Erythromycin and tetracycline resistant lactobacilli in the production of a typical dry sausage from the north of Italy

Lo scopo di questo lavoro di tesi era di stabilire la frequenza di lattobacilli resistenti all’eritromicina e alla tetraciclina sia nel prodotto finito che nella filiera di produzione di un salame DOP del Nord Italia. Il controllo della filiera produttiva comprendeva la pelle, la carne macinata e le feci di otto suini mentre il prodotto finito era analizzato a 0, 21, 35 e 45 giorni di stagionatura. Sono state isolate colonie di lattobacilli da terreni di coltura selettivi addizionati di antibiotici. Le specie antibiotico resistenti più frequentemente isolate erano Lactobacillus sakei, Lactobacillus curvatus and Lactobacillus plantarum. I geni che con maggior frequenza sono stati evidenziati nei ceppi isolati dalla filiera e nel prodotto finito erano tet(M) ed erm(B) mentre tet(W) ed erm(B) lo erano per i ceppi isolati dalle feci. Questo studio ha consentito di fornire dati circa la presenza e la diffusione di lattobacilli tetraciclina ed eritromicina resistenti in un salame tipico del Nord Italia. I lattobacilli antibiotico resistenti potrebbero trasferire ad altri batteri, patogeni o non patogeni, i geni responsabili della resistenza stessa. Alla luce di tutto questo l’80% dei salami analizzati possono essere considerati sicuri da questo punto di vista mentre il 20% può essere valutato come un prodotto a rischio anche se la situazione non è ancora preoccupante.The scope of this study was to assess the frequency of erythromycin and tetracycline resistant lactobacilli in the production chain (skin, minced meat, and stools of eight swine, the natural casing, and the final product at days 0, 21, 35, and 45 of ripening) of a Protected Designation of Origin dry sausage from the North of Italy and in the end products. We isolated colonies of lactobacilli from selective medium supplemented with erythromycin or tetracycline and the most frequently antibiotic resistant species isolated were Lactobacillus sakei, Lactobacillus curvatus and Lactobacillus plantarum The most frequent resistance genes in process line strains were tet(M) and erm(B) while tet(W) and erm(B) were common in strains isolated from swine stools. This study provides evidence of the presence of tetracycline and, to a lesser extent, erythromycin resistant lactobacilli in fermented dry sausages produced in Northern Italy. Although these antibiotic resistant lactobacilli could serve as reservoir organisms, in our study 80% of salami could be considered as safe even though 20% could represent a border line situation regarding the possibility of transferring antibiotic resistant genes to pathogens

Erythromycin- and tetracycline-resistant lactobacilli in Italian fermented dry sausages

Aims: To assess the frequency of erythromycin- and tetracycline-resistant lactobacilli in Italian fermented dry sausages. Methods and Results: We isolated lactobacilli colonies from 20 salami from the north of Italy (Piacenza province) using selective medium supplemented with erythromycin or tetracycline; we determined the minimum inhibitory concentration and searched for selected erythromycin and tetracycline resistance genes. A total of 312 lactobacilli colonies were genetically ascribed to 60 different strains belonging to seven Lactobacillus species. Lactobacillus sakei, Lactobacillus curvatus and Lactobacillus plantarum were the most frequently found species. Thirty strains (50%) were phenotypically resistant to erythromycin, 45 (75%) to tetracycline and 27 (45%) were resistant to both. The most frequently detected resistance genes were tet(M) and erm(B). Conclusions: This study provides evidence of the presence of tetracycline- and, to a lesser extent, erythromycin-resistant lactobacilli in fermented dry sausages produced in northern Italy. Significance and Impact of the Study: Although these antibiotic-resistant lactobacilli could serve as reservoir organisms, in our study, 16 of 20 salami could be considered safe in regard to possible antibiotic resistance gene transfer to pathogens, whereas 4 of 20 could represent a borderline situation

A randomized double-lind trial on perioperative administration of probiotics in colorectal cancer patients

