Anne's Midterms [CLBact Lec] MT'25 - Y3T1 - 10-20 PDF

● AUTOMATED APPROACH FOR DEFINITIVE IDENTIFICATION BACT/ALERT uses colorimetric detection, other fluids can be applicable here BIOCHEMICAL METHODS BIOCHEMICAL METHODS ○ Is a kind of phenotype since it only studies the gene product such as proteins, metabolic tests etc BD BACTEC Automated Blood Cu...

● AUTOMATED APPROACH FOR DEFINITIVE IDENTIFICATION BACT/ALERT uses colorimetric detection, other fluids can be applicable here BIOCHEMICAL METHODS BIOCHEMICAL METHODS ○ Is a kind of phenotype since it only studies the gene product such as proteins, metabolic tests etc BD BACTEC Automated Blood Culture System ANALYTICAL PROFILE INDEX (API) 20E ○ ○ ○ Is a biochemical panel for identification and differentiation of members of the family Enterobacteriaceae. Can also be used for gram positive acea and gram positive cocci but mainly for the family Enterobacteriaceae Has 20 miniature wells with different chemical substrates STEPS 1. Bacterial suspension is used to rehydrate each of the wells and the strips are incubated. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. 2. All positive and negative test results are compiled to obtain a profile number, which is then compared with profile numbers in a commercial codebook to determine the identification of the bacterial species. a. Lahat ng database sa automated, duon nakalagay lahat ng profiles ng mga clinically important or known microorganisms MOLECULAR METHODS PCR Bio fire multiplex nested pcr (panels yung nasa lower right) Has panels Multiplex nested Real time pcr Puwede rin for other fluid testing Detection of Mycobacterium tuberculosis Detect Rifampicin resistance Detect CNTG (chlamydia, neisseria, trachomatis, and gardnerella vaginalis) Film Array GeneXpert ● ● ● ● ● ● ● ● Biomerieux BacT / ALERT Microbial Detection System CULTURE BOTTLES Culture bottles are not solely used for blood specimens since it can be used for other bodily fluids Anticoagulant uses SPS (Sodium polyanethol Sulfonate) Green Aerobic S Orange Anaerobic Yellow Sterile Body Fluids Has 21 microwells - 21 tests NOTE: Yung profile numbers is yung binary code and then from there dun ma-determine yung commercial code or the octal code Binary code contains 21 digits (As seen in the table the binary code is: 101100001101111010) it will be converted into the octal code which has 7 digits (As seen in the table 5144572 which is a code for E.Coli) (pls watch video for this AUTOMATED CULTURE METHODS BACTEC and BACT/ALERT are both automated microbial detection systems based on the carbon dioxide production of growing microorganisms. BACTEC uses fluorescent technology whilst, more on blood ● ● MALDI-TOF Proteomics using matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF-MS) has emerged as a reliable method for the rapid identification of microbial pathogens. This technology involves mixing of the pathogen with a suitable chemical matrix on a solid surface, pulsing the sample with a laser for ablation and desorption, followed by ionization using time-of-flight technology to create a mass spectrum of the samples. ○ This mass spectrum is then compared with a database of known spectra to allow for organism identification. ○ Fluid tube - vacuumed tube sa Maldi - Tof ms ○ Proteomics - study of proteins inside the cell BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 10/ 33 WEEK 8: GRAM-POSITIVE COCCI (STAPHYLOCOCCUS SPP.) INTRODUCTION ● ● ● GRAM-POSITIVE COCCI: natural inhabitants - skin and mucous membranes (humans and animals) ; ubiquitous in the environment ○ Ubiquitous - found in the environment and always nasa paligid natin Among the medically significant gram positive cocci: ○ Staphylococcus - gram positive, they are color purple because of HIGH PEPTIDOGLYCAN CONTENT AND LOW LIPID CONTENT (CELL WALL) ○ Streptococcus ○ Enterococcus High peptidoglycan content and low lipid content (cell wall) ○ Alcohols and solvents do not penetrate (tighten / cenate) ; trap the primary stain (crystal violet) ○ Pagkakaiba sa gram negative: an outer membrane ● ● Columbia CNA - commonly used due to addition ng blood Facultative anaerobes SALT TOLERANT ● ○ ○ ○ ○ ○ ● ○ ○ GENERAL CHARACTERISTICS BERGEY’S MANUAL OF SYSTEMATIC BACTERIOLOGY (1996) ● Micrococcus Luteus Produces light yellow Micrococcus Rosaceous Pink to red, appeared in tetrads pairs or irregular clusters but never in chains, but appear in the same morphology as the gram stain smear of staphylococcus Micrococcaceae included 4 genera: Micrococcus, Staphylococcus, Planococcus, and Stomatococcus ○ Micrococcus and Staphylococcus clinically significant, the rest are sa animals and plants na MOLECULAR PHYLOGENETIC ANALYSIS 1. 2. 3. 4. Staphylococcus and Micrococcus are not closely related because only based on Phenotypic Staphylococcaceae are now combined with the Bacillaceae, Planococcaceae, and Listeriacee in the order Bacillales Micrococcus species have been reclassified into the genera Kocuria, Nesterenkonia, Kytococcus, Dermacoccus - nice to know because of the increasing use of molecular analysis 49 SPECIES; 25 SUBSPECIES ○ Staphylococcus - Facultative anaerobes but have species that are obligate anaerobes an may be catalase positive ■ S. aureus, subspecies anaerobious ■ S. saccharolyticus (?) - gram positive cocci, catalase positive ■ BOTH S. aureus subspecies anaerobious and S. saccharolyticus are obligate anaerobes and catalase negative ● ● ● Important human species : S. aureus, S. epidermidis, s. saprophyticus, S.lugdunensis, S. hominis, S. hemolyticus, S. warneri ○ S. aureus, S. epidermidis, s. saprophyticus, S.lugdunensis - most common that causes disease Catalase positive, non-motile, non-spore forming ○ CATALASE TEST - determines the ability or the presence of the catalase in the organism that converts hydrogen peroxide into water and oxygen ■ differentiates staph from strep ○ Non-motile - no flagella Gram positive cocci in single, pairs, tetrads, chains (liquid cultures); medium to large colonies (1.0 mm - 2.0 mm), raised, convex, döme-shaped, creamy colonies (BAP) ; white, cream, or golden (CNA) can tolerate up to 7.5% - 10% NaCI a.k.a. bacteria salt lover called Halophilic or halophiles Reduction of nitrates to nitrites Normal component ng katawan - nitrates Bacterial infection changes it into nitrites Mode of Transmission: ■ Direct contact - infected individuals (carriers) ; contaminated objects ■ Carriers can become reservoir, they can both stay and proliferate "Tolerance" : inhibited by a drug, but not killed (lack of activation of autolytic enzymes in the cell wall Micrococcus species has the same morphology kaya siya pinaghihiwalay w ur staphylococcus; colony and morphology resemble micrococcus but Micrococcus has circular smooth convex colonies + pigmented Staphylococcus - grapelike irregular clusters; column shape not yellow (gaslit) BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 11/ 33 Fig. 13.1 (A) Indirect Gram stain of Staphylococcus aureus demonstrating characteristic clusters of gram- positive cocci. (B) Indirect Gram stain of Micrococcus /uteus demonstrating characteristic tetrads or small clusters of gram-positive cocci. ● Staphylococcus - nagcclusters sila, grape-like irregular clusters, called that due to arrangement and shape ● They are differentiated using biochemical using different techniques and antibiotics ● ● ● EPIDEMIOLOGY Associated with