Antibiotic-induced microbiome depletion promotes intestinal colonization by Campylobacter jejuni in mice.

BACKGROUND: To establish a method to induce Campylobacter jejuni colonization in the intestines of C57BL/6 mice through antibiotic-induced microbiome depletion. RESULTS: Fifty-four female C57BL/6 mice were divided into the normal, control, and experimental groups. The experimental group was administered intragastric cefoperazone sodium and sulbactam sodium (50 mg/mL) for 2 days; then, the experimental and control mice were intragastrically administered 200 µL C. jejuni, which was repeated once more after 2 days. Animal feces were collected, and the HipO gene of C. jejuni was detected using TaqMan qPCR from day 1 to day 14 after modeling completion. Immunofluorescence was used to detect intestinal C. jejuni colonization on day 14, and pathological changes were observed using hematoxylin and eosin staining. Additionally, 16S rDNA analyses of the intestinal contents were conducted on day 14. In the experimental group, C. jejuni was detected in the feces from days 1 to 14 on TaqMan qPCR, and immunofluorescence-labeled C. jejuni were visibly discernable in the intestinal lumen. The intestinal mucosa was generally intact and showed no significant inflammatory-cell infiltration. Diversity analysis of the colonic microbiota showed significant inter-group differences. In the experimental group, the composition of the colonic microbiota differed from that in the other 2 groups at the phylum level, and was characterized by a higher proportion of Bacteroidetes and a lower proportion of Firmicutes. CONCLUSIONS: Microbiome depletion induced by cefoperazone sodium and sulbactam sodium could promote long-term colonization of C. jejuni in the intestines of mice.

Modulation of Campylobacter jejuni adhesion to biotic model surfaces by fungal lectins and protease inhibitors.

Campylobacter jejuni, a Gram-negative bacterium, is one of the most common causes of foodborne illness worldwide. Its adhesion mechanism is mediated by several bacterial factors, including flagellum, protein adhesins, lipooligosaccharides, proteases, and host factors, such as surface glycans on epithelial cells and mucins. Fungal lectins, specialized carbohydrate-binding proteins, can bind to specific glycans on host and bacterial cells and thus influence pathogenesis. In this study, we investigated the effects of fungal lectins and protease inhibitors on the adhesion of C. jejuni to model biotic surfaces (mucin, fibronectin, and collagen) and Caco-2 cells as well as the invasion of Caco-2 cells. The lectins Marasmius oreades agglutinin (MOA) and Laccaria bicolor tectonin 2 (Tec2) showed remarkable efficacy in all experiments. In addition, different pre-incubations of lectins with C. jejuni or Caco-2 cells significantly inhibited the ability of C. jejuni to adhere to and invade Caco-2 cells, but to varying degrees. Pre-incubation of Caco-2 cells with selected lectins reduced the number of invasive C. jejuni cells the most, while simultaneous incubation showed the greatest reduction in adherent C. jejuni cells. These results suggest that fungal lectins are a promising tool for the prevention and treatment of C. jejuni infections. Furthermore, this study highlights the potential of fungi as a rich reservoir for novel anti-adhesive agents.

Antimicrobial resistance patterns and genes of Campylobacter jejuni isolated from chickens in Pasuruan, Indonesia.

Background: Poultry is one of the most prominent sources of Campylobacter jejuni, which is also a major means of transmission to people. Campylobacter jejuni contamination in chicken meat comes from chicken feces because it naturally exists in the intestines of chickens. Aim: The purpose of this study is to identify the antibiotic resistance patterns and genes of C. jejuni, which was found in chickens in Pasuruan, Indonesia. Methods: The samples used in this study were 200 contents of the small intestine of broiler chickens from 40 farms in Pasuruan Regency. The enriched sample was streaked on the selective media of modified charcoal cefoperazone deoxycholate agar containing the CCDA selective supplement. Antimicrobial susceptibility test utilizing the Kirby-Bauer diffusion test method in accordance with Clinical and Laboratory Standards Institute standards. The polymerase chain reaction (PCR) method was used to detect the (hipO), which encodes the C. jejuni strain, fluoroquinolone resistance (gyrA), beta-lactam resistance (blaOXA-61), and tetracycline resistance (tetO) genes. Results: The findings revealed a 14% (28/200) prevalence of C. jejuni in the small intestine of broiler chickens. These isolates showed high resistance to enrofloxacin (92.9%). All isolates (100%) were susceptible to amoxicillin-clavulanate. The PCR results showed all C. jejuni isolates (100%) detected the gyrA gene, 96.4% detected the blaOXA-61 gene, and 50% detected the tetO gene. Conclusion: The findings of antimicrobial resistance at a high level from the small intestine of broiler chickens illustrate the potential threat to human health. To lessen the effects now and in the future, coordinated and suitable action is needed, as well as steps to guarantee the poultry industry's economic survival and public health insurance.

