MICROBIAL DRUG RESISTANCE

Volume 00, Number 00, 2019

ª Mary Ann Liebert, Inc.
DOI: 10.1089/mdr.2018.0282

Molecular Characteristics of ‘‘BlaB-Like’’ Chromosomal

Inducible Cephalosporinase of Yersinia enterocolitica
Biotype 1A Strains

Neelja Singhal,1 Deeksha Pandey,2 Nambram Somendro Singh,1 Manish Kumar,2 and Jugsharan Singh Virdi1

Yersinia enterocolitica biotype 1A strains are emerging pathogens, frequently isolated from clinical samples
across the globe. Y. enterocolitica strains produce two chromosomal b-lactamases, BlaA and BlaB. BlaA is a
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constitutively expressed, Ambler class A, penicillinase, whereas BlaB is Ambler class C-type, inducible ce-
phalosporinase. An earlier study from our laboratory indicated that instead of BlaB, Y. enterocolitica biotype
1A produced a ‘‘BlaB-like’’ enzyme. The objective of this work was to study the molecular characteristics of
‘‘Bla-B like’’ b-lactamases produced by biotype 1A strains to discern their similarity with AmpC-type
b-lactamases and the basis of varied levels of minimum inhibitory concentration (MIC) to cefotaxime. Thus, the
promoters and blaB genes were investigated in four strains of biotype 1A. Three-dimensional structures of the
‘‘BlaB-like’’ enzymes were modeled, and docked in silico with cefotaxime to understand how specific sub-
stitutions in gene sequences affect antibiotic susceptibilities. Our results indicated that all the reported key
catalytic residues were present in variants of ‘‘Bla-B-like’’ enzymes, discerned in biotype 1A strains, but at
different positions. Molecular docking of enzyme variants with cefotaxime revealed that lesser was the number
of the H-binding residues with cefotaxime in a strain, lower was the MIC of cefotaxime in that strain. To the
best of our knowledge, this is the first study in which the molecular characteristics and enzymatic interactions of
‘‘BlaB-like’’ cephalosporinases of Y. enterocolitica biotype 1A strains have been reported.

Keywords: cefotaxime, AmpC type, Yersinia enterocolitica

Introduction boring chromosomal, inducible AmpC b-lactamases have

significant clinical relevance.6 Many strains, when isolated

Y ersinia enterocolitica, an enteropathogenic bacte-

rial species has been classified into more than 60 sero-
types and 6 biotypes, with varied geographical distribution,
ecological niche, and pathogenic potential.1 Y. en-
terocolitica biotype 1B is highly pathogenic; biotypes 2–5
are moderately pathogenic, whereas the pathogenicity of
biotype 1A has been controversial.1 However, in the recent
past biotype 1A strains have been regarded as emerging
pathogens as these are being isolated frequently from clin-
ical samples across the globe.2–5
Y. enterocolitica strains produce two types of chromo-
somal b-lactamases named b-lactamase A (BlaA) and b-
lactamase B (BlaB). BlaA is a constitutively expressed,
Ambler class A, Bush group III penicillinase, whereas BlaB
is Ambler class C, Bush group I, AmpC-type inducible ce-
phalosporinase. The expression of blaB in Y. enterocolitica
is regulated by ampR and ampD. The ampR is transcrip-
tional regulator of ampC and, AmpD is a cytoplasmic N-
acetyl-anhydromuarmyl-l-alanine amidase. Bacteria har-
from the patient, are initially susceptible to broad-spectrum
cephalosporins. But when exposed to these antibiotics in vivo
during treatment of the patient, the genes get derepressed,
imparting resistance to the broad-spectrum antibiotics and
consequent treatment failures.7
Several investigators have reported the minimum inhibi-
tory concentration (MIC) and/or enzymatic data supporting
the presence of AmpC-type b-lactamases in species of
Yersinia.8–11 However, molecular data for AmpC-type b-
lactamases, except for a strain of serotype O: 5b has not
been reported for most of the bioserotypes of Y. en-
terocolitica.6,12 An earlier study from our laboratory indi-
cated that BlaB of Y. enterocolitica biotype 1A was different
from BlaB of other biotypes, as it revealed multiple bands of
pIs 6.8 and 7.1 on isoelectric focusing. This suggested that
Y. enterocolitica biotype 1A produced a ‘‘BlaB-like’’ en-
zyme.13 The Y. enterocolitica biotype 1A strains harboring
‘‘Bla-B like’’ cephalosporinase also showed high MIC for
cefoxitin (this study) and resistance to b-lactam/b-lactamase

Departments of 1Microbiology, 2Biophysics, University of Delhi South Campus, New Delhi, India.