human colonization and infection (community and healthcare settings) ○ Staph infection - centered at the healthcare setting but rn theres an increasing number found in community ○ Di na basta species, they are multidrug resistant species Infections = colonizing strains gain access to a normally sterile site ○ Surgical wound/operation ○ Trauma Epidemiology ○ Pyogenic infections - accumulation of neutrophils, bacterial cells & fluid (nagnana) THREE TYPES OF NASAL CARRIER STATE ● ○ All important, but main test = COMG Catalase Staph are all catalase positive Staph and strep are differentiated by the use of catalase test Strep - negative Staph - gram positive To differentiate the two, check the oxygen requirements 1. Staphylococcus also have obligate anaerobe a. Micrococcus is aerobic Oxygen Staphylococcus are facultative anaerobe requireme Micrococcus are aerobic nts Glucose utilization Fermentative Staphylococcus - presence in manitol agar Oxidative Micrococcus Nonsaccharolytic - hindi ginagamit ung sugar Modified Staphylococcus - Negative oxidase Micrococcus - Positive (using of Microdase) ● MICROCOCCUS (LABORATORY TECHNIQUES) MICRODASE ○ Detection of oxidase ○ Procedure: Rub a small amount of colonies ○ Incubate : 2 mins at room temp ○ Positive Result: Development of blue to purple-blue color ○ Negative Result: No color change ■ due to absenc of cytochrome ○ Persistent carriers - harbor a single strain for extended period ■ Mears Intermittent carriers - harbor different strains over time ■ “Mahirap both sa persistent carriers and intermittent carriers” Non Carriers - do not harbor any organisms PREVALENCE OF S. AUREUS CARRIERS ● ○ ○ ● ● ● Health care providers HAI - healthcare associated infection Staphylococcus reside in your nares (aka NOSE) NOTE: MOT : Person to person Spread rapidly as a commensal organism All staphylococci isolated from normally sterile sites and medical devices - identified to species level ○ Sample is either normal sterile sites - BLOOD, urine, csf, suprapubic aspirates ○ BLOOD once it is positive with any organisms normally should not have any bacteria ○ Medical devices - central catheter lines, shunts: intrathecal (spinal shunt) or AV shunt (atrioventricular), prosthesis, metals ○ Once ma-isolate, tas tumubo, importante na gawin is that you identify them to a species level decline attached to your brachial arm Peripherally inserted central catheter line dialysis na wala pang fistula, di makabitan ng swero Pigsa - caused by stapohylococcus specui species Spaghetti - pagkapanis is from pagkagamit ng carbohydrates ng staph BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 12/ 33 Shunts - intrathecal; tubo sa likod ng spinal duon nilalagay yung anesthesia AV shunts - atrioventricular shunt; for dialysis patients ■ ● STAPHYLOCOCCUS AUREUS Resistant to drying, heat (50C for 30 mins), 10% NaCl ○ Halophile at 10% sodium chloride Peptostreptococcus & Peptophilus - similar to staphylococi bukod sa micrococcus: TEST PERFORMED DIFFERENTIATES STAPHYLOCOCCUS 1. ● ● ● Tube coagulation a. Positive; presence of bubbles (white part) 2. Catalase test 3. Slide coagulation AUREUS - because of the color yellow when it grows The most virulent species of staphylococci Major human pathogen NARROW ZONE OF ß - HEMOLYSIS (BAP) ● ● YELLOW - HALO NARROW ○ Nagddilaw ung BAP, yellow halo - nahhemolyze ung blood ○ Narrow - gram + cocci - produce a wide zone of b hemolysis (malapad) Colonies : smooth, butyrous, creamy, raised / dome shaped with an entire edge; pigmented white, cream, or golden yellow ○ Small colony variants (SCVs) - limited nutrients / other selective pressures (revert to normal after subculture) ■ Prone to misidentification ■ Di namemeet