Euna Oh

Role of Alkyl Hydroperoxide Reductase (AhpC) in the Biofilm Formation of Campylobacter jejuni

Biofilm formation of Campylobacter jejuni, a major cause of human gastroenteritis, contributes to the survival of this pathogenic bacterium in different environmental niches; however, molecular mechanisms for its biofilm formation have not been fully understood yet. In this study, the role of oxidative stress resistance in biofilm formation was investigated using mutants defective in catalase (KatA), superoxide dismutase (SodB), and alkyl hydroperoxide reductase (AhpC). Biofilm formation was substantially increased in an ahpC mutant compared to the wild type, and katA and sodB mutants. In contrast to the augmented biofilm formation of the ahpC mutant, a strain overexpressing ahpC exhibited reduced biofilm formation. A perR mutant and a CosR-overexpression strain, both of which upregulate ahpC, also displayed decreased biofilms. However, the introduction of the ahpC mutation to the perR mutant and the CosR-overexpression strain substantially enhanced biofilm formation. The ahpC mutant accumulated more total reactive oxygen species and lipid hydroperoxides than the wild type, and the treatment of the ahpC mutant with antioxidants reduced biofilm formation to the wild-type level. Confocal microscopy analysis showed more microcolonies were developed in the ahpC mutant than the wild type. These results successfully demonstrate that AhpC plays an important role in the biofilm formation of C. jejuni.

Enhanced Transmission of Antibiotic Resistance in Campylobacter jejuni Biofilms by Natural Transformation

ABSTRACT Campylobacter jejuni is a leading food-borne pathogen, and its antibiotic resistance is of serious concern to public health worldwide. C. jejuni is naturally competent for DNA transformation and freely takes up foreign DNA harboring genetic information responsible for antibiotic resistance. In this study, we demonstrate that C. jejuni transfers antibiotic resistance genes more frequently in biofilms than in planktonic cells by natural transformation.

Regulation of oxidative stress resistance in Campylobacter jejuni, a microaerophilic foodborne pathogen

Campylobacter jejuni is one of the leading bacterial causes of human gastroenteritis. Due to the increasing rates of human campylobacteriosis, C. jejuni is considered as a serious public health concern worldwide. C. jejuni is a microaerophilic, fastidious bacterium. C. jejuni must overcome a wide range of stress conditions during foodborne transmission to humans, such as food preservation and processing conditions, and even in infection of the gastrointestinal tracts of humans. Particularly, this microaerophilic foodborne pathogen must survive in the atmospheric conditions prior to the initiation of infection. C. jejuni possesses unique regulatory mechanisms for oxidative stress resistance. Lacking OxyR and SoxRS that are highly conserved in other Gram-negative foodborne pathogens, C. jejuni modulates the expression of genes involved in oxidative stress resistance mainly via the peroxide resistance regulator and Campylobacter oxidative stress regulator. Based on recent findings of ours and others, in this review, we described how C. jejuni regulates the expression of oxidative stress defense.

High Prevalence of Hyper-Aerotolerant Campylobacter jejuni in Retail Poultry with Potential Implication in Human Infection

Campylobacter jejuni is a leading cause of foodborne illnesses around the world. Since C. jejuni is microaerophilic and sensitive to oxygen, aerotolerance is important in the transmission of C. jejuni to humans via foods under aerobic conditions. In this study, 70 C. jejuni strains were isolated from retail raw chicken meats and were subject to multilocus sequence typing (MLST) analysis. In the aerotolerance testing by aerobic shaking at 200 rpm, 50 (71.4%) isolates survived after 12 h (i.e., aerotolerant), whereas 20 (28.6%) isolates did not (i.e., aerosensitive). Interestingly, further aerobic cultivation showed that 25 (35.7%) isolates still survived even after 24 h of vigorous aerobic shaking (i.e., hyper-aerotolerant). Compared to aerosensitive strains, the hyper-aerotolerant strains exhibited increased resistance to oxidative stress, both peroxide and superoxide. A mutation of ahpC in hyper-aerotolerant strains significantly impaired aerotolerance, indicating oxidative stress defense plays an important role in hyper-aerotolerance. The aerotolerant and hyper-aerotolerant strains were primarily classified into MLST clonal complexes (CCs)-21 and -45, which are known to be the major CCs implicated in human gastroenteritis. Compared to the aerosensitive strains, CC-21 was more dominant than CC-45 in aerotolerant and hyper-aerotolerant strains. The findings in this study revealed that hyper-aerotolerant C. jejuni is highly prevalent in raw chicken meats. The enhanced aerotolerance in C. jejuni would impact human infection by increasing possibilities of the foodborne transmission of C. jejuni under aerobic conditions.