Combined application of bacteriophages with a competitive exclusion culture and carvacrol with organic acids can reduce Campylobacter in primary broiler production.

For reducing Campylobacter (C.) in the food production chain and thus the risk to the consumer, the combined application of different measures as a multiple-hurdle approach is currently under discussion. This is the first study to investigate possible synergistic activities in vivo, aiming at reducing intestinal C. jejuni counts by administering (i) bacteriophages (phages) in combination with a competitive exclusion (CE) product and (ii) carvacrol combined with organic acids. The combined application of the two selected phages (Fletchervirus phage NCTC 12673 and Firehammervirus phage vB_CcM-LmqsCPL1/1) and the CE product significantly reduced C. jejuni loads by 1.0 log10 in cecal and colonic contents as well as in cloacal swabs at the end of the trial (33 and 34 days post hatch). The proportion of bacterial isolates showing reduced phage susceptibility ranged from 10.9% (isolates from cecal content) to 47.8% (isolates from cloacal swabs 32 days post hatch) for the Fletchervirus phage, while all tested isolates remained susceptible to the Firehammervirus phage. The use of carvacrol combined with an organic acid blend (sorbic acid, benzoic acid, propionic acid, and acetic acid) significantly reduced Campylobacter counts by 1.0 log10 in cloacal swabs on day 30 only.

Development of Fluoroquinolone Resistance through Antibiotic Tolerance in Campylobacter jejuni.

Antibiotic tolerance not only enables bacteria to survive acute antibiotic exposures but also provides bacteria with a window of time in which to develop antibiotic resistance. The increasing prevalence of Campylobacter jejuni isolates resistant to clinically important antibiotics, particularly fluoroquinolones (FQs), is a global public health concern. Currently, little is known about antibiotic tolerance and its effects on resistance development in C. jejuni. Here, we show that exposure to ciprofloxacin or tetracycline at concentrations 10 and 100 times higher than the MIC induces antibiotic tolerance in C. jejuni, whereas gentamicin or erythromycin treatment causes cell death. Interestingly, FQ resistance rapidly develops in C. jejuni after tolerance induction by ciprofloxacin and tetracycline. Furthermore, after tolerance is induced, alkyl hydroperoxide reductase (AhpC) plays a critical role in reducing FQ resistance development by alleviating oxidative stress. Together, these results demonstrate that exposure of C. jejuni to antibiotics can induce antibiotic tolerance and that FQ-resistant (FQR) C. jejuni clones rapidly emerge after tolerance induction. This study elucidates the mechanisms underlying the high prevalence of FQR C. jejuni and provides insights into the effects of antibiotic tolerance on resistance development. IMPORTANCE Antibiotic tolerance compromises the efficacy of antibiotic treatment by extending bacterial survival and facilitating the development of mutations associated with antibiotic resistance. Despite growing public health concerns about antibiotic resistance in C. jejuni, antibiotic tolerance has not yet been investigated in this important zoonotic pathogen. Here, our results show that exposure of C. jejuni to ciprofloxacin or tetracycline leads to antibiotic tolerance development, which subsequently facilitates the emergence of FQR C. jejuni. Importantly, these antibiotics are commonly used in animal agriculture. Moreover, our study suggests that the use of non-FQ drugs in animal agriculture promotes FQ resistance development, which is crucial because antibiotic-resistant C. jejuni is primarily transmitted from animals to humans. Overall, these findings increase our understanding of the mechanisms of resistance development through the induction of antibiotic tolerance.

Antimicrobial Resistance and Antibiogram of Thermotolerant Campylobacter Recovered from Poultry Meat in Baghdad Markets, Iraq.

