Bacillus subtilis is described as a promising production strain for lipopeptides. In the case of B. subtilis strains JABs24 and DSM10T, surfactin, and plipastatin are produced. Lipopeptide formation is controlled, among others, by the DegU response regulator. The activating phospho-transfer by the DegS sensor kinase is stimulated by the pleiotropic regulator DegQ, resulting in enhanced DegU activation. In B. subtilis 168, a point mutation in the degQ promoter region leads to a reduction in gene expression. Corresponding reporter strains showed a 14-fold reduced expression. This effect on degQ expression and the associated impact on lipopeptide formation was examined for B. subtilis JABs24, a lipopeptide-producing derivative of strain 168, and B. subtilis wild-type strain DSM10T, which has a native degQ expression. Based on the stimulatory effects of the DegU regulator on secretory protease formation, the impact of degQ expression on extracellular protease activity was additionally investigated. To follow the impact of degQ, a deletion mutant was constructed for DSM10T, while a natively expressed degQ version was integrated into strain JABs24. This allowed strain-specific quantification of the stimulatory effect of degQ expression on plipastatin and the negative effect on surfactin production in strains JABs24 and DSM10T. While an unaffected degQ expression reduced surfactin production in JABs24 by about 25%, a 6-fold increase in plipastatin was observed. In contrast, degQ deletion in DSM10T increased surfactin titer by 3-fold but decreased plipastatin production by 5-fold. In addition, although significant differences in extracellular protease activity were detected, no decrease in plipastatin and surfactin produced during cultivation was observed.

Bromus tectorum (cheatgrass) is an invasive annual grass that has colonized large portions of the Intermountain Western United States. Cheatgrass stand failures have been observed throughout the invaded region, the cause of which may be related to the presence of several species of pathogenic fungi in the soil or surface litter. In this metagenomic study, we compared the fungal communities between sites that have and have not experienced stand failure. Samples were taken from the soil and surface litter near Winnemucca, Nevada and in Skull Valley, Utah. Our results show distinct fungal communities associated with stand failure based on both geography and sample type. In both the Winnemucca and Skull Valley surface litter, there was an elevated abundance of the endophyte Ramimonilia apicalis in samples that had experienced a stand failure. Winnemucca surface litter stand failure samples had increased abundance of a potential pathogen in the genus Comoclathris. Skull Valley surface litter stand failure samples had increased abundance of the known cheatgrass pathogen Clarireedia capillus-albis while the soils had increased abundance of potential pathogens in the genera Olpidium and Monosporascus.

The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S rRNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony-forming units (cfu) and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. The different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were re-sequenced in triplicates (n=120). The most reproducible results for alpha- and beta-diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pre-treatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pre-treatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols.

One hundred fecal samples from hooded vultures in the Gambia (Banjul area) were investigated for the presence of bacteria with extended-spectrum cephalosporin- (ESBL/AmpC), carbapenemases, and colistin resistance. No Enterobacteriales carrying carbapenemases or resistance against colistin were detected. Fifty-four ESBL-producing Escherichia coli and five ESBL-producing Klebsiella pneumoniae isolates were identified in 52 of the samples, of which 52 E. coli and 4 K. pneumoniae yielded passed sequencing results. Fifty of the E. coli had ESBL phenotype and genotype harboring blaCTX-M genes, of which 88.5% (n=46) were the blaCTX-M-15 gene, commonly found on the African continent. Furthermore, the genetic context around blaCTX-M-15 was similar between isolates, being co-localized with ISKpn19. In contrast, cgMLST analysis of the E. coli harboring ESBL genes revealed a genetic distribution over a large fraction of the currently known existing E. coli populations in the Gambia. Hooded vultures in the Gambia thus have a high ESBL E. coli-prevalence (>50%) with low diversity regarding key resistance genes. Furthermore, given the urban presence and frequent interactions between hooded vultures and humans, data from this study implies hooded vultures as potential vectors contributing to the further dissemination of antibiotic-resistance genes.

Cyanobacterial carbonate precipitation induced by cells and extracellular polymeric substances (EPS) enhances mortar durability. The percentage of cell/EPS attachment regulates the effectiveness of the mortar restoration. This study investigates the cell coverage on mortar and microbially induced carbonate precipitation. Statistical analysis of results from scanning electron and fluorescence microscopy shows that the cell coverage was higher in the presence of UV-killed cells than living cells. Cells preferably attached to cement paste than sand grains, with a difference of one order of magnitude. The energy-dispersive X-ray spectroscopy analyses and Raman mapping suggest cyanobacteria used atmospheric CO2 to precipitate carbonates.

