CASE #15

A 27-year-old woman who presents to the urgent care walk-in clinic with dysuria and polyuria for 3-day
duration.

History
She was married a year ago. Since then, she reports having experienced four times symptoms of dysuria,
increased frequency, and urgency and that went away after a course of antibiotics. She denies blood in her
urine, fevers, chills, flank pain, and vaginal discharge. She reports no past pregnancies or sexually
transmitted diseases.

Signs & Symptoms

For the past three days, she has once again been experiencing dysuria, increased frequency, and urgency,
so she goes to see her physician. She denies blood in her urine, fevers, chills, flank pain, and vaginal
discharge.
Her vital signs are T = 37.2oC, P = 100/min, RR = 18/min, and BP = 110/70 mmHg.
Physical examination reveals a mild tenderness to palpation in the suprapubic area, but no other
abnormalities. A bimanual pelvic examination reveals a normal-sized uterus and adnexae with no apparent
adnexal tenderness. No vaginal discharge is noted. The cervix appears normal. Her last menstrual was 1
week ago.

PATIENT ASSESSMENT
Profile 27 years old
Female
Chief Complaint Dysuria, Polyuria, Pollakiuria
Physical Temp: 37oC
Examination P: 100/min
PR: 18/min
BP: 110/70 mmHg
Supapubic area: mild ternderness to palpation
Normal size uterus
No adnexal tenderness
No vaginal discharge
Normal cervix
Patient History Maried, no past pregnancies, no STD, last menstruation a week ago
History of Illness Dysuria, Polyuria, Pollakiuria → went away after taking antibiotics

DEFINITION OF RELEVANT TERMS

Dysuria Any pain or discomfort associated with urination
Polyuria Polyuria is an excessive or an abnormally large production or passage of urine
Pollakiuria Aka extraordinary daytime urinary frequency
Suprapubic Area Anatomy of the abdominal area of the human body can be divided into 9 regions
including the suprapubic region, aka hypogastric region
Suprapubic is from latin “supra”, meaning above, and “pubis” meaning the front bone
of pelvis
Adnexae Parts adjoining an organ

INTERPRETATION OF LABORATORY RESULTS

CBC Results Reference range Interpretation
Hemoglobin 13.6 g/dL 12.0-15.0 g/dL Normal
Hematocrit 40.7% 35-49% Normal
MCV 84 fL 80-100 fL Normal
WBC 10,910/microliter 3.6-10.6 x103 / microliter Normal
URINALYSIS
Color Yellow Pale yellow, yellow, dark Normal
yellow
Transparency Cloudy Clear Abnormal
Leukocyte Large leukocytes Negative Abnormal
Nitrite Positive Negative Abnormal
pH 8 4.5-8.0 Normal
Hemoglobin Negative Negative Normal
Protein Negative Negative Normal
Glucose Negative Negative Normal
Ketones Negative Negative Normal
Bilirubin Negative Negative Normal
Specific Gravity 1.012 1.016-1.035 Normal
WBCs >100/hpf <5 cells / hpf Abnormal
RBCs 0-2/hpf 0-2 cells / hpf Normal
Squamous epithelial Rare Few Normal
cells
Bacteria TNTC Absent-few Abnormal
Others Few WBC clumps Abnormal
URINE CULTURE
A urine culture indicates approximately 106 bacterial cells/mL. A gram stain of the urine reveals gram-
positive cocci. The gram-positive bacterium is isolated and is found to be catalase positive and coagulase
negative.

