Correction: Liu et al. The Discovery of Novel Agents against Staphylococcus aureus by Targeting Sortase A: A Combination of Virtual Screening and Experimental Validation. Pharmaceuticals 2024, 17, 58.

In the original publication [...].

Evaluation of Hygiene Practice for Reducing Campylobacter Contamination on Cutting Boards and Risks Associated with Chicken Handling in Kitchen Environment.

Cutting boards can serve as potential carriers for the cross-contamination of pathogens from chicken to other surfaces. This study aimed to assess chefs' handling practices of cutting boards across five provinces in China and identify the key factors contributing to unsafe cutting board usage, including cleaning methods and handling practices. Handling practices associated with cutting boards were examined through a web-based survey (N = 154), while kitchen environment tests were conducted to investigate the splashing or survival of Campylobacter, inoculated in chicken or on cutting boards, to mimic the practices of chefs. Among chefs in the five provinces of China, wood and plastic cutting boards were the most commonly used for preparing chicken meat. Approximately 33.7% of chefs washed boards with running tap water, 31.17% of chefs washed boards with detergent, and 24.03% of chefs cleaned boards by scraping them with a knife after preparing other meats or chicken. The study tested 23 cutting boards from commercial kitchens for Campylobacter presence before and after chicken preparation and cleaning. Among these, 17 were cleaned with a knife, 5 with running tap water, and only 1 with disinfectant. Results showed that cleaning with a knife significantly reduced Campylobacter presence on cutting boards (p < 0.05), while the three main cleaning methods were inadequate in eliminating contamination to a safe level. In kitchen environment tests, contaminated chicken was chopped on cutting boards, with a maximum distance of 60 cm for low contamination, and 120 cm for medium and high contamination levels. This suggested a contamination risk exposure area ranging from 60 cm to 120 cm. Campylobacter survival on surfaces of wood, plastic, and stainless steel was also tested, with plastic surfaces showing the longest survival time (4.5 h at 15 °C and 3.5 h at 25 °C) In comparison, survival time on stainless steel or wood surfaces was only 3 h, implying a cross-contamination risk exposure period of 3 to 4.5 h after chicken preparation. In conclusion, based on the current study data, the practices employed by chefs play an important role in Campylobacter transfer in the kitchen environment. The presence of Campylobacter on cutting boards even after wiping or droplet splashing highlights its potential as a source of cross-contamination in the kitchen environment. So, chefs in China should reinforce their hygiene culture and adopt effective cutting board cleaning practices to prevent pathogen contamination.

The Discovery of Novel Agents against Staphylococcus aureus by Targeting Sortase A: A Combination of Virtual Screening and Experimental Validation.

Staphylococcus aureus (S. aureus), commonly known as "superbugs", is a highly pathogenic bacterium that poses a serious threat to human health. There is an urgent need to replace traditional antibiotics with novel drugs to combat S. aureus. Sortase A (SrtA) is a crucial transpeptidase involved in the adhesion process of S. aureus. The reduction in virulence and prevention of S. aureus infections have made it a significant target for antimicrobial drugs. In this study, we combined virtual screening with experimental validation to identify potential drug candidates from a drug library. Three hits, referred to as Naldemedine, Telmisartan, and Azilsartan, were identified based on docking binding energy and the ratio of occupied functional sites of SrtA. The stability analysis manifests that Naldemedine and Telmisartan have a higher binding affinity to the hydrophobic pockets. Specifically, Telmisartan forms stable hydrogen bonds with SrtA, resulting in the highest binding energy. Our experiments prove that the efficiency of adhesion and invasion by S. aureus can be decreased without significantly affecting bacterial growth. Our work identifies Telmisartan as the most promising candidate for inhibiting SrtA, which can help combat S. aureus infection.

Feeding Malic Acid to Chickens at Slaughter Age Improves Microbial Safety with Regard to Campylobacter.

