Proteomic analysis reveals the damaging role of low redox laccase from Yersinia enterocolitica strain 8081 in the midgut of Helicoverpa armigera.

OBJECTIVE: Earlier, we have found that the enteropathogenic Yersinia enterocolitica have evolved the survival mechanisms that regulate the expression of laccase-encoding genes in the gut. The present study aims to characterize the purified recombinant laccase from Y. enterocolitica strain 8081 biovar 1B and understand its effect on the midgut of cotton bollworm, Helicoverpa armigera (Hübner) larvae. RESULTS: The recombinant laccase protein showed high purity fold and low molecular mass (~ 43 kDa). H. armigera larvae fed with laccase protein showed a significant decrease in body weight and damage in the midgut. Further, transmission electron microscopy (TEM) studies revealed the negative effect of laccase protein on trachea, malpighian tubules, and villi of the insect. The proteome comparison between control and laccase-fed larvae of cotton bollworm showed significant expression of proteolytic enzymes, oxidoreductases, cytoskeletal proteins, ribosomal proteins; and proteins for citrate (TCA cycle) cycle, glycolysis, stress response, cell redox homeostasis, xenobiotic degradation, and insect defence. Moreover, it also resulted in the reduction of antioxidants, increased melanization (insect innate immune response), and enhanced free radical generation. CONCLUSIONS: All these data collectively suggest that H. armigera (Hübner) larvae can be used to study the effect of microbes and their metabolites on the host physiology, anatomy, and survival.

Evaluation of Probiotic Characteristics of Lactic Acid Bacteria Isolated from Two Commercial Preparations Available in Indian Market.

The enormous health benefits associated with probiotics has resulted in an increased consumption of probiotic supplements. Several factors like regular sub-culturing, storage, unfavourable conditions etc. might compromise the efficacy and/or safety of lactic acid bacteria which are the major components of many probiotic preparations available in the market. The present study evaluated the probiotic characteristics and safety of probiotic bacteria isolated from two preparations available commercially in India. The products did not specify the genera, species or strains of the bacteria used. These were cultured using standard microbiological methods for cultivation of lactic acid bacteria. Bacteria were identified by PCR amplification and sequence analysis of 16S rRNA gene. Microbiological and molecular analyses revealed that both preparations contained homogenous population of Enterococcus faecium and Pediococcus acidilactici respectively. Assessment for several essential and desirable probiotic properties revealed that both the probiotic strains were safe and resistant to salt, lysozyme, bile salt and common antibiotics. The probiotic preparation containing P. acidilactici was better than that containing E. faecium as it survived in low pH and showed bile salt hydrolase activity. The probiotic preparation containing P. acidilactici also exhibited cholesterol-lowering activity.

MALDI-TOF MS in clinical parasitology: applications, constraints and prospects.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is currently being used for rapid and reproducible identification of bacteria, viruses and fungi in clinical microbiological laboratories. However, some studies have also reported the use of MALDI-TOF MS for identification of parasites, like Leishmania, Giardia, Cryptosporidium, Entamoeba, ticks and fleas. The present review collates all the information available on the use of this technique for parasites, in an effort to assess its applicability and the constraints for identification/diagnosis of parasites and diseases caused by them. Though MALDI-TOF MS-based identification of parasites is currently done by reference laboratories only, in future, this promising technology might surely replace/augment molecular methods in clinical parasitology laboratories.

Molecular modeling and docking of novel laccase from multiple serotype of Yersinia enterocolitica suggests differential and multiple substrate binding.

Multi-copper oxidases (MCOs) are widely distributed in bacteria, where they are responsible for metal homeostasis, acquisition and oxidation. Using specific primers, yacK coding for MCO was amplified from different serotypes of Yersinia enterocolitica biovar 1A. Homology modeling of the protein followed by docking with five well-known substrates for different MCO's (viz., 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid [ABTS], syringaldazine, L-tyrosine, ammonium ferrous sulfate and guaiacol), lignin monomers (Coniferyl alcohol, p-coumaryl alcohol and sinapyl alcohol) and two inhibitors i.e., kojic acid and N-hydroxyglycine was done. The docking gave maximum GoldScore i.e., 91.93 and 72.64 with ammonium ferrous sulfate and ABTS, respectively. Similarly, docking with ICM gave -82.10 and -83.61 docking score, confirming the protein to be true laccase with ferroxidase activity. Further, validation with ammonium ferrous sulfate as substrate gave laccase activity of 0.36Units/L/min. Guaiacol, L-tyrosine, and lignin monomers showed good binding affinity with protein models with GoldScores of 35.89, 41.82, 40.41, 41.12 and 43.10, respectively. The sequence study of all the cloned Yack genes showed serotype specific clade in dendrogram. There was distinct discrimination in the ligand binding affinity of Y. enterocolitica laccase, among strains of same clonal groups, suggesting it as a tool for phylogenetic studies.

