Assessment of the hypothetical protein BB0616 in the murine infection of Borrelia burgdorferi.

bb0616 of Borrelia burgdorferi, the Lyme disease pathogen, encodes a hypothetical protein of unknown function. In this study, we showed that BB0616 was not surface-exposed or associated with the membrane through localization analyses using proteinase K digestion and cell partitioning assays. The expression of bb0616 was influenced by a reduced pH but not by growth phases, elevated temperatures, or carbon sources during in vitro cultivation. A transcriptional start site for bb0616 was identified by using 5' rapid amplification of cDNA ends, which led to the identification of a functional promoter in the 5' regulatory region upstream of bb0616. By analyzing a bb0616-deficient mutant and its isogenic complemented counterparts, we found that the infectivity potential of the mutant was significantly attenuated. The inactivation of bb0616 displayed no effect on borrelial growth in the medium or resistance to oxidative stress, but the mutant was significantly more susceptible to osmotic stress. In addition, the production of global virulence regulators such as BosR and RpoS as well as virulence-associated outer surface lipoproteins OspC and DbpA was reduced in the mutant. These phenotypes were fully restored when gene mutation was complemented with a wild-type copy of bb0616. Based on these findings, we concluded that the hypothetical protein BB0616 is required for the optimal infectivity of B. burgdorferi, potentially by impacting B. burgdorferi virulence gene expression as well as survival of the spirochete under stressful conditions.

Aerobic spore-forming bacteria associated with ropy bread: Identification, characterization and spoilage potential assessment.

Aerobic spore-forming (ASF) bacteria have been reported to cause ropiness in bread. Sticky and stringy degradation, discoloration, and an odor reminiscent of rotting fruit are typical characteristics of ropy bread spoilage. In addition to economic losses, ropy bread spoilage may lead to health risks, as virulent strains of ASF bacteria are not uncommon. However, the lack of systematic approaches to quantify physicochemical spoilage characteristics makes it extremely difficult to assess rope formation in bread. To address this problem, the aim of this study was to identify, characterize and objectively assess the spoilage potential of ASF bacteria associated with ropy bread. Hence, a set of 82 ASF bacteria, including isolates from raw materials and bakery environments as well as strains from international culture collections, were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and their species identity confirmed by 16S rRNA and gyrA or panC gene sequencing. A standardized approach supported by objective colorimetric measurements was developed to assess the rope-inducing potential (RIP) of a strain by inoculating autoclaved bread slices with bacterial spores. In addition, the presence of potential virulence factors such as swarming motility or hemolysis was investigated. This study adds B. velezensis, B. inaquosorum and B. spizizenii to the species potentially implicated of causing ropy bread spoilage. Most importantly, this study introduces a standardized classification protocol for assessing the RIP of a bacterial strain. Colorimetric measurements are used to objectively quantify the degree of breadcrumb discoloration. Furthermore, our results indicate that strains capable of inducing rope spoilage in bread often exhibit swarming motility and virulence factors such as hemolysis, raising important food quality considerations.

Genomic Characterization and Safety Assessment of Bifidobacterium breve BS2-PB3 as Functional Food.

Our group had isolated Bifidobacterium breve strain BS2-PB3 from human breast milk. In this study, we sequenced the whole genome of B. breve BS2-PB3, and with a focus on its safety profile, various probiotic characteristics (presence of antibiotic resistance genes, virulence factors, and mobile elements) were then determined through bioinformatic analyses. The antibiotic resistance profile of B. breve BS2-PB3 was also evaluated. The whole genome of B. breve BS2-PB3 consisted of 2,268,931 base pairs with a G-C content of 58.89% and 2,108 coding regions. The average nucleotide identity and whole-genome phylogenetic analyses supported the classification of B. breve BS2-PB3. According to our in silico assessment, B. breve BS2-PB3 possesses antioxidant and immunomodulation properties in addition to various genes related to the probiotic properties of heat, cold, and acid stress, bile tolerance, and adhesion. Antibiotic susceptibility was evaluated using the Kirby-Bauer disk-diffusion test, in which the minimum inhibitory concentrations for selected antibiotics were subsequently tested using the Epsilometer test. B. breve BS2-PB3 only exhibited selected resistance phenotypes, i.e., to mupirocin (minimum inhibitory concentration/MIC >1,024 µg/ml), sulfamethoxazole (MIC >1,024 µg/ml), and oxacillin (MIC >3 µg/ml). The resistance genes against those antibiotics, i.e., ileS, mupB, sul4, mecC and ramA, were detected within its genome as well. While no virulence factor was detected, four insertion sequences were identified within the genome but were located away from the identified antibiotic resistance genes. In conclusion, B. breve BS2-PB3 demonstrated a sufficient safety profile, making it a promising candidate for further development as a potential functional food.

