Surveillance of listeriosis in Navarre, Spain, 1995-2005--epidemiological patterns and characterisation of clinical and food isolates.

We monitored the incidence of human listeriosis in Navarre, a region in north of Spain between 1995 and 2005, and carried out the characterisation of Listeria monocytogenes isolates obtained from clinical samples and ready-to-eat products (sliced cooked meat, smoked salmon and liver pate). The active surveillance requesting hospitals to notify all listeriosis cases (n=40) yielded higher incidence rates (average annual rate 0.65/100,000 inhabitants, range 0.18-1.18/100,000 inhabitants) than expected. Pregnant women were the largest group affected (n=13, 32.5% of the cases), with a peak in incidence during the last three years of the study period. From the 40 human cases we obtained 33 Listeria isolates. Serological and molecular characterisation by PFGE identified 20 different pulsotypes, which on three occasions enabled us to link sporadic cases into clusters. Although we could not identify the incriminated food product we found two clinical pulsotypes among smoked salmon and cooked meat isolates. Surveillance of listeriosis in Spain should be improved and coordinated with other European Union Member States in order to better estimate the burden of disease and to prevent foodborne outbreaks.

Characterization of Listeria monocytogenes and Listeria innocua from a vegetable processing plant by RAPD and REA.

The incidence of Listeria monocytogenes in a vegetable processing plant was investigated over a 23-month period. Frozen ready-to-eat vegetable samples, well as the plant environment, were sampled. The molecular subtyping techniques, Random Amplified Polymorphic DNA (RAPD) and Restriction Endonuclease Analyses (REA), were performed to help investigate the origin and routes of Listeria dissemination. The low and sporadic incidence of L. monocytogenes made it impossible to establish an epidemiological sequence in the processing plant, though a case of cross-contamination between tomato and ratatouille was detected. Listeria innocua subtyping, however, allowed us to determine the prevalence of several strains in vegetables, and their presence on machinery samples suggested the possibility of cross-contamination during processing. The low incidence of L. monocytogenes indicated that the risk of listeriosis transmission by vegetable consumption is low. On the other hand, the isolation of the same strain of L. innocua in several surveys pointed out the risk of colonisation on surfaces and machinery. The persistence of Listeria spp. is a cause for concern as can lead to future contamination of vegetables processed in the plant and to a possible increased risk for health. Therefore, periodic controls for the presence of Listeria spp. and a further review of the cleaning and disinfection procedures used in frozen vegetable plants are recommended.

Occurrence of Listeria monocytogenes in fresh and processed foods in Navarra (Spain).

The presence of Listeria spp. was investigated in a total of 3685 food samples obtained from different industries and markets of Northern Spain in the last 4 years. The samples analyzed include fresh raw products (meat, milk and poultry) and treated products (cooked and cured meats, frozen vegetables and smoked salmon). Occurrence of Listeria spp. varied from 8.1% in soft cheese to 76.3% in raw poultry samples. The highest incidence of L. monocytogenes also occurred in raw poultry (36.1% positive samples). Despite this high incidence of contamination, these kinds of products carry a low risk of listeriosis transmission because of the heat treatment prior to consumption. On the other hand, the ready-to-eat products (RTE) tested in this study showed incidences that could pose serious health problems, taking into account that the storage conditions may allow for rapid growth of the pathogen. It was also found that up to 75.5% of the L. monocytogenes strains isolated in this study belonged to serogroup 1, mainly serotype 1/2a, while the clinical cases observed in Navarra in the same period of time belonged mainly to serotype 4b/4bx.

Random amplified polymorphic DNA typing applied to the study of cross-contamination by Listeria monocytogenes in processed food products.

The presence of Listeria spp. was investigated in 369 samples of cooked meat products and 52 of smoked salmon. Incidences of 17.6% for cooked meat and 38.5% for smoked salmon samples were found. All Listeria monocytogenes isolates (34 from meat products and 16 from smoked salmon) were typed serologically and by random amplified polymorphic DNA (RAPD) typing using primers HLWL74 (5'-ACGTATCTGC-3'), HLWL85 (5'-ACAACTGCTC-3'), and OMP-01 (5'-GTI'GGTGGCT-3'). Strains from cooked meat products were characterized and compared in relation to their origin. The detection of identical strains in products of different type and brand packed on the same date suggested cross-contamination, probably during the slicing process. All L monocytogenes isolates from smoked salmon were indistinguishable by serotyping and RAPD, suggesting that this strain was highly disseminated and adapted to the treatment used for the preservation of this food. RAPD subtypes were analyzed using GelCompar version 4.1 software and the unweighted pair method using arithmetic averages, and six groups with at least 78% similarity were established. Serotyping and RAPD results were in concordance, although RAPD showed a higher discriminatory power with L. monocytogenes isolates from meat products. RAPD is an easy method that could be useful to detect cross-contamination occurring during postprocessing manipulations.

[Controlled liberation of active principles by means of new galenic formulations]. / Liberación controlada de principios activos mediante el empleo de formulaciones galénicas.

Drugs inside a conventional galenic form are distributed between specific biological targets and other anatomical tissues. With the aim to obtain a more rational and a better therapeutic, one of the most promising possibilities by using the concept of vectorization: association of an active principle to an appropriate vector with the object to increase its action efficiency and efficacy. By this means, they do not just increase the affinity of the drug to the target but also active principle gets protected from a potentially hostile environment (hydrolytic enzymes, acid pH, etc.). The success in the extension of the applications of the vectorización depends more and more of an appropriate design, for what the fundamental objective of this revision will be the one of presenting the general characteristics and some of the current applications in these new galenic forms.