Identification of family specific fingerprints in ß-lactamase families.

Beta-lactamases are a superfamily of enzymes which degrade the ß-lactam class of antibiotics. They are produced endogenously by the bacterial cells, which when exposed to the ß-lactam class of antibiotics inactivate them by cleaving the ß-lactam ring. Based on the presence or absence of metallic ligand, ß-lactamases have been divided into two broad functional classes. ß-Lactamases are a constitutively evolving and expanding superfamily of enzymes, which could be further subdivided on the basis of presence/absence of conserved motifs. In the present study we have used the MEME/MAST suit to identify the patterns/motifs which are specific to a particular family or subfamily of ß-lactamases. The family specific patterns/motifs can be also useful in recognizing and assigning newly discovered ß-lactamases to one or the other family or subfamily. Cross-validation showed that the proposed method is highly sensitive and specific. We have also designed a webserver, LactFP, for this purpose.

Proteomic analysis of arsenite - mediated multiple antibiotic resistance in Yersinia enterocolitica biovar 1A.

Arsenic is one of the most important global environmental pollutants. In the present study, fifty one clinical strains of Yersinia enterocolitica biovar 1A showed high resistance to arsenite and arsenate. The minimum inhibitory concentration (MIC) of arsenite (0.625-20 mM) was lower than arsenate (10-80 mM). Growth of Y. enterocolitica in 2 mM arsenite led to 2-8 fold increase in MICs of the five antibiotics (amikacin, ciprofloxacin, gentamycin, kanamycin and tetracycline), suggesting expression of arsenite-induced multiple antibiotic resistance among the strains. Proteomic analysis of Y. enterocolitica revealed differential expression of certain proteins following arsenite exposure, which included a putative outer membrane porin (OmpA) and a putative amino acid transporter protein. In conclusion, modulation of membrane permeability may be involved in the induction of arsenite-mediated expression of multiple antibiotic resistance in Y. enterocolitica.

Exploring the genetic mechanisms underlying amoxicillin-clavulanate resistance in waterborne Escherichia coli.

Escherichia coli is a human commensal and faecal indicator bacteria which is also the etiologic agent of several nosocomial- and community-acquired infections. Amoxicillin-clavulanate (AMC) is a widely prescribed ß-lactam/ß-lactamase inhibitor which is used against E. coli infections. Resistance to AMC in E. coli has been primarily attributed to point mutations in blaTEM-1 resulting in inhibitor-resistant TEM (IRT) ß-lactamases. In this study, we have explored the reasons underlying AMC-resistance in waterborne E. coli. Most of the studies regarding IRT-producing E. coli have been conducted on clinical samples and studies exploring genetic mechanisms underlying AMC-resistance in aquatic E. coli are scarce. Since, blaTEM-1 and several antimicrobial resistance determinants are located on mobile genetic elements they can easily disseminate among other microbes inhabiting urban waterbodies. Thus, it is important to understand the underlying mechanisms to check the dissemination of AMC-resistance in other waterborne pathogens. Our results indicated that AMC-resistant E. coli were susceptible to other ß-lactam/ß-lactamase inhibitors like, ampicillin/sulbactam and piperacillin/tazobactam. Though, blaTEM-1 was present, none of the strains harbored point mutations which could qualify as IRT and only one strain harbored both blaTEM-1 and blaOXA-1. Hyperproduction of blaTEM-1, presence of plasimd-mediated ampC or promoter/attenuator mutations in the chromososmal ampC might not be related to IRT-like phenotype or AMC-resistance. This suggests that other mechanisms like, increased plasmid copy numbers or gene amplification or deficiency in the expression/function of porins might be responsible for AMC-resistance in waterborne E. coli.

