Exploring Klebsiella pneumoniae in Healthy Poultry Reveals High Genetic Diversity, Good Biofilm-Forming Abilities and Higher Prevalence in Turkeys Than Broilers.

Klebsiella pneumoniae is a well-studied human pathogen for which antimicrobial resistant and hypervirulent clones have emerged globally. K. pneumoniae is also present in a variety of environmental niches, but currently there is a lack of knowledge on the occurrence and characteristics of K. pneumoniae from non-human sources. Certain environmental niches, e.g., animals, may be associated with high K. pneumoniae abundance, and these can constitute a reservoir for further transmission of strains and genetic elements. The aim of this study was to explore and characterize K. pneumoniae from healthy broilers and turkeys. A total of 511 cecal samples (broiler n = 356, turkey n = 155), included in the Norwegian monitoring program for antimicrobial resistance (AMR) in the veterinary sector (NORM-VET) in 2018, were screened for K. pneumoniae by culturing on SCAI agar. K. pneumoniae was detected in 207 (40.5%) samples. Among the broiler samples, 25.8% were positive for K. pneumoniae, in contrast to turkey with 74.2% positive samples (p < 0.01). Antibiotic susceptibility testing was performed, in addition to investigating biofilm production. Whole genome sequencing was performed on 203 K. pneumoniae isolates, and analysis was performed utilizing comparative genomics tools. The genomes grouped into 66 sequence types (STs), with ST35, ST4710 and ST37 being the most prevalent at 13.8%, 7.4%, and 5.4%, respectively. The overall AMR occurrence was low, with only 11.3% of the isolates showing both pheno- and genotypic resistance. Genes encoding aerobactin, salmochelin or yersiniabactin were detected in 47 (23.2%) genomes. Fifteen hypervirulent genomes belonging to ST4710 and isolated from turkey were identified. These all encoded the siderophore virulence loci iuc5 and iro5 on an IncF plasmid. Isolates from both poultry species displayed good biofilm-forming abilities with an average of OD595 0.69 and 0.64. To conclude, the occurrence of K. pneumoniae in turkey was significantly higher than in broiler, indicating that turkey might be an important zoonotic reservoir for K. pneumoniae compared to broilers. Furthermore, our results show a highly diverse K. pneumoniae population in poultry, low levels of antimicrobial resistance, good biofilm-forming abilities and a novel hypervirulent ST4710 clone circulating in the turkey population.

Plasmid-associated antimicrobial resistance and virulence genes in Escherichia coli in a high arctic reindeer subspecies.

OBJECTIVES: In extreme environments, such as the Arctic region, the anthropogenic influence is low and the presence of antimicrobial-resistant bacteria is unexpected. In this study, we screened wild reindeer (Rangifer tarandus platyrhynchus) from the Svalbard High Arctic Archipelago for antimicrobial-resistant Escherichia coli and performed in-depth strain characterisation. METHODS: Using selective culturing of faecal samples from 55 animals, resistant E. coli were isolated and subjected to minimum inhibitory concentration (MIC) determination, conjugation experiments and whole-genome sequencing. RESULTS: Twelve animals carried antimicrobial-resistant E. coli. Genomic analysis showed IncF plasmids as vectors both for resistance and virulence genes in most strains. Plasmid-associated genes encoding resistance to ampicillin, sulfonamides, streptomycin and trimethoprim were found in addition to virulence genes typical for colicin V (ColV)-producing plasmids. Comparison with previously reported IncF ColV plasmids from human and animal hosts showed high genetic similarity. The plasmids were detected in E. coli sequence types (STs) previously described as hosts for such plasmids, such as ST58, ST88 and ST131. CONCLUSION: Antimicrobial-resistant E. coli were detected from Svalbard reindeer. Our findings show that successful hybrid antimicrobial resistance-ColV plasmids and their host strains are widely distributed also occurring in extreme environmental niches such as arctic ecosystems. Possible introduction routes of resistant bacterial strains and plasmids into Svalbard ecosystems may be through migrating birds, marine fish or mammals, arctic fox (Vulpes lagopus) or via human anthropogenic activities such as tourism.

Over 2000-Fold Increased Production of the Leaderless Bacteriocin Garvicin KS by Increasing Gene Dose and Optimization of Culture Conditions.

