Plasmid Borne Research Articles

Biodegradation of bonny light crude oil by plasmid and non-plasmid borne soil bacterial strains using biostimulation and bioaugmentation techniques

A laboratory scale study was designed and conducted to assess the biodegradation of Bonny light crude oil by plasmid and non-plasmid borne soil bacterial strains using biostimulation and bioaugmentation techniques. The enrichment technique, turbidometric test, plasmid curing test, molecular identification method, biostimulation test, bioaugmentation test and gas chromatographic technique were carried out using standard analytical techniques. The physicochemical analysis result showed that the pH was slightly neutral, the organic carbon content was higher (2.32 to 4.34%), the conductivity was higher (0.41 to 0.44 μS/cm), and the water holding capacity was lower (0.27 percent and 10.11 kg, respectively). Based on their capacity to use crude oil, the results showed that 22 of the 60 isolated bacterial strains had higher pollutant degrading potentials (A600nm > 0.3).The identified potent hydrocarbon degraders includes: Bacillus spp., Pseudomonas aeruginosa KAVK01 and Ochrobacterium E85b strains. The highest degradation efficiency of 91% was found in soil that had been contaminated with 3 % (v/w) crude oil, amended with inorganic salts, and inoculated with plasmid-borne mixed cultures. The result further indicated that the consortium of plasmid borne isolates enhanced the reduction of the crude oil from the initial concentration of 10,318 ppm to 501 ppm (95 %) whereas 64 % decontamination was facilitated by the consortium of plasmid cured isolates. The information gathered from this investigation may be useful in choosing bacterial species, particularly plasmid-borne ones that can be employed to biodegrade soil contaminated by crude oil in Nigeria's Niger Delta region as well as the sample collection locations.

In-depth characterization of multidrug-resistant NDM-1 and KPC-3 co-producing Klebsiella pneumoniae bloodstream isolates from Italian hospital patients.

Bloodstream infection (BSI) caused by carbapenem-resistant Klebsiella pneumoniae (KP) poses significant challenges, particularly when the infecting isolate carries multiple antimicrobial resistance (AMR) genes/determinants. This study, employing short- and long-read whole-genome sequencing, characterizes six New Delhi metallo-β-lactamase (NDM) 1 and KP carbapenemase (KPC) 3 co-producing KP isolates, the largest cohort investigated in Europe to date. Five [sequence type (ST) 512] and one (ST11) isolates were recovered from patients who developed BSI from February to August 2022 or February 2023 at two different hospitals in Rome, Italy. Phylogenetic analysis revealed two distinct clusters among ST512 isolates and a separate cluster for the ST11 isolate. Beyond blaNDM-1 and blaKPC-3, various AMR genes, indicative of a multidrug resistance phenotype, including colistin resistance, were found. Each cluster-representative ST512 isolate harbored a blaNDM-1 plasmid (IncC) and a blaKPC-3 plasmid [IncFIB(pQil)/IncFII(K)], while the ST11 isolate harbored a blaNDM-1 plasmid [IncFII(pKPX1)] and a blaKPC-3 plasmid [IncFIB(K)/IncFII(K)]. The blaNDM-1 plasmids carried genes conferring resistance to clinically relevant antimicrobial agents, and the aminoglycoside resistance gene aac(6')-Ib was found on different plasmids. Colistin resistance-associated mgrB/pmrB gene mutations were present in all isolates, and the yersiniabactin-encoding ybt gene was unique to the ST11 isolate. In conclusion, our findings provide insights into the genomic context of blaNDM-1/blaKPC-3 carbapenemase-producing KP isolates.IMPORTANCEThis study underscores the critical role of genomic surveillance as a proactive measure to restrict the spread of carbapenemase-producing KP isolates, especially when key antimicrobial resistance genes, such as blaNDM-1/blaKPC-3, are plasmid borne. In-depth characterization of these isolates may help identify plasmid similarities contributing to their intra-hospital/inter-hospital adaptation and transmission. Despite the lack of data on patient movements, it is possible that carbapenem-resistant isolates were selected to co-produce KP carbapenemase and New Delhi metallo-β-lactamase via plasmid acquisition. Studies employing long-read whole-genome sequencing should be encouraged to address the emergence of KP clones with converging phenotypes of virulence and resistance to last-resort antimicrobial agents.

