Antibiotic Treatment Administered to Pigs and Antibiotic Resistance of Escherichia coli Isolated from Their Feces and Carcasses.

Antimicrobial resistance (AMR) in bacteria is a frequent and widespread phenomenon. The European Food Safety Authority (EFSA) reports that multidrug resistant (MDR) Escherichia coli is considered an important hazard to public health. The lack of data on the correlation between the administration of antibiotics to pigs and the diffusion of MDR E. coli necessitates an in-depth study. The aims of our study were first of all to determine the presence of MDR and/or extended spectrum ß-lactamase (ESßL) E. coli isolated from feces and carcasses of pigs; and second, to evaluate the correlation between antibiotic resistance and the antibiotic treatment administrated to the animals considered. The examined E. coli was isolated from 100 fecal swabs and 100 carcass sponges taken from farms and slaughterhouses located in Reggio Emilia province in Italy. The MDR isolates were tested following the protocol defined by EUCAST (2015). Subsequently, a real-time PCR and an endpoint-PCR were used for the genomic analysis. Data highlighted 76.5% of MDR E. coli with a marked presence of the ampicillin (AMP)-streptomycin (STRE)-tetracycline (TETRA) pattern. Moreover, 13 isolates were ESßL producers, and the blaCTXM gene was the most frequently observed in genomic analysis. Results confirm the complexity of the AMR phenomenon showing a partial correlation between the administration of antibiotics and the resistance observed. Pigs destined to the production of Protected Designation of Origin items are colonized by bacteria resistant to a wide range of antibiotic classes even if data are encouraging for colistin and third generation cephalosporin. Furthermore, in-depth study focused on food production could be useful in a view of high safety standards for consumers.

Escherichia coli Strains Isolated from Retail Meat Products: Evaluation of Biofilm Formation Ability, Antibiotic Resistance, and Phylogenetic Group Analysis.

ABSTRACT: Escherichia coli is a ubiquitous organism capable of forming a biofilm. This is an important virulence factor and is critical in certain diseases and in the development of antibiotic resistance, which is increased by biofilm synthesis. In the present study, the potential health risk associated with handling and consumption of foods of animal origin contaminated with E. coli-producing biofilm was evaluated. We analyzed the ability of 182 E. coli strains isolated from pork, poultry, and beef, purchased in three different supermarkets in the area of the "Italian Food Valley" (Parma, northern Italy), to form biofilms. Positive strains were also tested for the presence of 12 biofilm-associated genes. Moreover, the 182 E. coli were characterized for antibiotic resistance, presence of multidrug resistance, extended-spectrum ß-lactamase strains, and phylogenetic diversity through PCR. Twenty-five percent of the isolates produced biofilm. The majority showed weak adherence, five were moderate, and three were strong producers. E. coli with a strong adherence capability (three of three) harbored eight biofilm-associated genes, while weak and moderate producers harbored only five (frequencies ranging from 80 to 100%). Multidrug resistance was observed in 20 biofilm-producing E. coli, and 15 of these belonged to phylogenetic group D. Among nonbiofilm producers, the percentage of strains belonging to phylogenetic groups B2 and D was approximately 40%, highlighting a potential health risk for consumers and people handling contaminated products. The present study underlines the importance of monitoring the prevalence and characteristics of E. coli contaminating retail meat in relation to the potential virulence highlighted here.

Antibacterial activity of metal complexes based on cinnamaldehyde thiosemicarbazone analogues.

The development of microbial antibiotic resistance has become one of the biggest threats to global health and the search for new molecules active against resistant pathogenic strains is a challenge that must be tackled. In many cases nosocomial infections are caused by bacteria characterized by multi-drug resistance patterns and by their ability to produce biofilms. These properties lead to the persistence of pathogens in the hospital environment. This paper reports the synthesis and characterization of three thiosemicarbazone derivatives based on a compound containing the cinnamaldehyde natural scaffold but possessing different logPow values. These molecules are then used as ligands to prepare complexes of the Cu(II) and Zn(II) ions. All these compounds, ligands and complexes, were screened in vitro on stains of Escherichia coli and Klebsiella pneumoniae for their antibacterial activity. Despite their molecular similarity they revealed variegated behaviors. Only two of them present interesting antimicrobial properties and have also been studied to verify their stability in solution. The compound with the lowest partition coefficient is the most promising. The minimal bactericidal concentration on K. pneumoniae and E. coli of these substances are very interesting and demonstrate that the use of metalloantibiotics is a promising device to fight antibiotic resistance.

Occurrence and Antimicrobial Profile of Bacterial Pathogens in Former Foodstuff Meat Products Used for Pet Diets.

