Discovery of M Protease Inhibitors Encoded by SARS-CoV-2.

The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (Mpro, also called 3C-like protease [3CLpro]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting Mpro in the picornavirus-like supercluster, is a potent inhibitor for the Mpro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC50) of 26.4 ± 1.1 nM. In this study, we also show that GC376 inhibits SARS-CoV-2 replication with a half-maximum effective concentration (EC50) of 0.91 ± 0.03 µM. Only a small portion of SARS-CoV-2 Mpro was covalently modified in the excess of GC376 as evaluated by mass spectrometry analysis, indicating that improved inhibitors are needed. Subsequently, molecular docking analysis revealed that the recognition and binding groups of GC376 within the active site of SARS-CoV-2 Mpro provide important new information for the optimization of GC376. Given that sufficient safety and efficacy data are available for GC376 as an investigational veterinary drug, expedited development of GC376, or its optimized analogues, for treatment of SARS-CoV-2 infection in human is recommended.

Prevalence and risk factors for Salmonella spp. contamination of slaughtered chickens in Taiwan.

The present study was designed to estimate the prevalence of Salmonella contamination in Taiwanese broilers at slaughter and to identify risk factors associated with the presence of Salmonella in processed batches of broilers. Carcass rinse samples from 362 batches of broilers were collected from 45 chicken abattoirs in Taiwan between February 2013 and November 2014. Univariate analyses and multivariable logistic regression analyses were conducted to identify putative risk factors for contamination. Salmonella was detected in 32.6 % (95 % CI: 30.4-34.8) of individual broilers and 56.4 % (95 % CI: 51.1-61.5) of the sampled batches. The multivariable logistic regression model identified season (July to November) (OR = 1.95; 95 % CI: 1.2-3.2) as increasing the risk of infection. Abattoirs in the southern region (Taichung and Kaohsiung) (OR = 0.45; 95 % CI: 0.3-0.8); batches scalded for > 90 s (OR = 0.2; 95 % CI: 0.1-0.3) and batches of commercial white broilers (BR) (OR = 0.21; 95 % CI: 0.1-0.4) all had a decreased risk of contamination compared to abattoirs from the northern region, scalding < 90 s and Taiwan native chickens (TNC), respectively. This study highlights the influence of environmental conditions and poultry breed on the risk of Salmonella contamination of chickens during slaughter.

Actinobacillus pleuropneumoniae exotoxin ApxI induces cell death via attenuation of FAK through LFA-1.

ApxI exotoxin is an important virulence factor derived from Actinobacillus pleuropneumoniae that causes pleuropneumonia in swine. Here, we investigate the role of lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), a member of the ß2 integrin family, and the involvement of the integrin signaling molecules focal adhesion kinase (FAK) and Akt in ApxI cytotoxicity. Using Western blot analysis, we found that ApxI downregulated the activity of FAK and Akt in porcine alveolar macrophages (AMs). Preincubation of porcine AMs with an antibody specific for porcine CD18 reduced ApxI-induced cytotoxicity as measured by a lactate dehydrogenase release assay and decreased ApxI-induced FAK and Akt attenuation, as shown by Western blot analysis. Pretreatment with the chemical compounds PMA and SC79, which activate FAK and Akt, respectively, failed to overcome the ApxI-induced attenuation of FAK and Akt and death of porcine AMs. Notably, the transfection experiments revealed that ectopic expression of porcine LFA-1 (pLFA-1) conferred susceptibility to ApxI in ApxI-insensitive cell lines, including human embryonic kidney 293T cells and FAK-deficient mouse embryonic fibroblasts (MEFs). Furthermore, ectopic expression of FAK significantly reduced ApxI cytotoxicity in pLFA-1-cotransfected FAK-deficient MEFs. These findings show for the first time that pLFA-1 renders cells susceptible to ApxI and ApxI-mediated attenuation of FAK activity via CD18, thereby contributing to subsequent cell death.

Detection of chicken carcasses contaminated with Salmonella enterica serovar in the abattoir environment of Taiwan.