The leaderless bacteriocin Garvicin KS (GarKS) is a potent antimicrobial, being active against a wide range of important pathogens. GarKS production by the native producer Lactococcus garvieae KS1546 is, however, relatively low (80 BU/ml) under standard laboratory growth conditions (batch culture in GM17 at 30°C). To improve the production, we systematically evaluated the impact of different media and media components on bacteriocin production. Based on the outcomes, a new medium formulation was made that increased GarKS production about 60-fold compared to that achieved in GM17. The new medium was composed of pasteurized milk and tryptone (PM-T). GarKS production was increased further 4-fold (i.e., to 20,000 BU/ml) by increasing the gene dose of the bacteriocin gene cluster (gak) in the native producer. Finally, a combination of the newly composed medium (PM-T), an increased gene dose and cultivation at a constant pH 6 and a 50-60% dissolved oxygen level in growth medium, gave rise to a GarKS production of 164,000 BU/ml. This high production, which is about 2000-fold higher compared to that initially achieved in GM17, corresponds to a GarKS production of 1.2 g/L. To our knowledge, this is one of the highest bacteriocin production reported hitherto.

Inactivation of the arn operon and loss of aminoarabinose on lipopolysaccharide as the cause of susceptibility to colistin in an atypical clinical isolate of proteus vulgaris.

Colistin has become a last-line antibiotic for the treatment of multidrug-resistant bacterial infections; however, resistance to colistin has emerged in recent years. Some bacteria, such as Proteus and Serratia spp., are intrinsically resistant to colistin although the exact mechanism of resistance is unknown. Here we identified the molecular support for intrinsic colistin resistance in Proteus spp. by comparative genomic, transcriptomic and proteomic analyses of colistin-susceptible (CSUR P1868_S) and colistin-resistant (CSUR P1867_R) strains of an atypical Proteus vulgaris. A significant difference in outer membrane glycoside structures in both strains that was corroborated by MALDI-TOF/MS analysis was found, which showed an absence of 4-amino-4-deoxy-l-arabinose (L-Ara4N) in the outer membrane lipid A moiety of the susceptible strain. Comparative genomic analysis with other resistant strains of P. vulgaris available in a local database found a mutation in the arnBCADTEF operon of the susceptible strain. Transcriptomic analysis of genes belonging to the arnBCADTEF operon showed a significant decrease in mRNA expression level of these genes in the susceptible strain, supporting addition of L-Ara4N in the outer membrane lipid A moiety as an explanation for colistin resistance. Insertion of the arnD gene that was suggested to be altered in the susceptible strain by in silico analysis led to a 16-fold increase of colistin MIC in the susceptible strain, confirming its role in colistin resistance in this species. Here we show that constitutive activation of the arn operon and addition of L-Ara4N is the main molecular mechanism of colistin resistance in P. vulgaris.

soxRS induces colistin hetero-resistance in Enterobacter asburiae and Enterobacter cloacae by regulating the acrAB-tolC efflux pump.

Background: Colistin is the last drug option for the treatment of MDR Gram-negative bacterial infections. Several types of resistance to colistin have been identified, including hetero-resistance, which has been observed in several Gram-negative pathogens. During a routine surveillance project on antimicrobial resistance, we found abnormal colistin-resistant Enterobacter asburiae and Enterobacter cloacae isolates. E. cloacae is an intestinal commensal bacterium and a well-known opportunistic nosocomial pathogen. Objectives: To characterize the molecular mechanism of colistin hetero-resistance in Enterobacter spp. Methods: Several approaches (WGS, transposome mutagenesis and RT-PCR analysis) were used to discover the molecular mechanism of colistin hetero-resistance. Results: Genomic analysis of mutant clones generated by transposome mutagenesis suggests that hetero-resistance is linked with overexpression of the acrAB-tolC efflux pump. Transcriptional analysis further found that naturally elevated soxRS triggers the induction of the acrAB-tolC efflux pump proteins followed by the development of colistin hetero-resistance in E. asburiae and E. cloacae. Transcriptional analysis results were further verified as demonstrating the development of hetero-resistance in colistin-susceptible strains by plasmid-based overexpression of soxRS. Conclusions: Our observations highlight the importance of such findings, which previously were only superficially described because of the challenges associated with their detection, in the context of common modes of colistin resistance in Gram-negative bacteria. This study constitutes a unique demonstration of efflux-based high-level colistin hetero-resistance, controlled by a soxRS regulator in Gram-negative bacteria.

