capsule renders the organism avirulent Should be in the nasopharynx and deficient of the specific antibody against the capsule: To contract pneumococcal infection Opsonization of the capsule renders the organism avirulent. Avirulent Factors: Hemolysins, Immunoglobulin A Protease, Neuraminidase, Hyaluronidase Related Diseases Mode of infection: Person-to-person contact with infected respiratory droplets and secretions CDC Recommendation on Vaccination: LABORATORY DIAGNOSIS 13-valent Conjugated vaccine (PCV 13): Given starting at age 2 months as part of the pediatric immunization 23-valent Polysaccharide vaccine (PSV 23): For adults >65 years old: Given upon the advice of clinician Agent of bacteremia, endocarditis, peritonitis, and typical Hemolytic Uremic Syndrome LOBAR PNEUMONIA 12 Bacteriology Lec Midterms A., Gabrielle, A. Romero Antimicrobial Resistance and Vaccination Generally treated with Penicillin If resistant: Use Erythromycin or Chloramphenicol Vaccine: Against most common capsule antigens Recommended for asplenic individuals, older adults, cardiac patients Helps reduce incidence and severity pneumococcal vaccine (PCV)/PCV13 - protecting against 13 serotypes commonly affecting children. 4 doses beginning at 2 months of age. 23-valent pneumococcal polysaccharide vaccine (PPSV 23) - For adults, Older than 65 years/ individual with long-term health problem. NEISSERIA SPP. & MORAXELLA CATARRHALIS Abiotrophia and Granulicatella Gram negative (-) diplococci AKA pyridoxal-dependent; vitamin B6-dependent; thiol-dependent; symbiotic strep Disease: Bacteremia, Endocarditis, Otitis Media, Brest- implant-associated infections, Endophthalmitis, and Septic Arthritis Difficult to treat with Antibiotics Part of the human oral and gastrointestinal microbiota Thiol compounds: Cysteine, vitamin B6 and pyridoxal Formerly Nutritionally-Variant streptococci (NVS); gram variable Requires 10 mg/L of Pyridoxal Hydrochloride added to media (BAP or CAP) Members and key characteristics Culture: Grow as “satellites” around some bacteria that Members of the neisseriaceae family produce Pyridoxal such as S. aureus (“Staphylococcal ○ Neisseria, kingella, eikenella, simonsiella and streak test”) Resemble viridans streptococci alysiella Abiotrophia will not grow on BAP or CAP unless Most neisseria spp. Pyridoxal is supplied ○ Aerobic Exhibit alpha or gamma hemolysis Laboratory test: staphylococcal streak test; (+) PYR and ○ Non motile LAP ○ Non spore forming Microscopy: Gram variable, pleomorphic ○ Gram negative diplococci ○ Cytochrome oxidase (screening test) and catalase positive Exceptions Gram negative bacilli, rod shaped ○ N. elongata 13 Bacteriology Lec Midterms A., Gabrielle, A. Romero ○ N. weaver ○ Bind to pathogen and prevent binding of effective ○ N. bacilliformis antibodies (protective immune system) Catalase negative Major outer membrane porin proteins ○ N. elongate ○ Por A / B ○ N, nitroreducens Effective against inflammatory response and complement killing (natural defense) ○ N. bacilliformis (to lyse bacteria) (serum protein) Catalase positive ○ Protein II (opa) ○ N. weaver Facilitate adherence to phagocytic and epithelial cells Neisseria - taxonomy ○ Protein III Block host serum bactericidal action (IgG) Prevent phagocytosis Pili Only two species of these encapsulated ○ Five distinct colony types Gram negative diplococci are normally pathogenic for humans: T1 and T2 virulent ○ N. gonorrhoeae T3 through T5 have no pili (avirulent) ○ N. meningitidis ○ Functions to evade the immune system N. sicca ○ Aid in attachment to host tissues N. lactamica ○ Help prevent phagocytosis ○ Aid in exchange of genetic material from cell to cell N. flavescens (sex pili) N. mucosa Lipooligosaccharide (LOS) or endotoxin N. subflava ○ Lipid A moiety and core LOS that differentiates it from the lipopolysaccharide (LPS) General characteristic Immunoglobulin A (IgA) protease (cleaving enzyme Many neisseria spp. are capnophilic (requires CO2 for that can cut protein) growth) with optimal growth in humid atmosphere ○ Cleaves IgA on mucosal surfaces (hemidophilis) ○ Disseminated infections If alternative electron acceptors (e.g., nitrites) are ○ Anal canal present organisms can grow anaerobically (devoid of O2) Neisseria gonorrhoeae Natural habitat Humans