Cold atmospheric plasma: Its time-dependent effects on the elimination of bacterial colony on periodontal manual scalers.

Background: This in vitro study investigated the time-dependent bactericidal effects of cold atmospheric argon plasma treatment of periodontal hand scalers as well as the scanning electron microscopic view of the scaler tip surfaces before and after plasma treatment. Materials and Methods: The study used 34 periodontal hand scalers which were divided into test and control groups. The scaler tips were inoculated with Escherichia coli and Staphylococcus aureus bacteria, following which the scalers in the control and test groups were subjected to conventional sterilization and argon plasma sterilization, respectively. Varying exposure times of plasma treatment were done on the test group samples to evaluate the minimum time required for complete sterilization. Subsequently, streaks were made on plate count agar using each of these instruments. The agar plates were then kept in an incubator for 24 h, following which bacterial colony count was assessed (colony-forming units/mL). Furthermore, the scanning electron microscopic (SEM) view of the scaler tip was studied before and after plasma treatment. Results: A complete elimination of bacterial load (Gram-positive as well as Gram-negative) from the instrument surface was achieved by the plasma exposure time of 15-20 s. SEM analysis did not show a significant difference before and after plasma treatment as not many organic residues were present on the scaler tip. Conclusion: Cold atmospheric pressure plasma is an efficient and time-saving method of sterilization, capable of destroying both Gram-positive and Gram-negative bacteria.

Mechanistic and evolutionary insights into alkaline phosphatase superfamily through structure-function studies on Sphingomonas alkaline phosphatase.

Alkaline phosphatases (APs), represented by E. coli AP (ECAP), employ an arginine residue to stabilize the phosphoryl group in the active site; whereas, AP from Sphingomonas (SPAP) shows a unique combination of substrate-binding residues; Thr89, Asn110, Lys171, and Arg173. Although such combination has been observed only in SPAP, these residues are present separately in different members of the AP superfamily. Here, we establish the presence of two distinct classes of APs; ECAP-type and SPAP-type. Bioinformatic analyses show that SPAP-type of APs are widely distributed in the bacterial kingdom. The role of active site residues in the catalytic mechanism has been delineated through a set of crystal structures reported here. These structures, representing different stages of the reaction pathway provide wealth of information for the catalytic mechanism. Despite critical differences in the substrate binding residues, SPAP follows a mechanism similar to that of ECAP-type of APs. Structure-based phylogenetic analysis suggests that SPAP and ECAP may have diverged very early during the evolution from a common ancestor. Moreover, it is proposed that the SPAP-type of APs are fundamental members of the AP superfamily and are more closely related to other members of the superfamily as compared to the ECAP-type of APs.

Genome dynamics, codon usage patterns and influencing factors in Aeromonas hydrophila phages.

In the present study, genome characteristics and codon usage patterns of 44 Aeromonas hydrophila phages were studied. Phage genomes varied from 30.8 to 262.0 kb with mean±SD and median values of 111.3 ± 81.4 kb and 79.4 kb, respectively. Though the great variation in phage GC contents (35.1-62.2%) was observed, GC contents of all phages (except two phages) were significantly less than the GC content (62.4 ± 5.6%) of the host. The effective number of codons (ENC) values of phage genes ranged from 27.7 to 61 with a mean±SD value of 47.4 ± 6.8. Out of a total 5773 phage genes, 207 (3.6%), 3,528 (61.1%) and 2,012 (34.9%) genes had strong (ENC < 35), moderate (35 < ENC < 50) and low (ENC ≥ 50) codon usage bias, respectively. During relative synonymous codon usage (RSCU) analysis, shared usage of preferred codons was also observed between the phages and host. During codon adaptation index (CAI) analysis, 1028 (17.8%) phage genes showed significant adaptation towards the host. Among these genes, 797 (78.0%) genes encoded hypothetical proteins or proteins of unknown function; whereas 118 (12%) genes encoded the phage structural and packaging proteins. Segregation of ENC, RSCU and CAI analysis results based on genome size also indicated that codon usage bias was more prominent in phages with small genomes. Correlation, neutrality and GC3 versus ENC analyzes indicated a more dominant role of natural selection in shaping the codon usage patterns of A. hydrophila phages. The findings of the current study could be useful from evolutionary and host-pathogen interaction perspectives leading to efficient utilization of phages for therapeutic and other applications.

Poly(vinylidene fluoride)/partially alkylated poly(vinyl imidazole) interpolymer ultrafiltration membranes with intrinsic anti-biofouling and antifouling property for the removal of bacteria.

