JOURNAL CLUB

PRESENTER : Dr. RESHMA .V.P

Moderator : Dr. ESHWAR SINGH .R
Detection of Various Beta-Lactamases in Escherichia
coli and Klebsiella sp.: A Study from Tertiary Care
Centre of North India

Varsha Gupta , Meenakshi Singh , Priya Datta, Anku Goel , Sanjay Singh , Kashinath
Prasad, Jagdish Chander

Nov 2020
 INTRODUCTION
 Carbapenem‑resistant Escherichia coli and Klebsiella species (CREK) - global health concern
particularly in developing countries.
 Carbapenems - most effective antibiotics against multidrug‑resistant GNB.
 Carbapenem resistance- various enzymes.
 CREK strains can be transmitted among patients in a hospital setting
 The resistance genes can also be transferred to other susceptible bacteria within the same
infected host
 Infections due to Enterobacteriales possessing different types of enzymes
 Increased length of stay
 Limited therapeutic options
 Higher mortality rates
 Increased hospital costs
 INTRODUCTION (contd.,)
 The main mechanisms of resistance to carbapenems in GNB is due to
 Acquisition of genes coding for enzymes leading to breakdown of carbapenems
 Overexpression of a beta‑lactamase with weak carbapenemase activity coupled with
modifications in permeability of porins and/or overexpression of efflux pumps.
 AMBLER CLASSIFICATION :

Class A Serine carbapenemases

• Klebsiella pneumoniae carbapenemase [KPC]
• Non‑metallocarbapenemase/imipenem hydrolysing beta ‑lactamase
• Serratia marcescens enzyme
Class B Metallo‑β‑lactamases (MBL)
• Verona integron‑encoded MBL [VIM]
• Active on imipenem [IMP]
• New Delhi MBL [NDM])
Class C • Amp C
Class D class D β‑lactamases (OXA‑48, OXA‑181, etc.)
 INTRODUCTION (contd.,)
 Distribution of different carbapenemases produced by GNB vary according to geographical
areas
 For the detection of CREK - Phenotypic tests and molecular methods

 Aim of this study

 To compare two phenotypic methods: CDT and modified carbapenem inactivation method
(mCIM)/ethylene diamine tetraacetic acid mCIM (eCIM)
 To evaluate molecular method for the identification of types of ESBLs and
carbapenemases produced by E. coli and Klebsiella sp.
 Materials and Methods - Study design and setting

 Prospective study
 Department of Microbiology, Government Medical College Hospital, Chandigarh
 January to July 2019
 126 E. coli and Klebsiella species isolates from various clinical samples from symptomatic patients
 Urine
 Body fluids
 Pus
 Throat swab
 High vaginal swabs.
 The bacterial isolates were identified to species level according to standard microbiological procedures.
 Materials and Methods - Study design and setting (contd.,)
 The antibiotic susceptibility testing was done by Kirby–Bauer disk diffusion test

 The following drugs (Hi‑media, Mumbai) were tested: ceftriaxone (30 µg), cefotaxime (30 µg),
gentamicin (10 µg), amikacin (10 µg), nalidixic acid (30 µg), ciprofloxacin (5 µg), norfloxacin (10
µg), nitrofurantoin (30 µg) and co‑trimoxazole (25 µg).

 Second‑line agents- cefoperazone‑sulbactam (75/30 µg), piperacillin ‑tazobactam (100/10 µg),

imipenem (10 µg) and meropenem (10 µg).

 Those strains which showed reduced susceptibility (≤19 mm) based on disc diffusion test to
meropenem/imipenem were confirmed for carbapenem resistance by microbroth dilution as per
CLSI (MIC ≥4 μg/ml).
Materials and Methods - Phenotypic detection

 Extended‑spectrum β‑lactamases by disc diffusion test

 Carbapenemases by combined disc test
 Modified carbapenem inactivation method along with ethylene
diamine tetraacetic acid carbapenem inactivation method
 Materials and Methods-
Genotypic characterization of extended-spectrum β-lactamases and
carbapenemases
 Microbiology Department of Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGI),
Lucknow, Uttar Pradesh
 DNA extraction - boiling lysis method.
 Nucleic acid amplification test :
 Uniplex polymerase chain reaction (PCR) was performed for all genes encoding ESBLs (blaCTX-M ,
blaSHV and blaTEM) and carbapenemases (blaVIM , blaKPC ,blaNDM , blaIMP and blaOXA-48 type).

