Anna Maria Ferrini

Susceptibility of Listeria monocytogenes isolated from food in Italy to antibiotics

The susceptibility of 148 strains of Listeria monocytogenes isolated from food to antibiotics currently used in veterinary and human therapy was determined by standard agar dilution and disk diffusion methods. The antibiotics included amikacin, amoxicillin, cefazolin, chloramphenicol, erythromycin, flumequine, fosfomycin, gentamicin, kanamycin, lincomycin, oxytetracycline, rifampicin, spiramycin, streptomycin, tetracycline, tobramycin and vancomycin. Soussys breakpoints and MIC(50)-MIC(90) values were used to classify the strains into sensitive, moderately sensitive and resistant groups. This work is part of a wider surveillance program on listeriosis started in Italy in 1995.

Clostridium botulinum spores and toxin in mascarpone cheese and other milk products.

A total of 1,017 mascarpone cheese samples, collected at retail, were analyzed for Clostridium botulinum spores and toxin, aerobic mesophilic spore counts, as well as pH, a(w) (water activity), and Eh (oxidation-reduction potential). In addition 260 samples from other dairy products were also analyzed for spores and botulinum toxin. Experiments were carried out on naturally and artificially contaminated mascarpone to investigate the influence of different temperature conditions on toxin production by C. botulinum. Three hundred and thirty-one samples (32.5%) of mascarpone were positive for botulinal spores, and 7 (0.8%) of the 878 samples produced at the plant involved in an outbreak of foodborne botulism also contained toxin type A. The chemical-physical parameters (pH, a(w), Eh) of all samples were compatible with C. botulinum growth and toxinogenesis. Of the other milk products, 2.7% were positive for C. botulinum spores. Growth and toxin formation occurred in naturally and experimentally contaminated mascarpone samples after 3 and 4 days of incubation at 28 degrees C, respectively.

A CASE OF INFANT BOTULISM ASSOCIATED WITH HONEY FEEDING IN ITALY

A case of infant botulism in a 9 week-old female is described. A strain of C. botulinum type B was isolated from the feces of the baby. The epidemiologic study detected in a sample of home canned honey Clostridium botulinum spores of the same serotype that was isolated from the patient. The honey had been used only to sweeten the pacifier of the baby. This is the first case of infant botulism in Europe linked conclusively to honey.

Detection and Identification of β-Lactam Residues in Milk Using a Hybrid Biosensor

A novel application of a hybrid biosensor is here employed as an analytical method for the detection and presumptive identification of beta-lactam residues in milk. The method is based on measurements of carbon dioxide (CO2), the production of which is related to the microbial growth of the test microorganism Bacillus stearothermophilus var. calidolactis. The presence of beta-lactams in milk inhibits microbial growth and, consequently, the CO2 production rate. The analysis is based on the variation of CO2 between a milk sample spiked with beta-lactams and a twin milk sample containing beta-lactams plus a broad spectrum beta-lactamase, using an electrochemical device of biosensor. A blank milk sample is included as control. The result is obtained starting from the first 120 min. Moreover, the ability to recognize all of the beta-lactams speeds the total time of analysis when chemical identification and quantification are required. The analytical method appears to be adequate for milk control for qualitative screening purposes, complying with the requirements stated in Decision 2002/657/EC.

Combined Plate Microbial Assay (CPMA): A 6-plate-method for simultaneous first and second level screening of antibacterial residues in meat

This paper proposes an improved high throughput microbial method for the simultaneous performance of first and second level screening for antibacterial residues in meat. It is based on growth inhibition of B. subtilis on agar medium pH 6, 7.2 and 8, of B. cereus on agar medium pH 5.9, of M. luteus on agar medium pH 8 and of E. coli on agar medium pH 7.2 (research or first level screening) and on the use of confirmatory solutions (Pase, Paba, MgSO4) for the identification or second level screening. In kidney control samples, dialysis membranes were interposed between samples and the agar surface to both prevent the action of lysozyme and reduce false positive results. The proposed method detects β-lactams, sulfonamides, tetracyclines, aminoglycosides, macrolides and quinolones at MRL concentrations and reliably indicates the inhibitor family. Results are obtained in 18–24 h.

Presumptive identification of sulphonamide and antibiotic residues in milk by microbial inhibitor tests

Abstract A microbial procedure is described for the presumptive identification of some antibiotic and sulphonamide residues in milk. Penicillins, cephalosporins, sulphonamides and streptomycin residues are tested using three different agar media and two microorganisms. The identification is reached by enclosing in the media four different substances to reverse the normal action of the antibiotics and sulphonamides under investigation.

