Nontuberculous mycobacteria (NTM) are environmental organisms that can cause opportunistic pulmonary disease with species diversity showing significant regional variation. In the United States, Hawai’i shows the highest rate of NTM pulmonary disease. The need for improved understanding of NTM reservoirs led us to identify NTM from patient respiratory specimens and compare NTM diversity between outdoor and indoor locations in Hawai’i. A total of 545 water biofilm samples were collected from 357 unique locations across Kaua’i (n = 51), O’ahu (n = 202), Maui (n = 159), and Hawai’i Island (n = 133) and divided into outdoor (n = 179) or indoor (n = 366) categories. rpoB sequence analysis was used to determine NTM species and predictive modeling applied to develop NTM risk maps based on geographic characteristics between environments. M. chimaera was frequently identified from respiratory and environmental samples followed by M. chelonae and M. abscessus; yet significantly less NTM were consistently recovered from outdoor compared to indoor biofilms, as exemplified by showerhead biofilm samples. While the frequency of M. chimaera recovery was comparable between outdoor and indoor showerhead biofilms, phylogenetic analyses demonstrate similar rpoB gene sequences between all showerhead and respiratory M. chimaera isolates, supporting outdoor and indoor environments as possible sources for pulmonary M. chimaera infections. Full article

Lectins have been increasingly utilized as carriers for targeted drug delivery based on their specific binding to glycans located on mammalian cells. This study employed two lectins, B subunit of bacterial Shiga holotoxin (Stx1B) and fungal Clitocybe nebularis lectin (CNL), for surface display on the lactic acid bacterium Lactococcus lactis. The specific adhesion of these engineered, lectin-displaying L. lactis to cancer cells was evaluated. The expression and surface display of both lectins on L. lactis were demonstrated by western blotting and flow cytometry, respectively. MTS assays revealed that recombinant Stx1B had no effect on Caco-2 cell viability at concentrations of ≤25 µg/mL, whereas CNL was non-toxic even at relatively high concentrations of ≤250 µg/mL. Stx1B bound to Caco-2, HT-29 and HeLa cells after 1 h of incubation. CNL bound to Caco-2 cells and recognized several glycoproteins in HT-29 and Caco-2 cell homogenates of which a 70 kDa protein predominated. Confocal microscopy revealed adhesion of Stx1B-displaying L. lactis to HeLa, Caco-2, and, to a lesser extent, HT-29 cells; CNL-displaying L. lactis showed a relatively similar level of adherence to HT-29 and Caco-2 cells. Thus, lectin-displaying L. lactis might serve as a carrier in targeted drug delivery when coupled to a therapeutic moiety. Full article

Antimicrobial peptides (AMPs) found in the innate immune system of a wide range of organisms might prove useful to fight infections, due to the reported slower development of resistance to AMPs. Increasing the cationicity and keeping moderate hydrophobicity of the AMPs have been described to improve antimicrobial activity. We previously found a peptide derived from the Tribolium castaneum insect defensin 3, exhibiting antrimicrobial activity against several human pathogens. Here, we analyzed the effect against Staphyloccocus aureus of an extended peptide (TcPaSK) containing two additional amino acids, lysine and asparagine, flanking the former peptide fragment in the original insect defensin 3 protein. TcPaSK peptide displayed higher antimicrobial activity against S. aureus, and additionally showed antiproliferative activity against the MDA-MB-231 triple negative breast cancer cell line. A SWATH proteomic analysis revealed the downregulation of proteins involved in cell growth and tumor progression upon TcPaSK cell treatment. The dual role of TcPaSK peptide as antimicrobial and antiproliferative agent makes it a versatile molecule that warrants exploration for its use in novel therapeutic developments as an alternative approach to overcome bacterial antibiotic resistance and to increase the efficacy of conventional cancer treatments. Full article

Campylobacter is the most frequent foodborne zoonotic bacteria worldwide, with chicken meat being overwhelmingly the most important reservoir for human infections. Control measures implemented at the farm level (i.e., biosecurity or vaccination), which have been successfully applied to limit other pathogens, such as Salmonella, have not been effective in reducing Campylobacter occurrence. Thus, new approaches are needed to fully understand the ecological interactions of Campylobacter with host animals to effectively comprehend its epidemiology. The objective of this study was to analyse longitudinally the gut microbiota composition of Campylobacter-infected and non-infected farms to identify any difference that could potentially be indicative of gut colonization by Campylobacter spp. Differences in the colonization rate and timing were observed at the farms that became positive for Campylobacter jejuni over the investigated time points, even though in positive tests, the occurrence of Campylobacter jejuni gut colonization was not observed before the second week of the life of the birds. Significant differences were observed in the abundances of specific bacterial taxa between the microbiota of individuals belonging to farms that became Campylobacter positive during the study and those who remained negative with particular reference to Bacteroidales and Clostridiales, respectively. Moreover, Campylobacter colonization dramatically influenced the microbiota richness, although to a different extent depending on the infection timing. Finally, a key role of Faecalibacterium and Lactobacillus genera on the Campylobacter microbial network was observed. Understanding the ecology of the Campylobacter interaction with host microbiota during infection could support novel approaches for broiler microbial barrier restoration. Therefore, evidence obtained through this study can be used to identify options to reduce the incidence of infection at a primary production level based on the targeted influence of the intestinal microbiota, thus helping develop new control strategies in order to mitigate the risk of human exposure to Campylobacter by chicken meat consumption. Full article