Although a nation-wide microbiological screening program of chicken carcasses after chilling in Taiwanese chicken abattoirs has been undertaken since 2006, little is known regarding the potential sources of the Salmonella during the slaughter process. The present study provides data on the detection and serotypes of Salmonella isolated from broilers during processing and from the environment in six abattoirs in Taiwan. Overall, Salmonella were detected in 156 of 622 samples (25.1%; 95% CI: 21.7-28.7) collected. The prevalence of Salmonella varied between sampling sites with 5.8, 17.6, 31.3 and 35.5% of cloacal swabs, environmental samples prior to processing, environmental samples during processing and carcass rinse fluid, respectively, being positive (χ2 = 51.3, p < 0.0001). A total of 15 serotypes were identified from the 156 Salmonella isolates with S. Albany (41.7%) S. Schwarzengrund (20.5%), S. Kentucky (12.8%) and S. Tennessee (5.1%) being the most commonly isolated serotypes. Characterization of 156 isolates by Pulse Field Gel Electrophoresis (PFGE) identified 50 PFGE types. Typing confirmed the presence of the same PFGE type at multiple stages during processing including plucking, evisceration, chilling and post-chilling. The abattoir environment and intestinal contents of chickens are important sources of Salmonella in broiler chicken abattoirs, with the same PFGE types detected at different stages of processing both before and during slaughtering. It is concluded that Salmonella isolates present in the environment and intestinal contents of processed birds survived in the abattoir environment resulting in subsequent carcass contamination along the processing chain including plucking, evisceration, chilling and post-chilling.

Repurposing old drugs as antiviral agents for coronaviruses.

BACKGROUND: New therapeutic options to address the ongoing coronavirus disease 2019 (COVID-19) pandemic are urgently needed. One possible strategy is the repurposing of existing drugs approved for other indications as antiviral agents for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Due to the commercial unavailability of SARS-CoV-2 drugs for treating COVID-19, we screened approximately 250 existing drugs or pharmacologically active compounds for their inhibitory activities against feline infectious peritonitis coronavirus (FIPV) and human coronavirus OC43 (HCoV-OC43), a human coronavirus in the same genus (Betacoronavirus) as SARS-CoV-2. METHODS: FIPV was proliferated in feline Fcwf-4 cells and HCoV-OC43 in human HCT-8 cells. Viral proliferation was assayed by visualization of cytopathic effects on the infected Fcwf-4 cells and immunofluorescent assay for detection of the nucleocapsid proteins of HCoV-OC43 in the HCT-8 cells. The concentrations (EC50) of each drug necessary to diminish viral activity to 50% of that for the untreated controls were determined. The viabilities of Fcwf-4 and HCT-8 cells were measured by crystal violet staining and MTS/PMS assay, respectively. RESULTS: Fifteen out of the 252 drugs or pharmacologically active compounds screened were found to be active against both FIPV and HCoV-OC43, with EC50 values ranging from 11 nM to 75 µM. They are all old drugs as follows, anisomycin, antimycin A, atovaquone, chloroquine, conivaptan, emetine, gemcitabine, homoharringtonine, niclosamide, nitazoxanide, oligomycin, salinomycin, tilorone, valinomycin, and vismodegib. CONCLUSION: All of the old drugs identified as having activity against FIPV and HCoV-OC43 have seen clinical use in their respective indications and are associated with known dosing schedules and adverse effect or toxicity profiles in humans. Those, when later confirmed to have an anti-viral effect on SARS-CoV-2, should be considered for immediate uses in COVID-19 patients.

Artificial intelligence approach fighting COVID-19 with repurposing drugs.

