Pathogenic potential assessment of the Shiga toxin-producing Escherichia coli by a source attribution-considered machine learning model.

Instead of conventional serotyping and virulence gene combination methods, methods have been developed to evaluate the pathogenic potential of newly emerging pathogens. Among them, the machine learning (ML)-based method using whole-genome sequencing (WGS) data are getting attention because of the recent advances in ML algorithms and sequencing technologies. Here, we developed various ML models to predict the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) isolates using their WGS data. The input dataset for the ML models was generated using distinct gene repertoires from positive (pathogenic) and negative (nonpathogenic) control groups in which each STEC isolate was designated based on the source attribution, the relative risk potential of the isolation sources. Among the various ML models examined, a model using the support vector machine (SVM) algorithm, the SVM model, discriminated between the two control groups most accurately. The SVM model successfully predicted the pathogenicity of the isolates from the major sources of STEC outbreaks, the isolates with the history of outbreaks, and the isolates that cannot be assessed by conventional methods. Furthermore, the SVM model effectively differentiated the pathogenic potentials of the isolates at a finer resolution. Permutation importance analyses of the input dataset further revealed the genes important for the estimation, proposing the genes potentially essential for the pathogenicity of STEC. Altogether, these results suggest that the SVM model is a more reliable and broadly applicable method to evaluate the pathogenic potential of STEC isolates compared with conventional methods.

The pathogenic potential and genetic attributes of Escherichia coli in milk from dairy cows with subclinical mastitis.

The centrality of milk and dairy products to the human diet allows potential pathogens to pose a threat to human health. Pathogenic Escherichia coli is a zoonotic foodborne pathogen with many virulence genes which cause variations in its pathogenicity. The current study aimed to investigate the pathogenic potential of E. coli from milk of dairy cows with subclinical mastitis and evaluate the genetic relatedness to E. coli from human sources. The majority of the E. coli isolates belonged to the A (55.0%) and B2 (22.5%) phylogenetic groups and the most prevalent virulence genes were colV (90.0%), fyuA (75.0%) and vat (42.5%). Mice injected with G4-BD23 (P < 0.05) and G5-BD3 had lower survival rates than controls and visible pathological changes to lung and kidney. Nineteen MLST types were identified in 40 dairy E. coli isolates and three STs (ST10, ST48 and ST942) were shared with those from human sources. Some dairy E. coli isolates were phylogenetically related to human E. coli isolates indicating pathogenic potential.

High survival rates of Campylobacter coli under different stress conditions suggest that more rigorous food control measures might be needed in Brazil.

Campylobacter spp. have been the most commonly reported gastrointestinal bacterial pathogen in many countries. Consumption of improperly prepared poultry meat has been the main transmission route of Campylobacter spp. Although Brazil is the largest exporter of poultry meat in the world, campylobacteriosis has been a neglected disease in the country. The aim of this study was to characterize 50 Campylobacter coli strains isolated from different sources in Brazil regarding the frequency of 16 virulence genes and their survival capability under five different stress conditions. All strains studied presented the cadF, flaA, and sodB genes that are considered essential for colonization. All strains grew at 4 °C and 37 °C after 24 h. High survival rates were observed when the strains were incubated in BHI with 7.5% NaCl and exposed to acid and oxidative stress. In conclusion, the pathogenic potential of the strains studied was reinforced by the presence of several important virulence genes and by the high growth and survival rates of the majority of those strains under different stress conditions. The results enabled a better understanding of strains circulating in Brazil and suggest that more rigorous control measures may be needed, given the importance of contaminated food as vehicles for Campylobacter coli.

Virulence-related genes, adhesion and invasion of some Yersinia enterocolitica-like strains suggests its pathogenic potential.

