First report of Streptococcus agalactiae isolated from a healthy captive sichuan golden snub-nosed monkey (Rhinopithecus roxellana) in China.

Streptococcus agalactiae (S. agalactiae) is an opportunistic pathogen, and to date, studies have mainly focused on S. agalactiae strains isolated from humans, dairy cows, and fish. We reported one S. agalactiae strain, named CFFB, which was isolated from a healthy Sichuan golden snub-nosed monkey. Classical bacteriological approaches, as well as, next-generation sequencing, comparative genomics, and mice challenge test were used to characterize this strain. CFFB was identified as serotype III, ST19 combination which is a common type found in human strains. Phylogenetic analysis showed that the genome of CFFB was closely related to human clinical isolates, rather far away from animal strains. In total, CFFB contained fewer virulence-associated genes and antibiotic resistance genes than human isolates that were close to CFFB in evolutionary relationships. In the mice challenge test, CFFB had a relative weak virulence that just caused death in 33 % of ICR mice at a dose of 108 CFU by intraperitoneal injection, and CFFB was reisolated from the cardiac blood of the dead mice. Meanwhile, two intact prophages (prophage 1 and 2) were identified in the CFFB genome and shared high similarities with phage Javan52 and Javan29 which from human S. agalactiae isolate Gottschalk 1002A and RBH03, respectively. Moreover, the type II-A CRISPR-Cas system was detected in the CFFB genome, and the spacers from CFFB were the same to the streptococci isolates from human. These results suggest that CFFB isolated from healthy Sichuan golden snub-nosed monkeys may have its origin in human S. agalactiae. Our results suggested some genomic similarities between the S. agalactiae colonized in Sichuan golden snub-nosed monkey and those in infected humans.

Phenotypic and genotypic determination of resistance to common disinfectants among strains of Acinetobacter baumannii producing and non-producing biofilm isolated from Iran.

BACKGROUND: Nosocomial infections are a global problem in hospitals all around the world. It is considered a major health problem, especially in developing countries. The increase in the patient's stay in hospitals has increased the mortality rate, and consequently, the costs drastically increase. The main purpose of using disinfectants in the hospital environment is to reduce the risk of nosocomial infections. Ethylene diamine tetra acetic acid (EDTA) causes lysis and increases susceptibility to antimicrobial agents in the planktonic form of bacteria. This substance affects the permeability of the outer membrane of bacteria. It also prevents the formation of biofilms by bacteria. MATERIALS AND METHODS: In the current study, 120 isolates of Acinetobacter baumannii (A. baumannii) were confirmed by phenotypic and genotypic methods. Antibiogram was performed and then the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of isolates against 5% sodium hypochlorite, ethanol %70, sayasept-HP 2%, chlorhexidine 2%, dettol 4/8% were evaluated. In addition, the disinfectant effect was re-evaluated with the mixture of EDTA solution. All isolates were examined for biofilm presence by crystal violet staining method in triplicates and repeated three times for each strain. Also for all isolates detection of efflux pump genes (Qac-E, qacE-Δ1, SUG-E) by PCR technique was done. RESULTS: Antibiogram results of A. baumannii showed that 6.7% were Multi-drug-resistant (MDR), and 89.2% were Extensively drug-resistant (XDR) isolates. The highest effect of disinfectants was related to 5% sodium hypochlorite, and the least effect was 70% ethanol. EDTA increases the efficacy of selected disinfectants significantly. The highest prevalence of the efflux pump genes was related to SUG-E (95%) and Qac-E (91.7%), and, the qacE-Δ1 gene with 12.5%. The biofilm production rate was 91.3% among all isolates. CONCLUSION: The best and safest way to disinfect hospital floors and surfaces is to choose the right disinfectants, and learn how to use them properly. In this study, a mixture of disinfectants and EDTA had a significant effect on bactericidal activity. it was found that improper use of disinfectants, especially the use of sub-inhibitory dilutions, increases the resistance of bacteria to disinfectants.

Data analysis of patients with positive mould or dimorphic fungal cultures from sterile sites.

