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.

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.

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.

Denitrification plays a central role in the global nitrogen cycle, reducing and removing nitrogen from marine and terrestrial ecosystems. The flux of nitrogen species through this pathway has a widespread impact, affecting ecological carrying capacity, agriculture, and climate. Nitrite reductase (Nir) and nitric oxide reductase (NOR) are the two central enzymes in this pathway. Here we present a previously unreported Nir domain architecture in members of Phylum Chloroflexi. Phylogenetic analyses of protein domains within Nir indicate that an ancestral horizontal transfer and fusion event produced this chimeric domain architecture. We also identify an expanded genomic diversity of a rarely reported nitric oxide reductase subtype, eNOR. Together, these results suggest a greater diversity of denitrification enzyme arrangements exist than have been previously reported.

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.

Carotenoids are widely used in functional foods, cosmetics, and health supplements, and their importance and scope of use are continuously expanding. Here, we characterised carotenoid biosynthetic genes of the plant-pathogenic bacterium Pantoea ananatis, which carries a carotenoid biosynthetic gene cluster (including crtE, X, Y, I, B, and Z) on a plasmid. Reverse transcription–polymerase chain reaction (RT-PCR) analysis revealed that the crtEXYIB gene cluster is transcribed as a single transcript and crtZ is independently transcribed in the opposite direction. Using splicing by overlap extension with polymerase chain reaction (SOE by PCR) based on asymmetric amplification, we reassembled crtE–B, crtE–B–I, and crtE–B–I–Y. High-performance liquid chromatography confirmed that Escherichia coli expressing the reassembled crtE–B, crtE–B–I, and crtE–B–I–Y operons produced phytoene, lycopene, and β-carotene, respectively. We found that the carotenoids conferred tolerance to UV radiation and toxoflavin. Pantoea ananatis shares rice environments with the toxoflavin producer Burkholderia glumae and is considered to be the first reported example of producing and using carotenoids to withstand toxoflavin. We confirmed that the carotenoid production of P. ananatis is dependent on RpoS, which is positively regulated by Hfq/ArcZ and negatively regulated by ClpP, similar to an important regulatory network of E. coli (HfqArcZ → RpoS Ͱ ClpXP). We also demonstrated that Hfq-controlled quorum signalling de-represses EanR to activate RpoS, thereby initiating carotenoid production. Survival genes such as those responsible for the production of carotenoids of the plant-pathogenic P. ananatis must be expressed in a timely manner to overcome stressful environments and compete with other microorganisms. This mechanism is likely maintained by a brake with excellent performance, such as EanR.

The ability of S. aureus to infect bone and osteoblasts is correlated to its incredible virulence armamentarium that can mediate the invasion/internalization process, cytotoxicity, membrane damage and intracellular persistence. We comparatively analyzed the interaction, persistence and modulation of expression of selected genes as well as cell viability in an ex-vivo model using human MG-63 osteoblasts of two previously studied and well-characterized S. aureus clinical strains belonging to ST239-SCCmecIII-t037 and ST228-SCCmecI-t041 clones at 3h and 24h post-infection (p.i). ATCC12598 was used as a control strain. Using Imaging Flow Cytometry analysis, we found that strains differently invaded osteoblasts after 3h and 24h: ATCC12598 internalized in 70% and 50% of cells, ST239-SCCmecIII in 50% and 45% and ST228-SCCmecI in 30% and 20%, respectively. ST239-III, during the infection period, exerted a significative cytotoxic activity due to the over-expression of hla and psmA and the increased expression of the genes involved in adhesion, probably due to the release and re-entry of bacteria inside MG-63 at 24h p.i. The lower invasiveness of ST228-I was also correlated with the non-cytotoxic activity inside osteoblasts. This clone was not able to activate a sufficient cellular reaction and succumbed in-side the MG-63 cells.

To survive within complex environmental niches, including the human host, bacteria have evolved intricate inter-species communities driven by competition for limited nutrients, cooperation via complementary metabolic proficiencies, and establishment of homeostatic relationships with the host immune system. Such complex, interdependent relationships have hampered attempts to culture many bacterial strains in research settings, where standard readout of co-culture experiments are usually limited to the relative abundance of each species. Here, we utilize a microfluidic-based co-culture system to characterize dynamic interactions between multiple oral bacterial isolates. Using time-lapse imaging, we define species-specific effects on spatial community relationships during co-culture of Streptococcus species and Staphylococcus aureus with Actinomyces species. Co-culture of Streptococcus cristatus or S. salivarius in nanoliter compartments with Actinomyces graevenitzii revealed localized exclusion of Streptococcus and Staphylococcus from media immediately surrounding A. graevenitzii micro colonies. This community structure did not occur with S. mitis or S. oralis strains, or in co-cultures containing other Actinomycetaceae species such as S. odontolyticus or A. naeslundii. Moreover, fewer neutrophils were attracted to compartments containing both A. graevenitzii and Staphylococcus aureus than to equal number of either species alone, suggesting a possible survival benefit from the interaction.