Ocean pollution is a worldwide environmental challenge that could be partially tackled through microbial applications. To shed light on the diversity and applications of the bacterial communities that inhabit the sediments trapped in artificial containers, we analyzed residues (Polyethylene terephthalate (PET) bottles and aluminum cans) collected from the Mediterranean Sea by scanning electron microscopy and Next Generation Sequencing. Moreover, we set a collection of culturable bacteria from the plastisphere that were screened for their ability to use PET as a carbon source. Our results reveal that Proteobacteria are the predominant phylum in all the samples and that Rhodobacteraceae, Woeseia, Actinomarinales, or Vibrio are also abundant in these residues. Moreover, we identified marine isolates with enhanced growth in the presence of PET: Aquimarina intermedia, Citricoccus spp., and Micrococcus spp. Our results suggest that the marine environment is a source of biotechnologically promising bacterial isolates that may use PET or PET additives as carbon sources.

Necrotic enteritis (NE), caused by Clostridium perfringens, is an intestinal disease with devastating economic losses to the poultry industry. NE is a complex disease and predisposing factors that compromise gut integrity are required to facilitate C. perfringens proliferation and toxin production. NE is also characterized by drastic shifts in gut microbiota; C. perfringens is negatively correlated with Lactobacilli. Vaccines are only partially effective against NE and antibiotics suffer from the concern of resistance development. These strategies address only some aspects of NE pathogenesis. Thus, there is an urgent need for alternative strategies that address multiple aspects of NE biology. Here, we developed Limosilactobacillus (Lactobacillus) reuteri vectors for in situ delivery of nanobodies against NetB and α toxin, two key toxins associated with NE pathophysiology. We generated nanobodies and showed that these nanobodies neutralize NetB and α toxin. We selected L. reuteri vector strains with intrinsic benefits and demonstrated that these strains inhibit C. perfringens and secrete over 130 metabolites, some of which play a key role in maintaining gut health. Recombinant L. reuteri strains efficiently secreted nanobodies and these nanobodies neutralized NetB. The recombinant strains were genetically and phenotypically stable over 480 generations and showed persistent colonization in chickens. A two-dose in ovo and drinking water administration of recombinant L. reuteri strains protected chickens from NE-associated mortality. These results provide proof-of-concept data for using L. reuteri as a live vector for delivery of nanobodies with broad applicability to other targets and highlight the potential synergistic effects of vector strains and nanobodies for addressing complex diseases such as NE.

This study compared the antimicrobial resistance (AMR) among commensal Escherichia coli in the fecal microbiota of young calves raised on organic and on conventional dairy farms in Switzerland. Further, fecal carriage of extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae was assessed for calves from both farming systems. Where possible, data on antimicrobial usage (AMU) were obtained. Antimicrobial susceptibility testing was performed on a total of 71 isolates using the disk diffusion method. ESBL producers were characterized by PCR-based multilocus sequence typing and sequencing of the blaESBL genes. Organically raised calves were significantly more likely to harbor E. coli that showed antimicrobial resistance to ampicillin (OR 2.78, 95%CI 1.02–7.61, p=0.046), streptomycin (OR 3.22, 95%CI 1.17–8.92, p=0.046), kanamycin (OR 11.3, 95%CI 2.94–43.50, p<0.001), and tetracycline (OR 3.25, 95%, 95%CI 1.13–9.31, p=0.028). Calves with reported AMU were significantly more likely to harbor E. coli with resistance to ampicillin (OR 3.91, 95%CI 1.03–14.85, p=0.045), streptomycin (OR 4.35, 95%CI 1.13–16.7, p=0.045), and kanamycin (OR 8.69 95%CI 2.01–37.7, p=0.004). ESBL-producing Enterobacteriaceae (18 E. coli and 3 Citrobacter braakii) were detected exclusively among samples from conventionally farmed calves (OR infinity (∞), 95%CI 2.3–∞, p< 0.0013). The observations from this study suggest that AMR is highly prevalent among commensal E. coli in young dairy calves, irrespective of the farm management system, with proportions of certain resistance phenotypes higher among organic calves. By contrast, the occurrence of ESBL producers among young dairy calves may be linked to factors associated with conventional farming.