Antimicrobial resistance is a critical public health issue that affects people all over the world. Since bacteria have a proclivity for rapidly acquiring and propagating the resistance gene, antimicrobial-resistant Campylobacter has a negative impact on public health. As a result, the creation of new and highly pathogenic clones is facilitated, making antimicrobial treatment more challenging. This study aimed to determine the antimicrobial resistance pattern (ARP) models, multiple drug resistance (MDR) models, and multiple antibiotic resistance (MAR) index of Campylobacter species isolated from poultry meat sold in Baghdad markets, Iraq. By employing the disc diffusion test, 30 Campylobacter strains from chicken meat, including C. jejuni (n=10) and C. coli (n=20), were exposed to tetracycline (TET), erythromycin, Gentamicin, nalidixic acid, ciprofloxacin, and norfloxacin (NOR). The ARP of the Campylobacter isolates revealed up to five antibiotypes for two species, which revealed resistance to one or more antimicrobials, and 67% of them had MDR resistance to two or more experienced antimicrobials. The NOR-TET model is the most common MDR, having a prevalence of 30% among experienced isolates. In addition, the MAR index, equal to and lower than one, was found in 87% of the isolates. Antibiotic resistance in Campylobacter raises the probability of treatment failure in humans and animals, as well as the propagation of antimicrobial resistance genes. As a result, the presence of Campylobacter in meat could pose a risk of human infection and pollution of the environment.

Dihydroxy-Acid Dehydratases From Pathogenic Bacteria: Emerging Drug Targets to Combat Antibiotic Resistance.

There is an urgent global need for the development of novel therapeutics to combat the rise of various antibiotic-resistant superbugs. Enzymes of the branched-chain amino acid (BCAA) biosynthesis pathway are an attractive target for novel anti-microbial drug development. Dihydroxy-acid dehydratase (DHAD) is the third enzyme in the BCAA biosynthesis pathway. It relies on an Fe-S cluster for catalytic activity and has recently also gained attention as a catalyst in cell-free enzyme cascades. Two types of Fe-S clusters have been identified in DHADs, i.e. [2Fe-2S] and [4Fe-4S], with the latter being more prone to degradation in the presence of oxygen. Here, we characterise two DHADs from bacterial human pathogens, Staphylococcus aureus and Campylobacter jejuni (SaDHAD and CjDHAD). Purified SaDHAD and CjDHAD are virtually inactive, but activity could be reversibly reconstituted in vitro (up to ∼19,000-fold increase with kcat as high as ∼6.7 s-1 ). Inductively-coupled plasma-optical emission spectroscopy (ICP-OES) measurements are consistent with the presence of [4Fe-4S] clusters in both enzymes. N-isopropyloxalyl hydroxamate (IpOHA) and aspterric acid are both potent inhibitors for both SaDHAD (Ki =7.8 and 51.6 µM, respectively) and CjDHAD (Ki =32.9 and 35.1 µM, respectively). These compounds thus present suitable starting points for the development of novel anti-microbial chemotherapeutics.

Detection of Campylobacter jejuni and Salmonella typhimurium in chicken using PCR for virulence factor hipO and invA genes (Saudi Arabia).

Campylobacter jejuni and Salmonella typhimurium are the leading causes of bacterial food contamination in chicken carcasses. Contamination is particularly associated with the slaughtering process. The present study isolated C. jejuni and S. typhimurim from fifty chicken carcass samples, all of which were acquired from different companies in Riyadh, Saudi Arabia. The identification of C. jejuni was performed phenotypically by using a hippurate test and genetically using a polymerase chain reaction with primers for 16S rRNA and hippurate hydrolase (hipO gene). For the dentification of S. typhimurim, a serological Widal test was carried out using serum anti-S. typhimurium antibodies. Strains were genetically detected using invA gene primers. The positive isolates for C. jejuni showed a specific molecular size of 1448 bp for 16S rRNA and 1148 bp for hipO genes. However, the positive isolates of the invA gene exhibited a specific molecular size at 244 bp using polymerase chain reaction (PCR). Comparing sequencing was performed with respect to the invA gene and the BLAST nucleotide isolates that were identified as Salmonella enterica subsp. enterica serovar typhimurium strain ST45, thereby producing a similarity of 100%. The testing identified C.jejuni for hippuricase, GenBank: Z36940.1. While many isolates of Salmonella spp. that contained the invA gene were not necessarily identified as S. typhimurim, the limiting factor for the Widal test used antiS. typhimurum antibodies. The multidrug resistance (MDR) of C. jejuni isolates in chickens was compared with the standard C. jejuni strain ATCC 22931. Similarly, S. typhimurium isolates were compared with the standard S. typhimurium strain ATCC 14028.