1
 2 SINGHAL ET AL.
inhibitor combination,14 indicating the possibility of ‘‘Bla-B
like’’ produced by Y. enterocolitica biotype 1A strains to
be actually an AmpC or AmpC-type enzyme. Thus, the
objective of this work was to study the molecular char-
acteristics of ‘‘Bla-B-like’’ b-lactamase produced by Y.
enterocolitica biotype 1A strains and the basis of varied
levels of MIC to cefotaxime to discern its similarity to
AmpC-type b-lactamases. The promoter regions and the
complete coding sequences (CCDS) of blaB genes in four
strains of Y. enterocolitica biotype 1A were investigated.
Three-dimensional (3D) structures of the ‘‘BlaB-like’’ en-
zymes were modeled, and docked in silico with cefotaxime
to understand how specific substitutions in gene sequences
affect antibiotic susceptibilities. To the best of our knowl-
edge, this is the first study to investigate the molecular
characteristics of ‘‘BlaB-like’’ enzyme of Y. enterocolitica
biotype 1A.
Materials and Methods
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Bacterial strains
Four strains of Y. enterocolitica biotype 1A maintained in
our laboratory, (University of Delhi South Campus) at 4C
in trypticase soy agar, were examined. The details of the
strains are given in Table 1. These were well-authenticated
strains deposited with Yersinia National Reference Labora-
tory and WHO Collaborating Center, Pasteur Institute, Paris
(France) and at the national repository at Microbial Type
Culture Collection (MTCC) and Gene Bank located at In-
stitute of Microbial Technology (IMTECH), Chandigarh
(India).
Determination of antibiotic susceptibility for cefoxitin
and phenotypic detection of AmpC production
Antibiotic susceptibility testing for cefoxitin—a second
generation cephalosporin and phenotypic detection of
AmpC production—was carried out with E-test strips (bio-
Mérieux, Inc., MO) of cefoxitin and AmpC using methods
described previously.14 In brief, bacterial strains were grown
on Muller–Hinton (MH) agar plates at 28C, overnight.
Single colonies were picked from agar plates and suspended
in 1 mL of normal saline to reach a turbidity equivalent to
0.5 McFarland standards (*1–2 · 108 colony-forming
units/mL). A 100 mL of this cell suspension was spread on
the surface of MH agar plates and E-test strips were placed
over the surface. The MH agar plates were further incubated
at 28C and the MIC values were recorded. All strains were
induced with imipenem for production of AmpC-type ce-
phalosporinases using methods described previously.9 The
AmpC E-test strips contain cefotetan at one end and
cefotetan+cloxacillin at the other end. A ratio of MIC of
cefotetan and cefotetan+cloxacillin of ‡8 indicates produc-
tion of AmpC. The MICs of the antibiotics were interpreted
according to the guidelines of Clinical and Laboratory
Standards Institute, 2010.15
Isolation of genomic DNA, primer designing, and PCR
amplification of promoter region and CCDS of ampC
DNA was isolated from bacterial pellet collected from
1 mL of the bacterial culture grown overnight at 28C using
DNeasy Tissue kit (Qiagen, Hilden, Germany) following
manufacturer’s instructions. Primes B11f (5¢-CCTGACTTT
TTCACGTATTAT-3¢) and B12r (5¢-GGGGATAGTGATA
AAGGTAT-3¢) were designed for amplification of promot-
ers and partial gene regions of blaB genes, whereas B15 (5¢-
TGACGGAAAGCCGCAATTCT-3¢) and B16 (5¢-TCATA
GAAGCGTCAACGCAA-3¢) were designed for amplifica-
tion of CCDS of blaB genes, using Primer-BLAST.a The
primers were synthesized from Sigma-Aldrich. PCR am-
plification was performed in a My Cycler Thermal Cycler
(Bio-Rad, CA). The PCR mixture comprised 1 · PCR buffer
(10 mM Tris-HCl, 1.5 mM MgCl2, 1.5 mM KCl, and 0.1%
Triton X-100), 200 mM of each of the 4 dNTPs (MBI Fer-
mentas GmbH, Germany), 10 pmol each of forward and
reverse primers (Microsynth GmbH), 2 U of Taq DNA
polymerase (DyNAzyme; Finnzymes, Finland), and 50–
100 ng of genomic DNA in a total volume of 25 mL. The PCR
conditions were the same as described previously14 except