This study supplied malic acid-supplemented drinking water to flocks that were naturally Campylobacter-positive and assessed the effect of feeding malic acid to chickens on Campylobacter reduction and poultry production. In Experiment 1, chickens were provided with malic acid-supplemented drinking water for three weeks. The contamination loads of Campylobacter were decreased by 0.91-0.98 log after the first week of use (p < 0.05). However, this effect did not persist over time and significant decontamination could not be found in the second and third weeks of application. Thus, in Experiment 2 malic acid-supplemented drinking water was given to chickens for a period of five days at slaughter age. The Campylobacter carriage was found to be effectively decreased by 1.05-1.55 log (p < 0.05). Malic acid had no adverse effects on chicken body weight, weight gain, intestinal indices, or the microbiota. In addition, it could change the composition of chicken meat since the moisture content was increased by 5.12-5.92% (p < 0.05) and the fat content was decreased by 1.60% (p < 0.05). Our study provides an effective means for reducing the contamination of Campylobacter during the chicken rearing period and this method can be applied to promote the safe development of poultry farming and its products.

An Investigation into the Critical Factors Influencing the Spread of Campylobacter during Chicken Handling in Commercial Kitchens in China.

Campylobacteriosis is the most common cause of bacterial gastroenteritis worldwide. Consumption of chicken meat is considered the main route for human infection with Campylobacter. This study aimed to determine the critical factors for Campylobacter cross-contamination in Chinese commercial kitchens during chicken handling. Five commercial kitchens were visited to detect Campylobacter occurrence from 2019 to 2020. Chicken samples (n = 363) and cotton balls from the kitchen surfaces (n = 479) were collected, and total bacterial counts and Campylobacter spp. were detected. Genotypic characterization of 57 Campylobacter jejuni isolates was performed by multilocus sequence typing (MLST). In total, 77.41% of chicken carcass samples and 37.37% of kitchen surfaces showed Campylobacter spp. contamination. Before chicken preparation, Campylobacter spp. were already present in the kitchen environment; however, chicken handling significantly increased Campylobacter spp. prevalence (p < 0.05). After cleaning, boards, hands, and knives still showed high bacterial loads including Campylobacter spp., which related to poor sanitary conditions and ineffective handling practices. Poor sanitation conditions on kitchen surfaces offer greater opportunities for Campylobacter transmission. Molecular typing by MLST revealed that Campylobacter cross-contamination occurred during chicken preparation. The most prevalent sequence types, ST693 and ST45, showed strong biofilm formation ability. Consequently, sanitary condition of surfaces and biofilm formation ability of isolates were the critical points contributing to spread of Campylobacter in kitchen environment. These results provide insight into potential targeted control strategies along the farm-to-plate chain and highlight the necessity for improvements in sanitary conditions. The implementation of more effective cleaning measures should be considered to decrease the campylobacteriosis risk.

Characterization and Prevalence of Campylobacter spp. From Broiler Chicken Rearing Period to the Slaughtering Process in Eastern China.

Campylobacter is one of the most important foodborne pathogens worldwide, and poultry is regarded as the main reservoir of Campylobacter. The contamination of Campylobacter in broiler chickens at the farm level is closely related to the transmission of Campylobacter in the poultry production chain. This study identified 464 Campylobacter isolates from 1,534 samples from broiler rearing period and slaughtering process including 233 Campylobacter jejuni isolates and 231 Campylobacter coli isolates. We have observed a dynamic distribution of Campylobacter during broiler chicken production, that 66.3% of Campylobacter isolates were C. jejuni during broiler rearing period, while C. coli occupied 60.4% of Campylobacter isolates during the broiler slaughtering process. A tag-label method allowed us to track the dynamic of Campylobacter in each broiler chicken from 31-day age at rearing to the partition step in the slaughterhouse. At the 31-day during rearing, 150 broiler chicken were labeled, and was tracked for Campylobacter positive from rearing period to slaughtering process. Among the labeled broiler, 11 of the tracking broiler samples were able to detect Campylobacter from rearing period to slaughtering. All Campylobacter isolates from the 11 tracking samples were sequenced and analyzed. C. jejuni isolates were divided into four STs and C. coli isolates were divided into six STs. Isolates with identical core genome were observed from the same tag-labeled samples at different stages indicating a vertical transmission of Campylobacter in the early broiler meat production. Meanwhile, the core genome analysis elucidated the cross-contamination of Campylobacter during the rearing period and the slaughtering process. The virulotyping analysis revealed that all C. jejuni isolates shared the same virulotypes, while C. coli isolates were divided into three different virulotypes. The antimicrobial resistance gene analysis demonstrated that all Campylobacter isolates contained at least two antibiotic resistance genes (ARGs), and the ARG profiles were well-corresponding to each ST type. Our study observed a high prevalence of Campylobacter during the early chicken meat production, and further studies will be needed to investigate the diversity and transmission of Campylobacter in the poultry production chain.

