The occurrence of AmpC β-lactamase and ESBL producing Gram-negative bacteria by a simple and convenient screening method and its suitability in routine use

IOSR Journal of Dental and Medical Sciences (IOSR-JDMS)
e-ISSN: 2279-0853, p-ISSN: 2279-0861.Volume 14, Issue 12 Ver. I (Dec. 2015), PP 19-24
www.iosrjournals.org
DOI: 10.9790/0853-141211924 www.iosrjournals.org 19 | Page
The occurrence of AmpC β-lactamase and ESBL producing
Gram-negative bacteria by a simple and convenient screening
method and its suitability in routine use
Snehlata Singh, Dr. Abhineet Mehrotra
(Department of Microbiology, Mayo Institute Of Medical Sciences, Safedabad Barabanki, India),
Asst.Professor(Department of Microbiology, Career Institute of Medical Sciences and Hospital, Lucknow,
India)
Abstract:
Background: All clinical samples (e.g. Pus, urine, sputum, blood, tracheal aspirate, peritoneal fluid, catheter
tip, ET tip tracheostomy aspirate) etc are sent for culture and antibiotic sensitivity in a clinical microbiology
laboratory to achieve etiological diagnosis.
Aims: The study was done to detect the AmpC β-lactamase and ESBL producing gram negative bacteria from
different clinical samples. This study included AmpC disc screening test and found out that the modified three
dimensional tests using whole cell growth gives clearer result.
Setting and Design: A 6-month prospective analytical study was done in a tertiary care hospital.
Materials and Methods: A total of 141 sample, non-enteric Gram-negative clinical isolates obtained from
different clinical samples (e.g. Pus, urine, sputum, blood, tracheal aspirate, peritoneal fluid, catheter tip, ET tip
tracheostomy aspirate, etc) The organism included E.coli , Pseudomonas spp , Klebsiella pneomoniae ,
Klebsiela spp , Acinetobacter spp , Proteus spp ,Citrobacter spp , and Enterobacter spp . Antimicrobial
susceptibility of the strains were put according to CLSI guidelines, for ESBL and AmpC enzyme detection
source of the discs were HiMedia 19
Result: Among all the strains out of 141 isolates were tested 47 are AmpC producer and 94 are AmpC non-
producer. Maximal incidence of AmpC producers was found among E. coli (20) followed by Klebsiella
pneumonia (10). Isolates were tested for ESBL detection and 91 (64.53%) were found to be ESBL producer and
50 (35.46%) were ESBL non-producer. E. coli was the highest occurrence of ESBL producer (45.07%),
followed by Klebsiella pneumonia (29.57%).
Conclusion: Modified three dimensional test using whole cell growth in peptone water is well comparable to
the modified 3 dimensional test using cell extract method and is better than AmpC disc screening assay at the
same time is very cost effective and simple assay to be used for routine reporting of AmpC β-lactamase.
I. Introduction
The first bacterial enzyme reported to destroy penicillin was the AmpC β-lactamase of Escherichia coli
(1)
. Mutation with stepwise-enhanced resistance were termed as ampA and ampB (2,3)
. A mutation in an ampA
strain that resulted in reduced resistance was then designated as ampC. In the Ambler structural classification of
β-lactamases(4)
, AmpC enzyme belong to class C, while in the functional classification scheme of Bush et al. (5)
,
they were designated to group 1.
They are active on penicillins but even more active on cephalosporins and can hydrolyze cephmycins
such as cefoxitin and cefotetan; oxyiminocephalosporins such as ceftazidime, cefotaxime, and ceftriaxone; and
monobactams such as aztreonam but at slow rate (6)
. Inhibitor of class A enzyme such as clavulanic acid,
sulbactams, and tazobactam have much less effect on AmpC β-lactamase, although some are inhibited by
tazobactam and sulbactam(7,8,9)
. AmpC β-lactamase are poorly inhibited by p-chloromercuribenzoate and not at
all by EDTA. Cloxacillin, oxacillin, and aztreonam are good inhibitors (5)
.
The predominant mechanism for resistant to β-lactam antibiotics in gram-negative bacteria is the
synthesis of β-lactamase. To meet this challenge, β-lactamase with greater β-lactamase stability, including
cephalosporins, carbapenems, and monobactams, were introduced in the 1980s.
There is presently no CLSI or other approved criteria for AmpC β-lactamase detection (11)
, however
various workers have detected AmpC enzyme by three dimensional assay using cell extract, and AmpC disc
screening assay etc. The true rate of occurrence of AmpC β-lactamases in different organisms, including
members of Enterobacteriacae, remains unknown Coudron et al.(12)
used the standard disc diffusion breakpoint
for cefoxitin (zone diameter <18mm) to screen isolates, and used a three dimensional extract test as a
confirmatory test for isolates that harbour AmpC β-lactamases. The disc diffusion test was found to be non
specific and there is always a search for newer methods and the aim to make existing methods more user-