Yersina enterocolitica-like species have not been extensively studied regarding its pathogenic potential. This work aimed to assess the pathogenic potential of some Y. enterocolitica-like strains by evaluating the presence of virulence-related genes by PCR and their ability to adhere to and invade Caco-2 and HEp-2 cells. A total of 50 Y. frederiksenii, 55 Y. intermedia and 13 Y. kristensenii strains were studied. The strains contained the following genes: Y. frederiksenii, fepA(44%), fes(44%) and ystB(18%); Y. intermedia, ail(53%), fepA (35%), fepD(2%), fes(97%), hreP(2%), ystB(2%) and tccC(35%); Y. kristensenii, ail(62%), ystB(23%), fepA(77%), fepD(54%), fes(54%) and hreP(77%). Generally, the Y. enterocolitica-like strains had a reduced ability to adhere to and invade mammalian cells compared to the highly pathogenic Y. enterocolitica 8081. However, Y. kristensenii FCF410 and Y. frederiksenii FCF461 presented high invasion potentials in Caco-2 cells after five days of pre-incubation increased by 45- and 7.2-fold compared to Y. enterocolitica 8081, respectively; but, the ail gene was not detected in these strains. The presence of virulence-related genes in some of the Y. enterocolitica-like strains indicated their possible pathogenic potential. Moreover, the results suggest the existence of alternative virulence mechanisms and that the pathogenicity of Y. kristensenii and Y. frederiksenii may be strain-dependent.

Genotypic diversity and pathogenic potential of Yersinia enterocolitica biotype 2 strains isolated in Brazil.

AIMS: To investigate the pathogenic potential and genotypic diversity of Yersinia enterocolitica biotype 2 strains isolated in Brazil and to compare these strains with other Y. enterocolitica biotypes using ERIC-PCR and PFGE. METHODS AND RESULTS: Forty strains of Y. enterocolitica biotype 2 (B2) isolated from humans (5), the environment (34) and animal (1), in Brazil over 19 years were studied. In addition to these isolates, we also analysed 26 Y. enterocolitica strains belonging to the biotypes 1A, 1B, and 3-5. All of the B2 strains contained the genes inv, ail, ystA, hreP, tccC and myfA. The genes fepD and fes were detected in 39 (97·5%) strains, virF was found in three (7·5%) strains, and ystB and fepA were not detected in any strains. The B2 strains showed genotypic similarities of more than 84·8% by ERIC-PCR and of more than 69·0% by PFGE. CONCLUSIONS: The pathogenic potential of the B2 strains examined in this study was highlighted by the occurrence of the majority of the virulence markers searched. The results of the ERIC-PCR and PFGE showed that the B2 strains evaluated in this study had a high genotypic similarity, suggesting that these strains differed little over the 19 year study period and that the environment was a possible source of contamination of humans and animals in Brazil. Furthermore, the ERIC-PCR technique grouped the strains belonging to Y. enterocolitica biotypes 1A, 1B, 2, 3, 4 and 5 according to their pathogenicity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided new information about the pathogenic potential and genotypic similarity of Y. enterocolitica B2 isolated from diverse sources in Brazil. Furthermore, ERIC-PCR showed to be a valuable tool for grouping Y. enterocolitica of different biotypes according their pathogenicity.

Evaluation of the pathogenic potential, antimicrobial susceptibility, and genomic relations of Yersinia enterocolitica strains from food and human origin.

Yersinia enterocolitica is a food-borne pathogen that causes gastroenteritis with occasional postinfection sequels. This study was aimed to determinate the pathogenic potential, antimicrobial susceptibility, and genomic relationships of Y. enterocolitica strains of different bioserotypes (B/O) isolated from foods and human samples in San Luis, Argentina. Strains obtained by culture were bioserotyped and characterized by phenotypic and genotypic virulence markers, antimicrobial susceptibility, and pulsed-field gel electrophoresis (PFGE). Yersinia enterocolitica was detected in 9.2% of 380 samples, with a distribution of 10.6% (30/284) for food products and 5.2% (5/96) for human samples. Regarding the pathogenic potential, B1A strains of different serotypes were virF(-) ail(-), of which 72.0% (13/18) were ystB(+) with virulence-related phenotypic characteristics. Among B2/O:9 isolates, 75.0% (9/12) exhibited the genotype virF(+) ail(+) ystB(-) along with phenotypic traits associated with virulence; the same genotype was observed in 80.0% (4/5) of B3/O:3 and B3/O:5 strains. By PFGE, it was possible to separate Y. enterocolitica biotypes into 4 clonal groups (A to D) with 23 genomic types, generating a discriminatory index of 0.96. All isolates were susceptible to antimicrobials used for clinical treatment. This study highlights the presence of pathogenic bioserotypes and the high genomic diversity of the Y. enterocolitica strains isolated in our region.

Epidemiology and identification of potential fungal pathogens causing invasive fungal infections in a tertiary care hospital in northeast Thailand.