Background: Moulds and dimorphic fungi are increasingly recognised as pathogens carrying high morbidity and mortality in critically ill and immune-compromised patients. The lack of surveillance data limits our understanding of these infections. Objectives: To determine the distribution, patient characteristics, risk factors, therapy and treatment outcome in patients with positive mould or dimorphic fungal cultures from sterile sites at a tertiary hospital in central South Africa. Method: All moulds or dimorphic fungi cultured from sterile specimens from 1 July 2014 to 30 June 2017 were identified retrospectively. Laboratory and clinical records were reviewed. Information collected included gender and age, type of specimen collected for investigation, specific fungi isolated, underlying conditions, other contributing risk factors, treatment and outcome of the patients. Results: Forty-eight patient records were analysed. Male and female patients were equally distributed. The mean age was 40.5 years (range 7-78 years). Aspergillus spp. were most commonly isolated. The most common underlying condition was HIV infection, followed by haematological conditions. Twenty-six (54.2%) patients received treatment involving antifungal therapy alone (n = 19; 73.1%), surgery alone (n = 5; 19.2%) or a combined medical and surgical approach (n = 2; 7.7%). Twenty-two (45.8%) patients received no treatment. The overall mortality rate was 25.0% (n = 12). Conclusion: The diagnosis of fungal infections remains challenging. In the current study, moulds and dimorphic fungi were isolated from at-risk patients' specimens. Despite treatment with appropriate antifungal agents, the associated mortality rate was high. Contribution: This study contributes to the growing body of knowledge on these potentially life-threatening infections.

Grapevine Pinot gris virus spreads in infected vineyards: latent infections have no direct impact on grape production.

BACKGROUND: Grapevine Pinot gris virus (GPGV) infects grapevines worldwide and causes symptoms such as chlorotic mottling and deformations on leaves, stunted shoots and short panicles, or none of these symptoms if it appears as latent infection. So far, the consequences of GPGV infections for winegrowers are difficult to assess since important information such as plant performance at different GPGV infection levels and symptom expression are not fully clarified. METHODS: In order to investigate the course of GPGV spread, annual visual evaluations and ELISA tests were conducted over 3-4 consecutive years in four GPGV-infected vineyards in southern Germany: GEM, HEC, NIM, and REI. The program PATCHY was used to analyze spatial disease patterns. Sanger sequencing was used to determine virus isolates in vines at different GPGV infection levels, to test their respective influence on symptom expression. Yield and GrapeScan (FTIR) analyses were conducted to test the impact of different GPGV infection levels and isolates on fruit quantity and quality. RESULTS: GPGV infections significantly increased in all four vineyards (GEM 22-32%, HEC 50-99%, NIM 83-90%, REI 56-76%) with significant spreading patterns across and along rows. Specific symptom progression patterns were not observed. According to our results, the virus isolate has an influence on whether symptoms develop during a GPGV infection. While yield analyses revealed that yield losses only occur in symptomatic vines and range from 13 to 96% depending on the severity of symptoms, latent infections have no impact on grape production. No relevant effects of GPGV infections on must quality were observed. CONCLUSIONS: Secondary spread of GPGV was observed in all vineyards monitored, indicating vector-borne transmission that is likely to be accelerated by human viticultural management. GPGV should be further monitored to prevent the accumulation of detrimental symptomatic isolates. The results of this study can be used to assess the risk of GPGV to viticulture and should be considered when developing management strategies against the virus.

Exploring the effects of whey protein components on the interaction and stability of cyanidin-3-O-glucoside.

BACKGROUND: Anthocyanins are susceptible to degradation due to external factors. Despite the potential for improved anthocyanin stability with whey protein isolate (WPI), the specific effects of individual components within WPI on the stability of anthocyanins have yet to be studied extensively. This study investigated the interaction of WPI, ß-lactoglobulin (ß-Lg), bovine serum albumin (BSA), and lactoferrin (LF) with cyanidin-3-O-glucoside (C3G), and also considered their effects on stability. RESULTS: Fluorescence analysis revealed static quenching effects between C3G and WPI, ß-Lg, BSA, and LF. The binding constants were 1.923 × 103 L · mol⁻¹ for WPI, 24.55 × 103 L · mol⁻¹ for ß-Lg, 57.25 × 103 L · mol⁻¹ for BSA, and 1.280 × 103 L · mol⁻¹ for LF. Hydrogen bonds, van der Waals forces, and electrostatic attraction were the predominant forces in the interactions between C3G and WPI and between C3G and BSA. Hydrophobic interaction was the main binding force in the interaction between C3G and ß-Lg and between C3G and LF. The binding of C3G with WPI, ß-Lg, BSA, and LF was driven by different thermodynamic parameters. Enthalpy changes (∆H) were -38.76 kJ · mol⁻¹ for WPI, -17.59 kJ · mol⁻¹ for ß-Lg, -16.09 kJ · mol⁻¹ for BSA, and 39.50 kJ · mol⁻¹ for LF. Entropy changes (∆S) were -67.21 J · mol⁻¹·K⁻¹ for WPI, 3.72 J · mol⁻¹·K⁻¹ for ß-Lg, 37.09 J · mol⁻¹·K⁻¹ for BSA, and 192.04 J · mol⁻¹·K⁻¹ for LF. The addition of C3G influenced the secondary structure of the proteins. The decrease in the α-helix content suggested a disruption and loosening of the hydrogen bond network structure. The presence of proteins enhanced the light stability and thermal stability (stability in the presence of light and heat) of C3G. In vitro simulated digestion experiments demonstrated that the addition of proteins led to a delayed degradation of C3G and to improved antioxidant capacity. CONCLUSION: The presence of WPI and its components enhanced the thermal stability, light stability, and oxidation stability of C3G. Preheated proteins exhibited a more pronounced effect than unheated proteins. These findings highlight the potential of preheating protein at appropriate temperatures to preserve C3G stability and bioactivity during food processing. © 2024 Society of Chemical Industry.