Naphthenic acids (NAs) are carboxylic acids with the formula (CnH2n+ZO2) and are the toxic, persistent constituents of oil sands process-affected waters (OSPW), produced during oil sands extraction. Currently, the proteins and mechanisms involved in NA biodegradation are unknown. Using LC-MS/MS shotgun proteomics, we identified proteins overexpressed during the growth of Pseudomonas fluorescens Pf5 on a model NA (4-n-butylphenyl)-4-butanoic acid (n-BPBA) and commercial NA mixture (Acros). By day 11, >95% of n-BPBA was degraded. With Acros, a 17% reduction in intensity occurred with 10-18 carbon compounds of the Z family -2 to -14 (major NA species in this mixture). A total of 554 proteins (n-BPBA) and 631 proteins (Acros) were overexpressed during growth on NAs; including several transporters (e.g. ABC transporters), suggesting a cellular protective response from NA toxicity. Several proteins associated with fatty acid, lipid and amino acid metabolism were also overexpressed; including acyl-CoA dehydrogenase and acyl-CoA thioesterase II, which catalyze part of the fatty acid beta-oxidation pathway. Indeed, multiple enzymes involved in the fatty acid oxidation pathway were upregulated. Given the presumed structural similarity between alkyl-carboxylic acid side chains and fatty acids, we postulate that P. fluorescens Pf-5 was using existing fatty acid catabolic pathways (among others) during NA degradation.

Although Streptococcus agalactiae periprosthetic joint infection (PJI) is not as prevalent as staphylococcal PJI, invasive S. agalactiae infection has recently increased in incidence. Here, RNA-Seq was used to perform transcriptomic analysis of S. agalactiae PJI using fluid derived from sonication of explanted arthroplasties of subjects with S. agalactiae PJI, with results compared to those of S. agalactiae strain NEM316 grown in vitro. 227 genes with outlier expression were found (164 up-regulated and 63 down-regulated) between PJI sonicate fluid and in vitro conditions. Functional enrichment analysis showed genes involved in mobilome and inorganic ion transport and metabolism to be most enriched. Genes involved in nickel, copper, and zinc transport, were upregulated. Among known virulence factors, cyl operon genes, encoding beta-hemolysin/cytolysin, were consistently highly expressed in PJI versus in vitro. The data presented provide insight into S. agalactiae PJI pathogenesis and may be a useful resource for the identification of novel PJI therapeutics or vaccines against invasive S. agalactiae infections.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory infectious disease responsible for global economic losses in the pig industry. From a monitoring perspective as well as due to the different courses of disease associated with the various serovars, it is essential to distinguish them in different herds or countries. In this study, we developed a novel high resolution melting (HRM) assay based on reference strains for each of the 19 known serovars and additional 15 clinical A. pleuropneumoniae isolates. The novel HRM comprises the species-specific APP-HRM1 and two serovar-specific HRM assays (APP-HRM2 and APP-HRM3). APP-HRM1 allowed PCR amplification of apxIV resulting in an A. pleuropneumoniae specific melting curve, while nadV specific primers differentiated biovar 2 from biovar 1 isolates. Using APP-HRM2 and APP-HRM3, 13 A. pleuropneumoniae serovars can be determined by inspecting the assigned melting temperature. In contrast, serovar 3 and 14, serovar 9 and 11, and serovar 5 and 15 have partly overlapping melting temperatures and thus represent a challenge to accurately distinguish them. Consequently, to unambiguously ensure the correct assignment of the serovar, it is recommended to perform the serotyping HRM assay using a positive control for each serovar. This rapid and user-friendly assay showed high sensitivity with 1.25 fg - 125 pg of input DNA and a specificity of 100% to identify A. pleuropneumoniae. Characteristic melting patterns of amplicons might allow detecting new serovars. The novel HRM assay has the potential to be implemented in diagnostic laboratories for better surveillance of this pathogen.