Melanophryniscus admirabilis (admirable red-belly toad) is a microendemic and critically endangered species found exclusively along 700 m of the Forqueta River, in a fragment of the Atlantic Forest of southern Brazil. One of the greatest concerns regarding the conservation of this species is the extensive use of pesticides in areas surrounding their natural habitat. In recent years, the adaptation and persistence of animal species in human-impacted environments have been associated with microbiota. Therefore, the present study aimed to characterize the oral bacterial community of wild M. admirabilis and to address the question of how this community might contribute to this toad’s adaptation in the anthropogenic environment as well as its general metabolic capabilities. A total of 11 oral samples collected from wild M. admirabilis were characterized and analyzed via high-throughput sequencing. Fragments of the 16S rRNA variable region 4 (V4) were amplified, and sequencing was conducted using an Ion Personal Genome Machine (PGM) System with 316 chips. A total of 181,350 sequences were obtained, resulting in 16 phyla, 34 classes, 39 orders, and 77 families. Proteobacteria dominated (53%) the oral microbiota of toads, followed by Firmicutes (18%), Bacteroidetes (17%), and Actinobacteria (5%). No significant differences in microbial community profile from among the samples were reported, which suggests that the low dietary diversity observed in this population may directly influence the bacterial composition. Inferences of microbiome function were performed using PICRUSt2 software. Important pathways (e.g., xenobiotic degradation pathways for pesticides and aromatic phenolic compounds) were detected, which suggests that the bacterial communities may serve important roles in M. admirabilis health and survival in the anthropogenic environment. Overall, our results have important implications for the conservation and management of this microendemic and critically endangered species. Full article

Entomopathogenic fungi can be a useful resource for controlling insect vectors of citrus plant pathogens, such as the Asian citrus psyllid (Diaphorina citri) associated with huanglongbing or the citrus root weevil (Diaprepes abbreviatus) associated with the spread of Phytophtora spp. In this study, Cordyceps fumosorosea (Cfr) was investigated in planta as a potential endophytic entomopathogenic fungus and various inoculation techniques were used to determine if it would colonize the Carrizo citrange (Citrus × insitorum) seeds and plants. The four inoculation methodologies evaluated were seed soaking, stem injection, foliar spray, and soil drench. Seed immersion trials demonstrated that the roots of the Carrizo citrange plant can be inoculated successfully with Cfr. Stem injection, foliar spray, and soil drench also provided successful inoculation of Cfr. However, this fungus was only endophytic in the plant stem. Sand cores indicated that Cfr moved down through the sand column and was able to inoculate the roots. Given the prevalence of Cfr in the soil during the drench experiment, and that the fungus was able to colonize Carrizo citrange roots through seed immersion, this finding provides evidence of the potential endophytism of this fungus when applied to citrus plant species. Full article

Waste biomass coming from a local coffee company, which supplied burnt ground coffee after an incorrect roasting process, was employed as a starting material in the composting plant of the Experimental Station of the University of Naples Federico II at Castel Volturno (CE). The direct molecular characterization of compost using ¹³C-NMR spectra, which was acquired through cross-polarization magic-angle spinning, showed a hydrophobicity index of 2.7% and an alkyl/hydroxyalkyl index of 0.7%. Compost samples that were collected during the early “active thermophilic phase” (when the composting temperature was 63 °C) were analyzed for the prokaryotic community composition and activities. Two complementary approaches, i.e., genomic and predictive metabolic analysis of the 16S rRNA V3–V4 amplicon and culture-dependent analysis, were combined to identify the main microbial factors that characterized the composting process. The whole microbial community was dominated by Firmicutes. The predictive analysis of the metabolic functionality of the community highlighted the potential degradation of peptidoglycan and the ability of metal chelation, with both functions being extremely useful for the revitalization and fertilization of agricultural soils. Finally, three biotechnologically relevant Firmicutes members, i.e., Geobacillus thermodenitrificans subsp. calidus, Aeribacillus pallidus, and Ureibacillus terrenus (strains CAF1, CAF2, and CAF5, respectively) were isolated from the “active thermophilic phase” of the coffee composting. All strains were thermophiles growing at the optimal temperature of 60 °C. Our findings contribute to the current knowledge on thermophilic composting microbiology and valorize burnt ground coffee as waste material with biotechnological potentialities. Full article