yung requirements kaya ganon lang sila kasmall ■ Morphology: clustering FERMENT GLUCOSE ANAEROBICALLY (MSA) ○ ○ ○ S. aureus - fermentative species (note: basahin nalang book for other species) Ferments glucose MSA - Mannitol salt agar ■ Mannitol - carbohydrate alcohol ■ During fermentation - yellow halo; yellow colorization ■ Hemolysis - seen at around 2-3 days at room temp MANNITOL SALT AGAR ● ● ● ● Golden yellow for colonies Yellow colorization due to hemolysis Yellow halo due to fermented mannitol incubate: 48-72 hrs to grow BLOOD AGAR PLATE ● ● ● ● ● Sterile Fluid In BAP CAP MAC THIO- may depend on the broth ○ Incubated at 35 C in CO2 incubator IF NO incubator to produce CO2 ○ Alternative method is CANDLE JAR Other media: ○ Blood CS bottles - allows growth of wide variety ○ Thioglycolate ○ Dextrose ○ Brain Heart Infusion (BHI) STAPHYLOCOCCUS AUREUS (VIRULENCE FACTORS) Capsule production - slime layer / biofilm (11 serotypes ; type 5 & 8 - majority of infections) ○ Production of polysaccharide ■ Unless may ibang component (haemophilus) ○ Quorum sensing - nagcchikahan, nag-uusap-usap sila, nagkkumpulan then magkakaroon ng biofilm / slime layer; could evade phagocytosis ○ Type 5 & 8 - vaccines ○ Peptidoglycan (cell wall) attacked by some antibiotics From osteomyelitis patient BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 13/ 33 ○ MICROBIAL SURFACE COMPONENTS RECOGNIZING ADHESIVE MATRIX MOLECULES (MSCRAMMs) ● collective parts of staphylococcus ● PROTEIN A ● Bound to cytoplasmic membrane; high affinity for the FC receptor on lgG molecules, and complement ○ Itsura ng antibody “Y” ○ Protein A attaches to Fc receptor ○ FC portion is at the bottom ○ attach to the white blood cells ○ Fab - nagrrecognize ng antigen of the organisms ○ Usually used in the laboratory in a form of coagglutination; hindi makakadaan due to coag can be seen in coagulation negative: Staph epidermidis and staph haemolyticus ; they can produce virulence Gamma - produced by all strains; in association with Panton Valentine Leukocidin (PVL) (toxic to neutrophils; CA-MRSA) PANTON VALENTINE LEUKOCIDIN (PVL) ● Inhibits phagocytosis, toxic to neutrophils, binubutas WBC, granules sa loob ay lalabas. Ex: IL-8, leukotriene, histamine ○ PVL - toxic to WBC and works with gamma toxine to lyse ● Commonly found in CA-MRSA ○ CA - correlated with community acquired ○ LA - correlated with lifestyle acquired Note: Alpha and Beta works in combination CLUMPING FACTOR (A&B) ● Located at cell wall surface ; binds nonenzymatically the fibrinogen and platelets - lead to aggregation of the bacteria (highly immunogenic) ○ Would produce clots ○ Highly immunogenic - regarding the response of immune system; once it triggers, may cause autoimmune diseases ○ Sinisira ng immunity natin from our own body yung sarili nating katawan (autoimmune disease) ○ Type of coagulase (bound coagulase) ○ Coat something prevents something TWO TYPES OF COAGULASE METHICILLIN - RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) Beta and alpha work in conjunction SUPERANTIGENS BOUND COAGULASE (STAPHYCOAGULASE / CUMPLING FACTOR ● ● ● Detected using slide coagulase ○ Fibrin may coat neutrophils and prevent phagocytosis ■ Neutrophils react immediately to acute infections Staphylococcus logdonesis & Staphylococcus Sheferri could produce clumping factor or coagulase EXTRACELLULAR / FREE COAGULASE ● ● ● ● ● Detected by tube coagulase-Coagulase Reaction Factor(CRF) complex (resembles thrombin) ○ Activates the differentiation of from thrombin ○ Nasa fibrin clot yung organism Differentiated by coagulase test / coagulation formation ○ Some produce coagulase / clumping factor ○ Ex: staph lugdunensis and Staph schleiferi EXOTOXIN / HEMOLYSIN Alpha toxin - disrupts smooth muscle in blood vessels & toxic to RBCs, WBCs, hepatocytes, platelets, macrophages Beta toxins (heat-labile sphingomyelinase) - hydrolysis of membrane phospholipids (cell lysis) Delta - nonspecific membrane toxicity to other mammalian cells (cytolytic to erythrocytes) allow proliferation of WBCs, TCRs and APCs, cause cytokine storm Highly inflammatory (immune system causes destruction) ENTEROTOXINS ● ● GAStro (mga tiyan); food poisoning 75% related to staphylococcus aureus ○ caused by 15 enterotoxins (A-E, G-P) ○ All of them are Heat-stable (Hindi nasisira ng init) ■ Most staphylococcal-related food poisonings - A, B, D, H ■ Associated with enterocolitis - B and C - could produce the what?? (stomach creates another layer) ■ HEAT STABLE ■ Resistant to stomach and intestinal enzymes ■ Emetic effect - vomiting effect ■ Often found in milk products ■ Pseudomembranous enterocolitis - the stomach creates pseudomembrane ■ Genes - Pathogenicity Island ➢ genes that produce enterotoxins ■ Note: ❖ Gastro - sa tiyan ❖ 25 mg of enterotoxin B causes vomiting and diarrhea BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 14/ 33 ❖ Often found sa gatas Enterotoxin A to E Enterotoxin G to B? Endo gram negative Exotoxin Positive + EXFOLIATIVE TOXINS / EPIDERMOLYTIC TOXINS ● 2 distinct proteins of the same molecular weight ; superantigens ○ Exfoliative Toxin A : eta (phage) ; heat stable (resists boiling for 20 mins) ○ Exfoliative Toxin B : plasmid mediated ; heat labile ■ Heat labile - di kaya mainitan so nasisira ○ Generalized Desquamation of the Staphylococcal Scalded Skin Syndrome / Ritter's disease ■ look for book ○ Genes - Pathogenicity Island TOXIC SHOCK SYNDROME TOXIN - 1 (TSST-1) / PYROGENIC EXOTOXIN C ● ● ● ● Same as enterotoxin F; prototypical superantigen Common: women who use tampons during menstruation, surgical wound / focal infection, surgical procedure (nose/ sinuses) causes: Fever, desquamation, hypotension >> shock or death Pyrogenic - causes fever EXTRACELLULAR ENZYMES ● ● ● ● ● Catalase: inactivates hydrogen peroxide (neutrophils Fibrinolysin (Staphylokinase): dissolves fibrin clot, enable spread of infection Hyaluronidase (Spreading factor) : hydrolysis of hyaluronic acid; promote the spread of infection Lipase : hydrolyzes lipids in plasma & skin; initiation of skin infections Nuclease (Deoxyribonuclease / DNase) : degradation of DNA SLIDE COAGULASE TEST ● ● ● ● ● ● ● BOUND COAGULASE Procedure: bacterial colony + rabbit plasma Negative Control: Bacterial colony + Sterile saline/water Positive result: agglutination reaction (within 1 min)/ white fibrin clots ○ uses bacterial colony (+), sterile water or sterile saline Invalid: agglutination in both the test and negative control Negative Result: Absence of Agglutination; proceed with tube coagulase test ○ Negative test should proceed with tube coagulase test ○ To determine the presence of extracellular or free coagulase Note: ○ Preferred: Plasma (EDTA) - False positive if citrated plasma due to citrate utilizing organism ■ something di mabasa = vibrin organism di mabasa Photo: Dissolving of Mucopolysaccharide LABORATORY TECHNIQUES CATALASE TEST ● BOUND COAGULASE ● Procedure: bacterial colony + 30% or 3% hydrogen peroxide ● Positive result: copious bubbles ○ Effervescence - CO2 ● Negative result: No or Few bubbles BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 15/ 33 TUBE COAGULASE TEST ● ● ● ● Procedure: Bacterial colony + (0.5 mL) Rabbit plasma Incubate: 35 -37 degrees celsius water bath (4 hours; monitor every 30 min interval) ○ Monitored every 30 minutes in case of false negative in cases of fibrinolysin ○ Fibrinolysin - active in 35 - 37% celsius