Two outbreaks and sporadic occurrences of Candida auris from one hospital in China: an epidemiological, genomic retrospective study.

OBJECTIVES: To investigate the clinical relevance, origin, transmission, and resistance of Candida auris (C. auris) isolates from two outbreaks and sporadic occurrences from one hospital in China. METHODS: A total of 135 C. auris isolates were collected. Clinical characteristics were obtained and antifungal susceptibility testing (AFST) was performed using the method of broth microdilution. Phylogenetic tree, WGS analysis, and single nucleotide polymorphisms (SNPs) were used to determine the origin, transmission, and resistance mechanisms. RESULTS: A total of 31 patients (91.2%, 31/34) received invasive medical procedures and 13 patients (38.2%, 13/34) had antifungal agents before C. auris infection/colonization, except one patient whose clinical information was missing. Only 4 cases of C. auris candidemia were observed. 18 patients died, 13 patients recovered, and the outcomes of 3 patients were not available. A total of 35 C. auris isolates, which were successfully cultivated and the first isolated or harbored specific drug-resistant phenotype from each patient, were selected to be sequenced and further analyzed. C. auris isolates presented low genetic variability and belonged to clade I, possibly originating from BJ004-H7 in Beijing. All 35 isolates were resistant to Fluconazole (FCZ) and amphotericin B (AMB), and 3 isolates were resistant to caspofungin (CAS). Mutations in ERG11 and FKS1 were linked to reduced azole and echinocandin susceptibility, respectively. CONCLUSIONS: Two outbreaks of highly clonal, multidrug-resistant C. auris isolates within the medical facility were reported. The intensive performance of disinfection measures helped block in-hospital transmission. Understanding the epidemiology, drug resistance and management of C. auris will be helpful for implementing effective infection control and treatment strategies.

Effects of probiotics fermentation on physicochemical properties of plum (Pruni domesticae semen) seed protein-based gel.

The plum seed protein isolates (PSPI) were used to prepare a gel by probiotics fermentation. The effects of fermentation time (from 0 to 12 h) on the physicochemical properties of PSPI gel were evaluated. The results showed that PSPI started to form a gel after 6 h of fermentation, as evidenced by a decrease in pH from 6.6 to 5.2, an increase in particle size from 10 µm to 40 µm, appearance of a new peak with retention time of 10 min in gel filtration high-performance liquid chromatography, and formation of aggregation and porous structure observed by fluorescence and scanning electron microscope. The PSPI gel from 9 h of fermentation exhibited the highest viscosity (318 Pa.s), storage modulus (18,000 Pa), water holding capacity (37 %), and gel strength (21.5 g) due to stronger molecular interactions such as hydrogen bond, electrostatic, hydrophobic interaction and disulfide bond. However, increasing fermentation time over 9 h led to disrupture of PSPI gel. Furthermore, the subunit around 15 kDa of PSPI disappeared after fermentation, indicating that the formation of PSPI gel was induced by both acidification and partial hydrolysis. Our results suggest that PSPI can provide an alternative for developing plant-based gel products.

A Retrospective Study (2019-2023) on the Prevalence and Antimicrobial Resistance of Isolates from Canine Clinical Samples Submitted to the University Veterinary Hospital in Stara Zagora, Bulgaria.

