Sources of psychrophilic and psychrotolerant clostridia causing spoilage of vacuum-packed chilled meats, as determined by PCR amplification procedure.

AIMS: To determine possible preslaughter and processing sources of psychrophilic and psychrotolerant clostridia causing spoilage of vacuum-packed chilled meats. METHODS AND RESULTS: Molecular methods based on the polymerase chain reaction (PCR) amplification of specific 16S rDNA fragments were used to detect the presence of Clostridium gasigenes, Clostridium estertheticum, Clostridium algidicarnis and Clostridium putrefaciens in a total of 357 samples collected from ten slaughter stock supply farms, slaughter stock, two lamb-processing plants, their environments, dressed carcasses and final vacuum-packed meat stored at -0.5 degrees C for 5(1/2) weeks. Clostridium gasigenes, C. estertheticum and C. algidicarnis/C. putrefaciens were commonly detected in farm, faeces, fleece and processing environmental samples collected at the slaughter floor operations prior to fleece removal, but all these micro-organisms were detected in only 4 out of 26 cooling floor and chiller environmental samples. One out of 42 boning room environmental samples tested positive for the presence of C. gasigenes and C. estertheticum, but 25 out of 42 of these samples were positive for C. algidicarnis/C. putrefaciens. Nearly all of the 31 faecal samples tested positive for the presence of C. gasigenes and C. estertheticum; however, only two of these samples were positive for C. algidicarnis and/or C. putrefaciens. Clostridial species that were subject to this investigation were frequently detected on chilled dressed carcasses. CONCLUSIONS: The major qualitative and quantitative differences between the results of PCR detection obtained with the primers specific for 'blown pack' -causing clostridia (C. gasigenes and C. estertheticum) and those obtained with primers specific for C. algidicarnis and C. putrefaciens suggest that the control of meat spoilage caused by different groups of meat clostridia is best approached individually for each group. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides information significant for controlling meat spoilage-causing clostridia in the meat-processing plants.

Influence of peroxyacetic acid-based carcass rinse on the onset of "blown pack" spoilage in artificially inoculated vacuum-packed chilled beef.

"Blown pack" spoilage is an increasingly reported spoilage condition of vacuum-packed chilled meats. This spoilage condition is primarily caused by a psychrophilic obligately anaerobic microorganism, Clostridium estertheticum. The present study investigated whether peroxyacetic acid (POAA)-based carcass rinse can delay the onset of gas production in chilled vacuum-packed beef artificially inoculated with C. estertheticum spores. The variables studied were (i) two prepackaging meat rinses (water and POAA-based rinse); (ii) three levels of C. estertheticum spores (0, 4, and 40 spores per cm2); and (iii) three postpackaging storage temperatures (-1.5, 0, and 2 degrees C). Treatment with POAA-based rinse marginally delayed the onset of pack blowing in packs carrying high numbers of C. estertheticum spores but not in packs carrying low levels of inoculum or in uninoculated controls. The presence of as few as 4 spores per cm2 of meat surface effectively decreased by two-thirds the nominal shelf life of vacuum-packed chilled beef. Increasing the inoculum by 10-fold to 40 spores per cm2 resulted in the additional acceleration of the onset of pack blowing. The onset of gas production was significantly delayed by storing the packaged product at -1.5 degrees C rather than at 0 degrees C. The results of this study indicate that the POAA-based rinse tested will not eliminate the spoilage threat posed by clostridial blown pack spoilage spores present on meat surfaces. POAA-based rinse can be used alone to achieve some extension of shelf life of beef cuts heavily contaminated with C. estertheticum spores. Alternatively, the rinse may offer an opportunity for a more substantial extension of shelf life of contaminated cuts when used with additional hurdles.

Evaluation of molecular methods to determine enterotoxigenic status and molecular genotype of bovine, ovine, human and food isolates of Staphylococcus aureus.