Prevalence of Campylobacter spp. in Pig Slaughtering Line in Eastern China: Analysis of Contamination Sources.

Aiming at investigating the potential sources of Campylobacter spp. contamination in pig slaughterhouse in eastern China, a total of 2056 samples were collected in pork production chain stretching from the upstream farm to the slaughterhouse, including 54 cloacal swabs from 54 live pigs on farm, 1726 samples from 214 pig carcasses along the eight main steps in the slaughtering line, and 276 samples from slaughtering environment. Campylobacter spp. were found, may be propagated in each slaughtering step, with an average prevalence of 19.3% (333/1726). The isolation rate of Campylobacter spp. in samples collected before the slaughter (42.5%, 4.87 ± 1.23 log colony-forming units [CFU]/g), dehairing (28%, 2.40 ± 0.49 log CFU/500 cm2), and evisceration (29.4%, 2.50 ± 0.54 log CFU/500 cm2) were significantly higher than other slaughter processes (p < 0.05). The prevalence of Campylobacter spp. of pigs on farm was 18.5% (10/54), compared to the slaughtering environment, which was 27.9% (77/276). Campylobacter spp. isolates were obtained from a batch of samples in slaughterhouse and farm belonged to ST-828 complex. Interventions are required to minimize Campylobacter spp. contamination in slaughtering line, especially during dehairing and evisceration. The upstream farm was an additional and usually neglected source of contamination. These findings may provide a new perspective regarding the safety provision of Campylobacter spp. contamination in pork meat production.

Investigating the Role of FlhF Identifies Novel Interactions With Genes Involved in Flagellar Synthesis in Campylobacter jejuni.

FlhF is a key protein required for complete flagellar synthesis, and its deletion results in the complete absence of a flagella and thus motility in Campylobacter jejuni. However, the specific mechanism still remains unknown. In this study, RNA-Seq, EMSAs, ChIP-qPCR and ß-Galactosidase assays were performed to elucidate the novel interactions between FlhF and genes involved in flagellar synthesis. Results showed that FlhF has an overall influence on the transcription of flagellar genes with an flhF mutant displaying down-regulation of most flagellar related genes. FlhF can directly bind to the flgI promoter to regulate its expression, which has significant expression change in an flhF mutant. The possible binding site of FlhF to the flgI promoter was explored by continuously narrowing the flgI promoter region and performing further point mutations. Meanwhile, FlhF can directly bind to the promoters of rpoD, flgS, and fliA encoding early flagellin regulators, thereby directly or indirectly regulating the synthesis of class I, II, and III flagellar genes, respectively. Collectively, this study demonstrates that FlhF may directly regulate the transcription of flagellar genes by binding to their promoters as a transcriptional regulator, which will be helpful in understanding the mechanism of FlhF in flagellar biosynthetic and bacterial flagellation in general.

Insights into the impact of flhF inactivation on Campylobacter jejuni colonization of chick and mice gut.

BACKGROUND: Campylobacter jejuni (C. jejuni) is a leading cause of foodborne gastroenteritis worldwide. This bacterium lacks many of the classical virulence factors, and flagellum-associated persistent colonization has been shown to be crucial for its pathogenesis. The flagellum plays a multifunctional role in C. jejuni pathogenesis, and different flagellar elements make diverse contributions. The flhF gene encodes the flagellar biosynthesis regulator, which is important for flagellar biosynthesis. In this study, the influence of flhF on C. jejuni colonization was systematically studied, and the possible mechanisms were also analyzed. RESULTS: The flhF gene has a significant influence on C. jejuni colonization, and its inactivation resulted in severe defects in the commensal colonization of chicks, with approximately 104- to 107-fold reductions (for NCTC 11168 and a C. jejuni isolate respectively) observed in the bacterial caecal loads. Similar effects were observed in mice where the flhF mutant strain completely lost the ability to continuously colonize mice, which cleared the isolate at 7 days post inoculation. Characterization of the phenotypic properties of C. jejuni that influence colonization showed that the adhesion and invasion abilities of the C. jejuni flhF mutant were reduced to approximately 52 and 27% of that of the wild-type strain, respectively. The autoagglutination and biofilm-formation abilities of the flhF mutant strain were also significantly decreased. Further genetic investigation revealed that flhF is continuously upregulated during the infection process, which indicates a close association of this gene with C. jejuni pathogenesis. The transcription of some other infection-related genes that are not directly involved in flagellar assembly were also influenced by its inactivation, with the flagellar coexpressed determinants (Feds) being apparently affected. CONCLUSIONS: Inactivation of flhF has a significant influence on C. jejuni colonization in both birds and mammals. This defect may be caused by the decreased adhesion, invasion, autoagglutination and biofilm-formation abilities of the flhF mutant strain, as well as the influence on the transcription of other infection related genes, which provides insights into this virulence factor and the flagellum mediated co-regulation of C. jejuni pathogenesis.