The genus Diaporthe and its anamorph Phomopsis are distributed worldwide in many ecosystems. They are regarded as potential sources for producing diverse bioactive metabolites. Most species are attributed to plant pathogens, non-pathogenic endophytes, or saprobes in terrestrial host plants. They colonize in the early parasitic tissue of plants, provide a variety of nutrients in the cycle of parasitism and saprophytism, and participate in the basic metabolic process of plants. In the past ten years, many studies have been focused on the discovery of new species and biological secondary metabolites from this genus. In this review, we summarize a total of 335 bioactive secondary metabolites isolated from 26 known species and various unidentified species of Diaporthe and Phomopsis during 2010–2019. Overall, there are 106 bioactive compounds derived from Diaporthe and 246 from Phomopsis, while 17 compounds are found in both of them. They are classified into polyketides, terpenoids, steroids, macrolides, ten-membered lactones, alkaloids, flavonoids, and fatty acids. Polyketides constitute the main chemical population, accounting for 64%. Meanwhile, their bioactivities mainly involve cytotoxic, antifungal, antibacterial, antiviral, antioxidant, anti-inflammatory, anti-algae, phytotoxic, and enzyme inhibitory activities. Diaporthe and Phomopsis exhibit their potent talents in the discovery of small molecules for drug candidates. Full article

Cereals are still one of the most important food and feed sources, thus determining cereal’s safety, i.e., compliance with legislation, is extremely important. As systematic investigations of nowadays unavoidable secondary fungal metabolites and other common legally regulated contaminants occurrence in Croatian cereals are still lacking, this research aims to monitor the contamination levels of nation-wide crops by mycotoxins, pesticide residues, and heavy metals by employing UHPLC-MS/MS, GC-MS/MS, and atomic absorption spectrometer (AAS) validated analytical methods. The most common secondary fungal metabolites were found to be Fusarium mycotoxins, with DON being the most occurring present in 73.7% of the samples. At least one pesticide residue was found in 331.8% of the samples, and Hg and Cd were the most occurring heavy metals. A total of 8.5% of the samples was non-compliant to the European Union (EU) legislation for food regarding the found mycotoxins concentrations, 4.5% regarding pesticide residues and none regarding heavy metals. The unusual presence of certain pesticide residue and heavy metal indicates the importance of systematic control of the contaminant presence, in order to gather enough occurrence data for proper risk assessment that these contaminants represent for the consumer’s health. Full article

Cryptosporidium spp. is responsible for several food and waterborne disease outbreaks worldwide. Healthier lifestyles attract consumers to eat, notably, fresh food like fruits and vegetables. The consumption of raw or under-cooked food increases the risk of foodborne transmission of Cryptosporidiosis. The assessment of the consumer’s exposure to Cryptosporidium danger is crucial for public health. Still, the standardized method to detect this parasite in fresh leafy greens and berry fruits has only been available since 2016 and suffers from weaknesses. Consequently, in this study, we propose a method with minimum processing steps that combines cell culture and the quantitative PCR (CC–qPCR) for detecting infectious C. parvum oocysts recovered from lamb’s lettuce. This CC–qPCR is a rapid and easy method that can detect up to one oocyst, whereas it is undetectable by classic qPCR. Full article

A global increase in Escherichia coli (E. coli) resistant to cephalosporins (extended-spectrum β-lactamases (ESβLs) and AmpC β-lactamases) has been recorded in the last 20 years. Similarly, several studies have reported the spread of colistin resistance in Enterobacteriaceae isolated from food and the environment. The aim of the present study was to evaluate the prevalence of ESβL, AmpC and colistin-resistant E. coli isolated from pork and wild boar meat products in the Emilia Romagna region (North Italy). The isolates were analysed phenotypically (considering both resistant and intermediate profiles) and genotypically. The prevalence of genotypically confirmed ESβL and AmpC E. coli was higher in pork meat products (ESβL = 11.1% vs. AmpC = 0.3%) compared to wild boar meat (ESβL = 6.5% vs. AmpC = 0%). Intermediate profiles for cefotaxime (CTX) and ceftazidime (CAZ) were genotypically confirmed as ESβL in pork meat isolates but not for wild boar. Four E. coli from wild boar meat were resistant to colistin but did not harbour the mcr-1 gene. E. coli isolated from wild boar meat seem to show aspecific antimicrobial resistance mechanisms for cephalosporins and colistin. The prevalence of resistant isolates found in wild boar is less alarming than in pork from farmed domestic pigs. However, the potential risk to consumers of these meat products will require further investigations. Full article

