Detection of chloramphenicol resistance genes in broad
spectrum β lactamase-producing Escherichia coli strains
Mohamed Hamed Mohamed Al-Agamy
Microbiology and Immunology department, Faculty of Pharmacy
Al-Azhar University, Cairo, Egypt
Abstract:
Out of twenty one broad spectrum β-lactamase (BSBL) producing E. coli strains,
eighteen strains (85.71%) were found chloramphenicol (CHL) resistant. Existence of five
different CHL resistance genes i.e. catI, catII, catIII, catB and cmlA genes, were checked
using polymerase chain reaction (PCR) specific primer pairs. Only catI gene was
detected in eight strains (44.44%), whereas cmlA was found alone in one strain (5.55%).
The remainder nine strains (50%) showed coexistence of both catI and cmlA genes. These
findings proved that the enzymatic inactivation by chloramphenicol acetyl transferase
(CAT) due to catI gene was more frequent than non-enzymatic efflux mechanism by
cmlA gene. In addition, both mechanisms were more likely to coexist in the strain than to
present alone. Absence of catI gene was linked to comparatively low CHL resistance
phenotype (MIC = 16 ug/ml), whereas catI existence either in the absence of cmlA or in
its existence is tied to high CHL resistance phenotype (MIC 32 to > 128 µg/ml). This
investigation is devoted to studying the molecular genetic mechanisms for CHL
resistance in BSBL-producing E. coli strains in comparison with those in extended
spectrum β-lactamase (ESBL) producing E. coli strains (Al-Agamy, 2004).
Introduction:
Chloramphenicol is a broad spectrum bacteriostatic antibiotic, which is effective
in the treatment of a wide variety of bacterial infections including serious anaerobic
infections (Vassort-Brunean et al., 1996). Molecular mechanisms for CHL resistance
were found to be due to: plasmid or chromosomal encoded enzymatic CAT activity
(Gaffney et al., 1978 and Shaw, 1983) and non-enzymatic CHL resistance induced by
exposure to subinhibitory level of CHL (Gaffney et al., 1981; Bissonnette et al., 1991 and
Cloeckaert et al., 2001).
Chloramphenicol enzymatic inactivation is the more common resistance
mechanism, where CAT enzyme acetylates CHL rendering the antibiotic unable to
interfere with translation (Rogers et al., 2002). Several cat genes, both from Gram
positive and Gram negative organisms were sequenced. All sequenced cat genes showed
a good degree of similarity especially around the active site (Bannam and Rood, 1991).
Two groups of cat genes were identified, catA and catB groups respectively, catA group
comprises catI, catII and catIII genes, whereas catB group includes catB1 to catB9
(Tennigkeit and Matzura, 1991; Parent and Roy, 1992; Bunny et al., 1995; VassortBrunean et al., 1996 and Pai et al., 2003). On the other hand, non-enzymatic CHL
resistance mechanism was encoded on plasmids of different incompatibility groups in
Gram negative bacteria (Gaffney et al., 1978 and Gaffney et al., 1981). Basically, it is an
1
efflux mechanism which is present principally in Gram negative bacteria (Bissonnette et
al., 1991).
Materials and Methods:
Bacterial strains:
Twenty one clinical E. coli strains were collected from urinary tract infections and
burns at Sayed Galal and Al-Hussein Hospitals in Cairo during the period from June 2001
to September 2001.These strains were stored in brain heart infusion broth containing 20%
glycerol at -700C.These strains were selected on the criteria of susceptibility to extended
spectrum cephalosporins such as cefotaxime. The strains were resistant to ampicillin,
amoxicillin and susceptible to cefotaxime. The selected strains characterized
phenotypically as BSBL-producing E. coli strains.
Determination of minimum inhibitory concentrations (MICs):
The antimicrobial MICs for to BSBL-producing E. coli strains were determined
by broth micro-dilution method according to the recommendation of national committee
for clinical laboratory standards (NCCLS, 2001 and 2002) using microtiter plates
containing dehydrated antibiotics (Merlin Diagnostika, Germany) in two–fold dilution.
