A high oxfendazole dose to control porcine cysticercosis: pharmacokinetics and tissue residue profiles.

Oxfendazole (OFZ) is efficacious for porcine cysticercosis at 30 mg/kg. OFZ is not registered to be used at this dose. The assessment of the OFZ and metabolites [(fenbendazole sulphone (FBZSO2), fenbendazole (FBZ)] plasma pharmacokinetic and tissue residue profiles after its oral administration to pigs and the withdrawal period for human consumption were reported. Forty-eight pigs allocated into two groups received OFZ (30 mg/kg) orally as a commercial (CF) or as experimental formulation (SMF). Samples (blood, muscle, liver, kidney and fat) were collected over 30 days post-treatment and analyzed by HPLC. OFZ was the main compound recovered in plasma, followed by FBZSO2 and low FBZ concentrations. OFZ AUC0-LOQ (209.9±33.9 µg·h/ml) and Cmax (5.40±0.65 µg/ml) parameters for the CF tended to be higher than those for the SMF (AUC0-LOQ: 159.4±18.3 µg h/ml, Cmax: 3.80±0.35 µg/ml). The highest total residue (OFZ+FBZSO2+FBZ) concentrations were quantified in liver, followed by kidney, muscle and fat tissue. FBZSO2 residue levels were the highest found in muscle (0.68±0.39 µg/g) and fat (0.69±0.39 µg/g). In liver and kidney the highest residues corresponded to FBZ (5.29±4.36 µg/g) and OFZ (2.86±0.75 µg/g), respectively. A withdrawal time of 17 days post-treatment was established before tissues are delivered for human consumption.

Reduction of irrelevant and redundant data from TFRs for EEG signal classification.

Time-frequency representations (TFR) are one of the most popular characterization methods for non-stationary biosignals. Despite of their potential advantages, these representations suffer of large quantity of redundant and irrelevant data which makes them difficult to use for classification purposes. In this work, a methodology for reduction of irrelevant and redundant data is explored. This approach consists on removing irrelevant data, applying a relevance measure on the t-f plane that measures the dependence of each t-f point with the class labels. Then, principal component analysis (PCA) and partial least squares (PLS) are used as non-supervised and supervised linear decomposition approaches to reduce redundancy of remaining t-f points. Results show that the proposed methodology improves the performance of classifier up to 3% when no relevance and redundancy on TFRs is reduced.

Campylobacter spp. contamination of chicken carcasses during processing in relation to flock colonisation.

The presence and numbers of campylobacters on chicken carcasses from 26 slaughter groups, originating from 22 single-house flocks and processed in four UK plants, were studied in relation to the level of flock colonisation determined by examining the caecal contents of at least ten birds per group. The prevalence of campylobacters on carcasses from five campylobacter-negative flocks processed just after other negative flocks was low (8.0 log(10) cfu) than carcasses originating from low prevalence flocks (average of 2.3 log(10) cfu; range: <1.1 to 4.1 log(10) cfu). There was a reduction in the numbers of campylobacters on carcasses between plucking and chilling in eight of ten fully colonised flocks. In another eight flocks, a significant (P<0.001) decrease (0.8 log(10) cfu) in the number of campylobacters on carcasses from just before to after chilling was detected. Campylobacter spp. could be isolated from aerosols, particles and droplets in considerable numbers in the hanging-on, defeathering and evisceration areas but not in the chillers. This was the case even when campylobacters were not isolated from the target flock. Campylobacters on carcasses from two partly colonised flocks were either the same subtype, as determined by speciation, Multi-Locus Sequence Typing (MLST) and flaA Restricted Fragment Length Polymorphism (RFLP) typing, as those in the fully colonised flocks processed previously, although not necessarily the most prevalent ones; or were the same subtypes as those found in the caeca of the flock itself. The prevalences of the different campylobacter subtypes found on carcasses from two fully colonised flocks did not closely reflect those found in the caeca. MLST combined with flaA RFLP provided a good method for ascertaining the relatedness of strains isolated from carcasses and caecal contents. This study showed that carcass contamination is related to the within-flock prevalence of campylobacter colonisation, but that contamination from previously processed flocks was also significant, especially on carcasses from low prevalence flocks. Forced dry air cooling of carcasses reduced contamination levels.

Sources of Campylobacter spp. colonizing housed broiler flocks during rearing.