European legislation stipulates that food no longer intended for human consumption, due to commercial reasons, manufacturing problems, or some defect, can be used in pet feed. However, the presence of former foodstuffs in pet diets could constitute a public health issue because pets can act as reservoirs of antimicrobial resistance genes. In this study, for the first time, biological hazards due to the presence of Escherichia coli and Salmonella in former foodstuff meat products were evaluated. Among the 112 samples of packaged fresh meat (poultry, pork, and beef) collected from cold storage warehouses of a mass market retailer, the overall prevalence of E. coli and Salmonella, the prevalence of strains with multidrug resistance, the phenotypic and genotypic characteristics of strains that produce extended-spectrum ß-lactamase, and the presence of biofilm producers were assessed. A high prevalence of E. coli was observed in former foodstuffs of poultry (100%), pork (100%), and beef (93.3%). Salmonella Derby and Salmonella Typhimurium were found in 11.5% of the poultry samples, and Salmonella Typhimurium was found in 13.3% of the pork samples. Salmonella was not recovered from the beef samples. Multidrug resistance was found in E. coli and Salmonella isolates from poultry, pork, and beef. Overall, 18.2% of E. coli isolates and 20% of Salmonella isolates were resistant to various types of antibiotics with various mechanisms of action. Salmonella isolates from pork are an important source of extended-spectrum ß-lactamase production. Both E. coli and Salmonella were carriers of antibiotic resistance marker genes ( blaTEM, blaSHV, and blaCTX-M) associated with ß-lactamase production in poultry and pork. Approximately 30% of the E. coli isolates from the various types of meat were phenotypically biofilm producers, and all carried the fimH gene. These findings indicate that the use of former foodstuff meat products in pet diets can represent a risk for public health.

Antimicrobial resistance, biofilm synthesis and virulence genes in Salmonella isolated from pigs bred on intensive farms.

Salmonella is the second cause of food-borne infection in humans in the USA and Europe. Pigs represent the second most important reservoir for the pathogen and the consumption of pork meat is a major risk factor for human salmonellosis. Here, we evaluated the virulence patterns of eleven Salmonella isolated from pigs (carcasses and faces) bred in intensive farms in the north of Italy. The two serotypes identified were S. Typhimurium and its monophasic variant 1,4,5,12:i:-. None of the isolates was an ESBL producer, as confirmed also by PCR. However, the presence of a multi-drug resistant pattern was evident, with all the isolates being resistant to at least to five antimicrobial agents belonging to various classes. Moreover, six out of eleven isolates showed important resistance profiles, such as resistance against colistin and ciprofloxacin, with nine to twelve recorded resistances. The isolates were negative for the biofilm synthesis test, while four different virulotypes were characterized. All the isolates showed the presence of invA, hilA, stn, ssrA, sipC. One sample also harbored ssaR and spvC genes. One strain was positive for all the virulence genes tested and was resistant to 12 antimicrobial agents. The present study contributes new data to the surveillance program for antibiotic resistance. Furthermore, the presence of eleven highly virulent isolates poses concern for human health in relation to their diffusion in the environment.

Toxoplasma Gondii and Pre-treatment Protocols for Polymerase Chain Reaction Analysis of Milk Samples: A Field Trial in Sheep from Southern Italy.

Toxoplasmosis is a zoonotic disease caused by the protozoan Toxoplasma gondii. Ingestion of raw milk has been suggested as a risk for transmission to humans. Here the authors evaluated pre-treatment protocols for DNA extraction on T. gondii tachyzoite-spiked sheep milk with the aim of identifying the method that resulted in the most rapid and reliable polymerase chain reaction (PCR) positivity. This protocol was then used to analyse milk samples from sheep of three different farms in Southern Italy, including real time PCR for DNA quantification and PCR-restriction fragment length polymorphism for genotyping. The pre-treatment protocol using ethylenediaminetetraacetic acid and Tris-HCl to remove casein gave the best results in the least amount of time compared to the others on spiked milk samples. One sample of 21 collected from sheep farms was positive on one-step PCR, real time PCR and resulted in a Type I genotype at one locus (SAG3). Milk usually contains a low number of tachyzoites and this could be a limiting factor for molecular identification. Our preliminary data has evaluated a rapid, cost-effective and sensitive protocol to treat milk before DNA extraction. The results of the present study also confirm the possibility of T. gondii transmission through consumption of raw milk and its unpasteurised derivatives.

Meat Juice Serology for Toxoplasma Gondii Infection in Chickens.

Toxoplasma gondii is an important foodborne zoonosis. Free-range chickens are at particularly high risk of infection and are also excellent indicators of soil contamination by oocysts. In the present study, hearts of 77 free-range chickens were collected at slaughter. T. gondii meat juice enzyme-linked immunosorbent assay was performed with a commercial kit, following validation with positive controls, from experimentally infected chickens, and negative ones. Out of 77 samples, only 66 gave sufficient meat juice for serology. Of these, 24 (36.4%) were positive for T. gondii considering the 5*standard deviation values (calculated on the optical density of negative controls), while all the samples were negative considering sample/positive% values. Parasite-specific polymerase chain reaction was carried out on all samples obtained from heart tissue and none were positive for the presence of T. gondii DNA. Results would suggest that further study on the use of meat juice with a validated serological test to detect T. gondii in chickens could lead to widespread epidemiological studies in this important intermediate host. However, sample collection and test specificity require further evaluation.