produce clot formation; kapag hindi mo nakita after 30 minutes interval automatic; NEGATIVE Negative after 4 hours >> incubate at room temp for 16-18 hrs (slower coagulase producers) ○ 16-18 hrs: To check for slower coagulase NOTE: ○ SLIDE - DETECT BOUND ○ TUBE - DETECT FREE COAGULASE ● ○ Fluid filled, not pus Osteomyelitis : an infection in a bone that results in destruction of the whole infected area, usually destroys the whole marrow LATEX AGGLUTINATION TEST ● ● ● ● ● ● It is direct detection test Latex particles coated with plasma fibrinogen and IgG ○ Beads have fibrinogen and IgG Detection of clumping factor and protein A Procedure: Latex reagent + pure colonies Positive result: visible agglutination Note: ○ Protein A attaches to FC portion (bottom of Ig portion) ○ FAB (top portion of Ig) ○ For clumping factor → Fibrinogen ■ Becomes something with monoclonal antibodies STAPHYLOCOCCAL SCALDED SKIN SYNDROME ● ● Exfoliative toxin ; bullous dermatitis (often seen in newborns) Ritter's disease : extreme extensive peeling of the skin DEOXYRIBONUCLEIC ACID HYDROLYSIS (DNASE) TEST ● ● ● ● Procedure: inoculate Dnase agar with the test organism (Incubate 35 deg C - 37 deg C; 13-24 hours) Positive result: release of methyl green and colorless area around the test organism Negative result: no degradation and medium remains green Note: ○ 1.5???? ○ combines with highly polymerase A at 1.5? ○ kapag positive may clearing sa paligid ng colony METHICILLIN - RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) ● ● ● ● ● ● ● ● ● ● ● STAPHYLOCOCCUS AUREUS (PATHOGENESIS) Folliculitis : infection of the hair follicle / oil glands Boils : red lump that becomes more painful as it fills with pus Furuncles : small abscesses of the skin and subcutaneous tissue (neck & axillae) Carbuncles : group of furuncles / boils ; may be more invasive (deeper tissue) Bullous impetigo : superficial skin infection ; begins as a reddening of the skin and soft tissue then develops into fluid-filled & crusted lesions (sac) ● ● ● Resistant to staphylococcus aureus Resistant to ß-lactam antibiotics - penicillin, oxacillin, amoxicillin (some cephalosphorins) ○ some of these strains are resistant on penicillin antibiotics; ex: nafcilin, oxacilin, methicilin Became known as HA-MRSA (Hospital Acquired MRSA) Primary mechanism : production of altered penicillin-binding protein or beta lactamase ○ Beta lactamase destroys beta lactam ring mecA gene (SSSmec) (SCCmec) : encodes for the PBP2a (Penicillin Binding Protein 2A) ○ mecA gene ○ mec C gene - expresses penicillin binding protein A ○ SCCmec - carries me ß-lactamase (Penicillinase) : hydrolysis and inactivation cleavage of ß-lactam ring CA-MRSA (Community Acquired MRSA): athletes, correctional facilities, homeless, military personnel LA-MRSA (Livestock-associated MRSA) MRSA Screening Agar - functions differentiates between two population grows (resistant or susceptible results) ○ > 19milimeter; considered as resistant BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 16/ 33 CEFOTOXIN DISK DIFFUSION TEST MRSA LATEX TEST FOR PBP2 ● ● detection for resistance of Staphylococcus aureus and Staphylococcus lugdunensis (high mortality rate) ● ● ● ● ● ● ● ● NOTE: Penicillin - Beta lactam drug Have beta lactam ring - antibiotics MecC genes - encode for the expression of penicillin binding protein → PBP2A Community acquired - found within athletes because of closed area, correctional facilities, homeless areas, military personnel Staphylococcus has nature for tolerance against antibiotics but they have become resistant nowadays Superbugs - resistant to most of the antibiotics and other medications commonly used Antibiotic of choice: Vancomycin