Staphylococcus pseudintermedius is one of the major pathogens causing canine skin infection. In canine atopic dermatitis (AD), heterogeneous strains of S. pseudintermedius reside on the affected skin site. Because an increase in specific IgE to this bacterium has been reported, S. pseudintermedius is likely to exacerbate the severity of canine AD. In this study, the IgE reactivities to various S. pseudintermedius strains and the IgE-reactive molecules of S. pseudintermedius were investigated. First, examining the IgE reactivities to eight strains of S. pseudintermedius using 141 sera of AD dogs, strain variation of S. pseudintermedius showed 10-63% of the IgE reactivities. This is different from the expected result based on the concept of Staphylococcus aureus clonality in AD patients. Moreover, according to the western blot analysis, there were more than four proteins reactive to IgE. Subsequently, the analysis of the common IgE-reactive protein at ∼15 kDa confirmed that the DM13-domain-containing protein was reactive in AD dogs, which is not coincident with any S. aureus IgE-reactive molecules. Considering these, S. pseudintermedius is likely to exacerbate AD severity in dogs, slightly different from the case of S. aureus in human AD.

When Antoni van Leeuwenhoek began his work with microscopes in the late 17th century, western medicine was mostly based on the work of a Roman doctor called Galen (129-199 ad), theological interpretation, superstition, and folk remedies. During modern discussions of Van Leeuwenhoek's work, a common question from listeners is why it took so long for the link between Van Leeuwenhoek's discoveries and infectious disease to be accepted. Published literature, examples of which are discussed here, shows that many researchers, doctors, and others reported the link, even during Van Leeuwenhoek's lifetime. However, it was frequently not taken seriously by the most influential people. The scientific establishment included a faction of the Royal Society of London who called themselves the 'Mechanical Philosophers'. They ridiculed those reporting animalcule-linked infection, dismissing them as 'Contagionists'. The medical establishment also included many influential people with a lot to lose if they changed their established approaches, and many quack doctors. Most religious ministers were strongly orthodox, some even claiming that helping the sick angered God. A major problem, of course, was that technology and biological understanding also lagged far behind. Despite the fact that the use of vaccination was under active discussion in the Royal Society at the time of Van Leeuwenhoek's death and quarantine was in regular use, a possible microbial connection was apparently not considered. It was not until late in the 19th century, that Robert Koch (1843-1910) isolated Bacillus anthracis, proved that it caused anthrax, and was believed. This paper follows a lecture given during the online Microbe Forum in June 2021, and illustrates the difficulties of establishing the true link between Van Leeuwenhoek's animalcules and infectious disease in humans, animals, and plants.

Azotobacter vinelandii is a soil bacterium that produces alginates, a family of polymers of biotechnological interest. In A. vinelandii, alginate production is controlled by the two-component system GacS/GacA. GacS/GacA, in turn, regulates the Rsm post-transcriptional regulatory system establishing a cascade that regulates alginate biosynthesis by controlling the expression of the algD biosynthetic gene. In Pseudomonas aeruginosa, GacS/GacA is influenced by other histidine-kinases constituting a multicomponent signal transduction system. In this study, we explore the presence of GacS-related histidine-kinases in A. vinelandii and discover a novel histidine-kinase (Avin_34990, renamed HrgS). This histidin-kinase acts as a negative regulator of alginate synthesis by controlling the transcription of the sRNAs belonging to the Rsm post-transcriptional regulatory system, for which a functional GacS is required.

In the phylogenetic tree of RRM-type Rbps (RNA-binding proteins) in cyanobacteria, Rbp1 of Synechocystis 6803, with a single RRM (RNA recognition motif) region and a C-terminal glycine-rich region, and Rbp2, without the C-terminal region, both belong to the cluster I, whereas Rbp3 with a different type of C-terminal region is in the cluster II. Rbp1 is required for the cold adaptability of the cyanobacterium, and Rbp3 is for salt tolerance. Here, we report that the C-terminal region of Rbp1 is not required for the cold adaptability function but the C-terminal region of Rbp3 can direct the RRM of Rbp1 to the salt tolerance function. Bioinformatic and experimental analyses indicate that Rbps in cyanobacteria should be classified as two types. It is the first report for the distinct roles of C-terminal regions of Rbps in stress tolerance of cyanobacteria.