Filoviruses are zoonotic, negative-sense RNA viruses, most of which are capable of causing severe disease in humans and nonhuman primates, often with high case fatality rates. Among these viruses, those belonging to the Ebolavirus genus—particularly Ebola virus, Sudan virus, and Bundibugyo virus—represent some of the most pathogenic to humans. Taï Forest virus (TAFV) is thought to be among the least pathogenic ebolaviruses; however, only a single non-fatal case has been documented in humans, in 1994. With the recent success of the ferret as a lethal model for a number of ebolaviruses, we set out to evaluate its suitability as a model for TAFV. Our results demonstrate that, unlike other ebolaviruses, TAFV infection in ferrets does not result in lethal disease. None of the intramuscularly inoculated animals demonstrated any overt signs of disease, whereas the intranasally inoculated animals exhibited mild to moderate weight loss during the early stage of infection but recovered quickly. Low levels of viral RNA were detected in the blood and tissues of several animals, particularly the intranasally inoculated animals, and all animals mounted a humoral immune response, with high titers of GP-specific IgG detectable as early as 14 days post-infection. These data provide additional insight into the pathogenesis of TAFV. Full article

Post-operative endophthalmitis caused by Enterococcus spp. progresses rapidly and often results in substantial and irreversible vision loss. Therefore, novel alternative treatments that are effective against enterococcal endophthalmitis are required. Bacteriophage therapy has the potential to be an optional therapy for infectious diseases. Therefore, we investigated the therapeutic potential of three newly isolated enterococcal phages, phiEF7H, phiEF14H1, and phiEF19G, in E. faecalis-induced endophthalmitis. These phages could lyse the broad-range E. faecalis, including strains derived from endophthalmitis and vancomycin-resistant E. faecalis in vitro, as determined by the streak test. Morphological and genomic analyses revealed that these phages were classified into the Herelleviridae genus Kochikohdavirus. The whole genomes of these phages contained 143,399, 143,280, and 143,400 bp, respectively. Endophthalmitis was induced in mice by injection of three strains of E. faecalis derived from post-operative endophthalmitis or vancomycin-resistant strains into the vitreous body. The number of viable bacteria and infiltration of neutrophils in the eye were both decreased by intravitreous injection of phiEF7H, phiEF14H1, and phiEF19G 6 h after injection of all E. faecalis strains. Thus, these results suggest that these newly isolated phages may serve as promising candidates for phage therapy against endophthalmitis. Full article

In this study, we analyzed microbial community composition and the functional capacities of degraded sites and restored/natural sites in two typical wetlands of Northeast China—the Phragmites marsh and the Carex marsh, respectively. The degradation of these wetlands, caused by grazing or land drainage for irrigation, alters microbial community components and functional structures, in addition to changing the aboveground vegetation and soil geochemical properties. Bacterial and fungal diversity at the degraded sites were significantly lower than those at restored/natural sites, indicating that soil microbial groups were sensitive to disturbances in wetland ecosystems. Further, a combined analysis using high-throughput sequencing and GeoChip arrays showed that the abundance of carbon fixation and degradation, and ~95% genes involved in nitrogen cycling were increased in abundance at grazed Phragmites sites, likely due to the stimulating impact of urine and dung deposition. In contrast, the abundance of genes involved in methane cycling was significantly increased in restored wetlands. Particularly, we found that microbial composition and activity gradually shifts according to the hierarchical marsh sites. Altogether, this study demonstrated that microbial communities as a whole could respond to wetland changes and revealed the functional potential of microbes in regulating biogeochemical cycles. Full article

Bistable expression of the Salmonella enterica pathogenicity island 1 (SPI-1) and the flagellar network (Flag) has been described previously. In this study, simultaneous monitoring of OFF and ON states in SPI-1 and in the flagellar regulon reveals independent switching, with concomitant formation of four subpopulations: SPI-1^OFF Flag^OFF, SPI-1^OFF Flag^ON, SPI-1^ON Flag^OFF, and SPI-1^ON Flag^ON. Invasion assays upon cell sorting show that none of the four subpopulations is highly invasive, thus raising the possibility that Flag^OFF cells might contribute to optimal invasion as previously proposed for SPI-1^OFF cells. Time lapse microscopy observation indicates that expression of the flagellar regulon contributes to the growth impairment previously described in SPI-1^ON cells. As a consequence, growth resumption in SPI-1^ON Flag^ON cells requires switching to both SPI-1^OFF and Flag^OFF states. Full article