The following antibiotics were tested: ampicillin, amoxicillin, amoxicillin/clavulanic
acid, cefotaxime, cefotaxime/clavulanic acid, gentamicin, norfloxacin, and
chlorampheicol.
Preparation of plasmid:
Plasmids were prepared by a rapid alkaline lysis method according to Sambrook
and Russel (2001). Plasmids were stored at -200C and used as DNA template in PCRs.
DNA primers:
The primers used in the current research are listed in Table 1.
Table (1): Primers used in this study
Target
primer
Position
Size
Sequence 5’3’
Annealing
References
temp (0C)
CatI-F
F
CatI-R
R
catII
CatII-F
F
CatII-R
R
catIII
CatIII-F
F
CatIII-R
R
catB
CatB-FA
F
CatB-RC
R
catI
cmlA
cmlA-F
F
cmlA-R
R
585
GGCATTTCAGTCAGTTG
50
495
CCTGGAACCGCAGAGAAC
508
ATTGGCTTCGCCGTGAGC
280
TTYATBATGGCBGGBAATCARGGNC
VassortBruneau et al.
CCGCCCTGCCACTCATC
50
1996
CCTGCTGAAACTTTGCCA
50
AGTCTATCCCCTTCTTG
48
2003
GARCCDATCCAVACRTCATKDCC
698
CCGCCACGGTGTTGTTGTTATC
CACCTTGCCTGCCCATCATTAG
Sherwood,
50
Keyes et al.
2000
* Codon is degenerate codon built an aminoacid sequence preserved in all catB genes
2
Molecular detection of CHL resistance determinants by PCR:
PCR was used to detect catI, catII, catIII, catB and cmlA genes according to
references listed in Table 1. All of PCRs were conducted under standard conditions using
plasmid DNA as template, taq polymerase (Sigma), deoxynuclosides triphosphates
(Bohrenger manheim), HPLC-grade water (Merck). All of PCRs were done in i Cycler
(Bio-Rad, München, Germany). PCR amplified products were separated in 0.8% agarose
gels stained with ethidium bromide, detected with UV light and digitally recorded. PCRs
of E. coli plasmids of CHL-susceptible E. coli strains i.e. EC10 (MIC 4g/ml), EC13 (MIC
8 g/ml), and EC16 (MIC 4 g/ml) are used as negative controls.
Results:
Antimicrobial susceptibility testing:
The result of MICs of tested E. coli strains are listed in Table 2. Data showed that
that all the tested strains, i.e. 21 E. coli strains, were resistant to ampicillin (100%) and
amoxicillin (100%), whereas all strains were sensitive to cefotaxime and
cefotaxime∕clavulanic acid, i.e. all strains were BSBL type but not of the ESBL type
according to Westphal scheme for characterization of β-lactamases (Westphal et al.,
2002). When these strains were tested for CHL resistance eighteen strains (85.71%) were
found resistant and three strains (14.29%) were considered sensitive, i.e. E. coli strains
designated as EC10 (MIC 4g/ml), EC13 (MIC 8 g/ml), and EC16 (MIC 4 g/ml).
Phenotypic expression of resistance was varied between tested strains. For example, E.
coli strains EC18 and EC20 (9.52%) had MIC16 g/ml whereas strain EC16 and EC 17
(9.52%) had MIC = 32 g/ml. Other resistant strain (76.19%) showed MIC 128 g/ml.
interestingly, four strains designated as EC17-EC19 and EC21 were found to be resistant to
both gentamicin and norfloxacin, whereas strain EC20 was resistant to gentamicin only.
Molecular characterization of chloramphenicol resistance genes:
A. Characterization of enzymatic inactivation mechanism(s):
PCR was performed for detection of catI, catII, catIII and catB genes in the tested
E. coli strains. Results of catII, catIII and catB-PCRs (Data not shown) proved the
absence of catII, catIII, and catB genes in all tested E. coli strains. On the other hand, all
CHL resistant strains except strain EC16 harboured catI gene, i.e. 17 (94.44%) out 18
CHL-resistant E. coli strains designated as EC1-EC9, EC11, EC12, EC14, EC15, EC17 and
EC19 - EC21 were catI positive.