Background: The ongoing COVID-19 pandemic has caused more than 193,825 deaths during the past few months. A quick-to-be-identified cure for the disease will be a therapeutic medicine that has prior use experiences in patients in order to resolve the current pandemic situation before it could become worsening. Artificial intelligence (AI) technology is hereby applied to identify the marketed drugs with potential for treating COVID-19. Methods: An AI platform was established to identify potential old drugs with anti-coronavirus activities by using two different learning databases; one consisted of the compounds reported or proven active against SARS-CoV, SARS-CoV-2, human immunodeficiency virus, influenza virus, and the other one containing the known 3C-like protease inhibitors. All AI predicted drugs were then tested for activities against a feline coronavirus in in vitro cell-based assay. These assay results were feedbacks to the AI system for relearning and thus to generate a modified AI model to search for old drugs again. Results: After a few runs of AI learning and prediction processes, the AI system identified 80 marketed drugs with potential. Among them, 8 drugs (bedaquiline, brequinar, celecoxib, clofazimine, conivaptan, gemcitabine, tolcapone, and vismodegib) showed in vitro activities against the proliferation of a feline infectious peritonitis (FIP) virus in Fcwf-4 cells. In addition, 5 other drugs (boceprevir, chloroquine, homoharringtonine, tilorone, and salinomycin) were also found active during the exercises of AI approaches. Conclusion: Having taken advantages of AI, we identified old drugs with activities against FIP coronavirus. Further studies are underway to demonstrate their activities against SARS-CoV-2 in vitro and in vivo at clinically achievable concentrations and doses. With prior use experiences in patients, these old drugs if proven active against SARS-CoV-2 can readily be applied for fighting COVID-19 pandemic.

Inhibition of SARS-CoV-2 by Highly Potent Broad-Spectrum Anti-Coronaviral Tylophorine-Based Derivatives.

Tylophorine-based compounds and natural cardiotonic steroids (cardenolides and bufadienolides) are two classes of transmissible gastroenteritis coronavirus inhibitors, targeting viral RNA and host cell factors, respectively. We tested both types of compounds against two types of coronaviruses, to compare and contrast their antiviral properties, and with view to their further therapeutic development. Examples of both types of compounds potently inhibited the replication of both feline infectious peritonitis virus and human coronavirus OC43 with EC50 values of up to 8 and 16 nM, respectively. Strikingly, the tylophorine-based compounds tested inhibited viral yields of HCoV-OC43 to a much greater extent (7-8 log magnitudes of p.f.u./ml) than the cardiotonic steroids (about 2-3 log magnitudes of p.f.u./ml), as determined by end point assays. Based on these results, three tylophorine-based compounds were further examined for their anti-viral activities on two other human coronaviruses, HCoV-229E and SARS-CoV-2. These three tylophorine-based compounds inhibited HCoV-229E with EC50 values of up to 6.5 nM, inhibited viral yields of HCoV-229E by 6-7 log magnitudes of p.f.u./ml, and were also found to inhibit SARS-CoV-2 with EC50 values of up to 2.5-14 nM. In conclusion, tylophorine-based compounds are potent, broad-spectrum inhibitors of coronaviruses including SARS-CoV-2, and could be used for the treatment of COVID-19.

Staphylococcus aureus isolated from pork and chicken carcasses in Taiwan: prevalence and antimicrobial susceptibility.

Staphylococcus aureus is a cause of many diseases in both humans and animals. This pathogen is also a major target in the screening of slaughterhouse carcasses to monitor hygienic conditions during slaughter. During 2004 to 2006, S. aureus was recovered from 8.8% (38 of 430), 11.3% (77 of 680), and 4.3% (13 of 300) of pork carcass samples, respectively, collected at 53 slaughterhouses in Taiwan. During 2003 to 2005, it was recovered from 0.3% (1 of 305), 0.4% (1 of 260), and 7.8% (31 of 395) of rinse fluids from chicken carcasses, respectively, collected at 17 meat processing plants. The minimum dilution method was used to determine antimicrobial susceptibility (MICs) of these strains (n = 103) as well as those collected from pork and chicken carcasses (n = 104) in a previous study beginning in 2000. All 207 strains were sensitive to nitrofurantoin and vancomycin. Over 50% were resistant to clindamycin (MIC that inhibited 90% of strains [MIC90] = 32 microg/ml) and tetracycline (MIC90 = 64 microg/ml). The percentages resistant to methicillin (oxacillin), chloramphenicol, erythromycin, and tylosin were 19.4% (40 of 207), 18.8% (39 of 207), 23.2% (48 of 207), and 20.8% (43 of 207) with MIC90s of 8, 64, > or = 64, and > or = 128 microg/ml, respectively. The methicillin-resistant S. aureus (MRSA) strains exhibited resistance to more antibiotics than did the methicillin-susceptible strains, and 87.5% (35 of 40) of the MRSA strains carried the mecA gene sequence. Since MRSA infections have become a public health concern in both communities and hospitals, testing for the presence of MRSA in animal carcasses during slaughtering operations is warranted.

