Bacteriophage Therapy to Reduce Colonization of Campylobacter jejuni in Broiler Chickens before Slaughter.

Campylobacteriosis is the most commonly reported gastrointestinal disease in humans. Campybacter jejuni is the main cause of the infection, and bacterial colonization in broiler chickens is widespread and difficult to prevent, leading to high risk of occurrence in broiler meat. Phage therapy represents an alternative strategy to control Campylobacter in poultry. The aim of this work was to assess the efficacy of two field-isolated bacteriophages against experimental infections with an anti-microbial resistant (AMR) Campylobacter jejuni strain. A two-step phage application was tested according to a specific combination between chickens' rearing time and specific multiplicities of infections (MOIs), in order to reduce the Campylobacter load in the animals at slaughtering and to limit the development of phage-resistant mutants. In particular, 75 broilers were divided into three groups (A, B and C), and phages were administered to animals of groups B and C at day 38 (Φ 16-izsam) and 39 (Φ 7-izsam) at MOI 0.1 (group B) and 1 (group C). All broilers were euthanized at day 40, and Campylobacter jejuni was enumerated in cecal contents. Reductions in Campylobacter counts were statistically significant in both group B (1 log10 colony forming units (cfu)/gram (gr)) and group C (2 log10 cfu/gr), compared to the control group. Our findings provide evidence about the ability of phage therapy to reduce the Campylobacter load in poultry before slaughtering, also associated with anti-microbial resistance pattern.

A large food-borne outbreak of campylobacteriosis in kindergartens and primary schools in Pescara, Italy, May-June 2018.

Introduction. In May-June 2018, an outbreak of campylobacteriosis involved students and school staff from kindergartens and primary schools in Pescara, southern Italy.Aim. We present details of the epidemiological and microbiological investigation, and the findings of the analytical study, as well as the implemented control measures.Methodology. To identify possible risk factors associated with the observed outbreak, a case control study was conducted using a questionnaire to collect information on the date of symptoms onset, type and duration of symptoms, type of healthcare contact, school attendance, and food items consumed at school lunches during the presumed days of exposure. Attack rates were calculated for each date and school. Logistic regression models were used to estimate the odds ratios of being a case and the odds of illness by food items consumed, respectively. Moreover, we carried out a comparative genomic analysis using whole genome multilocus sequence typing (wgMLST) of Campylobacter jejuni strains isolated during the outbreak investigation to identify the source of the outbreak.Results. Overall, 222 probable cases from 21 schools were identified, and C. jejuni was successfully isolated from 60 patients. The meals in the schools involved were provided by two cooking centres managed by a joint venture between two food companies. Environmental and food sampling, epidemiological and microbiological analyses, as well as a case control study with 176 cases and 62 controls from the same schools were performed to identify the source of the outbreak. The highest attack rate was recorded among those having lunch at school on 29 May (7.8 %), and the most likely exposure was 'caciotta' cheese (odds ratio 2.40, 95 % confidence interval 1.10-5.26, P=0.028). C. jejuni was isolated from the cheese, and wgMLST showed that the human and cheese isolates belonged to the same genomic cluster, confirming that the cheese was the vehicle of the infection.Conclusion. It is plausible that a failure of the pasteurization process contributed to the contamination of the cheese batches. Timely suspension of the catering service and summer closure of the schools prevented further spread.

Campylobacter in chicken - Critical parameters for international, multicentre evaluation of air sampling and detection methods.

The present pilot study aimed at evaluating air sampling as a novel method for monitoring Campylobacter in poultry farms. We compared the bacteriological isolation of Campylobacter from boot swabs and air filter samples using ISO 10272-1:2017. A secondary aim was to evaluate the use of molecular methods, i.e. real time PCR, on the same sample set. Samples from 44 flocks from five European countries were collected, and included air samples, in parallel with boot swabs. Campylobacter spp. was isolated from seven of 44 boot swabs from three of five partners using the enrichment method. Two of these positive boot swab samples had corresponding positive air samples. Using enrichment, one positive air sample was negative in the corresponding boot swabs, but Campylobacter spp. was isolated from direct plating of the boot swab sample. One partner isolated Campylobacter spp. from six of 10 boot swabs using direct plating. Overall, 33 air filter samples were screened directly with PCR, returning 14 positive results. In conclusion, there was a lack of correspondence between results from analysis of boot swabs and air filters using ISO 10272-1:2017. In contrast, the combination of air filters and direct real-time PCR might be a way forward. Despite the use of the detailed ISO protocols, there were still sections that could be interpreted differently among laboratories. Air sampling may turn into a multi-purpose and low-cost sampling method that may be integrated into self-monitoring programs.

Campylobacter jejuni and Campylobacter coli: prevalence, contamination levels, genetic diversity and antibiotic resistance in Italy.