Molecular Epidemiology of Fluoroquinolones Resistance Genes among Non-typhoidal Salmonella Species from Humans and Poultry Faeces in Ido-Ekiti, Ekiti State

Non-typhoidal Salmonella (NTS) strains are among the most common bacterial pathogens associated with antimicrobial resistance (AMR) where animals are known to be the major reservoir of NTS despite improvements in hygiene and sanitation. This study aimed to investigate the prevalence and basis of fluoroquinolone resistance genes in non-typhoidal Salmonella (NTS) recovered from human and poultry fecal samples in Ido Ekiti. The study used a total of 300 fecal samples,100 from humans and 200 from poultry sources. Faecal samples were cultured using Selenite F broth and XLD agar. Isolates were identified using morphology, biochemical tests, and molecular characterization. Antibiotic susceptibility were determined using disc diffusion test. Plasmid extraction and curing were done using alkaline lysis and Ethidium Bromide. Phylogenetic relatedness was determined using the Unweighted Pair Group Method with Arithmetic Mean. The results revealed that 10.7% of NTS were identified, with 9% from human and 11.5% from poultry sources. High resistance to ofloxacin (77.8%) was observed in both human and poultry samples. S. enteritidis from human sources had a concomitant presence of two qnr genes (qnrA and qnrS) at 20%, while only qnrS (60%) was detected from poultry sources. Statistically significant risk factors for NTS infection among human included the consumption of poultry products and the use of untreated poultry droppings. The study identified Qnr resistance gene in ESBL producing S. enteritidis from both human and poultry sources in Ido Ekiti and are plasmid borne, establishing the role of plasmids in the acquisition, and spread of antibiotic resistance of fluoroquinolones.

Genomic Analysis of a Strain Collection Containing Multidrug-, Extensively Drug-, Pandrug-, and Carbapenem-Resistant Modern Clinical Isolates of Acinetobacter baumannii.

ABSTRACTIn this study, we characterize a new collection that comprises multidrug-resistant (MDR), extensively drug-resistant (XDR), pandrug-resistant (PDR), and carbapenem-resistant modern clinical isolates of Acinetobacter baumannii collected from hospitals through national microbiological surveillance in Belgium. Bacterial isolates (n = 43) were subjected to whole-genome sequencing (WGS), combining Illumina (MiSeq) and Nanopore (MinION) technologies, from which high-quality genomes (chromosome and plasmids) were de novo assembled. Antimicrobial susceptibility testing was performed along with genome analyses, which identified intrinsic and acquired resistance determinants along with their genetic environments and vehicles. Furthermore, the bacterial isolates were compared to the most prevalent A. baumannii sequence type 2 (ST2) (Pasteur scheme) genomes available from the BIGSdb database. Of the 43 strains, 40 carried determinants of resistance to carbapenems; blaOXA-23 (n = 29) was the most abundant acquired antimicrobial resistance gene, with 39 isolates encoding at least two different types of OXA enzymes. According to the Pasteur scheme, the majority of the isolates were globally disseminated clones of ST2 (n = 25), while less frequent sequence types included ST636 (n = 6), ST1 (n = 4), ST85 and ST78 (n = 2 each), and ST604, ST215, ST158, and ST10 (n = 1 each). Using the Oxford typing scheme, we identified 22 STs, including two novel types (ST2454 and ST2455). While the majority (26/29) of blaOXA-23 genes were chromosomally carried, all blaOXA-72 genes were plasmid borne. Our results show the presence of high-risk clones of A. baumannii within Belgian health care facilities with frequent occurrences of genes encoding carbapenemases, highlighting the crucial need for constant surveillance.