The occurrence of AmpC β-lactamase and ESBL producing Gram-negative bacteria by a simple …..
friendly to detect these enzymes for use in routine diagnostic laboratories. The main aim is to pass on the benefit
to the ultimate beneficiary, the patient, as quickly as possible and, obviously, at lowest possible cost(13)
.
The present study was designed to determine the occurrence of β-lactamases from Barabanki region. In
present study used whole cell growth in place of cell extract. The three dimensional test being made more user
friendly to be applied as a phenotypic screening method for the detection of AmpC-harbouring Gram negative
isolates.
II. Material And Method
A total of 141 sample, non-enteric Gram-negative clinical isolates obtained from different clinical
samples (e.g. Pus, urine, sputum, blood, tracheal aspirate, peritoneal fluid, catheter tip, ET tip tracheostomy
aspirate, etc) in clinical bacteriology laboratory Department of Microbiology Mayo Medical College and
Hospital, during June 2014 to January 2015 were included in this study.
The organism included E.coli (51 isolates), Pseudomonas spp (32 isolates), Klebsiella pneomoniae (23 isolates),
Klebsiela spp (18 isolates), Acinetobacter spp (10 isolates), Proteus spp (5 isolates),Citrobacter spp (one
isolate), and Enterobacter spp (one isolate).
The isolates were identified by standard microbiological techniques used in the laboratory.
Antimicrobial susceptibility of the strains were put according to CLSI guidelines, source of the discs were
HiMedia (19)
a. ESBL Detection
Irrespective of their antimicrobial susceptibility profile all isolate of E.coli and Klebsiella spp and
Proteus spp were tested for ESBL production using Ceftazidime (30µg) discs and Ceftazidime / Clavulanic acid
(30/10 µg) discs were used as, recommended by CLSI Guideline. Eschericia coli ATCC 25922 was included in
the study for ensuring quality control. Klebsiella pneumoniae 700603 ATCC were used as an ESBL Positive
control. Increase in zone diameter of ≥5 mm for Ceftazidime/Clavulanic acid versus its zone when tested alone
was a positive test for ESBL producer.
b. AmpC enzyme Detection : three-dimensional extract test
AmpC enzyme production was detected by a modified three-dimensional extract test
Fresh overnight growth from Mueller-Hinton agar was transferred in peptone water and incubated it for 2-4
hours at 37ºC.
Lawn culture of E. coli ATCC 25922 were prepared on Mueller-Hinton Agar plate and Cefoxitin (30 µg) disc
were placed on the plate.
Linear slits (3cm) were cut using a sterile lancet 3mm away from the periphery of Cefoxitin disc.
Small circular wells were made on the slits at 5mm distance, inside the outer edge of the slit, by stabbing the cut
end of micropipette tip.
The wells were loaded slowly with peptone water growth in 10 µL increments until the well was filled to the
top, taking care to not overflow.
The plates were kept upright for 5-10 minutes until the solution dried, and the plates were incubated at 37ºC.
overnight.
The isolates showing clear distortion of zone of inhibition of cefoxitin disc were taken as AmpC producers. The
isolate with no distortion were taken as AmpC non-producers.
III. Result
a. ESBL Profile
Among all the strains tested out of 141 isolates were tested for ESBL detection and 91 (64.53%) were found to
be ESBL producer and 50 (35.46%) were ESBL non-producer. E. coli was the highest occurrence of ESBL
producer (45.07%), followed by Klebsiella pneumoniae (29.57%).
ISOLATES
ESBL POSITIVE
N (%)
ESBL NEGATIVE
N (%)
E. col 42(46.15) 20(21.97)
Klebsiella pneumonia 25(16.48) 15(16.48)
Klebsiella spp. (other than K. pneumoniae) 20(21.97) 10(10.98)
Proteus mirabilis 2(2.19) 3(3.29)
Proteus spp. (other than P. mirabilis) 2(2.19) 2(2.19)

b. AmpC β-lactamase Profile
Among all the strains out of 141 isolates were tested 47 are AmpC producer and 94 are AmpC non-producer.
Maximal incidence of AmpC producers was found among E. coli (20) followed by Klebsiella pneumoniae (10).
ISOLATE
n = 141
AmpC producer
n = 47
AmpC
non-producer
n = 94
Total
E. coli 20 42 62
Klebsiella pneumonia 10 25 35
Klebsiella spp. 52 20 25
Pseudomonas spp. 5 2 7
Acinetobacter spp. 3 1 4
Proteus spp. 2 4 6
Citrobacter spp. 1 0 1
Enterobacter spp. 1 0 1
c. AmpC negative ESBL profile
A total of 94 AmpC non producer were tested for ESBL production and out of them 64 (68.08%) were found to
be ESBL producer while 30 (31.91%) were ESBL non producer.
IIssoollaattee
nn == 9944
AAmmppCC nneeggaattiivvee EESSBBLL ppoossiittiivvee
nn == 6644 ((6688..0088%%))
AAmmppCC nneeggaattiivvee EESSBBLL nneeggaattiivvee
nn == 3300 ((3311..9911%%))
EE.. CCoollii 2255 ((2266..5599%%)) 1155 ((1155..9955%%))
KKlleebbssiieellllaa ppnneeuummoonniiaaee 1155 ((1155..9955%%)) 1100 ((1100..6633%%))
KKlleebbssiieellllaa sspppp.. 1122 ((1122..7766%%)) 33 ((33..1199%%))
PPrrootteeuuss sspppp 88 ((88..5511%%)) 22 ((22..1122%%))

