Antimicrobial susceptibility of Clostridium difficile isolated from food animals on farms.

Clostridium difficile is commonly associated with a spectrum of disease in humans referred to as C. difficile-associated disease (CDAD) and use of antimicrobials is considered a risk factor for development of disease in humans. C. difficile can also inhabit healthy food animals and transmission to humans is possible. As a result of the complexity and cost of testing, C. difficile is rarely tested for antimicrobial susceptibility. A total of 376 C. difficile strains (94 each from swine and dairy feces, and 188 from beef cattle feces) were isolated from healthy food animals on farms during studies conducted by the National Animal Health Monitoring System. Using the Etest (AB Biodisk, Solna, Sweden), samples were tested for susceptibility to nine antimicrobials implicated as risk factors for CDAD (linezolid, amoxicillin-clavulanic acid, ampicillin, clindamycin, erythromycin, levofloxacin, metronidazole, rifampicin, and vancomycin). Vancomycin was active against all isolates of C. difficile (MIC90=3.0µg/ml) while almost all isolates (n=369; 98.1%) were resistant to levofloxacin. With the exception of vancomycin, resistance varied by animal species as follows: linezolid (8.5% resistance among swine versus 2.1 and 1.1% resistance among dairy and beef, respectively), clindamycin (56.4% resistance among swine versus 80% and 90.9% resistance among dairy and beef, respectively), and rifampicin (2.1% and 0% resistance among swine and dairy cattle isolates, respectively versus 14.3% resistance among beef isolates). Regardless of species, multiple drug resistance was observed most often to combinations of clindamycin and levofloxacin (n=195; 51.9%) and ampicillin, clindamycin and levofloxacin (n=41; 10.9%). The reason for the variability of resistance between animal species is unknown and requires further research.

Effect of Bacillus Subtilis-based Direct-fed Microbials on Immune Status in Broiler Chickens Raised on Fresh or Used Litter.

Type of dietary direct-fed microbials (DFMs) or poultry litter could directly influence the composition of gut microbiota. Gut microbiota plays an important role in shaping the developing immune system and maintaining the homeostasis of the mature immune system in mammal and chickens. The present study was carried out to investigate the interaction among litter, DFMs and immunity in broiler chickens exposed to a field-simulated environment. Immune status of broiler chickens was assessed by serum antibodies against Eimeria spp. and Clostridium spp. and intestinal cytokine mRNA expression. The current experimental design had a 3 ×2 factorial arrangement of treatments with three types of litter, i.e., fresh litter or used litter that was obtained from a farm with no disease outbreak (used litter) or a farm with history of a gangrenous dermatitis outbreak (GD litter), and two dietary treatments with or without DFMs. It was found that either DFM addition or type of litter significantly affected anticoccidial antibody levels of broiler chickens at d 42. In general, dietary DFMs increased the anticoccidial antibodies in the fresh-litter raised chickens, but lowered the levels in the GD-litter raised chickens. Serum antibodies against Clostridium perfringens α-toxin were significantly (p<0.05) higher in chickens raised on GD litter compared with those raised on fresh litter. Cytokine mRNA expression was significantly (p<0.05) altered by either the type of litter or DFMs. Of interest, dietary DFMs lowered interferon-γ, interleukin 1beta, and CXCLi2 cytokine mRNA expression in chickens raised on fresh litter but increased them in GD-litter raised chickens. In conclusion, dietary DFMs modulate various immune parameters of broiler chickens, but the DFM-mediated effects were dependent upon the type of litter on which chickens were raised.

Effect of dietary antimicrobials on immune status in broiler chickens.