Pharmacokinetics of tulathromycin in pregnant ewes (Ovis aries) challenged with Campylobacter jejuni.

The purpose of this study was to evaluate the pharmacokinetics of tulathromycin in the plasma and maternal and fetal tissues of pregnant ewes when administered within 24 hours of a single, IV Campylobacter jejuni (C. jejuni) challenge. Twelve, pregnant ewes between 72-92 days of gestation were challenged IV with C. jejuni IA3902 and then treated with 1.1 ml/45.36 kg of tulathromycin subcutaneously 18 hours post-challenge. Ewes were bled at predetermined time points and euthanized either at a predetermined time point or following the observation of vaginal bleeding or abortion. Following euthanasia, tissues were collected for bacterial culture, pharmacokinetics and histologic examination. The maximum (geometric) mean tulathromycin plasma concentration was estimated at 0.302 µg/mL, with a peak level observed at around 1.2 hours. The apparent systemic clearance of tulathromycin was estimated at 16.6 L/h (or 0.28 L/kg/h) with an elimination half-life estimated at approximately 22 hours. The mean tissue concentrations were highest in the uterus (2.464 µg/g) and placentome (0.484 µg/g), and were lowest in fetal liver (0.11 µg/g) and fetal lung (0.03 µg/g). Compared to previous reports, results of this study demonstrate that prior IV administration of C. jejuni appeared to substantially alter the pharmacokinetics of tulathromycin, reducing both the peak plasma concentrations and elimination half-life. However, additional controlled trials are required to confirm those observations.

Inhibition of phage-resistant bacterial pathogen re-growth with the combined use of bacteriophages and EDTA.

The combined effects of ethylenediaminetetraacetic acid (EDTA) and bacteriophage (phage) treatment of foodborne pathogens were investigated. Although viable counts for Campylobacter jejuni decreased by 1.5 log after incubation for 8 h in the presence of phage PC10, re-growth was observed thereafter. The combination of phage PC10 and 1 mM EDTA significantly inhibited the re-growth of C. jejuni. The viable counts for C. jejuni decreased by 2.6 log (P < 0.05) compared with that of the initial count after 24 h. Moreover, EDTA at 0.67 or 1.3 mM, combined with the specific lytic phages, also effectively inhibited the re-growth of phage-resistant cells of Campylobacter coli, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Typhimurium. In addition, the combined effects of lytic phages and EDTA were investigated on the viability of Campylobacter in BHI broth at low temperatures followed by the optimum growth temperature. The re-growth of C. coli was significantly inhibited by the coexistence of 1.3 mM EDTA, and the viable counts of surviving bacteria was about the same as the initial viable count after the incubation. This is the first study demonstrating the combined use of lytic phages and EDTA is effective in inhibiting the re-growth of phage-resistant bacteria in Gram-negative bacteria.

Antibiofilm Potential of Lavandula Preparations against Campylobacter jejuni.