d. Ward wise distribution
WWAARRDD // OOPPDD // IICCUU
NN == 114411
AAmmppCC PPOOSSIITTIIVVEE
nn == 4477 ((%%))
AAmmppCC NNEEGGAATTIIVVEE
nn == 9944 ((%%))
IICCUU 1155 ((3311..9911%%)) 2200 ((2211..2277%%))
WWAARRDD 2277 ((5577..4444%%)) 4433 ((4455..7744%%))
OOPPDD 55 ((1100..6633%%)) 3311 ((3322..9977%%))
e. Sample wise distribution
SSAAMMPPLLEE
nn == 114411 ((%%))
AAmmppCC ppoossiittiivvee
nn == 4477 ((3333..3333%%))
AAmmppCC nneeggaattiivvee
nn == 9944 ((6666..6666%%))
PPUUSS 2200 4400
UURRIINNEE 1133 3344
BBLLOOOODD 33 66
SSPPUUTTUUMM 22 44
PPEERRIITTOONNEEAALL FFLLUUIIDD 11 00
TTRRAACCHHEEAALL AASSPPIIRRAATTEE 66 22
EETT TTUUBBEE 11 33
CCAATTHHEETTEERR TTIIPP 11 33
VVAAGGIINNAALL SSAAWWBB 00 22

f. Antibiotic Sensitivity Profile
IV. Keywords:
Ak- Amikacin, Cef- Ceftazidime, Cef- Cefixime, Ctx- Ceftriaxone, Cip- Ciprofloxacin, Cpz- Cefoperazone, Cot-
Co-Tromoxazole, Dox- Doxicycline , Gati- Gatifloxacin, Gen- Gentamicin, Imi- Imipenem, Levo- Levofloxacin,
Mrp- Meropenem, Nit- Nitrofurantoin, , Pip- Pipercillin
V. DISCUSSION
AmpC and ESBL producing strains all over the world , it is necessary to know the prevalence of these
strains in hospitals.The occurrence of AmpC beta lactamase (33.33) in our isolate to be quite high. Also high
occorrence of ESBL (64.53%) is seen in our hospital. Maximal incidence of AmpC producers was found among
E. coli (20) followed by Klebsiella pneumoniae (10). Maximal incidence of ESBL producers was found among
E. coli (42) followed by Klebsiella pneumoniae (25). The highest incidence was found in the sample Pus 20
(42.55%), then in Urine 13 (27.65%), followed by Tracheal aspirate 6 (12.76%), then in blood 3 (6.3%). The
highest resistance rate was found in cephalosporins like Ceftazidime, Ceftriaxone, & CO-Trimoxazole (100%).
The lowest resistance rate was found in Imipenem 16.39% followed by Meropenem 36.68%. Highest
incidence was found in patient those who was admitted in ward i.e, 27(57.44%) followed by ICUs 15(31.91%),
then in OPD 5(10.63%). Out of all AmpC negative strains 94were tested for ESBL detection and from them 64
(68.08%) were ESBL positive and 30 (31.91%)were ESBL negative.
Among the ICUs the highest rate was found in SICU, followed by NICU and MICU.
VI. Conclusion
Various researchers have tried the three dimensional test as well as AmpC disc test for screening of
AmpC β lactamases but till date no satisfactory technique has been found for routine use. This study included
AmpC disc screening test and found out that the modified three dimensional test using whole cell growth gives
clearer result. Modified three dimensional test using whole cell growth in peptone water is well comparable to
the modified 3 dimensional test using cell extract method and is better than AmpC disc screening assay at the
same time is very cost effective and simple assay to be used for routine reporting of AmpC β-lactamase.
Total
(N=141) Imi Mrp Cef Pip Cef Ctx Cip Levo Gati Doxi Gen Amk Cpz Nit Cot
Resistant 15.38 36.58 100 55.81 100 100 90 61.9 55.82 82.92 91.89 64.7 68 66 100
Sensitive 84.61 63.42 0 44.19 0 0 10 38.1 44.18 17.08 8.1 34.3 32 34 0

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