The study aimed to identify sources of campylobacter in 10 housed broiler flocks from three United Kingdom poultry companies. Samples from (i) the breeder flocks, which supplied the broilers, (ii) cleaned and disinfected houses prior to chick placement, (iii) the chickens, and (iv) the environments inside and outside the broiler houses during rearing were examined. Samples were collected at frequent intervals and examined for Campylobacter spp. Characterization of the isolates using multilocus sequence typing (MLST), serotyping, phage typing, and flaA restriction fragment length polymorphism typing was performed. Seven flocks became colonized during the growing period. Campylobacter spp. were detected in the environment surrounding the broiler house, prior to as well as during flock colonization, for six of these flocks. On two occasions, isolates detected in a puddle just prior to the birds being placed were indistinguishable from those colonizing the birds. Once flocks were colonized, indistinguishable strains of campylobacter were found in the feed and water and in the air of the broiler house. Campylobacter spp. were also detected in the air up to 30 m downstream of the broiler house, which raises the issue of the role of airborne transmission in the spread of campylobacter. At any time during rearing, broiler flocks were colonized by only one or two types determined by MLST but these changed, with some strains superseding others. In conclusion, the study provided strong evidence for the environment as a source of campylobacters colonizing housed broiler flocks. It also demonstrated colonization by successive campylobacter types determined by MLST during the life of a flock.

DNA-based subtyping of verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2 strains from human and raw meat sources.

AIMS: To investigate subtyping methods for verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2. METHODS AND RESULTS: Eleven human and food strains isolated over a 15-year period were examined. All were intimin (eae)-negative, but all possessed enterohaemolysin and VT1-encoding sequences which in nine strains were vtx1c variant. Ten strains had VT2 genes which were all vtx2d. Plasmid profiles and randomly amplified polymorphic DNA-PCR were not discriminatory. Long-PCR restriction fragment length polymorphism of amplicons bound by the p gene and the VT2A subunit had screening potential. Pulsed field gel electrophoresis (PFGE) using XbaI gave fine discrimination although VT2 sequences were located on a 220 kbp fragment conserved in nine strains and on a 200 kbp fragment in the 10th. CONCLUSIONS: As a result of apparent clonality, PFGE proved essential for differentiation. Long-PCR has promise for screening but requires further evaluation of inter-strain variable sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: A combined phenotypic and genotypic screen, and PFGE for selected strains was effective.

Impact of transport crate reuse and of catching and processing on Campylobacter and Salmonella contamination of broiler chickens.

The influence of transport, catching, and processing on contamination of broiler chickens with Salmonella and Campylobacter was investigated. Transport crates were reused with high frequency and were often still contaminated with Salmonella and Campylobacter when they arrived at the farm despite the fact that they were washed at the factory, and thus they were a potential route of infection. These organisms contaminated the feathers of previously Campylobacter- and Salmonella-negative birds going to the processing plant and were isolated from processed carcasses, albeit at a low frequency. The Campylobacter types which were the predominant organisms on the live birds when they arrived at the processing plant were not necessarily the types that were most frequently isolated from processed carcasses. This finding may reflect cross-contamination that occurred during processing or differences in the tolerance of the strains to the hostile environments that the bacteria experienced. The process of catching and putting the birds in crates significantly increased the chance of contamination with Campylobacter (P < 0.001).

Changes in the carriage of Campylobacter strains by poultry carcasses during processing in abattoirs.

The recent development of simple, rapid genotyping techniques for Campylobacter species has enabled investigation of the determinative epidemiology of these organisms in a variety of situations. In this study we have used the technique of fla typing (PCR-restriction fragment length polymorphism analysis of the flaA and flaB genes) to identify the sources of strains contaminating the carcasses of five campylobacter-positive and two campylobacter-negative broiler flocks during abattoir processing. The results confirmed that, in the United Kingdom, individual broiler flocks are colonized by a limited number of subtypes of Campylobacter jejuni or C. coli. In some but not all cases, the same subtypes, isolated from the ceca, contaminated the end product as observed in carcass washes. However, the culture methodology, i.e, use of direct plating or enrichment, affected this subtype distribution. Moreover, the number of isolates analyzed per sample was limited. fla typing also indicated that some campylobacter subtypes survive poultry processing better than others. The extent of resistance to the environmental stresses during processing varied between strains. The more robust subtypes appeared to contaminate the abattoir environment, surviving through carcass chilling, and even carrying over onto subsequent flocks. From these studies it is confirmed that some campylobacter-negative flocks reach the abattoir but the carcasses from such flocks are rapidly contaminated by various campylobacter subtypes during processing. However, only some of these contaminating subtypes appeared to survive processing. The sources of this contamination are not clear, but in both negative flocks, campylobacters of the same subtypes as those recovered from the carcasses were isolated from the crates used to transport the birds. In one case, this crate contamination was shown to be present before the birds were loaded.