In naturally competent bacteria, DNA transformation through horizontal gene transfer is an evolutionary mechanism to receive extracellular DNA. Bacteria need to maintain a state of competence to accept foreign DNA and this is an energy-driven phenomenon that is tightly controlled. In Streptococcus, competence development is a complex process that is not fully understood. In this study, we used Streptococcus mutans, an oral bacterium, to determine how cell density affects competence development. We found that in S. mutans the transformation efficiency is maximum when the transforming DNA was added at low cell density and incubated for 2.5 h before selecting for transformants. We also found that S. mutans cells remain competent until the mid-logarithmic phase, after which the competence decreases drastically. Surprisingly, we observed that individual components of Clp proteolytic complexes differentially regulate competence. If the transformation is carried out at the early growth phase, both ClpP protease and ClpX ATPase are needed for competence. In contrast, we found that both ClpC and ClpE negatively affect competence. We also found that if the transformation is carried out at the mid-logarithmic growth phase ClpX is still required for competence but ClpP negatively affects competence. While the exact reason for this differential effect of ClpP and ClpX on transformation is currently unknown, we found that both ClpC and ClpE have a negative effect on transformation, which was not reported before.

Microbially influenced corrosion (MIC) may contribute significantly to overall corrosion risks, especially in the gas and petroleum industries. In this study, we isolated four Prolixibacter strains, which belong to the phylum Bacteroidetes, and examined their nitrate-respiration- and Fe0-corroding activities, together with two previously isolated Prolixibacter strains. Four of the six Prolixibacter strains reduced nitrate under anaerobic conditions, while the other two strains did not. The anaerobic growth of the four nitrate-reducing strains was enhanced by nitrate, which was not observed in the two nitrate-non-reducing strains. When the nitrate-reducing strains were grown anaerobically in the presence of Fe0 or carbon steel, the corrosion of the materials was enhanced by more than 20-fold compared to that in aseptic controls. This enhancement was not observed in cultures of the nitrate-non-reducing strains. The oxidation of Fe0 in the anaerobic cultures of nitrate-reducing strains occurred concomitantly with the reduction of nitrite. Since nitrite chemically oxidized Fe0 under anaerobic and aseptic conditions, the corrosion of Fe0- and carbon-steel by the nitrate-reducing Prolixibacter strains was deduced to be mainly enhanced via the biological reduction of nitrate to nitrite, followed by the chemical oxidation of Fe0 to Fe2+ and Fe3+ coupled to the reduction of nitrite.

M-protein (PAM) largely contributes to the pathogenesis of Pattern D Group A Streptococcus pyogenes (GAS). However, the mechanism of complex formation is unknown. In a system consisting of a Class II PAM from Pattern D GAS isolate NS88.2 (PAMNS88.2), with one K2hPg binding a-repeat in its A-domain, we employed biophysical techniques to analyze the mechanism of the K2hPg/PAMNS88.2 interaction. We show that apo-PAMNS88.2 is a coiled-coil homodimer (M.Wt. ~80 kDa) at 4°C - 25°C, and is monomeric (M.Wt. ~40 kDa) at 37°C, demonstrating a temperature-dependent dissociation of PAMNS88.2 over a narrow temperature range. PAMNS88.2 displayed a single tight binding site for K2hPg at 4°C, which progressively increased at 25°C through 37°C. We isolated the K2hPg/PAMNS88.2 complexes at 4°C, 25°C, and 37°C and found molecular weights of ~50 kDa at each temperature, corresponding to a 1:1 (m:m) K2hPg/PAMNS88.2 monomer complex. hPg activation experiments by streptokinase demonstrated that the hPg/PAMNS88.2 monomer complexes are fully functional. The data show that PAM dimers dissociate into functional monomers at physiological temperatures or when presented with the active hPg module (K2hPg) showing that PAM is a functional monomer at 37°C.

Background: Candida auris is an emerging fungal pathogen of worldwide interest. It is associated with high mortality rates and exhibits increased resistance to antifungals. Ultraviolet-C (UVC) light can be used to disinfect surfaces to mitigate its spread. In this study, we analyzed the performance of different UVC wavelengths against C. auris to determine its wavelength sensitivity and UVC dose requirements and evaluated biofilm prevention dose requirements on most used materials in healthcare settings. Objectives: 1. To investigate UVC disinfection performances and wavelength sensitivity of C. auris; 2. To evaluate the UVC dose required for prevention of biofilm prevention on stainless steel. Methods: C. auris was grown following standard procedures. The study utilized six different UVC LED arrays with wavelengths between 252 and 280 nm. Arrays were set at similar intensities, to obtain doses of 5-40 mJcm-2 and similar irradiation time. Disinfection performance for each array was determined using log reduction value (LRV) and percentage reduction by comparing the controls against the irradiated treatments. Evaluation of the ability of 267 nm UVC LEDs to prevent C. auris biofilm formation was investigated using stainless steel, plastic coupons, and poly-cotton fabric. Results: Peak sensitivity to UVC disinfection was between 267 and 270 nm. With 20 mJcm-2, the study obtained LRV 3. On steel coupons, 30 mJcm-2 was sufficient to prevent biofilm formation, on plastic only 10 mJcm-2. A dose of 60 mJcm-2 reduced biofilms on poly-cotton fabric significantly. Conclusions: Results revealed that C. auris was most susceptible at 267-270 nm. Additional highlights from the study allow for the design and implementation of disinfection systems.

