Microbiological challenge of four protective devices for the reconstitution of cytotoxic agents.

AIMS: To evaluate the susceptibility to microbial contamination that occurs during simulated handling of protective devices for the preparation of cytotoxic drug solutions. METHODS AND RESULTS: Four devices, i.e. Chemoprotect spike, Clave connector, PhaSeal and Securmix were challenged with low and high inocula of micro-organisms. The cells, transferred to the connected vials during repeated manipulations of the devices were counted by means of solid-phase cytometry. Of the four devices, PhaSeal afforded the lowest transfer of micro-organisms. Secondly, the efficiency of procedures for the disinfection of an artificially contaminated rubber stopper was compared prior to connection of the vial to the PhaSeal device. Spraying or swabbing alone was inadequate, as opposed to a combination of spraying [0.5% or 2.0% (w/v) chlorhexidine in isopropanol] and swabbing [70% (v/v) isopropanol]. CONCLUSIONS: Although Phaseal afforded the lowest transfer of micro-organisms, adequate disinfection of the vial prior to connection remains required. SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike aspects of operator protection, which are well documented, the microbiological safety of protective devices for the preparation of cytotoxic drugs has not been addressed in the literature. This study estimates the susceptibility to microbial contamination during handling of four commonly used devices.

Solid phase cytometry as a tool to detect viable but non-culturable cells of Campylobacter jejuni.

Solid phase cytometry (SPC) in conjunction with fluorescent viability staining has been investigated as a tool to detect viable but non-culturable Campylobacter jejuni in drinking water. Inoculated water samples were filtered over a polyester membrane filter and the retained cells were stained using a carboxyfluorescein ester as a substrate for intracellular esterases. The number of green fluorescent bacteria was automatically counted by an Ar laser scanning device (ChemScan) in 3 min. In parallel, the plate count was determined on Columbia Blood Agar. The number of culturable cells decreased below the detection limit of plate counting in less than 50 days. In contrast, the number of fluorescent bacteria remained at its initial level for at least 85 days. The discrepancy between the two results can be attributed to the transition of culturable C. jejuni cells into VBNC C. jejuni cells. Furthermore, as SPC can distinguish between low numbers of dividing and non-dividing cells of Campylobacter it has the potential to monitor attempts to resuscitate VBNC cells.

A survey for Mycobacterium avium subspecies paratuberculosis in the Royal Zoological Society of Antwerp.

Paratuberculosis is a chronic intestinal disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (Map). Very little is known about the status of paratuberculosis in European zoos. In this study, the presence of Map in the animal collection of the Royal Zoological Society of Antwerp (RZSA) was investigated. Faecal and post mortem samples from 48 ruminants were used to set up cultures. DNA from faeces, tissue and positive cultures were tested by IS900 polymerase chain reaction (PCR). Additionally, 448 serum samples were tested with an ELISA kit. All culture samples were negative whereas PCR gave three positives on biopsy samples and one positive on faecal samples. With the ELISA, 21 sera could be classified as positive. There is evidence that Map is present in the RZSA but no high level faecal shedders could be detected. Further investigations are required in other European Zoos in order to complete the picture of Map infections.

Persistence of Campylobacter jejuni on surfaces in a processing environment and on cutting boards.

AIMS: The objectives of the study were to determine the spread and persistence of Campylobacter in a poultry processing plant and to provide a quantitative estimate of the survival of Campylobacter jejuni on the surface of a cutting board. METHODS AND RESULTS: Several contact surfaces in a poultry processing plant were sampled before the start of processing, after 30 min and after 120 min. Next, the survival of four C. jejuni strains was studied on a beech and polypropylene cutting board during 120 min. CONCLUSIONS: A rapid introduction and spread of Campylobacter in a well cleaned processing plant as well as a significant survival in time on the example of a cutting board is shown. SIGNIFICANCE AND IMPACT OF THE STUDY: The need to prevent cross-contamination in the food processing and preparation area and the importance of an integrated approach throughout the whole food chain to control transmission of Campylobacter is highlighted.

Effect of different sterilisation methods on the properties of bioadhesive powders and ocular minitablets, and clinical evaluation.