are the only natural host ○ Mucous membranes of respiratory and urogenital tracts ○ Gonorrhea — meaning “flow of seed” (when urethral discharge was mistaken for semen) Clinical significances ○ Also called clap, from french word for brothel (clapoir) Excuse as usual flora in the upper respiratory and urogenital tracts Reportable disease known as gonorrhea ○ Meaning most isolates are not pathogenic and are routine normal flora Neisseria gonorrhoeae epidemiology ○ N. meningitidis can be commensal inhabitant in Most commonly transmitted by sexual contact carriers (asymptomatic) Primary reservoir Primary human pathogens ○ Asymptomatic carriers National reportable disease Gram negative diplococci ○ Confirmed cases must be reported to state health ○ Coffee bean shaped laboratories N. gonorrhoeae (often called gonococci) Highest rates of infection ○ Always pathogenic when present ○ Men and women between the ages of 20 and 24 N. meningitidis (meningcocci) ○ May be present as a commensal inhabitant of Neisseria gonorrhoeae clinical infections upper respiratory tract of carriers Short incubation period (2-7 days) ○ May become an invasive pathogen (epidemic of Gonorrhea meningitis and meningococcemia) ○ Acute pyogenic infection of the (epithelial cells: Both pathogens Urogenital tract ○ Fastidious (requires additional supplements for growth usually in the form of blood) Pharynx ○ Require enriched media (BAP & CAP) for optimal Conjunctiva of the eye recovery ○ Less likely infections Disseminated infections Pathogenic Neisseria virulence factors Anal canal Receptors for human transferrin Neonatal blindness ○ Allow it to compete for iron ○ 1% AgNO3 Capsule Neisseria gonorrhoeae clinical infections in Men ○ Prevent phagocytosis Cell outer membrane proteins Incubation period 2- 7 days ○ Gram negative Transmitted only by intimate sexual contact ○ Antigenic variation Asymptomatic gonococcal infection is uncommon ○ Cause production of useless antibodies 90% of infected men show symptoms of acute infection 14 Bacteriology Lec Midterms A., Gabrielle, A. Romero Symptoms Infection is preventable with the ○ Dysuria application of eye drops at birth (erythromycin) ○ Urethral discharge Every infant at birth, by law, must Complications be treated ○ Epididymitis — inflammation of small coiled tube of testicle N. gonorrhoeae specimen collection and transport ○ Urethral stricture — narrowing of the urethral ○ Prostatitis — inflammation of the prostate gland Clinical specimens ○ Genital sites Normal handling Neisseria gonorrhoeae clinical infections in Women Urethra in males As many as 50% are asymptomatic Collect purulent discharge Endocervix is most common site of infection If no discharge, insert swab Symptoms (if symptomatic) (dacron or rayon) 2 cm in urethra ○ Dysuria and slowly rotate ○ Cervical discharge Endocervix in females ○ Lower abdominal pain ○ Rectal culture Complications Insert swab 4 to 5 cm into the anal canal ○ Pelvic inflammatory disease (PID) ○ Oral / pharyngeal ○ Sterility ○ Eye ○ Ectopic pregnancy ○ Blood / joint fluids ○ Perihepatitis (fitz hugh curtis syndrome) ○ When possible, notifications of pending Neisseria samples should be made to the laboratory Preferred swabs for specimen collection ○ Dacron or rayon Fitz hugh curtis syndrome ○ Calcium alginate and cotton swabs are inhibitory Complications of pelvic inflammatory disease (PID) to N. gonorrhoeae Perihepatitis Direct plating of specimen to gonococcal selective Inflammation of hepatic capsule & diaphragm media gives optimal results Pleuritic chest pain Transport systems are available that contain selective media and a CO2 capnophilic atmosphere May or may not have signs / syndrome of PID ○ JEMBEC, Gono-pak transgrow Violin string sign Specimen rolled in a “2” pattern ○ Adhesion b/w liver and abdominal wall on media 24 hour growth of N. gonorrhoeae N. gonorrhoeae clinical infections in other sites Blood borne dissemination (4.5 ○ Occurs after inoculation of the organism into skin or 1 drop 10% KOH cutaneous tissues Diagnostic Tests ○ Infection begins as a localized subcutaneous abscess (Actinomycotic mycetoma) ○ Nugent Scoring System for Gram-Stained Vaginal Smears ○ Progress of infection: Burrowing sinuses open to the skin surface and drain pus ○ Pus is pigmented and