Combined comparative genomics and clinical modeling reveals plasmid-encoded genes are independently associated with Klebsiella infection

Members of the Klebsiella pneumoniae species complex frequently colonize the gut and colonization is associated with subsequent infection. To identify genes associated with progression from colonization to infection, we undertook a case-control comparative genomics study. Concordant cases (N = 85), where colonizing and invasive isolates were identical strain types, were matched to asymptomatically colonizing controls (N = 160). Thirty-seven genes are associated with infection, 27 of which remain significant following adjustment for patient variables and bacterial phylogeny. Infection-associated genes are not previously characterized virulence factors, but instead a diverse group of stress resistance, regulatory and antibiotic resistance genes, despite careful adjustment for antibiotic exposure. Many genes are plasmid borne, and for some, the relationship with infection is mediated by gut dominance. Five genes were validated in a geographically-independent cohort of colonized patients. This study identifies several genes reproducibly associated with progression to infection in patients colonized by diverse Klebsiella.

Transmissibility and Persistence of the Plasmid-Borne Mobile Colistin Resistance Gene, mcr-1, Harbored in Poultry-Associated E. coli.

Colistin, a last-resort antibiotic, is used to treat infections caused by multi-drug-resistant Gram-negative bacteria. Colistin resistance can emerge by acquiring the mobile colistin gene, mcr-1, usually plasmid borne. Studies on mcr-1 and its transmissibility are limited in the Middle East and North Africa (MENA) region. Here, we investigated the occurrence of mcr-1 in 18 previously collected Escherichia coli isolates collected from chicken samples in Qatar; whole-genome sequencing was performed to determine the location (plasmid-borne and chromosomal) of mcr-1 in the isolates. Additionally, we assessed the transmissibility of plasmid-borne mcr-1 and its cost on fitness in E. coli biofilms. Our results showed that the E. coli isolates belonged to different sequence types, indicating that mcr-1 was occurring in strains with diverse genetic backgrounds. In silico analysis and transformation assays showed that all the isolates carried mcr-1 on plasmids that were mainly IncI2 types. All the mcr-1 plasmids were found to be transmissible by conjugation. In biofilms, a significant reduction in the number of CFU (≈0.055 logs CFU/mL) and colistin resistance (≈2.19 log CFU/mL) was observed; however, the reduction in resistance was significantly larger, indicating that the plasmids incur a high fitness cost. To our knowledge, this is the first study that investigates mcr-1 transmissibility and persistence in Qatar. Our findings highlight that mcr has the potential to spread colistin resistance to potentially disparate strains and niches in Qatar, posing a risk that requires intervention.

Antibiogram and Plasmid Mediated Resistance in Bacteria Isolated from Infected Wounds

Bacterial infection of wound plays an important role in the development of chronicity and delayed healing. In this study, a total of 50 wound swabs were aseptically collected from patients attending specialist hospital Jimeta Yola, Adamawa State and were screened for bacteria. The isolates were identified using Gram-staining and biochemical tests. Eight different bacterial species were identified with Staphylococcus aureus having the highest occurrence with 11(26.19%), followed by Escherichia coli 8(19.05%), Klebsiella pneumoniae 6(14.29%), Pseudomonas aeruginosa and Staphylococcus epidermidis 5(11.9%), Proteus vulgaris, Streptococcus pyogenes 3(7.14%) and lastly, Bacillus subtilis with 1(2.38%). Antibiotic susceptibility test using Kirby-Bauer disk diffusion method revealed that most of the Gram-positive isolates significantly resisted oxacillin, penicillin and amoxicillin. Most Gram negatives significantly resisted septrin, chloramphenicol, amoxicillin, augmentin and pefloxacin. Ciprofloxacin was 100% effective against both Gram positive and Gram-negative isolates. Plasmid curing of resistant isolates using 10% sodium dodecyl sulphate (SDS) revealed that resistance to penicillin, oxacillin, amoxicillin, augmentin and pefloxacin were plasmid borne whereas chloramphenicol and septrin (trimethoprim) were not. Bacteria associated with wound infections encompass both Gram-negative and Gram-positive bacteria in nearly equal proportions with high rate of resistance among the isolates.

