Comparison of broth microdilution and disk diffusion test for antimicrobial resistance testing in Yersinia enterocolitica 4/O:3 strains.

The aim of this study was to compare the results obtained from two methods for the determination of antimicrobial resistance in 110 Yersinia enterocolitica 4/O:3 strains. Ten antimicrobial agents were tested using broth microdilution and disk diffusion. Similar results were determined for six antimicrobials. Very major errors (false-susceptible by disk diffusion test) were detected for ampicillin (at a rate of 1.8%). Major errors (false-resistant by disk diffusion test) were found for streptomycin (0.9%) and sulfamethoxazole (1.8%). Minor errors (intermediate susceptible by disk diffusion and resistant or susceptible by microdilution) were obtained for ampicillin (2.7%) and sulfamethoxazole (13.6%). All Y. enterocolitica were resistant to at least one antimicrobial agent. Resistances to three classes of antimicrobial agents were obtained by 3% of the strains included in the study. A slightly higher frequency of multiresistance was obtained by disk diffusion (3%) compared with broth microdilution (1%). Resistance to streptomycin was found frequently (13%); in contrast, resistance to tetracycline was rare (1%). The disk diffusion test produced unacceptably high rates of very major errors for ampicillin and a high frequency of minor errors for sulfamethoxazole. Susceptibility tests should thus be carried out by the more reliable method of microdilution. Most of the antimicrobials that can be used for therapy were very effective when tested against Y. enterocolitica. In order to identify changes in susceptibilities as early as possible, antimicrobial resistance in Y. enterocolitica should be regularly surveyed.

Salmonella in raw meat and by-products from pork and beef.

After campylobacteriosis, salmonellosis is the second main cause of human bacterial enteritis in Germany. Salmonella is known to colonize the gastrointestinal tract of animals without producing any clinical signs. Therefore, carcasses can become contaminated with Salmonella at the time of slaughter. During an 11-month period, a total of 4,170 raw meat samples and by-products from beef and pork, obtained from seven different slaughterhouses in Southern Germany, were screened by the VIDAS system for Salmonella in this study. Positive results were confirmed by isolation of the pathogen on selective agars. The overall percentage of Salmonella-positive samples was 1.4% by the VIDAS system and 0.7% by culture confirmation. Salmonella was detected in 1.8% of pork samples by the VIDAS system and in 1.1% of samples by culture. In beef samples the presence of Salmonella was verified in 0.6% of samples by the VIDAS system and in 0.1% by culture on selective agars. The highest contamination rates were found in porcine and bovine tongue samples. Salmonella was detected in porcine samples throughout the year, except in samples collected in July, and a slight increase was observed in the colder months. The VIDAS system was shown to be an efficient screening method for the detection of Salmonella, with the advantage of a reduced analysis time.

Wild boars as an important reservoir for foodborne pathogens.

One hundred fifty-three wild boars shot in the canton of Geneva, Switzerland, were studied for the occurrence of foodborne pathogens. Tonsils and fecal samples of the animals were examined using real-time polymerase chain reaction, enzyme-linked fluorescent immunoassay, and cultural methods. The detection rate of Salmonella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis, stx-positive Escherichia coli, and Listeria monocytogenes was 12%, 35%, 20%, 9%, and 17%, respectively, when tonsil samples were studied. Only Y. enterocolitica (5%) and L. monocytogenes (1%) were detected in fecal samples. None of the samples was positive for Campylobacter spp. Females (71%) and young animals (61%) carried more frequently one or more pathogens than males (53%) and older ones (44%). In total, 8 Salmonella spp., 14 Y. enterocolitica, 4 Y. pseudotuberculosis, and 26 L. monocytogenes strains were further characterized. Most of the Salmonella spp. strains were of serotype Salmonella Enteritidis (75%) followed by serotypes Salmonella Stourbridge (13%) and Salmonella Veneziana (13%). L. monocytogenes strains belonged to serotypes 1/2a (42%), 1/2b (19%), and 4b (38%). Serotypes O:3 (36%), O:5,27 (21%), and O:9 (29%) were identified among Y. enterocolitica strains and serotypes O:1 (75%) and O:2 (25%) among Y. pseudotuberculosis strains. This study shows that wild boars are frequent carriers of foodborne pathogens. High wild boar densities and increasing popularity of outdoor ranging of pigs may intensify the risk of transmission of these pathogens to fattening pigs.

High bacterial contamination of pig tonsils at slaughter.

Food-borne zoonoses have a major health impact in industrial countries. Campylobacter spp., Salmonella enterica, Yersinia enterocolitica and Listeria monocytogenes are high-risk food-borne zoonotic hazards in finishing pigs. The objectives of this work were (1) to study the isolation rate of pathogenic Y. enterocolitica, Salmonella spp., Campylobacter spp. and L. monocytogenes in the tonsils and feces and (2) to determine the number of mesophilic aerobic bacteria (MAB) and Escherichia coli in the tonsils of fattening pigs at slaughter. The samples, which were collected from one slaughterhouse on five occasions, originated from 50 pigs and 15 farms. The number of MAB varied from 6.40 to 7.82 log(10) CFU/g and E. coli from 4.38 to 6.53 log(10) CFU/g. Additionally, 31 (62%) of the tonsils were colonized with Y. enterocolitica and 16 (32%) with L. monocytogenes. Campylobacter spp. were more frequently excreted in feces and only 3 (6%) of the pigs carried Campylobacter spp. in the tonsils. No Salmonella spp. were isolated. The pig tonsils were shown to be colonized with a high number of bacteria including E. coli, which is the most important indicator for fecal contamination, and with Y. enterocolitica and L. monocytogenes, which are important food-borne pathogens. This study demonstrates that the tonsils are highly contaminated with micro-organisms and can be a very important source of contamination in the slaughterhouse.

