Urinary tract infections
Background
 UTI is one of the most common community-acquired
infections
 in the hospital environment it is the most common cause of
health care associated infections
 UTI is an attack on the urinary tract tissue by one or more
microorganisms, generating inflammatory response and
symptoms
 Can be involved:
 urethra,
 urinary bladder,
 renal pelvis
 kidneys.
 clinical symptomatology is having
positive prediction for UTI ~ 50%.
 sometimes, UTI can be
asymptomatic;
 for children until 2 years old the
symptoms are unspecific.
The cytological and bacteriological
analysis are the most commonly
performed analysis in the laboratory
Indications for bacteriological culture
and cytological examination
 Children
 Young adults
 Men
 Women with diabetes, immunosuppression, pregnancy,
recurrent UTI
 Patients with known abnormalities of the urinary tract
 When pyelonephritis can not be ruled out
 When therapy fails.
Algorithm of urinary tract infection diagnosis for adults
Etiopathogeny
 Ascending: the most UTI with
conditionated pathogenic bacteria
 Hematogenous: primary pathogenic
bacteria – M. tuberculosis.
 Predisposing factors:
 short urethra (woman)
 catheterization
 obstacle in the way of urinary flux:
stones, anatomic abnormalities.
 refluxe bladder – urethra.
 Etiologic agents:
 E. coli (80% UTI)
 Proteus mirabilis
 Klebsiella pneumoniae
 Enterococci
 Enterobacter aerogenes
 Staphylococcus saprophyticus
 S. aureus
 Candida albicans
 Corynebacterium urealyticum
Etiopathogeny
 Nosocomial UTI
 Pseudomonas aeruginosa
 Stenotrophomonas maltophilia
 Acinetobacter spp.
 Dissuria caused by urethritis
 Neisseria gonorrhoeae
 Chlamydia trachomatis
 Ureaplasma urealyticum
 Herpes simplex virus
Urine sampling
Customary sampling
•
•
•
Midstream urine
Intensive care unit (ICU) immobile patients – minimum
precautions to avoid contamination.
Special sampling
•
•
•
•
through catheter: only for patients from intensive therapy
children: urine collecting bag
suprapubic aspiration.
Urine sampling
 Sterile recipient
 Screwed lid
Sampling
 Sampling time:
 first urine in the morning;
 3 hours after the last micturition.
 necessary quantity:
 20 ml for quantitative identification of conditionated pathogens
microorganisms (e.g., E. coli)
 3 x 50 ml for specific pathogens identification (M. tuberculosis)
On the requesting paper should be
specified:
 Sample type: midstream specimen urine
 clean or
 whithout special de-contamination of genital organs
 sampling by catheter
 antimicrobial treatment.
Urine: transporting and preservation
 Urine specimen must be examined in maximum one hour
after sampling.
 If the time can’t be respected, the sample must be
refrigerated at 4°C until examination.
Microscopic counting of cells
 Haemocytometer is used to count the leucocytes
 Normal urine contains less than 1000 leucocytes or red
blood cell /ml
Microscopic examination using
Gram staining
 For specific clinical situations
 It gives the medical microbiologist rapid guidance for the
diagnostic approach: choice of culture media or specific culture
conditions
 It easily demonstrate contamination of the urine by local flora
 Epithelial cells, lactobacilli: suggestive for contamination
 Limitation of microscopy: is insufficiently sensitive to detect
bacteriuria of 103 -104 CFU/ml.
Smear from urine, Gram stain
Culture
 Techniques for semiquantitation by culture
 Innoculation choosing the agar
 Identification:
 Chromogenic agars: 16 – 18 hours of incubations
 MALDI-TOF: rapidly replacing classical biochemical
techniques.
 (Matrix-assisted laser desorption/ionization)
Chromogenic agar
MALDI-TOF
 Matrix-assisted laser desorption ionization time-of-flight
mass spectrometry (MALDI-TOF MS) is a
 rapid,
 accurate, and
 cost-effective method for microbial identification.