AIM: To investigate whether probiotic bacteria, given perioperatively, might adhere to the colonic mucosa, reduce concentration of pathogens in stools, and modulate the local immune function. METHODS: A randomized, double-blind clinical trial was carried out in 31 subjects undergoing elective colorectal resection for cancer. Patients were allocated to receive either a placebo (group A, n = 10), or a dose of 107 of a mixture of Bifidobacterium longum (BB536 ) and Lactobacillus johnsonii (La1 ) (group B, n = 11), or the same mixture at a concentration of 109 (group C, n = 10). Probiotics, or a placebo, were given orally 2 doses/d for 3 d before operation. The same treatment continued postoperatively from day two to day four. Stools were collected before treatment, during surgery (day 0) and 5 d after operation. During the operation, colonic mucosa samples were harvested to evaluate bacterial adherence and to assess the phenotype of dendritic cells (DCs) and lymphocyte subsets by surface antigen expression (flow cytometry). The presence of BB536 and La1 was evaluated by the random amplified polymorphism DNA method with specific PCR probes. RESULTS: The three groups were balanced for baseline and surgical parameters. BB536 was never found at any time-points studied. At day 0, La1 was present in 6/10 (60%) patients in either stools or by biopsy in group C, in 3/11 (27.2%) in group B, and none in the placebo group (P = 0.02 C vs A). There was a linear correlation between dose given and number of adherent La1 (P = 0.01). The rate of mucosal colonization by enterobacteriacae was 30% (3/10) in C, 81.8% (9/11) in B and 70% (7/10) in A (P = 0.03 C vs B). The Enterobacteriacae count in stools was 2.4 (log10 scale) in C, 4.6 in B, and 4.5 in A (P = 0.07 C vs A and B). The same trend was observed for colonizing enterococchi. La1 was not found at day +5. We observed greater expression of CD3, CD4, CD8, and naive and memory lymphocyte subsets in group C than in group A with a dose response trend (C > B > A). Treatment did not affect DC phenotype or activation, but after ex vivo stimulation with lipopolysaccharides, groups C and B had a lower proliferation rate compared to group A (P = 0.04). Moreover, dendritic phenotypes CD83-123, CD83-HLADR, and CD83-11c (markers of activation) were significantly less expressed in patients colonized with La1 (P = 0.03 vs not colonized). CONCLUSION: La1 , but not BB536 , adheres to the colonic mucosa, and affects intestinal microbiota by reducing the concentration of pathogens and modulates local immunity

Susceptibility to tetracycline and erythromycin of Lactobacillus paracasei strains isolated from traditional Italian fermented foods

The aim of this study was to evaluate the susceptibility of 197 isolates of Lactobacillus paracasei, isolated from Italian fermented products coming from different geographical areas, to tetracycline and erythromycin, two antimicrobials widely used in clinical and animal therapy. Isolation media were supplemented with antibiotics according to the microbiological breakpoints (BPs) defined by European Food Safety Authority (EFSA). Isolates were identified at the species level and were typed by rep-PCR using the (GTG)5 primer. A total of 121 genotypically different strains were detected and their phenotypic antimicrobial resistance to tetracycline and erythromycin was determined as the minimum inhibitory concentration (MIC) using the broth microdilution method. The presence of the genes ermB, ermC and tetL, tetM, tetS, tetW, in the phenotypically resistant isolates was investigated by PCR. Tetracycline induction of tetM expression on representative resistant strains, grown in medium either lacking or containing the antibiotic, was also analyzed by RT-PCR. Among the 121 tested strains, 77.7% were susceptible to tetracycline (MIC 64 4 \ub5g/ml; BP = 4 \ub5g/ml) and 94.2% to erythromycin (MIC 64 1 \ub5g/ml; BP = 1 \ub5g/ml). Overall, 27 strains appeared to be resistant to tetracycline (16 64 MIC 64 128 \ub5g/ml ) (TetR), and 7 of them were also resistant to erythromycin (MIC 65 1024 \ub5g/ml) (ErmR). The tetM and ermB genes were the most frequently detected in the TetR and/or ErmR strains. The tetM expression was induced by antibiotic addition to the growth medium. Our study confirmed that L. paracasei is quite sensitive to tetracycline and erythromycin, but the high level of resistance of ErmR strains suggested that acquired resistance took place. Further investigations are required to analyze whether the genes identified in L. paracasei isolates might be horizontally transferred to other species. Since \u201ccommensal\u201d bacteria, which L. paracasei belongs to, may play an active role in the spreading of antibiotic resistance, a series of measures inspired from a principle of precaution should be taken before they are used as commercial starters or probiotic cultures in food products, complemented by a more prudent use of antibiotics in agriculture, veterinary, and human medicine