The purpose of this study was to evaluate the influence of gamma-irradiation and dry heat sterilisation on the properties of a bioadhesive powder mixture containing ciprofloxacin and its corresponding ocular minitablets. The molecular weight characteristics of drum dried waxy maize starch (DDWM), employed as major component of the bioadhesive formulation, the decay kinetics of radicals, the rheological properties of the bioadhesive polymers and the microbial activity of ciprofloxacin were studied. The influence of the different sterilisation methods on the characteristics of the ocular minitablets was investigated by measuring the crushing strength, the friability, and the in vitro release of ciprofloxacin from the minitablets. Finally, the clinical value of the selected sterilised minitablets was evaluated in seven healthy volunteers. Both sterilisation methods similarly affected the properties of the bioadhesive formulation by inducing stable radicals and decreasing the molecular weight of DDWM, although no changes in the microbiological activity of ciprofloxacin were measured. An obvious influence of both sterilisation methods was observed in the in vitro release study. The crushing strength and friability of the minitablets were not significantly influenced by gamma-irradiation. Based on these data, gamma-irradiation was more adequate as sterilisation method for the bioadhesive ocular minitablets than dry heat sterilisation, because it affected the least the physical properties of the minitablets. Therefore, the gamma-sterilised minitablets were selected for an in vivo evaluation in seven volunteers. The concentration of ciprofloxacin in the tear film remained above its MIC value for the most common ocular pathogens for at least 8 h. Consequently, the gamma-irradiated minitablets containing ciprofloxacin can be considered as a promising formulation to treat bacterial keratitis and conjunctivitis.

Survival of Campylobacter jejuni strains of different origin in drinking water.

AIMS: The aim of the study was to measure the survival of 19 Campylobacter jejuni strains of different origins, including two reference strains, four poultry-derived isolates, nine human isolates and four water isolates, in sterilized drinking water. METHODS AND RESULTS: Pure cultures of 19 C. jejuni strains were inoculated in sterile drinking water and incubated at 4 degrees C for 64 days. Survival was determined by culturability on both selective (Karmali agar) and non-selective [Columbia blood agar (CBA)] media. Culturability was shown to be strain and origin-dependent. Campylobacter jejuni showed prolonged survival on a non-selective than on a selective medium. CONCLUSIONS: The origin of the strain is a determining factor for the survival of C. jejuni in drinking water at 4 degrees C. Poultry isolates showed a prolonged survival, which could be an indication that these strains could play an important role in the transmission of campylobacteriosis through water. In addition, culture conditions are an important factor for evaluating the survival of C. jejuni in drinking water at 4 degrees C. The non-selective agar (CBA) allowed growth of C. jejuni over a longer period of time than the selective agar (Karmali). Furthermore, an enrichment broth (Bolton) allowed the recovery of all 19 C. jejuni strains during the 64 days of incubation at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted differences in culturability depending on culture conditions and on strain origin.

Laser scanning cytometry to detect viable but non-culturable C. jejuni in drinking water.

Laser scanning cytometry has been investigated as a tool to detect viable but non-culturable C. jejuni in drinking water. After suspending the cells in sterile drinking water, a sample was taken every seven days, the (see text) were retained on a polyester membrane filter and labelled using a fluorescein derivative as a substrate for intracellular esterases. The number of green fluorescent bacteria was automatically counted by an Ar laser scanning device in three minutes. In parallel, the number of culturable cells was determined on a non-selective medium. The number of culturable cells decreased to below the detection limit for cultivation in less then 50 days. At the contrary, fluorescent bacteria remained at the initial level during the 71 days of incubation. The discrepancy between the two results can be assigned to the presence of VBNC C. jejuni cells. Therefore laser scanning cytometry can be used as a fast and sensitive tool to detect viable but non-culturable C. jejuni in drinking water.

Determination of somatic cells in milk by solid phase cytometry.

The somatic cell count of milk is routinely determined by the fluoro-opto-electron method and sometimes by the direct epifluorescent filter technique (DEFT). This paper investigates the potential of solid phase cytometry (SPC), a novel technique combining aspects of both the fluoro-opto-electronic method and epifluorescence microscopy for somatic cell counting. In SPC, cells are retained on a membrane filter, fluorescently labelled and automatically detected on the entire membrane filter by means of a laser scanning instrument ChemScan). Fluorescent spots can be visually inspected by an epifluorescence microscope with a computer-driven moving stage. The performance of SPC was compared with that of the fluoro-opto-electronic method using a Fossomatic 360 instrument for 68 milk samples with varying somatic cell counts (10(3)-10(6)/ml). The sample throughput and repeatability of SPC were inferior to those of' the Fossomatic method and statistical analysis of the method comparison data using the approach of J. M. Bland & D. G. Altman (The Lancet 1986 February 8 pp 307-310) revealed a poor comparability between the two methods. Moreover, problems of milk filterability and the interference of fluorescent particles presently hamper the routine application of SPC. Nevertheless, this method represents the first example of the application of SPC to milk.

Effect of antibiotics on viability staining of Escherichia coli in solid phase cytometry.

Solid phase cytometry (SPC) has been investigated as a tool to assess the effect of antibiotics on the viability of Escherichia coli. After exposure of the cells to the antibiotic, they are retained on a polyester membrane filter and labelled using a fluorescein derivative as a substrate for intracellular esterases. The number of fluorescent bacteria is automatically counted in an Ar laser scanning device. In the presence of nutrients, all antibiotics tested in concentrations exceeding the MIC inhibited the multiplication of cells but not the labelling per se. However, when no nutrients were added, the cells did not multiply, and inhibition of the fluorescent staining was only observed for membrane permeabilizing antibiotics, even at sub-MIC concentrations. The selective detection by SPC of membrane-permeabilizing antibiotics corroborates the requirement of membrane integrity for viability labelling of bacteria. This selectivity has been exploited to develop a method for the detection of colistin residues in milk.