Detection of Colistin Resistance in Uropathogenic Carbapenem Resistant Klebsiella pneumoniae

Background: Colistin resistance among carbapenem-resistant Klebsiella pneumoniae (CRKp) strains at an accelerated rate poses a serious global threat which limits therapeutic options. Objective: In this study, 203 laboratory-archived uropathogenic K. pneumoniae isolates were screened for carbapenem and colistin resistance. Methods: Carbapenam resistance screening was performed using combined disc test. Colistin resistance was determined for CRKp isolates, according to clinical and laboratory standards institute- European Committee on Antimicrobial Susceptibility Testing guidelines. Polymerase Chain Reaction for carbapenamase genes (blaNDM-1, blaKPC-2 and blaOXA-1) and plasmid-mediated colistin resistance genes (mcr-1-2) were performed for the resistant isolates. Results: About 30% of the CRKp isolates were resistant to colistin; 57.1% and 35.7% of the isolates carried blaNDM-1 and blaOXA-1 gene, respectively. None of the isolates showed positive for blaKPC-2 and mcr (1-2) genes. Conclusion: Colistin resistance in the absence of a plasmid borne transfer mechanism was observed among our CRKp strains. Escalating colistin resistance is a crucial obstacle in treatment of K. pneumoniae infections and repurposing their usage is important to combat and control the resistance.

Plasmid Borne Antibiotic Resistance Factors in Indigenous Campylobacter spp. Isolated from Humans in Azad Jammu and Kashmir, Pakistan

Plasmid Borne Antibiotic Resistance Factors in Indigenous Campylobacter spp. Isolated from Humans in Azad Jammu and Kashmir, Pakistan

Mutations in porin LamB contribute to ceftazidime-avibactam resistance in KPC-producing Klebsiella pneumoniae

Ceftazidime-avibactam (CAZ-AVI) shows promising activity against carbapenem-resistant Klebsiella pneumoniae (CRKP), however, CAZ-AVI resistance have emerged recently. Mutations in KPCs, porins OmpK35 and/or OmpK36, and PBPs are known to contribute to the resistance to CAZ-AVI in CRKP. To identify novel CAZ-AVI resistance mechanism, we generated 10 CAZ-AVI-resistant strains from 14 CAZ-AVI susceptible KPC-producing K. pneumoniae (KPC-Kp) strains through in vitro multipassage resistance selection using low concentrations of CAZ-AVI. Comparative genomic analysis for the original and derived mutants identified CAZ-AVI resistance-associated mutations in KPCs, PBP3 (encoded by ftsI), and LamB, an outer membrane maltoporin. CAZ-AVI susceptible KPC-Kp strains became resistant when complemented with mutated bla KPC genes. Complementation experiments also showed that a plasmid borne copy of wild-type lamB or ftsI gene reduced the MIC value of CAZ-AVI in the induced resistant strains. In addition, bla KPC expression level increased in four of the six CAZ-AVI-resistant strains without KPC mutations, indicating a probable association between increased bla KPC expression and increased resistance in these strains. In conclusion, we here identified a novel mechanism of CAZ-AVI resistance associated with mutations in porin LamB in KPC-Kp.

Molecular mechanisms driving the in vivo development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR Pseudomonas aeruginosa infections.

