Bacteriology of urine specimen Mrs. Dalia Kamal Eldien Msc in Microbiology lecture NO(4)

Objectives Definition of Urinary tract infection –UTI Definition of some terms related to UTI Possible pathogens of UTI Laboratory diagnosis of UTI Commonly used antibiotic in UTI Causes of sterile pyuria Case study of patient suffering from UTI

Urinary tract infection –UTI- Also known as acute cystitis or bladder infection, is an infection that affects part of the urinary tract. When it affects the lower urinary tract it is known as a cystitis (a bladder infection) and when it affects the upper urinary tract it is known as pyelonephritis (a kidney infection). Symptoms from a lower urinary tract include dysuria (painful urination) and either frequent urination or urge to urinate (or both), while those of pyelonephritis include fever and flank pain in addition to the symptoms of a lower UTI. In the elderly and the very young, symptoms may be vague or non-specific.

Innate immunity against UTI: Urinary tract infections occur more commonly in women than men, due the short female urethra. In men, the infection is low as a result of antibacterial activity of prostatic secretions Innate immunity against UTI: Protection against infection is normally given by the constant flow of urine and regular bladder emptying. Urine is a poor culture medium for many bacteria due to its acidity, high urea concentration and variable osmolality

The following is a list of terms used in UTI: Bacteriuria : The presence of bacteria in urine. Pyuria: present of WBCs (pus cell) in the urine Symptomatic patients: may be bacteriuric or abacteriuric. The diagnosis of UTI should be CLNICAL, based on symptoms and signs, and laboratory testing.

Laboratory diagnosis Possible pathogens Gram positive Staphylococcus Saprophyticus Haemolytic streptococci Gram negative Escherichia coli Proteus species Pseudomonas aeruginosa Klebsiella strains Salmonella Typhi& Paratyphi Neisseria gonorrhoeae

Also Mycobacterium tuberculosis, Leptospira interrogans, Chlamydia, Mycoplasma and Candida species. PARASITES Schistosoma haematobium, Trichomonas vaginalis, and occasionally Enterobius vermicularis, Wuchereria bancrofti and Onchocerca volvulus.

Commensals The bladder and urinary tract are normally sterile. The urethra however may contain a few commensals and also the perineum (wide variety of Gram positive and Gram negative organisms) which can contaminate urine when it is being collected. With female patients, the urine may become contaminated with organisms from the vagina. Vaginal contamination is often indicated by the presence of epithelial cells (moderate to many) and a mixed bacterial flora.

E. coli is the commonest urinary pathogen causing 60–90% of infections. UTIs caused by Pseudomonas, Proteus, Klebsiella species and S. aureus, are associated with hospital-acquired infections, often following catheterization or gynaecological surgery. Proteus infections are also associated with renal stones.

S. saprophyticus infections are usually found in sexually active young women. Infection of the anterior urinary tract (urethritis) is mainly caused by N. gonorrhoeae (especially in men), staphylococci, streptococci, and chlamydia. M. tuberculosis is usually carried by the blood to the kidney from another site of infection. It is often suspected in a patient with chronic fever when there is pyuria but the routine culture is sterile.

Laboratory diagnosis Specimen: Give the patient a sterile, dry, leak-proof container with instructions on how to collect a clean-catch MSU. Urine for culture must not be preserved with a bactericidal chemical such as thymol, hydrochloric acid, or chloroform. Macroscopical Examination: Describe the appearance of the specimen – Color of specimen – Whether it is clear or cloudy

Microscopical Exam: 1. Wet preparation Pus cell Red blood cells casts crystal yeast cells T. vaginalis motile trophozoites S. haematobium eggs bacteria (providing the urine is freshly collected) 2. Gram stained smear of the urine when bacteria present

Test the specimen biochemically Leukocyte esterase , this enzyme is specific for polymorphonuclear neutrophils (pus cells).  Is a urine test for the presence of white blood cells and other abnormalities associated with infection. It detects the enzyme from both active and lyzed WBCs. LE testing is an alternative method of detecting pyuria when it is not possible to examine fresh urine microscopically

Culture the specimen It is not necessary to culture urine which is microscopically and biochemically normal, except when screening for asymptomatic bacteriuria Estimating bacterial numbers It is necessary to estimate the approximate number of bacteria in urine because normal specimens may contain small numbers of contaminating organisms, usually less than 10,000 (104) per ml of urine. Urine from a person with an untreated acute urinary infection usually contains 100,000 (105) or more bacteria per ml.