High Prevalence of Drug Resistance and Class 1 Integrons in Escherichia coli Isolated From River Yamuna, India: A Serious Public Health Risk.

Globally, urban water bodies have emerged as an environmental reservoir of antimicrobial resistance (AMR) genes because resistant bacteria residing here might easily disseminate these traits to other waterborne pathogens. In the present study, we have investigated the AMR phenotypes, prevalent plasmid-mediated AMR genes, and integrons in commensal strains of Escherichia coli, the predominant fecal indicator bacteria isolated from a major urban river of northern India Yamuna. The genetic environment of bla CTX-M-15 was also investigated. Our results indicated that 57.5% of the E. coli strains were resistant to at least two antibiotic classes and 20% strains were multidrug resistant, i.e., resistant to three or more antibiotic classes. The multiple antibiotic resistance index of about one-third of the E. coli strains was quite high (>0.2), reflecting high contamination of river Yamuna with antibiotics. With regard to plasmid-mediated AMR genes, bla TEM-1 was present in 95% of the strains, followed by qnrS1 and armA (17% each), bla CTX-M-15 (15%), strA-strB (12%), and tetA (7%). Contrary to the earlier reports where bla CTX-M-15 was mostly associated with pathogenic phylogroup B2, our study revealed that the CTX-M-15 type extended-spectrum ß-lactamases (ESBLs) were present in the commensal phylogroups A and B1, also. The genetic organization of bla CTX-M-15 was similar to that reported for E. coli, isolated from other parts of the world; and ISEcp1 was present upstream of bla CTX-M-15. The integrons of classes 2 and 3 were absent, but class 1 integron gene intI1 was present in 75% of the isolates, denoting its high prevalence in E. coli of river Yamuna. These evidences indicate that due to high prevalence of plasmid-mediated AMR genes and intI1, commensal E. coli can become vehicles for widespread dissemination of AMR in the environment. Thus, regular surveillance and management of urban rivers is necessary to curtail the spread of AMR and associated health risks.

Rhizospheric Lactobacillus plantarum (Lactiplantibacillus plantarum) strains exhibit bile salt hydrolysis, hypocholestrolemic and probiotic capabilities in vitro.

Lactobacillus plantarum (renamed as Lactiplantibacillus plantarum) has been isolated from many sources but very rarely from rhizospheric soil. This is the first report on isolation and assessment of probiotic capabilities of L. plantarum strains isolated from rhizospheric soil. The isolates were confirmed by 16S rRNA gene sequencing and named as NS14, NS16 and NGG. All the isolates were evaluated for bile salt hydrolysis, hypocholestrolemic potential and probiotic attributes. Our results indicated that all the strains harboured bsh and showed in vitro cholesterol assimilation capabilities which increased when bile salts were also present in the culture medium. Also, all the strains remained viable at high temperatures and in the presence of NaCl, lysozyme, simulated gastric juice, bile salts and, exhibited auto- and co-aggregation capabilities. Additionally, L. plantarum strain NS14 survived in the presence of phenols, acidic environment (pH 2-3) and was resistant to many clinically relevant antibiotics. Since, L. plantarum NS14 exhibited most of the desirable and essential characteristics of a probiotic it should be further investigated as a potent probiotic with an additional benefit as a hypocholesterolemic biotherapeutic. Moreover, rhizosphere can be explored as a useful ecological niche for isolating microorganisms with biotechnological and probiotic potential.

Draft Genome Sequence of a Poly-γ-Glutamic Acid-Producing Isolate, Bacillus paralicheniformis Strain bcasdu2018/01.

Bacillus paralicheniformis bcasdu2018/01 was isolated from the indoor environment of a chemistry laboratory. As part of the extracellular matrix, this isolate produces copious amounts of poly-γ-glutamic acid (γ-PGA). Here, we report the 4.25-Mbp draft genome assembly of the organism with an average G+C content of 45.92%.

Comparative Proteomics of Commensal and Pathogenic Strains of Escherichia coli.