B. Characterization of non-enzymatic inactivation mechanism:
PCR was performed for detection of cmlA gene in the tested E. coli strains. Ten E.
coli strains (55.55%) showed existence of cmlA gene (figure 2), namely strains
designated as EC2-EC9, and EC18-EC20. All of cmlA efflux mechanism was coexisting
with catI enzymatic inactivation mechanism except strain EC18 showed cmlA efflux
mechanism alone (Table 3).
3
Table (2): MICs data of BSBLs-Producing E. coli strains.
Antibiotics
AMP
AMX
AMX/C
CEFT
CEFT/C
GEN
NOR
CHL
MIC (µg/ml)
EC1
EC2
EC3
EC4
EC5
EC6
EC7
EC8
EC9
EC10
EC11
512
>2048
512
1024
512
1024
>2048
>2048
>2048
>2048
>2048
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16/4
16/4
16/4
8/4
8/4
8/4
32/4
32/4
32/4
16/4
32/4
0,5
<0,25
<0,5
<0,5
<0,5
<0,5
<0,5
<0,5
<0,5
<0,5
<0,5
<0,5/2
<0,5/2
<0,5/2
<0,5/2
<0,5/2
<0,5/2
<0,5/2
<0,5/2
<0,5/2
<0,5/2
<0,5/2
1
1
1
2
2
2
2
2
1
2
1
0,5
0,5
0,125
0,5
0,5
0,25
0,25
0,25
0,25
0,25
0,25
>128
>128
>128
>128
>128
>128
>128
>128
>128
4
>128
Antibiotics
MIC (µg/ml)
E. coli
EC12
EC13
EC14
EC15
EC16
EC17
EC18
EC19
EC20
EC21
2
>2048
>2048
2048
512
2048
1024
>2048
2048
512
2048
ATCC 25925
AMP
AMX
AMX/C
CEFT
CEFT/C
GEN
NOR
CHL
2
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
<4/4
64/4
16/4
64/4
16/4
32/4
32/4
16/4
32/4
4/4
16/4
<0,5
<0,5
<0,5
<0,5/2
<0,5
<0,5
<0,5
<0,5
<0,5
<0,5
<0,5
<0,5/2
<0,5/2
<0,5/2
<0,25
<0,5/2
<0,5/2
<0,5/2
<0,5/2
<0,5/2
<0,5/2
<0,5/2
0.5
1
2
2
2
1
32
32
32
32
>32
<,03
0,25
0,25
0,5
2
>32
1
32
>32
0,25
32
0,25
>128
8
>128
>128
4
32
16
>128
>128
>128
AMP = ampicillin
.AMX = amoxicillin
CEFT = cefotaxime
M
GEN = gentamicin
1
2
3
C = clavulanic acid
NOR = norfloxacin
4
5
Band size
bp
catI
1000
800
600
400
200
A
4
6
CHL = chloramphenicol
7
8
9
10
11
M
12
13
14
15
16
17
18
19
20
21
NC
catI
600 
400
200
B
Fig. 1: catI-PCR of the tested BSBL-producing E. coli strains using catI-F and catI-B primers.
Lane M contained 5 l of1kb DNA ladder and lanes 1-21 contained 10 l (each) of catI-PCR
products BSBL-producing E. coli strains designated as EC1 to EC21 respectively. Lane NC
contained 10 l of catI-PCR product of negative control. All were showing catI resistant
plasmid except EC10, EC13, EC16 and EC18 (lanes 10, 13, 16 and 18 respectively).
M
1
2
3
4
5
6
7
8
9
10
11
18
19
20
cmlA
A
M
NC
12
13
14
15
16
17
21
cmlA
B
Fig. 2: cmlA-PCR of the tested ESBL-producing E. coli strains using cmlA-F and cmlA-B
primers. Lane M contained 5 l of1kb DNA ladder and lanes 1-21 contained 10 l (each) of
cmlA-PCR products BSBL-producing E. coli strains designated as EC1 to EC21 respectively.