Invasive fungal infections (IFIs) are life threatening and associated with a high mortality rate. Here, we describe the distribution of pathogens, host risk factors, and significance of fungi isolated from patients with IFIs. The study included 861 fungal isolates recovered between 2006 and 2011 from 802 patients at Srinagarind Hospital, Thailand. Based on the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group 2008 criteria, 28.5% (245/861 isolates) of the fungal isolates were considered to be causative agents of IFIs. The most common fungus was Candida albicans (46%, 396/861 isolates). However, the most common yeast causing IFIs was Cryptococcus neoformans (34.7%, 85/245 isolates), while the most common mould was Penicillium marneffei (10.6%, 26/245 isolates). Cryptococcosis was significantly associated with human immunodeficiency virus infections (P < 0.001). Trend analysis revealed that there was no significant increase in IFI cases (P = 0.34) from 2006 to 2011 or from 2007 to 2011 (P = 0.05), but there was a trend toward significant increases in candidiasis (P = 0.04). The fungal isolates were categorized according to the positive predictive value of their recovery in cultures as being true (>95%), moderate (5%-95%), and rare (<5%) pathogens. This classification system could facilitate the prediction of the likelihood of diseases caused by the isolated fungi.

Lactobacillus gasseri associated severe soft tissue infection of the lower extremity: A case report.

Background: Lactobacilli are gram-positive, lactic acid-producing, facultative anaerobes of the human microbiota located in the human gastrointestinal tract, genitourinary tract, and the oral cavity and are considered non-pathogenic. When certain risk factors are present, they have the potential to cause serious infections. The incidence of localized infections associated with Lactobacilli are rare and to our knowledge we present the first known case of severe soft tissue infection of the extremity linked to a Lactobacillus strain. Case presentation: We describe the case of a 41-year-old man with a history of type 2 Diabetes Mellitus (DM), arterial hypertension and schizophrenia, who was admitted for weakness, high fever of 39.7 °C (103.5°F) and an abscess formation of the left thigh caused by an infection with Lactobacillus gasseri (L.gasseri). Conclusion: While infections caused by Lactobacilli are rare, it is crucial not to underestimate the potential of typically non-pathogenic bacteria like L. gasseri to act as infectious agents in immunocompromised patients. Abscess drainage and antibiotic treatment were successful treatment strategies for this rare case of soft tissue infection cause by L.gasseri.

Heterotrophic bacteria in drinking water: evaluating antibiotic resistance and the presence of virulence genes.

Heterotrophic bacteria, impacting those with infections or compromised immunity, pose heightened health risks when resistant to antibiotics. This study investigates heterotrophic plate count bacteria in water from North West-C (NWC) and North West-G (NWG) facilities, revealing prevalent ß-hemolysis (NWC 82.5%, NWG 86.7%), enzyme production (98%), and antibiotic resistance, especially in NWC. NWG exhibits variations in hemolysin (P = 0.013), lipase (P = 0.009), and DNase activity (P = 0.006). Antibiotics, including ciprofloxacin, persist throughout treatment, with high resistance to ß-lactams and trimethoprim (47%-100%), predominantly in NWC. Multiple antibiotic resistance index indicates that 90% of values exceed 0.20, signifying isolates from high antibiotic usage sources. Whole genome sequencing reveals diverse antibiotic resistance genes in heterotrophic strains, emphasizing their prevalence and health risks in water.IMPORTANCEThis study's findings are a stark reminder of a significant health concern: our water sources harbor antibiotic-resistant heterotrophic bacteria, which can potentially cause illness, especially in individuals with weakened immune systems or underlying infections. Antibiotic resistance among these bacteria is deeply concerning, as it threatens the effectiveness of antibiotics, critical for treating various infections. Moreover, detecting virulence factors in a notable proportion of these bacteria highlights their elevated risk to public health. This research underscores the immediate need for enhanced water treatment processes, rigorous water quality monitoring, and the development of strategies to combat antibiotic resistance in the environment. Safeguarding the safety of our drinking water is imperative to protect public health and mitigate the spread of antibiotic-resistant infections, making these findings a compelling call to action for policymakers and public health authorities alike.

Experimental comparison of pathogenic potential of two sibling species Anisakis simplex s.s. and Anisakis pegreffii in Wistar rat.