BACKGROUND: Most of the proteomic studies in Escherichia coli have focussed on pathogenic strains, while very few studies have studied the commensal strains. It is important to study the commensal strains because under the selective pressure of their habitat, commensal strains might serve as reservoirs of virulent and pathogenic strains. OBJECTIVE: In this study, we have performed a comparative proteomic analysis of commensal and pathogenic strains of E. coli isolated from a major river flowing through northern India. METHODS: Proteins were resolved by two dimensional gel electrophoresis and the differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time of flight mass-spectrometry (MALDI-TOF MS). RESULTS: Many proteins of the commensal strain showed an increased expression compared to the pathogenic strain, of which seventeen proteins were identified by MALDI-TOF MS. Functional classification of these proteins revealed that they belonged to different functional pathways like energy metabolism, nucleotide and nucleoside conversions, translation, biosynthesis of amino acids and motility and energytaxis/chemotaxis. CONCLUSION: As per the best of our knowledge, this is the first report on comparative proteomic analysis of E. coli commensal and pathogenic strains of aquatic origin. Our results suggest that the increased production of these proteins might play an important role in adaptation of E. coli to a commensal/pathogenic lifestyle. However, further experiments are required to understand the precise role of these proteins in regulating the pathogenicity/commensalism of E. coli.

Exploring the genetic determinants underlying the differential production of an inducible chromosomal cephalosporinase - BlaB in Yersinia enterocolitica biotypes 1A, 1B, 2 and 4.

Yersinia enterocolitica is an enteric bacterium which can cause severe gastroenteritis. Beta-lactams are the most widely used antibiotics against Y. enterocolitica. Y. enterocolitica produces two chromosomal ß-lactamases, BlaA and BlaB. BlaB is an Ambler Class C inducible broad spectrum cephlaosporinase which showed differential enzyme activity in different biotypes of Y. enterocolitica. The expression of blaB is mainly regulated by ampR- the transcriptional regulator and, ampD - which helps in peptidoglycan recycling. The aim of this study was to identify and characterize genetic determinants underlying differential enzyme activity of BlaB in Y. enterocolitica biotypes 1 A, IB, 2 and 4. Thus, ampR, blaB and ampD were PCR-amplified and modeled in silico. The intercistronic region containing promoters of ampR and blaB was also investigated. Our results indicated that blaB was more inducible in biotypes 2 and 4, than in biotypes 1 A and 1B. Superimposition of in silico modeled proteins suggested that variations in amino acid sequences of AmpR, BlaB and AmpD were not responsible for hyper-production of BlaB in biotypes 2 and 4. Analysis of promoter regions of ampR and blaB revealed variations at -30, -37 and -58 positions from blaB transcription start site. Studies on relative expression levels of blaB in different biotypes by qRT-PCR indicated that nucleotide variations at these positions might contribute to a higher enzyme activity of BlaB in biotypes 2 and 4. However, this is a preliminary study and further studies including more strains of each biotype are required to strengthen our findings. Nevertheless, to the best of our knowledge, this is the first study which has investigated the genetic determinants underlying differential inducible production of BlaB in different biotypes of Y. enterocolitica.

Molecular analysis of ampR and ampD to understand variability in inducible expression of "BlaB-like" cephalosporinase in Yersinia enterocolitica biotype 1A.

Yersinia enterocolitica strains produce two chromosomal ß­lactamases, BlaA - a constitutively produced penicillinase, and BlaB - an inducible "AmpC-type" cephalosporinase. As in other members of Enterobacteriaceae, expression of ampC in Y. enterocolitica is regulated by the genes - ampR and ampD. The ampR encodes a transcriptional regulator which represses the expression of ampC and, ampD encodes a cytoplasmic N­acetyl­anhydromuramyl­l­alanine amidase which participates in recycling of peptidoglycan. Exposure of bacteria to antibiotics like imipenem and cefoxitin results in generation and accumulation of large quantities of muropeptides in cytoplasm which is beyond the recycling capability of AmpD. These muropeptides bind to AmpR, converting it into an activator of ampC expression (ampC de-repression). Earlier studies from our laboratory indicated that instead of BlaB, Y. enterocolitica biotype 1A strains produced a "BlaB-like" enzyme which was non-heterogeneous and showed a differential expression when induced with imipenem. The detection of "BlaB-like" cephalosporinase which was also induced differentially in Y. enterocolitica biotype 1A strains presented an opportunity to discern newer mechanisms, if any, which may underlie inducible expression of "AmpC-type" cephalosporinases. Thus, the objective of the present study was to understand the role of ampR and ampD in regulating differential expression of "BlaB-like" cephalosporinases in biotype 1A strains. Analysis of promoters and amino acid sequences of AmpR revealed that these were conserved in all strains of biotype 1A. Analysis of AmpD amino acid sequences revealed that five variants of AmpD were present which did not contribute to hyper-inducible production of "BlaB-like" enzyme. In-silico prediction of the mRNA secondary structures of ampD revealed significant differences, which might have affected the rate of translation of ampD and accumulation of un-recycled muropeptides inside the cell leading to hyper production of "BlaB-like" cephalosporinases in some Y. enterocolitica biotype 1A strains. The findings provide newer insights to our understanding of the mechanisms underlying regulation of expression of "AmpC-type" ß­lactamases.