FINAL DIAGNOSIS: LOWER URINARY TRACT INFECTION

Causative agent: Staphylococcus saprophyticus
Criteria for UTI:
At least 1 of the following signs/symptoms:
1. Acute dysuria or pain, swelling or ternderness of testes, epididymis or prostate
2. Fever or leukocytosis + at least 1 of these localizing criteria:
a) Acute CVA pain or Tenderness
b) Suprapubic pain
c) Gross hematuria
d) New or marked increase in incontinence
e) New or marked increase in urgency
f) New or marked increase in frequency
3. If no fever or leukocytosis, 2 or more items from a-f, are documented

Note: Does not include change in behavior, urine odor or color, or appearance of sediment in urine

EPIDEMIOLOGY
 UTIs in women are very common
 Cystitis occurs in 0.3-1.3% of pregnancies
 Acute pyelonephritis occurs in 1-2% of pregnancies
 Rates of infection are high in postmenopausal women
 Neonates specifically boys have high risk of developing UTI

PATHOPHYSIOLOGY
Urinary Tract Infections
 Most common hospital and health care-associated infection
 Urinary tract - source for the primary occurrence of bacteremia
Routes of Infection
 Ascending Route
 Most common course of infection in females
 Organisms in the GIT must be able to colonize the vaginal cavity / periurethral area
 Once organisms gain access to the bladder, they may multiply and then pass up the ureters to the
kidneys
 Occurs more often in females = short female urethra and its proximity to the anus
 Sexual activity increases the chances of bacterial contamination of female urethra
 Catheter-associated UTI
 Hematogenous / blood borne route
 Occurs as a result of bacteremia
 Less than 5% of UTIs
 Lymphatic pathways
 Increased pressure on the bladder can cause lymphatic flow into the kidneys = UTI
 Host-Pathogen Relationship
 Urine - inhibitory to some of the urethral microbiota - anaerobes
 Low pH, high or low osmolality, high urea concentration, high organic acid - inhibits growth
 Constant flushing of contaminated urine:
 Eliminates bacteria
 Maintains their numbers at low levels
 Mechanical obstruction - promote development of UTI
 Valve-like mechanism at the junction of ureter and bladder - prevents reflux of urine
 If function is compromised - urine reflux provides a direct route for organisms to reach the kidney
 Hormonal changes during pregnancy - increase the chance for urine reflux to upper urinary tract

Interstitial Disorders
 Tubulointerstitial disorders
 Disorders affecting the interstitium also affect the tubules; due to close proximity between the
renal tubules and renal interstitium

 Urinary Tract Infections

 Most common renal disease
 Lower urinary tract - urethra & bladder
 Upper urinary tract - renal pelvis, tubules, interstitium

 Cystitis
 Most frequently encountered infection of the bladder
 Can progress to a more serious upper UTI if untreated
 Seen more often in women and children
 Dysuria, frequency, urgency
 Tenderness and pain over the area of bladder
 Urine - cloudiness & bad odor
 Presence of numerous WBCs and bacteria
 Mild proteinuria, hematuria, increased pH

PYELONEPHRITIS
 Inflammation of the kidney parenchyma, calices and pelvis

Acute Pyelonephritis
 Enlarged kidneys with surface abscess
 Result of ascending movement of bacteria from lower UTI into the renal tubules and interstitium
 Ascending movement of bacteria from bladder - enhanced with conditions that interfere with the
downward flow of urine or the incomplete emptying of bladder
 Renal calculi, pregnancy, reflux of urine from bladder back to ureter (vesicoureteral reflux)
 Symptoms
 Urinary frequency
 Burning on urination
 Lower back pain
 Numerous leukocytes and bacteria
 Mild proteinuria & hematuria
 WBC cast & bacterial cast

Chronic Pyelonephritis
 Scarring on one or both kidneys and interstitial fibrosis on the pelvic wall
 Can result in permanent damage to the renal tubules and possible progression to chronic renal
failure
 Cause: congenital urinary structural defects producing reflux nephropathy
 Children; may not be suspected until tubular damage has become advanced
 Inflammatory infiltrate of WBCs, predominantly lymphocytes
 Tubules in kidneys may be dilated or constricted and contain colloid casts (crystallized mucous
secretions)
 Granular, waxy, broad casts
 Increased proteinuria & hematuria and renal concentration is decreased
Acute Interstitial Nephritis
 Inflammation of the renal interstitium followed by inflammation of the renal tubules
 Symptoms:
 Oliguria
 Edema
 decreased renal concentrating ability
 decreased GFR
 fever, skin rash
 Primarily associated with allergic reaction to medications
 Penicillin, methicillin, ampicillin, cephalosporins, sulfonamides, NSAIDS, thiazide diuretics
 Hematuria, mild to moderate proteinuria, numerous WBCs, WBC casts without bacteria, increased
eosinophils