if it becomes resistant? Mixed colonies: perform MRSA mix agar ○ 2 to 4 % sodium chloride to grow organism ○ 35 degrees something ○ Cyfoxitin disk diffusion - For Staphylococcus aureus ● ● Latex reagent sensitized with monoclonal antibody against PBP2 Gene extracted from the organism's cell membrane Presumptive Positive Result: visible clumping DOUBLE DISK DIFFUSION /D ZONE TEST ● ● ● ● ● Modified Kirby Bauer test Detect inducible clindamycin resistance; erythromycin-resistant strains of S. aureus ○ Increased resistance is usually due to the erythromycin Bacteria will grow around the erythromycin disk and in the area between the 2 antibiotic disk Flattened clindamycin zone (resistance) on the side closest to the erythromycin disk NOTE: Types of Resistance ○ Macrolides: ■ Efflux pump (msRA gene) ■ Albercal ribosomal binding site (erm gene) ■ Target: 50S or 30S ribosome STAPHYLOCOCCUS AUREUS (Detection of Antibiotic Resistant) CHROMagar ● ● ● ● ● ● ● ● Has chromogen and cefoxitin Alain Rambach MRSA strains will grow in the presence of cefoxitin Selective and Differential media Direct detection of nasal colonization ○ Nasal colonization: nasal swab Mauve-colored colonies Other medium may utilize different chromogenic substrates Note: ○ 1 CHROMOGEN + CEFOXITIN CEFINASE (BD-BBL) ● ● ● Qualitative test to detect β-lactamase production Positive Result: Deep pink color Negative Result: No color change VANCOMYCIN - RESISTANT STAPHYLOCOCCUS AUREUS (VRSA) ● ● ● ● 1986 (Japan) ; 2002 (USA) MIC greater than or equal to 16 mg/mL Microdilution techniques vanA : derived from enterococci ○ enterococci has van c VANCOMYCIN - INTERMEDIATE SUSCEPTIBLE STAPHYLOCOCCUS AUREUS (VISAs) ● ● ● ● ● MIC 4-8 pg/mL Prolonged vancomycin therapy Vancomycin treatment failure Structural alterations within the organism's cell wall and increase cell wall synthesis Resistant to teicoplanin BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 17/ 33 ● ● ● ● ● ● ● ● ● NOTE: Similar in action to vancomycin VISAs ○ Unreliable and underreported ○ Not recommended ● ● ● ● ● NOTE: If di maidentify: Positive for Coagulase-Negative ● ● ● ● ● ● ● ● STAPHYLOCOCCUS EPIDERMIS (General Characteristics, Virulence Factors & Pathogenesis) Gram stain :gram positive cocci in clusters Colony: medium-sized, creamy, white to grey colonies, nonhemolytic (BAP) 50% of all clinical isolates Cannot ferment mannitol Catheter-related blood infections Exopolysaccharide "slime" - forms slime layer “biofilm” Delta Toxin NOTE: Mannitol Salt Agar no halo Blood agar plate white creamy to white and non-hemolytic BOX 9-3 IDENTIFYING CHARACTERISTICS OF S. EPIDERMIDIS ● Gram stain: Gram-positive cocci in clusters ● Blood agar and CNA: Medium colonies that are white to grey in color, creamy, raised growth; nonhemolytic ● Growth, but lack of fermentation, on MSA ● Coagulase negative ● DNase negative ● Susceptible to novobiocin ● ● ● ● ● NOTE: IV line connected to central artery IMPORTANT CHARACTERISTICS OF S. AUREUS Blood agar and CNA : Medium to large, raised colonies with cream to golden yellow pigmentation; ß-hemolysis Gram stain: Gram-positive cocci arranged in clusters Bound coagulase (clumping factor): Positive Free coagulase: Positive MSA: Growth and fermentation DNase: Positive Thermostable nuclease: Positive COAGULASE-NEGATIVE STAPHYLOCOCCI (CoNS) Contaminants, normal flora, nonpathogenic / limited pathogenicity Important causes of bloodstream infections prosthetic devices, infective endocarditis, UTI (frequently HAI) Foreign body-related bacteremia : medical devices Any CoNS isolated from a sterile site - identified to the species level Several species isolated from humans 1. S. epidermidis, 2. S. saprophyticus, 