Functional genomics to discover antibiotic resistance genes: The paradigm of resistance to colistin mediated by ethanolamine phosphotransferase in Shewanella algae MARS 14.

Shewanella algae MARS 14 is a colistin-resistant clinical isolate retrieved from bronchoalveolar lavage of a hospitalised patient. A functional genomics strategy was employed to discover the molecular support for colistin resistance in S. algae MARS 14. A pZE21 MCS-1 plasmid-based genomic expression library was constructed in Escherichia coli TOP10. The estimated library size was 1.30×10(8) bp. Functional screening of colistin-resistant clones was carried out on Luria-Bertani agar containing 8 mg/L colistin. Five colistin-resistant clones were obtained after complete screening of the genomic expression library. Analysis of DNA sequencing results found a unique gene in all selected clones. Amino acid sequence analysis of this unique gene using the Integrated Microbial Genomes (IMG) and KEGG databases revealed that this gene encodes ethanolamine phosphotransferase (EptA, or so-called PmrC). Reverse transcription PCR analysis indicated that resistance to colistin in S. algae MARS 14 was associated with overexpression of EptA (27-fold increase), which plays a crucial role in the arrangement of outer membrane lipopolysaccharide.

Multifunctional cellulolytic auxiliary activity protein HcAA10-2 from Hahella chejuensis enhances enzymatic hydrolysis of crystalline cellulose.

The modular auxiliary activity (AA) family of proteins is believed to cause amorphogenesis in addition to oxidative cleavage of crystalline cellulose although the supporting evidence is limited. HcAA10-2 is a modular AA10 family protein (58 kDa) composed of a AA10 module and a family two carbohydrate binding module (CBM2), joined by a long stretch of 222 amino acids of unknown function. The protein was expressed in Escherichia coli and purified to homogeneity. Scanning electron microscopy and X-ray diffraction analysis of Avicel treated with HcAA10-2 provided evidence for the disruption of the cellulose microfibrils ("amorphogenesis") and reduction of the crystallinity index, resulting in a twofold increase of cellulase adsorption on the polysaccharide surface. HcAA10-2 exhibited weak endoglucanase-like activity toward soluble cellulose and cello-oligosaccharides with an optimum at pH 6.5 and 45 °C. HcAA10-2 catalyzed oxidative cleavage of crystalline cellulose released native and oxidized cello-oligosaccharides in the presence of copper and an electron donor such as ascorbic acid. Multiple sequence alignment indicated that His1, His109, and Phe197 in the AA10 module formed the conserved copper-binding site. The reducing sugar released from Avicel by the endoglucanase Cel5 and Celluclast accompanying HcAA10-2 was increased by four- and sixfold, respectively. Moreover, HcAA10-2 and Celluclast acted synergistically on pretreated wheat straw biomass resulting in a threefold increase in reducing sugar than Celluclast alone. Taken together, these results suggest that HcAA10-2 is a novel multifunctional modular AA10 protein possessing amorphogenesis, weak endoglucanase, and oxidative cleavage activities useful for efficient degradation of crystalline cellulose.

Engineering of family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.

Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.

Construction and characterization of chimeric cellulases with enhanced catalytic activity towards insoluble cellulosic substrates.

The chimeric proteins viz. CBM3-Cel9A, CBM4-Cel9A and CBM30-Cel9A, are constructed by fusion of family 3, 4, and 30 cellulose binding modules (CBMs) to N-terminus of family 9 endoglucanase (Cel9A) from Alicyclobacillus acidocaldrious. The chimeric enzymes were successfully expressed in Escherichia coli and purified to homogeneity. The chimeric enzymes showed significant increase in Avicel (8-12 folds) and filter paper (7-10 folds) degradation activities compared to Cel9A endoglucanase. Computational protein modeling and simulation on the chimeric enzymes were applied to analyze the fused CBMs effect on the increased insoluble cellulosic substrates degradation activity. Thin layer chromatography analysis of the enzymatic hydrolysis products and distribution of reducing sugars between soluble and insoluble fractions indicated processive cleavage of insoluble cellulosic substrates by the chimeras. The fused CBMs played a critical accessory role for the Cel9A catalytic domain and changed its character to facilitate the processive cleavage of insoluble cellulosic substrates.

Significant reduction in toxicity, BOD, and COD of textile dyes and textile industry effluent by a novel bacterium Pseudomonas sp. LBC1.