annealing temperature which was 49C and 54C for primer
pairs B11f, B12r and B15f, and B16r, respectively. The PCR
products were analyzed by electrophoresis on 1% (w/v)
agarose gel. The gels were stained with ethidium bromide
(0.5 mg/mL) and visualized under UV transilluminator.
Sequencing of promoter regions and CCDS
of blaB genes
PCR amplicons containing partial coding sequences of
blaB genes including the promoter region and CCDS were
purified using HiYield extraction kit (RBC Bioscience,
New Taipei City, Taiwan) and sequenced following San-
ger’s method at a commercial facility (Invitrogen BioServices
India Pvt. Ltd., Bangalore, India). The sequences obtained
were analyzed further for similarity by homology search us-
ing NCBI-BLAST.b The nucleotide sequences amplified us-
ing primer pair B11f, B12r and B15f, and B16r were aligned
together to construct the CCDS of blaB genes.
Analysis of ‘‘BlaB-like’’ proteins by conserved domain
database, InterProScan and, multiple
sequence alignment
The functional classification of ‘‘BlaB-like’’ proteins into
families was carried out using the web tools—Conserved
Domain Database and InterProScan.16 Multiple sequence
alignment (MSA) of promoters and translated CCDS of
‘‘BlaB-like’’ proteins of biotype 1A strains was carried out
by Clustal Omega.c
Homology modeling of ‘‘BlaB-like’’ cephalosporinases:
3D structure prediction, modeling, and validation
MSA revealed that a few point substitutions were present
in amino acid sequences of ‘‘BlaB-like’’ proteins; hence, pro-
tein structures of all four Y. enterocolitica strains were modeled
in silico. The 3D structures of the proteins were predicted
using MODELLER 9.10 (http://salilab.org/modeller). Of the
predicted models, the best model was selected based on
the lowest modeler objective function and validated using
a
http://www.ncbi.nlm.nih.gov/tools/primer-blast
b
https://blast.ncbi.nlm.nih.gov/Blast.cgi
c
http://www.ebi.uc.ak/clustal
 BLAB-LIKE CEPHALOSPORINASE OF Y. ENTEROCOLITICA 1A
Table 1. Details of Yersinia enterocolitica Biotype 1A Strains and Their Antimicrobial
Susceptibilities Determined by E-Test
Strain no. Source Country Serotype Amoxicillin-clavulanic acid a Cefotaximea (mg/L) Cefoxitin (mg/L)
10 Wastewater India O:6, 30–6, 31
20 Clinical India O:6, 30
28 Wastewater India O:15
E7 Clinical Europe O:6, 30
a
The data on MIC for amoxicillin–clavulanic acid and cefotaxime has been retrieved from Singhal et al.14
CLSI, 2010 guidelines for MIC breakpoints: amoxicillin–clavulanic acid, £8/4(S), 16/8(I), ‡32/16(R); cefotaxime, £1(S), 2(I), ‡4(R);
cefoxitin—£2(S), 10(I), ‡32(R).
Letters in parentheses indicate antibiotic susceptibility: I, intermediate; S, sensitive; R, resistant.
MIC, minimum inhibitory concentration.
the programs–PROCHECK,17 ERRAT,18 and Verify 3D.19
The protein models were visualized using PyMol.d
Molecular docking
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Molecular docking is a method that is used to determine
preferred binding orientations of two molecules to form a
stable complex, which in turn predicts their binding affinity.
The binding affinity of ‘‘BlaB-like’’ protein variants with
cefotaxime, molecular docking of ‘‘BlaB-like’’ protein
variants was performed using AutoDock 4.0. The PDB
structure of cefotaxime (drug bank ID: DB00493) was
downloaded from the database, DrugBank.e Protein struc-
tures were preprocessed for docking using AutoDockTools
(ADT) version 1.5.6 by adding polar hydrogen atoms and
Kollman charges, removing all nonprotein molecules, and
converting the PDB file into PDBQT format. Ligand
structures were also prepared for docking by adding hy-
drogen and charges and, converting it into PDBQT format.
The docking search space was determined visually by cen-
tering the Grid Box at the known binding sites of the ligand
and expanding the dimensions of the cubic search space so
that it completely encompassed that site. Docking was
performed at default parameters, other than the search space
specifications mentioned previously. The maximum number