This study evaluated the use of PFGE and single enzyme AFLP techniques for the determination of the genetic relationships between Staphyloccocus aureus isolates from human, bovine, ovine and food related sources and reports the prevalence of 'classic' (sea to see) and 'new' (seg, seh, sei, sej, sem, sen and seo) staphylococcal enterotoxin (se) genes in 92 S. aureus strains. A sub-set of the se genotyping results was confirmed by ELISA and the presence of SE toxin determined in isolates from different sources. A 100% correlation was observed, between detection of enterotoxin genes sea-see and expression of corresponding enterotoxin proteins in vitro. The se genotyping data generated from 90 of the S. aureus isolates showed that many of the S. aureus strains producing identical se genotypes correlated with both AFLP and PFGE pattern types. However, single enzyme AFLP technique did not possess the discriminatory power of the PFGE method, but similar clonal relationships were observed by both techniques in many of the isolates tested. Results reported here include the first comprehensive study using a single enzyme AFLP technique to investigate the genetic background of S. aureus isolates from a wide distribution including animal, human and food related sources.

The Hawaiian Archipelago: a microbial diversity hotspot.

The Hawaiian Archipelago is a "biodiversity hotspot" where significant endemism among eukaryotes has evolved through geographic isolation and local topography. To address the absence of corresponding region-wide data on Hawaii's microbiota, we compiled the first 16S SSU rDNA clone libraries and cultivated bacteria from five Hawaiian lakes, an anchialine pool, and the Lo'ihi submarine volcano. These sites offer diverse niches over approximately 5000 m elevation and approximately 1150 nautical miles. Each site hosted a distinct prokaryotic community dominated by Bacteria. Cloned sequences fell into 158 groups from 18 Bacteria phyla, while seven were unassigned and two belonged in the Euryarchaeota. Only seven operational taxonomic units (each OTU comprised sequences that shared > or =97% sequence identity) occurred in more than one site. Pure bacterial cultures from all sites fell into 155 groups (each group comprised pure cultures that shared > or =97% 16S SSU rDNA sequence identity) from 10 Bacteria phyla; 15 Proteobacteria and Firmicutes were cultivated from more than one site. One hundred OTUs (60%) and 52 (33.3%) cultures shared <97% 16S SSU rDNA sequence identity with published sequences. Community structure reflected habitat chemistry; most delta-Proteobacteria occurred in anoxic and sulfidic waters of one lake, while beta-Proteobacteria were cultivated exclusively from fresh or brackish waters. Novel sequences that affiliate with an Antarctic-specific clade of Deinococci, and Candidate Divisions TM7 and BRC1, extend the geographic ranges of these phyla. Globally and locally remote, as well as physically and chemically diverse, Hawaiian aquatic habitats provide unique niches for the evolution of novel communities and microorganisms.

Abattoir sources of psychrophilic clostridia causing blown pack spoilage of vacuum-packed chilled meats determined by culture-based and molecular detection procedures.

AIMS: To identify the abattoir source(s) of psychrophilic clostridia causing 'blown pack' spoilage of vacuum-packed chilled meats. METHODS AND RESULTS: Molecular procedures were used to detect the presence of specific 16S rRNA gene fragments of blown pack-causing clostridia in samples collected from a commercial abattoir and its environs. Blown pack-causing clostridia were consistently detected in hide, soil and faecal samples, as well as in samples collected at slaughter plant locations associated with handling of animals and animal carcasses prior to pelt removal. CONCLUSIONS: The data indicate that pelts per se or soil particles/faecal material attached thereto are the most probable primary reservoir of blown pack clostridia in the abattoir. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper provides information critical for controlling blown pack spoilage in commercial meat-processing plants.

PCR detection of psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled meats.

