Development of Antimicrobial Therapies Against Campylobacter jejuni in Poultry

Campylobacter jejuni is a Gram negative bacterium and well-recognised as a major etiologic agent for acute gastro-intestinal diseases in humans, particularly in the elderly and in children. C. jejuni can be isolated from a variety of wild and domestic animals, especially poultry and is primarily transmitted through uncooked or raw foods of animal origin. The consumption and mishandling of poultry products is the main cause of this illness in humans, but no effective control measures are available at present. Development of strategies to control colonisation of Campylobacter in poultry may be effective in reducing the incidence of this disease. The major aim of the present study is to improve food safety in the chicken industry by the reduction or prevention of C. jejuni colonization and carriage in poultry. To achieve this, the present study has focused on the antimicrobial potential of Australian native plants and plant derived compounds for their potent feed additives for their potential in reducing colonization and carriage of C jejuni in poultry. In addition, selected phage-displayed peptides were also tested for potential antimicrobial activity against C. jejuni. Antimicrobial activity of 115 extracts from 109 Australian plant species was screened against two C. jejuni strains using an in vitro broth microdiluton assay. The effects of plant extracts against various phases of C. jejuni growth were compared. C. jejuni cells were more susceptible in the exponential growth phase compared to the stationary phase. Most of the plant extracts (93%) showed activity at a concentration between 32 and 1024 μg/mL against at least one C. jejuni strain. Seventeen plant extracts that had activity at concentration less than 256 μg/mL were selected for further testing against six additional C. jejuni strains, as well as Campylobacter coli, and also against a panel of enteric gut pathogens (Escherichia coli, Salmonella typhimurium, Bacillus cereus, Proteus mirabilis and Enterecoccus faecalis). The best antimicrobial activity was obtained from the extract of Eucalyptus occidentalis, with an inhibitory concentration of 32 μg/mL against C. jejuni and B. cereus. In addition, three essential oils (EOs) and five terpenoid compounds were examined for their antimicrobial potential against two representatives of C. jejuni strains, C. coli and the other gut bacteria. Antimicrobial activity was determined by the use of disc diffusion and broth dilution techniques. Additionally, the antimicrobial activity of neem oil (Azadirachta indica) in different formulations with some of these active components was investigated for its synergistic activity towards the same bacteria in disc diffusion assay. The essential oil from Melaleuca alternifolia showed the most prominent activity against all bacteria with inhibitory concentrations in the range 0.001-0.25% v/v. In these in vitro studies, Campylobacter spp. was found to be the most susceptible organism. The usefulness of in vitro fermentation technique to test antimicrobial activity of natural compounds towards C. jejuni in mixed culture was also explored. Mixed caecal bacteria spiked with C. jejuni were incubated in anaerobic media for 48 h with chicken feed as the substrate. The number of C. jejuni was determined and gas production was recorded throughout the incubation period. At the end of the experiment, methane and volatile fatty acids (VFAs) concentration was determined as well. Test agents, which reduced the number of C. jejuni had no adverse effects on total gas and VFAs production, were selected for in vivo trial. Phage display technology was also used to identify antimicrobial peptides that would inhibit C. jejuni. This technique allows the peptides and proteins to be displayed on the surface of filamentous phage by inserting them into phage major and minor coat proteins. Using this approach, seven ligands with 15-mer peptide interacting with C. jejuni cells were selected and tested to determine their antimicrobial activity against C. jejuni. All phage peptides had high growth-inhibitory potency against C. jejuni. Among them, DT 3/15 showed particularly a high bactericidal activity in in vitro assay. The peptides were also tested towards the other gut bacteria given above and did not show any activity. In addition, the effect of DT/15 on the fermentation kinetics, C. jejuni counts and VFAs production was monitored using an in vitro fermentation method. DT3/15 had no effects on fermentation parameters and no inhibition of C. jejuni. Regardless of the results obtained in vitro fermentation assay, broth dilution study demonstrated that the isolated clones displayed high specificity against Campylobacter cells as no activity was observed on a panel of other bacteria. The use of plant derived agents as C. jejuni inhibitor in chicken feed was tested in an animal trial. Over a seven-week, birds were fed a normal basal diet or basal diet supplemented with the plant extracts derived from Acacia decurrens and Eremophila glabra, lemon myrtle oil, terpinene-4-ol, formulation of compounds named α-tops and antibiotic, virginiamycin. The number of C. jejuni was determined by using the traditional culture and RTQ-PCR methods from the faecal and caecal samples. In addition, body weight gain (BWG) and feed intake (FI) were recorded weekly, and feed conversion efficacy (FCE) were calculated. The mean log10 counts of C. jejuni tended to be lower in faecal and caecal samples obtained from α-tops supplemented group than other treatments and control diet. However, other supplementations did not (P>0.05) cause differences in C. jejuni numbers, even though observably less C. jejuni was counted in the first faecal shedding. No difference (P>0.05) in broiler performance (BWG, FI and FCE) were obtained for dietary supplementation, except E. glabra extracts which had negative impact (

