Lecture : Practical UTI

important Extra notes "ﻻ ﺣول وﻻ ﻗوة إﻻ ﺑﺎ اﻟﻌﻠﻲ اﻟﻌظﯾم" وﺗﻘﺎل ھذه اﻟﺟﻣﻠﺔ إذا داھم اﻹﻧﺳﺎن أﻣر ﻋظﯾم ﻻ notes Doctors ﯾﺳﺗطﯾﻌﮫ ، أو ﯾﺻﻌب ﻋﻠﯾﮫ اﻟﻘﯾﺎم ﺑﮫ . Objectives: 1- to Know the important steps in specimen collection and transport to the lab. 2. How to process urine Specimens in the lab. – Urine microbiological and biochemical analysis. – Organisms culture and identification. – Antibiotic susceptibilitytesting. – Results interoperation. 3. Know the clinically important etiological Organisms associated with UTI, their identification and susceptibility testing.

• اﻟﻌﻣل ﻣﻌﺗﻣد و ﺷﺎﻣل ﻋﻠﻰ ﻣﺣﺎﺿرات اﻟدﻛﺎﺗرة : Overview • ﻻ ﺗﺧﺎﻓون ﻣن ﻋدد اﻟﺳﻼﯾدز , اﻟﺳﻼﯾدز ﺧﻔﯾﻔﺔ و ﻏﺎﻟﺑﯾﺔ اﻟﻣﻌﻠوﻣﺎت ﻣﻌروﻓﺔ وﺳﮭﻠﺔ اﻟﻣﺣﺎﺿرة ﺗﺗﻛﻠم ﻋن أرﺑﻊ ﻣﺣﺎور أﺳﺎﺳﯾﺔ : إن ﺷﺎء ﷲ collecting urine 1- • ﻓﻲ ﻧﮭﺎﯾﺔ اﻟﻣﺣﺎﺿرة ﻓﯾﮫ ﻛﯾﺳﯾز ﻣن ﺳﻼﯾد اﻟدﻛﺎﺗرة , ﻟﻛن اﻟﺣل ﻣن اﻟﺗﯾم analysis urine 2- • رﻛزوا ﻋﻠﻰ .. groth + stain + culture their and the 3- (وﺑﺎﻟﺗوﻓﯾﯾق اﺟﻣﻌﯾﯾن) Testing Susceptibility Antimicrobial 4- Important aspects of Microbiologic Examination of UTI: • Urine collection • Urine analysis • Interpretation of microbiology • laboratory result 1- Urine collection Midstream urine (MSU): • The urine collected in a wide mouthed container from patients a mid stream specimen is the most ideal for processing • Female patients pass urine with a labia separated and mid stream sample is collected Adhesive bag

Types of specimens: Suprapubic aspiration (for children)

Clean catch Catheter sample: (used with elderly) • Urine specimens for laboratory investigations can be collected from catheterized patients as shown (left). The second port is for putting fluids into the bladder (right). • Urine from the drainage bag should not be tested because it may have been standing for several hours. 1- Urine collection Transport media

Dip slide (one side is Cled Sterile urine media and the other is container MacConkey agar or blood agar) 2- Urine processing SPECIMEN PROCESSING:

C-Antimicrobial B-Culture and D-Results A-Urine analysis Susceptibility identification (ID) interpretation testing

Microbiological

Biochemical 2- Urine processing Urine analysis is subdivided into biochemical and microbiological testing: A- Urine analysis

Biochemical Microbiological (dipstick)

leukocyte esterase Microscopic Macroscopic (next slide) Microbiological Physical (with naked Nitrate test (next slide) (Microscopic Urine Examination (WET eyes) MOUNT)) Macroscopic: Microscopic: • Color +(blood) PH A- Cell-counting chamber (WBC,RBC) • Odor • Turbidity

Glucose B-Parasite (Ovum, Trichomonas, Yeast) A C- Casts Bilirubin

Protein* *For kidney impairment A B C 2- Urine processing Testing for UTI: o Midstream clean catch with dipstickanalysis: It is negative in case of gram positive • Nitrite possitvie → for gram-negative bacteria which can convert nitrate ü to nitrite (sensitivity 92-100%, lowspecificity); ü false negative with bacteria that do not reduce nitrate ü gram-positive bacteria ü excess dietary Vitamin C