The identification of local susceptibility patterns is important for the elaboration of effective local antimicrobial use guidelines and improvement in treatment outcomes. This retrospective study investigated the prevalence of microbial pathogens in dogs over a five-year period (2019-2023) and their antimicrobial resistance patterns with an emphasis on multidrug-resistant strains on the basis of 896 swab samples submitted to the microbiological laboratory at the University Veterinary Hospital, Stara Zagora, Bulgaria. A total of 1247 strains-1046 bacteria and 201 yeasts-were isolated. An increased proportion of Staphylococcus spp. as an agent of infections in dogs along with significant decrease in the share of Streptococcus spp. (from 16.2% in 2019 to 7.7% in 2023) was found. The occurrence of Staphylococcus spp. in otitis externa increased from 53.4% in 2019 to 84.5% in 2023 (p < 0.0001). The resistance of Staphylococcus spp. isolates to amoxicillin/clavulanic acid and cephalexin increased significantly in 2023 vs. 2022. At the same time, increased susceptibility to amikacin was observed in 2023 vs. 2019. For Enterobacteriaceae, significantly decreased resistance against amikacin and marbofloxacin was demonstrated in 2023 compared to 2019. Multidrug resistance (MDR) was present in 405 of 1046 bacterial isolates (38.7%). More than 50% of streptococci and pseudomonads were MDR. Of the MDR staphylococci, 41.7% were isolated from skin lesions and 28.3% were isolated from otitis. More than half of the strains resistant to seven, eight and nine groups of antimicrobial drugs (AMDs) were from wounds/abscesses. The results highlighted the importance of regular local monitoring of the spread of bacterial strains in veterinary clinics and their susceptibility to AMDs with regard to successful therapy outcomes and control on MDR spread.

Tracing Acinetobacter baumannii's Journey from Hospitals to Aquatic Ecosystems.

BACKGROUND: This study provides a comprehensive analysis of Acinetobacter baumannii in aquatic environments and fish microbiota by integrating culture-dependent methods, 16S metagenomics, and antibiotic resistance profiling. METHODS: A total of 83 A. baumannii isolates were recovered using culture-dependent methods from intra-hospital infections (IHI) and wastewater (WW) and surface water (SW) samples from two southern Romanian cities in August 2022. The antibiotic susceptibility was screened using disc diffusion, microdilution, PCR, and Whole Genome Sequencing assays. RESULTS: The highest microbial load in the analyzed samples was found in Glina, Bucharest, for both WW and SW samples across all investigated phenotypes. For Bucharest isolates, the resistance levels corresponded to fluoroquinolones > aminoglycosides > ß-lactam antibiotics. In contrast, A. baumannii from upstream SW samples in Târgoviște showed the highest resistance to aminoglycosides. The blaOXA-23 gene was frequently detected in IHI, WW, and SW isolates in Bucharest, but was absent in Târgoviște. Molecular phylogeny revealed the presence of ST10 in Târgoviște isolates and ST2 in Bucharest isolates, while other minor STs were not specifically correlated with a sampling point. Using 16S rRNA sequencing, significant differences in microbial populations between the two locations was identified. The low abundance of Alphaproteobacteria and Actinobacteria in both locations suggests environmental pressures or contamination events. CONCLUSIONS: These findings indicate significant fecal contamination and potential public health risks, emphasizing the need for improved water quality monitoring and management.

Biofilm Formation and Antibiotic Resistance Profiles in Carbapenemase-Producing Gram-Negative Rods-A Comparative Analysis between Screening and Pathological Isolates.

(1) Background: Carbapenem-resistant (CR) bacteria pose a significant global public health challenge due to their ability to evade treatment with beta-lactam antibiotics, including carbapenems. This study investigates the biofilm-forming capabilities of CR clinical bacterial isolates and examines the impact of serum on biofilm formation. Additionally, the study evaluates the resistance profiles and genetic markers for carbapenemase production. (2) Methods: Bacterial isolates were collected from the microbiology laboratory of Mures County Clinical Hospital between October 2022 and September 2023. Pharyngeal and rectal swabs were screened for carbapenem-resistant bacteria using selective media. Lower respiratory tract samples were also analyzed for CR Gram-negative bacteria. The isolates were tested for their ability to form biofilms in the presence and absence of fetal bovine serum at 24 and 48 h. Carbapenemase production was detected phenotypically and confirmed via PCR for relevant genes. (3) Results: Out of 846 screened samples, 4.25% from pharyngeal swabs and 6.38% from rectal swabs tested positive for CR bacteria. Acinetobacter baumannii and Klebsiella pneumoniae were the most common species isolated. Biofilm formation varied significantly between clinical isolates and standard strains, with clinical isolates generally showing higher biofilm production. The presence of serum had no significant effect on biofilm formation in Klebsiella spp., but stimulated biofilm formation for Acinetobacter spp. Carbapenemase genes blaKPC, blaOXA-48-like, and blaNDM were detected in various isolates, predominantly in Klebsiella spp., but were not the main determinants of carbapenem resistance, at least in screening isolates. (4) Conclusions: This study highlights the variability in biofilm formation among CR clinical isolates and underscores the differences between the bacteria found as carriage versus infection. Both bacterial species and environmental factors variably influence biofilm formation. These insights are crucial for the development of effective treatment and infection control strategies in clinical settings.