Lane NC contained 10 l of catI-PCR product of negative control.
5
Discussion:
This study showed that the frequency of CHL resistance in BSBL strains was
85.71%. A previous study showed that 29 out of 30 ESBL-producing E. coli strains
(96.66%) were resistant to CHL (Al-Agamy, 2004). CHL resistance could be genetically
related to two major mechanisms. First: antibiotic inactivation by CAT enzymes
produced from two groups of cat genes, i.e. catA genes which include catI, catII and
catIII genes as well as catB genes which comprise catB1-9. Second: alteration in
permeability to antibiotics through an efflux mechanism mediated by cmlA gene product
(Gaffney et al., 1978; Burns et al., 1986; Bissonnette et al., 1991 and Bissonnette and
Roy, 1992).
Results of this study showed that the frequency of detecting a gene for enzymatic
inactivation in BSBL-producing E. coli strains was 94.44%, whereas the frequency for
detecting a gene for efflux mechanism was 55.55%. A previous study on ESBLproducing E. coli strains demonstrated that the frequency for finding a gene for
enzymatic inactivation was 100%, whereas the frequency for a gene for efflux
mechanism was 20.7% (Al-Agamy, 2004). This is in accordance with previous findings
that the efflux resistance gene, cmlA, is a less frequent mechanism for CHL resistance
(Burns et al., 1986; Bissonnette et al., 1991; White et al., 2000; Cloeckaert et al., 2001;
George and Hall, 2002 and Al-Agamy, 2004). In addition, several researchers reported
that CHL inactivation mechanism is the most frequent mechanism for CHL resistance
(Vassort-Brunean et al., 1996; White et al., 2000; Cloeckaert et al., 2001; Kehrenberg
and Schwarz, 2001 and Al-Agamy, 2004).
As shown in Table (3) catI gene was more tied with high CHL resistance
phenotype (MIC 32≥128 g/ml), whereas cmlA gene might link to intermediate
resistance to CHL (MIC = 16 g/ml). Further studies have be done to prove this issue.
The present study showed absence of catII, catIII, and catB genes as a source of
enzymatic inactivation mechanism. Which was solely related to catI gene existence. It is
known that catI gene is the most widely distributed however since CHL use in human
therapy is limited because of concerns about its toxicity; the evolution and prevalence of
these mechanisms of resistance are not well documented (Vassort-Brunean et al., 1996).
As for existence of more than one mechanism for CHL resistance in the same
strain, results of this study showed that 9 out of 18 E. coli CHL resistant strains (50%)
harbour genes for both CHL enzymatic inactivation and CHL efflux mechanisms (Table
3) followed by presence of CHL enzymatic inactivation gene (catI) alone, i.e. 8 out of 18
E. coli CHL resistance strains (44.44%), while presence of cmlA gene alone is the least
frequent (one strain, 5.56%).
In a study of CHL resistance genetic mechanisms in ESBL-producing E. coli
strains, it was fond that catI gene was the most frequent gene to be found alone (12 out of
29 strains  41.38%). This was followed by existence of catB gene alone in 9 out of 29
strains (31.03%). On the other hand, the efflux gene, cmlA, was never found alone, it was
always either in coexistence with catB gene (6 strain = 20.86%) or in coexistence with
catI gene (2 strains = 6.89%). Presence of two genes with the same mechanism for CHL
resistance was the least frequent mechanism, e.g. catI and catB in one strain (3.44%). In
all situations, presence of catI and catB was linked to high CHL resistance phenotype
whether cmlA gene coexist with one of them (Al-Agamy, 2004).
The existence of catB gene in 16 out of 29 ESBL-producing strains (55.17%) as
6
the most frequent gene for CHL resistance followed by catI gene in 15 out of 29
(51.72%) ESBL-producing strains (Al-Agamy, 2004) showed more diversity in the
molecular genetic mechanisms for CHL resistance in ESBL-producing E. coli strains than
in BSBL producing E. coli strains even though they were isolated from the same
Egyptian hospitals. Also the coexistence of two genes of different CHL resistance
mechanism is more frequent than coexistence of the two genes with the CHL resistance
mechanism in the same strain (27.75% vs. 3.44%) (Al-Agamy, 2004). This is considered
as another feature for efficient combination between the two mechanisms for CHL
resistance.