Nematode-trapping fungi are natural enemies of nematodes in nature. Arthrobotrys oligospora, a typical nematode-trapping fungus with a clear genetic background, can capture and infect nematodes by forming adhesive three-dimensional networks. Lectins, a class of glycoproteins containing glycosyl-specific recognition domains, play an important role in biological recognition. However, the fucose-specific lectins have rarely been studied regarding the process of preying on nematodes. In this study, we characterized the biological role of the fucose-specific lectin-encoding gene AOL_s00054g276 (g276) in A. oligospora. The gene g276 was first deleted based on homologous recombination, then the phenotype and nematocidal activity of the Δg276 mutant was evaluated. The results showed that the deletion of gene g276 delayed trap formation and weakened its nematocidal activity; however, mycelial growth, conidia production, conidial germination rates and adaption to environmental stresses were not affected. Our results suggest that the fucose-specific lectin-encoding gene g276 might be associated with the morphogenesis of this fungus, and its deletion resulted in a significantly low density of three-dimensional traps (P < 0.05) and a significantly low nematode-trapping efficiency (P < 0.001). These findings provide a basis for further elucidating the mechanism of A. oligospora preying on nematodes and lay a foundation for the development and utilization of fungal-derived lectins for nematode control in the future.

CTX-M-15 is a major extended-spectrum beta-lactamase disseminated throughout the globe. The roles of amino acids present in the active-site are widely studied though little is known about the role of the amino acids lying at the close proximity of the CTX-M-15 active-site. Here, by using site-directed mutagenesis we attempted to decipher the role of individual amino acids lying outside the active-site in imparting the beta-lactamase activity of CTX-M-15. Based on the earlier evidence, three amino acid residues namely, Glu169, Asp173 and Arg277 were substituted with alanine. The antibiotic susceptibility of E. coli cells harboring E169A and N173A substituted CTX-M-15 were enhanced by ∼ >32 fold for penicillins and ∼ 4-32 fold for cephalosporins, in comparison to CTX-M-15. However, cells carrying CTX-M-15_R277A did not show a significant difference in antibiotic susceptibility as compared to the wild-type. Further, the catalytic efficiency of the purified CTX-M-15_E169A and CTX-M-15_N173A were compromised when compared with the efficient beta-lactam hydrolysis of purified CTX-M-15. Moreover, the thermal stability of the mutated proteins CTX-M-15_E169A and CTX-M-15_N173A were reduced as compared to the wild type CTX-M-15. Therefore, we conclude that E169 and N173 are crucial non-active-site amino acids that are able to govern the CTX-M-15 activity.

The aim of this investigation was to discover the promoters that drive expression of the sig genes encoding sigma factors of RNA polymerase in Rhodococcus erythropolis CCM2595 and classify these promoters according to the sigma factors which control their activity. To analyze the regulation of major sigma factors, which control large regulons that also contain genes expressed under exponential growth and non-stressed conditions, we used the R. erythropolis CCM2595 culture, which grew rapidly in minimal medium. The transcriptional start sites (TSSs) of the genes sigA, sigB, sigD, sigE, sigG, sigH, sigJ, and sigK were detected by primary 5'-end-specific RNA sequencing. The promoters localized upstream of the detected TSSs were defined by their -35 and -10 elements, which were identical or closely similar to these sequences in the related species Corynebacterium glutamicum and Mycobacterium tuberculosis. Regulation of the promoter activities by different sigma factors was demonstrated by two independent techniques (in vivo and in vitro). All analyzed sig genes encoding the sigma factors with extracytoplasmic function (ECF) were found to be also driven from additional housekeeping promoters. Based on the classification of the sig gene promoters, a model of the basic sigma transcriptional regulatory network in R. erythropolis was designed.

The secondary metabolites produced by microorganisms are a source of novel compounds with antitumor activities. In this study, we isolated biologically active secondary metabolites produced by microorganisms in the intestinal tract of Periplaneta americana. Based on the 16S rRNA gene sequencing, Gordonia hongkongensis WA12-1-1 was identified as the main microorganisms in the intestinal tract of P. americana. The obtained sequence was deposited in the National Center for Biotechnology Information (NCBI) database under the accession number MZ348554. The isolated secondary metabolites were separated and purified by thin layer chromatography, silica gel column chromatography, Sephadex column chromatography, open octadecyl silane column chromatography, high-performance liquid chromatography (HPLC), and semipreparative HPLC. Next, the structure of individual compounds was determined by ultraviolet spectroscopy, nuclear magnetic resonance, and mass spectrometry. A total of 20 compounds were isolated from the secondary metabolites produced by G. hongkongensis WA12-1-1. A total of 12 compounds were obtained from the crude ethyl acetate extract of the culture supernatant and eight from the cellular fraction. Compound 1 was identified as a triterpenoid named gordonterpene and showed cytotoxicity against A549 and HepG2 cell lines. These findings form a basis for further studies on the bioactivity of gordonterpene to tumor cells.