AIMS: To develop a practical molecular procedure that directly, without isolation, and specifically detects the presence of clostridia which cause 'blown pack' spoilage of vacuum-packed meat. METHODS AND RESULTS: Primer sets and PCR amplification procedures were developed that detect the presence of 16S rDNA gene and/or 16S-23S rDNA internal transcribed spacer fragments of 'blown pack' causing clostridia in meat. The specificity of the developed procedures was evaluated with DNA obtained from close phylogenetic neighbours of 'blown pack' causing clostridia, food clostridia and common meat spoilage microorganisms. The sensitivity of detection was assessed in non-enriched and low-temperature-enriched beef mince inoculated with serially diluted pure cultures of Clostridium estertheticum DSMZ 8809T and Cl. gasigenes DB1AT. The efficacy of detection procedures was evaluated for naturally contaminated vacuum-packed meat samples. Three primer sets, 16SE, 16SDB and EISR, produced amplicons of the expected size with DNA templates from target clostridia, but failed to yield PCR products with DNAs from any other microorganisms tested. With 16SE and 16SDB primers, minimum levels of detection were 104 CFU g(-1) for non-enriched, and 102 CFU g(-1) for enriched meat samples. Based on the established specificity of these primers, as well as DNA sequencing of amplicons, Cl. gasigenes was confirmed as the causative agent of 'blown pack' spoilage in two packs, and Cl. estertheticum as the causative agent in the third. CONCLUSIONS: The developed method can be used for rapid detection of 'blown pack' causing clostridia in commercial blown packs, or following low temperature enrichment, for detection of these microorganisms in meat containing as few as 100 clostridial cells per gram. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper reports practical procedures that can be used for rapid confirmation of the causative agents of clostridial 'blown pack' spoilage in commercial spoiled packs, or for detection of psychrophilic clostridia in epidemiological trace back of 'blown pack' spoilage incidents in meat processing plants.

PCR detection of psychrotolerant clostridia associated with deep tissue spoilage of vacuum-packed chilled meats.

AIMS: To develop a practical molecular procedure that directly (without isolation) and specifically detects the presence of clostridia, which cause the deep tissue spoilage condition. METHODS AND RESULTS: A primer set was designed and a PCR amplification procedure developed to detect the presence of Clostridium algidicarnis and Cl. putrefaciens 16S rDNA gene fragments in meat. The procedure yielded amplicons of the expected size with homologous DNA templates, but failed to give PCR products with DNAs from 47 food clostridia and common meat spoilage micro-organisms. The minimum level of detection was 10(4) cfu g-1 for nonenriched meat samples. Based on the established specificity of these primers, as well as DNA sequencing of amplicons, the presence of Cl. algidicarnis and/or Cl. putrefaciens was confirmed in a swab sample taken from the cartilage of an ovine stifle joint, which on opening exhibited strong offensive odours. CONCLUSIONS: The developed method can be used for rapid detection of clostridia causing deep tissue spoilage in commercial vacuum packs. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper reports practical procedures that can be used for rapid confirmation of the causative agents of deep tissue clostridial spoilage in commercial vacuum-packed chilled meats.

The abattoir source of culturable psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled venison.

AIMS: To identify the abattoir source(s) of culturable psychrophilic clostridia causing 'blown pack' spoilage of vacuum-packed chilled meats. METHODS AND RESULTS: Psychrophilic and psychrotolerant clostridia were isolated from hides, faeces and tonsils of deer slaughter stock, and from a meat plant environment. The isolates were differentiated using restriction fragment length polymorphism analysis of the 16S rDNA gene (PCR-RFLP) and 16S-23S rDNA internal transcribed spacer (ITS) analysis. PCR-RFLP group I clostridia were found to have restriction patterns indistinguishable from the patterns of 'blown pack'-causing Clostridium gasigenes DB1A(T) and R26. Gas production in packs inoculated with vegetative cells of PCR-RFLP group I clostridia was first evident after 14 days at 2 degrees C. The prevalence of these clostridia was similar in hide and faecal samples from slaughter animals, but these micro-organisms were absent from tonsils and the meat plant environment. Banding patterns of PCR-RFLP group II clostridia showed some cross-similarity with patterns of the 'blown pack'-causing micro-organism Cl. estertheticum DSM 8809(T) and Cl. estertheticum-like meat strains. The majority of clostridia in PCR-RFLP group II were found in the faeces of slaughter animals. Isolates representing PCR-RFLP group II did not, however, produce gas in vacuum packs stored at 2 degrees C for 84 days. CONCLUSIONS: The data suggest that soil particles attached to hide or present in faeces are the most probable primary reservoir from which 'blown pack' clostridia are introduced onto carcasses. Therefore, dressing procedure hygiene remains paramount in order to control the spread of psychrophilic Clostridium spp. in a meat plant. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper provides information critical for controlling 'blown pack' spoilage in meat processing plants. It reports on the use of molecular techniques for determination of abattoir sources of 'blown pack'-causing clostridia.