3. S. lugdunensis ● ● ● ● ● STAPHYLOCOCCUS SAPROPHYTICUS (General Characteristics, Virulence Factors & Pathogenesis) Variable fermentation - mannitol Resistant to novobiocin Virulence : ability to adhere to epithelial cells & urethral cells UTI - pyelonephritis & cystitis (sexually active young women), catheter-associated UTI ○ Initial colony counts to confirm : <1.0 x 105/mL ○ Epidermis is Found in medical devices central lines and shunts ○ But this one is found in the kidneys and urinary tract ■ pyelonephritis (kidney) and cystitis (urethra & bladder) foley catheter can also be NOTE: Common cause of UTI is Escherichia coli (E.coli) BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 18/ 33 STAPHYLOCOCCUS EPIDERMIDIS VS STAPHYLOCOCCUS SAPROPHYTICUS PYR BROTH TEST ● NOVOBIOCIN TEST ● ● ● ● ● ● ● ● ● ● ● ● ● Performed on CoNS isolated from urine specimens Procedure : 0.5 McFarland standard is prepared and streaked on a BAP ○ 5 mg Novobiocin disk is applied Incubate : 18-24 hrs at 35 oc Susceptible : > 16 mm (Staphylococcus epidermidis) Resistant : 6-12 mm (Staphylococcus saprophyticus) Performed on CoNS isolated from urine specimens Procedure : 0.5 McFarland standard is prepared and streaked on a BAP ○ 5 mg Novobiocin disk is applied Incubate : 18-24 hrs at 35 oc Susceptible : > 16 mm (Staphylococcus epidermidis) Resistant : 6-12 mm (Staphylococcus saprophyticus) ORNITHINE DECARBOXYLASE TEST ● ● ● ● ● ● ● ● ● ● ● Procedure : Loopful of colonies and emulsify with the Ornithine Decarboxylase broth medium Incubate : 24 hrs at 350C-370C Positive Result : purple color change Negative Result : failure to change change color after 24 hrs or change back to purple after 48hrs VOGES-PROSKAUER TEST ● Staphylococcus lugdunensis (General Characteristics, Virulence Factors & Pathogenesis) Opportunistic pathogen - aggressive infections with high mortality rate catheter-associated bacteremia, Venous prosthetic joint infections, shunt infections brain abscess & meningitis Infective endocarditis - prosthetic valves Osteomyelitis, UTI Positive : 2-hour Pyrrolidonyl arylamidase (P YR)and Ornithine decarboxylase (ODC) Production of acetoin Procedure : Loopful of colonies and emulsify with the PYR broth Incubate : 4 hrs at 350C Add 1 drop of PYR reagent after Positive Result : brilliant red-fuchsia color Negative Result : yellow or weakly orange color ● ● ● ● ● ● ● Procedure : Loopful of colonies and emulsify with the MRVP broth Incubate : 48 hrs at 350C-370C ○ Add 6 drops of alpha-naphthol and 2 drops of KOH Positive Result : red color Negative Result : yellow color PREVENTION No approved antistaphylococcal vaccines Healthcare workers (intranasal carriers) treated with topical mupirocin and rifampin (in some cases) Hospital nurseries (umbilical cord stump) (pusod) gentian violet, acriflavine, chlorhexidine, or bacitracin ○ Epidemics : all full-term infants bathed with 3% hexachlorophene (birth until discharge) CDC: screening of patients for MRSA prior admission and a variety of contact isolation procedures Note: lugdunensis aggressive mortality LAH Staphylococcus lugdunensis (Laboratory Techniques) PYR DISC TEST ● ● ● ● Procedure : Smear the colonies on the PYR disk Incubate : 2 mins ○ Add 1 drop of cinnamaldehyde after Positive Result : Deep cherry red color Negative Result : No color change NOTE: ● May automated identification ○ Vitech -colorimetric, card detects for the enzymes, ○ Maldi Tof - matrix assisted, more accurate, employs laser and detection is based on protein particles BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 19/ 33 CORRECTION SA PIC: Micrococcus Lysostaphin resistant NOT susceptible BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 20/ 33