The human microbiome has begun to emerge as a potential forensic tool, with varied applications ranging from unique identification to investigative leads that link individuals and/or locations. The relative abundance of the combined DNA of the microbiome, compared to human nuclear DNA, may expand potential sources of biological evidence, especially in cases with transfer or low-copy number DNA samples. This work sought to determine the optimal swab type for the collection and analysis of the microbiome. A bacterium (Proteus mirabilis) was deposited by pipette onto four swab types (cotton, flocked, dental applicators, and dissolvable), and extraction and real-time PCR quantitation of the bacterial DNA were performed, which allowed for absolute microbial DNA recovery and comparison of yields across the four sampling substrates. Flocked swabs had the highest yield (~1240 ng) compared to the cotton swabs (~184 ng), dental applicators (~533 ng), and dissolvable swabs (~430 ng). The collection efficiency was further evaluated for cotton and flocked swabs using dried microbial samples spotted onto non-porous surfaces (treated wood, glass, plastic, and tile). Flocked swabs performed consistently better across wood, glass, and tile, but showed decreased recovery from plastic. The cotton swabs failed in the recovery of P. mirabilis DNA across all surfaces. Knowing the appropriate sampling substrate will be useful as others continue to investigate the use of the microbiome as a forensics tool.

Due to the emerging rise of multi-drug resistant bacteria, the discovery of novel antibiotics is of high scientific interest. Through their high chemodiversity of bioactive secondary metabolites, cyanobacteria have proven to be promising microorganisms for the discovery of antibacterial compounds. These aspects make appropriate antibacterial screening approaches for cyanobacteria crucial. Up to date, screenings are mostly carried out using a phenotypic methodology, consisting of cyanobacterial cultivation, extraction, and inhibitory assays. However, the parameters of these methods highly vary within the literature. Therefore, the common choices of parameters and inhibitory assays are summarized in this review. Nevertheless, less frequently used method variants are highlighted, which lead to hits from antimicrobial compounds. In addition to the considerations of phenotypic methods, this review provides an overview of developments in the genome-based screening area, be it in-vivo using PCR technique or in-silico using the recent genome-mining method. Though, up to date these techniques are a lot less applied than phenotypical screening.

The genome of Halobacterium strain 63-R2 was recently reported and provides the opportunity to resolve long-standing issues regarding the source of two widely used model strains of Hbt. salinarum, NRC-1 and R1. Strain 63-R2 was isolated in 1934 from a salted buffalo hide (epithet ‘cutirubra’), along with another strain from a salted cowhide (91-R6T, epithet ‘salinaria’, the type strain of Halobacterium salinarum). Both strains belong to the same species according to genome-based taxonomy analysis (TYGS), with chromosome sequences showing 99.64% identity over 1.85 Mb. The chromosome of strain 63-R2 is 99.99% identical to the two laboratory strains NRC-1 and R1, with only 5 indels, excluding the mobilome. The two reported plasmids of strain 63-R2 share their architecture with plasmids of strain R1 (pHcu43/pHS4, 99.89% identity; pHcu235/pHS3, 100.0% identity). We detected and assembled additional plasmids, using PacBio reads deposited at the SRA database, further corroborating that strain differences are minimal. One plasmid, pHcu190 (190,816 bp) corresponds to pHS1 (strain R1) but is even more similar in architecture to pNRC100 (strain NRC-1). Another plasmid, pHcu229, assembled partially and completed in silico (229,124 bp), shares most of its architecture with pHS2 (strain R1). In deviating regions, it corresponds to pNRC200 (strain NRC-1). Further architectural differences between the laboratory strain plasmids are not unique but are present in strain 63-R2, which contains characteristics from both of them. Based on these observations, it is proposed that the early 20th-century isolate 63-R2 is the immediate ancestor of the twin laboratory strains NRC-1 and R1.