A research was carried out in Italy with the aim of assessing Campylobacter contamination in broilers from breeding to slaughter, of defining the genetic diversity of isolates and their antibiotic resistance. Sampling was carried out in a slaughterhouse, and in farms representative of the most common broiler production in Italy. At farm, the 78.8% (95% C.I.: 74.5%­82.5%) of cloacal samples tested positive for Campylobacter spp. C. jejuni showed higher prevalence in winter than in spring and summer (p < 0.00001, χ2 = 32.9), while C. coli showed an opposite trend (p < 0.00001, χ2= 41.1). At slaughterhouse, the 32.3% (95% C.I.: 30.2%­35.2%) and the 23.9% (95% C.I.: 21.7%­26.3%) of skin samples tested positive for C. jejuni for C. coli, respectively. C. coli showed higher prevalence than C. jejuni at washing (p < 0.05, χ2 = 11.11) and at chilling (p < 0.05, χ2 = 9.26). PFGE revealed high heterogeneity among isolates. Some clones were identified within the same farm in more than one season, suggesting environmental conditions able to support their persistence; other clones resulted to be spatially distant, suggestive of cross­contamination. Both Campylobacter species showed high resistance to nalidixic acid and ciprofloxacin, while resistance to erythromycin was more frequent in C. coli than C. jejuni (p < 0.05; χ2 test).

Correction: Antimicrobial resistance genotypes and phenotypes of Campylobacter jejuni isolated in Italy from humans, birds from wild and urban habitats, and poultry.

[This corrects the article DOI: 10.1371/journal.pone.0223804.].

Antimicrobial resistance genotypes and phenotypes of Campylobacter jejuni isolated in Italy from humans, birds from wild and urban habitats, and poultry.

Campylobacter jejuni, a common foodborne zoonotic pathogen, causes gastroenteritis worldwide and is increasingly resistant to antibiotics. We aimed to investigate the antimicrobial resistance (AMR) genotypes of C. jejuni isolated from humans, poultry and birds from wild and urban Italian habitats to identify correlations between phenotypic and genotypic AMR in the isolates. Altogether, 644 C. jejuni isolates from humans (51), poultry (526) and wild- and urban-habitat birds (67) were analysed. The resistance phenotypes of the isolates were determined using the microdilution method with EUCAST breakpoints, and AMR-associated genes and single nucleotide polymorphisms were obtained from a publicly available database. Antimicrobial susceptibility testing showed that C. jejuni isolates from poultry and humans were highly resistant to ciprofloxacin (85.55% and 76.47%, respectively), nalidixic acid (75.48% and 74.51%, respectively) and tetracycline (67.87% and 49.02%, respectively). Fewer isolates from the wild- and urban-habitat birds were resistant to tetracycline (19.40%), fluoroquinolones (13.43%), and quinolone and streptomycin (10.45%). We retrieved seven AMR genes (tet (O), cmeA, cmeB, cmeC, cmeR, blaOXA-61 and blaOXA-184) and gyrA-associated point mutations. Two major B-lactam genes called blaOXA-61 and blaOXA-184 were prevalent at 62.93% and 82.08% in the poultry and the other bird groups, respectively. Strong correlations between genotypic and phenotypic resistance were found for fluoroquinolones and tetracycline. Compared with the farmed chickens, the incidence of AMR in the C. jejuni isolates from the other bird groups was low, confirming that the food-production birds are much more exposed to antimicrobials. The improper and overuse of antibiotics in the human population and in animal husbandry has resulted in an increase in antibiotic-resistant infections, particularly fluoroquinolone resistant ones. Better understanding of the AMR mechanisms in C. jejuni is necessary to develop new strategies for improving AMR programs and provide the most appropriate therapies to human and veterinary populations.

The prevalence, characterisation, and antimicrobial resistance of Yersinia enterocolitica in pigs from Central Italy.

Widely spread in nature, Yersinia enterocolitica (YE) is a foodborne pathogen of major health and economic significance in developed countries. The aim of this study is to analyse YE strains isolated from 400 slaughtered pigs from the Abruzzo region, Italy, using biochemical tests and a multiplex polymerase chain reaction PCR detecting 6 chromosomal genes (ystA, irp2, 16s, ail, inv, hemR) and one plasmid-borne virulence gene (yadA). Antimicrobial susceptibility was evaluated and pulsed-field gel electrophoresis (PFGE) was also performed in order to assess phylogenetic diversity. In total, 56 samples of porcine tonsils (14%) were found to be positive for the presence of pathogenic YE. All YE belonged to the pathogenic bioserotype 4/O:3. All YE samples were positive for the chromosomal virulence genes ystA, ail, and inv, whereas results for the presence of yadA and hemR were variable. This study found that YE isolates were resistant to ampicillin (100%), streptomycin (26.79%), sulfisoxazole (19.65%), tetracycline (16.08%), nalidixic acid (14.30%), and chloramphenicol (10.72%). The strains characterised by PFGE showed a high similarity. This study demonstrates the usefulness of multiplex polymerase chain reaction (PCR) compared with conventional phenotypic assays for the identification of pathogenic YE isolates and the limitations of PFGE for the molecular typing of YE bioserotype 4/O:3.

Evidence of Campylobacter jejuni reduction in broilers with early synbiotic administration.