New approaches for the control of Campylobacter jejuni biofilms in the food industry are being studied intensively. Natural products are promising alternative antimicrobial substances to control biofilm production, with particular emphasis on plant extracts. Dried flowers of Lavandula angustifolia were used to produce essential oil (LEO), an ethanol extract (LEF), and an ethanol extract of Lavandula postdistillation waste material (LEW). The chemical compositions determined for these Lavandula preparations included seven major compounds that were selected for further testing. These were tested against C. jejuni for biofilm degradation and removal. Next-generation sequencing was used to study the molecular mechanisms underlying LEO actions against C. jejuni adhesion and motility. Analysis of LEO revealed 1,8-cineol, linalool, and linalyl acetate as the main components. For LEF and LEW, the main components were phenolic acid glycosides, with flavonoids rarely present. The MICs of the Lavandula preparations and pure compounds against C. jejuni ranged from 0.2 mg/ml to 1 mg/ml. LEO showed the strongest biofilm degradation. The reduction of C. jejuni adhesion was ≥1 log10 CFU/ml, which satisfies European Food Safety Authority recommendations. Lavandula preparations reduced C. jejuni motility by almost 50%, which consequently can impact biofilm formation. These data are in line with the transcriptome analysis of C. jejuni, which indicated that LEO downregulated genes important for biofilm formation. LEW also showed good antibacterial and antibiofilm effects, particularly against adhesion and motility mechanisms. This defines an innovative approach using alternative strategies and novel targets to combat bacterial biofilm formation and, hence, the potential to develop new effective agents with biofilm-degrading activities. IMPORTANCE The Lavandula preparations used in this study are found to be effective against C. jejuni, a common foodborne pathogen. They show antibiofilm properties at subinhibitory concentrations in terms of promoting biofilm degradation and inhibiting cell adhesion and motility, which are involved in the initial steps of biofilm formation. These results are confirmed by transcriptome analysis, which highlights the effect of Lavandula essential oil on C. jejuni biofilm properties. We show that the waste material from the hydrodistillation of Lavandula has particular antibiofilm effects, suggesting that it has potential for reuse for industrial purposes. This study highlights the need for efforts directed toward such innovative approaches and alternative strategies against biofilm formation and maintenance by developing new naturally derived agents with antibiofilm activities.

First report of the optrA-carrying multidrug resistance genomic island in Campylobacter jejuni isolated from pigeon meat.

Campylobacter spp., such as Campylobacter jejuni and Campylobacter coli, are important zoonotic Gram-negative pathogens that cause acute intestinal diseases in humans. The optrA gene, encoding an ATP-binding cassette F (ABC-F) protein that confers resistance to oxazolidinones and phenicols, has been found in C. coli in China. In this study, the optrA gene was first identified in C. jejuni collected from retail meat in China from 2013 to 2016. Nine strains, isolated from a pigeon meat sample, carry the optrA gene. The molecular characteristics of the optrA-positive strains were determined by whole genome sequencing. Pulsed-field gel electrophoresis, multilocus sequence typing, and single nucleotide polymorphism analyses demonstrated that the nine optrA-positive isolates were genetically homogeneous. Phylogenetic characteristics and sequence comparison revealed that optrA was located on a chromosome-borne multidrug resistance genomic island. The optrA gene along with the tet(O) gene formed two different translocatable units (TUs), thereby supporting the transmission of TU-associated resistance genes. The emergence and spread of such TUs and strains are of great concern in terms of food safety, and measures must be implemented to avoid their dissemination in other Gram-negative bacteria and food chains.

Lactic acid bacteria that activate immune gene expression in Caenorhabditis elegans can antagonise Campylobacter jejuni infection in nematodes, chickens and mice.

BACKGROUND: Campylobacter jejuni is the major micro-bacillary pathogen responsible for human coloenteritis. Lactic acid bacteria (LAB) have been shown to protect against Campylobacter infection. However, LAB with a good ability to inhibit the growth of C. jejuni in vitro are less effective in animals and animal models, and have the disadvantages of high cost, a long cycle, cumbersome operation and insignificant immune response indicators. Caenorhabditis elegans is increasingly used to screen probiotics for their anti-pathogenic properties. However, no research on the use of C. elegans to screen for probiotic candidates antagonistic to C. jejuni has been conducted to date. RESULTS: This study established a lifespan model of C. elegans, enabling the preselection of LAB to counter C. jejuni infection. A potential protective mechanism of LAB was identified. Some distinct LAB species offered a high level of protection to C. elegans against C. jejuni. The LAB strains with a high protection rate reduced the load of C. jejuni in C. elegans. The transcription of antibacterial peptide genes, MAPK and Daf-16 signalling pathway-related genes was elevated using the LAB isolates with a high protection rate. The reliability of the lifespan model of C. elegans was verified using mice and chickens infected with C. jejuni. CONCLUSIONS: The results showed that different LAB had different abilities to protect C. elegans against C. jejuni. C. elegans provides a reliable model for researchers to screen for LAB that are antagonistic to C. jejuni on a large scale.

Nutraceutical intervention protects against bacterial and chemical-induced gastrotoxicity in a non-mammalian model, Galleria mellonella.