Acute Urethral Syndrome

 Young, sexually active females
 Dysuria, frequency, urgency
 Yield fewer organisms than 105 CFU/mL on culture
 >105 CFU/mL - highly indicative of infection

STAPHYLOCOCCUS
General Characteristics
 Gram-positive cocci; catalase-positive; oxidase (-)
 Non-motile; nonsporeforming organisms; glucose fermenter
 Reduces nitrates to nitrites
 Grows in 7.5-10% NaCl
 Facultatively aerobic (except S. saccharolyticus - obligate anaerobe); grow best in aerobic conditions
 Resistant to 0.04 U of bacitracin
 Susceptible to furazolidone
 Microdase (-)
 Facultatively anaerobic
 Normal microbiota of skin, mucosal surfaces, and intestine
 Common isolates and responsible for several suppurative types of infections
 Rare strains are fastidious requiring CO2, hemin, or menadione for growth (small colony variants with
1/10 the size of the wild type strains after 48 hours of incubation)
 Microscopy:
 Spherical cells; singy, in pairs, in clusters
 Culture:
 4-8 mm
 Creamy, white or light gold
 Buttery looking
 Gray colonies (other spp.)
 Some spp are β-hemolytic (S. aureus)

Chief source of infection:

1. Shedding from human lesions
2. Fomites
3. Contaminated lesion esp. Respiratory tract and skin
4. Asymptomatic carriers among hospital staff & patient

Mode of Transmission:
1. Spread of patient’s endogenous strain to normally sterile site by traumatic induction
(surgical/microabrasions) or as a result of implantation of medical devices (shunts / prosthetic devices)
2. Person to person transmission by fomites, air
3. Transmitted from infected skin lesion

Antigenic Structure:

1. Capsule
 Slime layer / biofilm
 Allows the organisms to adhere to inorganic surfaces & impairs or inhibits the penetration of
antibiotics
 2. Peptidoglycan
 polysaccharide polymer which provide rigid exoskeleton of the cell wall; constitute 40 – 60%
of the cell weight
 Functions:
 Elicits production of interleukin-1 and opsonic antibodies by monocytes
 it can be a chemoattractant, have endotoxin-like activity, and activate complement
 composed of acetylglucosamine and acetylmuramic acid
3. Teichoic acid
 polymers of glycerol phosphate (S. epidermidis) or ribitol phosphate (S. aureus)
 Functions:
 plays important role in maintenance of normal physiologic function of the cell
 regulate the cationic environment of cell thus control the activity of autolytic enzyme
responsible for growth of cell wall
 Antiteichoic acid antibodies detectable by gel diffusion*
4. Protein A
 bacterial surface protein that has been characterized among a group of adhesins called
microbial surface components recognizing adhesive matrix molecules (MSCRAMMS)
 major protein component of cell wall
 major antigenic determinant unique to Staphylococcus aureus
 antiphagocytic (through binding to the Fc portion of IgG)

Staphylococcus aureus
 Most virulent
 aureus - gold
 Coagulase (+)
 Culture
 Golden yellow pigment (lipochrome)
 β-hemolytic - BAP
 Can be cultivated with added 7.5-10% NaCl (halophilic microorganism)
 Chiefly responsible for the various skin, wound, and deep tissue infections
 Responsible for 80% of suppurative infection in human
 Capacity to produce disease not diminised even with antibiotics - MRSA
 Dominant site of colonization: anterior nares, axilla, perineum and 10-15% human skin (MOIST
AREAS)
 Principal virulence: coagulase