OBJECTIVES: There are little data available on the pathology caused by the sibling species Anisakis simplex s.s. and Anisakis pegreffii. The differences shown in their ability to penetrate the muscle of fish may also be manifested in humans. The purpose of this study is to confirm possible differences in pathogenicity between A. simplex s.s. and A. pegreffii using an experimental model which simulates infection in humans. METHODS: Female Wistar rats were infected with 190 Anisakis type I L3 larvae from the Iberian coastline. After the animal was sacrificed, these L3 larvae were then recovered and identified via PCR-RFLP of the ITS1-5.8S-ITS2. A logistic regression analysis was performed searching for association between experimental pathogenic potential and species. RESULTS: The distribution of A. simplex s.s. and A. pegreffii between Atlantic and Mediterranean waters of the Iberian Peninsula showed statistically significant differences (P < 0.001) which were not observed in the hybrid genotypes (P > 0.3). 21.6% showed pathogenic potential, interpreted as the capacity of the larvae to cause lesions, stick to the gastrointestinal wall or penetrate it. The species variable showed association with the pathogenic role of the larva (P = 0.008). Taking A. simplex s.s. as our reference, the OR for A. pegreffii is 0.351 (P = 0.028). CONCLUSIONS: Despite this difference, A. pegreffii is also capable of causing anisakiasis, being responsible for 14.3% of the penetrations of the gastric mucosa found in rats, which justifies both species being considered aetiologic agents of this parasitic disorder.

Pathogenic Potential and Antibiotic Susceptibility: A Comprehensive Study of Enterococci from Different Ecological Settings.

The pathway and the lifestyle of known enterococcus species are too complicated. The aim of the present study is to trace the path of pathogenicity of enterococci isolated from seven habitats (Cornu aspersum intestine; Bulgarian yoghurt; goat and cow feta cheese-mature and young, respectively; Arabian street food-doner kebab; cow milk; and human breast milk) by comparing their pathogenic potential. In total, 72 enterococcal strains were isolated and identified by MALDI-TOF, sequencing, and PCR. Hemolytic and gelatinase activity were biochemically determined. PCR was carried out for detection of virulence factors (cylB, esp, gls24, nucl, psaA, agg, gelE, and ace) and antibiotic resistance (erm, ermB, blaZ, vanA, aphA, mefA, gyrA, catpIP501, and aac6'-aph2″). Phenotypic antibiotic resistance was assigned according to EUCAST. Eleven representatives of the genus Enterococcus were identified: E. mundtii, E. casseliflavus, E. gilvus, E. pseudoavium, E. pallens, E. malodoratus, E. devriesei, E. gallinarum, E. durans, E. faecium, and E. faecalis. Twenty-two strains expressed α-hemolysis. Thirteen strains had the cylB gene. Only two strains expressed α-hemolysis and possessed the cylB gene simultaneously. Positive amplification for gelE was found in 35% of the isolates, but phenotypic gelatinase activity was observed only in three strains. All isolates showed varying antibiotic resistance. Only E. faecalis BM15 showed multiple resistance (AMP-HLSR-RP). Correlation between genotypic and phenotypic macrolide resistance was revealed for two E. faecalis strains.

Health risk posed by direct ingestion of yeasts from polluted river water.

River water is an essential human resource that may be contaminated with hazardous microorganisms. However, the risk of yeast infection through river water exposure is unclear because it is highly dependant on individual susceptibility and has therefore not been well-studied, to date. To evaluate this undefined risk, we analysed the fungal communities in less polluted (LP) and highly polluted (HP) river water, as determined using principal coordinate analysis of pollution indicators. We enumerated culturable yeasts using a thermally selective isolation procedure (37 °C) and thus promoted the growth of potentially opportunistic species. Yeast species identified as clinically relevant were then tested for antifungal resistance. In addition, we propose a quantitative microbial risk assessment (QMRA) framework to quantitatively assess the potential risk of yeast infection. Our results indicated that pollution levels significantly altered fungal communities (p = 0.007) and that genera representing opportunistic and pathogenic members were significantly more abundant in HP waters (p = 0.038). Additionally, the yeast species Candida glabrata and Clavispora lusitaniae positively correlated with other pollution indicators, demonstrating the species' indicator potential. Our QMRA results further indicate that higher risk of infection is associated with increased water pollution levels (considering both physicochemical and bacterial indicators). Furthermore, yeast species with higher pathogenic potential present an increased risk of infection despite lower observed concentrations in the river water. Interestingly, the bloom of Meyerozyma guilliermondii during the wet season suggests that other environmental factors, such as dissolved oxygen levels and water turbulence, might affect growth characteristics of yeasts in river water, which consequently affects the distribution of annual infection risks. The presence of antifungal resistant yeasts, observed in this study, could further contribute to variation in risk distribution. Research on the ecophysiology of yeasts in these environments is therefore necessary to ameliorate the uncertainty and sensitivity of the proposed QMRA model. In addition to the vital knowledge on opportunistic and pathogenic yeast occurrence in river water and their observed association with pollution, this study provides valuable methods and insights to initiate future QMRAs of yeast infections.