Lactic acid bacteria (LAB) have long been used to extend the shelf life and improve the taste and texture of fermented milk. In this study, we investigated the texturing potential of LAB in plant-based fermentation by high-throughput screening of 1232 Lactococcus lactis strains for texture in milk and liquid soybean matrices. We found that most strains with texturing abilities in fermented milk were also capable of enhancing the texture in fermented soybean, despite the large differences in composition of the two matrices. Exocellular polysaccharide production is believed to contribute positively to fermented milk and plant-base texture. It appeared as if it was the properties of the polysaccharides rather than their protein interaction partners that were responsible for the enhanced texture in both matrices. We mined whole genome sequences of texturing strains for polysaccharide biosynthesis (eps) gene clusters. The comparative genomics approach revealed 10 texturing strains with novel eps gene clusters. Currently, the relationship between the novel genes and their functionality in milk and plant matrices is unknown.

Erratum in FEMS Microbiol Lett. 2022 Apr 9;369(1):

The carbapenem-resistant Escherichia coli has aroused increasing attention worldwide, especially in terms of imipenem (IMP) resistance. The molecular mechanism of IMP resistance remains unclear. This study aimed to explore the resistance mechanisms of IMP in E. coli. Susceptible Sx181-0-1 strain was induced into resistance strains by adaptive laboratory evolution. The drug resistance spectrum was measured using the disk diffusion and microbroth dilution methods. Whole-genome sequencing and resequencing were used to analyze the nonsynonymous single-nucleotide polymorphisms (nsSNPs) between the primary susceptible strain and resistant strains. The expression levels of these genes with nsSNPs were identified by real-time quantitative PCR (RT-qPCR). Resistance phenotype appeared in the induced 15th generation (induction time = 183 h). Sx181-32 and Sx181-256, which had the minimum inhibitory concentrations of IMP of 8 and 64 µg ml-1, were isolated during continuous subculture exposed to increasing concentrations of IMP, respectively. A total of 19 nsSNPs were observed both in Sx181-32 and Sx181-256, distributed in rpsU, sdaC, zwf, ttuC, araJ, dacC, mrdA, secF, dacD, lpxD, mrcB, ftsI, envZ, and two unknown function genes (orf01892 and orf01933). Among these 15 genes, five genes (dacC, mrdA, lpxD, mrcB, and ftsI) were mainly involved in cell wall synthesis. The mrdA (V338A, L378P, and M574I) and mrcB (P784L, A736V, and T708A) had three amino acid substitutions, respectively. The expression levels of rpsU, ttuC, and orf01933 were elevated in both Sx181-32 and Sx181-256 compared to Sx181-0-1. The expression levels of these genes were elevated in Sx181-256, except for araJ. Bacteria developed resistance to antimicrobials by regulating various biological processes, among which the most involved is the cell wall synthesis (dacC, mrdA, lpxD, mrcB, and ftsI). The combination mutations of mrdA, envZ, and ftsI genes may increase the resistance to IMP. Our study could improve the understanding of the molecular mechanism of IMP resistance in E. coli.

The table grape is a non-climateric fruit that is very susceptible to fungal contamination, in addition to suffering an accelerated loss of quality during storage. The in vitro and in grape antifungal and antiocratoxigenic effects of the essential oils from Alpinia speciosa and Cymbopogon flexuosus against Aspergillus carbonarius and Aspergillus niger were studied. The oils were encapsulated in poly(lactic acid) (PLA) nanofibers as a potential active packaging to be applied to control the degradation of grapes stored during the post-harvest period. Fungal proliferation and ochratoxin A synthesis in A. carbonarius and A. niger decreased in the presence of the active packaging. However, the nanofiber containing the essential oil from C. flexuosus was more efficient in providing a fungicidal effect against A. carbonarius (10% and 20%) and A. niger (20%). In addition, weight loss and color changes were controlled and the parameters of acidity, °Brix, softening and the texture of the grape were maintained. A very small mass loss of the essential oils encapsulated in nanofibers was observed by thermogravimetric analysis, showing that the nanofiber was efficient in enabling the controlled release. The quality and safety of table grapes were maintained for longer periods of storage in the presence of active packaging, so the incorporation of these oils in nanofibers can be a promising way to increase the shelf life of grapes.

