Microbial agents of

Urinary Tract Infections

A.R. Sultan, MD., DMM., MScI&I

MEDICAL FACULTY
UNIVERSITY OF NUSA CENDANA
2013
 Incidence of UTI
• Third most common after respiratory and gastrointestinal infections
• The second most common cause of bloodstream infections
• Approximately 36% of all hospital-acquired infections, and 80% are
associated with urinary catheterization
• Referred to as the largest source for antibiotic resistant organisms

Klevens RM., et al 2007

 Urinary tract infections

• Invasion and colonization of microbes on urinary tract – Bacteriuria

• Wide clinical range: from asymptomatic bacteria to fulminant sepsis

Transmission
• Ascending infection
from normal flora of skin
and gastrointestinal
tract (most frequently)
• Descending infections
(hematogenous)
 Predisposing factors
Blockage → Bacterial colonization → UTI

Woman has high risk for UTIs

 Anatomical factors
(anatomical factor)
• Short urethra - Woman
• Congenital malformation
In healthy women;
 Mechanical factors
 Bacteriuria increases with age:
• Scars on urinary tracts
• Tumor – 1 % among 5-14 years old
• Stone – >20% among >80 years old
• Urinary catheterization  Maybe initiated by:
 Hormonal or associated - Post-menopausal (Low
diseases estrogen) decreases commensal
• Diabetic flora growth
• Pregnancy - Flora alteration by medication
• Immunosuppression
• Age
 Urine Taking
Morning urine and before antibiotics medication

How:
1. Mid-stream urine
- OUE are sterilized
- Discard the first flow
- Collect the next urine flow
2. Plastic bag
- Children
- Non-cooperative adult people
3. Catheterization-Not recommended
4. Supra-pubic punction
- Sterilization of suprapubic area
- Aspirate urine at 1 cm upper of os pubic
using 10ml spoit
 Transportation of specimen

• Normally, urine is sterile

• But urine is an excellent culture
media
• As quickly as possible
• Culture within one1-2 hours of
collection
• Preserve urine if delay in
processing
– Store at 4oC
– Preservative e.g. boric acid
– Appropriate transport device
 Taking, Storing and Transportation of
Urine specimen

MID-STREAM URINE URINE BAG

URINE SUPRAPUBIC ASPIRATION

Collecting STERILE ASPIRATION BY SPOIT URINE BAG

Storing STERILE BOTTLE STERILE BOTTLE URINE BAG

4oC 4oC 4 oC

Transportation
BRING IMMEDIATELY
IN COOL BOX

Lab
Identification SENTIRIFUGE
Start within 2 ISOLATION & BACTERIAL
hours IDENTIFICATION COUNT
GRAM STAINING
 Bacteriological examination of urine
• Urine inspection
– Crystal clear urine – rarely infected
– Cloudy urine does not always imply
infection
– Hemorrhagic/blood stain may indicate
other cause

• Rapid test
– Detection of specific markers reflecting the
presence of bacteria and WBC

• Microscopic examination
– To look for and to count WBC and RBC
– Cast made of protein, WBC or RBC
– Predominant organism

• Culture
– Identification of Microbes
 Microscopic examination
• Wet smear or gram stain
• Using high-power lens
– WBC
– Squamous epithelial cells and
mix flora indicate contaminated
urine
– Positive gram stain but
negative culture may indicate
• Fastidious organism /
anaerobe
• Mix up of specimen
 Urine culture
Selection of culture media
Routinely – media that support
the growth of commonly
isolated pathogen
- Nutrient agar
- Blood agar,
- Mac Conkey agar

Incubation
– Aerobically overnight
– Count the number of colony
formed, e.g:
• Pour plate method
– Significant count proceed to
bacterial identification and
sensitivity testing
 URINE

SEMIQUANTITATIVE COUNT MICROSCOPIC EXAMINATION

Use a calibrated loop to spread
onto BA and Mac Conkey plate
Incubate overnight at
35-37C
COLONY COUNT
Shake urine
Place a drop of urine on slide and
let dry without spreading – Gram
stain
NB: If smear show plenty of pus
cells and one type of bacteria
– Do primary sensitivity

REPORT: (General guideline)

