Actinobacteria are important cave inhabitants, but knowledge of how anthropization and anthropization-related visual marks affect this community on cave walls is lacking. We compared Actinobacteria communities among four French limestone caves (Mouflon, Reille, Rouffignac, Lascaux) ranging from pristine to anthropized, and within Lascaux Cave between marked (wall visual marks) and unmarked areas in different rooms (Sas-1, Passage, Apse, Diaclase). In addition to the 16S rRNA gene marker, 441 bp fragments of the hsp65 gene were used and an hsp65-related taxonomic database was constructed for identification of Actinobacteria to the species level by Illumina-MiSeq analysis. The hsp65 marker revealed higher resolution for species and higher richness (99% OTU cutoff) versus 16S rRNA gene; however, more taxa were identified at higher taxonomic ranks. Actinobacteria communities varied between Mouflon and Reille caves (both pristine), and Rouffignac and Lascaux (both anthropized). Rouffignac displayed high diversity of Nocardia, suggesting human inputs, and Lascaux exhibited high Mycobacterium relative abundance, whereas Gaiellales were typical in pristine caves and the Diaclase (least affected area of Lascaux Cave). Within Lascaux, Pseudonocardiaceae dominated on unmarked walls and Streptomycetaceae (especially Streptomyces mirabilis) on marked walls, indicating a possible role in mark formation. A new taxonomic database (https://zenodo.org/record/5576074) was developed. Although not all Actinobacteria species were represented, the use of the hsp65 marker enabled species-level variations of the Actinobacteria community to be documented based on the extent of anthropogenic pressure. This approach proved effective when comparing different limestone caves or specific conditions within one cave.
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.
The goal of this study was to design genus-specific primers for rapid evaluation of the most abundant bacterial genera identified using amplicon-based sequencing of the 16S rRNA gene in fish-related samples and surrounding water. Efficient genus-specific primers were designed for eleven bacterial genera including Alkalimarinus, Colwellia, Enterovibrio, Marinomonas, Massilia, Oleispira, Phaeobacter, Photobacterium, Polarbacerium, Pseudomonas, and Psychrobium. The specificity of the primers was confirmed by the phylogeny of the sequenced polymerase chain reaction (PCR) amplicons that indicated primers were genus-specific except in the case of Colwellia and Phaeobacter. Copy number of the 16S rRNA gene obtained by quantitative PCR using genus-specific primers and the relative abundance obtained by 16S rRNA gene sequencing using universal primers were well correlated for the five analyzed abundant bacterial genera. Low correlations between quantitative PCR and 16S rRNA gene sequencing for Pseudomonas were explained by the higher coverage of known Pseudomonas species by the designed genus-specific primers than the universal primers used in 16S rRNA gene sequencing. The designed genus-specific primers are proposed as rapid and cost-effective tools to evaluate the most abundant bacterial genera in fish-related or potentially other metagenomics samples.
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.
Antibacterial properties of toothpastes enable chemical plaque control in limited-access tooth regions that are mechanically not sufficiently reached by toothbrushes. Therefore, this study aimed to compare different microbial methods to assess antimicrobial toothpaste properties and evaluate different toothpastes in terms of their antibacterial efficacy against different oral microorganisms in vitro. Six toothpaste suspensions with varying antibacterial supplements were applied to a multi-species biofilm model (Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans) as well as to each microorganism. A culture method was used to assess the anti-biofilm effects and two different agar diffusion assays were performed for testing the antimicrobial effect on each microorganism. The measurements of the culture and diffusion analyses were statistically normalized and compared and toothpastes were ranked according to their antimicrobial efficacy. The results of both agar diffusion assays showed a high correlation across all tested species (Spearman correlation coefficients ρs > 0.95). The results of the multi-species biofilm model, however, substantially differed in its assessment of antibacterial properties (ρs ranging from 0.22 to 0.87), compared to the results of both diffusion assays. Toothpastes with amine fluoride with and without stannous fluoride, and triclosan resulted in the highest antimicrobial efficacy, while activated carbon supplements were comparable to the negative control NaCl. The appropriate selection of a broad range of oral microorganisms seems crucial when testing the chemical impact of toothpastes and toothpaste supplements.
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.
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.
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.
Listeria monocytogenes is a human pathogen. It is the causative agent of listeriosis, the leading cause of bacterial-linked foodborne mortality in Europe and elsewhere. Outbreaks of listeriosis have been associated with the consumption of fresh produce including vegetables and fruits. In this review we summarize current data providing direct or indirect evidence that plants can serve as habitat for L. monocytogenes, enabling this human pathogen to survive and grow. The current knowledge of the mechanisms involved in the interaction of this bacterium with plants is addressed, and whether this foodborne pathogen elicits an immune response in plants is discussed.
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.
Interspecific interactions within biofilms determine relative species abundance, growth dynamics, community resilience, and success or failure of invasion by an extraneous organism. However, deciphering interspecific interactions and assessing their contribution to biofilm properties and function remain a challenge. Here, we describe the constitution of a model biofilm composed of four bacterial species belonging to four different genera (Rhodocyclus sp., Pseudomonas fluorescens, Kocuria varians, and Bacillus cereus), derived from a biofilm isolated from an industrial milk pasteurization unit. We demonstrate that the growth dynamics and equilibrium composition of this biofilm are highly reproducible. Based on its equilibrium composition, we show that the establishment of this 4-species biofilm is highly robust against initial, transient perturbations but less so towards continuous perturbations. By comparing biofilms formed from different numbers and combinations of the constituent species and by fitting a growth model to the experimental data, we reveal a network of dynamic, positive, and negative interactions that determine the final composition of the biofilm. Furthermore, we reveal that the molecular determinant of one negative interaction is the thiocillin I synthesized by the B. cereus strain, and demonstrate its importance for species distribution and its impact on robustness by mutational analysis of the biofilm ecosystem.