Growth of Salmonella enteritidis in artificially contaminated eggs: the effects of inoculum size and suspending media.

Growth profiles of two isolates of Salmonella enteritidis phage type (PT) 4 inoculated into either the albumen of whole shell eggs or into separated albumen were found to be markedly affected by the size of the inoculum and the composition of the medium used to suspend the cells prior to inoculation. Using our model with an inoculum of two cells, multiplication of the Salmonella was not seen in 93% of eggs held at 20 degrees C for 8 days. In approximately 7% of eggs, however, growth occurred during the 8 days of storage. If the inoculum equaled or exceeded 25 cells per egg when eggs were subsequently stored at 20 degrees C, or 250 cells per egg when eggs were stored at 30 degrees C, high levels of growth of Salmonella in the egg occurred significantly more frequently than when the inoculum was two cells. High levels of growth were also seen more frequently if the inoculum was suspended in buffered peptone water or maximal recovery diluent rather than in phosphate buffered saline. Growth of Salmonella in separated albumen occurred very infrequently (1.1% of samples) at low inoculum levels and did not become significant until the inoculum was 250 cells or greater. Growth in the albumen was unaffected by the composition of the suspending medium. Provided that the inoculum was approximately 2 cells per egg and the bacteria were suspended in PBS, observed growth profiles of S. enteritidis inoculated into the albumen of whole eggs resembled those in naturally contaminated eggs.

Transmission of Vero cytotoxin producing Escherichia coli O157 infection from farm animals to humans in Cornwall and west Devon.

A study of Vero cytotoxin producing Escherichia coli (VTEC) O157 infections in Cornwall and West Devon was conducted to identify associations between human infection and contact with farm animals. In three years from November 1994 to October 1997, 63,000 stool specimens were submitted to four participating microbiology laboratories and screened for E. coli O157. Sixty-nine confirmed cases were interviewed to assess the extent of any direct or indirect contact with farm animals. Nine out of 22 investigations conducted on farms--in which animal rectal swabs, faecal specimens, fore-stream milk samples (first draw-off from teats), and various environmental samples were tested--yielded VTEC O157. In seven incidents one or more isolates from animals were indistinguishable from the isolate(s) from the human case(s) using phenotypic and genotypic subtyping. Cases associated with animal contact included farm visitors, holidaymakers, and members of farming families and farm workers.

Prostatitis.

The laboratory diagnosis of acute bacterial prostatitis is straightforward and easily accomplished in clinical laboratories. Chronic bacterial prostatitis, and especially chronic idiopathic prostatitis (most often referred to as abacterial prostatitis), presents a real challenge to the clinician and clinical microbiologist. Clinically, the diagnosis of chronic idiopathic prostatitis is differentiated from that of acute prostatitis by a lack of prostatic inflammation and no "significant" (controversial) leukocytes or bacteria in the expressed prostatic secretions. Despite these diagnostic criteria, the etiology of chronic idiopathic prostatitis is unknown. While this review covers the entire spectrum of microbially caused acute prostatitis (including common and uncommon bacteria, viruses, fungi, and parasites) and microbially associated chronic prostatitis, a special focus has been given to chronic idiopathic prostatitis. The idiopathic syndrome is commonly diagnosed in men but is poorly treated. Recent data convincingly suggests a possible bacterial etiology for the condition. Provocative molecular studies have been published reporting the presence of 16S rRNA bacterial sequences in prostate biopsy tissue that is negative for ordinary bacteria by routine culture in men with chronic idiopathic prostatitis. Additionally, special culture methods have indicated that difficult-to-culture coryneforms and coagulase-negative staphylococci are present in expressed prostatic secretions found to be negative by routine culture techniques. Treatment failures are not uncommon in chronic prostatitis. Literature reports suggest that antimicrobial treatment failures in chronic idiopathic prostatitis caused by organisms producing extracellular slime might result from the virulent properties of coagulase-negative staphylococci or other bacteria. While it is difficult to definitively extrapolate from animal models, antibiotic pharmokinetic studies with a murine model have suggested that treatment failures in chronic prostatitis are probably a result of the local microenvironment surrounding the persistent focal and well-protected small bacterial biofilms buried within the prostate gland. These conclusions support the molecular and culture data implicating bacteria as a cause of chronic idiopathic prostatitis.