The development of resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of Pseudomonas aeruginosa infections is concerning. Characterization of the mechanisms leading to the development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR P. aeruginosa infections. Four paired ceftolozane/tazobactam- and ceftazidime/avibactam-susceptible/resistant isolates were evaluated. MICs were determined by broth microdilution. STs, resistance mechanisms and genetic context of β-lactamases were determined by genotypic methods, including WGS. The OXA-10 variants were cloned in PAO1 to assess their impact on resistance. Models for the OXA-10 derivatives were constructed to evaluate the structural impact of the amino acid changes. The same XDR ST253 P. aeruginosa clone was detected in all four cases evaluated. All initial isolates showed OprD deficiency, produced an OXA-10 enzyme and were susceptible to ceftazidime, ceftolozane/tazobactam, ceftazidime/avibactam and colistin. During treatment, the isolates developed resistance to all cephalosporins. Comparative genomic analysis revealed that the evolved resistant isolates had acquired mutations in the OXA-10 enzyme: OXA-14 (Gly157Asp), OXA-794 (Trp154Cys), OXA-795 (ΔPhe153-Trp154) and OXA-824 (Asn143Lys). PAO1 transformants producing the evolved OXA-10 derivatives showed enhanced ceftolozane/tazobactam and ceftazidime/avibactam resistance but decreased meropenem MICs in a PAO1 background. Imipenem/relebactam retained activity against all strains. Homology models revealed important changes in regions adjacent to the active site of the OXA-10 enzyme. The blaOXA-10 gene was plasmid borne and acquired due to transposition of Tn6746 in the pHUPM plasmid scaffold. Modification of OXA-10 is a mechanism involved in the in vivo acquisition of resistance to cephalosporin/β-lactamase inhibitor combinations in P. aeruginosa.

Plasmid-Mediated Biodegradation of Chlorpyrifos and Analysis of Its Metabolic By-Products.

Organophosphate pesticide persistence is an emerging menace to the environment and despite this fact, its use has been enhanced due to its high efficiency. Bioremediation using microorganisms would be the only means by which these hazardous compounds could be wiped out without disturbing the environmental harmony. The current work studied the molecular mechanism of degradation of Chlorpyrifos (CP) by a bacterial consortium C5 comprising of three soil isolates Staphylococcus warneri (CPI 2), Pseudomonas putida (CPI 9) and Stenotrophomonas maltophilia (CPI 15), which unveiled that the property is plasmid borne. All the isolates were found to possess a 4kb plasmid which could be cured only by using sodium azide. The Escherichia coli JM109 cells when transformed individually with the plasmid of the isolates showed CP degradation in mineral salts medium (MSM) that contained CP as the sole carbon source. The degradative enzyme organophosphorus hydrolase (~ 60 KDa) of the isolates was extracted and purified to 31.85, 26 and 37.74 fold, respectively. The possible metabolic by-products of CPdegradation by the consortium C5, were also analysed. The LC-Q-Tof MS analysis revealed the presence of the major metabolite 3, 5, 6 -trichloropyridine (TCP) with the formation of chlorpyrifos oxon as the intermediate. The isolates also showed trichloropyridine degradation (> 80%) individually in MSM-TCP medium proving its efficiency to remediate both CP and TCP.

Efficient production of S-adenosyl-l-methionine from dl-methionine in metabolic engineered Saccharomyces cerevisiae.

S-Adenosyl-l-methionine (SAM) is an important small molecule compound widely used in treating various diseases. Although l-methionine is generally used, the low-cost dl-methionine is more suitable as the substrate for industrial production of SAM. However, d-methionine is inefficient for SAM formation due to the substrate-specificity of SAM synthetase. In order to increase the utilization efficiency of dl-methionine, intracellular conversion of d-methionine to l-methionine was investigated in the type strain Saccharomyces cerevisiae BY4741 and an industrial strain S. cerevisiae HDL. Firstly, via disruption of HPA3 encoding d-amino acid-N-acetyltransferase, d-methionine was accumulated in vivo and no N-acetyl-d-methionine production was observed. Further, codon-optimized d-amino acid oxidase (DAAO) gene from Trigonopsis variabilis (Genbank MK280686) and l-phenylalanine dehydrogenase gene (l-PheDH) from Rhodococcus jostii (Genbank MK280687) were introduced to convert d-methionine to l-methionine, SAM concentration and content was increased by 110% and 72.1% in BY4741 (plasmid borne) and increased by 38.2% and 34.1% in HDL (genome integrated), by feeding 0.5 g/L d-methionine. Using the recently developed CRISPR tools, the DAAO and l-PheDH expression cassettes were integrated into the HPA3 and SAH1 loci while SAM2 expression was integrated into the SPE2 and GLC3 loci of HDL, and the resultant strain HDL-R2 accumulated 289% and 192% more SAM concentration and content, respectively, by feeding 0.5 g/L dl-methionine. Further, in a 10 L fed-batch fermentation process, 10.3 g/L SAM were accumulated with the SAM content of 242 mg/g dry cell weight by feeding 16 g/L dl-methionine. The strategies used here provided a promising approach to enhance SAM production using low-cost dl-methionine.