Bacterial contaminated water causes potential health issues. Conventional chlorine treatment has shortcomings of environmental hazards and chlorine adoptability by the bacterial cells. Ultrafiltration membrane can intercept bacterial species from feed water. Membrane having anti-biofouling/antifouling properties is needed for the removal of bacteria from feed water. Herein, interpolymer membranes with inherent antimicrobial activity and fouling release property have been prepared by the blend of poly(vinylidene fluoride) (PVDF), poly(vinyl pyrrolidone) and partially long chain alkylated (C12 chain) poly(vinyl imidazole) copolymer (PVIm-co-PVIm-C12) followed by cross-linking of the remaining VIm groups with an activated di-halide compound. The membranes obtain with copolymers of degree of alkyl substitution (DSC12) in the range of 0.75-0.85 and amount in the range of 0.9-3.5% w/w in the casting solutions exhibit good antimicrobial activity (>99 % of inhibition) and dynamic anti-biofouling property. The membrane prepared with 0.9% w/w of the copolymer (DSC12=0.85) shows higher flux recovery ratio (91 % for bacterial filtration and 88 % for protein filtration) compare to a pristine membrane (57 % for bacterial filtration and 58 % for protein filtration). The membrane is able to reject the bacteria completely. Use of small amount of copolymer and facile fabrication of stable anti-biofouling/antifouling membranes show potential for the purification of bacterial contaminated water.

Expression of virulence and stress response genes in Aeromonas hydrophila under various stress conditions.

Aeromonas hydrophila has emerged as an important human pathogen as it causes gastroenteritis and extra-intestinal infections. Information regarding the influence of environmental stresses on gene expression profile of A. hydrophila is lacking. The impact of nutrient replenishment, nutrient deprivation, acid stress, and cold shock on housekeeping, general stress-response, and virulence genes was studied using quantitative real-time PCR in two A. hydrophila strains, CECT 839T , and A331. These sub-lethal stresses invoked significant changes in the expression of these genes in a strain-dependent manner. Overall, nutrient replenishment and deprivation significantly induced the expression of housekeeping (rpoD), general stress regulators (uspA and rpoS), and virulence (aer) genes, indicating their importance in regulating the survival and virulence of A. hydrophila under these stress conditions. rpoS gene was significantly induced under cold shock; whereas, acid stress significantly induced the expression of uspA gene. This is the first study to investigate the effect of environmental parameters on the expression of stress-response and virulence genes in A. hydrophila strains.

Increasing genomic diversity and evidence of constrained lifestyle evolution due to insertion sequences in Aeromonas salmonicida.

BACKGROUND: Aeromonads make up a group of Gram-negative bacteria that includes human and fish pathogens. The Aeromonas salmonicida species has the peculiarity of including five known subspecies. However, few studies of the genomes of A. salmonicida subspecies have been reported to date. RESULTS: We sequenced the genomes of additional A. salmonicida isolates, including three from India, using next-generation sequencing in order to gain a better understanding of the genomic and phylogenetic links between A. salmonicida subspecies. Their relative phylogenetic positions were confirmed by a core genome phylogeny based on 1645 gene sequences. The Indian isolates, which formed a sub-group together with A. salmonicida subsp. pectinolytica, were able to grow at either at 18 °C and 37 °C, unlike the A. salmonicida psychrophilic isolates that did not grow at 37 °C. Amino acid frequencies, GC content, tRNA composition, loss and gain of genes during evolution, pseudogenes as well as genes under positive selection and the mobilome were studied to explain this intraspecies dichotomy. CONCLUSION: Insertion sequences appeared to be an important driving force that locked the psychrophilic strains into their particular lifestyle in order to conserve their genomic integrity. This observation, based on comparative genomics, is in agreement with previous results showing that insertion sequence mobility induced by heat in A. salmonicida subspecies causes genomic plasticity, resulting in a deleterious effect on the virulence of the bacterium. We provide a proof-of-concept that selfish DNAs play a major role in the evolution of bacterial species by modeling genomes.

The members of M20D peptidase subfamily from Burkholderia cepacia, Deinococcus radiodurans and Staphylococcus aureus (HmrA) are carboxydipeptidases, primarily specific for Met-X dipeptides.

Three members of peptidase family M20D from Burkholderia cepacia (BcepM20D; Uniprot accession no. A0A0F7GQ23), Deinococcus radiodurans R1 (DradM20D; Uniprot accession no. Q9RTP6) and Staphylococcus aureus (HmrA; Uniprot accession no. Q99Q45) were characterized in terms of their preference for various substrates. The results thus reveal that all the enzymes including HmrA lack endopeptidase as well as aminopeptidase activities and possess strong carboxypeptidase activity. Further, the amidohydrolase activity exerted on other substrates like N-Acetyl-Amino acids, N-Carbobenzoxyl-Amino acids and Indole acetic acid (IAA)-Amino acids is due to the ability of these enzymes to accommodate different types of chemical groups other than the amino acid at the S1 pocket. Further, data on peptide hydrolysis strongly suggests that all the three enzymes are primarily carboxydipeptidases exhibiting highest catalytic efficiency (kcat/Km 5-36 × 10(5) M(-1) s(-1)) for Met-X substrates, where -X could be Ala/Gly/Ser/Tyr/Phe/Leu depending on the source organism. The supportive evidence for the substrate specificities was also provided with the molecular docking studies carried out using structure of SACOL0085 and homology modelled structure of BcepM20D. The preference for different substrates, their binding at active site of the enzyme and possible role of these enzymes in recycling of methionine are discussed in this study.

Diverse Profiles of N-acyl Homoserine L-Lactones, Biofilm, Virulence Genes and Integrons in Food-Borne Aeromonas Isolates.