Preparations of the fungus Cordyceps sinensis and bovine colostrum are considered nutraceuticals due to their anti-inflammatory, repair and gut alimentation properties in mammalian models. To reduce the reliance on rodents in routine experimentation, we gauged the capacity of nutraceuticals to alleviate gastric damage in an insect surrogate, Galleria mellonella. Larvae were reared on standard or supplemented diets - 10% (w/w) colostrum, 10% (w/w) C. sinensis, or 5% + 5% each - prior to receiving an oral dose of the NSAID indomethacin (30 mg/kg) or challenged with the bacterial pathogen Campylobacter jejuni (1-3 x106) via two inoculation routes. Insects reared on a cordyceps-supplemented diet proved most resistant to indomethacin-induced gut leakiness, and displayed stable health indices after C. jejuni challenge (~77% survival). Insects reared on a colostrum-supplemented diet also showed recalcitrance in the gut, but were more sensitive to C. jejuni when injected directly into the body cavity (50% survival). The nutraceutical blend yielded improved health outcomes when compared to the standard diet, but was not as effective as either nutraceutical alone. Our findings represent clear evidence that insects were more resistant to known chemical and microbial agitators when reared on nutraceutical-supplemented diets - toxicological endpoints that are shared with vertebrate studies.

Coexistence of optrA and fexA in Campylobacter.

Previous studies indicated that Campylobacter has developed several mechanisms that confer resistance to florfenicol, which is used in food animal production. This study describes the coexistence of optrA and fexA in Campylobacter jejuni and Campylobacter coli isolates from pigs and poultry. Moreover, whole-genome sequencing data showed that the two genes are located in various multidrug resistance genomic islands within different regions of the Campylobacter genomes. The emergence of optrA and fexA may support the spread of florfenicol-resistant Campylobacter strains of animal origin.IMPORTANCE Florfenicol is widely used for the treatment of respiratory infections and as a feed additive in food animal production. As a foodborne pathogen, Campylobacter is constantly exposed to florfenicol, and resistance to this antimicrobial agent has increased in recent years. Previous studies indicated that Campylobacter has developed several mechanisms that confer resistance to florfenicol. This study describes for the first time the coexistence of the florfenicol exporter FexA and the ribosomal protective protein OptrA in Campylobacter jejuni isolated from pigs. The two genes were located in various multidrug resistance genomic islands within different regions of the Campylobacter genomes. Although phenicols are not commonly used for the treatment of Campylobacter infections, the extensive use of florfenicol in food animals may play a role in the coselection of multidrug resistance genomic island (MDRGI)-carrying Campylobacter isolates which also exhibited resistance to critically important antimicrobial agents (macrolides, aminoglycosides, and tetracyclines) commonly used for the treatment of human campylobacteriosis.

Campylobacter jejuni ST50, a pathogen of global importance: A comparative genomic analysis of isolates from Australia, Europe and North America.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis globally, and infections are often transmitted through consumption of raw or undercooked poultry. Campylobacter jejuni ST50 is among the top ten sequence types (STs) reported in the collected isolates listed at PubMLST records from poultry, food and clinical sources for Asia, Europe, North America, Oceania and South America. This study was designed to determine the most commonly reported C. jejuni STs globally using the PubMLST database and assess similarities between genomes of C. jejuni ST50 isolates from geographically distinct locations. To gain a better understanding of C. jejuni diversity, we compared draft genome sequences of 182 ST50 isolates recovered from retail or caecal poultry samples in Oceania, Europe and North America that were collected over a period of 9 years (2010 to 2018). Overall, phylogenetic analysis revealed that isolates from geographically distinct locations tended to cluster based on the continent where the sample was collected. Among ST50 isolates from Europe and North America, we identified resistance determinants associated with phenotypic resistance to beta-lactams (EU: 55%; GB: 43.1%), tetracyclines (CA: 77.3%; EU: 37.5%; GB: 9.8%; US: 43.5%) and fluoroquinolones (EU: 60.0%; GB: 15.7%); no resistance determinants were identified in isolates from Australia. In general, the majority of the virulence genes, with rare exceptions such as wlaN, cj1138, hddA and rfbC, were evenly distributed throughout the genomes of all ST50 isolates in this study. Genomic-based characterization of C. jejuni ST50 isolates from poultry on three continents highlighted that geographically distinct isolates have evolved independently but only represent a glimpse into the diversity of C. jejuni.