Cryptic bacterial infection in chronic prostatitis: diagnostic and therapeutic implications.

Chronic idiopathic prostatitis, sometimes called prostatodynia or abacterial prostatitis, is a commonly diagnosed and poorly treated urological syndrome. Clinically, this condition frustrates the patient and physician due to its chronicity and resistance to therapy. Recent studies suggest that the etiology of chronic idiopathic prostatitis may be of bacterial origin. Three types of provocative data have demonstrated bacterial presence from prostatic specimens (tissue and secretions) that were negative by traditional clinical microbiologic tests: (i) presence of bacterial gene sequences in prostatic tissue encoding 16S rRNA and tetracycline resistance (tetM-tetO-tetS); (ii) controlled cultural findings showing coagulase-negative staphylococci as the most common isolates (68%) in prostatodynia (chronic idiopathic prostatitis); and (iii) culture of difficult-to-grow coryneforms in expressed prostatic secretions (EPS) on enriched culture media and direct microscopic observation of these pleomorphic bacteria in EPS. Additionally, earlier experimental studies in a rat model support the concept that antibiotic therapy in chronic bacterial prostatitis may not be due to altered antibiotic pharmacokinetics in the chronically inflamed prostate gland. Rather, ineffective antimicrobial eradication might result from protected bacterial micro-colonies within an infection-induced altered micro-environment deep within the prostate gland. We postulate that extracellular slime substances produced by bacteria that are buried in prostatic tissues could impair host defenses by their anti-phagocytic and anti-chemotactic properties that affect neutrophils as well as anti-proliferative characteristics that affect lymphocytes. These extracellular slime substances could also have cytoprotective properties which can conceal bacteria from otherwise bactericidal levels of antibiotics and lead to recrudescent infections resistant to therapy. Persistence of bacterial antigens might initiate a cascade of cellular immunologic events resulting in chronic inflammation of the prostate gland.

Bacterial persistence and expression of disease.

A considerable body of experimental and clinical evidence supports the concept that difficult-to-culture and dormant bacteria are involved in latency of infection and that these persistent bacteria may be pathogenic. This review includes details on the diverse forms and functions of individual bacteria and attempts to make this information relevant to the care of patients. A series of experimental studies involving host-bacterium interactions illustrates the probability that most bacteria exposed to a deleterious host environment can assume a form quite different from that of a free-living bacterium. A hypothesis is offered for a kind of reproductive cycle of morphologically aberrant bacteria as a means to relate their diverse tissue forms to each other. Data on the basic biology of persistent bacteria are correlated with expression of disease and particularly the mechanisms of both latency and chronicity that typify certain infections. For example, in certain streptococcal and nocardial infections, it has been clearly established that wall-defective forms can be induced in a suitable host. These organisms can survive and persist in a latent state within the host, and they can cause pathologic responses compatible with disease. A series of cases illustrating idiopathic conditions in which cryptic bacteria have been implicated in the expression of disease is presented. These conditions include nephritis, rheumatic fever, aphthous stomatitis, idiopathic hematuria, Crohn's disease, and mycobacterial infections. By utilizing PCR, previously nonculturable bacilli have been identified in patients with Whipple's disease and bacillary angiomatosis. Koch's postulates may have to be redefined in terms of molecular data when dormant and nonculturable bacteria are implicated as causative agents of mysterious diseases.

Bacterial doubling time modulates the effects of opsonisation and available iron upon interactions between Staphylococcus aureus and human neutrophils.

Staphylococcus aureus was grown exponentially at two doubling times (DT), one related to in vivo (DT 60 min) and one typical of laboratory conditions (DT 24 min), and under iron-poor and iron-rich conditions. Relative to the fast-grown phenotypes, both slow-grown phenotypes exhibited low surface hydrophobicity and low protein A expression, induced poorly in non-opsonised and opsonised chemiluminescence, and survived well in whole blood killing. In particular, slow-grown, iron-poor cocci demonstrated enhanced survival in whole blood killing which correlated with significant reduction in their association with polymorphonuclear leukocytes, compared to the three other phenotypes; iron sufficiency increased the ability to stimulate polymorphonuclear leukocytes irrespective of opsonisation status. Staphylococcal DT may, by influencing surface hydrophobicity, modify interactions with immune system components.