This study evaluated the effects of dietary anticoccidial drugs plus antibiotic growth promoters (AGPs) on parameters of immunity in commercial broiler chickens. Day-old chicks were raised on used litter from a farm with endemic gangrenous dermatitis to simulate natural pathogen exposure and provided with diets containing decoquinate (DECX) or monensin (COBN) as anticoccidials plus bacitracin methylene disalicylate and roxarsone as AGPs. As a negative control, the chickens were fed with a non-supplemented diet. Immune parameters examined were concanavalin A (ConA)-stimulated spleen cell proliferation, intestine intraepithelial lymphocyte (IEL) and spleen cell subpopulations, and cytokine/chemokine mRNA levels in IELs and spleen cells. ConA-induced proliferation was decreased at 14 d post-hatch in DECX-treated chickens, and increased at 25 and 43 d in COBN-treated animals, compared with untreated controls. In DECX-treated birds, increased percentages of MHC2(+) and CD4(+) IELS were detected at 14 d, but decreased percentages of these cells were seen at 43 d, compared with untreated controls, while increased TCR2(+) IELs were evident at the latter time. Dietary COBN was associated with decreased fractions of MHC2(+) and CD4(+) IELs and reduced percentages of MHC2(+), BU1(+), and TCR1(+) spleen cells compared with controls. The levels of transcripts for interleukin-4 (IL-4), IL-6, IL-17F, IL-13, CXCLi2, interferon-γ (IFN-γ), and transforming growth factorß4 were elevated in IELs, and those for IL-13, IL-17D, CXCLi2, and IFN-γ were increased in spleen cells, of DECX- and/or COBN-treated chickens compared with untreated controls. By contrast, IL-2 and IL-12 mRNAs in IELs, and IL-4, IL-12, and IL-17F transcripts in spleen cells, were decreased in DECX- and/or COBN-treated chickens compared with controls. These results suggest that DECX or COBN, in combination with bacitracin and roxarsone, modulate the development of the chicken post-hatch immune system.

Multilocus sequence typing subtypes of poultry Clostridium perfringens isolates demonstrate disease niche partitioning.

Clostridium perfringens is a ubiquitous and versatile pathogenic bacterium and is implicated in the etiology of the poultry diseases necrotic enteritis (NE) and poultry gangrene (PG). In this study, multilocus sequence typing was used to investigate genotypic relationships among 139 C. perfringens isolates from 74 flocks. These isolates had multiple disease, host, and environmental origins. The results indicated a polymorphic yet highly clonal population, with 79.6% of all isolates partitioning into one of six clonal complexes or two dominant sequence types, ST-9 and ST-31. The most prolific clonal complex, CC-1, contained 27.3% of all isolates and was not clearly associated with one particular disease. The subtypes CC-4 and ST-31 were highly associated with NE and represented 9.4% and 7.2% of the total isolates, respectively. No PG-associated and NE-associated C. perfringens isolates shared the same sequence type or clonal complex. NE-associated subtypes were more clonal and appeared more evolutionarily divergent than PG-associated subtypes, which tended to cluster in the more ancestral lineages alongside isolates from asymptomatic chickens and turkeys. Toxin gene screening identified cpb2 throughout these isolates and correlated the presence of netB with NE pathology. Previous investigations into the genetic basis of C. perfringens pathogenicity have focused on toxins and other variable genetic elements. This study presents the first sequence-based comparison of C. perfringens isolates recovered in clinical cases of PG and NE and demonstrates that niche specialization is observable in the core genomes of poultry-associated C. perfringens isolates, a concept with both epidemiological and evolutionary significance.

Clostridium difficile from healthy food animals: optimized isolation and prevalence.

Two isolation methods were compared for isolation of Clostridium difficile from food animal feces. The single alcohol shock method (SS) used selective enrichment in cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate, followed by alcohol shock and isolation on tryptic soy agar supplemented with 5% sheep blood, and cycloserine-cefoxitin fructose agar. The double alcohol shock method (DS) used alcohol shock prior to and after selective enrichment in cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate, followed by isolation on tryptic soy agar supplemented with 5% sheep blood and cycloserine-cefoxitin fructose agar. A total of 55 (15.9%, n = 345) swine fecal samples, 32 (2.4%, n = 1,325) dairy cattle fecal samples, and 188 (6.3%, n = 2,965) beef cattle fecal samples were positive for C. difficile by either method. However, the DS was significantly better than the SS for the recovery of C. difficile from swine feces, while the SS was significantly better than the DS for the recovery of C. difficile from beef cattle feces. There was no significant difference between methods for the recovery of C. difficile from dairy cattle feces. This study suggests that food animals might harbor C. difficile and it provides critical information that isolation methods might not have universal application across animal species.

Quantitative real-time PCR assay for Clostridium septicum in poultry gangrenous dermatitis associated samples.