Significant contribution of the CmeABC Efflux pump in high-level resistance to ciprofloxacin and tetracycline in Campylobacter jejuni and Campylobacter coli clinical isolates.

BACKGROUND: Campylobacter resistance to antimicrobial agents is regarded as a major concern worldwide. The aim of this study was to investigate the expression of the CmeABC efflux pump and the RAPD-PCR pattern in drug-resistant Campylobacter isolates. METHODS: A total of 283 stool specimens were collected from children under the age of five with diarrhea. The minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin was determined by broth microdilution method and E-test, respectively. Detection of tetracycline and ciprofloxacin determinants was done by amplification of tetO gene and PCR-sequencing of the gyrA gene. The cmeABC transcriptional expression was analyzed by Real-time (RT)-PCR. Clonal correlation of resistant strains was determined by RAPD-PCR genotyping. RESULTS: Out of 283 fecal samples, 20 (7.02%) samples were positive for Campylobacter spp. Analysis of duplex PCR assay of the cadF gene showed that 737 and 461 bp amplicons were corresponding to Campylobacter jejuni and Campylobacter coli, respectively. All of the 17 phenotypically tetracycline-resistant Campylobacter isolates harbored the tetO gene. Also, four phenotypically ciprofloxacin-resistant Campylobacter isolates had a point mutation at codon 257 of the gyrA gene (ACA to ATA; Thr > Ile). High-level expression of the cmeA gene was observed in ciprofloxacin-resistant and high-level tetracycline-resistant Campylobacter isolates, suggesting a positive correlation between the cmeA gene expression level and tetracycline resistance level. Moreover, a statistically significant difference was observed in the cmeA gene expression between ciprofloxacin-resistant and ciprofloxacin-susceptible strains, which signifies the crucial contribution of the efflux pump in conferring multiple drug resistance phenotype among Campylobacter spp. RAPD analysis of Campylobacter isolates exhibited 16 different patterns. Simpsone`s diversity index of RAPD-PCR was calculated as 0.85, showing a high level of homogeneity among the population; however, no clear correlation was detected among tetracycline and/or ciprofloxacin resistant isolates. CONCLUSION: Significant contribution of the CmeABC efflux pump in conferring high-level resistance to tetracycline and ciprofloxacin was observed in C. jejuni and C. coli clinical isolates. The resistant phenotype is suggested to be mediated by CmeABC efflux pumps, the tetO gene, and point mutation of the gyrA gene. Genotyping revealed no clonal correlation among resistant strains, indicating distinct evolution of tetracycline and ciprofloxacin resistant genotypes among the isolates.

Campylobacter jejuni Antimicrobial Resistance Profiles and Mechanisms Determined Using a Raman Spectroscopy-Based Metabolomic Approach.

Rapid identification of antimicrobial resistance (AMR) profiles and mechanisms is critical for clinical management and drug development. However, the current AMR detection approaches take up to 48 h to obtain a result. Here, we demonstrate a Raman spectroscopy-based metabolomic approach to rapidly determine the AMR profile of Campylobacter jejuni, a major cause of foodborne gastroenteritis worldwide. C. jejuni isolates with susceptible and resistant traits to ampicillin and tetracycline were subjected to different antibiotic concentrations for 5 h, followed by Raman spectral collection and chemometric analysis (i.e., second-derivative transformation analysis, hierarchical clustering analysis [HCA], and principal-component analysis [PCA]). The MICs obtained by Raman-2nd derivative transformation agreed with the reference agar dilution method for all isolates. The AMR profile of C. jejuni was accurately classified by Raman-HCA after treating bacteria with antibiotics at clinical susceptible and resistant breakpoints. According to PCA loading plots, susceptible and resistant strains showed different Raman metabolomic patterns for antibiotics. Ampicillin-resistant isolates had distinctive Raman signatures of peptidoglycan, which is related to cell wall synthesis. The ratio of saturated to unsaturated fatty acids in the lipid membrane layer of ampicillin-resistant isolates was higher than in susceptible ones, indicating more rigid envelope structure under ampicillin treatment. In comparison, tetracycline-resistant isolates exhibited prominent Raman spectral features associated with proteins and nucleic acids, demonstrating more active protein synthesis than susceptible strains with the presence of tetracycline. Taken together, Raman spectroscopy is a powerful metabolic fingerprinting technique for simultaneously revealing the AMR profiles and mechanisms of foodborne pathogens. IMPORTANCE Metabolism plays the central role in bacteria to mediate the early response against antibiotics and demonstrate antimicrobial resistance (AMR). Understanding the whole-cell metabolite profiles gives rise to a more complete AMR mechanism insight. In this study, we have applied Raman spectroscopy and chemometrics to achieve a rapid, accurate, and easy-to-operate investigation of bacterial AMR profiles and mechanisms. Raman spectroscopy reduced the analysis time by an order of magnitude to obtain the same results achieved through traditional culture-based antimicrobial susceptibility approaches. It offers great benefits as a high-throughput screening method in food chain surveillance and clinical diagnostics. Meanwhile, the AMR mechanisms toward two representative antibiotic classes, namely, ampicillin and tetracycline, were revealed by Raman spectroscopy at the metabolome level. This approach is based on bacterial phenotypic responses to antibiotics, providing information complementary to that obtained by conventional genetic methods such as genome sequencing. The knowledge obtained from Raman metabolomic data can be used in drug discovery and pathogen intervention.