Acquisition of plasmids from Shiga toxin-producing Escherichia coli strains had low or neutral fitness cost on commensal E. coli.

Although it has been hypothesized that the acquisition of plasmids-especially those bearing virulence factors and antimicrobial resistance genes-increases the energetic burden and reduces the fitness of a bacterium in general, some results have challenged this view, showing little or no effect on fitness after plasmid acquisition, which may lead to change in the view that there are evolutionary barriers for a wide spread of such plasmids among bacteria. Here, to evaluate the fitness impact of plasmid-encoded antibiotic resistance and virulence genes, plasmids from O26:H11, O111:H8, and O118:H16 Shiga toxin-producing Escherichia coli (STEC) human and bovine isolates were transferred to the non-virulent E. coli HS and K-12 MG1655 strains. Sequencing and PCR were used to characterize plasmids, and to identify the presence of antimicrobial resistance and/or virulence genes. The fitness impact of plasmids encoding virulence and antimicrobial resistance upon bacterial hosts was determined by pairwise growth competition. Plasmid profile analysis showed that STEC strains carried one or more high and low molecular weight plasmids belonging to the B/O, F, I, K, P, Q, and/or X incompatibility groups encoding virulence genes (SPATE-encoding genes) and/or antimicrobial resistance genes (aadA1, strAB, tetA, and/or tetB). Competition experiments demonstrated that the biological cost of carriage of these plasmids by the commensal E. coli strain HS or the laboratory strain E. coli K-12 MG1655 was low or non-existent, ranging from - 4.7 to 5.2% per generation. This suggests that there are few biological barriers-or, alternatively, it suggests that there are biological barriers that we were not able to measure in this competition model-against the spread of plasmid encoding virulence and resistance genes from STEC to other, less pathogenic E. coli strains. Thus, our results, in opposition to a common view, suggest that the acquisition of plasmids does not significantly affect the bacteria fitness and, therefore, the theorized plasmid burden would not be a significant barrier for plasmid spread.

Complete genome sequence and potential pathogenic assessment of Flavobacterium plurextorum RSG-18 isolated from the gut of Schlegel's black rockfish, Sebastes schlegelii.

Flavobacterium plurextorum is a potential fish pathogen of interest, previously isolated from diseased rainbow trout (Oncorhynchus mykiss) and oomycete-infected chum salmon (Oncorhynchus keta) eggs. We report here the first complete genome sequence of F. plurextorum RSG-18 isolated from the gut of Schlegel's black rockfish (Sebastes schlegelii). The genome of RSG-18 consists of a circular chromosome of 5,610,911 bp with a 33.57% GC content, containing 4858 protein-coding genes, 18 rRNAs, 63 tRNAs and 1 tmRNA. A comparative analysis was conducted on 11 Flavobacterium species previously reported as pathogens or isolated from diseased fish to confirm the potential pathogenicity of RSG-18. In the SEED classification, RSG-18 was found to have 36 genes categorized in 'Virulence, Disease and Defense'. Across all Flavobacterium species, a total of 16 antibiotic resistance genes and 61 putative virulence factors were identified. All species had at least one phage region and type I, III and IX secretion systems. In pan-genomic analysis, core genes consist of genes linked to phages, integrases and matrix-tolerated elements associated with pathology. The complete genome sequence of F. plurextorum RSG-18 will serve as a foundation for future research, enhancing our understanding of Flavobacterium pathogenicity in fish and contributing to the development of effective prevention strategies.