Target optimization for peptide nucleic acid (PNA)-mediated antisense inhibition of the CmeABC multidrug efflux pump in Campylobacter jejuni

Objectives CmeABC is a resistance-nodulation-cell division (RND)-type multidrug efflux pump conferring resistance to clinically important antibiotics in Campylobacter. This study aimed to identify the optimal target sites for the inhibition of CmeABC with antisense peptide nucleic acid (PNA). Methods Eighteen PNAs were designed to bind to the translational initiation regions of cmeABC, spanning the ribosome-binding site (RBS) and the start codon of the cmeABC genes. Campylobacter jejuni was treated with CmeABC-specific PNAs (CmeABC-PNAs) at various concentrations and subjected to western blotting to measure changes in the level of CmeABC expression. The MICs of ciprofloxacin and erythromycin were measured to evaluate the impact of CmeABC knockdown on antibiotic susceptibility. Results While antisense PNA significantly affected CmeA and CmeB expression, interestingly, CmeC expression was not altered by any of the CmeC-PNAs used in this study. A CmeA-PNA targeting the RBS of cmeA and its upstream region reduced CmeA expression most efficiently, and CmeB expression was most significantly decreased by PNA binding to the RBS of cmeB and its downstream region. CmeA- and CmeB-PNAs increased the susceptibility of C. jejuni to ciprofloxacin and erythromycin in proportion to the inhibition levels observed in western blotting. Conclusions The cmeA gene is the best target to knockdown CmeABC with antisense PNA. The RBS is the major target for the PNA-mediated antisense inhibition of CmeABC. However, regions in its vicinity also significantly influence the effectiveness of the PNA-based knockdown of CmeABC.

Synergistic anti-Campylobacter jejuni activity of fluoroquinolone and macrolide antibiotics with phenolic compounds.

The increasing resistance of Campylobacter to clinically important antibiotics, such as fluoroquinolones and macrolides, is a serious public health problem. The objective of this study is to investigate synergistic anti-Campylobacter jejuni activity of fluoroquinolones and macrolides in combination with phenolic compounds. Synergistic antimicrobial activity was measured by performing a checkerboard assay with ciprofloxacin and erythromycin in the presence of 21 phenolic compounds. Membrane permeability changes in C. jejuni by phenolic compounds were determined by measuring the level of intracellular uptake of 1-N-phenylnaphthylamine (NPN). Antibiotic accumulation assays were performed to evaluate the level of ciprofloxacin accumulation in C. jejuni. Six phenolic compounds, including p-coumaric acid, sinapic acid, caffeic acid, vanillic acid, gallic acid, and taxifolin, significantly increased the susceptibility to ciprofloxacin and erythromycin in several human and poultry isolates. The synergistic antimicrobial effect was also observed in ciprofloxacin- and erythromycin-resistant C. jejuni strains. The phenolic compounds also substantially increased membrane permeability and antibiotic accumulation in C. jejuni. Interestingly, some phenolic compounds, such as gallic acid and taxifolin, significantly reduced the expression of the CmeABC multidrug efflux pump. Phenolic compounds increased the NPN accumulation in the cmeB mutant, indicating phenolic compounds may affect the membrane permeability. In this study, we successfully demonstrated that combinational treatment of C. jejuni with antibiotics and phenolic compounds synergistically inhibits C. jejuni by impacting both antimicrobial influx and efflux.