The approximate number of bacteria per ml of urine, can be estimated by using a calibrated loop or a measured piece of filter paper. Both methods are based on accepting that a single colony represents one organism. For example, if an inoculums of1/500 ml produces 20 colonies, the number of organisms represented in1/500 ml of urine is 20, or 10.000 in 1 ml (500 X 20).

Reporting bacterial numbers Count the approximate number of colonies. Estimate the number of bacteria, i.e. colony-forming units (CFU) per ml of urine. Report the bacterial count as: Less than 10.000/ml organisms/ml (104/ml), non significant. 10.000–100.000/ml (104–105/ml), doubtful significance (suggest repeat specimen) More than 100.000/ml (105/ml), significant bacteriuria.

Example If 25 E. coli colonies are counted and a 1/500ml loop was used, the approximate number of CFU per ml of urine: 500 x 25 12.500, Such a count would be reported as: 10.000–100.000 E. coli/ml

Culture media BA & MAC or CLED, incubate aerobically at 37C, if mycobacterium suspected add LJ Colonial morphology: Appearance of some urinary pathogens on CLED agar E. coli: Yellow (lactose-fermenting) opaque, moderate, smooth colonies. Klebsiella : Large mucoid yellow colonies. Proteus : Transluscent blue-grey colonies. P. aeruginosa: Green colonies with rough periphery (characteristic color and smell). E. faecalis: Small yellow colonies. S. aureus: Deep yellow colonies of uniform color. S. saprophyticus and other coagulase negative staphylococci Yellow to white colonies.

Gram stain Biochemical tests according to the result of Gram stain Antimicrobial sensitivity test

Antimicrobial susceptibility testing Perform susceptibility testing on urines with significant bacteriuria, particularly from patients with a history of recurring UTI. Results of the testing are usually reported as: Susceptible — category implies that isolates are inhibited by the usually achievable concentrations of antimicrobial agent when the recommended dosage is used for the site of infection, it is an appropriate choice for treatment Intermediate — may be effective at a higher dosage, or more frequent dosage, or effective only in specific body sites where the antibiotic penetrates to provide adequate concentrations Resistant — not effective at inhibiting the growth of the organism; may not be an appropriate choice for treatment

Commonly used antibiotic in UTI:- Amoxicillin/Clavulanic Acid (Augmentin) 20ug/10ug Cephalothin 30ug Ciprofloxacin 5ug Nitrofurantoin 300ug Trimethoprim 1.25ug/23.75ug

Sensitivity test

Causes of sterile pyuria A recently (within last 2 weeks) treated urinary tract infection UTI with 'fastidious' organism, eg Neisseria gonorrhoeae Renal tract tuberculosis, chlamydia urethritis. False negative culture due to contamination with antiseptic. Contamination of the sample with vaginal leukocytes. Urinary tract neoplasm, including renal cancer and bladder cancer.

E.Coli in MAC

EMB media

Gram stain

Indole test

Biochemical test

Analytical profile index (API)

For more reading: 1. Nicolle LE, Bradley S, Colgan R, et al For more reading: 1. Nicolle LE, Bradley S, Colgan R, et al. Infectious Diseases Society of America guidelines for the diagnosis and treatment of asymptomatic bacteriuria in adults. Clin Infect Dis 2005;40:643-54 2. District Laboratory Practice in Tropical Countries Part 2 Second Edition-Monica Cheesbrough-