Food risk associated with vegetable consumption, exposure to antimicrobial-resistant strains and pesticide residues.

This preliminary study aimed to detect biological and chemical contaminants in vegetables sold in Sicily for human consumption, assess the spread of antimicrobial-resistant (AMR) strains in these foods, and characterize their antimicrobial-resistance genes. A total of 29 fresh and ready-to-eat samples were analyzed. Microbiological analyses were performed for the detection of Salmonella spp. and the enumeration of Enterococci, Enterobacteriaceae, and Escherichia coli. Antimicrobial resistance was assessed by the Kirby-Bauer method, according to the Clinical and Laboratory Standards Institute guidelines. Pesticides were detected by high-performance liquid chromatography and gas chromatography coupled with mass spectrometry. No samples were contaminated by Salmonella spp., E. coli was detected in 1 sample of fresh lettuce at a low bacterial count (2 log cfu/g). 17.24% of vegetables were contaminated by Enterococci and 65.5% by Enterobacteriaceae (bacterial counts between 1.56 log cfu/g and 5.93 log cfu/g and between 1.6 log cfu/g and 5.48 log cfu/g respectively). From 86.2% of vegetables, 53 AMR strains were isolated, and 10/53 isolates were multidrug resistant. Molecular analysis showed that the blaTEM gene was detected in 12/38 ß-lactam-resistant/intermediate-resistant isolates. Genes conferring tetracycline resistance (tetA, tetB, tetC, tetD, tetW) were detected in 7/10 isolates. The qnrS gene was detected in 1/5 quinolone-resistant isolates, the sulI gene was detected in 1/4 sulfonamide- resistant/intermediate-resistant isolates and the sulIII gene was never detected. Pesticides were detected in 27.3% of samples, all of which were leafy vegetables. Despite the satisfactory hygienic status of samples, the high percentage of AMR bacteria detected stresses the need for an effective monitoring of these foods as well as adequate strategies to counteract the spread of AMR bacteria along the agricultural chain. Also, the chemical contamination of vegetables should not be underestimated, especially considering that leafy vegetables are commonly consumed raw and that no official guidelines about maximum residue limits of pesticides in ready-to-eat vegetables are available.

A Two-Component regulatory system with opposite effects on glycopeptide antibiotic biosynthesis and resistance.

The glycopeptide A40926, produced by the actinomycete Nonomuraea gerenzanensis, is the precursor of dalbavancin, a second-generation glycopeptide antibiotic approved for clinical use in the USA and Europe in 2014 and 2015, respectively. The final product of the biosynthetic pathway is an O-acetylated form of A40926 (acA40926). Glycopeptide biosynthesis in N. gerenzanensis is dependent upon the dbv gene cluster that encodes, in addition to the two essential positive regulators Dbv3 and Dbv4, the putative members of a two-component signal transduction system, specifically the response regulator Dbv6 and the sensor kinase Dbv22. The aim of this work was to assign a role to these two genes. Our results demonstrate that deletion of dbv22 leads to an increased antibiotic production with a concomitant reduction in glycopeptide resistance. Deletion of dbv6 results in a similar phenotype, although the effects are not as strong as in the Δdbv22 mutant. Consistently, quantitative RT-PCR analysis showed that Dbv6 and Dbv22 negatively regulate the regulatory genes (dbv3 and dbv4), as well as some dbv biosynthetic genes (dbv23 and dbv24), whereas Dbv6 and Dbv22 positively regulate transcription of the single, cluster-associated resistance gene. Finally, we demonstrate that exogenously added acA40926 and its precursor A40926 can modulate transcription of dbv genes but with an opposite extent: A40926 strongly stimulates transcription of the Dbv6/Dbv22 target genes while acA40926 has a neutral or negative effect on transcription of those genes. We propose a model in which glycopeptide biosynthesis in N. gerenzanensis is modulated through a positive feedback by the biosynthetic precursor A40926 and a negative feedback by the final product acA40926. In addition to previously reported control systems, this sophisticated control loop might help the producing strain cope with the toxicity of its own product. This work, besides leading to improved glycopeptide producing strains, enlarges our knowledge on the regulation of glycopeptide biosynthesis in actinomycetes, setting N. gerenzanensis and its two-component system Dbv6-Dbv22 apart from other glycopeptide producers.

Phosphate-controlled regulator for the biosynthesis of the dalbavancin precursor A40926.

The actinomycete Nonomuraea sp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of the novel antibiotic dalbavancin. Previous studies have shown that phosphate limitation results in enhanced A40926 production. The A40926 biosynthetic gene (dbv) cluster, which consists of 37 genes, encodes two putative regulators, Dbv3 and Dbv4, as well as the response regulator (Dbv6) and the sensor-kinase (Dbv22) of a putative two-component system. Reverse transcription-PCR (RT-PCR) and real-time RT-PCR analysis revealed that the dbv14-dbv8 and the dbv30-dbv35 operons, as well as dbv4, were negatively influenced by phosphate. Dbv4 shows a putative helix-turn-helix DNA-binding motif and shares sequence similarity with StrR, the transcriptional activator of streptomycin biosynthesis in Streptomyces griseus. Dbv4 was expressed in Escherichia coli as an N-terminal His(6)-tagged protein. The purified protein bound the dbv14 and dbv30 upstream regions but not the region preceding dbv4. Bbr, a Dbv4 ortholog from the gene cluster for the synthesis of the glycopeptide balhimycin, also bound to the dbv14 and dbv30 upstream regions, while Dbv4 bound appropriate regions from the balhimycin cluster. Our results provide new insights into the regulation of glycopeptide antibiotics, indicating that the phosphate-controlled regulator Dbv4 governs two key steps in A40926 biosynthesis: the biosynthesis of the nonproteinogenic amino acid 3,5-dihydroxyphenylglycine and critical tailoring reactions on the heptapeptide backbone.

Expression in Streptomyces lividans of Nonomuraea genes cloned in an artificial chromosome.

A bacterial artificial chromosomal library of Nonomuraea sp. ATCC39727 was constructed using Escherichia coli-Streptomyces artificial chromosome (ESAC) and screened for the presence of dbv genes known to be involved in the biosynthesis of the glycopeptide A40926. dbv genes were cloned as two large, partially overlapping, fragments and transferred into the host Streptomyces lividans, thus generating strains S. lividansColon, two colonsNmESAC50 and S. lividansColon, two colonsNmESAC57. The heterologous expression of Nonomuraea genes in S. lividans was successfully demonstrated by using combined RT-PCR and proteomic approaches. MALDI-TOF analysis revealed that a Nonomuraea ABC transporter is expressed as two isoforms in S. lividans. Moreover, its expression may not require a Nonomuraea positive regulator at all, as it is present at similar levels in both clones even though S. lividansColon, two colonsNmESAC57 lacks regulatory genes. Considered together, these results show that S. lividans expresses Nonomuraea genes from their own promoters and support the idea that S. lividans can be a good host for genetic analysis of Nonomuraea.