 MALDI-TOF MS generates characteristic mass spectral
fingerprints that are unique signatures for each
microorganism and are thus ideal for an accurate microbial
identification at the genus and species level.
Wrong procedures:
 Sampling in a tube;
 Return passage of the urine form a big recipient in a tube;
 Sampling from women with vaginal discharge, without vulva
toilet and buffer used for vagina obstruction;
 Sampling during antimicrobial therapy, without indication of the
antibiotic used;
 Sampling from the drainage bag;
 Sending for culture of the catheter ’s top:
 colonization with bacteria from urethra;
 contaminated at catheter recession.
Qualitative criteria of urine sample





Time and temperature until examination
The absence of antibiotic substances
The absence of squamos epithelial cell
The presence of a single microbial type on Gram stain
Significant bacteriuria with one single bacterial species.
Interpretation
 Symptoms
 Preanalytical phase – covered
 Parameters:
 epidemiological circumstances (community-acquired /
healthcare, history, localisation)
 risk factors (urinary catheter, urinary tract procedure)
 urinary symptoms or fever
 current / previous antibiotic treatment
 level of leukocituria
 absence of local flora on the gram stain
 level of bacteriuria and the type of microorganisms isolated.
Results interpretation
Cytobacterioscopy
 Pyuria: leukocyte count in Fuchs Rosenthal county chamber
 „present pyuria ” = >10 leukocyte/ml urine
 „absent pyuria” = < 3 leukocyte/ml urine
 „possible pyuria”: 3-10 leukocyte/ ml urine
 Cylinder type (leukocyte, erythrocyte) – suggestive for pyelonephritis;
 Epithelial cell:
 squamoas: contamination from vagina;
 bladder, kidney: inflammation in this anatomic sites.
Results interpretation
Quantitative culture
 Single gram negative species is isolated in concentration of 105 CFU/ml or
staphylococci ≥ 5x104 UFC/ml means:
 + pyuria = UTI
 - pyuria = abortive colonization of the urine / late examination of the
urine
 One single species of coliform bacilli - 105 CFU/ml:
 For men: + pyuria – clinical significant.
 For women: + pyuria +dysuria and increasing urinary frequency- is significant;
isolation of E. coli in concentration of 103-4 CFU/ml is significant.
Interpretation of the results
 > 2 bacterial species - 104-5 CFU/ml – sample contamination prior
examination / late examination.
 Pressence of < 103 CFU/ml:
 in the absence of pyuria = excluded UTI;
 in the presence of pyuria and clinical signs and symptoms (+):
 renal elimination of antimicrobial substances;
 treatment with a β-lactamine - chronic infection „L” type bacteria wall.
 infection with Ureaplasma urealyticum
 kidney T.B.
Case 1
Woman, 46 years old: dyssuria, increasing urinary frequency, flank pain,
fever 38,5-39,2°C. Urine sample – middle clean stream is collected.
Cytology: 58 leukocyte/mm3
Bacterioscopy: 2-3 gram negative bacilli / microscopic field (1000 x)
Quantitative culture of urine:
- 1,27x 105 CFU/mL urine Enterobacter aerogenes,
- 1,5 x 104 CFU/ mL urine Staphylococcus spp.
Case 2
Woman, 26 years old, pregnant - 20 weeks, without clinical symptomathology.
During periodical examination, a sample of urine was taken (middle clean
urine sample).
Cytology: 6 leukocyte/mm3, 4 epithelial squamos cell/field.
Bacterioscopy: 5 gram negative bacilli and 1-2 yeasts/ microscopic field (1000x)
Quantitative urine culture:
- 1,31x105 CFU/mL urine Escherichia coli;
- 8.000 CFU/ mL urine Candida albicans
Case 3
Woman, 56 years old, present increasing urinary frequency,
dysuria.
Cytology : 87 leukocyte/mm3, increased number of
erythrocytes.
Bacterioscopy: 1-2 gram negative bacilli / microscopic field
(1000x)
Quantitative urine culture: 8 x 103 CFU/mL urine - Escherichia
coli
Case 4
Woman, 21 years old, student, present dysuria and increasing
urinary frequency. She send a middle clean urine sample to a
private laboratory, after the courses at the University.