Protective effect of liposomal gentamicin against systemic acute murine brucellosis.

Liposomes of the stable multilamellar type, which previously demonstrated great efficiency in antibiotic transport [Vitas et al: Antimicrob Agents Chemother 1996;40:146-151] were used in this study as carriers of gentamicin for treatment of mice lethally infected with Brucella abortus. Treatment with gentamicin in positively charged liposomes produced a protective effect when it was administered 1 day after lethal challenge (70% of protection). On the other hand, the use of free gentamicin or in liposomes with a negative net charge did not produce a protective effect. Moreover, the results reported here also indicated that cationic liposomes themselves had a therapeutic effect on the course of the infection (up to 50% of protection). In conclusion, we observed that cationic liposomal encapsulation of gentamicin resulted in an enhancement of the therapeutic activity of free gentamicin in this mouse model of acute infection.

Effect of composition and method of preparation of liposomes on their stability and interaction with murine monocytes infected with Brucella abortus.

The success of the use of liposomes as drug carriers depends on both their formulation and the method of preparation. We have carried out a series of in vitro studies using different formulations and preparation methods, with the aim of obtaining a type of liposome which is efficient in the treatment of brucellosis. On the basis of results obtained in studies of stability at 37 degrees C in the presence of serum lipoproteins and of the activation of phagocytic cells and antibiotic transport to the interior of monocytes infected with Brucella abortus, we conclude that the most suitable vesicles are positively charged, stable plurilamellar vesicles (phosphatidylcholine, 30% cholesterol, and 10% stearylamine). Gentamicin incorporated into these cationic liposomes completely eliminated all of the intracellular Brucella organisms (4.6 logs), while free gentamicin was capable of reducing the number of intracellular bacteria by only 0.3 log.

Comparative activity of azithromycin and doxycycline against Brucella spp. infection in mice.

The activities of a short therapeutic regimen with azithromycin and the classic treatment doxycycline with streptomycin were compared and evaluated in mice infected with Brucella melitensis. In a chronic model, starting therapy 31 days after challenge, azithromycin (10 days, 50 mg/kg/day) significantly reduced the infection (2.9 logs, day 48 post-infection). The effectiveness of doxycycline (21 days, 50 mg/kg/12 hourly) was greater than azithromycin (4.1 logs of reduction, day 48 post-infection), and when doxycycline was administered for a period of 45 days, all the animals were bacteriologically cured from day 78. The combination with streptomycin (14 days, 10 mg/kg/day) did not improve the effect of any of the regimens. In an acute model infection, treatments with doxycycline or doxycycline-streptomycin, for a period of 3 days, starting 1 day after lethal challenge, were able to protect all the mice. In contrast, only 50% of the mice treated with azithromycin survived the challenge. In conclusion, although a short oral treatment with azithromycin was able to reduce the infection significantly, it was not able to cure the animals as effectively as the classic regimen with doxycycline administered for a longer period of time.

Protective effect of Brucella outer membrane complex-bearing liposomes against experimental murine brucellosis.

Liposomes of stable multilamellar type, which previously demonstrated great efficiency in antibiotic transport, were used in this study as transport vehicles of antigenic extracts of Brucella melitensis (HS: complex of lipopolysaccharide/phospholipids/outer membrane proteins). The incorporation of HS into positively charged liposomes produced a protective effect against experimental murine brucellosis when they were administered 1 day before or 2 days after infection, as the number of colony-forming units in the spleen was reduced in relation to the untreated control group (P < 0.01). On the other hand, the use of HS-free or bound in liposomes with negative net charge did not produce a significant effect. Moreover, the incorporation of HS into cationic liposomes eliminated the toxicity of the lipopolysaccharide.

Factors affecting detection of Brucella melitensis by BACTEC NR730, a nonradiometric system for hemocultures.

The detection of Brucella bacteremia by subculture does not always correlate with a positive signal in the BACTEC NR730 nonradiometric system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.). The effect of the inoculum size, pH, sodium polyanetholesulfonate, carbon sources (i-erythritol, sodium pyruvate, monosodium glutamate, D-glucose, and L-alanine), and urea in the release of CO2 was evaluated by using the reference strain Brucella melitensis 16M. In standard NR6 vials with or without blood, inocula 5 to 10 times larger (at least 265 CFU per vial) than those usually found in the blood of patients with brucellosis were necessary to produce a positive growth value (GV) in 4 days or less, and similar results were obtained with vials supplemented with the substrates listed above. GVs were consistently lower in vials with sodium polyanetholesulfonate than in vials without this agent. Vials with no blood inoculated with 265 CFU per vial showed turbidity 1 day before GVs became positive, proving that the major limiting detection factor was the low level of release of CO2 and not an inadequate growth medium. In NR6 vials buffered to pH 6.2, GVs became positive faster and were higher than those in standard vials. NR6 vials at pH 6.2 with 0.3% sodium pyruvate yielded a positive GV in the first day of bacterial turbidity.

Brucella group 3 outer membrane proteins contain a heat-modifiable protein.

Brucella melitensis and B. ovis outer membrane blebs contained a protein displaying a temperature-dependent molecular mass upshift from 25 kDa to 30 kDa. A fraction of the protein tightly bound to LPS did not show the molecular mass upshift which was also blocked by exposure of the protein to Zwittergent 314. The B. melitensis heat-modifiable protein and Escherichia coli OmpA shared antigenic determinants. These data indicate that the Brucella group 3 outer membrane proteins belonged to the OmpA family of proteins.