100.000 CFU/ml – Significant bacteriuria
10.000 – 100.000 cfu/ml – borderline significant, suggest repeat
<10.000 cfu/ml – clinically not significant
Any number in suprapubic urine - significant
Mixed growth – suggest repeat
 Microbial Agents
1. Enterobacteriaceae
• E. coli (80–85% of UTIs)
• Urinary tract abnormalities:
• Klebsiella
• Proteus
• Enterobacter sp
2. Pseudomonas
3. Other Gram negative bacteria
Recurrent / nosocomial UTIs :
• Proteus sp,
• Serratia sp
• Pseudomonas sp,
Diabetic patients:
Serratia marcescens
Hematogenous:
• S.. Pyogenes & S. aureus,

4. Gram positive cocci Part of STD:

1. S. saprophyticus (5–10%) • N. gonorrhoea,
• T. pallidum,
2. S. aureus (secondary to
• Chlamydia and
blood-borne infections) • Trichomonas
5. Candida albicans: woman, R/AB,
DM, on Catheter Contaminant: S. epidermidis
Microbial Agents

Mahesh E., et al. 2011

 Escherichia coli
Characteristics
- Short generation time
- Facultative anaerobe
- Motile negative-gram cocco-bacil
- Catalase +
Virulence factors:
- Hemolysin producing
- Adhesion factor for urinary tract epithelia:
- Upper UTIs: Ag O
- Lower UTIs: Ag K
- Pyelonephritis: Ag P (fimbriae)
Uropathogenic E. coli:
• P-fimbriae (pyelonephritis-associated pili)
• alpha- and beta-hemolysins (which cause
lysis of urinary tract cells)
• High related to Anal sex
• Causing hemolytic-uremic syndrome (HUS)
 Klebsiella sp
• Nosocomial infections
• Urinary catheters
• Risk factor: renal failure and older
persons

Characteristic
- Gram-negative basil,
- Facultative anaerobe
- Non-motile

Virulence factors:
- Capsule
- Plasmid exchange – Antibiotic
resistance
- Carbapenem-Resistant Klebsiella
pneumoniae (CRKP)
 Proteus
Proteus mirabilis

Can be found in environment, particularly in

soil, sewage and vegetables

Characteristic:
• Gram-negative bacilli
• High motility (swarming pattern)
• Non-capsulated
• Produce H2S
• Urease producing - alkalic urine:
- Inactivation of complements
- Mineralization (struvite stone /
kidney stone)
 Pseudomonas aeruginosa
The most frequent colonizer of medical devices
Particularly on area with high humidity

o Obligate aerobe, negative-gram bacilli

o Polysaccharide capsulated
o Produces pigments:
- Pyocyanine (blue):
 Inhibit other bacteria growth
 Cytotoxic
 Bluish colony
- Pyoverdin
 yellow-green
 Flourescent
- Pyorubin (red-brown)
o Virulence factors
- Low antibiotic susceptibility
- Exotoxin A: inhibit proteins
synthetize, lead to necrotize
- Exoenzyme ExoU: plasma membrane
degradation of eukaryotic cells
 Staphylococcus saphrophyticus

• Normal flora of mucosa and skin

• 10-20% of UTIs
• 2nd most common causative agent
of acute UTIs among young woman
• Causing honeymooner's UTI
• Symptomatic cystitis

Virulence factors:
• Catalase +
• Capacity for selective adherence to
human urothelium
• It causes direct hemagglutination
 Characteristic of Staphylococcci

Characteristic S. aureus S. epidermidis S. saprophyticus

Pigment Yellow to White to pale

white White gray

Hemolysis + ± -

Yes No No
Coagulase production

No No
Mannitol fermentation Yes

Novobiocin sensitivity Sensitive Sensitive Resistance

 Species Virulence Source & Diseases
• Lactamase + • Nosocomial infections
Enterobacter • Catalae + • Venous catheter insertions
Aerogenes • Causing Sepsis

• Catalase + • Nosocomial infections

Serratia • Prodigiosin (reddish-orange • Catheter-associated bacteremia
marcescens tripyrrole pigment) • Commonly growing in bathrooms
• AB Multiresistant (R-factor)

• Catalase + • Nosocomial infections

Morganella • Emergence of highly resistant to • Post-operative infections
morganii 3rd generation cephalosporins