Surface assembly of poly(I:C) on polyethyleneimine-modified gelatin nanoparticles as immunostimulatory carriers for mucosal antigen delivery.

The mucosal immune system is the host's first line of defense against invasion by foreign pathogens. Gelatin nanoparticles (GNPs) are suitable carriers for the delivery of antigens via various routes of administration. In the present study, GNPs were modified with polyethyleneimine (PEI), a positively charged polymer. Then, ovalbumin (OVA) and polyinosinic:polycytidylic acid (poly(I:C)), an immunostimulant, were adsorbed onto the surface of the positively charged GNPs. We assessed whether GNPs could act as an effective mucosal vaccine that is capable of inducing both mucosal and systemic immune responses. The results showed that GNPs effectively adsorbed OVA/poly(I:C), facilitated cellular uptake by RAW 264.7 macrophage cells and murine bone marrow-derived dendritic cells (BMDCs) in vitro, and led to increased expression of the maturation markers CD80 and CD86 on BMDCs. Furthermore, GNPs induced increased secretion of proinflammatory cytokines in both RAW 264.7 and BMDCs. C57BL/6 mice that were intranasally twice-immunized with OVA/poly(I:C)-loaded GNPs produced high levels of serum OVA-specific IgG antibodies and secretory IgA in nasal and lung lavage. Spleen cells from immunized mice were collected and re-stimulated with OVA, and results showed significantly augmented production of IFN-γ, IL-4, IL-5, and IL-6 in mice that received OVA/poly(I:C)-loaded GNPs. Moreover, intranasal immunization with OVA/poly(I:C)-loaded GNPs resulted in the inhibition of EG7 tumor growth in C57BL/6 mice. Taken together, these results indicate that nasal administration of OVA/poly(I:C)-loaded GNPs elicited effective mucosal and systemic immune responses, which might be useful for further applications of antigen delivery. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1228-1237, 2019.

Primers specific for the fimbrial major subunit gene stdA can be used to detect Salmonella enterica serovars.

The feasibility of using two primers internal to the stdA gene (which encodes the fimbrial major subunit of the std fimbrial gene cluster in Salmonella enterica serovar Typhi) to detect Salmonella by PCR was explored. The 518-bp stdA specific sequence was conserved among 268 strains from 45 serovars of S. enterica. One Salmonella bongori CCUG 30042 strain and 34 non-Salmonella strains did not possess this sequence. A sensitivity test revealed that the stdA-specific primer set detected 3.4 x 10(-1) pg of genomic DNA and 3.0 x 10(5) CFU/ml with serial dilutions of Salmonella Typhimurium cells. In vitro testing for specificity using pig carcass sponge samples contaminated with Salmonella Typhimurium also was performed. An initial Salmonella Typhimurium inoculum of 4.4 x 10(1) CFU/ml in pig carcass exudates reached the stdA primer detection level after preenrichment in buffered peptone water at 37 degrees C for 18 h in the presence of indigenous non-Salmonella flora at 4.0 X 10(7) CFU/ml, but the detection level decreased to 4.4 x 10(0) CFU/ml after selective enrichment in Rappaport-Vassiliadis R10 broth for 18 h at 42 degrees C. The PCR method with primers specific for stdA is a quick and sensitive tool for detecting S. enterica, which is an important cause of foodborne disease.

Molecular cloning, expression, and functional characterization of the ß-agarase AgaB-4 from Paenibacillus agarexedens.

In this study, a ß-agarase gene, agaB-4, was isolated for the first time from the agar-degrading bacterium Paenibacillus agarexedens BCRC 17346 by using next-generation sequencing. agaB-4 consists of 2652 bp and encodes an 883-amino acid protein with an 18-amino acid signal peptide. agaB-4 without the signal peptide DNA was cloned and expressed in Escherichia coli BL21(DE3). His-tagged recombinant AgaB-4 (rAgaB-4) was purified from the soluble fraction of E. coli cell lysate through immobilized metal ion affinity chromatography. The optimal temperature and pH of rAgaB-4 were 55 °C and 6.0, respectively. The results of a substrate specificity test showed that rAgaB-4 could degrade agar, high-melting point agarose, and low-melting point agarose. The Vmax and Km of rAgaB-4 for low-melting point agarose were 183.45 U/mg and 3.60 mg/mL versus 874.61 U/mg and 9.29 mg/mL for high-melting point agarose, respectively. The main products of agar and agarose hydrolysis by rAgaB-4 were confirmed to be neoagarotetraose. Purified rAgaB-4 can be used in the recovery of DNA from agarose gels and has potential application in agar degradation for the production of neoagarotetraose.