A Community-Curated DokuWiki Resource on Diagnostics, Diversity, Pathogenicity, and Genetic Control of Xanthomonads.

Xanthomonads, including Xanthomonas and Xylella species, constitute a large and significant group of economically and ecologically important plant pathogens. Up-to-date knowledge of these pathogens and their hosts is essential for the development of suitable control measures. Traditional review articles or book chapters have inherent limitations, including static content and rapid obsolescence. To address these challenges, we have developed a Web-based knowledge platform dedicated to xanthomonads, inspired by the concept of living systematic reviews. This platform offers a dynamic resource that encompasses bacterial virulence factors, plant resistance genes, and tools for diagnostics and genetic diversity studies. Our goal is to facilitate access for newcomers to the field, provide continuing education opportunities for students, assist plant protection services with diagnostics, provide valuable information to breeders on sources of resistance and breeding targets, and offer comprehensive expert knowledge to other stakeholders interested in plant-pathogenic xanthomonads. This resource is available for queries and updates at https://euroxanth.ipn.pt. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Prevalence and In Vivo Assessment of Virulence in Shiga Toxin-Producing Escherichia coli Clinical Isolates from Greater Cairo Area.

Background: Shiga toxin-producing Escherichia coli (STEC) has been identified as an important etiologic agent of human disease in Egypt. Aims: To investigate the occurrence and describe the characterization as well as prevalence of STEC in Greater Cairo hospitals as well as molecular characterization of virulence and resistance genes. Methods: Four hundred seventy E. coli clinical isolates were collected from eight hospitals and analyzed by genotypic and phenotypic methods for STEC, followed by histopathological examination and scoring of different organs lesions. Results: The highest proportion of isolates was from urine (151 isolates), whereas the lowest was from splenic drain (3 isolates). In tandem, when serogrouping was performed, 15 serogroups were obtained where the most prevalent was O157 and the least prevalent was O151. All isolates were positive when screened for identity gene gad A, while only typable strains were screened for seven virulence genes stx1 (gene encoding Shiga toxin 1), stx2 (gene encoding Shiga toxin 2), tsh (gene encoding thermostable hemagglutinin), eaeA (gene encoding intimin), invE (gene encoding invasion protein), aggR (gene encoding aggregative adherence transcriptional regulator), and astA (aspartate transaminase) where the prevalence was 48%, 30%, 50%, 57%, 7.5%, 12%, and 58%, respectively. Of 254 typable isolates, 152 were STEC carrying stx1 or stx2 genes or both. Conclusions: Relying on in vivo comparison between different E. coli pathotypes via histopathological examination of different organs, E. coli pathotypes could be divided into mild virulent, moderate virulent, and high virulent strains. Statistical analysis revealed significant correlation between different serogroups and presence of virulence genes.

In vitro and in silico assessment of anti-quorum sensing activity of Naproxen against Pseudomonas aeruginosa.

Serious infections caused by Pseudomonas aeruginosa are usually related to quorum sensing (QS)-dependent virulence factors. Hence, QS inhibition is a promising approach to overcoming P. aeruginosa infections. This study aimed to investigate the effect of naproxen on biofilm formation and QS-related virulence traits of P. aeruginosa. Furthermore, the anti-QS potential of naproxen was evaluated using real-time PCR and molecular docking analysis. Our findings supported the anti-QS activity of naproxen, as evidenced by down-regulation of the lasI and rhlI genes expression as well as the attenuation of bacterial protease, hemolysin, pyocyanin, biofilm, and motility. Additionally, the high binding affinity of naproxen with QS regulatory proteins was determined in the molecular docking simulation. Altogether, these findings suggest that naproxen has a promising potential in inhibiting QS-associated traits of P. aeruginosa.