• Opportunistic pathogen on severe burns

or long-term indwelling urinary catheters
Providencia
• Elderly individuals
Stuartii
• Common cause of purple urine-bag
syndrome
• Ability to accumulate uranium by • Rarely as the source of illnesses, except
building phosphate complexes for urinary tract infections of
Citrobacter
• Have inducible ampC
freundii
• Plasmid-encoded resistance
genes
• Catalase + Normally found in environments in
Alkaligenes • Alpha hemolytic association with humans
Faecalis • Urine alkalization
 Biochemical Characteristics

TSI Gas H2S Indol Mo MR VP Citrat Urea

E. coli A/A + - + + + - - -
K. pneumoniae A/A + - - - -/+ + + +

E. aerogenes A/A + - - + + + -

E. cloacae A/A + - - + + + +/-

Serratia A/A + - - + -/+ + + -
P. vulgaris A/A +/- + + +s + - -/+ +
P. mirabilis Al/A + + - +s + +/- +/- +
Morganella Al/A + - + + + - - +
P. stuartii Al/A - - + + + - + +
P. alkalifaciens Al/A -/+ - + + + - + -

Citrobacter Al/A + + - + - + +/-

P. aeruginosa Al/A - - - + - - + +/-

A.faecalis Al/A - - - + - - + +
Negative culture result, possibly:

• Recent antibiotic use

• Chlamydial infections
• Fungal infections
• Improper culture methods
• Stored urine specimen
Sexually Transmitted
Diseases (STD)
with urinary tract symptoms
 Microbial agents
Bacteria Virus
Neisseria gonorrhoea Human (alpha) herpes virus 1 or 2
Chlamydia trachomatis (herpes simplex virus)
Treponema pallidum Human (beta) herpes virus 5 (formerly
cytomegalovirus)
Haemophilus ducreyi
Mycoplasma hominis Hepatitis virus B

Ureaplasma urealyticum Human papilloma virus

Calymmatobacterium granulomatis Molluscum contagiosum virus
Shigella Sps. Human immunodeficiency virus
Campylobacter sp
Staphylococcus sp Fungi
Group B streptococcus Candida albicans
Bacterial vaginosis-associated
organisms
 Most Frequent!

Syphilis, Gonorrhea, Herpes,

Chlamydia &
HIV
 Neisseria gonorrhoeae
o Overall rates falling, but incidence
in certain groups remains high
o Most common in the reproductive
age group (menarche to
menopause)
o Men are usually symptomatic
(urethra), women are commonly
asymptomatic
o Incubation period 2 to 10 days
o Resistance to medication is an
spreading problem
 Neisseria gonorrhoeae
SIFAT-SIFAT
o Negative-gram diplococci
o Non motile
o No spore
o Encapsulated
o Has villi and microphilic
o Produce oxidase
 Identification
Specimens
o Pus or secretions from urethral,
prostate, endocervix,
o Swab from mucosal rectal,
oropharynx, rectal swab and
conjunctiva
o Synovial fluid and CSF
o Blood (systemic infection)

Tests
o Direct gram staining
o Immunofluorescence
o Isolation and identification
o Antibiotic sensitivity test
o Nucleic acid hybridization
 Treponema pallidum
SIFAT-SIFAT
o Causing Syphilis = Raja Singa
o Can be very serious and can cause brain damage
What T. pallidum can do to your body?
 Treponema pallidum
Characteristic:
SIFAT-SIFAT
• Negative-gram bacteria
• Spiral shaped
• High motile
• Microaerophilic
• Non cultivable
• Inactivated by drying or heating.
• Survive in blood or plasma at 4oC
for 24 hours

Virulence factors:
o Outer membrane protein (OPA)
o Endoflagella
o Hyalurinidase
o 100 Ag proteins
 Identification
DIAGNOSE LABORATORIUM
Specimens: Sera, lesion and body liquor

Identification tests
Microscopic assay:
– Dark field microscopic; motility
– Negative staining: morphology