Evaluation of MMV Pandemic Response Box compounds to identify potent compounds against clinically relevant bacterial and fungal clinical isolates in vitro.

Background: Multidrug resistant bacterial and fungal pathogens are resistant to a number of significant front-line drugs, hence, identification of new inhibitory agents to combat them is crucial. In this study, we aim to evaluate the activity of Pandemic Box compounds from Malaria Medicines Venture (MMV) against A. baumannii and P. aeruginosa bacterial, C. auris, C. albicans and A. niger fungal clinical isolates. Methods: Isolates were initially screened with 201 antibacterial and 46 antifungal compounds (10 µM) using a microbroth dilution in triplicates to determine MIC. A persister assay was performed for bacterial pathogens. Results: Out of 201 antibacterial compounds, twenty-nine and seven compounds inhibited the growth of A. baumannii and P. aeruginosa at 10 µM, respectively. MMV1580854, MMV1579788, eravacycline and epetraborole inhibited both the bacterial test isolates. In a persister assay, MMV1634390 showed complete bactericidal effect against A. baumannii. With antifungal activity compounds, C. auris responded to15 compounds, Six compounds inhibited C. albicans and one was effective against A. niger at 10 µM. The ratio of Minimum Fungicidal Concentration (MFC): Minimum Inhibitory Concentration (MIC) of MMV1782110 was 2 against C. auris. Eberconazole, amorolfine and luliconazole are fungicidal targeting C. albicans at a MFC: MIC ratio of 2. Conclusion: Five compounds from MMV Pandemic Box were found to be inhibiting colistin and ceftazidime resistant A. baumannii clinical isolate, also against colistin and ß-lactam resistant P. aeruginosa clinical isolate. MMV1634390 showed complete bactericidal effect against A. baumannii in a persister assay. MMV1782110, Eberconazole, amorolfine and luliconazole exhibited potent anti-fungal activity. Further investigations are warranted to identify the targets and mechanism.

Changing Paradigm of Yeast Isolates in HIV-Seropositive Patients with Oropharyngeal Candidiasis (OPC).

Background Oropharyngeal candidiasis (OPC) is a common fungal infection in HIV-seropositive patients. Understanding the spectrum of yeast isolates and their antifungal susceptibility patterns is crucial for effective management. This study aimed to determine the yeast isolates, antifungal susceptibility patterns, and associated factors in HIV-seropositive patients with OPC. Material and methods A prospective observational study was conducted on 350 HIV-seropositive patients attending an Integrated Counselling and Testing Centre (ICTC) at the Indira Gandhi Institute of Medical Sciences (IGIMS), Patna, Bihar. Yeast isolates from oropharyngeal lesions were identified, and their antifungal susceptibility was determined by automated method VITEK 2. Demographic characteristics, highly active antiretroviral therapy (HAART) status, and CD4+ cell count categories were analyzed for associations. Results This study of 350 HIV-seropositive patients revealed that 100 tested positive for Candida, with distinct differences between HAART (n=67) and non-HAART (n=33) groups. HAART patients had a younger age distribution and higher median CD4+ cell counts (350 vs. 250 cells/mm³, U = 175, p < 0.05) compared to non-HAART patients. Candida albicans was the most common species in both groups, but significant variations in species distribution (χ² = 9.23, p < 0.05) and antifungal susceptibility were noted. Specifically, susceptibility differences were significant for flucytosine (χ² = 7.21, p = 0.027) and voriconazole (χ² = 8.64, p = 0.013), emphasizing the influence of HAART on managing immune function and antifungal resistance in HIV patients. Conclusion This study provides insights into the spectrum of yeast isolates and their antifungal susceptibility patterns in HIV-seropositive patients with OPC. The findings emphasize the importance of considering multiple factors, such as Candida species, HAART status, and individual patient characteristics, in treatment decisions. The results will aid in the development of evidence-based management protocols for this vulnerable population. Further research is warranted to explore additional factors influencing antifungal susceptibility and optimize treatment strategies for this patient population.