Cytology: 4 leukocyte/mm3, numerous erythrocytes.
Bacterioscopy: 1-2 gram negative bacilli / microscopic field
(1000x)
Quantitative urine culture: 1,5x105 UFC/mL urine Escherichia
coli, 118.000 UFC/mL urine Proteus spp.
Case 5
Woman, 26 years old, the clinical suspicion of lower UTI was confirmed
with E.coli with the next sensitivity results of difuzimetric antibiogram:
Amoxicillin - R
Amoxicillin + clavulanic acid- I
Ticarcilin - R
Piperacilin - I
Ceftazidim - S
Ceftriaxon - S
Gentamicin - S
Amikacin - S
Cloramfenicole - S
Cotrimoxazole - R
Acid nalidixic - R
Norfloxacin - S
Nitrofurantoine - S
Example of natural resistance
Antimicrobial drugs:
why do they sometimes fail ?
Proportion of 3rd gen. cephalosporins Resistant
(R) Escherichia coli Isolates in Participating
Countries in 2011
(This report has been generated from data submitted to TESSy, The European
Surveillance System on 2013-08-25. Page: 1 of 1. The report reflects the state of
submissions in TESSy as of 2013-08-25 at 09:00)
Proportion of Fluoroquinolones Resistant (R)
Escherichia coli Isolates in Participating Countries
in 2011
(This report has been generated from data submitted to TESSy, The European Surveillance System on
2013-08-25. Page: 1 of 1. The report reflects the state of submissions in TESSy as of 2013-08-25 at
09:00)
Proportion of Fluoroquinolones Resistant (R)
Escherichia coli Isolates in Participating Countries
in 2011
(This report has been generated from data submitted to TESSy, The European Surveillance System on
2013-08-25. Page: 1 of 1. The report reflects the state of submissions in TESSy as of 2013-08-25 at
09:00)
The main resistance phenotypes of E. coli
Antibiotic
Wild phenotype
Low level
penicinilase
High level
penicinilase
Aminopenicillins
S
R
R
Aminopenicillins +IBL
S
S
I/R
Carboxipenicillins
S
R
R
Ureidopenicillins
S
I/R
I/R
First generation
cephalosporins
Second generation
cephalosporins
Third generation
cephalosporins
Third generation
cephalosporins + IBL
Cefamicins
S
I
I/R
S
S
S/R
S
S
S
S
S
S
S
S
S
Broad spectrum
cephalosporins
carbapenems
S
S
S
S
S
S
Francois Jehl, Monique Chomarat, Michele Weber, Alain Gerard, “De l’antibiogramme a
prescription”. Edition bioMerieux, ISBN 973 – 86485-2-1, 2010.
Extended-spectrum beta-lactamase
(ESBL)-producing Enterobacteriaceae
 ESBLs are enzymes that hydrolyze most penicillins and
cephalosporins, including
 Oxyimino- beta-lactam compounds (cefuroxime, third- and
fourth-generation cephalosporins and aztreonam) but not
cephamycins or carbapenems.
 Most ESBLs belong to Ambler class A of beta-lactamases and
are inhibited by beta lactamase - inhibitors (clavulanic acid,
sulbactam and tazobactam)
Clinical and/or epidemiological
importance
 The first ESBL-producing strains were identified in 1983, and
since then have been observed worldwide.
 This distribution is a result of the clonal expansion of
producer organisms, the horizontal transfer of ESBL genes on
plasmids and, less commonly, their emergence de novo.
 By far the most clinically important groups of ESBLs are CTX-M
enzymes, followed by SHV- and TEM-derived ESBLs.
 Certain class D OXA derived enzymes are also included
within ESBLs, although inhibition by class A- beta-lactamase
inhibitors is weaker than for other ESBLs.
Clinical and/or epidemiological
importance
 ESBL production has been observed mostly in Enterobacteriaceae,
first in hospital environments, later in nursing homes, and since
around 2000 in the community (outpatients, healthy carriers, sick
and healthy animals, food products).