The measurement of fungal cell growth in submerged culture systems containing insoluble compounds is essential yet difficult due to the interferences from the insoluble compounds like biopolymers. Here, we developed a fluorescent strategy based on chitosan-modified fluorescein isothiocyanate (GC-FITC) to monitor the cell growth of lignocellulosic fungi cultivated on biopolymers. GC-FITC could stain only lignocellulosic fungi (Tricoderma reesei, Penicillium oxalicum, Aspergillus nidulans, and Neurospora crassa), but not biopolymers (cellulose, xylan, pectin, or lignin), excluding the interferences from these insoluble biopolymer. Moreover, a linear relationship was observed between the fluorescence intensity of GC-FITC absorbed by lignocellulosic fungi and the biomass of lignocellulosic fungi. Therefore, GC-FITC was leveraged to monitor the cell growth of lignocellulosic fungi when using biopolymers like cellulose as the carbon sources, which is faster, more convenient, time-saving, and cost-effective than the existing methods using protein/DNA content measurement. GC-FITC offers a powerful tool to detect fungal growth in culture systems with insoluble materials.

Engineering transporter for improved efflux efficiency provides a powerful strategy to alleviate product-associated cytotoxicity and promote microbial production of desired compounds. However, the scarcity of efficient transporters limits its application in the construction of microbial cell factories. Here, we sought to improve the transport performance of the EamB transporter, a L-cysteine exporter from Escherichia coli. A total of four EamB variants (A31V, I83M, G156A, and N157M) were firstly obtained by random mutagenesis and screening, and two other improved mutant (G156S and N157S) were also identified by site-specific saturation mutagenesis. The transport assays revealed that the G156S and N157S mutants had increased L-cysteine export capacity relative to the native EamB transporter. A combinatorial mutagenesis approach was used to generate the best mutant G156S/N157S, which conferred cells optimal resistance to L-cysteine and highest yields of L-cysteine in shake flask fermentation. Taken together, our results offer several EamB mutants with improved efflux properties, highlighting the potential of these exporters in L-cysteine fermentative production.

This work compared response surface methodology (RSM) and genetic algorithm (GA) analysis to optimize the lactic acid content by Lactobacillus rhamnosus PTCC 1637 and Lactobacillus acidophilus PTCC 1643 in a medium based on date syrup. Three parameters including concentrations of sucrose [10 and 20% (w/w)] and yeast extract [1, 2 and 3% (w/w)] along with different amplitudes of ultrasound (30 kHz, 25 and 50%) were investigated in terms of their impacts on both viable cell counts and lactic acid production. Regarding RSM and GA, optimized samples were selected by achieving high lactic acid concentration. The results indicated that an increase in the amounts of sucrose and yeast extract led to increasing the cell growth and lactic acid production. Application of ultrasound at 25% amplitude significantly (P < 0.05) stimulated the fermentation process. However, increasing the amplitude to 50% significantly (P < 0.05) decreased the lactic acid production compared with the control samples. The best treatment was observed at 20% sucrose, 3% yeast extract and 25% ultrasound amplitude. The present results indicate that the best productivity of lactic acid can be achieved at appropriate fermentation conditions, including a suitable amplitude of ultrasound and supplementation of date syrup.

There is increasing interest in gluten-degrading enzymes for use during food and drink processing. The industrially available enzymes usually work best at low to ambient temperatures. However, food manufacturing is often conducted at higher temperatures. Therefore, thermostable gluten-degrading enzymes are of great interest. We have identified a new thermostable gluten-degrading proline-specific prolyl endoprotease from the archaea Thermococcus kodakarensis. We then cloned and expressed it in Escherichia coli. The prolyl endoprotease was found to have a size of 70.1 kDa. The synthetic dipeptide Z-Gly-Pro-p-nitroanilide was used to characterize the prolyl endoprotease and it had maximum activity at pH 7 and 77°C. The Vmax, Km and kcat values of the purified prolyl endoprotease were calculated to be 3.14 mM/s, 1.10 mM and 54 s-1, respectively. When the immunogenic gluten peptides PQPQLPYPQPQLPY (α-gliadin) and SQQQFPQPQQPFPQQP (γ-hordein) were used as substrates, the prolyl endoprotease was able to degrade these. Furthermore, gluten in wort was reduced when the prolyl endoprotease was used during mashing of barley malt. The discoveries open up new food processing possibilities and further the understanding of proline-specific protease diversity.