Pectate Lyase from Fusarium sacchari Induces Plant Immune Responses and Contributes to Virulence.

Fusarium sacchari is one of the primary pathogens causing Pokkah Boeng disease (PBD) in sugarcane in China. Pectate lyases (PL), which play a critical role in pectin degradation and fungal virulence, have been extensively studied in major bacterial and fungal pathogens of a wide range of plant species. However, only a few PLs have been functionally investigated. In this study, we analyzed the function of the pectate lyase gene, FsPL, from F. sacchari. FsPL is a key virulence factor of F. sacchari and can induce plant cell death. FsPL also triggers the pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) response in Nicotiana benthamiana, as reflected by increases in reactive oxygen species (ROS) production, electrolyte leakage, and callose accumulation, as well as the upregulation of defense response genes. In addition, our study also found that the signal peptide of FsPL was necessary for induced cell death and PTI responses. Virus-induced gene silencing showed that FsPL-induced cell death in Nicotiana benthamiana was mediated by leucine-rich repeat (LRR) receptor-like kinases BAK1 and SOBIR1. Thus, FsPL may not only be a critical virulence factor for F. sacchari but may also induce plant defense responses. These findings provide new insights into the functions of pectate lyase in host-pathogen interactions. IMPORTANCE Pokkah Boeng disease (PBD) is one of the main diseases affecting sugarcane in China, seriously damaging sugarcane production and economic development. Therefore, it is important to clarify the pathogenic mechanisms of this disease and to provide a theoretical basis for the breeding of PBD-resistant sugarcane strains. The present study aimed to analyze the function of FsPL, a recently identified pectate lyase gene from F. sacchari. FsPL is a key virulence factor of F. sacchari that induces plant cell death. Our results provide new insights into the function of pectate lyase in host-pathogen interactions.

Assessment of the influence of selected stress factors on the growth and survival of Listeria monocytogenes.

BACKGROUND: Listeria monocytogenes are Gram-positive rods, which are the etiological factor of listeriosis. L. monocytogenes quickly adapts to changing environmental conditions. Since the main source of rods is food, its elimination from the production line is a priority. The study aimed to evaluate the influence of selected stress factors on the growth and survival of L. monocytogenes strains isolated from food products and clinical material. RESULTS: We distinguished fifty genetically different strains of L. monocytogenes (PFGE method). Sixty-two percent of the tested strains represented 1/2a-3a serogroup. Sixty percent of the rods possessed ten examined virulence genes (fbpA, plcA, hlyA, plcB, inlB, actA, iap, inlA, mpl, prfA). Listeria Pathogenicity Island 1 (LIPI-1) was demonstrated among 38 (76.0%) strains. Majority (92.0%) of strains (46) were sensitive to all examined antibiotics. The most effective concentration of bacteriophage (inhibiting the growth of 22 strains; 44.0%) was 5 × 108 PFU. In turn, the concentration of 8% of NaCl was enough to inhibit the growth of 31 strains (62.0%). The clinical strain tolerated the broadest pH range (3 to 10). Five strains survived the 60-min exposure to 70˚C, whereas all were alive at each time stage of the cold stress experiment. During the stress of cyclic freezing-defrosting, an increase in the number of bacteria was shown after the first cycle, and a decrease was only observed after cycle 3. The least sensitive to low nutrients content were strains isolated from frozen food. The high BHI concentration promoted the growth of all groups. CONCLUSIONS: Data on survival in stress conditions can form the basis for one of the hypotheses explaining the formation of persistent strains. Such studies are also helpful for planning appropriate hygiene strategies within the food industry.

Assessment of virulence factors and antimicrobial resistance among the Pseudomonas aeruginosa strains isolated from animal meat and carcass samples.