 The most frequently encountered ESBL-producing species are
Escherichia coli and K. pneumoniae.
 all other clinically-relevant Enterobacteriaceae species are also common
ESBL-producers.
 The prevalence of ESBL-positive isolates depends on a range of
factors including species, geographic locality, hospital/ward,
group of patients and type of infection, and large variations have
been reported in different studies.
ESBL screening methods for
Enterobacteriaceae
Algorithm for phenotypic detection of ESBLs
EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or
epidemiological importance, July 2013
Case 5
Woman, 26 years old, the clinical suspicion of lower UTI was confirmed
with E.coli with the next sensitivity results of difuzimetric antibiogram:
Amoxicillin - R
Amoxicillin + clavulanic acid- I
Ticarcilin - R
Piperacilin - I
Ceftazidim - S
Ceftriaxon - S
Gentamicin - S
Amikacin - S
Cloramfenicole - S
Cotrimoxazole - R
Acid nalidixic - R
Norfloxacin - S
Nitrofurantoine - S
The main resistance phenotypes of E. coli
Antibiotic
Wild phenotype
Low level
penicinilase
High level
penicinilase
Aminopenicillins
S
R
R
Aminopenicillins +IBL
S
S
I/R
Carboxipenicillins
S
R
R
Ureidopenicillins
S
I/R
I/R
First generation
cephalosporins
Second generation
cephalosporins
Third generation
cephalosporins
Third generation
cephalosporins + IBL
Cefamicins
S
I
I/R
S
S
S/R
S
S
S
S
S
S
S
S
S
Broad spectrum
cephalosporins
carbapenems
S
S
S
S
S
S
Francois Jehl, Monique Chomarat, Michele Weber, Alain Gerard, “De l’antibiogramme a
prescription”. Edition bioMerieux, ISBN 973 – 86485-2-1, 2010.
Case 5: comments
• Strain which produce beta – lactamase (penicillinase type) – high resistance
level;
• Resistance to nalidixic acid indicate a risk for mutants selection under
fluorochinolones treatment;
• Cloramphenicole is NOT useful for testing (no efficient concentration in
urine);
• Useful: large board spectrum cephalosporines (oral administration) or
nitrophurantoine;
• Fluoroquinolone administration – according with results of antibiogram
(before and after treatment end).
Case 6
A 64 years old patient was diagnosed with prostatitis and E.
coli was isolated.
Susceptibility test results:
Amoxicilin - R
Amoxicilin + Acid clavulanic - S
Ticarcilin - R
Piperacilin - I
Ceftazidim - S
Gentamicin - S
Amikacin - S
Cotrimoxazole - R
Nalidixic acid- S
Norfloxacin - S
Nitrofurantoin - S
Ceftriaxon - S
Case 6 – comments:
• Penicillinase producing strain (low level), resistant to penicillin and
sensitive to other antibiotics;
• From active antibiotics, fluoroquinolones (ofloxacine, ciprofloxacine)
reach high concentration in prostatic tissue;
• Long treatment period (4 – 6 weeks) – we must test the sensitivity
during therapy.
Case 7
3 month old patient; history: UTI – treatment with ceftriaxone (E. coli – 2 month old).
Cistoscopy for urinary tract investigation: fever and cloudy urine.
Quantitative urine-culture: iatrogenic infection with P. aeruginosa.
Susceptibility test results:
Ticarcilline - R
Piperacilline - I
Piperacilline+Tazobactam - S
Ceftazidime - S
Cefepime - S
Imipenem - R
Meropenem - R
Gentamicyn - R
Amikacine - S
Netilmicine - R
Ciprofloxacine - S
Cotrimoxazole - R
Case 7: comments
• Low level resistance to penicilline by penicillinase production;
• Resistance to carbapenems by D porines modification (sensitivity to 3rd
and 4th generation cephalosporines it is not change);
• Ciprofloxacine, even active, can not be use at such small child;
• Cotrimoxazole – not useful to test (P. aeruginosa is natural resistant).