The detection and differentiation of canine respiratory pathogens using oligonucleotide microarrays.

Canine respiratory diseases are commonly seen in dogs along with co-infections with multiple respiratory pathogens, including viruses and bacteria. Virus infections in even vaccinated dogs were also reported. The clinical signs caused by different respiratory etiological agents are similar, which makes differential diagnosis imperative. An oligonucleotide microarray system was developed in this study. The wild type and vaccine strains of canine distemper virus (CDV), influenza virus, canine herpesvirus (CHV), Bordetella bronchiseptica and Mycoplasma cynos were detected and differentiated simultaneously on a microarray chip. The detection limit is 10, 10, 100, 50 and 50 copy numbers for CDV, influenza virus, CHV, B. bronchiseptica and M. cynos, respectively. The clinical test results of nasal swab samples showed that the microarray had remarkably better efficacy than the multiplex PCR-agarose gel method. The positive detection rate of microarray and agarose gel was 59.0% (n=33) and 41.1% (n=23) among the 56 samples, respectively. CDV vaccine strain and pathogen co-infections were further demonstrated by the microarray but not by the multiplex PCR-agarose gel. The oligonucleotide microarray provides a highly efficient diagnosis alternative that could be applied to clinical usage, greatly assisting in disease therapy and control.

Involvement of NF-κB in regulation of Actinobacillus pleuropneumoniae exotoxin ApxI-induced proinflammatory cytokine production in porcine alveolar macrophages.

Actinobacillus pleuropneumoniae is a crucial respiratory pathogen that causes fibrinous, hemorrhagic, necrotizing pleuropneumonia in pigs. A. pleuropneumoniae exotoxins (ApxI to IV) are the major virulence factors contributing to A. pleuropneumoniae pathogenesis. Previously, we demonstrated that ApxI induces the expression of proinflammatory cytokines in porcine alveolar macrophages (PAMs) via the mitogen-activated protein kinases (MAPKs) p38 and cJun NH2-terminal kinase (JNK). Nonetheless, the role of nuclear factor (NF)-κB-a transcription factor widely implicated in immune and inflammatory responses-in ApxI-elicited cytokine production has yet to be defined. In the present study, we examined the involvement of NF-κB in ApxI-elicited production of interleukin (IL)-1ß, IL-8, and tumor necrosis factor (TNF)-α in PAMs and investigated the correlation between NF-κB and MAPK (p38 and JNK) pathways in this event. The results of Western blot analysis, confocal microscopy, and a DNA binding activity assay revealed that the classical NF-κB pathway was activated by ApxI, as evidenced by the decreased levels of IκB and subsequent NF-κB translocation and activation in ApxI-stimulated PAMs. Moreover, the blocking of ApxI-induced NF-κB activation significantly attenuated the levels of mRNA and protein secretion of IL-1ß, IL-8, and TNF-α in PAMs. Notably, the attenuation of JNK activation by a specific inhibitor (SP600125) reduced ApxI-induced NF-κB activation, whereas a p38 blocker (SB203580) had no effect on the NF-κB pathway. Further examination revealed that the level of phosphorylation at serine 536 on the NF-κB p65 subunit was dependent on JNK activity. Collectively, this study, for the first time, demonstrates a pivotal role of NF-κB in ApxI-induced IL-1ß, IL-8, and TNF-α production; JNK, but not p38, may positively affect the activation of the classical NF-κB pathway.

One-year (2003) nationwide pork carcass microbiological baseline data survey in Taiwan.

