Genetic relationships between clinical and non-clinical strains of Yersinia enterocolitica biovar 1A as revealed by multilocus enzyme electrophoresis and multilocus restriction typing

<p>Abstract</p> <p>Background</p> <p>Genetic relationships among 81 strains of <it>Y. enterocolitica </it>biovar 1A isolated from clinical and non-clinical sources were discerned by multilocus enzyme electrophoresis (MLEE) and multilocus restriction typing (MLRT) using six loci each. Such studies may reveal associations between the genotypes of the strains and their sources of isolation.</p> <p>Results</p> <p>All loci were polymorphic and generated 62 electrophoretic types (ETs) and 12 restriction types (RTs). The mean genetic diversity (<it>H</it>) of the strains by MLEE and MLRT was 0.566 and 0.441 respectively. MLEE (DI = 0.98) was more discriminatory and clustered <it>Y. enterocolitica </it>biovar 1A strains into four groups, while MLRT (DI = 0.77) identified two distinct groups. BURST (Based Upon Related Sequence Types) analysis of the MLRT data suggested aquatic serotype O:6,30-6,31 isolates to be the ancestral strains from which, clinical O:6,30-6,31 strains might have originated by host adaptation and genetic change.</p> <p>Conclusion</p> <p>MLEE revealed greater genetic diversity among strains of <it>Y. enterocolitica </it>biovar 1A and clustered strains in four groups, while MLRT grouped the strains into two groups. BURST analysis of MLRT data nevertheless provided newer insights into the probable evolution of clinical strains from aquatic strains.</p

Molecular and biochemical characterization of urease and survival of Yersinia enterocolitica biovar 1A in acidic pH in vitro

<p>Abstract</p> <p>Background</p> <p><it>Yersinia enterocolitica</it>, an important food- and water-borne enteric pathogen is represented by six biovars <it>viz</it>. 1A, 1B, 2, 3, 4 and 5. Despite the lack of recognized virulence determinants, some biovar 1A strains have been reported to produce disease symptoms resembling that produced by known pathogenic biovars (1B, 2-5). It is therefore imperative to identify determinants that might contribute to the pathogenicity of <it>Y. enterocolitica </it>biovar 1A strains. <it>Y. enterocolitica </it>invariably produces urease and the role of this enzyme in the virulence of biovar 1B and biovar 4 strains has been reported recently. The objective of this work was to study genetic organization of the urease (<it>ure</it>) gene complex of <it>Y. enterocolitica </it>biovar 1A, biochemical characterization of the urease, and the survival of these strains under acidic conditions <it>in vitro</it>.</p> <p>Results</p> <p>The <it>ure </it>gene complex (<it>ureABCEFGD</it>) of <it>Y. enterocolitica </it>biovar 1A included three structural and four accessory genes, which were contiguous and was flanked by a urea transport (<it>yut</it>) gene on the 3' side. Differences were identified in <it>ure </it>gene complex of biovar 1A strain compared to biovar 1B and 4 strains. This included a smaller <it>ureB </it>gene and larger intergenic regions between the structural genes. The crude urease preparation exhibited optimal pH and temperature of 5.5 and 65°C respectively, and Michaelis-Menten kinetics with a K_mof 1.7 ± 0.4 mM urea and V_maxof 7.29 ± 0.42 μmol of ammonia released/min/mg protein. The urease activity was dependent on growth temperature and growth phase of <it>Y. enterocolitica </it>biovar 1A, and the presence of nickel in the medium. The molecular mass of the enzyme was > 545 kDa and an isoelectric point of 5.2. The number of viable <it>Y. enterocolitica </it>biovar 1A decreased significantly when incubated at pH 2.5 for 2 h. However, no such decrease was observed at this pH in the presence of urea.</p> <p>Conclusions</p> <p>The <it>ure </it>gene cluster of biovar 1A strains though similar to biovar 1B and 4 strains, exhibited important differences. The study also showed the ability of biovar 1A strains of <it>Y. enterocolitica </it>to survive at highly acidic pH <it>in vitro </it>in the presence of urea.</p

Characteristics of β-lactamases and their genes (blaA and blaB) in Yersinia intermedia and Y. frederiksenii

BACKGROUND: The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India. RESULTS: The enzymes, Bla-A (a constitutive class A penicillinase) and Bla-B (an inducible class C cephalosporinase) were found to be present in all the clinical and non-clinical strains of Y. intermedia and Y. frederiksenii by double disc diffusion method. The results showed differential expression of Bla-A as indicated by presence/absence of synergy whereas expression of Bla-B was quite consistent. The presence of these enzymes was also reflected in the high minimum inhibitory concentrations, MIC(50 )(126–1024 mg/L) and MIC(90 )(256–1024 mg/L) of β-lactam antibiotics against these species. Restriction fragment length polymorphism (RFLP) revealed heterogeneity in both blaA and blaB genes of Y. intermedia and Y. frederiksenii. The blaA gene of Y. intermedia shared significant sequence identity (87–96%) with blaA of Y. enterocolitica biovars 1A, 1B and 4. The sequence identity of blaA of Y. frederiksenii with these biovars was 77–79%. The sequence identity of blaB gene of Y. intermedia and Y. frederiksenii was more (85%) with that of Y. enterocolitica biovars 1A, 1B and 2 compared to other species viz., Y. bercovieri, Y. aldovae and Y. ruckeri. Isoelectric focusing data further revealed that both Y. intermedia and Y. frederiksenii produced Bla-A (pI 8.7) and "Bla-B like" (pI 5.5–7.1) enzymes. CONCLUSION: Both Y. intermedia and Y. frederiksenii showed presence of blaA and blaB genes and unequivocal expression of the two β-lactamases. Limited heterogeneity was detected in blaA and blaB genes as judged by PCR-RFLP. Phylogenetic relationships showed that the two species shared a high degree of identity in their bla genes. This is the first study reporting characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii isolated from Asian region

MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis

Currently microorganisms are best identified using 16S rRNA and 18S rRNA gene sequencing. However, in recent years Matrix Assisted Laser Desorption Ionization-Time of Flight mass spectrometry (MALDI-TOF MS) has emerged as a potential tool for microbial identification and diagnosis. During the MALDI-TOF MS process, microbes are identified using either intact cells or cell extracts. The process is rapid, sensitive and economical in terms of both labor and costs involved. The technology has been readily imbibed by microbiologists who have reported usage of MALDI-TOF MS for a number of purposes like, microbial identification and strain typing, epidemiological studies, detection of biological warfare agents, detection of water- and food-borne pathogens, detection of antibiotic resistance and detection of blood and urinary tract pathogens etc. The limitation of the technology is that identification of new isolates is possible only if the spectral database contains peptide mass fingerprints of the type strains of specific genera/species/subspecies/strains. This review provides an overview of the status and recent applications of mass spectrometry for microbial identification. It also explores the usefulness of this exciting new technology for diagnosis of diseases caused by bacteria, viruses and fungi

Genetic Environment of blaTEM-1, blaCTX-M-15, blaCMY-42 and Characterization of Integrons of Escherichia coli Isolated From an Indian Urban Aquatic Environment

The presence of antibiotic resistance genes (ARGs) including those expressing ESBLs and AmpC-β-lactamases in Escherichia coli inhabiting the aquatic environments is a serious health problem. The situation is further complicated by the fact that ARGs can be easily transferred among bacterial species with the help of mobile genetic elements – plasmids, integrons, insertion sequences (IS), and transposons. Therefore, the analysis of genetic environment and mobile genetic elements associated with ARGs is important as these provide useful information about the epidemiology of these genes. In our previous study, we had reported presence of various β-lactam resistance genes present in E. coli strains inhabiting the river Yamuna traversing the National Capital Territory of Delhi (India). In the present study, we have analyzed the genetic environment of three ARGs blaTEM-1, blaCTX-M-15, and blaCMY -42 of those E. coli strains. The structure of class 1 integrons and their gene cassettes was also analyzed. Insertion sequence IS26 was present upstream of blaTEM-1, ISEcp1 was present upstream of blaCTXM-15 gene and orf477 was present downstream of blaCTXM-15. ISEcp1 was also present upstream of blaCMY -42 and, blc and sugE genes were present in the downstream region of this gene. Thus, the overall genetic environment surrounding these genes was similar to that reported from E. coli strains isolated globally. Conjugation assays, isolation and analysis of plasmid DNA of the transconjugants indicated that blaTEM-1, blaCTX-M-15, blaCMY -42 and class 1 integron were plasmid-mediated and possibly transmit between genera through horizontal gene transfer (HGT). This might lead to dissemination of antimicrobial resistance genes in aquatic environment. The work embodied in this paper is the first describing the genetic environment of bla and integrons in aquatic E. coli isolated from India

Preparation and antimicrobial action of three tryptic digested functional molecules of bovine lactoferrin.

Lactoferrin is an 80 kDa bilobal, iron binding glycoprotein which is primarily antimicrobial in nature. The hydrolysis of lactoferrin by various proteases in the gut produces several functional fragments of lactoferrin which have varying molecular sizes and properties. Here, bovine lactoferrin has been hydrolyzed by trypsin, the major enzyme present in the gut, to produce three functional molecules of sizes approximately 21 kDa, 38 kDa and 45 kDa. The molecules have been purified using ion exchange and gel filtration chromatography and identified using N-terminal sequencing, which reveals that while the 21 kDa molecule corresponds to the N2 domain (21LF), the 38 kDa represents the whole C-lobe (38LF) and the 45 kDa is a portion of N1 domain of N-lobe attached to the C-lobe (45LF). The iron binding and release properties of 21LF, 38LF and 45LF have been studied and compared. The sequence and structure analysis of the portions of the excision sites of LF from various species have been done. The antibacterial properties of these three molecules against bacterial strains, Streptococcus pyogenes, Escherichia coli, Yersinia enterocolitica and Listeria monocytogenes were investigated. The antifungal action of the molecules was also evaluated against Candida albicans. This is the first report on the antimicrobial actions of the trypsin cleaved functional molecules of lactoferrin from any species

The entire sequence of bovine native LF.

<p>Arrows [blue (21LF), green (38LF), red (45LF)] indicate the origin sites of the three fragments respectively. The amino acid sequence of the first fifteen residues of 21LF, 38LF and 45LF is indicated against their names towards the left,</p

Zone of diameters of LF, 21LF, 38LF and 45LF against bacterial species.

<p>Zone of diameters of LF, 21LF, 38LF and 45LF against bacterial species.</p

LC₅₀ values of LF, 21LF, 38LF and 45LF against bacterial species.

<p>All the data were expressed as mean values ± standard deviations; <i>P</i><0.05, student's <i>t-</i> test.</p><p>*Highest concentration tested.</p