Aeromonas are regarded as opportunistic as well as primary pathogens of humans and fish, and are associated with gastroenteritis and septicemia in humans. Production of N-acyl-homoserine lactone (AHL) signal molecules and biofilm was determined in 22 Aeromonas isolates, from different food products in India, using thin-layer chromatography (TLC) analysis and microtiter-plate assay, respectively. Overall, highly heterogeneous patterns of AHL production were observed, with the production of N-butanoyl homoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL) by the majority (81.8%) of Aeromonas food isolates. Moreover, putative N-pentanoyl homoserine lactone (C5-HSL), N-heptanoyl homoserine lactone (C7-HSL), and N-octanoyl homoserine lactone (C8-HSL) were produced by 72.7%, 27.3%, and 9.1% of isolates, respectively. This is the 1st report of production of C7-HSL by Aeromonas species. Aeromonas food isolates were highly variable in their biofilm forming abilities with majority of them as weak biofilm producers in 2 different media, TSB and M9 minimal medium supplemented with 0.4% glucose. The genes encoding for putative virulence factors, glycerophospholipid cholesterol acyltransferase (gcat), heat-labile cytotonic enterotoxin (alt), heat-stable cytotonic enterotoxin (ast), serine protease (ser), polar flagella (fla), and lateral flagella (lafA) were present in 95.5%, 59.1%, 22.7%, 81.8%, 77.3%, and 22.7% of the strains, respectively. Class 1 integrons (100 to 3000 bp) were found in 68.2% of food isolates; whereas, 50% isolates contained class 2 integrons (150 to 1600 bp). This study provides a baseline data on the diversity of AHLs, biofilm forming ability and presence of virulence genes and integrons in Aeromonas food isolates from India.

Characterization of Aeromonas strains isolated from Indian foods using rpoD gene sequencing and whole cell protein analysis.

Aeromonas are responsible for causing gastroenteritis and extra-intestinal infections in humans. Twenty-two Aeromonas strains isolated from different food sources were re-identified up to species level using rpoD gene sequence analysis. Biochemical tests and 16S rRNA gene sequencing were insufficient to identify Aeromonas till species level. However, incorporation of additional biochemical tests lead to correct identification of 95.5 % strains up to species level. The 16S rRNA gene sequencing was useful to identify Aeromonas isolates at the genus level only. Sequences of the rpoD gene showed greater discriminatory power than 16S rRNA gene and provided conclusive discrimination of the strains for which the phenotypic species identification was uncertain. All these 22 strains were accurately identified up to species level by rpoD gene as A. salmonicida (6), A. veronii bv. veronii (4), A. caviae (3), A. hydrophila (2), A. veronii bv. sobria (2), A. jandaei (1), A. trota (1), A. sobria (1), A. allosaccharophila (1) and A. bivalvium (1). All these strains were also characterized using whole cell protein (WCP) analysis by gradient SDS-PAGE and showed different whole cell protein (WCP) profile [22-28 polypeptide bands (~10 to >97 kDa)], indicating high genetic diversity. The present work emphasizes the use of molecular methods such as rpoD gene sequencing along with comprehensive biochemical tests for the rapid and accurate identification of Aeromonas isolates till species level. The WCP profile can be subsequently used to characterize Aeromonas isolates below species level.

Prevalence, characterization, and antimicrobial resistance of Aeromonas strains from various retail food products in Mumbai, India.

A total of 154 food samples (chicken, fish, and ready-to-eat sprouts) from various retail outlets in Mumbai, India, were analyzed for the presence of Aeromonas spp. over a period of 2 y (January 2006 to March 2008). Twenty-two Aeromonas isolates belonging to 7 different species were isolated from 18 (11.7%) food samples. The highest percentages of isolation were from chicken (28.6%) followed by fish (20%) and sprout (2.5%) samples. Aeromonas caviae, A. veronii bv. sobria, and A. salmonicida were the most frequently isolated species from sprouts, chicken, and fish samples, respectively. The genes encoding for putative virulence factors, cytotoxic enterotoxin (act), hemolysin (hly), aerolysin (aer), elastase (ahyB), and lipase (lip) were detected using polymerase chain reaction method in 59.1%, 40.9%, 22.7%, 54.5%, and 31.8% of the strains, respectively. The isolated Aeromonas strains were found to be positive for virulence factors, that is, amylase, DNase, gelatinase, protease, and lipase production. More than 60% isolates were also positive for ß-hemolytic activity. All these food isolates were found to be resistant to ampicillin and bacitracin, and sensitive to gentamicin, 3rd-generation cephalosporins (ceftazidime, cephotaxime, ceftriaxone), and chloramphenicol. Seventeen (77.2%) isolates harbored single and/or multiple plasmids (approximately 5 to >16 kb). The XbaI digestion patterns of chromosomal DNA of these isolates, using pulsed field gel electrophoresis, showed high genetic diversity among these isolates. Our results demonstrate the presence of various Aeromonas spp. with virulence potential and antimicrobial resistance in different food products marketed in Mumbai, India. The potential health risks posed by consumption of these raw or undercooked food products should not be underestimated.