From January through December 2003, swab samples from 1,650 pork carcasses were collected from 39 slaughter plants in Taiwan. These samples were analyzed for the prevalence of indicator microorganisms and specific pathogens. Viable aerobic bacteria, total coliforms, and Escherichia coli were recovered from 100, 95.3, and 87.5% of these carcasses, respectively. Of those carcasses that harbored bacteria, the mean aerobic plate, total coliform, and Escherichia coli counts were 4.0, 0.6, and 0.1 log CFU/cm2, respectively. Staphylococcus aureus, Clostridium perfringens, Campylobacter jejuni, Campylobacter coli, Listeria monocytogenes, and Salmonella were recovered from 4.8, 0.3, 13.8, 0.7, and 1.7 of 1,038 carcasses, respectively. E. coli O157:H7 was not detected from any carcass. When positive for a specific pathogen, the mean carcass concentration was 0.57 log CFU/cm2 for S. aureus, 0.66 most probable number (MPN)/cm2 for C. jejuni and C. coli, and 0.18 MPN/cm2 for Salmonella. The findings of this study will help provide a reference for establishing hygienic standards and a criterion for evaluating the effects of slaughtering operations in Taiwan.

FimY of Salmonella enterica serovar Typhimurium functions as a DNA-binding protein and binds the fimZ promoter.

Salmonella enterica serovar Typhimurium produces type 1 fimbriae with binding specificity to mannose residues. Elements involved in fimbrial structural biosynthesis, transport, and regulation are encoded by the fim gene cluster. FimZ, FimY, FimW, STM0551, and an arginine transfer RNA (fimU) were previously demonstrated to regulate fimbrial expression. The amino acid sequences of the C-terminal portion of FimY revealed similarity with those of LuxR-like proteins. Electrophoretic mobility shift assays indicated that FimY possessed DNA-binding capacity and bound a 605-bp DNA fragment spanning the intergenic region between fimY and fimZ, while a FimY protein harboring a double mutation in the C-terminal helix-turn-helix region containing a glycine (G) to aspartate (D) substitution at residue 189 and isoleucine (I) to lysine (K) substitution at residue 195 lost its ability to bind this DNA fragment. A lux box sequence (5'-TCTGTTATTACATAACAAATACT-3') within the fimZ promoter was required for binding. None of the DNA fragments derived from the promoters for fimA, fimY, or fimW was shifted by FimY. Pull-down assays showed that there were physical protein/protein interactions between FimY and FimZ. We propose that in the regulatory circuit of type 1 fimbriae, FimY functions as a DNA-binding protein to activate fimZ, and a FimY-FimZ protein complex may form to regulate other fim genes. Confirming these proposals requires further study.

Elucidating the role of ApxI in hemolysis and cellular damage by using a novel apxIA mutant of Actinobacillus pleuropneumoniae serotype 10.

Exotoxins produced by Actinobacillus (A.) pleuropneumoniae (Apx) play major roles in the pathogenesis of pleuropneumonia in swine. This study investigated the role of ApxI in hemolysis and cellular damage using a novel apxIA mutant, ApxIA336, which was developed from the parental strain A. pleuropneumoniae serotype 10 that produces only ApxI in vitro. The genotype of ApxIA336 was confirmed by PCR, Southern blotting, and gene sequencing. Exotoxin preparation derived from ApxIA336 was analyzed for its bioactivity towards porcine erythrocytes and alveolar macrophages. Analysis results indicated that ApxIA336 contained a kanamycin- resistant cassette inserted immediately after 1005 bp of the apxIA gene. Phenotype analysis of ApxIA336 revealed no difference in the growth rate as compared to the parental strain. Meanwhile, ApxI production was abolished in the bacterial culture supernatant, i.e. exotoxin preparation. The inability of ApxIA336 to produce ApxI corresponded to the loss of hemolytic and cytotoxic bioactivity in exotoxin preparation, as demonstrated by hemolysis, lactate dehydrogenase release, mitochondrial activity, and apoptosis assays. Additionally, the virulence of ApxIA336 appeared to be attenuated by 15-fold in BALB/c mice. Collectively, ApxI, but not other components in the exotoxin preparation of A. pleuropneumoniae serotype 10, was responsible for the hemolytic and cytotoxic effects on porcine erythrocytes and alveolar macrophages.