Enzymes and Toxins Produced (Virulence Factors)

1. Catalase
 Heme enzyme; catalyzes the decomposition of H2O2 to water and oxygen
 Differentiates staphylococci (+) from streptococci (-)
 Aerobic catalase test - 3% H2O2
 Anaerobic catalase test - 15% H2O2
 (+): bubble formation / effervescence
 Pseudocatalase reaction - Aerococcus, Enterococcus, Rothia

2. Coagulase
 Coagulates fibrinogen in plasma
 Promotes formation of fibrin layer around staphylococcal abscess, thereby protecting the
bacteria from phagocytosis
 Reacts with thermostable thrombin-like molecule - coagulase-reacting factor (CRF) to form
coagulase-CRF complex
 97% of Staphylococcus aureus isolated in human produce the enzyme

2 types:
a. Cell-bound coagulase / clumping factor
 Rapid slide test
 Bound to cell wall
 Causes bacterial cells to agglutinate in plasma
 Clots human, rabbit, or pig plasma
 b. Unbound coagulase / free coagulase
 Tube coagulase test
 Extracellular enzyme; free from cell wall
 Causes clot formation when bacterial cells are incubated with plasma

3. Hyaluronidase (spreading-factor enzyme)

 Enhances invasion and survival in tissue
 found in the intracellular ground substance of connective tissue
 promote invasion of organism by digesting the intracellular ground substance “glue” (hyaluronic
acid) that binds connective tissue in host tissue
 Breaks down hyaluronic acid present in intracellular ground substance of connective tissues,
resulting to spread of bacteria
 Binds cells together and renders the intercellular spaces passable to pathogen

4. Staphylokinase (fibrinolysin)
 Has fibrinolytic activity
 Dissolves fibrin clot after 4 hours of incubation at 35 C
 Production by most strain of Staphylococcus

5. Lipase (fat-splitting enzyme)

 lipid – hydrolyzing enzyme
 counteracts the action of fatty substances secreted by the body
 help in the colonization of organism – oily skin surfaces
 Produced by both coagulase (+) and coagulase (-) staphylococci
 Essential for survival in sebaceous areas of the body
 Important for the formation of furuncles, carbuncles and boils

6. Deoxyribonuclease (DNAse) and Phosphatase

 heat – resistant protein
 Lowers viscosity of exudates, giving the pathogen more mobility
 Destroys DNA
 most specific test for Staphylococcus aureus
 found in cell surfaces of 90% - 96% of Staphylococcus aureus
7. Beta-lactamase
 Breaks down penicillin and other beta-lactam drugs
 90% or more of clinical staphylococci isolates are resistant to penicillin as a result of enzyme
production
8. Enterotoxins (heat-stable)
 Toxins act as neurotoxins that stimulate vomiting through the vagus nerve
 Produced by 30%-50% of isolates
 Resistant to hydrolysis by gastric and intestinal enzyme
 Produce when Staphylococcus aureus grows in carbohydrate and protein foods
 Most often EXCRETED from the cell; but some accumulate inside the cell are either injected
directly or are relased by cell lysis
 Superantigens (like TSST-1); have the ability to interact with many T cells, activating an
aggressive immune response
 Stable to heating at 100 C for 30 minutes (reheating contaminated food will not prevent
disease)
 They do not cause any detectable odor or change in the appearance or taste of the food
9. Leukocidin / Panton-Valentine Leukocidin (Cytolytic Toxin)
 Toxic to WBCs
 Prevents clearance of organism by the immune system
 Attacks and kills WBC (PMN, macrophage, monocytes)
 Pore-forming exotoxin that kills WBC; suppresses phagocytosis
 Responsible for necrotizing skin & soft tissue infections
 composed of two (2) protein components that can kills white blood cells of humans and
incapacitate the phagocytic line of the defense of the host. (cause pore formation in the cellular
membraneànecrosis and severe inflammation)
 important virulence factor in CA – MRSA infections
 Encoded on a mobile phage
10. Hemolysin (Cytolytic Toxin)
 lyse rbc by destroying their membrane
 producing tissue damage;
 abscess formation
 Chromosomal mediated
 Causes anemia - make iron availble for microbial growth