Prevalence, Virulence Potential, and Growth in Cheese of Bacillus cereus Strains Isolated from Fresh and Short-Ripened Cheeses Sold on the Italian Market.

This study investigated B. cereus presence in 122 samples belonging to 34 typologies of fresh or short-ripened cheeses made from cow, sheep, goat, or buffalo pasteurized milk, and sold on the Italian market. B. cereus was isolated at a prevalence of 9.8%, with a marked variability among cheese categories, and at low counts (always below 2.26 Log CFU/g). Twelve isolates were identified by MALDI-TOF analysis and typified by RAPD PCR as belonging to different B. cereus strains. All the strains were tested for the production of hemolysin BL, phosphatidylcholine-specific phospholipase C, proteases, and biofilm formation, and for the presence of chromosomal toxin-encoding genes (sph, plcA, cytK, entFM, bcet, nheA, nheB, nheC). Overall, 92% of strains harbored bcet, 75% the three genes nheA, nheB, and nheC, as well as plcA and sph, 67% entFM, and 33% cytK. All strains showed biofilm-forming ability. A chemical-physical characterization of the cheeses was also performed to show their suitability as substrates for B. cereus growth, showing high heterogeneity in terms of pH, aw, salt content, and concentration of organic acids. Finally, the ability to support spore germination and vegetative cell growth of a selected cheese was investigated in spores-inoculated samples maintained at 10 °C and 15 °C, showing the inhibitory effect of low storage temperatures.

Thermal and pigment characterization of environmental fungi in the urban heat island of Baltimore City.

One of the major barriers of fungal infections of mammals is the inability to grow and/or survive at mammalian body temperature, typically around 37°C. This has provided mammals an advantage over fungi. However, environmental fungi may soon adapt to persist at higher temperatures, consistent with mammalian body temperature, due to thermal selection pressures imposed by climate change, global warming, and increased frequency of extreme heat events. Consequently, there is a need for more updated information about the thermal tolerance range of fungi near humans, such as in urban areas. The heat island effect suggests that cities are up to 8°C warmer than their suburban counterparts because of increased heat production, asphalt coatings and reduced greenspace among other factors, and it is more common in lower income and marginalized urban communities. Thus, urban centers are at increased risk for the emergence of heat tolerant fungi. In this study, we developed a methodology to collect and archive fungal isolates from sidewalk and soil samples in both warmer and cooler neighborhoods in Baltimore, Maryland. We demonstrate a novel methodology for fungal sample collection from sidewalks, employing the use of standardized and commercially available taffy. Analysis of fungal isolates collected from warmer neighborhoods revealed greater thermal tolerance and lower pigmentation, suggesting local adaptation to heat. Lower pigmentation in hotter areas is consistent with the notion that fungi use pigmentation to help regulate their temperature. Further, we identified the robust presence of the polyextremotolerant fungus Aureobasidium pullalans from the warmest neighborhood in Baltimore, further showing that the extreme conditions of cities can drive proliferation of extremotolerant fungi. This study develops new techniques for environmental fungal collection and provides insight on the fungal census in an urban setting that can inform future work to study how urban environments may drive stress/thermotolerance in fungi, which could alter fungal interactions with humans and impact human health.

In vivo characterization of the novel ebolavirus Bombali virus suggests a low pathogenic potential for humans.