A PCR survey of psychrotrophic Clostridium botulinum-like isolates for the presence of BoNT genes.

Isolates (259) of psychrotrophic Clostridium spp. associated with either blown pack spoilage (five isolates) or slaughter stock (254 isolates) were screened for the presence of botulinum neurotoxin (BoNT) genes using degenerate PCR primers capable of amplifying A, B, E, F and G BoNT genes. No BoNT gene amplification products were detected using DNA templates from the 259 psychrotrophic isolates, including 249 isolates that showed the same 16S rRNA gene Restriction Fragment Length Polymorphism (RFLP) patterns as authentic Cl. botulinum type B. It is concluded that although the growth of such micro-organisms in vacuum-packed chilled meat leads to product spoilage, it does not prejudice product safety.

An assessment of temafloxacin in the treatment of chronic bacterial prostatitis.

Based on its in-vitro activity against the majority of organisms associated with bacterial prostatitis and its excellent penetration into prostatic tissue, prostatic secretions and seminal fluid, temafloxacin appears to be a suitable agent for the treatment of prostatic infections. The efficacy and safety of temafloxacin 400 mg bd for 28 days were assessed in 61 patients from ten centres in Germany with symptomatic bacterial prostatitis diagnosed by segmented localizing cultures. Urine and prostatic secretions were obtained for culture. Clinical signs and symptoms were evaluated at two weeks during treatment, and at 5 to 9 days and 26 to 30 days after treatment. Safety was monitored during and at the end of treatment. Escherichia coli and Enterococcus spp. were the most frequent pathogens. In 41 clinically and bacteriologically evaluable patients, 37 (90%) were successfully treated at 5 to 9 days; four of these patients did not return for follow-up at the final visit and the remaining patients (33) continued to be clinically cured or improved. Thirty-seven patients (90.2%) had eradication of pre-treatment pathogens at 5 to 9 days after treatment; three of these patients did not return for this final follow-up visit. There were six patients with persistent or recurrent pathogens isolated and six patients with reinfecting pathogens. Thus, 26 of 38 (68%) evaluable patients at visit 5 were free from infection (from either a pre-treatment pathogen or any subsequent new infecting pathogen) up to 26 to 30 days after treatment. One clinically evaluable but bacteriologically non-evaluable patient was classified as a therapeutic failure after nine days of treatment and was not included in the final assessment. Improvement in the severity of specific signs and symptoms was observed in greater than 90% of cases. Mild to moderate adverse events, mostly occurring during the first or second weeks of long-term therapy, were reported in 11.5% of patients. Temafloxacin 400 mg bd, for four weeks was very effective and well tolerated by the majority of patients with documented bacterial prostatitis.

Penetration of rufloxacin into human prostatic tissue and fluid.

The penetration of rufloxacin into prostatic tissue and prostatic fluid was investigated in 16 patients undergoing elective transurethral prostate resection. Rufloxacin 400 mg was given orally approximately 16 h before surgery with a further dose of 200 mg approximately 5 h before surgery. Sampling was inadequate in three patients. One patient reported transient flushing and headache after the first dose of rufloxacin and was hence withdrawn from the study. In the remaining 12 evaluable patients, the mean ratios of rufloxacin concentrations (determined by HPLC) in prostatic tissue and fluid to the plasma concentration were 1.9 and 1.5, respectively. There were no significant differences between the tissue penetration ratios in different parts of the prostate. The levels of rufloxacin found in prostate tissue and fluid, in this study, exceeded the MICs for most micro-organisms causing chronic bacterial prostatitis.

An open multicentre study on the efficacy and safety of rufloxacin in patients with chronic bacterial prostatitis.