of poses generated per ligand was set to 10. The final output
was selected on the basis of binding global energy (kcal/-
mol) associated with each docked pose The hydrogen
bonding and hydrophobic interactions in the enzyme–ligand
complex were analyzed using LigPlot and PyMoL version
2.0 (Schrödinger, LLC).
Results and Discussion
It has been reported that bacteria which produce AmpC
b-lactamases show reduced susceptibility to b-lactam/
b-lactamase inhibitors like amoxicillin–clavulanic acid.7
Thus, four strains of Y. enterocolitica biotype 1A that were
resistant to amoxicillin–clavulanic acid were included in this
study.14 The production of BlaA was observed to be similar
in these strains.14 Resistance to cefoxitin - a cephamycin and
second-generation cephalosporin - is considered as indica-
tive of production of inducible cephalosporinases7; hence,
all Y. enterocolitica strains were also tested for susceptibility
to second-generation cephalosporin - cefoxitin, and for
d
https://pymol.org/2
e
http://www.drugbank.ca/
3
>256(R) 0.38(S) 16(I)
>256(R) 0.064(S) 16(I)
>256(R) 0.64(S) 32(I)
>256(R) 0.38(S) 16(I)
AmpC production, using E-test strips of cefotetan/cefotetan+
cloxacillin. E-test indicated that all the four biotype 1A
strains showed intermediate susceptibility to second-
generation cephalosporin-cefoxitin (Table 1). In addition, it
was noted that E-test strips of cefotetan/cefotetan+cloxacillin
were not suitable for phenotypic detection of AmpC produc-
tion in Y. enterocolitica, as all strains tested negative for AmpC
production, whereas enzymatic assays9 and PCR amplification
indicated that ‘‘BlaB-like’’ inducible cephalosporinases were
present in Y. enterocolitica strains of biotype 1A.
Various studies have reported that Y. enterocolitica strains
of the same biotype do not display similar antimicrobial sus-
ceptibilities.20,21 Similar findings were reported by us while
studying susceptibilities of Y. enterocolitica biotype 1A strains
for various cephalosporins.14 In this study, we also tried to
understand the molecular basis underlying differential inter-
actions of ‘‘BlaB-like’’ b-lactamase of Y. enterocolitica bio-
type 1A strains with cefotaxime. Mutations and insertions in
the promoter regions of b-lactamase genes have been associ-
ated with hyperproduction of b-lactamases.22,23 To understand
if variations in promoter sequences or in amino acid sequences
of ‘‘BlaB-like’’ proteins were responsible for differential sus-
ceptibilities of biotype 1A strains to cefotaxime, the corre-
sponding gene sequences were analyzed. Primer pair B11f and
B12r resulted in the expected amplicon of 1,076 bp in all
strains. BLAST analysis confirmed that the amplicons encoded
promoters and partial gene sequences of blaB. MSA of the
promoter regions revealed that promoter sequences of blaB
were identical in all strains (Fig. 1). Thus, these might not be
responsible for differential susceptibilities of biotype 1A
strains to cefotaxime.
To investigate if variations in gene sequence were re-
sponsible for differential antibiotic susceptibilities of biotype
1A strains, genes of ‘‘BlaB-like’’ b-lactamase were PCR
amplified, sequenced, and their CCDS were translated. The
primer pair B15 and B16 resulted in the expected amplicons
of 1,002 bp in all strains. Gene sequencing and BLAST
analysis confirmed these to encode for ‘‘BlaB-like’’ b-
lactamases of Y. enterocolitica. The key catalytic residues
and sites, substitutions at which reportedly lower the enzy-
matic activity of AmpC enzymes are S64, K67, Y150, N152,
K315, and A318.24 Analysis of amino acid sequences of
‘‘BlaB-like’’ cephalosporinases of biotype 1A strains indi-
cated that all the reported key catalytic residues were present
in variants of ‘‘Bla-B-like’’ enzymes, but at different posi-
tions. The positions of the catalytic site residues in BlaB-like
cephalosporinases were S-89, K-92, Y-175, N-177, K-342,
and A-345. The two significant motifs conserved across the
 4 SINGHAL ET AL.