contain sulfur granules (Yellow or orange with granular appearance) LABORATORY DIAGNOSIS for Nocardia SPECIMEN AND STAINING Specimen Biopsy or Drainage material Sputum, Exudates, Skin/Abscess aspirates Staining Modified Ziehl Neelsen or Kinyoun Stain Modified AFS (0.5-1% H 2 SO4 ) to observe branching filaments NON-SPORE FORMING, BRANCHING AEROBIC Gomori’s Methenamine silver stain - Histopathology ACTINOMYCETES Wet mount: Crush granules to see cellular morphology CULTURE AEROBIC Actinomycetes BAP, CAP, SDA without Chloramphenicol, BHI, LJ ”Fungi-like bacteria” Medium, Middlebrook Media, Litmus Milk Broth, PDA, Aerobic, Branched, Beaded: Filaments extend along the TMA, Tap Water Agar agar (aerial hyphae) and into the agar (substrate hyphae) Enhance Acid Fastness before AFB stain: 4 days growth Ability to degrade Amyl Alcohol, Paraffin, and Rubber on Middlebrook 7H10 agar or Litmus Milk Culture: Hair-like strands like molds Also grows on BCYE (for Legionella spp.) Tap Water Agar – Observe morphology of Actinomycetes; differentiate branching Nocardia from nonbranching Rhodococcus Substrate Hydrolysis: Casein, Hypoxanthine, Xanthine, and Tyrosine Positive Casein and Tyrosine: N. brasiliensis Growth at 45 ̊C: N. farcinica Acid production from Rhamnose: (+/-) N. asteroides Gelatin Hydrolysis: (+) N. brasiliensis Nocardia species Opacification of Middlebrook Agar: (+) N. farcinica Strict Aerobic G+ bacilli with TREATMENT OF NOCARDIOSIS long, thin, beaded, branching Involves drainage and surgery along with antimicrobials filaments Antimicrobial profiles vary ○ Beads: Not spaced at Resistant to Penicillin; Susceptible to Sulfonamides consistent intervals Antifungal agents: No effect Partially acid-fast Nocardia often present along with other organisms, ○ Cell Wall: Peptidoglycan including fungi Proper testing and antimicrobial treatment essential (NAM and NAG), m-DAP, Arabinose, Galactose Differential Test: Resistance Rhodococcus equi (The Pink Bacterium) to Lysozyme Salmon-pink pigment at RT (SBA): Resemble Klebsiella Partially acid-fast: Has mycolic Primarily affect acid with longer carbon Immunocompromised chains Colonial growth: Can transform from rod to cocci ○ Wrinkled, dry, chalky-white to orange-tan pigment Replicate within Macrophages: colonies (“Breadcrumbs looking”), or Facultative intracellular organism ○ Waxy, bumpy, velvety rugose forms with yellow to Can infect immunocompromised orange pigment in 5-20 days patients (HIV): Granulomatous pneumonia ○ Under dissecting microscope, may reveal presence of Microscopy: Coccobacilli in aerial hyphae (first clue to identity of colony) “zigzag” pattern; appear as diphtheroids Nocardia species Clinical Infection Culture: Nocardiosis: Pulmonary Infections ○ BAP: Pale Pink or Yellow ○ Initial lesion in Lung (“Coral-like, Slimy”) ○ Often focus of Pneumonitis that advances to colonies Necrosis ○ Resembles Klebsiella with ○ Tissue involvement may occur resulting in damage salmon-pink colonies at room temperature ○ Little inflammatory response or scarring Differential test: ○ Sputum: Thick and Purulent Susceptible to Lysozyme ○ No sulfur granules upon staining. Masses of filamentous organisms bound by Gordonia species Calcium Phosphate Gram Positive/Gram variable, Partially Acid-fast, 25 Bacteriology Lec Midterms A., Gabrielle, A. Romero Non-motile, Catalase (+) Considered “Nocardioforms”: Fragment into rod- shaped or coccoid elements Culture: Slimy colonies, glossy to irregular edges; absence of Mycelia Differential test: Susceptible to Lysozyme Tsukamurella species Gram +, Long Rods, Fragment to 3 parts; No aerial Hyphae, (+) Catalase Slightly acid-fast Culture: circular colonies with rhizoid edges and white/orange pigment NON-ACID FAST, AEROBIC GRAM-POSITIVE ACTINOMYCETES Streptomyces Culture: Dry to Chalky heaped colonies, gray-white colonies “Musty Basement Odor” S. somaliensis: Human Pathogen Actinomadura Cause wound infection for persons walking barefooted (tropical countries): Mycetomas Culture: Waxy, Cerebriform colored colonies (“Molar Tooth appearance”) Species: A. madurae, A. pelletieri Tropheryma whipplei Agent of Whipple’s Disease (middle-aged men; presence of PAS-staining macrophages) G+ actinomycete, facultatively intracellular pathogen Cultivated in cell lines 26