 Amplification was carried out in C1000 Touch™ Thermal Cycler (Bio‑Rad, USA) in 25 µl
reaction volume containing 2 µl of genomic DNA, 1 X DreamTaq PCR master mix (Thermo
Fisher, USA), and forward and reverse primers (0.8 µM) specific for each gene were used.
 The resulting PCR products were analyzed by 1.5% agarose gel electrophoresis and images
were documented by a Bio‑Rad Gel Doc imaging system.
 RESULTS
 Out of a total of 126 carbapenem resistant isolates with (MIC ≥4 µg/ml)
 E. coli were (n = 76)
K. pneumoniae were (n = 44)
Klebsiella oxytoca (n = 6).
 67 (53%) isolates were phenotypically positive for ESBLs (40 E. coli, 24 K. pneumoniae and
3 K. oxytoca)
 RESULTS (contd.,)
 On molecular analysis, 121 (96%) of the total isolates were ESBL producers based on the
presence of various ESBL genes.

 The most prevalent β‑lactamase was bla

CTX-M (81%), followed by blaSHV (54%) and

blaTEM(52%)

 Thirty‑seven of the isolates carried all the three ESBL genes, while 24 isolates carried only
combination of two ESBL genes
RESULTS (contd.,)
 RESULTS (contd.,)
 The results of the phenotypic CDT showed a total of (112/126) KPC, MBL, AmpC and Class D
carbapenemases
 All the phenotypically positive ESBL producers were also showing MBL production
RESULTS (contd.,)
 RESULTS (contd.,)

 mCIM showed (102/126) positive isolates

 RESULTS (contd.,)
The results of the PCR showed:
 Ninety (71%) of the isolates harboured blaNDM gene

 Three of the phenotypically negative MBL strains carried blaNDM-1 gene.

 The isolated gene of Class D was found in 44/126 (35%) of the isolates.

 Nine of the blaNDM positive isolates also possessed blaOXA-48 gene.

 None of the isolate was positive for blaVIM , blaKPC and blaIMP

 Furthermore amongst these, many of the strains harboured multiple genes.

 Twenty‑two of the isolates also harboured different combinations of any of the three ESBL types
(CTX‑M, SHV and TEM) and one carbapenemase type of gene (blaNDM )
RESULTS (contd.,)
 DISCUSSION
 Based on CLSI phenotypic detection test, 53% of E. coli and Klebsiella were found to be ESBL
producers.
 On genotypic analysis, 96% of the isolates were carrying ESBL genes.
 This is in agreement with other studies where the prevalence ranges from 4.7% to 90%.
 This variation could be due to
 Existence of multiple enzymes which can mask the inhibition of clavulanic acid in the medium.
 Some β‑lactamases fail to reach a level to be detectable by disk diffusion tests and it may be based
on the quality of Muller–Hinton agar used.
 Gene is present but not expressed due to the absence of effective promoter or presence of repressor
genes which may lead to varied levels of enzyme expression and the poor specificity of some
antibiotic combinations.
 DISCUSSION (contd.,)
 Three main genes of ESBLs i.e., blaCTX-M , blaSHV and blaTEMwere studied.