This study examined the water-to-air transfer and viability of the fecal indicator bacteria, enterococci, and compared capture performance of an impactor and aerosol filter. Results show that concentration of viable enterococci collected by the impactor (70.1 colony-forming units [CFU]/L) was lower than that using the filter (171.2 CFU/L) at 95% significance. Between the impactor and filter, coefficients of variation equaled 13% and 14%, respectively. Hence, for the collection of aerosolized enterococci in a controlled environment, use of the aerosol filter yielded significantly higher recovery relative to impaction, though equally variable data were collected by both methods. This work confirms that viable enterococci transfer across a simulated air-sea interface and that aerosol filters perform well in capturing viable bacteria. Results from this study are relevant to studies that measure environmentally generated aerosols such as those that occur via wave breaking from sewage-contaminated waters.

Thiobencarb is a herbicide globally used in the agricultural sector, and its extensive application leads to severe environmental pollution. In this study, the thiobencarb supplementation caused a significant shift in the bacterial community in the sediment slurry. An analysis of the degradation metabolites of microorganisms from the sediment indicated that deschlorothiobencarb, S-4-chlorobenzyl ethylthiocarbamate, 4-chlorobenzyl mercaptan, 4-chlorobenzyl alcohol, 4-chlorobenzoic acid and chlorobenzene were the main intermediates. The degradation rates were significantly enhanced using a horizontal-flow anaerobic reactor with immobilized cells in polyurethane foam. The degradation rates at 2.6, 12.9 and 25.6 mg L-1 concentrations by suspended microorganisms from the sediment in the mineral medium supplemented with glucose were 0.085 ± 0.000, 0.383 ± 0.010 and 0.500 ± 0.045 mg day-1, respectively. The corresponding data for degradation in the reactor were 2.54 ± 0.03, 11.69 ± 0.72 and 18.58 ± 1.83 mg day-1 at the sixth operation period. Moreover, COD removal efficiencies were >90% achieved in the reactor. The proposed method facilitates degradation using a horizontal-flow anaerobic immobilized biomass bioreactor. Moreover, this study reveals the degradation of metabolites of thiobencarb under anaerobic conditions.

Multidrug-resistant (MDR) Escherichia coli in food animals such as chickens is an emerging public health concern in Zambia. Additionally, the country's high demand for poultry products necessitates further investigation into the link between poultry and human MDR E. coli. Twenty cefotaxime-resistant E. coli isolates collected from poultry in Lusaka, Zambia, were screened for multidrug resistance and sequenced on MiSeq and MinION platforms. Genomes were assembled de novo and compared with 36 previously reported cefotaxime-resistant E. coli isolates from inpatients at the University Teaching Hospital, Lusaka. All (20/20, 100%) poultry isolates exhibited resistance to ampicillin, chloramphenicol and doxycycline. Phylogenetic analysis and hierarchical clustering showed a high degree of genetic relatedness between E. coli O17:H18-ST69 from poultry and humans. The E. coli O17:H18-ST69 clone accounted for 4/20 (20%) poultry- and 9/36 (25%) human-associated isolates that shared two plasmids harboring 14 antimicrobial resistance (AMR) genes. However, comparison analysis showed that the isolates also had other AMR plasmids distinct for each niche. Our results suggested clonal transmission of MDR E. coli between poultry and humans, with the potential acquisition of niche-specific AMR plasmids. Thus, the control of MDR E. coli requires a One Health approach involving both human and animal health sectors.

Cells can be affected by several causes of osmotic stress, by which they are forced to adapt. An essential aspect of adaptation is ion regulation, and many insights into such complex processes can be obtained through measurement of the plasma membrane potential (PMP) of cells during stress. We recently established genetically encoded voltage indicator proteins that could be utilized to report the yeast PMP change in real time. In this work, we employed this method to monitor the early change in the PMP of yeast Saccharomyces cerevisiae with intact cell wall, immediately following hyperosmotic up-shock due to various stress agents. The results pointed to differential effects of NaCl, sorbitol and polyethylene glycol (PEG) 6000. Yeast PMP was more responsive toward PEG 6000 than NaCl and sorbitol at comparable osmotic pressure, and PEG 6000 stimulated the largest response magnitude, followed by sorbitol and NaCl, respectively. After prolonged treatment, PEG 6000 also instigated distinct cell morphology from NaCl and sorbitol. Accordingly, this study presents new evidence supporting multiple pathways underlying yeast adaptation to varying hyperosmotic conditions, enabled through the optical physiology approach. Our findings promote better understanding of the yeast cellular response to hyperosmotic stress, with tenable relevance to the physiologically related plant cells.