Genome sequencing of Bacillus thuringiensis isolate T414 toxic to pink bollworm (Pectinophora gossypiella Saunders) and its insecticidal genes

Bacillus thuringiensis is a spore-forming bacterium that is pathogenic towards a range of insect and nematode species and had been widely used as a biopesticide. In this study, we present the morphological, molecular and genetic characteristics of an indigenous Bt isolate T414 which displayed an effective toxicity against Pectinophora gossypiella. Scanning electron microscopy revealed the presence of bipyramidal, spherical and cubic shaped protein crystals in its spore-crystal suspension. SDS-PAGE analysis of its spore-crystal mixture showed the presence of two major protein bands viz.130 and 65 kDa. Whole genome sequencing with MiSeq divulged that it contains a chromosome and many plasmids. The assembled genome finally contained 6493494bp. Automated annotation of this genome draft predicted 6877 coding sequences and 152 RNAs (rRNAs, tRNAs and ncRNAs). NCBI blast analysis showed that assembled genome was distributed in a chromosome and 15 different types of plasmids. Further analysis of draft sequence revealed it harbors parasporal crystal protein genes (cry1Aa, cry1Ab, cry1Ac, cry1IAa, cry2Aa, cry2Ab and cyt1), vegetative insecticidal protein gene (vip3Aa), all plasmid borne and various additional virulence factors such as chitinases, proteases, bacteriocins and hemolysins. From the analysis it is evident that all the Cry, Cyt or Vip toxins are plasmid borne and are present on two types of plasmids named as p414A and p414E in the present study. A cry2A type gene was cloned and sequenced. It was named as cry2Aa21 by Bt nomenclature committee.

Rapid formylation of the cellular initiator tRNA population makes a crucial contribution to its exclusive participation at the step of initiation.

Initiator tRNAs (i-tRNAs) possess highly conserved three consecutive GC base pairs (GC/GC/GC, 3GC pairs) in their anticodon stems. Additionally, in bacteria and eukaryotic organelles, the amino acid attached to i-tRNA is formylated by Fmt to facilitate its targeting to 30S ribosomes. Mutations in GC/GC/GC to UA/CG/AU in i-tRNACUA/3GC do not affect its formylation. However, the i-tRNACUA/3GC is non-functional in initiation. Here, we characterised an Escherichia coli strain possessing an amber mutation in its fmt gene (fmtam274), which affords initiation with i-tRNACUA/3GC. Replacement of fmt with fmtam274 in the parent strain results in production of truncated Fmt, accumulation of unformylated i-tRNA, and a slow growth phenotype. Introduction of i-tRNACUA/3GC into the fmtam274 strain restores accumulation of formylated i-tRNAs and rescues the growth defect of the strain. We show that i-tRNACUA/3GC causes a low level suppression of am274 in fmtam274. Low levels of cellular Fmt lead to compromised efficiency of formylation of i-tRNAs, which in turn results in distribution of the charged i-tRNAs between IF2 and EF-Tu allowing the plasmid borne i-tRNACUA/3GC to function at both the initiation and elongation steps. We show that a speedy formylation of i-tRNA population is crucial for its preferential binding (and preventing other tRNAs) into the P-site.

A plasmid borne, functionally novel glycoside hydrolase family 30 subfamily 8 endoxylanase from solventogenic Clostridium.

