Vol. 25 No. 3 129Ampc β-Lactamase Production in Gram-Negative Bacilli:-Chaudhary U, et al.

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Original Article

Detection of Inducible AmpC βββββ-Lactamase-ProducingGram-Negative Bacteria in a Teaching Tertiary CareHospital in North India

ABSTRACT

AmpC β-lactamase enzymes confer resistance to a wide variety of β-lactam antibiotics, except

carbapenems. AmpC β-lactamase-producing bacteria are known to be causative pathogens of nosocomial

infections, which are difficult to be treated. Hence, this study was aimed out to determine the prevalence of

AmpC β-lactamase enzyme production among Gram-negative bacteria. A total of 500 multidrug-resistant

isolates were tested for cefoxitin susceptibility during a one-year period of study. All the isolates were

tested for AmpC β-lactamase production by both the disk approximation and AmpC disk methods. The

isolates positive for AmpC β-lactamase production were also tested for antimicrobial susceptibility by the

disk diffusion method. AmpC β-lactamase production was noted in 101 (20.20%) isolates. All the isolates

were 100-percent susceptible to imipenem and meropenem with variable susceptibilities to other antimicrobial

agents. (J Infect Dis Antimicrob Agents 2008;25:129-33.)

Uma Chaudhary, M.D., FIAMS*,Ritu Aggarwal, M.D.*Sanjeev Ahuja, M.D.**

*Department of Microbiology, Pt. B.D. Sharma, PGIMS, Rohtak, Pt. B.D. Sharma, PGIMS, Rohtak, India.**Senior Resident, Super Specialty Hospital Janakpuri, New Delhi, India.Received for publication: July 4, 2008.Reprint request: Dr. Ritu Aggarwal, M.D., H. No. 717, Sector-1, HUDA, Rohtak-124001, Haryana, India.

E-mail: drritu252@yahoo.com

Keywords: AmpC β-lactamase, multidrug resistance, carbapenems, Gram-negative bacteria

INTRODUCTION

β-lactamase enzyme production is one of the most

common mechanism of drug resistance to β-lactam

antibiotics in Gram-negative bacteria, and class C

(AmpC) enzymes are very important and clinically

significant.1

AmpC β-lactamases are cephalosporinases that

are poorly inhibited by clavulanic acid. They can be

differentiated from the extended-spectrum β-

lactamases (ESBLs) by their ability to hydrolyze

cephamycins and other extended-spectrum cephalo-

sporins. AmpC β-lactamases, either chromosomally

or plasmid mediated, have been described in various

pathogens including Klebsiella pneumoniae, Escherichia

coli, Salmonella spp., Proteus mirabilis, Citrobacter

freundii, Acinetobacter spp., Enterobacter spp., and

Pseudomonas aeruginosa.2 The enzyme production

can be constitutive or inducible.3

130 J INFECT DIS ANTIMICROB AGENTS Sep.-Dec. 2008

The prevalence of AmpC β-lactamase-producing

Gram-negative bacteria appears to be increasing, and

these bacteria has been responsible for some nosocomial

infections. It has been stated that the detection of

AmpC β-lactamase production is challenging since the

hyperproduction of chromosomal AmpC in associated

with OMP F porin loss in E. coli or porin deficiency in

K. pneumoniae can produce similar resistance

phenotypes.1

There is no clear consensus regarding the

guidelines for performing tests for the phenotypic

screening or confirmatory tests for the bacterial isolates

that harbour AmpC β-lactamases.4 In addition, the

Clinical Laboratory Standards Institute (CLSI) do not

recommend any method for the detection of AmpC β-

lactamase production.5 This study was aimed to

determine the occurence of inducible AmpC β-

lactamase-producing Gram-negative bacteria in Pt.

B.D. Shama PGIMS, Rohtak, North India, a tertiary

hospital, using the standard phenotypic methods

presently available for their detection.