FIG. 1. MSA of promoter region of blaB of Yersinia enterocolitica biotype 1A strains. MSA, multiple sequence alignment.

Ambler class C b-lactamase, that is, 151SXXK154 and
342
KTG344 (where X represents any amino acid) were present
in BlaB-like cephalosporinase of all biotype 1A strains. A 24
amino acid long signal peptide was present at the terminal
end in all ‘‘BlaB-like’’ enzyme variants. As the signal peptide
is cleaved before the mature enzyme is released in the peri-
plasmic space, variations in it were excluded from compar-
ative studies. MSA results indicated that although the amino
acid sequences were similar, variations were present at a few
places (Fig. 2). Variations like Q30L and K38N were ob-
served in Y. enterocolitica strain 20; V56I, T74A, and
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individual strains are given in Figure 2. Submission of
‘‘BlaB-like’’ enzyme sequences to Conserved Domain Data-
base (CCD) and Interproscan retrieved no results.
The variants of ‘‘BlaB-like’’ enzymes discerned in biotype
1A strains were 3D modeled and docked with cefotaxime
in silico. The top three templates (PDB ID: 5GGW_A,
1FSW_A, 2BLS_A) with a sequence identity >65% were
chosen as templates to model the 3D structures. The predicted
normalized Dope scores for the final selected models of
‘‘BlaB-like’’ enzyme variants in Y. enterocolitica strains 20,
10, 28, and E7 were -1.165, -1.198, -1.159, and -1.224,