Prevalence of antibiotic resistance and serotypes in pneumococci in England and Wales: results of observational surveys in 1990 and 1995.

OBJECTIVE: To assess the prevalence of antibiotic resistance and serotype distribution among pneumococci in England and Wales in 1990 and 1995. DESIGN: Observational surveys in March 1990 and March 1995. During two weeks in each survey period all pneumococci isolated in public health laboratories in England and Wales were collected and assessed for sensitivity to antibiotics and the distribution of serogroups or serotypes. SETTING: The network of public health laboratories throughout England and Wales. SUBJECTS: 1127 individual patient isolates of Streptococcus pneumoniae obtained during the two surveys. MAIN OUTCOME MEASURES: Sensitivity or resistance to a range of antibiotics; serogroup or serotype. RESULTS: The prevalence of intermediate or full resistance to penicillin increased from 1.5% in 1990 to 3.9% in 1995 and resistance to erythromycin increased from 2.8% to 8.6%. About 92% of isolates belonged to serogroups or serotypes included in the currently available pneumococcal vaccine. CONCLUSION: Resistance to penicillin and erythromycin has increased among pneumococci in England and Wales. Continued surveillance to assess further increases in the prevalence of pneumococcal resistance to antibiotics is essential.

Bacterial infection in prostatodynia.

PURPOSE: We investigated a possible bacterial etiology for prostatodynia. MATERIALS AND METHODS: We evaluated segmented urine specimens from 22 patients and 16 controls by bacteriological localization studies. Immunological studies were performed on patient and control sera. RESULTS: Nine patients had positive cultures from prostatic secretions. When compared to controls, this novel finding was statistically significant (p < 0.025). Coagulase-negative staphylococci were the most common isolates (68%). No humoral (IgG) immune differences were found between patients and controls. CONCLUSIONS: In a subset of prostatodynia patients bacteria may have an etiological role. Antibiotic treatment demonstrated clinical efficacy.

Dormant microbes in interstitial cystitis.

Interstitial cystitis (IC) is an inflammatory disease of the urinary bladder that has no known etiology. A microbial association with this disease has not been supported since routine cultures of urine from IC patients are usually negative. However, we have demonstrated the presence of bacterial 16S rRNA genes in bladder biopsies from 29% of patients with IC, but not from control patients with other urological diseases. The ability to identify the presence of bacterial DNA in these patients was accomplished using a sensitive and specific nested PCR method capable of amplifying 16S rRNA genes from a wide variety of bacterial genera. Cloning and sequencing of 16S rRNA gene fragments amplified from bladder tissue of IC patients showed that these genes were derived from genera representing Gram-negative bacteria. In addition to the molecular data, a novel finding of 0.22 micron. filterable forms has been isolated in culture from the biopsy tissue of 14 of 14 IC patients and from 1 of 15 controls. The forms contain nucleic acids and resemble cell wall-deficient bacteria in gross morphology; however, their swirled myelin-like ultrastructure is unusual and suggests a heretofore unclassified microbe. These results demonstrate for the first time an association of Gram-negative bacterial DNA and filterable forms with affected bladder tissue from patients with IC.

Antibodies to Escherichia coli 06 porins cross-react with urinary pathogens.

Antibodies to partially purified E. coli 06 35-40 KDa porin trimers recognized the reactive epitopes in the intact porin surface molecule present in various wild-type, heterologous, urinary pathogens. The presence of lipopolysaccharide in the membrane did not shield the antibody binding sites. The reactivity was shown to be specific for porins since LPS-absorbed porin antisera reacted with porins on immunoblots and showed no reactivity with LPS. Additionally, the cross-reactions were abolished by absorption of the porin antisera with E. coli 06 containing porin trimers. These data strengthen the rationale for exploring the enhancement of immunoprotection by monoclonal antibodies to specific immunoreactive antigens in the porin molecule.

Testing antimicrobial susceptibilities of adherent bacteria by a method that incorporates guidelines of the National Committee for Clinical Laboratory Standards.

Membrane filters (0.22-microns pore size) were colonized with 10(4) CFU of logarithmic-phase bacteria per filter under laminar flow conditions in a Modified Robbins Device. Colonized filters were then placed upon agar dilution plates for MIC determinations. Subsequently, filters were transferred to control plates and incubated to obtain MBCs.