Influence of Pigskin on Salmonella Contamination of Pig Carcasses and Cutting Lines in an Italian Slaughterhouse.

Ninety pig carcasses and twenty one food contact surfaces (FCSs) were tested for Salmonella in a slaughterhouse processing ca. 380 pigs/h between 2014-2015. Sampling was performed during seven sessions. Four carcass sites of 100 cm2 each (back, belly, jowl externally, and the diaphragmatic area internally) were swabbed after evisceration. Meat conveyors and dressing tables were tested swabbing areas of 200 to 400 cm2. After pre-enrichment in buffered peptone water, samples were tested by Salmonella MDS® assay and the presumptive positives were confirmed by the ISO 6579 method. Salmonella isolates were serotyped following the Kauffman-White-Le Minor scheme and genotyped by XbaI pulsed field gel electrophoresis. Salmonella was isolated from 16/90 [17.8%; confidence interval (CI) 95%=11.2-26.9] carcasses and 4/21 (19.0%; CI 95%=7.7-40.0) FCSs. Four serovars were identified on carcasses. S. enterica 4,[5],12:i:-was the most prevalent (43.75%), followed by S. Rissen (31.25%), S. Derby (12.5%) and S. Bovismorbificans (12.5%). Two serovars were found on FCSs, namely S. Derby (75%) and S. Livingstone (25%). During one sampling session, a failure in carcass dehairing occurred and caused significantly higher prevalence of carcass contamination (60%) than in the remaining sessions. Moreover, in the same session, Salmonella prevalence was marginally significantly higher on FCSs than in the remaining sampling days, suggesting that dehairing affects contamination not only on carcasses, but also on the working surfaces.

Detection of Salmonella enterica in pigs at slaughter and comparison with human isolates in Italy.

In 2013-2014, 201 pigs belonging to 67 batches were tested for Salmonella in their mesenteric lymph nodes (MLN) in one abattoir of Northern Italy. For each batch, faecal material was collected at lairage by swabbing the pen floor for approximately 1600 cm(2). The aim of this study was to investigate the prevalence of Salmonella in MLN of pigs at slaughter, to assess Salmonella contamination at lairage and to evaluate the effect of lairage duration on its prevalence. Serotyping, XbaI PFGE typing and antimicrobial testing of the isolates were performed. Pig and human Salmonella isolates of the same region of Italy were compared to evaluate possible correlations. Salmonella enterica was isolated from 19.9% of the MLN and 49.3% of the environmental faecal samples. Nine different serovars were identified among 75 S. enterica isolates. In MLN Salmonella Derby was the most common (52.5%), followed by S. enterica 4,[5],12:i:- (17.5%) and Salmonella Rissen (10.0%). In faecal samples S. Derby was prevalent (51.4%), followed by S. enterica 4,[5], 12:i:- (20.0%) and Salmonella Brandenburg (14.3%). Lairage holding varied between 1 and ≥ 12 h (median value: 2.5h). In pigs held for 1-3h, 14.1% were positive for Salmonella in MLN but the prevalence reached 31.8% when they were held for ≥ 12 h. The contamination of MLN was statistically different (p=0.0045) between the two groups, thus confirming the role of long-lasting lairage in Salmonella contamination of pigs. XbaI PFGE typing detected 36 PFGE types. Twenty-three PFGE types were identified among the 40 MLN isolates and 22 PFGE types among the 35 faecal isolates. A total of 11 PFGE types were shared between the MLN of pigs and the lairage environment. Among S. Derby, 6 shared PFGE types between MLN and faeces were found and among S. enterica 4,[5],12:i:- one PFGE type was common between MLN and the faecal samples. Shared profiles between human and swine isolates of S. Derby, S. enterica 4,[5],12:i:-, S. Rissen, Salmonella Manhattan, S. Brandenburg, Salmonella Livingstone, Salmonella London and Salmonella Muenchen were identified. Among S. Derby and S. enterica 4,[5],12:i:- isolates found in pigs, 6/15 profiles (40.0%) and 8/10 (80.0%) were shared with human isolates. High resistance rates to streptomycin (97.3%), sulphonamide compounds (84.0%) and tetracycline (56.0%) were observed. No resistance was detected to ertapenem and meropenem. High proportions of isolates showed intermediate sensitivity to ciprofloxacin (85.3%) and cefotaxime (66.7%). High sensitivity rates were found to chloramphenicol (96.0%) and trimethoprim/sulfamethoxazole (81.3%).