Ebolaviruses cause outbreaks of haemorrhagic fever in Central and West Africa. Some members of this genus such as Ebola virus (EBOV) are highly pathogenic, with case fatality rates of up to 90%, whereas others such as Reston virus (RESTV) are apathogenic for humans. Bombali virus (BOMV) is a novel ebolavirus for which complete genome sequences were recently found in free-tailed bats, although no infectious virus could be isolated. Its pathogenic potential for humans is unknown. To address this question, we first determined whether proteins encoded by the available BOMV sequence found in Chaerephon pumilus were functional in in vitro assays. The correction of an apparent sequencing error in the glycoprotein based on these data then allowed us to generate infectious BOMV using reverse genetics and characterize its infection of human cells. Furthermore, we used HLA-A2-transgenic, NOD-scid-IL-2γ receptor-knockout (NSG-A2) mice reconstituted with human haematopoiesis as a model to evaluate the pathogenicity of BOMV in vivo in a human-like immune environment. These data demonstrate that not only does BOMV show a slower growth rate than EBOV in vitro, but it also shows low pathogenicity in humanized mice, comparable to previous studies using RESTV. Taken together, these findings suggest a low pathogenic potential of BOMV for humans.

Invasion of intestinal cells by Staphylococcus warneri, a member of the human gut microbiota.

Coagulase negative staphylococci (CoNS) are a heterogeneous group of bacteria that colonize different types of human epithelia. These bacteria have a highly variable pathogenic potential ranging from avirulent species to major nosocomial pathogens. Staphylococcus warneri is a CoNS species considered to be nonpathogenic. Here, we identify that S. warneri is a natural member of both human and mouse gut microbiota. In addition, we demonstrate that this bacterium is able to get internalized into human cells. We show that S. warneri efficiently invades several human cell types and, more specifically, intestinal epithelial cells, using actin-dependent mechanisms. In contrast to bona fide pathogens, S. warneri does not actively replicate within intestinal cells or resist killing by macrophages. Together, our results highlight that bacteria from the human gut microbiota that are not associated with a high pathogenic potential, can actively invade intestinal cells and may, in this way, impact intestinal physiology.

Protective Properties of S-layer Protein 2 from Lactobacillus crispatus 2029 against Candida albicans Infections.

Previously, the protective role of the S-layer protein 2 (Slp2) of the vaginal Lactobacillus crispatus 2029 (LC2029) strain against foodborne pathogens Campylobacter jejuni, Salmonella enterica serovar Enteritidis, and Escherichia coli O157:H was demonstrated. We demonstrate the new roles of the Slp2-positive LC2029 strain and soluble Slp2 against C. albicans infections. We show that LC2029 bacteria can adhere to the surface of the cervical epithelial HeLa cells, prevent their contact with C. albicans, and block yeast transition to a pathogenic hyphal form. Surface-bound Slp2 provides the ability for LC2029 to co-aggregate with various C. albicans strains, including clinical isolates. C. albicans-induced necrotizing epithelial damage is reduced by colonization with the Slp2-positive LC2029 strain. Slp2 inhibits the adhesion of various strains of C. albicans to different human epithelial cells, blocks yeast transition to a pathogenic hyphal form, and prevents the colonization and pathogenic infiltration of mucosal barriers. Only Slp2 and LC2029 bacteria stimulate the production of protective human ß-defensin 3 in various epithelial cells. These findings support the anti-Candida albicans potential of the probiotic LC2029 strain and Slp2 and form the basis for further research on their ability to prevent and manage invasive Candida infections.

In Vitro Cytopathogenic Activities of Acanthamoeba T3 and T4 Genotypes on HeLa Cell Monolayer.

Amoebic keratitis and encephalitis are mainly caused by free-living amoebae of the genus Acanthamoeba, which consists of both pathogenic and nonpathogenic species. The global distribution, amphizoic properties and the severity of the disease caused by Acanthamoeba species have inspired the scientific community to put more effort into the isolation of Acanthamoeba, besides exploring the direct and indirect parameters that could signify a pathogenic potential. Therefore, this study was performed to characterize the pathogenic potential of Acanthamoeba isolated from contact lens paraphernalia and water sources in Malaysia. Various methodologies were utilized to analyze the thermotolerance and osmotolerance, the secretion level of proteases and the cytopathic effect of trophozoites on the cell monolayer. In addition, the in vitro cytopathogenicity of these isolates was assessed using the LDH-release assay. A total of 14 Acanthamoeba isolates were classified as thermo- and osmotolerant and had presence of serine proteases with a molecular weight of 45-230 kDa. Four T4 genotypes isolated from contact lens paraphernalia recorded the presence of serine-type proteases of 107 kDa and 133 kDa. In contrast, all T3 genotypes isolated from environmental samples showed the presence of a 56 kDa proteolytic enzyme. Remarkably, eight T4 and a single T3 genotype isolates demonstrated a high adhesion percentage of greater than 90%. Moreover, the use of the HeLa cell monolayer showed that four T4 isolates and one T3 isolate achieved a cytopathic effect in the range of 44.9-59.4%, indicating an intermediate-to-high cytotoxicity level. Apart from that, the LDH-release assay revealed that three T4 isolates (CL5, CL54 and CL149) and one T3 isolate (SKA5-SK35) measured an exceptional toxicity level of higher than 40% compared to other isolates. In short, the presence of Acanthamoeba T3 and T4 genotypes with significant pathogenic potential in this study reiterates the essential need to reassess the functionality of other genotypes that were previously classified as nonpathogenic isolates in past research.