BACKGROUND: Pseudomonas aeruginosa bacteria are emerging causes of food spoilage and foodborne diseases. Raw meat of animal species may consider a reservoir of P. aeruginosa strains. OBJECTIVES: The present survey was done to assess the prevalence, antibiotic resistance properties and distribution of virulence factors among the P. aeruginosa strains isolated from raw meat and carcass surface swab samples of animal species. METHODS: Five hundred and fifty raw meat and carcass surface swab samples were collected from cattle and sheep species referred to as slaughterhouses. P. aeruginosa bacteria were identified using culture and biochemical tests. The pattern of antibiotic resistance was determined by disk diffusion. The distribution of virulence and antibiotic resistance genes was determined using polymerase chain reaction. RESULTS: Forty-seven of 550 (8.54%) examined samples were contaminated with P. aeruginosa. The prevalence of P. aeruginosa in raw meat and carcass surface swab samples were 6.57 and 12%, respectively. P. aeruginosa isolates showed the maximum resistance rate toward penicillin (87.23%), ampicillin (85.10%), tetracycline (85.10%), gentamicin (65.95%) and trimethoprim (57.44%). The most commonly detected antibiotic resistance genes were BlaCTX-M (53.19%), blaDHA (42.55%) and blaTEM (27.65%). The most commonly detected virulence factors was ExoS (42.55%), algD (31.91%), lasA (31.91%), plcH (31.91%) and exoU (25.53%). CONCLUSIONS: Meat and carcass surface swab samples may be sources of resistant and virulent P. aeruginosa, which pose a hygienic threat in their consumption. However, further investigations are required to identify additional epidemiological features of P. aeruginosa in meat and carcass surface samples.

Assessment of stn, sipB and sopB Virulence Genes in Various Salmonella Serovars.

Salmonella is a zoonotic bacterium that is considered to be one of the most common causes of foodborne infections worldwide. Bearing in mind the genes involved in its virulence, identifying these genes can enable experts to better understand bacterial pathogenicity, which could subsequently help develop more efficient means to control and prevent infections. This study aimed to analyze stn, sipB, and sopB genes in various Salmonella serovars. To carry out this study, 103 Salmonella serovars were extracted from livestock, poultry, and humans from existing samples at the Department of Microbiology of the Razi Serum and Vaccine Research Institute in Karaj, Iran. These samples were cultured in selection and differential media, and their serovars were identified using specific antibodies based on Kaufman-White Tables. Utilizing PCR and specific primers, stn, sopB, and sipB genes were detected among these serovars. In this investigation, the most common human serovars were Salmonella paratyphi A, Salmonella paratyphi B, and Salmonella enteritidis; the most common serovars among livestock consisted of Salmonella dublin and Salmonella typhimurium and the most common Salmonella serovars among poultry consisted of Salmonella infantis and Salmonella enteritidis. The results of PCR on stn, sipB, and sopB genes demonstrated segments with 617bp, 875 bp, and 220 bp on agar gel, respectively. Based on the obtained findings, stn, sipB, and sopB genes were detected in 96.11%, 99.02%, and 98.05% of Salmonella serovars, respectively. Considering the fact that the aforementioned genes play significant roles in bacterial virulence, they can be used to develop diagnostic ELISA kits and recombinant vaccines.

Phenotypic Assessment of Clinical Escherichia coli Isolates as an Indicator for Uropathogenic Potential.