Transferable tetracycline resistance in Listeria monocytogenes from food in Italy.

Mechanisms of tetracycline resistance were investigated in two recent Listeria monocytogenes isolates from food, with L. innocua 52P tet(r) as a control. Tetracycline resistance was transferred conjugatively from all three strains to L. ivanovii and from one isolate and the control to Enterococcus faecalis. Molecular analysis demonstrated a chromosomal location for the tet determinant, which was identified as tetM in all cases. These studies are the first to show that L. monocytogenes from food could be a source of tetracycline resistance genes able to spread to other micro-organisms.

Wound botulism : clinical and microbiological findings of an Italian case

A case of wound botulism in a 41-year old man is reported. The patient had accidently been wounded when he fell on an iron bar. Some days later he developed typical clinical manifestations of botulism. Wound botulism was confirmed by detection and quantification of type B botulinal toxin in the serum. Ventilatory supportive care was necessary and botulinal antitoxin was not given. The patient was hospitalized for 30 days and recovery was complete.

Microbial screening for quinolones residues in cow milk by bio-optical method

The use of antibiotics on lactating cows should be monitored for the possible risk of milk contamination with residues. Accordingly, Maximum Residue Levels (MRLs) are established by the European Commission to guarantee consumers safety. As pointed out by Dec 2002/657/EC, screening is the first step in the strategy for antibiotic residue control, thus playing a key role in the whole control procedure. However, current routine screening methods applied in milk chain still fail to detect residues of quinolones at concentrations of interest. This paper reports the findings of a new bio-optical method for the screening of quinolones residues in bovine milk, based on E. coli ATCC 11303 growth inhibition. The effect of blank and spiked cow milk samples (aliquots equivalents to 0.8%, v/v) is evaluated in Mueller Hinton Broth (MHb) and MHb enriched with MgSO4 2% (MHb-Mg) inoculated with the test strain at the concentration of 10(4)CFU/mL. The presence of quinolones inhibits the cellular growth in MHb, while this effect is neutralized in MHb-Mg allowing both detection and presumptive identification of quinolones. Growth of the test strain is monitored at 37 °C in a Bioscreen C automated system, and Optical Density (OD) at 600 nm is recorded every 10 min after shaking for 10s. Growth curves (OD vs. time) of E. coli ATCC 11303 are assessed in milk samples, with and without quinolones, and their differences in terms of ΔOD (ΔOD600nm=ODMHb-Mg-ODMHb) are calculated. The presence of quinolones is detected by the cellular growth inhibition (OD vs time, none increase in the value OD) and presumptively identified through the increase of the slope of ΔOD600nm curve (ΔOD vs. time), after about 3h of incubation. The detection limit for ciprofloxacin and enrofloxacin is at the level of MRL, for marbofloxacin is at 2-fold the MRL whereas for danofloxacin is at 4-fold the MRL. Although the sensitivity of the method could be further improved and the procedure automated, it is a promising step forward to integrate screening assays into the control process and, in particular, to fill in the gap for quinolones; moreover, these technological developments contribute to the One Health perspective through the monitoring of safe and correct use of veterinary antibiotics.

Depletion of long-acting ampicillin in goat milk following intramuscular administration.

Although goat milk production represents today a very small percentage of the world milk market, this percentage has been growing continuously during the past 20 years. Goat milk is the basic milk supply in many developing countries and provides tasteful derivative products in developed countries. Goats, as well as all milk-producing animals, can be affected by mastitis, but goats being considered a minor species, few drugs are specifically registered for these animals; most, at least for mastitis treatment, are usually tested and registered for use in cows. This situation leads often to the adoption for goat milk of withdrawal periods defined for cows even if these extrapolations prove almost never valid for goats. In the present study, the elimination of the β-lactam antibacterial agent ampicillin in goat milk was investigated. Ampicillin was chosen because it is one of the most common antibiotics used by goat farmers against mastitis due to the fact that it is well tolerated and has short elimination times in cows. Goats were treated with long-acting ampicillin at 15 mg (kg of body weight)(-1) by double intramuscular injection at 72 h interval. Milk was collected in a 12 h milking scheme. The method used to determine the levels of ampicillin in goat milk was based on a liquid-liquid extraction of this drug from the matrix, successive derivatization with formaldehyde, and final separation by HPLC with fluorescence detection. The results point out a slow depletion of ampicillin and, consequently, a withdrawal period (13 milkings) longer than that extrapolated and authorized for cows and sheep.