Acanthamoeba spp. are free-living amoebae that can cause life-threatening brain infections and can also infect the eyes and skin. Amoebas/cysts of this species have previously been isolated from the River Soar, which runs through the city of Leicester, England, and is frequently used by the public. It has also been found in grass samples collected from public parks and green spaces across the city. Acanthamoeba can host endosymbionts, which can include important human microbes, acting as Trojan horses. Although their zoonotic role is poorly understood, cysts of Acanthamoeba spp. and other free-living amoebae have been found in the faeces and/or the gastrointestinal tract of various animals, including domestic livestock (pigs, cows) and wildlife (squirrels, pigeons), suggesting a potential source of transmission. To investigate their zoonotic potential in pigeons, we analysed fresh droppings from 628 pigeons collected from various urban parks and green spaces in the city of Leicester and surrounding rural areas as follows: 136 samples in summer 2017, 140 in autumn, 124 in winter 2017/18, 135 in spring, and 94 in summer 2018. After appropriate pre-concentration, thin smears were stained with trichrome and examined by light microscopy. DNA was extracted using a Fast DNA® Spin Kit. PCR inhibitors were removed using the QIAamp® micro DNA extraction kit. The extracts were screened for Acanthamoeba spp. using a triplex real-time TaqMan PCR assay with an appropriate positive control. All samples tested microscopically and molecularly were negative. Although their zoonotic role is not well understood, a potential and unknown animal reservoir could explain the moderate to high distribution found in both the grass samples and the river, posing a potential risk to people involved in sports such as rowing, football, or rugby. Culture techniques should be used to better investigate the potential interaction of Acanthamoeba with urban pigeons.

The US Environmental Protection Agency's Contaminant Candidate List includes Acanthamoeba spp. and Naegleria fowleri due to their ability to withstand harsh environmental conditions and their function as reservoirs for various important human pathogens. These human-pathogenic free-living amoebas (FLAs) pose significant challenges for their elimination from water systems because of their resistance to commonly used disinfectants, including those employed in treating drinking water. Our research group has previously isolated and detected cysts/amoebas of Acanthamoeba spp. in water from various open water systems across Bombali and Tonkolili Districts in Sierra Leone (SL), emphasising a potential public health risk to the Sierra Leonean population. We have used the emerging guidelines to decontaminate different environments, including open and drinking water systems, available in the UK Recovery Handbook for Biological Incidents (UKRHBI) to identify applicable recovery options (ROs) to mitigate the presence of FLAs in water systems in SL. The UKRHBI provides customised ROs for decontaminating/managing environments affected by any biological hazard, which are appropriately selected according to the physiological characteristics of the biological hazard (in this case, FLAs) and the characteristics of the site, following an accessible methodology. The following ROs might be applicable to the characteristics of the water systems and areas monitored: restricting water use, encouraging bottled water for drinking, removing contamination sources, modifying water treatment systems (e.g., integrating ozone-based treatments), and sludge treatment (use of ultrasonic disintegration treatment and topsoil stabilisation treatment). Emerging technologies, such as photolysis of sodium chloride and sodium hypochlorite, have demonstrated efficacy in inactivating Acanthamoeba castellanii and have the potential to be adapted to target other chlorine-resistant pathogens. The adoption of these interventions by the relevant authorities in SL could lead to the effective protection of vulnerable populations, including children, the elderly, immunocompromised individuals, and Ebola survivors.

The pig is susceptible to numerous zoonoses and represents the main reservoir for some zoonoses. These include Paslahepevirus balayani (formerly called HEV), Mycobacterium avium paratuberculosis (MAP), and Brucella suis. These infections cause mild symptoms in pigs (the only one to cause truly evident symptoms is Brucella suis, which causes reproductive disorders) but have serious consequences in humans. The purpose of this study was to assess the swine population's exposure to three zoonotic infections (Brucella suis, Mycobacterium avium, and HEV) in the Campania region (Southern Italy). A total of 370 blood samples were collected and tested with commercial indirect ELISAs to evaluate the circulation of these pathogens. Prevalences of 41.4%, 3.5%, and 0% were found for HEV, MAP, and Brucella suis, respectively. The data obtained are in line with what has been found in other European countries or in other industrialized countries undergoing eradication plans for Brucella. The data relating to HEV have also been confirmed in other epidemiological studies in the literature (pigs and deer are the main reservoirs of the infection, and therefore, the presence of anti-HEV antibodies in these species is very frequent). The evaluation of risk factors highlighted how higher seroprevalences were obtained in animals at the end of their production careers. This study highlights the spread of some zoonoses in the pig population and the need for continuous surveillance in reservoir species.