In fungal pathogens, the transcription factor SrbA (a sterol regulatory element-binding protein, SREBP) and CBC (CCAAT binding complex) have been reported to regulate azole resistance by competitively binding the TR34 region (34 mer) in the promoter of the drug target gene, erg11A. However, current knowledge about how the SrbA and CBC coordinately mediate erg11A expression remains limited. In this study, we uncovered a novel relationship between HapB (a subunit of CBC) and SrbA in which deletion of hapB significantly prolongs the nuclear retention of SrbA by increasing its expression and cleavage under azole treatment conditions, thereby enhancing Erg11A expression for drug resistance. Furthermore, we verified that loss of HapB significantly induces the expression of the rhomboid protease RbdB, Dsc ubiquitin E3 ligase complex, and signal peptide peptidase SppA, which are required for the cleavage of SrbA, suggesting that HapB acts as a repressor for these genes which contribute to the activation of SrbA by proteolytic cleavage. Together, our study reveals that CBC functions not only to compete with SrbA for binding to erg11A promoter region but also to affect SrbA expression, cleavage, and translocation to nuclei for the function, which ultimately regulate Erg11A expression and azole resistance.
To understand antibiotic resistance in pathogenic bacteria, we need to monitor environmental microbes as reservoirs of antimicrobial resistance genes (ARGs). These bacteria are present in the air and can be investigated with the whole metagenome shotgun sequencing approach. This study aimed to investigate the feasibility of a method for metagenomic analysis of microbial composition and ARGs in the outdoor air. Air samples were collected with a Harvard impactor in the PM10 range at 50 m from a hospital in Budapest. From the DNA yielded from samples of PM10 fraction single-end reads were generated with an Ion Torrent sequencer. During the metagenomic analysis, reads were classified taxonomically. The core bacteriome was defined. Reads were assembled to contigs and the ARG content was analyzed. The dominant genera in the core bacteriome were Bacillus, Acinetobacter, Leclercia and Paenibacillus. Among the identified ARGs best hits were vanRA, Bla1, mphL, Escherichia coli EF-Tu mutants conferring resistance to Pulvomycin, BcI, FosB, and mphM. Despite the low DNA content of the samples of PM10 fraction, the number of detected airborne ARGs was surprisingly high.
Identification, source tracking, and surveillance of food pathogens is a crucial factor for the food-producing industry. Over the last decade, the techniques used for this have moved from conventional enrichment methods, through species-specific detection by PCR to sequencing-based methods, whole-genome sequencing (WGS) being the ultimate method. However, using WGS requires the right infrastructure, high computational power, and bioinformatics expertise. Therefore, there is a need for faster, more cost-effective, and more user-friendly methods. A newly developed method, ON-rep-seq, combines the classical rep-PCR method with nanopore sequencing, resulting in a highly discriminating set of sequences that can be used for species identification and also strain discrimination. This study is essentially a real industry case from a salmon processing plant. Twenty Listeria monocytogenes isolates were analyzed both by ON-rep-seq and WGS to identify and differentiate putative L. monocytogenes from a routine sampling of processing equipment and products, and finally, compare the strain-level discriminatory power of ON-rep-seq to different analyzing levels delivered from the WGS data. The analyses revealed that among the isolates tested there were three different strains. The isolates of the most frequently detected strain (n=15) were all detected in the problematic area in the processing plant. The strain level discrimination done by ON-rep-seq was in full accordance with the interpretation of WGS data. Our findings also demonstrate that ON-rep-seq may serve as a primary screening method alternative to WGS for identification and strain-level differentiation for surveillance of potential pathogens in a food-producing environment.
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.
Vibrio alginolyticus and Vibrio (Aliivibrio) fischeri are Gram-negative bacteria found globally in marine environments. During the past decade, studies have shown that certain Gram-negative bacteria, including Vibrio species (cholerae, parahaemolyticus, and vulnificus) are capable of using exogenous polyunsaturated fatty acids (PUFAs) to modify the phospholipids of their membrane. Moreover, exposure to exogenous PUFAs has been shown to affect certain phenotypes that are important factors of virulence. The purpose of this study was to investigate whether V. alginolyticus and V. fischeri are capable of responding to exogenous PUFAs by remodeling their membrane phospholipids and/or altering behaviors associated with virulence. Thin-layer chromatography (TLC) analyses and ultra-performance liquid chromatography-electrospray ionization mass spectrometry (UPLC/ESI-MS) confirmed incorporation of all PUFAs into membrane phosphatidylglycerol and phosphatidylethanolamine. Several growth phenotypes were identified when individual fatty acids were supplied in minimal media and as sole carbon sources. Interestingly, several PUFAs acids inhibited growth of V. fischeri. Significant alterations to membrane permeability were observed depending on fatty acid supplemented. Strikingly, arachidonic acid (20:4) reduced membrane permeability by approximately 35% in both V. alginolyticus and V. fischeri. Biofilm assays indicated that fatty acid influence was dependent on media composition and temperature. All fatty acids caused decreased swimming motility in V. alginolyticus, while only linoleic acid (18:2) significantly increased swimming motility in V. fischeri. In summary, exogenous fatty acids cause a variety of changes in V. alginolyticus and V. fischeri, thus adding these bacteria to a growing list of Gram-negatives that exhibit versatility in fatty acid utilization and highlighting the potential for environmental PUFAs to influence phenotypes associated with planktonic, beneficial, and pathogenic associations.
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.