A. Alpha hemolysin
 Disrupts smooth muscle in blood vessels
 Toxic to erythrocytes, leukocytes, hepatocytes, and platelets
 A heterogenous protein that acts on a broad spectrum of eukaryotic cell membranes
 most powerful toxin that lyses rbc of various animals (platelets and macrophages= tissue
damage)
 produce hemolysis in blood agar medium
 Predominant hemolysin produced by S. aureus
 Destroys RBC, platelets, macrophages
 Causes severe tissue damage
B. Beta hemolysin (hot-cold lysin / sphingomyelinase C)
 Destroys sphingomyelin and RBC around nerves
 Catalyzes the hydrolysis of membrane phospholipids resulting in cell lysis
 Has enhanced hemolytic activity on incubation at 37 C and subsequent exposure to 4 C
 Heat labile
C. Delta hemolysin
 Less toxic as compared to α and β lysins
 Cytolytic to erythrocytes and demonstrates nonspecific membrane toxicity
 Causes injury in RBC in culture and produces edematous lesions
 aureus, epidermidis, haemolyticus
D. Gamma hemolysin
 Destroys RBC
 Associated with the Panton-Valentine leukocidin (PVL)
 Produced by all strains of S. aureus
11. Exfoliatin A & B (superantigens) / Epidermolytic toxin A & B
 Serine protease
 Splits the intracellular bridges of the epidermidis → sloughing of epidermidis
 Burn like effect on patients
 Causes epidermal layer of the skin to slough off - stratum granulosum
 Causes scalded-skin syndrome / Ritter disease
12. Toxic Shock Syndrome 1 (TSST-1) / Enterotoxin F / Pyrogenic exotoxin C
 Chromosomal-mediated toxin
 SUPERANTIGEN
 binds to major histocompatibility class (MHC) Class II molecules, yielding T – cell stimulation,
which promotes the protean manifestations of the TOXIC SHOCK SYNDROME
 Causes almost all cases of menstruating-associated TSS
 Stimulates production of large amount of cytokines that are responsible for the symptoms
 Absorbed through the vaginal mucosa, allowing the systemic effects seen in TSS
 associated with: fever, shock, multisystem involvement, including desquamative skin rash
13. Protein A
 Immunologically active substance found in the cell wall
 Antiphagocytic by competing with neutrophils for the Fc portion of specific opsonins

Differential Tests for Staphylococcus aureus

1. Coagulase test
 Best single criterion of pathogenecity of S. aureus
 Reagent: rabbit plasma

a) Slide method
 Used to screen catalase (+) colonies
 Detects all cell bound coagulase / clumping factor on the surface of the cell wall which
reacts with the fibrinogen in the plasma
 Any negative slide tests should be confirmed with the tube method, because about 5% of
S. aureus do not produce clumping factor
  (+): clot / coagulum formation within 30 seconds
 Other slide coagulase (+): S. lugdunensis & S. schleiferi
b) Tube method
 Sensitive method; definitve test
 Detects extracellular / unbound / free coagulase
 Procedure: inoculate a tube containing plasma & incubate at 35 C
 (+): clot / coagulum formation after 1-4 hours of incubation
 If no clot appears after 4 hours of incubation, the tube should be left at RT for an
additional 20 hours of incubation
 Other tube coagulase (+): S. hyicus, intermedius, delphini, schleiferi subsp. coagulans

2. Mannitol Fermentation test

 Used to differentiate pathogenic from nonpathogenic staphylococci
 Pathogenic staphylococci ferment mannitol and produce acid
 MSA (1% mannitol + 7.5 NaCl) is both a selective and differential medium
 pH indicator: phenol red (will turn to yellow color)
 (+): yellow color - S. aureus (colonies surrounded by yellow halo)
 S. Saprophyticus - some strains also ferments mannitol (resembles S. aureus on MSA)