Clostridium septicum is a spore-forming anaerobe frequently implicated in cases of gangrenous dermatitis (GD) and other spontaneously occurring myonecrotic infections of poultry. Although C. septicum is readily cultured from diseased tissues it can be difficult to enumerate due to its tendency to swarm over the surface of agar plates. In this study a quantitative real-time PCR assay was developed in order to more accurately measure the levels of C. septicum in healthy as well as GD associated poultry samples. The assay was specifically designed to target the C. septicum alpha toxin gene, csa, which is, to our knowledge, carried by all strains of C. septicum and has been shown to be essential for virulence. Genomic DNAs from a diverse collection of bacterial species, including closely related Clostridium chauvoei, Clostridium carnis, Clostridium tertium as well as several strains of Clostridium perfringens, all failed to produce a positive reaction. An approximate reproducible limit of detection in spiked extracts of at least 10(3) cfu/g of C. septicum was observed for a variety of different sample types. C. septicum levels in broiler chicken field samples estimated from the results of qPCR were statistically correlated to culture based enumerations obtained from those same tissues.

Effects of direct-fed microbials on growth performance, gut morphometry, and immune characteristics in broiler chickens.

This study was conducted to compare growth performance, gut morphometry, and parameters of local and systemic immunity in broiler chickens fed for 22 consecutive days with a diet supplemented with Bacillus spp. as direct-fed microbials (DFM), a commercial product incorporating 3 DFM, or a nonsupplemented diet. Direct-fed microbials did not significantly modify BW gain and most failed to affect serum antibody levels in response to immunization with a recombinant Eimeria protein. However, altered intestinal morphometric measurements were readily apparent in DFM-fed chickens as revealed by increased villus height and crypt depth compared with non-DFM-fed controls. In addition, serum levels of alpha-1-acid glycoprotein as an inflammatory marker were reduced in DFM-fed birds, whereas splenic lymphocyte proliferation, intestine intraepithelial lymphocyte subpopulations, and cytokine mRNA levels in intraepithelial lymphocytes were increased, decreased, or unchanged compared with controls depending on the DFM used. These results provide a rational scientific basis for future studies to investigate DFM as immunomodulating agents to enhance host protective immunity against enteric pathogens in broiler chickens.

Antimicrobial activity of lupulone against Clostridium perfringens in the chicken intestinal tract jejunum and caecum.

OBJECTIVES: Owing to the spread of antibiotic resistance among human infectious agents, there is a need to research antibiotic alternatives for use in animal agricultural systems. Antibiotic-free broiler chicken production systems are known to suffer from frequent outbreaks of necrotic enteritis due in part to pathogenic type A Clostridium perfringens. Hop (Humulus lupulus) bitter acids are known to possess potent antimicrobial activity. Lupulone was evaluated for in vivo antimicrobial activity to inhibit C. perfringens in a chick gastrointestinal colonization model. METHODS: Using a week-2 per os inoculated C. perfringens chicken colonization model, C. perfringens counts in mid-intestinal and caecal contents were compared between chickens administered lupulone at 62.5, 125 and 250 ppm in drinking water versus 0 ppm control. Results At day 22, post-hatch intestinal C. perfringens counts of lupulone-treated chickens were significantly lower (P < 0.05) than water-treated control groups in both jejunal and caecal sampling sites across all lupulone dosages tested. CONCLUSIONS: Lupulone administered through water inhibits gastrointestinal levels of inoculated pathogenic clostridia within the chicken gastrointestinal tract. Lupulone was effective within the chemically complex mixture of material within the gastrointestinal tract, thereby making this agent a target of further research as an antibiotic alternative for this and possibly other intestinal infections.

Isolation and purification of enterocin E-760 with broad antimicrobial activity against gram-positive and gram-negative bacteria.