Could FlhF be a key element that controls Campylobacter jejuni flagella biosynthesis in the initial assembly stage?

The disordered arrangement of flagella biosynthetic genes, combined with a simplified regulatory mechanism, has made elucidating the process of Campylobacter jejuni flagellation difficult. FlhF is a recently identified element that controls the assembly of the flagella, although its function mechanism and regulatory preference are not well defined at present. In this study, we found that inactivation of FlhF caused the transcription of most flagella genes down-regulated. The importance of FlhF was systematically evaluated by analyzing changes in the transcription profiles between wild-type and flhF mutant strains, which showed that FlhF affects late flagella genes obviously. FlhF is constitutively expressed during C. jejuni growth, demonstrating that it is a class I flagella element that participates in early flagella assembly. In addition, the early flagella component FlhB was not localized to the cell pole in the flhF mutant. Thus, flagella assembly was impeded at the initial stage. We propose a model in which FlhF helps target the early flagella components to the cell pole, functioning prior to the formation of the flagella export apparatus, and thus places FlhF at the top of the flagella regulatory cascade hierarchy. Inactivation of FlhF impeded flagella assembly at the initial stage and decreased transcription of flagella genes through a feed-back control mechanism, leading to FlhF having a significant influence on the expression of late flagella components and resulting in the aflagellate C. jejuni phenotype. Our present study has uncovered how FlhF influences C. jejuni flagella biosynthesis, which will be helpful in understanding the C. jejuni flagella biosynthetic pathway and bacterial flagellation in general.

Insights into Campylobacter jejuni colonization and enteritis using a novel infant rabbit model.

A lack of relevant disease models for Campylobacter jejuni has long been an obstacle to research into this common enteric pathogen. Here we used an infant rabbit to study C. jejuni infection, which enables us to define several previously unknown but key features of the organism. C. jejuni is capable of systemic invasion in the rabbit, and developed a diarrhea symptom that mimicked that observed in many human campylobacteriosis. The large intestine was the most consistently colonized site and produced intestinal inflammation, where specific cytokines were induced. Genes preferentially expressed during C. jejuni infection were screened, and acs, cj1385, cj0259 seem to be responsible for C. jejuni invasion. Our results demonstrates that the infant rabbit can be used as an alternative experimental model for the study of diarrheagenic Campylobacter species and will be useful in exploring the pathogenesis of other related pathogens.

cj0371: A Novel Virulence-Associated Gene of Campylobacter jejuni.

Campylobacter jejuni is the major cause of human bacterial diarrhea worldwide. Its pathogenic mechanism remains poorly understood. cj0371 is a novel gene that was uncovered using immunoscreening. There have been no previous reports regarding its function. In this study, we constructed an insertion mutant and complement of this gene in C. jejuni and examined changes in virulence. We observed that the cj0371 mutant showed significantly increased invasion and colonization ability. We also investigated the role of cj0371 in motility, chemotaxis, and growth kinetics to further study its function. We found that the cj0371 mutant displays hypermotility, enhanced chemotaxis, and enhanced growth kinetics. In addition, we localized the Cj0371 protein at the poles of C. jejuni by fluorescence microscopy. We present data that collectively significantly proves our hypothesis that cj0371 is a new virulence-associated gene and through the influence of chemotaxis plays a negative role in C. jejuni pathogenicity.

[Preparation and characterization of monoclonal antibodies against cytolethal distending toxin protein of Campylobacter jejuni].