Prevalence, Antimicrobial Resistance, and Whole Genome Sequencing Analysis of Shiga Toxin-Producing Escherichia coli (STEC) and Enteropathogenic Escherichia coli (EPEC) from Imported Foods in China during 2015-2021.

Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) are foodborne pathogens that cause hemolytic uremic syndrome and fatal infant diarrhea, respectively, but the characterization of these bacteria from imported food in China are unknown. A total of 1577 food samples from various countries during 2015-2021 were screened for STEC and EPEC, and the obtained isolates were tested for antimicrobial resistance and whole genome sequencing analysis was performed. The prevalence of STEC and EPEC was 1.01% (16/1577) and 0.51% (8/1577), respectively. Antimicrobial resistances to tetracycline (8%), chloramphenicol (8%), ampicillin (4%), ceftazidime (4%), cefotaxime (4%), and trimethoprim-sulfamethoxazole (4%) were observed. The antimicrobial resistance phenotypes corresponded with genotypes for most strains, and some resistance genes were related to mobile genetic elements. All 16 STEC isolates were eae negative, two solely contained stx1 (stx1a or stx1c), 12 merely carried stx2 (stx2a, stx2d, or stx2e), and two had both stx1 and stx2 (stx1c + stx2b, stx1a + stx2a + stx2c). Although they were eae negative, several STEC isolates carried other adherence factors, such as iha (5/16), sab (1/16), and lpfA (8/16), and belonged to serotypes (O130:H11, O8:H19, and O100:H30) or STs (ST297, ST360), which have caused human infections. All the eight EPEC isolates were atypical EPEC; six serotypes and seven STs were found, and clinically relevant EPEC serotypes O26:H11, O103:H2, and O145:H28 were identified. Two STEC/ETEC (enterotoxigenic E. coli) hybrids and one EPEC/ETEC hybrid were observed, since they harbored sta1 and/or stb. The results revealed that food can act as a reservoir of STEC/EPEC with pathogenic potential, and had the potential ability to transfer antibiotic resistance and virulence genes.

Hortaea werneckii isolates exhibit different pathogenic potential in the invertebrate infection model Galleria mellonella.

Hortaea werneckii is a black yeast with a remarkable tolerance to salt. Most studies have been dedicated to understanding how H. werneckii adapts to hypersaline environments. H. werneckii has an unconventional cell cycle in which it alternates between fission and budding, which is modulated by cell density. Additionally, H. werneckii can cause superficial mycosis of the palm and sole of humans. Here, we determine the impact of salt concentration on the EXF-2000 strain's cell division pattern and morphology by performing timelapse microscopy at different salt concentrations. At low density and no salt, EXF-2000 primarily grows as pseudohyphae dividing mainly by septation. When grown in the presence of salt at a similar concentration to saltwater or hypersaline environments, we observe it grows first by undergoing fission followed by budding at the poles. Then, we examined a collection of 16 isolates in the presence of 0.6M NaCl, including isolates from marine and hypersaline environments and isolates from patients. These isolates exhibit a wide diversity in colony shape and cellular morphology. The isolates grew as yeast, pseudohyphae, and true hyphae, indicating that isolates can exhibit various cell morphologies under similar environmental conditions. We used the insect larvae Galleria mellonella to determine the pathogenic potential of our isolates. We observe that only a subset of isolates can cause death in our model, and there was no correlation between H. werneckii morphology and capacity to cause disease. Taken together, H. werneckii genomic and phenotypic diversity can serve as a model to better understand how phenotypes and pathogenic potential evolve in environmental fungi.