C. jejuni is considered a food safety concern to both public health authorities and consumers since it is the leading bacterial cause of food-borne gastroenteritis in humans. A high incidence of C. jejuni in broiler flocks is often correlated to pathogen recovery in retail poultry meat, which is the main source of human infection. In this work broiler chickens were fed with a synbiotic product mixed with conventional feed using two different administration strategies. The synbiotic was formulated with the microencapsulated probiotic Bifidobacterium longum PCB133 and a xylo-oligosaccharide (XOS). 1-day old chicks were infected with C. jejuni strain M1 (105 cells) and the synbiotic mixture was then administered starting from the first and the 14th day of chicken life (for animal groups GrpC and GrpB respectively). The goal of this study was to monitor C. jejuni load at caecum level at different sampling time by real-time PCR, identifying the best administration strategy. The microbiological analysis of the caecal content also considered the quantification of Campylobacter spp., Bifidobacterium spp. and B. longum. The supplemented synbiotic was more successful in reducing C. jejuni and Campylobacter spp. when administered lifelong, compared to the shorter supplementation (GrpB). Bifidobacterium spp. quantification did not show significant differences among treatments and B. longum PCB133 was detected in both supplemented groups evidencing the successful colonization of the strain. Moreover, the samples of the control group (GrpA) and GrpC were analysed with PCR-denaturing gradient gel electrophoresis (PCR-DGGE) to compare the caecal microbial community profiles at the beginning and at the end of the trial. Pattern analysis evidenced the strong influence of the early synbiotic supplementation, although a physiological change in the microbial community, occurring during growth, could be observed. Experimental results demonstrate that the synbiotic approach at farm level can be an effective strategy, combined with biosecurity measures, to improve the safety of poultry meat.

Characterization of antimicrobial resistance patterns and detection of virulence genes in Campylobacter isolates in Italy.

Campylobacter has developed resistance to several antimicrobial agents over the years, including macrolides, quinolones and fluoroquinolones, becoming a significant public health hazard. A total of 145 strains derived from raw milk, chicken faeces, chicken carcasses, cattle faeces and human faeces collected from various Italian regions, were screened for antimicrobial susceptibility, molecular characterization (SmaI pulsed-field gel electrophoresis) and detection of virulence genes (sequencing and DNA microarray analysis). The prevalence of C. jejuni and C. coli was 62.75% and 37.24% respectively. Antimicrobial susceptibility revealed a high level of resistance for ciprofloxacin (62.76%), tetracycline (55.86%) and nalidixic acid (55.17%). Genotyping of Campylobacter isolates using PFGE revealed a total of 86 unique SmaI patterns. Virulence gene profiles were determined using a new microbial diagnostic microarray composed of 70-mer oligonucleotide probes targeting genes implicated in Campylobacter pathogenicity. Correspondence between PFGE and microarray clusters was observed. Comparisons of PFGE and virulence profiles reflected the high genetic diversity of the strains examined, leading us to speculate different degrees of pathogenicity inside Campylobacter populations.

Brucella ceti from two striped dolphins stranded on the Apulia coastline, Italy.

Since 1994, when Brucella ceti was first isolated from an aborted dolphin fetus, several cases have been reported worldwide. The first case of B. ceti in the Mediterranean (and in Italy), however, was recorded only in 2012, off the coast of Tuscany. Extensive studies, using serological and microbiological methods, have documented this bacterium in dolphins and demonstrated its zoonotic potential. We describe the typing of two B. ceti strains isolated from striped dolphins (Stenella coeruleoalba) stranded on the southern Apulia coastline. B. ceti isolates were conventionally typed, and then genotyped by both the multilocus sequence typing (MLST) and the multilocus variable number of tandem repeats typing (MLVA) methodologies to infer phylogeny and potential epidemiological links between the two cases. The two isolates were identified through MLST analysis as belonging to the common sequence type 26 (ST26), while MLVA analysis, having established that the two isolates have identical profiles, assigned them to a novel genotype within cluster A - a unique representative of a new Mediterranean subcluster. The results thus revealed a link between the two cases studied, demonstrating the usefulness of MLST and MLVA for the epidemiological investigation of brucellae among marine mammals.

Monitoring of polycyclic aromatic hydrocarbons in bees (Apis mellifera) and honey in urban areas and wildlife reserves.

The honeybee is a good biological indicator that quickly reflects chemical impairment of the environment by its high mortality and the presence of pollutants in its body or in beehive products. In this work the honeybee (Apis mellifera) and honey were used to detect the presence of polycyclic aromatic hydrocarbons (PAHs) in several areas with different degrees of environmental pollution. All sampling sites showed the presence of PAHs. Benzo(a)pyrene was never detected. Fluorene, phenanthrene, anthracene, fluoranthene, benz(a)anthracene, benzo(b)fluoranthene, and benzo(k)fluoranthene were the PAHs detected in bees, whereas the honey contained only phenanthrene, anthracene, and chrysene. Phenanthrene showed the highest mean values in honeybees and honey. Independent from the season and location the pattern of PAHs in honeybees and honey was dominated by the presence of the lowest molecular weight PAHs. Furthermore, the mean PAH concentrations in honey samples were lower than those reported in honeybees, and no positive correlation was found between the compounds detected in bees and those in honey.