Strain NRRL B-30745, isolated from chicken ceca and identified as Enterococcus durans, Enterococcus faecium, or Enterococcus hirae, was initially identified as antagonistic to Campylobacter jejuni. The isolate produced a 5,362-Da bacteriocin (enterocin) that inhibits the growth of Salmonella enterica serovar Enteritidis, S. enterica serovar Choleraesuis, S. enterica serovar Typhimurium, S. enterica serovar Gallinarum, Escherichia coli O157:H7, Yersinia enterocolitica, Citrobacter freundii, Klebsiella pneumoniae, Shigella dysenteriae, Pseudomonas aeruginosa, Proteus mirabilis, Morganella morganii, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Campylobacter jejuni, and 20 other Campylobacter species isolates. The enterocin, E-760, was isolated and purified by cation-exchange and hydrophobic-interaction chromatographies. The proteinaceous nature of purified enterocin E-760 was demonstrated upon treatment with various proteolytic enzymes. Specifically, the antimicrobial peptide was found to be sensitive to beta-chymotrypsin, proteinase K, and papain, while it was resistant to lysozyme and lipase. The enterocin demonstrated thermostability by retaining activity after 5 min at 100 degrees C and was stable at pH values between 5.0 and 8.7. However, activity was lost below pH 3.0 and above pH 9.5. Administration of enterocin E-760-treated feed significantly (P < 0.05) reduced the colonization of young broiler chicks experimentally challenged and colonized with two strains of C. jejuni by more than 8 log(10) CFU. Enterocin E-760 also significantly (P < 0.05) reduced the colonization of naturally acquired Campylobacter species in market age broiler chickens when administered in treated feed 4 days prior to analysis.

Quantitative analysis of the intestinal bacterial community in one- to three-week-old commercially reared broiler chickens fed conventional or antibiotic-free vegetable-based diets.

AIMS: To explore the effect of drug-free poultry production on the intestinal microflora of broiler chickens, the bacterial community of this environment was quantitatively profiled in both conventionally reared birds and birds reared without antibiotic growth promotants (AGPs) on a vegetable-based diet. METHODS AND RESULTS: Quantitative, real-time PCR with group-specific 16S rDNA primer sets was used to enumerate the abundance of the following chicken gastrointestinal (GI) tract phylogenetic groups: the Clostridium leptum-Faecalibacterium prausnitzii subgroup (Clostridium genus cluster IV), the Clostridium coccoides - Eubacterium rectale subgroup (Clostridium cluster XIVa and XIVb), the Bacteroides group (including Prevotella and Porphyromonas), Bifidobacterium spp., the Enterobacteriaceae, the Lactobacillus group (including the genera Leuconostoc, Pediococcus, Aerococcus and Weissella), the Clostridium perfringens subgroup (Clostridium cluster I), Enterococcus spp., Veillonella spp., Atopobium spp., Campylobacter spp. and the domain Bacteria. A species-specific 5'-nuclease (Taqman) assay was also employed to specifically assess Cl. perfringens abundance. Ten birds were sampled from each of two commercial chicken houses, one in which feed was supplemented with AGPs and exogenous animal protein, and the other vegetable-based and drug-free, at 7, 14 and 21 days of age. The ileal community was dominated by two large populations, the lactobacilli and the Enterobacteriaceae, with those taxa much more numerous in drug-free vegetable-based diet fed birds than those conventionally reared at the 7- and 14-day time periods. The progressive changes in microflora in both the conventional and drug-free caeca were similar to each other, with the Enterobacteriaceae sequences dominating at day 7, but being replaced by obligate anaerobe signature sequences by day 14. Of note was the finding that all the day 14 and day 21 replicate caecal samples from the drug-free house were positive for Campylobacter spp. averaging >10(8) 16S rDNA gene copies per gram wet weight. CONCLUSIONS: Quantitative, real-time PCR indicates that the effects of drug-free rearing on the chicken GI tract microbial community are most pronounced in the ileal region, but AGPs may be important in controlling Campylobacter colonization of the caecum. SIGNIFICANCE AND IMPACT OF THE STUDY: A quantitative taxonomic understanding of the shifting microbial ecology of the broiler chicken gut microbiota is important in the light of AGP withdrawal. AGP withdrawal has occurred in response to concerns over the transfer of antimicrobial-resistant bacteria to humans via the food production chain.

Campylobacter spp. subtype analysis using gel-based repetitive extragenic palindromic-PCR discriminates in parallel fashion to flaA short variable region DNA sequence analysis.