For women in the United States, urinary tract infections (UTIs) are the most frequent diagnosis in emergency departments, comprising 21.3% of total visits. Uropathogenic Escherichia coli (UPEC) causes ~80% of uncomplicated UTIs. To combat this public health issue, it is vital to characterize UPEC strains as well as to differentiate them from commensal strains to reduce the overuse of antibiotics. It has been challenging to determine a consistent genetic signature that clearly distinguishes UPEC from other E. coli strains. Therefore, we examined whether phenotypic data could be predictive of uropathogenic potential. We screened 13 clinical strains of UPEC, isolated from cases of uncomplicated UTI in young otherwise healthy women, in a series of microbiological phenotypic assays using UPEC prototype strain CFT073 and nonpathogenic E. coli strain MG1655 K-12 as controls. Phenotypes included adherence, iron acquisition, biofilm formation, human serum resistance, motility, and stress resistance. By use of a well-established experimental mouse model of UTI, these data were able to predict the severity of the bacterial burden in both the urine and bladders. Multiple linear regression using three different phenotypic assays, i.e., growth in minimal medium, siderophore production, and type 1 fimbrial expression, was predictive of bladder colonization (adjusted R2 = 0.6411). Growth in ex vivo human urine, hemagglutination of red blood cells, and motility modeled urine colonization (adjusted R2 = 0.4821). These results showcase the utility of phenotypic characterization to predict the severity of infection that these strains may cause. We predict that these methods will also be applicable to other complex, genetically redundant, pathogens. IMPORTANCE Urinary tract infections are the second leading infectious disease worldwide, occurring in over half of the female population during their lifetime. Most infections are caused by uropathogenic Escherichia coli (UPEC) strains. These strains can establish a reservoir in the gut, in which they do not cause disease but, upon introduction to the urinary tract, can infect the host and elicit pathogenesis. Clinically, it would be beneficial to screen patient E. coli strains to understand their pathogenic potential, which may lead to the administration of prophylactic antibiotic treatment for those with increased risk. Others have proposed the use of PCR-based genetic screening methods to detect UPEC strains and differentiate them from other E. coli pathotypes; however, this method has not yielded a consistent uropathogenic genetic signature. Here, we used phenotypic characteristics such as growth rate, siderophore production, and expression of fimbriae to better predict uropathogenic potential.

Molecular Assessment of Staphylococcus Aureus Strains in STAT3 Hyper-IgE Syndrome Patients.

Hyper-IgE syndromes (HIES) are a group of inborn errors of immunity (IEI) caused by monogenic defects such as in the gene STAT3 (STAT3-HIES). Patients suffering from HIES show an increased susceptibility to Staphylococcus aureus (S. aureus) including skin abscesses and pulmonary infections. To assess if the underlying immune defect of STAT3-HIES patients influences the resistance patterns, pathogenicity factors or strain types of S. aureus. We characterized eleven S. aureus strains isolated from STAT3-HIES patients (n = 4) by whole genome sequencing (WGS) to determine presence of resistance and virulence genes. Additionally, we used multi-locus sequence typing (MLST) and protein A (spa) typing to classify these isolates. Bacterial isolates collected from this cohort of STAT3-HIES patients were identified as common spa types in Germany. Only one of the isolates was classified as methicillin-resistant S. aureus (MRSA). For one STAT3 patient WGS illustrated that infection and colonization occurred with different S. aureus isolates rather than one particular clone. The identified S. aureus carriage profile on a molecular level suggests that S. aureus strain type in STAT3-HIES patients is determined by local epidemiology rather than the underlying immune defect highlighting the importance of microbiological assessment prior to antibiotic treatment.

Phenotype-Based Threat Assessment.

Bacterial pathogen identification, which is critical for human health, has historically relied on culturing organisms from clinical specimens. More recently, the application of machine learning (ML) to whole-genome sequences (WGSs) has facilitated pathogen identification. However, relying solely on genetic information to identify emerging or new pathogens is fundamentally constrained, especially if novel virulence factors exist. In addition, even WGSs with ML pipelines are unable to discern phenotypes associated with cryptic genetic loci linked to virulence. Here, we set out to determine if ML using phenotypic hallmarks of pathogenesis could assess potential pathogenic threat without using any sequence-based analysis. This approach successfully classified potential pathogenetic threat associated with previously machine-observed and unobserved bacteria with 99% and 85% accuracy, respectively. This work establishes a phenotype-based pipeline for potential pathogenic threat assessment, which we term PathEngine, and offers strategies for the identification of bacterial pathogens.

Safety assessment and probiotic characteristics of Enterococcus lactis JDM1.