The 16S rRNA sequence analysis and biochemical characteristics were confirmed that the isolated bacterium is Pseudomonas sp. LBC1. The commonly used textile dye, Direct Brown MR has been used to study the fate of biodegradation. Pseudomonas sp. LBC1 showed 90% decolorization of Direct Brown MR (100 mg/L) and textile industry effluent with significant reduction in COD and BOD. The optimum condition for decolorization was 7.0 pH and 40°C. Significant increase in a activity of extracellular laccase suggested their possible involvement in decolorization of Direct Brown MR. Biodegradation metabolites viz. 3,6-dihydroxy benzoic acid, 2-hydroxy-7-aminonaphthol-3-sulfonic acid, and p-dihydroperoxybenzene were identified on the basis of mass spectra and using the 1.10 beta Shimadzu NIST GC-MS library. The Direct Brown MR and textile industry effluent were toxic to Sorghum bicolor and Vigna radiata plants as compared to metabolites obtained after decolorization. The Pseudomonas sp. LBC1 could be useful strain for decolorization and detoxification of textile dyes as well as textile industry effluent.

Biochemical characterization of laccase from hairy root culture of Brassica juncea L. and role of redox mediators to enhance its potential for the decolorization of textile dyes.

In vitro transgenic hairy root cultures provide a rapid system for physiological, biochemical studies and screening of plants for their phytoremediation potential. The hairy root cultures of Brassica juncea L. showed 92% decolorization of Methyl orange within 4 days. Out of the different redox mediators that were used to achieve enhanced decolorization, 2, 2'-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was found to be the most efficient. Laccase activity of 4.5 U mg(-1) of protein was observed in hairy root cultures of Brassica juncea L., after the decolorization of Methyl orange. Intracellular laccase produced by B. juncea root cultures grown in MS basal medium was purified up to 2.0 fold with 6.62 U mg(-1) specific activity using anion-exchange chromatography. Molecular weight of the purified laccase was estimated to be 148 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme efficiently oxidized ABTS which was also required for oxidation of the other tested substrates. The pH and temperature optimum for laccase activity were 4.0 and 40°C, respectively. The purified enzyme was stable up to 50°C and was stable in the pH range of 4.0-6.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol and L: -cysteine. The purified enzyme decolorized various textile dyes in the presence of ABTS as an efficient redox mediator. These findings contribute to a better understanding of the enzymatic process involved in phytoremediation of textile dyes by using hairy roots.

Decolorization of adsorbed textile dyes by developed consortium of Pseudomonas sp. SUK1 and Aspergillus ochraceus NCIM-1146 under solid state fermentation.

The objective of this study was to develop consortium using Pseudomonas sp. SUK1 and Aspergillus ochraceus NCIM-1146 to decolorize adsorbed dyes from textile effluent wastewater under solid state fermentation. Among various agricultural wastes rice bran showed dye adsorption up to 90, 62 and 80% from textile dye reactive navy blue HE2R (RNB HE2R) solution, mixture of textile dyes and textile industry wastewater, respectively. Pseudomonas sp. SUK1 and A. ochraceus NCIM-1146 showed 62 and 38% decolorization of RNB HE2R adsorbed on rice bran in 24h under solid state fermentation. However, the consortium of Pseudomonas sp. SUK1 and A. ochraceus NCIM-1146 (consortium-PA) showed 80% decolorization in 24h. The consortium-PA showed effective ADMI removal ratio of adsorbed dyes from textile industry wastewater (77%), mixture of textile dyes (82%) and chemical precipitate of textile dye effluent (CPTDE) (86%). Secretion of extracellular enzymes such as laccase, azoreductase, tyrosinase and NADH-DCIP reductase and their significant induction in the presence of adsorbed dye suggests their role in the decolorization of RNB HE2R. GCMS and HPLC analysis of product suggests the different fates of biodegradation of RNB HE2R when used Pseudomonas sp. SUK1, A. ochraceus NCIM-1146 and consortium PA.

Biochemical characteristics of a textile dye degrading extracellular laccase from a Bacillus sp. ADR.