Pharmacokinetics of florfenicol after treatment of pigs with single oral or intramuscular doses or with medicated feed for three days.

The pharmacokinetics of florfenicol, a structural analogue of thiamphenicol, were studied in six pigs after single oral and intramuscular doses of 15 mg/kg bodyweight, and after feeding them with medicated feed containing 250 mg/kg for three days, a concentration which provided approximately the same dose rate of the drug. The oral doses contained a specially prepared pelleted formulation of the drug. The bioavailability of the drug was similar for the oral and intramuscular doses. Florfenicol was absorbed rapidly from the feed and its concentration in plasma remained between 2 and 6 microg/ml - above the minimum inhibitory concentration values for common pig pathogens - during the three days.

Evaluation of a modified enzymatic test for the detection of tetracyclines in milk.

The tetracycline galactosidase (TG) test, a new method for the detection of tetracycline residues in raw milk based on the inhibition of beta-galactosidase biosynthesis in Escherichia coli, was previously validated with spiked milk samples. It has now been applied to milk from cows treated with oxytetracycline. In view of the occurrence of false positives, related to highly elevated somatic cell counts (>10(6)/ml), the improved TG test was developed, in which a heating step (80 degrees C, 15 min) preceded the original TG test protocol. A good agreement with other assays (Delvotest SP, the Bacillus cereus microtiter test, the LacTek tetracycline milk screening test, the Charm HVS-8100 tetracycline test) as well as with high-pressure liquid chromatography was obtained. No false negatives were observed with reference to the established maximum residue limit for tetracyclines of 100 microg/kg milk.

Chemiluminometric beta-galactosidase detection as a basis for a tetracycline screening test in milk.

The observation that tetracyclines inhibit the biosynthesis of beta-galactosidase in Escherichia coli to a greater extent than other antibacterials was exploited for the development of a chemiluminometric method to detect residues of this class of antibiotics in milk. The procedure involves the incubation of a milk sample with 10(7) CFU/ml of an E. coli strain in the presence of IPTG, an inducer of beta-galactosidase, and of EGTA, a chelator of calcium ions, followed by a 1000-fold dilution and measurement of the residual enzymatic activity using the chemiluminogenic substrate Galacton. Chemiluminometry proved an essential tool in this procedure because the extensive dilution of the sample, necessary to avoid light quenching by turbidity, results in an insufficient level of beta-galactosidase activity to be measurable by colorimetry. This tetracycline galactosidase (TG) test has been validated and compared in the field to existing commercial screening assays for antibiotics. Its detection limit for tetracyclines ranges between 40 and 65 micrograms/kg, which is below the European maximum residue limit (MRL = 100 micrograms/kg) in milk. No other antibacterials, at concentrations commonly expected in milk, were found to interfere with the TG test. Strategies to avoid false positive reactions possibly arising from very high somatic cell counts will be reported elsewhere.

Inhibition of beta-galactosidase biosynthesis in Escherichia coli by tetracycline residues in milk.

Low levels of tetracyclines found as residues in milk inhibited the biosynthesis of beta-galactosidase in Escherichia coli. To produce the same effect, other antibacterials had to occur in concentrations that were more than 10-fold higher. This relative selectivity was exploited for the development of a screening test for tetracyclines in milk based on a chemiluminometric assay of beta-galactosidase. The method was validated with spiked samples of raw milk and applied to field samples contaminated with tetracyclines.

Pharmacokinetics of florfenicol in cerebrospinal fluid and plasma of calves.

Florfenicol, a fluorinated analog of thiamphenicol, is of great value in veterinary infectious diseases that formerly responded favorably to chloramphenicol. In view of the treatment of meningitis in calves, we studied its pharmacokinetics in the cerebrospinal fluid (CSF) and plasma of six animals. To this end, a new high-performance liquid chromatography method was developed which, unlike previous ones, uses solid-phase instead of double-phase extraction to isolate the drug. After a single intravenous dose of 20 mg/kg of body weight, a maximum concentration in CSF of 4.67 +/- 1.51 microg/ml (n = 6) was reached, with a mean residence time of 8.7 h. The decline of florfenicol in both CSF and plasma fitted a biexponential model with elimination half-lives of 13.4 and 3.2 h, respectively. Florfenicol penetrated well into CSF, as evidenced from an availability of 46% +/- 3% relative to plasma. The levels remained above the MIC for Haemophilus somnus over a 20-h period. Our results provide evidence indicating the effectiveness of florfenicol in the treatment of bacterial meningitis of calves.