Bacterial wilt of tomato is caused by the soilborn bacterium Ralstonia solanacearum (Rs). The disease is difficult to manage and produces serious yield losses. Natural products, such as plant extracts, represent new environmentally sustainable control strategies. The aim of this study was to evaluate the antibacterial efficiency of Melia azedarach bark extracts against the phytopathogenic bacteria Rs. Three bark extracts were performed using water and ethanol as solvents. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of bark extract against Rs were defined using the broth microdillution method. Motility inhibition and changes in colony morphology were assessed, and bacterial survival was evaluated on humid substrate supplemented with different bark extract amounts. Bark extract affected the viability, colony morphology (where changes in the shape, cell density, and edge morphology of the colony were observed), and motility (with swarming motility reduced by 47.61% with ½ bark extract MIC, and 66.67% with bark extract MIC) on Rs. The application of the extract on the humid substrate controlled Rs in fewer amounts than MIC (0.176 g/l). Bacterial motility has been described as a key feature for pathogenicity in Rs. In this context, the inhibitory effect of M. azedarach bark extract on bacterial motility, and its bactericidal activity when applied on the substrate (where Rs survives between growing seasons), showed that this natural product is a promising tool to be used as an efficient sustainable strategy for the control of bacterial wilt of tomato.

Rhizobia are scattered across 18 genera, in which several taxa are known to have conserved genetic structure across rRNA operons. Recent metagenomic studies have revealed that the 16S rRNA gene and corresponding variable regions may have insufficient taxonomic resolution for the accurate identification of rhizobia and related plant-associated bacteria such as agrobacteria. Therefore, some housekeeping genes, including gyrB and rpoB, are used as alternative markers to 16S to analyze rhizobia communities. However, the extent to which the targeted genes and their corresponding primers could be suitable in metagenomic studies of all the genera of rhizobia remains elusive. This work evaluates in silico the taxonomic resolution of partial regions of two housekeeping and 16S rRNA genes to differentiate between rhizobia and agrobacteria communities. For the evaluation, nine V-regions (V1 to V9) spanning the entire 16S rRNA gene and commonly targeted in bacterial metagenomic-based studies were selected. The full-length 16S rRNA gene sequences retrieved from genome data archived in GenBank were first aligned for 19 rhizobia and agrobacteria genera, and the alignment was adjusted to the total number of positions that correspond to those of each V-region. The number of positions in each edited dataset was used to calculate pairwise similarity distances, which were used to identify uniquely distinguishable taxa for a given V-region, mainly at 97% (OTU) and 100% (ASV) cut-offs. The same uniquely distinguishable taxa approach was applied to rpoB (two regions) and gyrB (one region) at ~98% and 100% thresholds to allow for a comparative evaluation. This study suggested V5-V7 as the best 16S rDNA v-region for differentiating all genera at a 100% threshold. However, the rpoB and gyrB markers outcompeted the 16S rDNA in terms of taxonomic resolution regardless of the threshold, possibly replacing the use of 16S rDNA V-regions in the metagenomics of rhizobia and agrobacteria.

Wolbachia pipientis is the most widespread endosymbiotic bacterium, known for its ability to alter host reproduction. In Naupactini weevils, their reproductive mode appears to be linked to Wolbachia density, whereby species with low levels or an absence of Wolbachia reproduce sexually, while high-density infections would promote parthenogenesis. Although the factors driving high Wolbachia loads remain unclear, evidence suggests that the diversity of the host’s microbiota may influence Wolbachia abundance. This study explored the bacterial microbiota of Naupactini species with contrasting reproductive modes and Wolbachia densities using targeted metagenomics. The species analysed included Naupactus dissimulator and Naupactus xanthographus (sexually reproducing), as well as Naupactus versatilis and Pantomorus postfasciatus (parthenogenetic), all of which were infected with the dominant strain within the tribe, i.e., wNau1. Notably, P. postfasciatus comprises both parthenogenetic (PpP) and sexually reproducing populations (PpS), providing unique insights into factors influencing Wolbachia colonization.