Prevalence, Characterization, and Control of Campylobacter jejuni Isolated from Raw Milk, Cheese, and Human Stool Samples in Beni-Suef Governorate, Egypt.

Our study aimed to determine the prevalence of Campylobacter jejuni isolated from raw milk, cheese, and human stool samples in Beni-Suef Governorate, Egypt, and to characterize the antibiotic resistance profile and virulence genes of the isolates. An additional objective was to evaluate the effectiveness of cinnamon oil and Lactobacillus acidophilus La5 for controlling C. jejuni in cheese. A total of 200 samples of raw milk and dairy products, including 50 samples of raw milk and 150 samples of three different types of cheese were used. Fifty-three human stool samples were also collected. The samples were tested for the presence of C. jejuni using culture and molecular methods. Campylobacter spp. were isolated from 9.5% (19/200) of the raw milk and cheese samples. The highest prevalence was observed in milk samples (18%), followed by Kareish cheese (14%) and Talaga cheese (6%). In contrast, C. jejuni was not found in any of the Feta cheese samples. Of the human stool samples, 21 (39.6%) were positive for C. jejuni. Of the isolates, 60-90% were highly resistant to the antimicrobial agents tested, that is, nalidixic acid, ciprofloxacin, and tetracycline. Virulent cadF and cdtA genes were detected in all isolates. As milk and dairy products are important sources of contamination, reducing the level of C. jejuni in them will lower the risk to consumers. We showed that L. acidophilus La5 was able to control C. jejuni in Kareish cheese, but cinnamon oil was less effective.

Nettle-Leaf Extract Derived ZnO/CuO Nanoparticle-Biopolymer-Based Antioxidant and Antimicrobial Nanocomposite Packaging Films and Their Impact on Extending the Post-Harvest Shelf Life of Guava Fruit.

Green synthesized metal oxide nanoparticles (NPs) have prominent applications in antimicrobial packaging systems. Here we have attempted for the fabrication of chitosan-based nanocomposite film containing Urtica dioica leaf extract derived copper oxide (CuO) and zinc oxide (ZnO) NPs for shelf-life extension of the packaged guava fruits. Electron microscopy and spectroscopy analysis of the CuO and ZnO NPs exhibited nano-scale size, spherical morphologies, and negative ζ-potential values. The NPs possessed appreciable antioxidant and antimicrobial activity (AMA) in order of CuO NPs > ZnO NPs >nettle extract. Therefore, this work establishes for the first time the successful synthesis of CuO NPs and compares its antimicrobial and antioxidant properties with ZnO NPs. On incorporation in chitosan, the polymer nanocomposite films were developed by solvent casting technique. The developed films were transparent, had low antioxidant but substantial AMA. The NP supplementation improved the film characteristics as evident from the decrease in moisture content, water holding capacity, and solubility of the films. The nanocomposite films improved the quality attributes and shelf life of guava fruits by one week on packaging and storage compared to unpackaged control fruits. Therefore, this study demonstrates the higher antimicrobial potential of the nettle leaf extract derived CuO/ZnO NPs for development of antimicrobial nanocomposite films as a promising packaging solution for enhancing the shelf life of various perishable fruits.