Molecular modeling and MD-simulation studies: Fast and reliable tool to study the role of low-redox bacterial laccases in the decolorization of various commercial dyes.

Synthetic dyes are toxic and carcinogenic in nature, which also causes environmental pollution. The present study was aimed to decolorize various commercial dyes using purified recombinant bacterial laccases. Laccase gene from Yersinia enterocolitica strain 8081 (yacK), Y. enterocolitica strain 7 (yacK) and Bacillus pumilus DSKK1 was cloned in vector pET28a and overproduced in host Escherichia coli BL21. The high yield of recombinant laccase protein resulted in the formation of inclusion bodies, which were further solubilized, refolded, and purified. The purified recombinant laccases were alkali-tolerant and thermostable, with pH optima at 7-8, temperature optima at 60-70 °C and low redox potential. For in silico studies, laccase protein models of B. pumilus DSKK1, Y. enterocolitica strain 7 and Y. enterocolitica strain 8081 were docked with commercial dyes. This is the first and foremost study where the stability of docked complexes of pathogenic and non-pathogenic microorganism has been explored via molecular dynamics (MD) simulations using Gromacs version 4.5.5 with the gromos96 43a force field. Finally, the in silico results were validated experimentally and it was found that purified laccases from B. pumilus DSKK1 and Y. enterocolitica strain 7 efficiently decolorized rose bengal (90.4%), malachite green (77.7%), and congo red (74.5%) dyes.

Evaluation of Bile Salt Hydrolases, Cholesterol-Lowering Capabilities, and Probiotic Potential of Enterococcus faecium Isolated From Rhizosphere.

Bile salt hydrolase (BSH) activity, hypo-cholesterolemic effect, and probiotic properties have been reported for Enterococcus strains isolated from animal and human gut and fermented foods but not for strains isolated from environmental niches, like aquatic and terrestrial plants, soil, and water. The present study is the first report on isolation of Enterococcus faecium from rhizospheric soils that harbor the bsh gene, remove cholesterol in vitro, and possess essential and desirable probiotic attributes. Fifteen samples were collected from different sites located in northern, southern, and central regions of India, of which five yielded pure colonies that were named LR2, LR3, ER5, LR13, and VB1. These were identified by 16S rRNA gene sequencing as E. faecium and evaluated for BSH activity, cholesterol-lowering potential in vitro, and probiotic properties. Our results indicated that all the strains were capable of surviving the harsh conditions of the gastrointestinal tract and did not harbor any of the virulence genes. Though all strains showed the presence of bsh and potential for cholesterol removal, E. faecium strain LR13 showed a remarkable cholesterol removal capability and vancomycin susceptibility and possessed most of the desirable and essential attributes of a probiotic. Hence, it seems to be a fairly promising probiotic candidate that needs to be further evaluated in in vivo studies, especially for its hypo-cholesterolemic potential.

Virulence-associated traits and in vitro biofilm-forming ability of Escherichia coli isolated from a major river traversing Northern India.