Pathogenicity is associated with population structure in a fungal pathogen of humans.

Aspergillus flavus is a clinically and agriculturally important saprotrophic fungus responsible for severe human infections and extensive crop losses. We analyzed genomic data from 250 (95 clinical and 155 environmental) A. flavus isolates from 9 countries, including 70 newly sequenced clinical isolates, to examine population and pan-genome structure and their relationship to pathogenicity. We identified five A. flavus populations, including a new population, D, corresponding to distinct clades in the genome-wide phylogeny. Strikingly, > 75% of clinical isolates were from population D. Accessory genes, including genes within biosynthetic gene clusters, were significantly more common in some populations but rare in others. Population D was enriched for genes associated with zinc ion binding, lipid metabolism, and certain types of hydrolase activity. In contrast to the major human pathogen Aspergillus fumigatus, A. flavus pathogenicity in humans is strongly associated with population structure, making it a great system for investigating how population-specific genes contribute to pathogenicity.

Conversion of polyploid and alloploid Saccharomyces sensu stricto strains to leu2 mutants by genome DNA editing.

A large number of recombinant plasmids for the yeast Saccharomyces cerevisiae have been constructed and accumulated over the past four decades. It is desirable to apply the recombinant plasmid resources to Saccharomyces sensu stricto species group, which contains an increasing number of natural isolate and industrial strains. The application to the group encounters a difficulty. Natural isolates and industrial strains are exclusively prototrophic and polyploid, whereas direct application of most conventional plasmid resources imposes a prerequisite in host yeast strains of an auxotrophic mutation (i.e., leu2) that is rescued by a selection gene (e.g., LEU2) on the recombinant plasmids. To solve the difficulty, we aimed to generate leu2 mutants from yeast strains belonging to the yeast Saccharomyces sensu stricto species group by DNA editing. First, we modified an all-in-one type CRISPR-Cas9 plasmid pML104 by adding an antibiotic-resistance gene and designing guide sequences to target the LEU2 gene and to enable wide application in this yeast group. Then, the resulting CRISPR-Cas9 plasmids were exploited to seven strains belonging to five species of the group, including natural isolate, industrial, and allopolyploid strains. Colonies having the designed mutations in the gene appeared successfully by introducing the plasmids and assisting oligonucleotides to the strains. Most of the plasmids and resultant leu2- mutants produced in this study will be deposited in several repository organizations. KEY POINTS: • All-in-one type CRISPR-Cas9 plasmids targeting LEU2 gene were designed for broad application to Saccharomyces sensu stricto group species strains • Application of the plasmids generated leu2 mutants from strains including natural isolates, industrial, and allopolyploid strains • The easy conversion to leu2 mutants permits free access to recombinant plasmids having a LEU2 gene.

Biochemical characterisation and production kinetics of high molecular-weight (HMW) putative antibacterial proteins of insect pathogenic Brevibacillus laterosporus isolates.

BACKGROUND: Bacterial genomes often encode structures similar to phage capsids (encapsulins) and phage tails which can be induced spontaneously or using genotoxic compounds such as mitomycin C. These high molecular-weight (HMW) putative antibacterial proteins (ABPs) are used against the competitive strains under natural environment. Previously, it was unknown whether these HMW putative ABPs originating from the insect pathogenic Gram-positive, spore-forming bacterium Brevibacillus laterosporus (Bl) isolates (1821L, 1951) are spontaneously induced during the growth and pose a detrimental effect on their own survival. Furthermore, no prior work has been undertaken to determine their biochemical characteristics. RESULTS: Using a soft agar overlay method with polyethylene glycol precipitation, a narrow spectrum of bioactivity was found from the precipitated lysate of Bl 1951. Electron micrographs of mitomycin C- induced filtrates showed structures similar to phage capsids and contractile tails. Bioactivity assays of cell free supernatants (CFS) extracted during the growth of Bl 1821L and Bl 1951 suggested spontaneous induction of these HMW putative ABPs with an autocidal activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of spontaneously induced putative ABPs showed appearance of ~ 30 kDa and ~ 48 kDa bands of varying intensity across all the time intervals during the bacterial growth except in the initial hours. Statistically, spontaneously induced HMW putative ABPs of Bl 1951 exhibited a significant decrease in the number of viable cells of its producer strain after 18 h of growth in liquid. In addition, a significant change in pH and prominent bioactivity of the CFS of this particular time period was noted. Biochemically, the filtered supernatant derived from either Bl 1821L or Bl 1951 maintained bioactivity over a wide range of pH and temperature. CONCLUSION: This study reports the spontaneous induction of HMW putative ABPs (bacteriocins) of Bl 1821L and Bl 1951 isolates during the course of growth with potential autocidal activity which is critically important during production as a potential biopesticide. A narrow spectrum of putative antibacterial activity of Bl 1951 precipitate was found. The stability of HMW putative ABPs of Bl 1821L and Bl 1951 over a wide range of pH and temperature can be useful in expanding the potential of this useful bacterium beyond the insecticidal value.

