1. Clinico-Pathological Conference (CPC) Meet
Karpagam Medical College Hospital

2. Dr. SHREERAM A. DESHPANDE, MBBS, MD., Dr. R. SOMESHWARAN, MBBS, MD.,
DEPARTMENT OF MICROBIOLOGY KARPAGAM FACULTY OF MEDICAL SCIENCES AND RESEARCH
Dr. SHREERAM A. DESHPANDE, MBBS, MD.,
Dr. R. SOMESHWARAN, MBBS, MD.,

3. Clinical Scenario
A clinical isolate of Klebsiella pneumoniae Extended Spectrum Beta Lactamase (ESBL) and Carbapenemase producer in Urine in a 39 years old female.
9/16/2018

4. Clinical Profile Patient name: Mrs. XXXXXXXXX
Age: 39 years Sex: Female
Occupation: Housewife
OP No: O
Lab No: / 2015
Sample: Mid Stream Urine (MSU)
Test: Culture & Sensitivity
Diagnosis: XXX
Department: OB & GY
Antibiotic details: XXX
Co-morbid conditions: XXX
Patient data could not be retrieved as she was an Out patient (OP)
XXX – Not known
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5. DAY 0
Sample: Mid stream urine (MSU) in a wide mouthed sterile plastic container
Date of sample collection: / am
Transported immediately to lab and processed
5µl
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6. Culture & Sensitivity Wet mount performed by Microscopy
Sample inoculated on: BAP, MAC for Culture and Incubated 37C.
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7. Urine Wet Mount
Pus cells: 20-40/HPF
Bacteria: Present
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8. DAY 1 Culture plates read for Colony Morphology BAP: Blood Agar
Non hemolytic
Mucoid colonies
MAC: Mac Conkey’s Agar
Pink Lactose fermenting
mucoid colonies
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9. Day 1 Biochemical reaction - Incubated @ 37°C X 24 hrs
Antibiotic Susceptibility Testing (AST) performed on Mueller Hinton Agar with 0.5 Mac Farland’s standard of bacterial suspension – Incubated at 37°C X 24 hrs.
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10. Isolate: Klebsiella pneumoniae
DAY 2
Bio-chemical reaction
Indole
Citrate
Urease
MMM
TSI MR VP
Negative
Positive
+/-
A+/A-
Isolate: Klebsiella pneumoniae
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11. Antibiotic Susceptibility Test
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12. Identification discs Antibiotic discs Dose in µg Zone diameter
AST report
Ceftazidime (Ca) 30
30 mm
RESISTANT
Ceftazidime Clavulanate (Cac)
30/10
40 mm
Cefotaxime (Ce)
Cefotaxime Clavulanate (Cec)
Cefoxitin (Cn)
21 mm
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13. I & II line discs Antibiotic discs Dose in µg Zone diameter AST report
Ampicillin 10
6 mm
RESISTANT
Amoxy-Clavanate 30
Piperacillin Tazobactum
100/10
10 mm
Ceftriaxone
Cefpodoxime
Cephalothin
Cefaclor
Cefixime
Cefipime
9/16/2018

14. I & II line discs (Cont.) Antibiotic discs Dose in µg Zone diameter
AST report
Imipenem (I) 10
15 mm
RESISTANT
Nitrofurantoin
300
6 mm
Co-trimoxazole
1.25/23.75
Ciprofloxacin 5
Norfloxacin
Ofloxacin
Amikacin 30
Gentamicin
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15. What to do ???
No sensitive drugs observed in I & II line antibiotic list
Isolate was Klebsiella pneumoniae ESBL producer with
? Carbapenemase.
We subjected the isolate for III line antibiotic
susceptibility to confirm the diagnosis of Carbapenemase
production.
9/16/2018