MATERIALS AND METHODS

A total of 500 multidrug-resistant Gram-negative

bacteria, non-repetitive clinical isolates obtained from

the pus (100 isolates), the urine (100 isolates), the blood

(100 isolates), the cerebrospinal fluid, the other body

fluids (100 isolates), and the sputum (100 isolates) were

studied over a one-year period from April 2005 to March

2006. All isolates were identified, using the standard

conventional microbiological techniques.6 All isolates

were resistant to one or more than 3 classes of

antibiotics on antimicrobial susceptibility, testing

determined by the disk diffusion method. The isolates

were also tested for cefoxitin susceptibility, using the

30-μg cefoxitin disk diffusion method. The results were

interpreted as suggested by the CLSI 2006 guidelines,

and the ingibition zone diameter of less than 18 mm

was considered as resistant.5

All isolates of both groups were tested for AmpC

β-lactamase production by both the disk approximation7

and AmpC disk methods.2

Regarding the disk approximation method, the

isolate was inoculated onto a Mueller-Hinton Agar

(MHA) plate as recommended by the CLSI. The

cefoxitin (inducer) disk was placed at 2.5 cm (center-

to-center) distance from the cefotaxime or ceftazidime

disc for Enterobacteriaceae and P. aeruginosa,

respectively. After an overnight incubation, a flattening

of the inhibition zone around the antibiotic disk towards

cefoxitin disk equal or more than 1 mm was recorded

as the positive test for AmpC β-lactamase production.

Regarding the AmpC disk method, a lawn culture

of E. coli ATCC 29522 was prepared onto a MHA

plate. The sterile disks (6 mm in diameter) were

moistened with the sterile saline (20 mL) and inoculated

with several colonies of the test bacteria. The inoculated

disk was then placed besides a cefoxitin disk (almost

touching) onto the inoculated plate. The plates were

incubated overnight at 35�C. A positive test appeared

as a flattening or indentation of the cefoxitin inhibition

zone in the vicinity of the test disk.

All the AmpC β-lactamase-producing isolates

were tested for antimicorbial susceptibility by the

Stoke’s disk diffusion method.8 Briefly, 0.5 McFarland

turbidity suspension was prepared from the test isolates

and the standard control strains. The control inoculum

was spread in two bands on either side of the plate,

leaving a central band uninoculated. The test isolate

inoculum was streaked onto the central area of the plate.

An uninoculated gap of 2-3 mm wide was left between

the test and control isolates. The antimicrobial agents

tested for susceptibility included levofloxacin (5 μg),

sparfloxacin (5 μg), enrofloxacin (5 μg), ofloxacin (5

μg), tobramycin (30 μg), sisomycin (10 μg), netilmicin

(30 μg), fosfomycin (50 μg), aztreonam (30 μg),

Vol. 25 No. 3 131Ampc β-Lactamase Production in Gram-Negative Bacilli:-Chaudhary U, et al.

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cefepime (30 μg), imipenem (10 μg), meropenem (10

μg). The control strains E. coli NCTC 10418 and P.

aeruginosa NCTC 10662 were used.

RESULTS

A total of 500 multidrug-resistant Gram-negative

bacterial isolates, 340 and 160, respectively were

susceptible and resistant to cefoxitin. All 340 cefoxitin-

susceptible isolates were also susceptible to

cefotaxime and ceftazidime. Of 160 cefoxitin-

resistant isolates, 101 (20.20 %) Gram-negative

isolates were found to be positive for AmpC β-

lactamase production by the both phenotypic detection

methods. AmpC β-lactamase-producing isolates were

obtained from the urine (35 %), followed by the pus

(33 %), the blood (23 %), and others (Table 1). AmpC

β-lactamase production was most commonly noted in

P. aeruginosa (25.8 %), followed by Acinetobacter

spp. (21.43 %), and E. coli (19.05 %) (Table 2).

These AmpC β-lactamase producing isolates

were 100-percent susceptible to carbapenems including

meropenem and imipenem. The high rate of resistance

was observed in all other antimicrobial agents, ranging

from 36.63 percent to 99.01 percent (Table 3).

Table 1. Distribution of inducible AmpC βββββ-lactamase-producing isolates according to different

clinical specimens.