Serologic tests:
– Non-treponemal Ab = reagin
o VDRL test (Venereal Disease
Research Laboratories)
o RPR test (Rapid Plasma Reagin)
– Treponemal Ab
o TPHA (Treponema pallidum
Hemagglutination) test
 Chlamydia trachomatis
• Most common reportable disease in
the U.S.
• Estimated 3 million cases annually
– Incidence is highest among
sexually active adolescents and
young adults
• Most infections are asymptomatic
• Leading cause of preventable
infertility in women or vertical
transmission during child birth leads
to conjunctivitis in newborn
• Men: urethritis with possible
epididymitis
 Chlamydia trachomatis
- obligate intracellular
pathogen
- Has inclusion bodies
- Uncultivable
- Resistant to -lactamase
- Unique life-cycle

Biovarian Serotype Diseases

Trachoma
A, B, Ba & C Urethritis No-go
Trachoma
D to K Conjunctivitis mucopurulent
Conjunctivitis & pneumoniae on neonates

LGV L 1 , L 2 & L3 Lymphogranuloma venerum (LGV)

 Chlamydia Infections
in Women, Men, and Neonates

• Genitals •Genitals • Eye (conjunctivitis)

Cervicitis (Urethritis) • Lungs (pneumonia)
PID
(Epididymitis)
Urethritis
• Eye (Conjunctivitis) •Rectum (Proctitis)
• Throat (Pharyngitis) •Throat (Pharyngitis)
• Rectum (Proctitis) • Eye (Conjunctivitis)
70-80% ASYMPTOMATIC •Systemic (Reiter’s Syndrome)
>50% ASYMPTOMATIC
 Identification
DIAGNOSIS LABORATORIUM
Specimens:
• Cervix/urethra secretion
• Conjunctival scraping

Laboratory tests:
1. Direct staining:
– Giemsa 10% staining: inclusion bodies
– Lugol (iodium) staining: eyes secretions
and sputum
2. Species/serotypes identification
3. Tissue culture
4. Antigen or Antibody identification
1. Direct Immunofluorescent-Antibody
(DFA)
2. Enzyme Immunoassay (EIA) Test
3. Rapid test
5. Nucleic acid detection (DNA)
 Herpes

• Caused by Herpes Simplex Viruses

– HSV 1: orolabial herpes
– HSV 2: genital herpes
• Both symptomatic & asymptomatic
infections are common
• Asymptomatic shedding is well
documented
 Human Herpes Virus

Biologic Properties
Subfamily Official Name Common
“-herpes Genus “Human
Growth Cycle & Latent “-virus” Name
virinae” herpesvirus”
Cytophatology infections

Alpha Short, cytolytic Neurons Simplex 1 Herpes simplex virus

type 1
2 Herpes simplex virus
type 2
Long, cytomegalic Glands, kidneys Varicello 3 Varicella-zoster virus
Beta Long, lympho- Lymphoid tissue Cytomegalo 5 Cytomegalovirus
proliferative Roseolo 6 Human herpesvirus 6
7 Human herpesvirus 7

Gamma Variable, lympho- Lymphoid tissue Lymphocrypto 4 Epstein-Barr virus

proliferative Rhadino 8 Kaposi’s sarcoma
associated herpesvirus
 Human Herpes Virus
• Morphologically identical to herpes
simplex virus
• The virus causes chickenpox and
zoster  same, no significant genetic
variation
• Virion : spherical, 150-200 nm in
diameter (icosahedral)
• Genome : dsDNA, linear, 124-235 kbp

Outstanding characteristics:
– Establish latent infections
– Persist indefinitely in infected hosts
– reactivated in immunosuppressed
hosts
– some are cancer-causing
Transmission
Major routes: sexual & mother-to-infant
Most sexual transmission probably occurs
when index case is asymptomatic

Initial infection
Virus enters through microscopic breaks
in skin or mucose

Establishes chronic infection

Virus becomes latent in nerves cells
along spinal cord and can be reactivated

Virus can reactivate

– Virus reproduces and moves along nerve
axon to skin or mucosa, and recurrent
lesions can occur
– Reactivation (shedding) can also be
asymptomatic
 Laboratory Identification
Laboratory tests include:
• Culture of the virus,
• Direct Fluorescent Assay (DFA) studies to detect virus,
• Skin biopsy,
• Polymerase chain reaction (PCR) to test for presence of viral DNA,
• Serological test for antibodies to HSV:
Immunodot glycoprotein G-specific (IgG) HSV) accurately
distinguish type-specific glycoproteins gG1 and gG2 (98%
specificity)
Specimen and Lab Diagnostic of STD
Thank you