 The most common β‑lactamase was blaCTX-M (81%), followed by blaSHV (54%) and blaTEM (52%).
 Among ESBLs, blaCTX-M enzymes are most prevalent across the world as compared to other
enzymes.
 Prevalence of individual ESBL genes differ among other study centres.
 Until the year 2000, blaTEM was the most prevalent ESBL gene in the Indian bacterial population
but was replaced by blaCTX-M in the following decade.
 Thirty seven (30.6%) of the isolates carried all the three ESBL genes.
 24 (19.8%) isolates carried only combination of two ESBL genes.
 DISCUSSION (contd.,)
 In India, the prevalence of carbapenem resistance in Enterobacteriales varies widely from 12% to 97%
 The study for carbapenemases based on CLSI phenotypic evaluation showed overall positivity of 81%
(102/126) by mCIM/eCIM.
 Other phenotypic methods as per CLSI :The Modified Hodge Test ,CarbaNP
 mCIM is used in the study based on the in vitro inactivation of meropenem by hydrolysis and eCIM
which is used together with mCIM to differentiate MBLs from serine carbapenemases in
Enterobacteriales.
 Eighty‑seven out of 126 (69%) strains showed positive MBL results by both phenotypic methods
(CDT and mCIM/eCIM) and molecular analysis revealed blaNDM gene in all of these isolates by PCR
in addition to three more positives 90/126 (71%).
 New Delhi MBLs are more prevalent in the Indian subcontinent and Eastern Europe
 KPC serine carbapenemases are mostly found in the Americas, the Mediterranean countries and China.
 In the present study also, 71% of the isolates harboured blaNDM gene and no other MBL gene
 DISCUSSION (contd.,)
 CDT method is better for identifying CREK as compared to mCIM method due to
simultaneous detection of various classes of carbapenemases.
 The CDT method is based on inhibitors and has the advantage of showing excellent
performance in discrimination of KPC, MBL and OXA‑48 carbapenemases.
 The results of the CDT method showed higher number of total (89%) carbapenemases in
comparison to mCIM.
 Among these none of the isolates were positive for KPC both by phenotypic and genotypic
methods which are reflected in other studies also.
 DISCUSSION (contd.,)
 Class C beta‑lactamases, AmpC, are chromosomal enzymes that are produced by many species
of GNB. But in E. coli and Klebsiella species, they are present on mobile elements.
 In the present study, 5 (0.04%) of the isolates were found to be positive for AmpC production
by phenotypic method using cloxacillin as inhibitor.
 The AmpC‑enzyme is usually produced at low levels in E. coli and Klebsiella and thus rarely
contributes to β‑lactam resistance.
 Few plasmidic AmpC enzymes may contribute to carbapenem resistance in porin deficient
Enterobacteriales.
 DISCUSSION (contd.,)
 Class D carbapenemases, Oxa‑type conferring resistance to carbapenems are most commonly
present in Acinetobacter spp. and occasionally in Enterobacteriales and Pseudomonas spp.
 Oxacillinases hydrolyse carbapenems weakly and are poorly inhibited by clavulanate. These
enzymes have been previously reported in Mediterranean and European countries but now they are
being progressively disseminated to other geographical areas including India.
 Currently there is no available inhibitor for class D carbapenemases -> an antibiotic temocillin
which is highly resistant to Oxa‑48 strains has been incorporated in CDT to demonstrate Oxa ‑48
like enzymes.
 In the present study, only 16% of the isolates were presumed to be class D carbapenemases by
CDT but this percentage was raised upto35% for Oxa‑48 isolates on molecular characterization.
 Combination of blaNDM-1 and blaOXA-48 gene was seen in nineteen isolates of CREK as has been
reported earlier.
 The emergence of blaOXA carbapenemases in the Enterobacteriales, particularly Klebsiella spp., is
of major significance because these bacteria are true pathogens that are able to infect
immunocompetent individuals.
 DISCUSSION (contd.,)
 The phenotypic test based on CLSI and EUCAST both are missing some strains carrying blaNDM-1
gene though number is very less, i.e., only three for MBLs and also blaOXA-48 genes.
 The phenotypic testing has its limitation over isoelectric focusing and PCR which are considered
as the gold standard methods.
 Thus, attempts are going on to improve phenotypic testing with better sensitivity and specificity
as doing molecular techniques on routine basis is not feasible in low budget diagnostic
laboratories.
 There is a limit of using molecular techniques in identifying different carbapenemases as they
may lack sequence similarity to genes already described.
 Other drawbacks of using molecular techniques for end users are lack of trained professionals,
labour intensive and time consuming.
 CONCLUSION
 Carbapenems are the last drug of choice for multidrug‑resistant strains
 It has become important to keep a track on the surveillance of carbapenem resistant
Enterobacteriales.
 It is necessary to determine the various classes of enzymes as treatment can vary for different
classes of enzymes like KPC and MBL‑producing K. pneumoniae can be treated by
combination of carbapenems with colistin/tigecycline/ aminoglycosides and for Oxa ‑48,
carbapenems may not be a reliable treatment option.
 Further, monitoring of various carbapenemases prevalent in the regions is of concern to see
the change in trend from one type to another like NDM to OXA ‑48 group shift in enzymes as
is being suggested in some studies.
THANK YOU…..