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

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

Characterization of an Alkali- and Halide-Resistant Laccase Expressed in E. coli: CotA from <i>Bacillus clausii</i>

The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain BL21(DE3) and characterized fully in parallel for strict benchmarking. We report activity on ABTS, SGZ, DMP, caffeic acid, promazine, phenyl hydrazine, tannic acid, and bilirubin at variable pH. Whereas ABTS, promazine, and phenyl hydrazine activities vs. pH were similar, the activity of B. clausii cotA was shifted upwards by ~0.5-2 pH units for the simple phenolic substrates DMP, SGZ, and caffeic acid. This shift is not due to substrate affinity (K(M)) but to pH dependence of catalytic turnover: The k(cat) of B. clausii cotA was 1 s⁻¹ at pH 6 and 5 s⁻¹ at pH 8 in contrast to 6 s⁻¹ at pH 6 and 2 s⁻¹ at pH 8 for of B. subtilis cotA. Overall, k(cat)/K(M) was 10-fold higher for B. subtilis cotA at pH(opt). While both proteins were heat activated, activation increased with pH and was larger in cotA from B. clausii. NaCl inhibited activity at acidic pH, but not up to 500-700 mM NaCl in alkaline pH, a further advantage of the alkali regime in laccase applications. The B. clausii cotA had ~20 minutes half-life at 80°C, less than the ~50 minutes at 80°C for cotA from B. subtilis. While cotA from B. subtilis had optimal stability at pH~8, the cotA from B. clausii displayed higher combined salt- and alkali-resistance. This resistance is possibly caused by two substitutions (S427Q and V110E) that could repel anions to reduce anion-copper interactions at the expense of catalytic proficiency, a trade-off of potential relevance to laccase optimization

Synergistic degradation of diazo dye Direct Red 5B by Portulaca grandiflora and Pseudomonas putida

Plants and bacterial consortium of Portulaca grandiflora and Pseudomonas putida showed complete decolorization of a sulfonated diazo dye Direct Red 5B within 72 h, while in vitro cultures of P. grandiflora and P. putida independently showed 92 and 81 % decolorization within 96 h, respectively. A significant induction in the activities of lignin peroxidase, tyrosinase, 2,6-dichlorophenol indophenol reductase and riboflavin reductase was observed in the roots of P. grandiflora during dye decolorization; whereas, the activities of laccase, veratryl alcohol oxidase and 2,6-dichlorophenol indophenol reductase were induced in the cells of P. putida. Plant and bacterial enzymes in the consortium gave an enhanced decolorization of Direct Red 5B synergistically. The metabolites formed after dye degradation analyzed by UV-Vis spectroscopy, Fourier transformed infrared spectroscopy and high performance liquid chromatography confirmed the biotransformation of Direct Red 5B. Differential fate of metabolism of Direct Red 5B by P. grandiflora, P. putida and their consortium were proposed with the help of gas chromatography-mass spectroscopy analysis. P. grandiflora metabolized the dye to give 1-(4-diazenylphenyl)-2-phenyldiazene, 7-(benzylamino) naphthalene-2-sulfonic acid, 7-aminonaphthalene-2-sulfonic acid and methylbenzene. P. putida gave 4-hydroxybenzenesulfonic acid and 4-hydroxynaphthalene-2-sulfonic acid and benzamide. Consortium showed the formation of benzenesulfonic acid, 4-diazenylphenol, 6-aminonaphthalen-1-ol, methylbenzene and naphthalen-1-ol. Consortium achieved an enhanced and efficient degradation of Direct Red 5B. Phytotoxicity study revealed the nontoxic nature of metabolites formed after parent dye degradation. Use of such combinatorial systems of plant and bacteria could prove to be an effective and efficient strategy for the removal of textile dyes from soil and waterways

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

International audienceBackground: 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

Mathematical modeling, simulation and validation of the dynamic yarn path in a superconducting magnet bearing (SMB) ring spinning system

The new concept of a superconducting magnetic bearing (SMB) system can be implemented as a twisting element instead of the existing one in a ring spinning machine, thus overcoming one of its main frictional limitations. In the SMB, a permanent magnet (PM) ring rotates freely above the superconducting ring due to the levitation forces. The revolution of the PM ring imparts twists similarly to the traveler in the existing twisting system. In this paper, the forces acting on the dynamic yarn path resulting from this new technology are investigated and described with a mathematical model. The equation of yarn movement between the delivery rollers and the PM ring is integrated with the Runge-Kutta method using MATLAB. Thus, the developed model can estimate the yarn tension and balloon form according to different spindle speeds considering the dynamic behavior of the permanent magnet of the SMB system. To validate the model, the important relevant process parameters, such as the yarn tension, are measured at different regions of the yarn path, and the balloon forms are recorded during spinning with the SMB system using a high speed camera

Matematyczne modelowanie dynamicznej trajektorii przędzy z uwzględnieniem pierścienia sterującego balonem i elastyczności przędzy w procesie przędzeniaobrączkowego opartego na nadprzewodzącym elemencie skręcającym łożyska

The productivity of the conventional ring spinning process is currently limited by the frictional heat that occurs in the ring/traveler twisting system. In the framework of a fundamental research project from the German Research Foundation (DFG), the levitation principle of superconducting magnetic bearing (SMB) was implemented as a twisting element in order to eliminate the frictional problem and thus aim, at least, to double the productivity. A mathematical model of the dynamic yarn path has already been presented considering the friction free SMB system up to an angular spindle speed of 25.000 r.p.m. In this paper, the existing theoretical model, which was developed up to 25.000 r.p.m, was further modified considering the balloon control ring and yarn elasticity at a higher angular spindle speed, such as 50.000 r.p.m. The model was solved numerically using the Runge-Kutta method. With this model, it is possible to estimate the yarn tension distribution and balloon form considering the above-mentioned parameters. The model established was further validated by comparing the yarn tension and balloon forms predicted with measured ones up to an angular spindle speed of 15.000 r.p.m in a ring spinning tester based on superconducting magnetic bearing.Wydajność konwencjonalnego procesu przędzenia obrączkowego jest ograniczana przez ciepło tarcia występujące w układzie skręcania pierścień /wahadło. W ramach projektu badawczego z Niemieckiej Fundacji Badawczej (DFG) wprowadzono zasadę lewitacji nadprzewodzącego łożyska magnetycznego (SMB) jako elementu skręcającego w celu wyeliminowania problemu tarcia. Celem pracy było przynajmniej podwojenie wydajność. Przedstawiono model matematyczny dynamicznej trajektorii przędzy z uwzględnieniem systemu SMB bez tarcia, przy prędkości obrotowej wrzeciona 25.000 obr./min. Nastęnie istniejący del teoretyczny został dodatkowo zmodyfikowany, a przy modyfikacji wzięto pod uwagę pierścień kontrolny balonu i elastyczność przędzy przy wyższej prędkości obrotowej wrzeciona tj. 50.000 obr./min. Model został rozwiązany numerycznie za pomocą metody RUNGE-KUTTA. W tym modelu możliwe jest oszacowanie rozkładu naprężenia przędzy i kształtu balonu z uwzględnieniem wyżej wymienionych parametrów. Ustalony model został dodatkowo zweryfikowany poprzez porównanie naprężenia przędzy i przewidywanych form balonu. W tym celu użyto testera przędzenia obrączkowego opartego na nadprzewodzącym łożysku magnetycznym