Extended-Spectrum b-Lactamase, AmpC,and MBL-Producing Gram-Negative Bacteria on Fresh Vegetables and Ready-to-Eat Salads Sold in Local Markets

We investigated the occurrence of extended-spectrum b-lactamase (ESBL), AmpC, and carbapenemase- producing Gram-negative bacteria isolated from 160 samples of fresh vegetables (n =80) and ready-to-eat (RTE) prepacked salads (n =80). Phenotypic and genotypic analyses were carried out on the isolates in terms of the species present and relative resistance. Resistance to b-lactam antibiotics was found in only 44 (24 from fresh vegetables and 20 from RTE salads) of a total of 312 Gram-negative strains (14.1%). The prevalence of ESBL-producing strains from fresh vegetables was 83.3% (20/24) and 16.7% (4/24) for AmpC. Among the 20 bacterial isolates from RTE salads, 80% (16/20) were identified as ESBL-producing strains and the remaining 20% (4/20) as MBL-producing strains. PCR and sequencing confirmed the presence of blaSHV-12, blaCTX-M-1, blaCTX-M-15, blaRHAN-1, blaACC-1, blaDHA-1, blaVIM-1, and blaIMP-1. Seven different replicons were identified, where IncHI1, FIA, and I1 were the most representative types; when compared with the Inc types, isolates from fresh vegetables and RTE salads were similar. The location of genes on a conjugative plasmid was confirmed by positive results obtained with conjugation assays. Our study has demonstrated the occurrence and distribution of ESBL/AmpC and MBL strains in fresh vegetables and RTE salads in Italy and possible public health risks associated with consumption of these fresh products

Emerging Microbial Concerns in Food Safety and New Control Measures

The editorial collects a brief summary of the topics discussed in the articles that are published in Emerging Microbial Concerns in Food Safety and New ControlMeasures. We hope that readers of this special issue will find some information of interest in order to expand their knowledge in this field and to increase their level of attention on matters here reported

Extended spectrum ß-lactamase and AmpC-producing Enterobacteriaceae in raw vegetables

Antibiotic resistance in bacteria is a global problem exacerbated by the dissemination of resistant bacteria via uncooked food, such as green leafy vegetables. The attribution of fresh produce to the overall community-associated exposure of humans to ESBL- or AmpC- producing. The aim of the current study was carried out to assess the of extended spectrum β-lactamase and AmpC type β-lactamase in Enterobacteriaceae isolated from fresh vegetables sold in the local markets of Modena town. Methods: A total of 80 samples comprising carrot, spring onion, tomatoes, arugula, chicory, endive and frisee salad were processed microbiologically to isolate Enterobacteriaceae. The vegetables were mostly obtained from supermarkets. Species identification and antimicrobial susceptibility testing were carried out using the Vitek 2 system and the AST-GN041 card (bioMérieux). Phenotypic testing by using the Double Synergy Differential Test (DSDT) for ESBL/AmpC-positive strains was confirmed by PCR and DNA sequence analysis. The localization of β-lactamase genes was established by conjugation experiments. Results: Overall, of the 160 analyzed strains ESBLs/AmpC were detected in 20 isolates (12,5%) for the double-disk synergy test. The major ESBL determinant was the CTX-M type (7,5% isolates) and two SHV (1,25%). DHA and ACC (2,5 % of isolates) were AmpC-β lactamases type. Additionally, we identified newly recognized ESBL blaRAHN-2 sequences from Rahnella aquatilis. The conjugation has demonstrated the transmissibility of this resistance determinant. Conclusion: In conclusion, we report a highter and a low prevalence of ESBL and AmpC β-lactamase determinants respectively mostly in arugula and frisee salad . Public health risks associated with exposure to ESBL- and AmpC-producing bacteria through consumption of uncooked fresh produce are diverse. They range from occasional ingestion of 3GC-resistant opportunistic pathogens which may result in difficult-to-treat infections, to frequent ingestion of relatively harmless ESBL producing environmental bacteria that may therewith constitute a continuously replenished intestinal reservoir facilitating dissemination of ESBL genes to (opportunistic) pathogens

Molecular engineering of fluorescent penicillins for molecularly imprinted polymer assays