• Leukocyte esterase + → indicates presence of white blood cells,

ü (sensitivity 75-95%, specificity 94-98%)– ü Dipstick results may be affected by medications/dyes, ie pyridium, nitrofurantoin, metronidazole, bilirubin, methylene blue, Vitamin B complex 2- Urine processing B- CULTURE AND IDENTIFICATION: 1- Culture media 2- Urine inoculation and reading of culture 3- Identification of cultured organisms Culture media :

1-Blood Agar 2-MacConkey Agar 3-CLED Agar Selective culture medium, for Enriched culture medium, for culturing Differential culture medium, showing both detection and fastidious* microorganism and observing the lactose and non- lactose fermenting colonies. of E. coli and coliform bacteria in hemolytic reaction (Beta,Alpha,Gama). LFC = Pink NLFC = Colorless or appear same as the medium urine.

Useful for gram positive Useful for gram negative Useful for gram negative

*ﯾﻌﻧﻲ ﺗﺗطﻠب اﺣﺗﯾﺎﺟﺎت ﺧﺎﺻﺔ وﻣﻌﻘدة ﻣن ﻧﺎﺣﯾﺔ اﻟﻐذاء ﻋﺷﺎن ﺗﻧﻣو. 2- Urine processing Urine inoculation: Labartory examination of urin Smi-Quantitative Culture of Urine Sample Quantitative (Colony counts): + 1 + 2

+ 3 1. a urine sample is streaked on surface of Blood and CLED agar / McConkey agar with a special loop calibrated to deliver a known volume.

Over night incubation

2. Isolation of colonies, Biochemical tests, Drug susceptibility test. Over night incubation

results 2- Urine processing ID of cultured organisms Quantitative (Colony counts): A urine sample is streaked We identify the causative agents by using on surface of blood agar these methods : 1 plate and Cled agar/Mc Conkey agar • Biochemical tests

• Type of

2 Isolation of colonies • Serological tests

3 Results 3-The Microorganism Only in Male’s slides

Organism Culture (colony) Oxidase

Lactose fermentative - E.coli Negative bacilli (pink) Indole +

Lactose fermentative Klebsiella (mucoid)

Lactose non- _ Proteus Negative bacilli fermentative + (colorless) Lactose non- Pseudomonas Negative bacilli fermentative + (greenish)

Bile novabiocin S.saprophyticus Cocci in clusters White () Esculin

+ - R N/A Gray (blood culture) Enterococcus Cocci in chains - N/A N/A + 3-The Microorganism Etiological Agents of UTI: Bacteria Parasite Fungi

GRAM NEGATIVE GRAM POSITIVE Other Escherichia coli Enterococcus Candida albicans Klebsiella Staphylococcus Schistosoma haematobium saprophyticus Proteus Streptococcus agalactiae Tricomonas vaginalis (group B) Other Enterobacteriaceae Staphylococcus aureus1 (Enterobacter, Citrobacter….) (Associated with staphylococcemia) Pseudomonas aeruginosa 3-The Microorganism Etiological Agents of UTI:

Causes of UTI's Outpatients Inpatients (%) (%) Escherichia coli 53-72 18-57 Coagulase negative 2-8 2-13 Staphylococcus Klebsiella 6-12 6-15 Proteus 4-6 4-8 Morganella 3-4 5-6 Enterococcus 2-12 7-16 Staphylococcus 2 2-4 aureus Staphylococcus 0-2 0.4 saprophyticus Pseudomonas 0-4 1-11 Candida 3-8 2-26 3-The Microorganism

Complicated aetiology of UTI:

The Microorganism Percentage % Escherichia coli 21 – 54 Klebsiella pneumoniae 1.9 – 17 Enterobacter species 1.9 – 9.6 Citrobacter species 4.7 – 6.1 Proteus mirabilis 0.9 – 9.6 Providencia species 18 Pseudomonas aeruginosa 2 – 19 Enterococci species 6.1 – 23 3-The Microorganism Etiological Agents of UTI:

Gram-positive cocci Gram-negative bacilli

Staphylococci Streptococci Enterobacteriaceae Non-Enterobacteriaceae Catalase + Catalase - Nitrate +

Coagulase* + Coagulase - Streptococcus (group B) Escherichia coli Enterococci Klebseilla Lactose fermentator & Capsulated Proteus Staph. Aureus Staph. Saprophyticus Enterobacter Hematogenous Staph. Epidermidis Citrobacter infection only Sexually active women Childbearing age

*Coagulase test done only for staph. 3-The Microorganism 3-The Microorganism Escherichia Coli

Morphology Microscopic appearance Gram negative bacilli

MacConkey agar showing growth of CLED* agar showing growth of Lactose fermenter Lactose fermenter yellow Culture Pink colonies colonies LFC LFC

Indole Identification Reactions Test: Positive APE 20 E test***

*CLED= Cystine lactose electrolyte deficient agar. **Video 3-The Microorganism Klebseilla spp. (Klebsiella pneumoniae)

Morphology Microscopic appearance Gram negative bacilli

MacConkey agar showing CLED agar showing growth growth of Lactose of Lactose fermenter Fermenter Mucoid Pink Mucoid yellow colonies Culture colonies LFC LFC

*Indole Reactions Identification Test: Negative APE 20 E test

*The is a biochemical test performed on bacterial species to determine the ability of the organism to convert tryptophan into the indole. 3-The Microorganism Proteus spp.

Morphology Microscopic appearance Gram negativebacilli

Blood culture plate CLED {cystine-lactose- Culture showing warm of electrolyte-deficient} Proteus Inhibits The Proteus Swarming

Proteus is * Urease positive. Urease splits urea into ammonia and alkalinizes Identification the urine with production of APE 20 E test crystals.

*Urease test, is a rapid diagnostic test for diagnosis of Helicobacter pylori. The basis of the test is the ability of H. pylori to secrete the urease enzyme, which catalyzes the conversion of urea to ammonia and carbon dioxide. 3-The Microorganism Pseudomonas spp

Morphology Microscopic appearance Gram negative bacilli

MacConkey agar showing Nutrient Agar showing growth of Non-Lactose growth of Pseudomonas Culture Fermenter pigmentation Mucoid Pink colonies LFC

Identification

*Oxidase positive test APE 20 E test

*The is used to identify bacteria that produce cytochrome c oxidase, an enzyme of the bacterial electron transport chain. (note: All bacteria that are oxidase positive are aerobic). 3-The Microorganism Three API 20E strips : Only in Male’s slides Staphylococcus aureus Staphylococcus epidermidis Golden colonies (yellowish) white colonies

a.Immediately after inoculation b.After 24 hours incubation c.That in (b) after addition of reagents to certain wells. The organisms here is Escherichia coli. Here the first carbohydrate well (glucose). Staphylococcus spp. is also used for the nitrate reduction test. 3-The Microorganism Tests explanation

NOVOBIOCIN TEST CATALASE TEST**extra COAGULASE TEST

Staphylococcus Staphylococcus Coagulase is a protein enzyme made by several saprophyticus epidermidis microorganisms that enables the conversion of (resistant- (sensitive- fibrinogen to fibrin. In the laboratory, it is used ) Novobiocin ) to distinguish protein enzyme produced between different types of Staphylococcus isolates. Importantly, S. aureus is generally coagulase-positive, meaning that coagulase negativity usually excludes S. aureus. **extra

*Coagulase test is used to differentiate Staphylococcus aureus (positive) from Coagulase Negative Staphylococcus (CONS). Coagulase is an enzyme produced by S. aureus that converts (soluble) fibrinogen in plasma to (insoluble) fibrin. 3-The Microorganism Gram Positive Cocci

Gram Positive Cocci staphylococci Streptococcus enterococci (group B) Coagulase- positive (Staph. aureus) very dangerous Coagulase negative (Staph. saprophyticus)