In an open study, 27 patients with chronic bacterial prostatitis were treated for four weeks with rufloxacin. They received 400 mg on the first day, and then 200 mg once daily. The patients were studied for up to one month after completion of therapy. One patient was lost to follow-up, but was included in the safety analysis. One patient did not meet all the inclusion criteria (WBC less than 10/HPF in the prostatic fluid sample) and was evaluated separately (cure). One month after treatment, clinical success (cure and improvement) was obtained in 23 of 25 patients (92%) and bacteriological eradication was achieved in 19 of 24 patients (79%). The only adverse event possibly related to the study drug was a case of transient mild tiredness and nervousness. Rufloxacin did not accumulate in plasma during therapy. Hence, a single daily dose of rufloxacin 200 mg appears to be a safe treatment for chronic bacterial prostatitis.

Rufloxacin once daily in acute exacerbations of chronic bronchitis.

In this open study the efficacy and tolerability of rufloxacin in a single dose of 400 mg the first day and 200 mg the nine consecutive days was studied in 26 patients with an acute exacerbation of chronic bronchitis. Twenty-two patients were evaluable for efficacy. Four patients stopped treatment prematurely after five days because of clinical cure. At the enrollment visit a pathogen was isolated in the sputum sample in 19 of 22 evaluable patients. The predominant pathogens were Streptococcus pneumoniae and Moraxella catarrhalis. In 17 of these 19 bacteriologically evaluable patients the initial infecting organism was eradicated from specimens obtained within 48 hours after the end of therapy. There was one case of persistent infection caused by S. pneumoniae (MIC 4 mg/l), one patient had a superinfection with Serratia marcescens (MIC 1 mg/l) susceptible to rufloxacin and therapy was stopped after five days due to clinical failure. One week after the end of therapy, 15 patients remained free from infection whilst one patient experienced reinfection with Klebsiella pneumoniae (MIC 0.5 mg/l). Clinical cure or improvement was observed in 21 of 22 patients. Mild adverse events were reported by two of 26 enrolled patients. In one patient, complaining of headache and dizziness, the adverse events were considered possibly study drug related. No abnormal laboratory findings were reported. Nadir plasma levels of rufloxacin were measured and no accumulation in plasma was observed during treatment. A ten day course of an oral single dose of rufloxacin proved efficacious and was well tolerated in patients with an acute exacerbation of chronic bronchitis.(ABSTRACT TRUNCATED AT 250 WORDS)

Fosfomycin trometamol in a single dose versus norfloxacin for seven days in the treatment of uncomplicated urinary infections in general practice.

The efficacy and tolerability of fosfomycin trometamol in a single dose of 3 g was compared with norfloxacin 400 mg b.i.d. for seven days in the treatment of adult female patients with uncomplicated urinary infections. 158 female patients with a mean age of 30 years who presented symptoms of dysuria and frequency with documented pyuria and bacteriuria on urinalysis (greater than or equal to 10(5) cfu/ml of urine) were initially included in the study. The total number of clinically and bacteriologically evaluable patients was 111, of which 61 received fosfomycin trometamol and 50 norfloxacin. One to two days after the double blind medication schedule for seven days, 55 of 60 patients (92%) in the fosfomycin trometamol group and 48 of 50 patients (96%) in the norfloxacin group were clinically cured. 37 patients without significant bacteriuria showed a clinical cure rate of over 90% in both therapy groups. Two to three days after the single dose treatment with fosfomycin trometamol the initial infecting pathogen was eradicated in 60 of the 61 patients (98%). One to two days after a seven day treatment with norfloxacin 48 of 50 patients (96%) showed an eradication of the initial infecting pathogen. Six weeks after the start of therapy 39/60 patients (65%) and 32/49 (65%) in the fosfomycin trometamol and norfloxacin groups respectively, remained free from urinary infection. The reinfection rate in both treatment groups was approximately 25%. The relapse rate in the post treatment evaluation period of four weeks was relatively low in both therapy groups, 5/49 patients (10%) in the norfloxacin group and 3/55 patients (6%) in the fosfomycin trometamol group, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative human bioavailability study of macrocrystalline nitrofurantoin and two prolonged-action hydroxymethylnitrofurantoin preparations.