Enteroaggregative Escherichia coli (EAEC) isolates obtained from non-diarrheic children carry virulence factor-encoding genes from Extraintestinal Pathogenic E. Coli (ExPEC).

Enteroaggregative E. coli (EAEC) is one of the most frequent pathogens isolated from diarrheal patients as well as from healthy individuals in Brazil and has recently also been implicated as an extraintestinal pathogenic E. coli (ExPEC) associated with bloodstream and urinary tract infections. In this study, 37 EAEC isolates, obtained from fecal samples of non-diarrheic children, were molecularly and phenotypically characterized to access the pathogenic features of these isolates. The EAEC isolates were assigned into the phylogroups A (54.1%), D (29.7%), B1 (13.5%) and B2 (2.7%); and harbored genes responsible for encoding the major pilin subunit of the aggregative adherence fimbriae (AAFs) or aggregate-forming pili (AFP) adhesins as follows: aggA (24.3%), agg3A (5.4%), agg4A (27.0%), agg5A (32.4%) and afpA (10.8%). The most frequent O:H serotypes were O15:H2 (8.1%), O38:H25 (5.4%) and O86:H2 (5.4%). Twenty-one isolates (56.8%) produce the aggregative adherence (AA) pattern on HeLa cells, and biofilm formation was more efficient among EAEC isolates harboring the aggA and agg5A genes. PFGE analysis showed that 31 (83.8%) of the isolates were classified into 10 distinct clusters, which reinforces the high diversity found among the isolates studied. Of note, 40.5% (15/37) of the EAEC isolates have a genetic profile compatible with E. coli isolates with intrinsic potential to cause extraintestinal infections in healthy individuals, and therefore, classified as EAEC/ExPEC hybrids. In conclusion, we showed the presence of EAEC/ExPEC hybrids in the intestinal microbiota of non-diarrheic children, possibly representing the source of some endogenous extraintestinal infections.

Comparative membrane proteomic analysis of Tritrichomonas foetus isolates.

Tritrichomonas foetus is a flagellated and anaerobic parasite able to infect cattle and felines. Despite its prevalence, there is no effective standardized or legal treatment for T. foetus-infected cattle; the vaccination still has limited success in mitigating infections and reducing abortion risk; and nowadays, the diagnosis of T. foetus presents important limitations in terms of sensitivity and specificity in bovines. Here, we characterize the plasma membrane proteome of T. foetus and identify proteins that are represented in different isolates of this protozoan. Additionally, we performed a bioinformatic analysis that revealed the antigenicity potential of some of those proteins. This analysis is the first study to identify common proteins at the plasma membrane of different T. foetus isolates that could be targets for alternative diagnostic or vaccine techniques in the future.

Characterisation of Legionella Clinical Isolates in Spain from 2012 to 2022.

Although cases of Legionnaires' disease are notifiable, data on the phenotypic and genotypic characterisation of clinical isolates are limited. This retrospective study aims to report the results of the characterisation of Legionella clinical isolates in Spain from 2012 to 2022. Monoclonal antibodies from the Dresden panel were used for phenotypic identification of Legionella pneumophila. Genotypic characterisation and sequence type assignment were performed using the Sequence-Based Typing scheme. Of the 1184 samples, 569 were identified as Legionella by culture. Of these, 561 were identified as L. pneumophila, of which 521 were serogroup 1. The most common subgroups were Philadelphia (n = 107) and Knoxville (n = 106). The SBT analysis revealed 130 different STs, with the most common genotypes being ST1 (n = 87), ST23 (n = 57), ST20 (n = 30), and ST42 (n = 29). Knoxville has the highest variability with 32 different STs. ST23 is mainly found in Allentown/France (n = 46) and ST42 in Benidorm (n = 18), whereas ST1 is widely distributed. The results demonstrate that clinical isolates show high genetic diversity, although only a few sequence types (STs) are responsible for most cases. However, outbreaks can also occur with rare genotypes. More data on LD and associated epidemiological studies are needed to establish the risk of an isolate causing outbreak in the future.