Coagulase negative (Staph. epidermidis) 3-The Microorganism Staph. epidermidis Staph. aureus Rarely causes UTI Microscopic Gram positive Microscopic Gram positive cocci appearance cocci in clusters appearance in clusters

culture Blood culture plate culture Blood culture showing growth of plate showing white colonies growth of golden yellow colonies tests Coagulase test Negative

tests Coagulase test = Catalase test = Positive Positive

Novobiocin test Catalase test = Sensitive Positive 3-The Microorganism Seen in pregnant Staphylococcus saprophyticus Streptococcus agalactiae (group B)and neonate Microscopic Gram positive Microscopic Gram positive cocci in appearance cocci in clusters appearance chains

culture Blood culture culture Blood culture plate plate showing showing growth of Beta growth of white -haemolitic colonies colonies tests Coagulase test tests CAMP test positive =Negative

Catalase test = positive Catalase test = Negative

Novobiocin Test Streptics: Resistant Mix bacterial colony -group various with أھم اﺧﺗﺑﺎر ﻷﻧﮫ ﯾﻔرﻗﮭﺎ specific antisera on slide a ﻋن ﺳﺗﺎف اﺑﯾدﯾرﻣس 3-The Microorganism Enterococci From the previous slide: * to see it better* Microscopic Gram positive appearance cocci in chains

culture Blood culture plate showing growth of Beta- haemolitic colonies tests Catalase test = Negative

positive bile Esculin hydrolysis test

Both Group D streptococci and enterococci produce a positive (left) bile Esculin hydrolysis test. 3-The Microorganism Fungi and Parasites causing UTI:

Fungi Parasite

Candida albicans Schistosoma haematobium

It is a type of yeast that is dimorphic fungus since it (urine: eggs 115-170 x 45-65 micrometres) grows both as yeast and filamentous cells. (primates) 3-The Microorganism Candida albicans

Morphology Gram positive cocci in chains

Candida albicans on Candida albicans Culture on blood agar Sabouraud's Dextrose Media (SDA)

Identification

Chlamydospore= test Germ tube test Positive Positive*

*The bacteria elongates after applying the test. 4-AST Antimicrobial Susceptibility Testing (AST): Methods of AST: o Disk (agar) Diffusion Method:

ھذا اﻻﺧﺗﺑﺎر ﺑﺑﺳﺎطﺔ ﻓﯾﮫ ﺳﺗرب ﻓﻲ اﻟﻧص ﯾﺣﺗوي ﻋﻠﻰ اﻧﺗﻲ ﺑﺎﯾوﺗك Diffusion ﺑﺗﻛﯾز ﻣﻌﯾن اﻟﻠﻲ ھو 1 وﻛل ﻣﺎ ارﺗﻔﻊ اﻟﺗرﻛﯾز ﻋن رﻗم واﺣد ﻣﻌﻧﺎه ان اﻻورﻗﺎﻧزم أﻛﺛر رﯾزﯾﺳﺗﺎﻧس + ھذا اﻻﺧﺗﺑﺎر دﻗﯾق ﺟدا :test o E • Is a well-established method for antimicrobial resistance testing in microbiology laboratories. • Consists of a predefined gradient of antibiotic concentrations on a plastic strip • Used to determine the Minimum Inhibitory Concentration (MIC) of § Antibiotics E test § Antifungal agents § Antimycobacterial agents

ﯾﺣﺗوي ﻋﻠﻰ antibiotic واﺣد ﻟﻛن ب concentration ﻣﺧﺗﻠف Only in female’s slides The Antibiotic Sensitivity Testing Method:

1- Select well- isolated colonies 2- Inoculum suspension 3- Spread the inoculum

4-Apply antibiotics disks 5- Read the result Staphylococcus aureus

P P C C Gentamisin (CN) : 12 - 15 Chloramphenicol (C) : 12 - 18 Penicilin (P : 28 - 29

R (Resistant) ; S (Sensitive)

CN CN

اﻟدﻛﺗورة ﻗﺎﻟت : اﺣﻧﺎ ﻣﺎراح ﻧدﺧﻠﻛم ﺑﺎﻻﻧﺗﻲ ﺑﺎﯾوﺗﻛس , ﺑﯾﻛون .… growth the + stain the describe choice of drug the about ask will we maybe But ﺑس ﻣﺎراح ﯾﻛون دﯾب