C166W in Y. enterocolitica strains 20 and 10; E115K and respectively. The selected protein models were validated
I198M in Y. enterocolitica strain E7. The sites of variation in through various structure assessment programs. Results of the

FIG. 2. MSA of ‘‘BlaB-like’’ enzyme variants present in different strains of Y. enterocolitica biotype 1A. The key
catalytic residues are given in bold. The active site residues 89 S and 175 Y were present at their respective positions.
 BLAB-LIKE CEPHALOSPORINASE OF Y. ENTEROCOLITICA 1A
Table 2. Estimated Global Energy of Binding, H-Bond Interactions, and Hydrophobic Interactions
of Cefotaxime with ‘‘BlaB-like’’ Enzyme Variants Discerned in Yersinia enterocolitica Biotype 1A
BlaB-like Estimated free energy
enzyme variants of binding (kcal/mol)
BlaB10 -5.02 Hydrogen bonds
Hydrophobic
interactions
BlaB20 -6.27 Hydrogen bonds
Hydrophobic
interactions
BlaB28 -5.84 Hydrogen bonds
Hydrophobic
interactions
BlaBE7 -7.05 Hydrogen bonds
Hydrophobic
interactions
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PROCHECK program indicated that in ‘‘BlaB-like’’ enzyme
variants of Y. enterocolitica strains 20, 10, 28, and E7,
the residues present in the most favored regions of Rama-
chandran map were 93.4%, 92.8%, 91.6%, and 92.8%, re-
spectively. Calculation of the nonbonded atomic interactions of
the modeled protein variants by ERRAT program indicated an
average ERRAT score of 83.24, 79.77, 76.71, and 84.53 for
Y. enterocolitica strains 20, 10, 28, and E7, respectively. The
Verify-3D scores of the protein models were 87.3%, 89.69%,
89.69%, and 81.70% for the protein models of Y. enterocolitica
strains 8, 20, 10, 28, and E7, respectively. PROCHECK, ER-
RAT, and Verify-3D results confirmed that the predicted 3D
structures were reliable and within the acceptable range.
Molecular docking analysis of the modeled protein struc-
tures of ‘‘BlaB-like’’ enzymes were carried out to identify
their binding affinity for third-generation cephalosporin—
cefotaxime. Amino acid residue variations at sites other than
active sites of the enzyme might create local disturbances in
the 3D structure of enzyme and increase/decrease its confor-
mational flexibility, thereby affecting substrate binding. The
global free energy, hydrogen bond (H bond), and hydrophobic
interactions of cefotaxime with different ‘‘BlaB-like’’ enzyme
variants were analyzed and the details are given in Table 2.
In silico docking of the ‘‘BlaB-like’’ enzyme variants with
cefotaxime revealed that in each enzyme variant, different
amino acid residues were involved in H bonding and hydro-
phobic interactions (Fig. 3). It is well known that H bonds and
hydrophobic interactions not only play a crucial role in mo-
lecular recognition and overall stability of the protein struc-
FIG. 3. Molecular docking analysis of cefotaxime with the four variants of ‘‘BlaB-like’’ enzymes discerned in
Y. enterocolitica (a) strain 10, (b) strain 20, (c) strain 28, and (d) strain E7. Cefotaxime is represented by stick and the
interacting amino acids are given in dark color.
5
Interacting residues
Ser89, Gln145, Leu146, Asp148, Ala345, Asn373
Tyr175, Asn316, Leu320, Lys342, Thr343, Ala345
Ser89, Tyr246
Gln145, Leu146, Asp148, Tyr175, Asn177, Thr237,
Leu238, Gly239, Met240, Lys342, Thr343, Gly344,
Ala345, Thr346, Asn370, Asn373
Ser89, Arg173, Tyr246, Lys342, Thr343, Ala345
Gln145, Tyr175, Val236, Glu299, Asn316, Ala319,
Leu320, Gly344, Thr346, Asn347
Ser89, Glu299, Ala319, Ala345
Gly88, Lys92, Leu144, Arg173, Met292, Glu299, Tyr301,
Asp313, Leu320, His341, Lys342, Thr343, Gly344
ture, these also stabilize the protein–ligand complexes. In a
docked complex, the number of the residues involved in
H-bond formation indicates the binding affinity between an
enzyme and ligand.25 This implies that lesser the number of
H-binding residues in an enzyme–antibiotic complex, the
lesser is the hydrolysis of the antibiotic by the enzyme and,
lower is the MIC of the antibiotic. In this study it was observed
that in Y. enterocolitica strain 20, the number of residues of
‘‘BlaB-like’’ enzyme involved in H bonding with cefotaxime
was 2, and the MIC for cefotaxime in this strain was almost
10 times lesser than the MIC of cefotaxime observed for
Y. enterocolitica strain 28, in which the number of residues
involved in H-bond formation with cefotaxime was 6. A sim-
ilar observation was made while comparing the H-bonding
residues of other ‘‘BlaB-like’’ enzyme variants and their re-
spective MICs for cefotaxime. Lesser the number of H-bonding
residues with cefotaxime in a strain, lower was the MIC of
cefotaxime in that strain (Table 1). Thus, in silico analysis
explained the higher MIC for cefotaxime in strains 10, 28, and
E7. However, further experiments are required to strengthen
our findings that amino acid mutations affect the H-binding
residues of ‘‘BlaB-like’’ enzymes and their interaction with
cefotaxime. The foregoing data and discussion give further
evidence that ‘‘BlaB-like’’ inducible cephalosporinase of
Y. enterocolitica biotype 1A strains is an AmpC-type b-
lactamase. To the best of our knowledge, this is the first study
in which the molecular characteristics and enzymatic interac-
tions of ‘‘BlaB-like’’ cephalosporinase of Y. enterocolitica
biotype 1A strains have been reported.
 6 SINGHAL ET AL.
Acknowledgments
N.S. is supported by Science & Engineering Research
Board (SERB) of Department of Science & Technology
(SB/YS/LS-156/2014). D.P. is supported by fellowship from
DST-INSPIRE (IF160262). The authors thank Rajesh Ku-
mar Mahto for the technical help.
Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Manish Kumar, PhD
Department of Biophysics
University of Delhi South Campus
New Delhi 110021
India
E-mail: manish@south.du.ac.in
Jugsharan Singh Virdi, PhD
Department of Microbiology
University of Delhi South Campus
New Delhi 110021
India
E-mail: virdi_dusc@rediffmail.com