3. Tellurite Glycine Agar

 (+): jet black colonies of S. aureus

4. Polymyxin Sensitivity test

 (+): resistant - S. aureus

5. Voges-Proskauer test
 Differentiates S. aureus (+) from intermedius (-)
 (+): acetoin / acethylmethyl carbinol production - pink color

6. DNAse Test
 Used to detect DNAse activity of S. aureus
 Medium and reagent: DNA medium & methyl green dye
 (+): clearing of the dye (clear zone)

Methicillin-resistant Staphylococcus aureus (MRSA)

 Acquired after prolonged hospital stay (ICU & burn patients); proximity to patients with MRSA;
after receiving broad spectrum antibiotics; nasal carriage
 May also be resistant to other semisynthetic penicillins
 Controlled by proper isolation of the organism; rapid identification of the bacteria; hand hygiene;
control and treatment of sources
 Chromogenic test:
 (+) mauve-colored colonies within 24 hours & confirmed within 48 hours
 Rapid tests:
 IDI-MRSA test & BD Gene Ohm assay (results within 2 hours) using nasal swab as
specimen

Staphylococcus saprophyticus
 Associated with community-acquired UTI in young, sexually active females
 Adheres more effectively to the epithelial lining in the urogenital tract than other CoNS
 Rarely found on other mucous membranes or skin surfaces
 Habitat: human skin, peri-urethral and urethral area
 2nd most common pathogen associated with UTIs; next to E. coli
 Virulence factors:
 Adherence to urothelial cells by means of a surface-associated protein, lipoteichoic acid
 A hemagglutinin that binds to fibronecton, hemolysin
 Production of extracellular slime
 Infects humans through sexual intercourse / contact with animals
  Pathology:
 UTI in young sexually active female (CYSTITIS)
Symptoms:
 Burning sensation when passing urine
 Urge to urinate more often than usual
 Dripping effect after urination
 Weak bladder
 Bloated feeling with sharp razor pains in the lower abdomen around the bladder
and overy areas
 Razor-like pains during sexual intercourse
 Culture:
 White opaque; creamy white
 Pin-head slightly larger colonies
 50% of the strains produced yellow pigment
 Nonhemolytic - BAP
 Biochemical tests:
 MSA (-/+) (variable)
 Catalase (+)
 Coagulase (-) (CoNS)
 DNAse (-)
 Non-mannitol fermenter
 Novobiocin & Nalidixic acid resistant
 Urine culture
 10,000 CFU/mL (significant finding)
 Antimicrobial test
 Resistant to 5 ug Novobiocin (6-12mm ZOI)

Staphylococcus epidermis
 Normal flora of skin
 Represents 50-80% of all coagulase (-) staphylococci
 Skin contaminants; recovery from cultures doesn’t always indicate presence of disease
 Contaminant of medical instruments - catheters, CSF shunts and prosthetic heart valve implants
 Secretes poly-gamma-DL-glutamic acid which provides adherence to devices
 Cause hopsital-acquired infections
 Account for a large number of nosocomial & opportunistic pathogen in immunocompromised
patient
 Habitat: skin & mucous membrane
 Pathology:
 Bacteremia associated with the use of catheter, orthopedic/prosthetic implants, CNS shunt,
pacemaker
 Stitch abscess, endocarditis, UTIs
 Culture:
 Small-medium sized
 Nonhemolytic
 Nonpigmented
 White opaque
 Pinhead colonies - BAP
 Biochemical test:
 MSA (-)
 CoNS
 Antimicrobial test:
 Susceptible with 5 ug Novobiocin (16-27 mm)