Glycoside hydrolase family 30 subfamily 8 (GH30-8) β-1,4-endoxylanases are known for their appendage-dependent function requiring recognition of an α-1,2-linked glucuronic acid (GlcA) common to glucuronoxylans for hydrolysis. Structural studies have indicated that the GlcA moiety of glucuronoxylans is coordinated through six hydrogen bonds and a salt bridge. These GlcA-dependent endoxylanases do not have significant activity on xylans that do not bear GlcA substitutions such as unsubstituted linear xylooligosaccharides or cereal bran arabinoxylans. In the present study, we present the structural and biochemical characteristics of xylanase 30A from Clostridium acetobutylicum (CaXyn30A) which was originally selected for study due to predicted structural differences within the GlcA coordination loops. Amino acid sequence comparisons indicated that this Gram-positive-derived GH30-8 more closely resembles Gram-negative derived forms of these endoxylanases: a hypothesis borne out in the developed crystallographic structure model of the CaXyn30A catalytic domain (CaXyn30A-CD). CaXyn30A-CD hydrolyzes xylans to linear and substituted oligoxylosides showing the greatest rate with the highly arabinofuranose (Araf)-substituted cereal arabinoxylans. CaXyn30A-CD hydrolyzes xylooligosaccharides larger than xylotriose and shows an increased relative rate of hydrolysis for xylooligosaccharides containing α-1,2-linked arabinofuranose substitutions. Biochemical analysis confirms that CaXyn30A benefits from five xylose-binding subsites which extend from the −3 subsite to the +2 subsite of the binding cleft. These studies indicate that CaXyn30A is a GlcA-independent endoxylanase that may have evolved for the preferential recognition of α-1,2-Araf substitutions on xylan chains.

Pantoea ananatis Genetic Diversity Analysis Reveals Limited Genomic Diversity as Well as Accessory Genes Correlated with Onion Pathogenicity.

Pantoea ananatis is a member of the family Enterobacteriaceae and an enigmatic plant pathogen with a broad host range. Although P. ananatis strains can be aggressive on onion causing foliar necrosis and onion center rot, previous genomic analysis has shown that P. ananatis lacks the primary virulence secretion systems associated with other plant pathogens. We assessed a collection of fifty P. ananatis strains collected from Georgia over three decades to determine genetic factors that correlated with onion pathogenic potential. Previous genetic analysis studies have compared strains isolated from different hosts with varying diseases potential and isolation sources. Strains varied greatly in their pathogenic potential and aggressiveness on different cultivated Allium species like onion, leek, shallot, and chive. Using multi-locus sequence analysis (MLSA) and repetitive extragenic palindrome repeat (rep)-PCR techniques, we did not observe any correlation between onion pathogenic potential and genetic diversity among strains. Whole genome sequencing and pan-genomic analysis of a sub-set of 10 strains aided in the identification of a novel series of genetic regions, likely plasmid borne, and correlating with onion pathogenicity observed on single contigs of the genetic assemblies. We named these loci Onion Virulence Regions (OVR) A-D. The OVR loci contain genes involved in redox regulation as well as pectate lyase and rhamnogalacturonase genes. Previous studies have not identified distinct genetic loci or plasmids correlating with onion foliar pathogenicity or pathogenicity on a single host pathosystem. The lack of focus on a single host system for this phytopathgenic disease necessitates the pan-genomic analysis performed in this study.