16. III line discs Antibiotic discs Dose in µg Zone diameter AST report
Meropenem (Mr) 10
14 mm
RESISTANT
Tobramycin (Tb)
8 mm
Netilmicin 30
6 mm
Tigecycline 15
15 mm
SENSITIVE
Colistin
12 mm
Ceforperazone Sulbactum
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17. Antibiotic Susceptibility Test (AST)
Sensitive to:
Tigecycline, Colistin
Resistant to:
Ampicillin, Amoxyclav, Ceftazidime, Ceftazidime clavanate, Cefotaxime , Cefotaxime clavanate, Cefpodoxime, Cefoperazone sulbactum, Piperacillin tazobactum, Ceftriaxone, Cefuroxime, Cefpodoxime, Cefaclor, Cefipime, Cefoxitin, Imipenem, Meropenem, Ertapenem, Netilmicin, Gentamicin, Amikacin, Tobramycin, Ciprofloxacin, Ofloxacin, Nitrofurantoin, Co-trimoxazole, Norfloxacin.
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18. ESBL Detection
1. Screening test 2. Confirmation test
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19. ESBL Screening
Ceftazidime (Ca) and Ceftazidime – Clavulanate (Cac) identification antibiotic discs are used as per Clinical Laboratory Standards Institute (CLSI) guidelines 2014.
A zone difference of ≥ 5mm in Cac and Ca discs is suggestive of ESBLs.
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20. ESBL Screening Ca
Cac
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21. ESBL Screening
Ca – 30mm
Cac – 40 mm
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22. ESBL Confirmation
Cefotaxime (Ce) and Cefotaxime – Clavulanate (Cac) identification antibiotic discs are used as per Clinical Laboratory Standards Institute (CLSI) guidelines 2014.
A zone difference of ≥ 5mm in Cec and Ce discs is diagnostic of ESBLs.
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23. ESBL Detection Ce
Cec
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24. ESBL Detection
Ce – 30mm
Cec – 40 mm
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25. CLINICAL SIGNIFICANCE
ESBL producing bacteria are resistant to beta lactam antibiotics like Penicillins and 1st, 2nd, 3rd, 4th generation Cephalosporins and Monobactams like Aztreonam.
Note:
5th Generation antibiotics like Ceftabipirole and Ceftaroline are effective against MRSA & VRE but against ESBL.
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26. CEPHALOSPORINS
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27. CARBAPENEMASE DETECTION
Carbapenems like Imipenem, Meropenem, Ertapenem, Doripenem can be used for screening for Carbapenemase production by bacteria.
Disk diffusion - Zone size for Urine samples:
Carbapenem
Resistant (mm)
Intermediate (mm)
Sensitive (mm)
Imipenem 17
19-20 21
Meropenem 19
20-22 23
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28. Carbapenemase detectionMr
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29. Carbapenemase detection
I – 15mm
Mr – 14mm
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30. Carbapenemase confirmation
Modified Hodge test – will be a confirmatory test for Carbapenemase producer.
Lawn culture of Escherichia coli ATCC 25922, 10µg of Meropenem, isolate to be checked
“Clover-Leaf pattern of indentation at the intersection of the test organism and the ATCC strain within the zone of inhibition” is confirmatory for Carbapenemase production.
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31. MODIFIED HODGE TEST
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32. Treatment of Carbapenemase
Limited options left!!!
INJECTABLES (Intravenous)
Inj. Tigecycline
Inj. Colistin
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33. Final identification
Klebsiella pneumoniae ESBL + Carbapenemase producer 105 CFU/mL sensitive to Tigecycline and Colistin only in urine.
Klebsiella pneumoniae Carbapenemase (KPC)
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34. ESBLs and Carbapenemases
Extended spectrum Beta lactamases are enzymes produced by certain bacteria especially among Enterobacteriaceae members like Klebsiella pneumoniae, Escherichia coli, Salmonella and Proteus spp., and also observed in Pseudomonas aeruginosa and Acinetobacter baumannii causing Nosocomial infections.
ESBLs prevalence:
Hospital acquired/ Nosocomial – 80% Community acquired – 20%
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35. ESBLs and Carbapenemases
Carbapenemase producing bacteria are resistant to Carbapenems like Meropenem, Imipenem, Doripenem and or Ertapenem.
Ex: Klebsiella pneumoniae Carbapenemase (KPC)
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36. Significance of ESBL and Carbapenemases
ESBLs are resistant to beta lactam antibiotics like Penicillins, Cephalosporins (I, II, II Gen) and Monobactam (Ao) and Carbapenemase resistant to Carbapenems like Imipenem, Meropenem and etc.,
Risk of spread - Environment
Morbidity and Mortality - High
Nosocomial infection - Common
Treating physician is left with a only limited choice of antibiotics – poses a threat
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37. Preventive measures Hand washing Disinfection measures
Fumigation of wards
Isolation of patient
Educate nursing staff and other health care providers on Nosocomial infections, Multidrug resistant bugs, their treatment, Prevention.
Rationale use of Antibiotics by physicians
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38. TAKE HOME MESSAGE
If you see a report of ESBL producing Enterobacteriaceae – Don’t prescribe Beta Lactam Antibiotics like Penicillins and Cephalosporins.
Beta lactamase inhibitors like Piperacillin Tazobactum or Aminoglycosides or Fluoroquinolones may be used provided they are sensitive.
Hand washing is an easy, feasible remedy to prevent ESBL producers in the Hospitals.
9/16/2018