OBJECTIVE: The aims of this study were to evaluate the safety and probiotic characteristics of the newly isolated Enterococcus lactis strain JDM1. METHODS: Safety assessment of E. lactis JDM1 was accomplished by the combination of whole genome sequence information analysis and phenotypic assays, including antimicrobial susceptibility test, haemolysis assay, biogenic amine production assay, cytotoxicity assay. The bacteriostatic experiment and gastrointestinal tolerance experiment were also conducted to evaluate its applicability. RESULTS: E. lactis JDM1 possesses good gastrointestinal tolerance and can inhibit the growth of the pathogenic bacteria Clostridioides difficile and Listeria monocytogenes. The chromosome size of JDM1 was 2,570,998 bp with a GC content of 38.46%, which contained a plasmid. One intact prophage, 13 genomic islands and 19 IS elements were predicted in the JDM1 chromosome. Five resistance-related genes and seven virulence-related genes were predicted in the genome. Most resistance genes were conserved, and virulence factors were not related to functional pathogenicity. Antimicrobial susceptibility tests showed that JDM1 was sensitive to tedizolid, ciprofloxacin, levofloxacin, penicillin, ampicillin, vancomycin, linezolid, tetracycline, high-level gentamicin and high-level streptomycin. Genes encoding putative enzymes responsible for adverse metabolites were not found and JDM1 was unable to produce the six main biogenic amines. Cytotoxicity test showed that the JDM1 supernatant had no toxic effect. CONCLUSION: E. lactis JDM1 is expected to be developed as a probiotic, and its probiotic properties are worthy of further exploration.

Phenotypic and Genetic Characterization of Flavobacterium psychrophilum Recovered from Diseased Salmonids in China.

Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome, causes great economic losses in salmonid aquaculture worldwide. Recent molecular studies have uncovered important epidemiological and ecological aspects of this pathogen; however, such data are lacking for F. psychrophilum populations affecting aquaculture in China. Herein, F. psychrophilum phenotype, genotype, and virulence were characterized for isolates recovered from epizootics in multiple salmonid aquaculture facilities across China. Thirty-one F. psychrophilum isolates, originating from four provinces and three host fish species, were predominantly homogeneous biochemically but represented 5 sequence types (STs) according to multilocus sequence typing (MLST) that belonged to clonal complex CC-ST10 or 3 newly recognized singleton STs. PCR-based serotyping classified 19 and 12 F. psychrophilum isolates into molecular serotypes 1 and 0, respectively, showing an obvious relationship with host species. Antimicrobial susceptibility analysis via broth microdilution revealed reduced susceptibility to enrofloxacin, flumequine, and oxolinic acid, moderate susceptibility to gentamicin, erythromycin, and florfenicol, and variable susceptibility to ampicillin and oxytetracycline. In vivo challenge experiments confirmed the ability of two representative Chinese F. psychrophilum isolates to induce typical signs of BCWD and mortality in 1-year-old rainbow trout (Oncorhynchus mykiss). Findings collectively demonstrate (i) that BCWD outbreaks in China studied thus far are caused by F. psychrophilum lineages that are common on other continents (e.g., CC-ST10) and others that have not been reported elsewhere (e.g., ST355, ST356, ST357), (ii) that F. psychrophilum molecular serotypes distinguish isolates from different host fish species, even within STs, and (iii) reduced F. psychrophilum antimicrobial susceptibility against compounds used for BCWD control in China. IMPORTANCE Flavobacterium psychrophilum causes substantial economic losses in salmonid aquaculture worldwide. Although this bacterium is also believed to be a disease source in China, published reports of its presence do not yet exist. Herein, F. psychrophilum was linked to multiple disease outbreaks in several salmonid aquaculture facilities within four Chinese provinces, and polyphasic characterization revealed that most isolates were genetically distinct from strains recovered on other continents. Analyses further revealed the predominating molecular serotypes, antimicrobial susceptibility profiles, and pathogenic potential of two representative recovered isolates. Collectively, the results presented here provide important data on the epidemiology and disease ecology of F. psychrophilum in China and pave the way for targeted prevention and control methods to be pursued in the future.