Staphylococcus lugdunensis
 CoNS by tube method
 Can be confused with S. aureus if slide coagulase method is performed
 More aggressive than other CoNS in its ability to be infective - associated w/ catheter-related
bacteremia & endocarditis
 Can cause both community-associated & hospital-acquired infections
 Can be more virulent and can clinically mimic S. aureus infections
 Contains mecA gene which codes for oxacillin resistance
  Related infections:
 Infective endocarditis
 Meningitis
 Septicemia
 Skin & soft tissue infections
 UTI
 BAP:
 Smooth, glossy with slightly doomed center
 Cream to yellow color and may be beta hemolytic
Resistant Genes Produced by Staphylococci
 erm genes
 Class of enzymes inactivating genes, code for methylation of 23s rRNA, which results in
resistance to erythromycin and either inducible or constitutive resistance to clindamycin
 May not be detected in routine susceptibility testing
 Also confer cross resistance to the macrolides (erythromycin) and streptogramins (quinupristin)
 msr A gene
 Codes for efflux mechanism
 Results in resistance to erythromycin but susceptibility to clindamycin

TREATMENT & MANAGEMENT

Ciprofloxacin
 Inhibit DNA synthesis by binding DNA gyrase and topoisomerase IV

Nitrofurantoin
 Exact mechanism unknown
 Probable bacterial enzyme targets and direct DNA damage
 Treatment of UTI only
 100mg twice a day for 5 days or 7 days (complicated)

Trimethoprim
 Sulfamethoxadole (TMP-SMX)
 Interfere with folic acid pathyway
 S3 binds dihydropteroate synthase
 T binds dihydrofolate reductase
 160/800 mg twice a day given alternatively

Norfloxacin
 Inhibit DNA synthesis by binding DNA gyrase and topoisomerase IV

Resistant to: Novobiocin

The novobiocin susceptible CoNS: The novobiocin resistant CoNS:

S. Epidermidis S. Saprophyticus
S. capitis S. Cohnii
S. Haemolyticus S. Kloosii
S. hominis subsp hominis S. xylosus
S. Lugdunensis
S. Saccharolyticus
S. warneri

COMPLICATIONS
 Urethritis
 Acute Pyelonephritis

PREVENTIONS
 Healthy sanitations
 Avoid potentially irritating feminine products
 Deodorant sprays, douches, powders
 Avoid holding urine for long period of time
 Keep your genital area dry
 Change your birth control method
 Taking enough water
 Empty your bladders soon after intercourse

LABORATORY DIAGNOSIS
Differential Tests between Staphylococci & Micrococci
1. Bacitracin / Taxo A disk test
 0.04 units bacitracin
 Performed on BAP / MHA
 Result:
 Micrococci - susceptible (≥10 mm ZOI)
 Staphylococci - resistant
2. Furazolidone Susceptibility Test
 100 ug furazolidone
 Performed on BAP
 Result:
 Staphylococci - susceptible (≥15 mm ZOI)
 Micrococci - resistant (6-9 mm)
3. Modified Oxidase / Microdase Test
 Reagent paper: tetramethyl-p-phenylenediamine in dimethylsulfoxide
 (+): micrococci - blue within 2 minutes
4. Lysostaphin Sensitivity Test
 Staphylococci - 10-16 mm ZOI (sensitive)
 Micrococci - resistant
 S. Aureus lysed with lysostaphin
5. Growth on Furoxone-Tween 80-oil red O agar
 (+): micrococci
6. Acid production from Glycerol (with erythromycin)
 (+): staphylococci
7. Oxidation-Fermentation (OF) reaction
 Staphylococci ferment glucose while micrococci fail to produce acid under anaerobic condition