ﻟذﻟك ادرﺳوا ﺑذﻛﺎء (; Antibiotic Susceptibility Test: Only in female’s slides

o Enterobacteriacae: *You can READ it o Pseudomonas spp: • First line UTI treatment: – Ampicillin – TMP/SMX – Ciprofloxacin – Gentamicin – Amikacin – Nitrofunatoin Antibiotic Susceptibility Test: Only in female’s slides

o Staph. Aureus: *You can READ it o MRSA *You can READ it o Staph. saprophyticus

Methicillin Sensitive Staph Aureus (MSSA)

Novobiocin Test Resistant Bacterial Resistance to UTI Antibiotics: Results interpretation: Only in female’s slides *You can READ it • Nitrofurantoin • high probability of UTI requiring treatment ü E. coli <5% • If midstream clean catch positive for ü Other uropathogens 15-20% ü pyuria ü Not active against: ü microscopic hematuria, with or without positive nitrites § Proteus, ü + UTI symptoms § some Enterobacter • Presence of pyuria without bacteria on culture (sterile pyuria) § Klebsiella rule out: • TMP-SMX (Bactrim) ü Tuberculosis ü 10-22% varies geographically ü Interstitial cystitis (IC) • Fluoroquinolones (Cipro) ü Chlamydia urethritis ü therapy for acute uncomplicated cystitis ü Kidney disease (stone, glomerulonephritis) • Ampicillin ü 30% of E. coli resistant *You can READ it *You can READ it Only in female’s slides Only in female’s slides Emerging resistant to Ampicillin and TMP/SMX *You can READ it

Antibiotic MIC Interpretation Ampicillin ≥ 32 R Cephalexin ≤ 4 S Ciprofloxacin ≤ 4 S TMP/SMX ≥ 2 R Nitrofurantoin ≤ 16 S Gentamicin ≤ 8 S Ceftriaxone ≤ 1 S Imipenem ≤ 1 S Organism E coli Source urine Only in female’s slides Extended spectrum β-lactamase (ESBL) *You can READ it *You can READ it producing E coli:

Antibiotic MIC Interpretation Ampicillin ≥ 32 R Cephalexin ≥ 46 R Ciprofloxacin ≤ 4 S TMP/SMX ≥ 2 R Nitrofurantoin ≤ 16 S Gentamicin ≤ 8 S Ceftriaxone ≥ 46 R Ceftazidime ≥ 46 R Imipenem ≤ 1 S ESBL positive E coli Case 1 Only in Male’s slides

The blood agar plate and CLED plate provided were inoculated with a sample of urine from a patient with a suspected urinary tract infection. Examine the plates and photographs provided. •Identify the colonies on the blood agar plates and photographs. The photographs show the results of the Gram stain of each colony type. •Large colonies are Gram (Negative) .and small colonies are Gram (positive)

stain Gram agar Blood plate CLED اﻟﺣل اﻟﻠﻲ ﺑﺎﻟرﻣﺎدي ﺣل اﻟﺗﯾم Case 2 Only in Male’s slides

These Blood agar and CLED agar plates were inoculated with MSU from a 45 years old man suspected of having bladder stone and complaining of burning micturation. Urine examination showed : Moderate number of WBC and a PH of 8 Cled

A) What is the likely this pathogen? Proteus mirabilis B) How would you confirm the identity of this pathogen? Urease test Blood agar C) What is the role of this organism in forming stones? Urease splits urea into ammonia; and alkalinizes the urine with production of crystals

Mention one organism from each of the following which may cause urinary tract infection

A) Bacteria …………………………….. B) Parasites …………………………… C) Fungi ………………………………

اﻟﺣل اﻟﻠﻲ ﺑﺎﻟرﻣﺎدي ﺣل اﻟﺗﯾم GOOD LUCK! MICROBIOLOGY TEAM: • Nawaf alkhudhayri (leader) • Shrooq Alsomali (leader) • Yousef aljebrin • Hanin bashaikh • Saud alshenafi • Nada aldakheel • Mohammed alghandour The Editing File • Meshal alayidi

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