Specimen

Urine 100 35 (35%)

Pus 100 33 (33%)

Blood 100 23 (23%)

Cerebrospinal fluid and 100 5 (5%)

other body fluids

Sputum 100 5 (5%)

Total 500 101 (20.2%)

Isolates

Number

AmpC-producing isolates

Number (%)

Table 2. Distribution of inducible AmpC β-lactamase-producing isolates in different organisms.

Organism

Pseudomonas aeruginosa 183 47 (25.7%)

Escherichai coli 126 24 (19.1%)

Enterobacter spp. 79 13 (16.5%)

Acinetobacter spp. 42 9 (21.4%)

Klebsiella spp. 42 5 (11.9%)

Citrobacter spp. 18 3 (16.7%)

Proteus spp. 10 0 (0%)

Total 500 101 (20.2%)

Isolates

Number

AmpC-producing isolates

Number (%)

132 J INFECT DIS ANTIMICROB AGENTS Sep.-Dec. 2008

DISCUSSION

Despite the discovery of ESBLs and AmpC β-

lactamases at least two decades ago, there remains a

low level of awareness regarding their laboratory

detection and clinical significance. The confusion exists

about the importance of these resistance mechanisms,

the appropriate test methods, and the appropriate

reporting conventions. The failure to detect these

enzymes has contributed to their uncontrolled spread

and sometimes to therapeutic failures.2

In this study, 20.20 percent of multidrug-resistant

isolates were positive for AmpC β-lactamase

production. Our results are in concordance with the

previous studies.4-9 One study in Delhi, India, reported

a 20.7-percent AmpC β-lactamase production rate

among Gram-negative bacteria.9 The higher rate of

AmpC β-lactamase production was also reported by

several studies.10,11 However, some studies described

the low rate of AmpC β-lactamase production.7,12 This

difference may be due to the different selection criteria

of isolates, the variation in an ability to produce AmpC

β-lactamases among different Gram-negative bacteria,

and different clinical specimens.4

Of 160 cefoxitin-resistant isolates, 59 (36.97%)

were negative for AmpC β-lactamase production.

Cefoxitin resistance in these isolates could be due to

the lack of permeation porins.9 This result indicates

that eventhough the screening methods that use the

cefoxitin for the detection of AmpC-producing isolates

are easily performed, but they are not accurate.

This study had some limitations including the lack

of molecular epidemiologic analysis due to our

constrains, and no available detection of ESBL

production of the same isolates. However, there was

no evidence of clonal spread, based on the antimicrobial

susceptibility patterns of the isolates. In addition, the

antimicrobial susceptibility was carried out by the

Stoke’s disk diffusion method which is not suggested

by the CLSI. The therapeutic options for infections

caused by Gram-negative bacteria expressing AmpC

β-lactamases are limited because these organisms are

usually resistant to all β-lactam antibiotics except

carbapenems. In addition, plasmid-mediated AmpC β-

lactamase-producing isolates are typically resistant to

multiple classes of antimicrobial agents.1

This is a preliminary study designed with an

objective to detect the possible occurence of AmpC β-

lactamases in a tertiary care hospital and to institute

antibiotic policy to minimize the emergence of

antimicrobial resistance. This is perhaps the first report

of AmpC β-lactamase production among Gram-

negative clinical isolates from this state of India.

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Table 3. Antibiotiic susceptibility of inducible AmpC

βββββ-lactamase-producing isolates.

Antimicrobial agent

Imipenem 101 (100%)

Meropenem 101 (100%)

Cefepime 92 (91.10%)

Aztreonam 1 (0.99%)

Sparfloxacin 34 (33.66%)

Ofloxacin 54 (53.46%)

Levofloxacin 41 (40.59%)

Enrofloxacin 34 (33.66%)

Fosfomycin 55 (54.45%)

Sisomycin 9 (8.91%)

Tobramycin 15 (14.85%)

Netilmicin 17 (16.83%)

Susceptibility

Number (%)

Vol. 25 No. 3 133Ampc β-Lactamase Production in Gram-Negative Bacilli:-Chaudhary U, et al.

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