The interaction of seven novel fluorescent labeled beta-lactams with a library of six polymer materials molecularly imprinted (MI) with penicillin G (PenG) has been evaluated using both radioactive and fluorescence competitive assays. The highly fluorescent competitors (emission quantum yields of 0.4-0.95) have been molecularly engineered to contain pyrene or dansyl labels while keeping intact the 6-aminopenicillanic acid moiety for efficient recognition by the cross-linked polymers. Pyrenemethylacetamidopenicillanic acid (PAAP) is the tagged antibiotic that provides the highest selectivity when competing with PenG for the specific binding sites in a MI polymer prepared with methacrylic acid and trimethylolpropane trimethacrylate (10:15 molar ratio) in acetonitrile in the presence of PenG. Molecular modeling shows that recognition of the fluorescent analogues of PenG by the MI material is due to a combination of size and shape selectivity and demonstrates how critical the choice of label and tether chain is. PAAP has been applied to the development of a fluorescence competitive assay for PenG analysis with a dynamic range of 3-890 mu M in 99:1 acetonitrile-water solution. Competitive binding studies demonstrate various degrees of cross-reactivity for some antibiotics derived from 6-aminopenicillanic acid, particularly amoxicillin, ampicillin, and penicillin V (but not oxacillin, cloxacillin, dicloxacillin, or nafcillin). Other antibiotics, such as chloramphenicol, tetracycline, or cephapirin, do not compete with PAAP for binding to the imprinted polymer. The MI assay has successfully been tested for PenG analysis in a pharmaceutical formulation

Oxygen-sensitive biocompatible membranes for luminescent optical fiber sensors

One of the main problems associated with the application of sensors for continuous monitoring in the (aqueous) environment is related to biofouling of the sensitive membranes. Silicone rubber is the material of choice in optical and electrochemical sensors for oxygen monitoring. This material provides excellent oxygen permeability (300-400 x 10-13 cm2 s-1 Pa-1), but one of its main drawbacks arises from its high degree of biocompatibility as it behaves as an excellent support for all kinds of microorganisms originating a rapid fouling of the sensor head. We present herein the application of novel oxygen-sensitive luminescent membranes for optical fiber sensors aimed to overcome this undesirable limitation by integrating in their design new biofouling- resistant polymer coatings. Four different oxygen indicators have been synthesised and spectroscopically characterised for their application towards preparation of the sensitive membranes. The indicators belong to a family of luminescent ruthenium(II) and osmium(II) tris-chelate complexes with substituted and unsubstituted 1,10-phenanthroline. The indicator molecules have been immobilised into poly(dimethylsiloxane) or phosphorylcholine(PC)-containing polymer layers. Their emission lifetimes embedded into the different materials are comparable, pointing out a similar microenvironment of the luminescent probe in the tested polymers. Nevertheless the different oxygen sensitivity observed might be a consequence of a lower solubility of the analyte gas in the PC-containing materials compared to siloxanes. In a different study the effect of several siloxanes on the oxygen sensitivity of the indicator membranes has been evaluated, following the luminescence quenching process by steady-state and time-resolved measurements. The good agreement between both results (Stern-Volmer plots) indicates dynamic quenching of the photoexcited dye by the analyte. The selected silicone membranes have been coated with four different antibiofouling layers and their effect on the sensor response has been tested. Results will be presented showing the spectroscopic and analytical features of these optodes both in gas and aqueous phase. We have observed that the presence of PC coating decrease the oxygen response by 2-20% of that shown by the original (uncoated) membranes, regardless the type of coating applied. Several studies are in progress in order to evaluate the antifouling capabilities of the novel sensing layer

Analysis of alternariol and alternariol monomethyl ether in foodstuffs by molecularly imprinted solid-phase extraction and ultra-high-performance liquid chromatography tandem mass spectrometry

Molecularly imprinted porous polymer microspheres selective to Alternaria mycotoxins, alternariol (AOH) and alternariol monomethyl ether (AME), were synthesized and applied to the extraction of both mycotoxins in food samples. The polymer was prepared using 4-vinylpiridine (VIPY) and methacrylamide (MAM) as functional monomers, ethylene glycol dimethacrylate (EDMA) as cross-linker and 3,8,9-trihydroxy-6H-dibenzo[b, d] pyran-6- one (S2) as AOH surrogate template. A molecularly imprinted solid phase extraction (MISPE) method has been optimized for the selective isolation of the mycotoxins from aqueous samples coupled to HPLC with fluorescence (lambda(ex)= 258 nm;lambda(em) = 440 nm) or MS/MS analysis. The MISPE method was validated by UPLC-MS/MS for the determination of AOH and AME in tomato juice and sesame oil based on the European Commission Decision 2002/657/EC. Method performance was satisfactory with recoveries from 92.5% to 106.2% and limits of quantification within the 1.1-2.8 mu g kg(-1) range in both samples