OBJECTIVE: To express Campylobacter jejuni cytolethal distending toxin B protein (CdtB) in a prokaryote to prepare monoclonal antibodies (mAbs) against the protein, and to study their antitoxic effects. METHODS: The C. jejuni cdtB gene was amplified and inserted into the expression plasmids pET-30a( + ) and pGEX-6p-1. The purified rGST-CdtB protein was used as the immunogen to screen hybridoma cells for mAbs against the protein. The mAb titers were determined with an indirect enzyme-linked immunosorbent assay (ELISA), and their specificity with a Dot-ELISA and western blotting analysis. We determined the antitoxic properties of the mAbs in CaCo-2 and HD-11 cells. RESULTS: Recombinant expression plasmids pET-30a (+)-cdtB and pGEX-6p-l-cdtB were successfully constructed, and fusion proteins rHis-CdtB and rGST-CdtB expressed, respectively. Five hybridoma cell lines, designated 1F3, IF5, 2E4, 2E11, and 2F2, were screened for the stable secretion of mAbs against CdtB. The immunoglobulin subclass of 2E11 was IgG2b and that of the other mAbs was IgG1. The mAb titers in the ascites fluids were 1:1 x 10(8) on indirect ELISA. Dot-ELISA demonstrated that the five mAbs reacted specifically with C. jejuni. Western blotting analysis confirmed that the five mAbs reacted well with the rGST-CdtB fusion protein. The mAbs significantly reduced the adhesion and invasion capacities of the bacterium in CaCo-2 cells (P < 0.01). CONCLUSION: The successful preparation of five mAbs specific for the CdtB protein will allow further study of the biological characteristics of CdtB and the pathogenesis of C. jejuni.

Use of in vivo-induced antigen technology to identify in vivo-expressed genes of Campylobacter jejuni during human infection.

Campylobacter jejuni is a prevalent foodborne pathogen worldwide. Human infection by C. jejuni primarily arises from contaminated poultry meats. Genes expressed in vivo may play an important role in the pathogenicity of C. jejuni. We applied an immunoscreening method, in vivo-induced antigen technology (IVIAT), to identify in vivo-induced genes during human infection by C. jejuni. An inducible expression library of genomic proteins was constructed from sequenced C. jejuni NCTC 11168 and was then screened using adsorbed, pooled human sera obtained from clinical patients. We successfully identified 24 unique genes expressed in vivo. These genes were implicated in metabolism, molecular biosynthesis, genetic information processing, transport, and other processes. We selected six genes with different functions to compare their expression levels in vivo and in vitro using real-time RT-PCR. The results showed that the selected six genes were significantly upregulated in vivo but not in vitro. In short, these identified in vivo-induced genes may contribute to human infection of C. jejuni, some of which may be meaningful vaccine candidate antigens or diagnosis serologic markers for campylobacteriosis. IVIAT may present a significant and efficient method for understanding the pathogenicity mechanism of Campylobacter and for finding targets for its prevention and control.

[Preparation and characterization of monoclonal antibodies against FlhF protein of Campylobacter jejuni].

OBJECTIVE: To develop a prokaryotic expression system for Campylobacrer jejuni FlhF protein and prepare monoclonal antibodies(mAb) against this protein. METHODS: The C. jejuni flhF gene was amplified and inserted into the expression plasmids, pET-30a(+) and pGEX-6p-1. Then the rHis-FlhF protein and rGST-FlhF protein were expressed and purified by affinity column chromatography. They were subsequently used as immunogen and detecting antigen to screen antibodies produced by hybridoma cells against these proteins. The titers of the mAbs were measured by indirect ELISA, and the specificities of the mAbs was evaluated by Dot-ELISA and Western blot analysis. RESULTS: The recombinant expression plasmids pET-30a(+)-flhF and pGEX-6p-1-flhF were successfully constructed, and the fusion proteins rHis-FlhF and rGST-FlhF were produced at sufficient quantities. Two hybridoma cell lines were screened, designated 2C12 and 7A9, which secrete mAbs against FlhF stably. Their immunoglobulin subclasses were both IgG1. The titers of the ascites fluid were 1×10(9); and 2×10(7); respectively. Western blot analyses confirmed that the two mAbs both reacted with the rHis-FlhF fusion protein with good sensitivity. The Dot-ELISA results demonstrated that the two mAbs reacted specifically with Campylobacrer jejuni. CONCLUSION: The mAbs against Campylobacrer jejuni FlhF protein with high specificity were successfully prepared.

A novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens using pre-adsorbed sera from infected patients.

BACKGROUND: Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence. METHODS: In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni. RESULTS: Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI-TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro. CONCLUSION: We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients. GENERAL SIGNIFICANCE: This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.