A total of 1,630,618 high-quality reads yielded 923 operational taxonomic units (OTUs) across 106 bacterial genera. The four most abundant genera, Wolbachia (26.47%), Spiroplasma (20.35%), Enterobacter (13.88%), and Rickettsia (11.49%), accounted for 72% of all reads. Parthenogenetic populations showed a marked predominance of Wolbachia and Rickettsia, whereas Spiroplasma and Enterobacter were the most prevalent symbionts in sexual populations, which also exhibited a richer microbiota in terms of the genera identified. Principal Coordinate Analysis based on Bray–Curtis distances clearly distinguished populations by reproductive mode. PpS’ bacterial microbiota differed significantly from both PpP and other sexually reproducing species, exhibiting a profile that combined features of both groups. The low relative abundance of Wolbachia, coupled with the increased levels of Enterobacter and Spiroplasma, distinguished it from parthenogenetic populations, while elevated Rickettsia levels set it apart from other sexually reproducing species. These observations suggest that Enterobacter and Spiroplasma may inhibit Wolbachia proliferation, whereas Rickettsia might promote its growth.

The use of microorganisms with antagonistic activity against one or more pathogens can be an alternative to chemical control in agroecosystems. In this work, the potential for biocontrol by antagonism of a collection of environmental bacterial isolates against a phytopathogenic bacterium of Solanaceae, Xanthomonas vesicatoria, which causes bacterial spot in tomato and pepper, was evaluated. Control of this disease is usually limited by the lack of resistant hosts, the threat to the environment and the global health risks posed by the continued use of chemical agents.

Bacteria were isolated from different environmental samples from disease-free areas and tested for their in vitro antagonistic capacity against a reference strain of X. vesicatoria. The isolates with the greatest antagonistic activity were selected for initial ex vivo assays to determine this activity in plant material against the reference strain, using leaves from tomato plants of a variety sensitive to bacterial spot. The isolates with the greatest activity were further characterized for their capacity to produce hydrolytic enzymes, such as proteases, lipases, amylases and DNases, and for their nitrogen fixation capacity.

Overall, the results revealed the presence of bacterial isolates able to inhibit the growth of X. vesicatoria in vitro and reduce the severity of bacterial spot symptoms ex vivo, in addition to producing different hydrolases related to biological control and biotechnological use, and fixing molecular nitrogen. The prospects for improving biological control strategies against bacterial spot in tomatoes and peppers caused by X. vesicatoria are promising, thus promoting sustainable agricultural production that favors ecosystem balance.

Funding: This work is part of the R+D+i project PID2021-123600OR-C44, funded by MICIU/AEI/10.13039/501100011033 and by ERDF A way to do Europe, ERDF/EU.

Legionella spp. are pathogens of free-living protozoa that can cause a respiratory disease called legionellosis. Pneumonia caused by L. micdadei is mainly associated with hospital infections and primarily affects immunosuppressed patients. The infection of host cells by L. micdadei is facilitated by specialized mechanisms through which the bacteria modulate host cell physiology to create a replicative niche. Central to this process is the Dot/Icm Type IV secretion system (T4SS), complemented by the Type II secretion system (T2SS) and other host-manipulation strategies involving specific proteins.

This study aimed to analyze the composition of the outer (OM) and inner membrane (IM) proteins isolated from L. micdadei bacteria.

The bacterial mass collected from BCYE plates was digested with DNase and RNase and then disintegrated in a French press. A sucrose density gradient was used to isolate the outer and inner membranes of L. micdadei. Proteins isolated from these membranes were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS-MS). The L. micdadei proteome was investigated through diverse subtractive proteomics approaches, e.g., identification of pathogen-specific essential proteins, and draggability.