Pathogenic microbiological baseline survey of pork carcasses in Taiwan.

From 2004 to 2010, pork carcass swabs from state-inspected slaughter plants in Taiwan were intermittently analyzed to determine the prevalence of selected pathogenic microorganisms associated with foodborne illness. The prevalences of Staphylococcus aureus each year from 2006 to 2010 were 6.6, 10.8, 5.1, 6.4, and 7.4%, respectively, while those of Listeria monocytogenes were 1.2% in 2004, 1.3% in 2005, and 3.5% in 2008. The prevalences of Clostridium perfringens were 0.9% in 2004, 3.2% in 2005, and 1.1% in 2008. Campylobacter jejuni and Campylobacter coli had a higher recovery rate than the other surveyed microorganisms, with prevalences during 2004, 2005, and 2008 of 21.1, 13.7, and 8.1%, respectively. Salmonella strains were analyzed each year, and their prevalences ranged between 3.0 and 6.9%. Derby, Typhimurium, Anatum, Choleraesuis, and Agona were the five serovars most frequently identified among the Salmonella isolates. Escherichia coli O157:H7 was not detected in 2004, 2005, or 2010. Routine baseline surveying of pork carcasses to determine the prevalence of selected pathogens of concern for food safety can provide valuable information regarding the effectiveness of the slaughtering procedures or the need for interventions.

Comparative study of class 1 integron, ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline (ACSSuT) and fluoroquinolone resistance in various Salmonella serovars from humans and animals.

A total of 499 Salmonella isolates including 9 serovars from humans and various animal hosts were collected to compare prevalence of integron and antimicrobial resistance. The integron and gene cassette were detected by PCR, and then the gene cassette type was further determined by sequencing and restriction fragment length polymorphism (RFLP) analysis. The antimicrobial susceptibility test was conducted by disk diffusion method. The positivity percentage of class 1 integron and the diversity of gene cassettes carried by integron were quite different in various Salmonella serovars, especially comparing those from animals to humans. After sequencing and RFLP analysis, it was identified eight gene cassette types. The gene cassette type D carrying ampicillin/streptomycin resistance genes was the most common one (42.2%) in the integron-positive isolates. More diversity of gene cassette types was identified in humans comparing to that in animals. Several gene cassette types were identified for the first time in some Salmonella serovars. In this study, 31.5% (157/499) of the isolates were multi-resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (ACSSuT). S. Choleraesuis isolates with the cassette type A1, but S. Typhimurium isolates with the cassette type E1, were frequently associated with ACSSuT-resistant (80.6% and 72.7%, respectively). There was a significant association between the presence of class 1 integron and quinolone resistance in S. Choleraesuis isolates, but not in S. Typhimurium. Our findings imply that transmission efficiency of various gene cassettes through the integron could be different in various Salmonella serovars.

A low-pH medium in vitro or the environment within a macrophage decreases the transcriptional levels of fimA, fimZ and lrp in Salmonella enterica serovar Typhimurium.

Many Salmonella Typhimurium isolates produce type 1 fimbriae and exhibit fimbrial phase variation in vitro. Static broth culture favours the production of fimbriae, while solid agar medium inhibits the generation of these appendages. Little information is available regarding whether S. Typhimurium continues to produce type 1 fimbriae during in vivo growth. We used a type 1 fimbrial phase-variable strain S. Typhimurium LB5010 and its derivatives to infect RAW 264.7 macrophages. Following entry into macrophages, S. Typhimurium LB5010 gradually decreased the transcript levels of fimbrial subunit gene fimA, positive regulatory gene fimZ, and global regulatory gene lrp. A similar decrease in transcript levels was detected by RT-PCRwhen the pH of static brothmediumwas shifted frompH 7 to amore acidic pH 4. A fimA-deleted strain continued to multiply within macrophages as did the parental strain. An lrp deletion strain was unimpaired for in vitro growth at pH 7 or pH 4, while a strain harboring an lrp-containing plasmid exhibited impaired in vitro growth at pH 4. We propose that acidic medium, which resembles one aspect of the intracellular environment in a macrophage, inhibits type 1 fimbrial production by down-regulation of the expression of lrp, fimZ and fimA.