Specimen: aspirated secretions (best sample), purulent exudates, joint fluids

1. Gram stain
 Gram (+) spherical cells
 Singly, in pairs, in clusters
 Should be performed on young cultures
 Old cultures = decreased ability to retain CV → gram variable, negative
 Initial presumptive identification
2. Culture
 Culture media:
 BAP
 MSA
 PEA
 CNA
 CAP
 BHI
 thioglycollate
 MSA, CNA, and PEA - for heavily contaminated specimens
 CHROMagar staph aureus
 proprietary selective & differential medium for isolation of aureus
 Contains cefoxitin - MRSA is resistant
 Mauve-colored colonies
 Staphylococci grow easily on routine culture media
 CoNS recovered from sterile sites, and those sites associated with indwelling devices, should be
considered potential pathogens
 Low colony count of S. saprophyticus (urine culture) is considered significant
3. Catalase test
 Reagent: 3% H2O2
 (+): bubble formation / effervescence
 Differentiates members of family Micrococci / Staphylococci (+) from Streptococcus (-)
 The colonies which will be used for this test should not be taken from BAP because of the
presence of peroxidase in that medium
 Principle:
 H2O2 --> H2O + O2 (bubbles)
 Used to detect the presence of cytochrome oxidase
 Performed after gram staining
4. Coagulase test
 Reagent: rabbit plasma
 (+): clot / coagulum formation
 S. Aureus is frequently separated from less pathogenic species by being coagulase positive
 Isolates that do not produce either clumping factor or staphylocoagulase are reported as
coagulase (-) staphylococci
5. Mannitol Fermentation Test
 (+): yellow halo around colonies of mannitol fermenting-staphylococci
6. DNAse test
 0.1N HCl - agglutinate or precipitate protein
 0.1% toluidine blue
 (+): pink color - cell died
 (-): blue color - cell is alive
7. Pyrrolidonyl Arylamidase Test
 Differentiates coagulase-positive staphylococci
 Substrate: pyroglutamyl-B-naphthylamide (L-pyrrolidonyl-B-naphthylamide)
 Final reagent: p-dimethylaminocinnamaldehyde
 End products: L-pyrrolidone & B-naphthylamine (red color)
 (+): S. lugdunensis, intermedius, schleiferi
 (-): S. aureus
8. Voges-Proskauer (VP) test
 Differentiates coagulase-positive staphylococci
 Reagent: alpha naphthol and KOH
 End product: acetoin
 (+): S. aureus, lugdunensis, haemolyticus, schleiferi
 (-): S. intermedius
9. Antimicrobial Testing
 Drugs for treatment of staphylococcal infections: methicillin, oxacillin, nafcillin, cloxacillin, and
dicloxacillin (penicillinase-resistant penicillin drugs)
 Oxacillin - class representative most commonly used
 When an isolate shows resistance to one of the penicillinase-resistant penicillins, it must be
considered resistant to the entire group
A. Vancomycin agar screen plate
 screened using 6ug/ml VA that is incorporated to BHI agar
 (+) = any growth of the medium
B. Oxacillin screen plate (MHA w/ 4%NaCl and 6ug/ml oxacillin)
 used to screen MRSA clinical sample
 (+) growth of more than 1 colony
 (-) absence of growth on the agar plate
 resistant to oxacillin à Beta-lactam drugs report as resistant
C. Cefoxitin disc diffusion (30ug)
 preferred method for detecting methicillin resistance in both S. aureus and S. lugdunensis
D. Double-disc diffusion test (D –test)
 detect inducible clindamycin resistance in staphylococci
 15ug E and 2ug DA (15-26 mm apart on MHA or BAP)
 (+) = flattening on one side (near erythromycin disc) of the clindamycin ZOI, which gives
appearance of a “D-zone”
 (-) absence of blunting indicates E resistance
References:

Bacteriology by Rodriguez: Staphylococci page 100-110

Strasinger, Susan King., and Marjorie Schaub Di Lorenzo. Urinalysis and Body Fluids. 6th ed. Philadelphia:
F.A Davis, 200

Tille, P. (2017). Bailey & Scott’s Diagnostic Microbiology. St. Louis, Missouri :Elsevier

(For Renal Anatomy, Physiology, and Urinalysis, refer to AUBF notes)