A detailed analysis of the L. micdadei membrane proteome allowed for the identification of 1784 proteins, including virulence factors, proteins associated with the transport of substances across cell membranes and the synthesis of cell membrane components, proteins related to bacterial mobility, and signal transduction pathways. The virulence factors present in L. micdadei membranes included protein building type IVB and II secretion systems, as well as major outer membrane protein (MOMP), macrophage infectivity potentiator (Mip), periplasmic protein EnhC, RND efflux system, outer membrane lipoprotein, the NodT family, and phosphatidylglycerol-prolipoprotein diacylglyceryl transferase.

The identification of L. micdadei membrane proteins and the elucidation of their functions provide deeper insights into the mechanisms underlying the pathogenesis of these microorganisms.

Biofilms have a complex multicellular structure composed of bacteria and extracellular polymeric substances, which collectively display adhesive properties and allow bacterial cells to stick to each other and to given surfaces. Biofilms may be formed on the abiotic surfaces of medical instruments such as catheters or implants, as well as on biotic surfaces, e.g., living tissues. Biofilm structures are formed in order to increase bacteria’s chances of survival in unfavorable environmental conditions, prevent immune cells from eliminating bacterial cells, and to provide antibiotic resistance. Staphylococcus aureus is one of the most common sources of upper respiratory tract infections and is a procaryote with the ability to form biofilm structures. Biofilm is considered to be, among others, a virulence factorthat contributes to difficulties and therapeutic failures in treating antibiotic-resistant staphylococcal infections. This can pose a huge risk for individuals with an impaired function of the immune system, such as patients with CLL.

A total of 51 S. aureus strains were isolated from 34 patients with CLL treated in the Saint Jan of Dukla Oncology Centre of the Lublin Region (Poland). DNA from bacteria was extracted using the spin-column method and was later used to perform PCR reactions in order to detect the icaA, icaD, icaB, and icaC genes responsible for the formation of biofilm.

An analysis of the electrophoretic separation of amplification products in the studied S. aureus strains showed the presence of icaA genes in 49 (96.08%) isolates, while icaD, icaB, and icaC genes were detected in 45 (88.24%), 47 (92.16%), and 41 (80.4%) S. aureus isolates, respectively.

Our study found a very significant prevalence of all genes responsible for biofilm formation among isolated S. aureus strains, which, in turn, may contribute to chronic staphylococcal infections, difficulties in antibiotic therapies, and may also delay anticancer treatment, undermining its efficiency and even threatening the lives of patients.

Inga paterno produces seeds (Se) covered by a sarcotesta (Sa), both of which remain understudied. Meanwhile, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus (L-S) are commonly used in fermented products. This study investigated the potential of Se and Sa as substrates for probiotic fermentation using L-S.

Three formulations were prepared: 5%(w/v) Sa, 5%(w/v) Se, and a 1:1 mixture (Ae). These were sterilized, inoculated with L-S (7.67×10⁶ CFU/ml), fermented (72 hours at 35°C), and stored at refrigeration temperature (5°C). Antioxidant activity (AA), total phenolic content (TPC), condensed tannins (CT), flavonoids (FC), and phytic acid content (PAC) were measured at 0, 4, 11, and 25 days of storage. One-way ANOVA and Tukey's test (p < 0.05) were used to analyze the data.

L-S counts ranged from 6.69 to 9.41 log CFU/ml, with higher stability in Se (8.39 to 7.62 log CFU/ml) and Sa (8.44 to 8.01 log CFU/ml). AA ranged from 27.50 to 115.92 μg Trolox equivalents/ml, with Sa showing the highest and stable levels (84.07 to 113.46 log CFU/ml). PAC increased in Sa (from 34.22 to 42.54 μg of phytic acid /ml) but decreased in Se (from 40.13 to 37.73 μg of phytic acid /ml). CT decreased in all formulations (ranging from 1.53 to 0.70 mg of catechin equivalents/ml), and Sa presented a higher amount of FC (from 0.53 to 1.04 mg of quercetin equivalents/ml) than Se (from 0.03 to 0.08 mg of quercetin equivalents/ml) and Ea (from 0.09 to 0.21 mg of quercetin equivalents/ml). AA was mainly correlated with FC (R² = 0.6). L-S remained viable throughout storage in all formulations, while Sa provided a rich source of TPC and FC. These findings suggest that I. paterno sarcotesta can serve as promising substrates to produce a functional fermented beverage with enhanced antioxidant properties.