Non-selective regulation of peroxide and superoxide resistance genes by PerR in Campylobacter jejuni

Campylobacter jejuni is an important foodborne pathogen. The molecular mechanisms for the regulation of oxidative stress resistance have not yet been understood fully in this bacterium. In this study, we investigated how PerR (peroxide stress regulator) modulates the transcriptional regulation of both peroxide and superoxide resistance genes in C. jejuni, particularly under oxidative stress conditions. The transcriptional levels of ahpC, katA, and sodB were substantially increased by aeration and oxidant exposure. Interestingly, a perR mutation completely abrogated the transcriptional response of ahpC, katA and sodB to oxidants. Furthermore, we demonstrated that perR transcription was reduced by aeration and oxidant exposure. In contrast to the unique role of PerR homologs in peroxide stress regulation in other bacteria, C. jejuni PerR directly regulates the transcription of sodB, the most important gene in superoxide defense, as evidenced by the alteration of sodB transcription by the perR mutation and direct binding of rPerR to the sodB promoter. In addition, we also observed notable morphological changes in C. jejuni from spiral rods to cocoid morphology under aerobic conditions. Based on the intracellular ATP levels, C. jejuni entered a viable-but-non-culturable (VBNC) state under aerobic conditions. These findings clearly demonstrate that C. jejuni possesses a unique regulatory mechanism of oxidative stress defense that does not specifically distinguish between peroxide and superoxide defense, and PerR plays a pivotal role in this non-selective regulation of oxidative stress resistance in C. jejuni.

Differential Survival of Hyper-Aerotolerant Campylobacter jejuni under Different Gas Conditions

Campylobacter jejuni accounts for a significant number of foodborne illnesses around the world. C. jejuni is microaerophilic and typically does not survive efficiently in oxygen-rich conditions. We recently reported that hyper-aerotolerant (HAT) C. jejuni are highly prevalent in retail poultry meat. To assess the capabilities of HAT C. jejuni in foodborne transmission and infection, in this study, we investigated the prevalence of virulence genes in HAT C. jejuni and the survival in poultry meat in atmosphere at a refrigeration temperature. When we examined the prevalence of eight virulence genes in 70 C. jejuni strains from raw poultry meat, interestingly, the frequencies of detecting virulence genes were significantly higher in HAT C. jejuni strains than aerosenstive C. jejuni strains. This suggests that HAT C. jejuni would potentially be more pathogenic than aerosensitive C. jejuni. Under aerobic conditions, aerosensitive C. jejuni survived at 4°C in raw poultry meat for 3 days, whereas HAT C. jejuni survived in poultry meat for a substantially extended time; there was a five-log CFU reduction over 2 weeks. In addition, we measured the effect of other gas conditions, including N2 and CO2, on the viability of HAT C. jejuni in comparison with aerosensitive and aerotolerant strains. N2 marginally affected the viability of C. jejuni. However, CO2 significantly reduced the viability of C. jejuni both in culture media and poultry meat. Based on the results, modified atmosphere packaging using CO2 may help us to control poultry contamination with HAT C. jejuni.

Contribution of surface polysaccharides to the resistance of Campylobacter jejuni to antimicrobial phenolic compounds

Contribution of surface polysaccharides to the resistance of Campylobacter jejuni to antimicrobial phenolic compounds

An Improved Culture Method for Selective Isolation of Campylobacter jejuni from Wastewater

Campylobacter jejuni is one of the leading foodborne pathogens worldwide. C. jejuni is isolated from a wide range of foods, domestic animals, wildlife, and environmental sources. The currently available culture-based isolation methods are not highly effective for wastewater samples due to the low number of C. jejuni in the midst of competing bacteria. To detect and isolate C. jejuni from wastewater samples, in this study, we evaluated a few different enrichment conditions using five different antibiotics (i.e., cefoperazone, vancomycin, trimethoprim, polymyxin B, and rifampicin), to which C. jejuni is intrinsically resistant. The selectivity of each enrichment condition was measured with Ct value using quantitative real-time PCR, and multiplex PCR to determine Campylobacter species. In addition, the efficacy of Campylobacter isolation on different culture media after selective enrichment was examined by growing on Bolton and Preston agar plates. The addition of polymyxin B, rifampicin, or both to the Bolton selective supplements enhanced the selective isolation of C. jejuni. The results of 16S rDNA sequencing also revealed that Enterococcus spp. and Pseudomonas aeruginosa are major competing bacteria in the enrichment conditions. Although it is known to be difficult to isolate Campylobacter from samples with heavy contamination, this study well exhibited that the manipulation of antibiotic selective pressure improves the isolation efficiency of fastidious Campylobacter from wastewater.