Bacillus sp. ADR secretes an extracellular laccase in nutrient broth, and this enzyme was purified up to 56-fold using acetone precipitation and DEAE-cellulose anion exchange chromatography. The molecular weight of purified laccase was estimated to be 66 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified laccase oxidized 2,6-dimethoxy phenol, o-tolidine, hydroquinone, L-DOPA and guaiacol. The optimum pH for oxidation of o-tolidine, 2,6-dimethoxy phenol and guaiacol were 3.0, 4.0 and 5.0, respectively. The purified laccase contained 2.7 mol/mol of copper. The laccase was stable up to 40 °C and within the pH range of 7.0-9.0. Well-known inhibitors of multicopper oxidases such as, sodium azide, L-cysteine and dithiothreitol showed significant inhibition of laccase activity. The purified enzyme decolorized structurally different azo dyes with variable decolorization rates and efficiencies of 68-90%. This study is useful for understanding the precise use of Bacillus sp. ADR in the decolorization of textile dyes containing industrial wastewater.

Decolorization and detoxification of Congo red and textile industry effluent by an isolated bacterium Pseudomonas sp. SU-EBT.

The 16S rRNA sequence and biochemical characteristics revealed the isolated organism as Pseudomonas sp. SU-EBT. This strain showed 97 and 90% decolorization of a recalcitrant dye, Congo red (100 mg l(-1)) and textile industry effluent with 50% reduction in COD within 12 and 60 h, respectively. The optimum pH and temperature for the decolorization was 8.0 and 40 degrees C, respectively. Pseudomonas sp. SU-EBT was found to tolerate the dye concentration up to 1.0 g l(-1). Significant induction in the activity of intracellular laccase suggested its involvement in the decolorization of Congo red. The metabolites formed after decolorization of Congo red, such as p-dihydroxy biphenyl, 8-amino naphthol 3-sulfonic acid and 3-hydroperoxy 8-nitrosonaphthol were characterized using FTIR and GC-MS. Phytotoxicity study revealed nontoxic nature of the degradation metabolites to Sorghum bicolor, Vigna radiata, Lens culinaris and Oryza sativa plants as compared to Congo red and textile industry effluent. Pseudomonas sp. SU-EBT decolorized several individual textile dyes, dye mixtures and textile industry effluent, thus it is a useful strain for the development of effluent treatment methods in textile processing industries.

Influence of organic and inorganic compounds on oxidoreductive decolorization of sulfonated azo dye C.I. Reactive Orange 16.

An isolated bacterial strain is placed in the branch of the Bacillus genus on the basis of 16S rRNA sequence and biochemical characteristics. It decolorized an individual and mixture of dyes, including reactive, disperse and direct. Bacillus sp. ADR showed 88% decolorization of sulfonated azo dye C.I. Reactive Orange 16 (100 mg L(-1)) with 2.62 mg of dye decolorized g(-1) dry cells h(-1) as specific decolorization rate along with 50% reduction in COD under static condition. The optimum pH and temperature for the decolorization was 7-8 and 30-40 degrees C, respectively. It was found to tolerate the sulfonated azo dye concentration up to 1.0 g L(-1). Significant induction in the activity of an extracellular phenol oxidase and NADH-DCIP reductase enzymes during decolorization of C.I. Reactive Orange 16 suggest their involvement in the decolorization. The metal salt (CaCl2), stabilizers (3,4-dimethoxy benzyl alcohol and o-tolidine) and electron donors (sodium acetate, sodium formate, sodium succinate, sodium citrate and sodium pyruvate) enhanced the C.I. Reactive Orange 16 decolorization rate of Bacillus sp. ADR. The 6-nitroso naphthol and dihydroperoxy benzene were final products obtained after decolorization of C.I. Reactive Orange 16 as characterized using FTIR and GC-MS.

Biodegradation and detoxification of reactive textile dye by isolated Pseudomonas sp. SUK1.

An isolated bacterium from a textile disposal site, Pseudomonas sp. SUK1, has the ability to decolorize the reactive textile dyes and methyl orange. This bacterium showed the potential to decolorize the textile dye Reactive Blue 59 at a high concentration (5 g/L(-1)), which is frequently used in the textile industry of Solapur, India. Induction in the activities of lignin peroxidase, azoreductase, and dichlorophenol indophenol reductase was observed during the decolorization of Methyl Orange and Reactive Blue 59. Methyl Orange (as model azo dye) was used to understand the mechanism of biodegradation by Pseudomonas sp. SUK1. The final product was identified as 1,4-benzenediamine, N, N-dimethyl by gas chromatography-mass spectroscopy. Microbial and phytotoxicity studies revealed the nontoxic nature of the products of Reactive Blue 59.