AIMS: The repetitive extragenic palindromic-PCR (rep-PCR) subtyping technique, which targets repetitive extragenic DNA sequences in a PCR, was optimized for Campylobacter spp. These data were then used for comparison with the established genotyping method of flaA short variable region (SVR) DNA sequence analysis as a tool for molecular epidemiology. METHODS AND RESULTS: Uprime Dt, Uprime B1 or Uprime RI primers were utilized to generate gel-based fingerprints from a set of 50 Campylobacter spp. isolates recovered from a variety of epidemiological backgrounds and sources. Analysis and phenogram tree construction, using the unweighted pair group method with arithmetic mean, of the generated fingerprints demonstrated that the Uprime Dt primers were effective in providing reproducible patterns (100% typability, 99% reproducibility) and at placing isolates into epidemiological relevant groups. Genetic stability of the rep-PCR Uprime Dt patterns under nonselective, short-term transfer conditions revealed a Pearson's correlation approaching 99%. These same 50 Campylobacter spp. isolates were analysed by flaA SVR DNA sequence analysis to obtain phylogenetic relationships. CONCLUSIONS: The Uprime Dt primer-generated rep-PCR phenogram was compared with a phenogram generated from flaA SVR DNA sequence analysis of the same isolates. Comparison of the two sets of resulting genomic relationships revealed that both methods segregated isolates into similar groups. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that rep-PCR analysis performed using the Mo Bio Ultra Clean Microbial Genomic DNA Isolation Kit for DNA isolation and the Uprime DT primer set for amplification is a useful and effective tool for accurate differentiation of Campylobacter spp. for subtyping and epidemiological analyses.

Molecular subtyping of poultry-associated type A Clostridium perfringens isolates by repetitive-element PCR.

Clostridium perfringens strains (type A) isolated from an integrated poultry operation were subtyped using repetitive-element PCR with Dt primers. Isolates were obtained from fecal, egg shell, fluff, and carcass rinse samples as part of a previously reported temporally linked epidemiological survey. A total of 48 isolates of C. perfringens were obtained from different stages of the broiler chicken production chain from two separate breeder farms that supplied a single hatchery that in turn provided chicks to a single grow-out farm whose flocks were processed at a single plant. All 48 isolates were typeable (100% typeability) by repetitive-element PCR with Dt primers. This subtyping method was highly reproducible and discriminatory. By repetitive-element PCR with Dt primers, isolates were classified into four major branches with 12 subgroups or clades. The Simpson's index of discrimination was calculated to be 0.96 for groupings of >95% correlation. Toxin gene profiles of the isolates indicated that all of the isolates were C. perfringens alpha-toxin gene positive and 46 of 48 isolates were beta2-toxin gene positive. All strains were negative for beta- and epsilon-toxin genes. Repetitive sequence-based PCR was found to be a technically practical and reproducible means of subtyping C. perfringens libraries from specific epidemiological or production environment settings.

Bifidobacterium-selective isolation and enumeration from chicken caeca by a modified oligosaccharide antibiotic-selective agar medium.

AIMS: To determine the efficacy and selectivity of an acidified, antibiotic-selective, oligosaccharide-containing media for enumerating Bifidobacterium spp. from chicken caeca samples. METHODS AND RESULTS: Transoligosaccharide propionate agar medium (TOS) modified by addition of mupirocin (50 microg ml-1) and glacial acetic acid (1%, v/v), did not inhibit the growth of bifidobacteria compared with the control media yet inhibited the growth of Lactobacillus acidophilus, Lactobacillus gallinarum, Lactobacillus helveticus and Streptococcus gordonii. CONCLUSIONS: Addition of mupirocin (50 microg ml-1) and glacial acetic acid (1%, v/v) to TOS (TOS-AM50), is an effective selective medium for isolation and enumeration of Bifidobacterium spp. from chicken caeca samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of an intestinal bifidobacteria-selective media contributes to the study of probiotics and prebiotics in poultry and potentially other species.

Presence of naturally occurring Campylobacter and Salmonella in the mature and immature ovarian follicles of late-life broiler breeder hens.

