DEPARTMENT OF MICROBIOLOGYKARPAGAM FACULTY OF MEDICAL SCIENCES AND RESEARCH

Dr. SHREERAM A. DESHPANDE, MBBS, MD.,Dr. R. SOMESHWARAN, MBBS, MD.,

CLINICAL SCENARION

• A clinical isolate of Klebsiella pneumoniae ESBL + Carbapenemase in Urine in a 39 years old female.

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ESBLs and Carbapenemases

• ESBLs: 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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ESBLs and Carbapenemases

• Carbapenemases: Carbapenemase producing bacteria are resistant to

Carbapenems like Meropenem, Imipenem, Doripenem and or Ertapenem.

Ex: Klebsiella pneumoniae Carbapenemase (KPC)

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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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PATIENT DATA

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CLINICAL PROFILE• Patient name: Mrs. XXX XXX XXX• Age: 39 years• Sex: Female• Occupation: Housewife• OP No: O1404026256• Lab No: 105426 / 2015• Diagnosis: XXX• Department: OB & GY• Antibiotic details: XXX

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Patient data could not be retrieved as she was an Out patient (OP)

XXX – Not known

DAY 1

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MICROBIOLOGICAL DIAGNOSIS

• Sample: Mid stream urine (MSU)• Collected in: wide mouthed sterile plastic

container• Date of sample collection: 24-12-2014 / 10.30 am

• Transported immediately to lab and processed

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5µl

MICROBIOLOGICAL DIAGNOSIS

• Test: Culture & Sensitivity• Wet mount performed by Microscopy• Sample INOCULATED ON BAP, MAC for

Culture and Incubated overnight @ 37C.

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Urine Wet Mount

• Pus cells: 20-40/HPF• Bacteria: Present

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DAY 2

• Reading the culture plates and Reporting done

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Colony Morphology Reading

• BAP: Non hemolytic Mucoid colonies

• MAC: Pink Lactose fermenting mucoid colonies

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From the colonies

• Biochemical reactions – 37°C X 24 hrs• Antibiotic Susceptibility Testing performed on

Mueller Hinton Agar with 0.5 Mac Farland standard of bacterial suspension – Incubated at 37°C X 24 hrs.

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DAY 3

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Bio-chemical reaction

Indole Citrate Urease MMM TSI MR VP

Negative Positive Positive +/- A+/A- Negative Positive

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Antibiotic Susceptibility Test

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Identification discs

Antibiotic discs Dose in µg Zone diameter AST report

Ceftazidime (Ca) 30 30 mm RESISTANT

Ceftazidime Clavulanate (Cac) 30/10 40 mm RESISTANT

Cefotaxime (Ce) 30 30 mm RESISTANT

Cefotaxime Clavulanate (Cec) 30/10 40 mm RESISTANT

Cefoxitin (Cn) 30 21 mm RESISTANT

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I & II line discsAntibiotic discs Dose in µg Zone diameter AST report

Ampicillin 10 6 mm RESISTANT

Amoxy-Clavanate 30 6 mm RESISTANT

Piperacillin Tazobactum 100/10 10 mm RESISTANT

Ceftriaxone 30 6 mm RESISTANT

Cefpodoxime 30 6 mm RESISTANT

Cephalothin 30 6 mm RESISTANT

Cefaclor 30 6 mm RESISTANT

Cefixime 30 10 mm RESISTANT

Cefipime 30 10 mm RESISTANT

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I & II line discs (Cont.)Antibiotic discs Dose in µg Zone diameter AST report

Imipenem (I) 10 15 mm RESISTANT

Nitrofurantoin 300 6 mm RESISTANT

Co-trimoxazole 1.25/23.75 6 mm RESISTANT

Ciprofloxacin 5 6 mm RESISTANT

Norfloxacin 5 6 mm RESISTANT

Ofloxacin 10 6 mm RESISTANT

Amikacin 30 6 mm RESISTANT

Gentamicin 10 6 mm RESISTANT

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What to do ???

• No sensitive drugs observed in I & II line antibiotic list• Isolate was Klebsiella pneumoniae ESBL positive With ? Carbapenemase.• We subjected the isolate for III line disc antibiotic susceptibility to confirm the diagnosis of Carbapenemase production.

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III line discsAntibiotic discs Dose in

µgZone diameter AST report

Meropenem (Mr) 10 14 mm RESISTANT

Tobramycin (Tb) 10 8 mm RESISTANT

Netilmicin 30 6 mm RESISTANT

Tigecycline 15 15 mm SENSITIVE

Colistin 10 12 mm SENSITIVE

Ceforperazone Sulbactum 30 12 mm RESISTANT

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Antibiotic Susceptibility Test (AST)

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• 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.

ESBL Detection

1. Screening test2. Confirmation test

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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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ESBL Screening

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Ca Cac

ESBL Screening

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Ca – 30mm Cac – 40 mm

ESBL Confirmation

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ESBL Detection

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Ce Cec

ESBL Detection

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Ce – 30mm Cec – 40 mm

CLINICAL SIGNIFICANCE

• ESBL producing bacteria are resistant to beta lactam antibiotics like Penicillins and 1st, 2nd, 3rd, 4th generation Cephalosporinsm Aztreonam.

Note: 5th Generation antibiotics like Ceftabipirole and

Ceftaroline are effective against MRSA & VRE but against ESBL.

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CEPHALOSPORINS

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CARBAPENEMASE DETECTION

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Carbapenemase detection

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I Mr

Carbapenemase detection

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I – 15mm Mr – 14mm

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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MODIFIED HODGE TEST

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Treatment of Carbapenemase

Limited options left!!!! INJECTABLES (Intravenous)• Inj. Tigecycline• Inj. Colistin

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Final identification

• Klebsiella pneumoniae ESBL + Carbapenemase producer 105 CFU/mL sensitive to Tigecycline and Colistin only in urine.

05/03/23 43Klebsiella pneumoniae Carbapenemase (KPC)

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 physicians05/03/23 44

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.

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TROUBLE SHOOTING• Sample collection – As per SOP • Referral form Filling• Turn Around Time (TAT)• Chief complaints – Patient details• Working diagnosis• Treating physician name and signature• Antibiotic details• Communication – Intercom • Hospital Infection Control Committee (HICC)• Hospital Antibiotic Policy (HAP)• Clinical Audit at regular intervals• OP patients – Track record to be maintained (Publishing papers)

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QUERIES???

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What we expect!!!

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What happens actually!!!

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Thank you

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