Characterization of extended spectrum beta-lactamase ( ESBL)-producing Escherichia coli in Asi (Orontes) River in Turkey

In this study, the presence of extended spectrum beta-lactamase (ESBL)-producing Escherichia coli in aquatic environments (the Orontes River and an urban wastewater) was investigated. Fifty-four E. coli strains resistant to cefotaxime were isolated from the river waters and nearby waste water treatment plant and screened for ESBL gene variants, different classes of integrons and sulfonamide resistance genes. The ESBL-producing E. coli strains were further characterized by PhP-typing system, phylogenetic grouping and antimicrobial susceptibility testing. Of the 54 ESBL-producing strains, 14 (25.9%) belonged to four common PhP types and the remaining were of single types. CTX-M type ESBL genes were identified in 68% of the isolates. The most predominant specific CTX-M subtype identified was bla(CTX-M-15) (n=36), followed by bla(CTX-M-1) (n = 1). None of the isolates were SHV and OXA positive. Most of the ESBL positive isolates (n = 37; 68.5%) were harboring sul gene. This study indicates a widespread distribution of CTX-M-15 producing E. coli strains in the surface waters in part of Turkey, suggesting an aquatic reservoir for ESBL genes

Characterisation of Phenotypic and Genotypic Antibiotic Resistance Profile of Enterococci from Cheeses in Turkey

The aim of this study was to determine the prevalence of enterococci in cheese samples and to characterize their antimicrobial resistance profiles as well as the associated resistance genes. A total of 139 enterococci were isolated from 99 cheese samples, the isolates were identified as E. faecalis (61.2%), E. faecium (15.1%), E. gallinarum (12.9%), E. durans (5.0%), E. casseliflavis (2.9%) and E. avium (2.9%). The most frequent antimicrobial resistance observed in enterococci isolates was to lincomycin (88.5%), followed by kanamycin (84.2%), gentamycin (low level, 51.1%), rifampin (46.8%) and tetracycline (33.8%). Among the isolates, the frequencies of high level gentamycin and streptomycin resistant enterococci strains were 2.2% and 5.8%, respectively. Apart from the mentioned antibiotics, low levels of resistance to ciprofloxacin, erythromycin and chloramphenicol were found. Moreover no resistance was observed against penicillin and ampicillin. The antimicrobial resistance genes including tetM, tetL, ermB, cat, aph(3')-Illa, ant(6)-Ia and aac(6')-leaph(2 '')-Ia were found in enterococci from Turkish cheese samples. In the current study, we provided data for antibiotic resistance and the occurrence of resistance genes among enterococci. Regulatory and quality control programs for milk and other dairy products from farms to retail outlets has to be established and strengthened to monitor trends in antimicrobial resistance among emerging food borne pathogens in Turkey

Genomic analysis of sewage from 101 countries reveals global landscape of antimicrobial resistance

Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, evolution, and transmission of individual antibiotic resistance genes (ARGs) is essential to develop sustainable strategies combatting this threat. Here, we use metagenomic sequencing to analyse ARGs in 757 sewage samples from 243 cities in 101 countries, collected from 2016 to 2019. We find regional patterns in resistomes, and these differ between subsets corresponding to drug classes and are partly driven by taxonomic variation. The genetic environments of 49 common ARGs are highly diverse, with most common ARGs carried by multiple distinct genomic contexts globally and sometimes on plasmids. Analysis of flanking sequence revealed ARG-specific patterns of dispersal limitation and global transmission. Our data furthermore suggest certain geographies are more prone to transmission events and should receive additional attention