This single-blind crossover study compared the human bioavailability of macrocrystalline nitrofurantoin (Furadantine MC) and two prolonged-action hydroxymethylnitrofurantoin formulations (Urfadyn PL, bid, and Uridurine, tid), based on plasma nitrofurantoin concentrations and urinary nitrofurantoin excretion. The drugs were administered to 16 healthy females for a single day according to the recommended daily dosages. For comparison, Furadantine MC was administered both at the qid dosage recommended by the manufacturer and at tid dosage. Pharmacokinetic parameters determined were maximum plasma concentration after first dose, minimum plasma concentration after first dose, area under the plasma concentration-time curve (AUC), cumulative renal excretion over 30 hours (ARE), overall renal clearance, total body clearance, and bioavailability relative to Furadantine MC qid, based on plasma AUC (F) and ARE (Fren). F for Furadantine MC 100 mg tid was 108 +/- 25 percent (mean +/- SD); for Uridurine 100 mg tid and Urfadyn PL 100 mg bid, F equalled 86 +/- 33 percent and 53 +/- 20 percent (p less than 0.05), respectively. A similar relationship was observed between Fren for Furadantine MC 100 mg qid and the respective Fren of Furadantine MC 100 mg tid, Uridurine 100 mg tid, and Urfadyn PL 100 mg bid. No significant difference was found between the respective F and Fren of each of the drugs studied. Although bioavailability was comparable for Furadantine MC tid and qid, the single-day design of these studies precludes inferring that these dosage schedules are therapeutically equivalent. However, the significantly lower relative bioavailabilities for the prolonged-action hydroxymethylnitrofurantoin formulations suggest that Urfadyn PL 100 mg bid and Uridurine 100 mg tid are not pharmacokinetically equivalent to Furadantine MC.

Imipenem as monotherapy in the treatment of intensive care patients with severe infections.

In an open study, 24 intensive care patients were treated with imipenem/cilastatin as monotherapy for serious bacterial infections. Twenty-one patients were treated for bronchopulmonary infection, two patients for septicaemia, and one patient for an empyema. Initially all strains were susceptible to imipenem. Gram-negative bacilli accounted for 80% of these isolates. The most frequently isolated species were Proteus mirabilis, Escherichia coli and Pseudomonas aeruginosa. All 24 patients were considered clinically cured. Sixteen of these patients (67%) were both clinically and microbiologically cured. In eight of the 24 patients (33%), the strains isolated initially persisted. In eight of the 24 patients (33%), colonization of the respiratory tract developed. Two of the five Ps. aeruginosa isolates developed resistance during therapy but in none of these patients was therapy considered to have failed. In 12 patients (50%), transient elevations in hepatic function tests were observed and these were probably drug-related. The present study supports the view that imipenem/cilastatin may be useful as monotherapy in the treatment of severe infections in intensive care patients.

Efficacy and safety of pefloxacin in the treatment of patients with complicated urinary tract infections.

One hundred and four patients with complicated urinary tract infections (prolonged severe chronic infections or with complicated postoperative conditions) were treated for ten days with pefloxacin 400 mg bid. Bacteriological eradication of the initial pathogen was achieved in 98% of the patients. After six weeks 93% of the patients were still free of the initial infecting microorganism. Clinical improvement was achieved in 77% of the patients five to seven days after cessation of treatment. The side-effects which were definitely related to pefloxacin occurred in 9% of patients and were mostly of a gastro-intestinal, a neurological, or an allergic nature. No significant biochemical or haematological adverse reactions occurred.

New 4-quinolones in the treatment of urinary tract infections.

The new fluorinated quinolones norfloxacin, ciprofloxacin and pefloxacin were evaluated in urinary infections. Bacteriological cure rates in both uncomplicated and complicated urinary tract infections ranged from 85% to 99%. Clinical cure rates were often lower due to the underlying conditions in the urinary tract. Patients with neurological bladder disease were cured in a relatively high percentage of their Pseudomonas infection after three months treatment with norfloxacin. Pharmacokinetics of ciprofloxacin in prostatic tissue and fluid will probably offer an advance in the treatment of chronic urinary infections due to an infectious prostatic focus. Definitely drug related side effects (of gastro-intestinal, neurological or allergic nature) were mild in most cases. The new 4-quinolones should be followed with interest concerning their activity in urological infections in general as well as specifically. The minor influence on the natural human flora and the possibility to decrease plasmid-mediated resistance are of major importance.