Genetic diversity and antifungal susceptibilities of environmental Cryptococcus neoformans and Cryptococcus gattii species complexes.

Cryptococcosis is an opportunistic systemic mycosis caused by Cryptococcus neoformans and C. gattii species complexes and is of increasing global importance. Maintaining continued surveillance of the antifungal susceptibility of environmental C. neoformans and C. gattii isolates is desirable for better managing cryptococcosis by identifying resistant isolates and revealing the emergence of intrinsically resistant species. Relevant research data from Egypt are scarce. Thus, this study aimed to report the genetic diversity of C. neoformans and C. gattii species complexes originating from different environmental sources in Egypt, antifungal susceptibility profiles, antifungal combinations, and correlations of susceptibility with genotypes. A total of 400 environmental samples were collected, 220 from birds and 180 from trees. Cryptococcus spp. were found in 58 (14.5%) of the samples, 44 (75.9%) of the isolates were recovered from birds and 14 (24.1%) from trees. These isolates were genotyped using M13 polymerase chain reaction-fingerprinting and URA5 gene restriction fragment length polymorphism analysis. Of the 31 C. neoformans isolates, 24 (77.4%), 6 (19.4%) and one (4.4%) belonged to VNI, VNII, and VNIII genotypes, respectively. The 27 C. gattii isolates belonged to VGI (70.4%), VGII (18.5%), and VGIII (11.1%) genotypes. Non-wild type C. neoformans and C. gattii isolates that may have acquired resistance to azoles, amphotericin B (AMB), and terbinafine (TRB) were observed. C. gattii VGIII was less susceptible to fluconazole (FCZ) and itraconazole (ITZ) than VGI and VGII. C. neoformans isolates showed higher minimum inhibitory concentrations (MICs) to FCZ, ITZ, and voriconazole (VRZ) than those of C. gattii VGI and VGII. Significant (P < 0.001) correlations were found between the MICs of VRZ and ITZ (r = 0.64) in both C. neoformans and C. gattii isolates, FCZ and TRB in C. neoformans isolates, and FCZ and TRB (r = 0.52) in C. gattii isolates.There is no significant differences in the MICs of TRB in combination with FCZ (P = 0.064) or in combination with AMB (P = 0.543) and that of TRB alone against C. gattii genotypes. By calculating the fractional inhibitory concentration (FIC) index, the combination of FCZ + AMB was synergistic against all tested genotypes. These findings expand our knowledge of ecological niches, genetic diversity, and resistance traits of C. neoformans and C. gattii genotypes in Egypt. Further investigations into how they are related to clinical isolates in the region are warranted.

Occurrence of Enterococci in the Process of Artisanal Cheesemaking and Their Antimicrobial Resistance.

Enterococci are a group of microorganisms that have a controversial position from some scientific points of view. The species of the greatest clinical importance are E. faecalis and E. faecium, which are common agents of nosocomial infections. However, enterococci also have important applications in the dairy industry, as they are used as non-starter lactic acid bacteria (NSLAB) in a variety of cheeses, especially artisanal cheeses. The aim of this study was to determine the presence of representatives from the Enterococcus genus using PCR and MALDI-TOF MS methods on samples of raw milk, processing environment swabs, and cheese from four different artisanal dairy plants in Slovakia. Among the 136 isolates of enterococci, 9 species of genus Enterococci (E. faecalis, E. faecium, E. durans, E. devriesi, E. hirae, E. italicus, E. casseliflavus, E. malodoratus, and E. gallinarum) were identified and were tested for their antimicrobial resistance (AMR) to 8 antibiotics (amoxicillin, penicillin, ampicillin, erythromycin, levofloxacin, vancomycin, rifampicin, and tetracycline); most of them were resistant to rifampicin (35.3%), ampicillin (22.8%), and tetracycline (19.9%). A PCR analysis of vanA (4.41%) and tetM (14.71%) revealed that antimicrobial resistance genes were present in not only phenotypic resistant isolates of enterococci but also susceptible isolates. The investigation of antimicrobial resistance in enterococci during the cheesemaking process can be a source of valuable information for public health in the concept of "One Health".