Campylobacter and Salmonella are known to cause acute bacterial gastroenteritis in humans. Raw poultry products have been implicated as a significant source of these infections. Five trials were conducted to determine whether Campylobacter and Salmonella spp. exist naturally in the mature and immature ovarian follicles of late-life broiler breeder hens. Broiler breeder hens ranging from 60 to 66 wk of age were obtained from four different commercial breeder operations. For each trial, the hens were removed from the commercial operation and held overnight at the University of Georgia processing facility. The hens were euthanized, defeathered, and aseptically opened. To reduce the possibility of cross-contamination between samples, first the mature and immature ovarian follicles, then the ceca, were aseptically removed. Individual samples were placed in sterile bags, packed on ice, and transported to the laboratory for evaluation. Overall, Campylobacter was found in 7 of 55 immature follicles, 12 of 47 mature follicles, and 41 of 55 ceca. Campylobacter was found in at least one of each sample of mature follicles and in ceca in each of the five trials. Salmonella was found in 0 of 55 immature follicles, 1 of 47 mature follicles, and 8 of 55 ceca. In this study, the recovery rate of Salmonella from late-life broiler breeder hen ovarian follicles was relatively low. However, the recovery rate of Campylobacter from the hen ovarian follicles was reasonably high, suggesting that these breeder hens could be infecting fertile hatching eggs. Determining how Campylobacter contaminated these ovarian follicles and how many chicks could be colonized from this source are the next steps in helping to elucidate a better understanding of this ecology and the control of Campylobacter in poultry production.

Isomaltooligosaccharide increases cecal Bifidobacterium population in young broiler chickens.

A newly developed compound derived by fermentation, isomaltooligosaccharide (IMO), was hypothesized to enrich cecal bifidobacterial populations and reduce colonization levels of Salmonella in the ceca of broiler chickens. Broiler starter diets were prepared with final IMO concentrations of 1% (wt/wt), 2% (wt/wt), and 4% (wt/wt) and a control diet without IMO supplementation. Chickens were divided into 4 groups and challenged with 10(8) cell of Salmonlella enterica ser. Typhimurium with 200 microg/mL nalidixic acid resistant (S. Typhimurium Nalr) after 7 d of placement. The experiment was done in 3 replications. IMO-supplemented diets resulted in significantly higher cecal bifidobacteria compared with the control diet (P < 0.05). However, there was no significant difference in bifidobacteria counts among the treatment groups. Chickens fed diets with 1% IMO had a significant 2-log reduction in the level of inoculated S. Typhimurium Nalr (P < 0.05) present in, the ceca compared with the control group, but no differences were found between the control group and the groups fed 2 or 4% IMO for S. Typhimurium Nalr. No differences in feed consumption, feed conversion, or feed efficiency compared with the control group were observed; however, the result showed a significant reduction in weight for birds fed 1% IMO diet compared with those fed the control diet.

Stationary-phase acid resistance and injury of recent bovine Escherichia coli O157 and non-O157 biotype I Escherichia coli isolatest.

Stationary-phase acid resistance and the induction of acid resistance were assessed for recent bovine carcass isolates of Escherichia coli, including 39 serotype O157 strains and 20 non-O157 strains. When grown to stationary phase in the absence of glucose and without prior acid exposure, there was a range of responses to a pH challenge of 6 h at pH 2.5. However, populations of 53 of the 59 E. coli isolates examined were reduced by less than 2.00 log CFU/ml, and populations of 24 of these isolates were reduced by less than 1.00 log CFU/ml. In contrast, there was little variation in population reductions when the E. coli were grown with glucose and preadapted to acidic conditions. With few exceptions, acid adaptation improved survival to the acid challenge, with 57 of the 59 isolates exhibiting a log reduction of less than 0.50. Differences in acid resistance or the ability to adapt to acidic conditions between E. coli O157:H7 and non-O157 commensal E. coli were not observed. However, we did find that the E. coli O157 were disposed to greater acid injury after the low pH challenge than the non-O157 E. coli, both for cells that were and were not adapted to acidic conditions before the challenge. The enhancement of low pH survival after acid adaptation that was seen among these recent natural isolates of E. coli O157 further supports the idea that the previous environment of this pathogen should be a consideration when designing microbial safety strategies for foods preserved by low pH and acid.

Screening bovine carcass sponge samples for Escherichia coli O157 using a short enrichment coupled with immunomagnetic separation and a polymerase chain reaction-based (BAX) detection stept.