The frequent use of CHL as well as broad spectrum penicillins, e.g. amoxicillin as
cheap antibiotics in Egypt directed a strong selective pressure led to horizontal antibiotic
resistance gene transfer among bacteria. These genes are localized on genetic elements,
such as plasmids, transposons, and gene cassettes inserted into integrons (Lévesque et al.,
1995, and Fluit and Schmitz, 1999). This location favors dissemination of resistance
genes among nosocomial bacteria (Reyes et al., 2003). The diversity in mobile genetic
element combinations between different strains isolated from the same hospitals proposes
that mobile genetic elements play an essential part in the epidemic spread of antibiotic
resistances among bacterial populations, since the spread of the resistance genes is
greatly enhanced when they form part of a mobile gene cassette. (Collis and Hall, 1992
and Liebert et al., 1999).
Stokes and Hall (1989) have defined the elements borne on these multiresistance
plasmids and transposons as integron. Integrons contain the genetic determinants of the
compounds of site-specific recombination, i.e. integrase gene (int) accompanied with
attachment site (attI) which recognizes the 3' end of the gene cassette leading to sitespecific recombination with the gene incorporating it between 5' and 3' conserved
segments (Liebert et al., 1999). Several gene cassettes could be integrated into the same
integron leading the diversities in genetic elements between different strains (Stokes et
al., 1993, Arduino et al., 2002, Sabaté et al., 2002 and Wang et al., 2003). The integrated
gene cassette of integron encode the resistance determinants such as those for
aminoglycoside acetyltransferases and nucleotidyltransferases, β-lactamases and
enzymatic (acetyltransferases) and nonenzymatic resistance to CHL. CHL resistance
genes are usually encoded on mobile genetic elements such as transposons and integrons.
For example, the catI gene is encoded on Tn2424 (Parent and Roy, 1992) and catB3 is
encoded on integron in pBWH301 (Bunny et al., 1995). Finally cmlA gene is a part of
mobile gene cassette of In4 of Tn1696 (Stokes and Hall, 1991 and Bissonnette et al.,
1991).
The only way to decrease the dissemination of genes for antibiotic resistance is to
release the selective pressure for these resistance elements by both prohibiting usage of
cheap antibiotics as additives to animal and poultry feed, as well as preventing improper
use of antibiotic in human medication. Antibiotics must be prescribed in human
medication under proper and careful professional medical supervision where the proper
dosage and duration of therapy are strictly observed. Chloramphenicol was finally
forbidden in Europe for veterinary use in farm animal in 1994 (Vassort –Brunean et al.,
1996). In conclusion, the occurrence of CHL resistance genes either enzymatic or nonenzymatic gene on integron as gene cassette reported by many investigators, could
explain its distribution and prevalence in E. coli and several bacterial species.
7
Table (3): Detected genes of CHL-resistant BSBL-producing E. coli strains.
CHL detected genes
MIC
( g/ml)
E. coli
Enzymatic inactivation
Efflux
catI
catII
catIII
catB
cmlA
EC1
>128
+
-
-
-
-
EC2
>128
+
-
-
-
+
EC3
>128
+
-
-
-
+
EC4
>128
+
-
-
-
+
EC5
>128
+
-
-
-
-
EC6
>128
+
-
-
-
+
EC7
>128
+
-
-
-
+
EC8
>128
+
-
-
-
+
EC9
>128
+
-
-
-
+
EC11
>128
+
-
-
-
-
EC12
>128
+
-
-
-
-
EC14
>128
+
-
-
-
-
EC15
>128
+
-
-
-
-
EC17
32
+
-
-
-
-
EC18
16
-
-
-
-
+
EC19
>128
+
-
-
-
+
EC20
>128
+
-
-
-
+
EC21
>128
+
-
-
-
--
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