Several strains of Escherichia coli harbor virulence traits, resulting in E. coli-related intestinal and extra-intestinal infections. Various studies have reported that extra-intestinal pathogenic E. coli (ExPEC) strains were prevalent in nonhuman reservoirs, including environmental waterways. It is therefore important to identify the pathogenic potential and/or ExPEC status of E. coli strains inhabiting the aquatic environments associated with anthropogenic activities. Besides virulence-associated genes, biofilm production also helps in the survival of E. coli in environmental waterbodies. Thus, the aim of the current study was to assess the virulence potential, ExPEC status, and biofilm-producing capability of E. coli isolated from the River Yamuna, a major river traversing the National Capital Region of Delhi, India. We also tried to discern a co-relation, if any, between virulence, biofilm formation, and antimicrobial resistance in these strains. Our results indicated that virulence-associated genes were scarce and none of the strain qualified the molecular criteria essential for ExPEC. This suggested that E. coli strains which can presumably cause human extra-intestinal infections were not prominent in the River Yamuna. However, the fact that more than 80% of the aquatic E. coli isolates were moderate and strong biofilm producers suggests that E. coli in these environments might serve as opportunistic pathogens. Also, no unequivocal association was observed between biofilm production, virulence, and ß-lactamase genes in E. coli strains. As per the best of our knowledge, this is the first study where the relationship between virulence, biofilms, and antimicrobials has been examined in E. coli, isolated from an Indian urban aquatic waterbody.

ampD homologs in biotypes of Yersinia enterocolitica: Implications in regulation of chromosomal AmpC-type cephalosporinases.

Inducible 'AmpC-type' chromosomal cephalosporinases have been reported to be differentially expressed in different biotypes of Yersinia entercolocolitica. AmpD amidases are key regulators of the expression of ampC genes in Y. entercolocolitica as their inactivation results in hyper production of AmpC. To understand the differences in regulation of ampC expression in different biotypes of Y. enterocolitica, characteristics of ampD homologs were studied in strains of Y. enterocolitica belonging to five biotypes namely 1A, 1B, 2, 3 and 4. Our results indicated that the mechanisms which regulate expression of ampC might differ in different biotypes. While a three-step regulation mechanism seemed to be functional in biotypes 2, 3 and 4, a two-step regulation mechanism using other AmiD like proteins might be functional in biotypes 1A and 1B. The existence of ampD homolog(s)-mediated expression of ampC in other members of the family Enterobacteriaceae may provide further credence to our findings.

Molecular Characteristics of "BlaB-Like" Chromosomal Inducible Cephalosporinase of Yersinia enterocolitica Biotype 1A Strains.

Yersinia enterocolitica biotype 1A strains are emerging pathogens, frequently isolated from clinical samples across the globe. Y. enterocolitica strains produce two chromosomal ß-lactamases, BlaA and BlaB. BlaA is a constitutively expressed, Ambler class A, penicillinase, whereas BlaB is Ambler class C-type, inducible cephalosporinase. 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" ß-lactamases produced by biotype 1A strains to discern their similarity with AmpC-type ß-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 substitutions 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.

Antimicrobial resistance and its relationship with biofilm production and virulence-related factors in Yersinia enterocolitica biotype 1A.

The aim of the present study was to determine antimicrobial susceptibilities, biofilm production and, to discern a relationship between antimicrobial resistance, biofilm potential and virulence-related genes in strains of Yersinia entercocolitica biotype 1A. Thirty strains of Y. enterocolitica biotype 1A including clinical and non-clinical strains were investigated. Antimicrobial susceptibility for 15 antibiotics (representing different classes) was determined by disk-diffusion assay. Biofilm potential was determined on two different culture media using crystal violet assay. Also, a co-relation was studied between antimicrobial susceptibilities, biofilm production and virulence-related genes. All strains of biotype 1A produced biofilms and exhibited varied level of susceptibilities for different antibiotics. More than 60% of the strains were strong to moderate biofilm producers and, were exclusively associated with REP/ERIC clonal group B. Moderate and strong biofilm producers exhibited both sensitive and resistant phenotypes towards different antibiotics. Interestingly, weak biofilm producers were resistant to amoxicillin, amoxicillin-clavulanate and cefazolin. Analysis of antimicrobial susceptibilities, biofilm potential and virulence-related genes did not reveal any unequivocal relationships. The differential biofilm potential of Indian strains of Y. enterocolitica biotype 1A, suggests that biotype 1A strains are heterogeneous in nature.