Molecular characterization and phylogenetic analysis of Clostridium perfringens from animals and their environments by cpn60 UT sequencing analysis

Clostridium perfringens an ubiquitous environmental bacterium causes major food borne illnesses, digestive diseases and several soft tissue infections in humans and animals. In the present study, toxin typing of 91 C. perfringens isolates from animals with enteric diseases and their environments revealed the presence of type A and C strains. Enterotoxin gene (cpe), responsible for majority of the food poisoning incidences in humans and enteric infections in animals was present in 60.43 % of the isolates of which 76.3% and 23. 36% were chromosomal and plasmid borne respectively. Neighbour-joining tree inferred from cpn60 UT nucleotide sequences could differentiate the cpe+ve isolates from the cpe−ve isolates, provide clear distinction between the cpe-IS1470 and cpe-IS1151 genotypes and segregate type A and C strains in separate clusters. The present study is the first report on the utilization of cpn60 UT region for C. perfringens phylogeny analysis and demonstrates that cpn60 UT analysis alone, to a greater extent can be a simple, rapid and efficient method for differentiating between cpe+ve and cpe−ve strains or toxin types. The cpb2 gene was observed among 30 isolates of which 16.6% were from porcine sources while the rest were of non-porcine and environmental origin. The cpb2 sequences obtained in the present study though similar among them were diverse both from the consensus and atypical cpb2 sequences reported globally and formed a separate cluster. The study thus reports of novel cpb2 gene variant and warrants its characterization through further studies.

Genomic and Molecular Characterization of Clinical Isolates of Enterobacteriaceae Harboring mcr-1 in Colombia, 2002 to 2016.

Polymyxins are last-resort antimicrobial agents used to treat infections caused by carbapenem-resistant Enterobacteriaceae Due to the worldwide dissemination of polymyxin resistance in animal and human isolates, we aimed to characterize polymyxin resistance associated with the presence of mcr-1 in Enterobacteriaceae and nonfermenter Gram-negative bacilli, using isolates collected retrospectively in Colombia from 2002 to 2016. A total of 5,887 Gram-negative clinical isolates were studied, and 513 were found to be resistant to the polymyxins. Susceptibility to colistin was confirmed by broth microdilution for all mcr-1-positive isolates, and these were further subjected to whole-genome sequencing (WGS). The localization of mcr-1 was confirmed by S1 pulsed-field gel electrophoresis (S1-PFGE) and CeuI-PFGE hybridization. Transferability was evaluated by mating assays. A total of 12 colistin-resistant isolates recovered after 2013 harbored mcr-1, including 8 Escherichia coli, 3 Salmonella enterica serovar Typhimurium, and 1 Klebsiella pneumoniae isolate. E. coli isolates were unrelated by PFGE and belonged to 7 different sequence types (STs) and phylogroups. S Typhimurium and K. pneumoniae isolates belonged to ST34 and ST307, respectively. The mcr-1 gene was plasmid borne in all isolates but two E. coli isolates which harbored it on the chromosome. Conjugation of mcr-1 was successful in 8 of 10 isolates (8.2 × 10-5 to 2.07 × 10-1 cell per recipient). Plasmid sequences showed that the mcr-1 plasmids belonged to four different Inc groups (a new IncP-1 variant and the IncFII, IncHI1, and IncH families). Our results indicate that mcr-1 is circulating in clinical isolates of colistin-resistant Enterobacteriaceae in Colombia and is mainly harbored in transferable plasmids.

Genetic evidence for a novel competence inhibitor in the industrially important Bacillus licheniformis

Natural genetic competence renders bacteria able to take up and, in case there is sufficient homology to the recipient’s chromosome, integrate exogenously supplied DNA. Well studied in Bacillus subtilis, genetic competence is—in several aspects—known to be differently regulated in Bacillus licheniformis. We now report on the identification of a novel, chromosomally encoded homolog of a competence inhibitor in B. licheniformis (ComI) that has hitherto only been described as a plasmid borne trait in the ancestral B. subtilis NCIB3610. Bioinformatical analysis that included 80 Bacillus strains covering 20 different species revealed a ComI encoding gene in all of the examined B. licheniformis representatives, and was identified in few among the other species investigated. The predicted ComI of B. licheniformis is a highly conserved peptide consisting of 28 amino acids. Since deletion of comI in B. licheniformis DSM13 resulted in twofold increased transformation efficiency by genetic competence and overexpression resulted in threefold decreased transformability, the function as a competence inhibitor became evident.