A bovine carcass sponge sample screening protocol for detecting Escherichia coli O157:H7 was composed of a short selective enrichment followed by an immunomagnetic separation (IMS) and target detection using the BAX E. coli O157 polymerase chain reaction assay. This screening protocol was compared to a culture-based method for detection of the organism in carcass sponge samples. Enriched samples were subjected to IMS; the bead suspension was divided and plated on selected media or stored at -20 degrees C, then subjected to BAX analysis. The results showed a high degree of agreement between the plating method and the BAX system. Fifty-two of the 59 culture-positive samples were also positive using the BAX system (88.1% sensitivity). Of the 76 samples that appeared negative for the presence of E. coli O157:H7 by the culture method, 66 were determined as negative using the BAX system (86.8% specificity). Four of the 10 samples found negative by the initial culture method and positive by the BAX method were subsequently found to be culture positive upon reanalysis. Based on these data, the BAX system combined with a short, selective enrichment and IMS may be a rapid, reliable, and simple method to screen for E. coli O157:H7 in carcass sponge samples. Our data indicate that optimization and subsequent testing of this protocol for use as a carcass screening tool are warranted.

[Surgical treatment of varicocele in day hospital. Our experience]. / Il trattamento chirurgico del varicocele in regime di "day hospital". Nostra esperienza.

OBJECTIVE: The authors report their experience about surgical treatment of varicocele in Day Hospital (DH). DESIGN: Report of 10 cases; evaluation of effectiveness of the surgical treatment. SETTING: Operative Unit of General and Thoracic Surgery, Department of Surgical, Anatomical and Oncological Disciplines. Policlinico, University of Palermo. INTERVENTIONS: Surgical procedure according to Ivanissevich in all 8 patients. RESULT: 100% successful. CONCLUSION: Surgery in DH is a valid treatment option in varicocele, by the technical and economic point.

A rapid twofold dilution method for microbial enumeration and resuscitation of uninjured and sublethally injured bacteria.

AIMS: A rapid and simple method for enumerating uninjured and sublethally injured bacterial cells, the twofold dilution method (2FD), was developed and evaluated. METHODS AND RESULTS: Following twofold serial dilution of samples in a 96 well microtiter plate, double strength selective broth or nonselective broth was added to each well. For resuscitation of heat-injured (55 degrees C for 10 min) coliforms, the selective broth was added to the wells after 3 h preresuscitation time in buffered peptone water. The results of the 2FD were compared to plating methods for total and coliform plate counts from mixed cultures and beef carcass surface tissue samples. CONCLUSION: The 2FD method results were not significantly different for uninjured cells (P > 0.05) from those obtained using Petrifilm and standard plating. Correlation of the scatterplot of spread plating and 2FD indicated a high level of agreement between these two methods (R(2)=0.98 for total counts and R(2)=0.96 for coliforms from mixed cultures; R(2)=0.98 for total cell counts and R(2)=0.94 for coliforms from faeces inoculated beef carcasses). SIGNIFICANCE AND IMPACT OF THE STUDY: The twofold dilution method recovered significantly higher numbers of heat-injured coliforms compared to conventional plating methods (P < 0.05).

Improved antimicrobial activity of nisin-incorporated polymer films by formulation change and addition of food grade chelator.

AIMS: The following polymers were developed: polyethylene (PE), a PE and polyethylene oxide (70% PE and 30% PEO; PE + PEO) blend, PE and nisin (PE + nisin), PE, nisin, and EDTA (PE + nisin + EDTA), and PE + PEO with nisin (PE + PEO + nisin). METHODS AND RESULTS: Of the polymers tested, PE and PE + PEO did not exhibit any antimicrobial activity against Brochothrix thermosphacta (BT); however, PE + nisin, PE + nisin + EDTA, and PE + PEO + nisin did. Beef surfaces were experimentally inoculated with 3.50 log10 cfu/cm2 of BT, vacuum packaged with each of the five polymers, and held at 4 degrees C for 21 d. After 3 d at 4 degrees C, BT was reduced > 1.70 log(10) by PE + nisin and > 3.50 log(10) with PE + nisin + EDTA or PE + PEO + nisin. By 21 d at 4 degrees C, BT was reduced to 0.30 log(10) cfu/cm(2) when treated with PE + PEO + nisin. CONCLUSION: It appears that PE + PEO + nisin or PE + nisin + EDTA were more effective for reducing BT, as compared to polymers composed of PE + nisin. SIGNIFICANCE AND IMPACT OF THE STUDY: Nisin-incorporated polymers may control the growth of undesirable bacteria, thereby extending the shelf life and possibly enhancing the microbial safety of meats.