Safety Assessment of Lactiplantibacillus (formerly Lactobacillus) plantarum Q180.

The safety of the probiotic strain Q180, which exerts postprandial lipid-lowering effects, was bioinformatically and phenotypically evaluated. The genome of strain Q180 was completely sequenced, and single circular chromosome of 3,197,263 bp without any plasmid was generated. Phylogenetic and related analyses using16S rRNA gene and whole-genome sequences revealed that strain Q180 is a member of Lactiplantibacillus (Lp., formerly Lactobacillus) plantarum. Antimicrobial resistance (AMR) genes were bioinformatically analyzed using all Lp. plantarum genomes available in GenBank, which showed that AMR genes are present differently depending on Lp. plantarum strains. Bioinformatic analysis demonstrated that some mobile genetic elements such as prophages and insertion sequences were identified in the genome of strain Q180, but because they did not contain harmful genes such as AMR genes and virulence factor (VF)- and toxin-related genes, it was suggested that there is no transferability of harmful genes. The minimum inhibition concentrations of seven tested antibiotics suggested by the European Food Safety Authority guidelines were slightly lower than or equal to the microbiological cut-off values for Lp. plantarum. Strain Q180 did not show hemolytic and gelatinase activities and biogenic amine-producing ability. Taken together, this study demonstrated the safety of strain Q180 in terms of absence of AMR genes and VF- and toxin-related genes as a probiotic strain.

Identification and safety assessment of Enterococcus thailandicus TC1 isolated from healthy pigs.

Enterococci have the dual characteristics of being opportunistic pathogens and promising probiotics. The isolation from patients of CDC PNS-E2, a newly described Enterococcus species Enterococcus sanguinicola, may pose potential hazards. Enterococcus thailandicus from fermented sausage is a senior subjective synonym of E. sanguinicola. In this study, Enterococcus thailandicus TC1 was first isolated in healthy pigs in Tongcheng, China and identified by phenotypic analysis and 16S rRNA-based techniques. To evaluate the strain safety, an approach including virulence factors, antibiotic resistance, and animal experiments was adopted. The results show that cylA, gelE, esp, agg, ace, efaAfm, efaAfs, ptsD genes were undetected, and that the strain was sensitive or poorly resistant to some clinically relevant antibiotics. However, the isolated strain demonstrated ß-hemolytic activity in rabbit blood agar plates. Analysis of animal experiments revealed that the isolated strain had no adverse effect on translocation and the internal organ indices, though significant differences in histology (villi height, crypts height) of ileum were observed. The data acquired suggest that E. thailandicus TC1 may be associated with a potential health risk.

Safety assessment of Streptococcus salivarius DB-B5 as a probiotic candidate for oral health.

Streptococcus salivarius DB-B5 was previously isolated from the supragingival plaque of a healthy female adult and selected for development as a probiotic candidate for oral health. Probiotics are an important emerging therapeutic method for preventing, treating, and maintaining oral health. Although S. salivarius is a predominant member of the commensal oral microbiota and generally regarded as a safe species, it is recognized that each strain needs to be comprehensively assessed for safety. This study describes the in silico, in vitro, and clinical testing that were conducted to evaluate the safety of S. salivarius DB-B5. Both 16S rRNA and multi-gene phylogenetic reconstruction was used to confirm the taxonomic identity of this strain. Bioinformatic analysis of the genome demonstrated the absence of transmissible antibiotic resistance genes or virulence factors. Phenotypic testing further showed S. salivarius DB-B5 to be susceptible to clinically relevant antibiotics. S. salivarius DB-B5 displayed weak alpha-hemolysis, and does not produce biogenic amines. In a randomized, double-blind, placebo-controlled clinical study, consumption of S. salivarius DB-B5 at 10 billion CFU/day for 4 weeks by healthy adults was safe and well-tolerated (ClinicalTrials.gov registry number NCT04492631). This work has indicated that S. salivarius DB-B5 is a safe probiotic candidate.