Epidemiological data on pathogenic Yersinia enterocolitica in Southern Germany during 2000-2006.

Yersinia enterocolitica is the most common species causing enteric yersiniosis, which is still the third most frequently reported foodborne gastroenteritis in Europe. Y. enterocolitica generally causes sporadic human infections, and outbreaks are rare. The most important infection source of yersiniosis is believed to be contaminated pork and pork products. Data on the prevalence of pathogenic Y. enterocolitica in animals and foodstuffs are very limited and old; thus, more information on the extent and range of the prevalence of this enteropathogen in nonhuman sources is needed. In this work, prevalence of pathogenic Y. enterocolitica in different sources in Bavaria is presented. Further, the antimicrobial resistance of human and nonhuman strains is reported. The highest isolation rate of pathogenic Y. enterocolitica (67%) was found in tonsils of slaughter pigs. No pathogenic strains were isolated from cattle, sheep, turkey, and horses. ail-Positive Y. enterocolitica was detected in dogs (5%), cats (3%), and rodents (3%) by real-time PCR. Pathogenic Y. enterocolitica was isolated only from raw pork, especially from edible offal (51%). Surprisingly, 38% of game was contaminated with this pathogen when the samples were studied with PCR. Additionally, some raw pork sausages and one poultry sample were PCR positive. All pathogenic Y. enterocolitica isolates from nonhuman sources were belonging to bioserotype 4/O:3. Antimicrobial resistance of 60 human and 140 porcine strains of bioserotype 4/O:3 was tested by the agar disc diffusion method to 15 different antimicrobial agents. All Y. enterocolitica 4/O:3 strains were susceptible to most of the tested antibacterial agents.

Prevalence of pathogenic Yersinia enterocolitica in pigs slaughtered at a Swiss abattoir.

Human yersiniosis is the third most common enteric disease after campylobacteriosis and salmonellosis in many European countries. However, epidemiological data on the prevalence of pathogenic Yersinia enterocolitica in animals and humans is insufficient. Pigs are assumed to be the main reservoir of pathogenic Y. enterocolitica because pig is so far the only animal species from which pathogenic strains have frequently been isolated. This work was conducted to study the frequency of ail-positive Y. enterocolitica in pigs slaughtered at a Swiss abattoir. In total, 212 pig tonsils were screened by real-time PCR and culture methods. The prevalence rate of ail-positive Y. enterocolitica in pigs at slaughter was 88% and 34% with PCR and culture methods, respectively. The 148 ail-positive isolates from the 72 culture-positive tonsils were bio-and serotyped. The most common bioserotype was 4/O:3 found in 96% (69/72) of the culture-positive samples. However, pig was also shown to be a reservoir for ail-positive Y. enterocolitica belonging to bioserotypes 2/O:5,27 and 2/O:9, which were detected in 8% (6/72) and 1% (1/72) of the culture-positive samples, respectively. Using PFGE with NotI, only a limited number of different patterns was found. In all, 6 genotypes were obtained when 86 isolates of bioserotype 4/O:3 from 69 samples were characterised and two genotypes (N1 and N4) dominated. The biotype 4 differs clearly from biotype 2 with PFGE. Antimicrobial resistance testing of 77 ail-positive Y. enterocolitica isolates from 72 samples studied with disc-diffusion revealed that all strains were sensitive to cefotaxime, chloramphenicol, ciprofloxacin and tetracycline, which are antimicrobial agents used for treatment of human disease. The isolates of bioserotype 2/O:5,27 differed from the isolates of bioserotypes 2/O:9 and 4/O:3 in resistance to ampicillin and amoxicillin/clavulanic acid.

Yersiniosis in zoo marmosets (Callitrix jacchuss) caused by Yersinia enterocolitica 4/O:3.

The aim of this research was to describe two fatal cases of Yersinia enterocolitica bioserotype 4/O:3 infection in non-human primates and to characterise the isolates by PCR and PFGE. In July 2004, two marmosets (Callitrix jacchuss) born in captivity in Zagreb Zoo, died following a few days of intermittent diarrhoea in intervals of 2 weeks. The pathomorphological diagnosis of the female (born in 1997) and the male (born in 1995) marmoset, was disseminated miliary necrosis of the liver. Y. enterocolitica 4/O:3 was isolated from both livers showing that monkeys are susceptible to this bioserotype. The ail gene, which is an essential chromosomal virulence factor in pathogenic Y. enterocolitica isolates, was present in the marmoset isolates. Two different PFGE patterns were obtained from the isolates of the male liver with NotI enzyme. One genotype of the male marmoset isolate was indistinguishable from the genotype of the female marmoset isolate when NotI, ApaI and XhoI enzymes were used indicating a common infection source for the marmosets. The genotypes of the marmoset isolates differed only slightly from one human (of seven Croatian isolates) and from one pig isolate (representing a common genotype found among human and porcine isolates in Germany) suggesting that raw pork fed to the marmoset could have been the infection source.

Molecular epidemiology of Yersinia enterocolitica infections.

Yersinia enterocolitica is an important food-borne pathogen that can cause yersiniosis in humans and animals. The epidemiology of Y. enterocolitica infections is complex and remains poorly understood. Most cases of yersiniosis occur sporadically without an apparent source. The main sources of human infection are assumed to be pork and pork products, as pigs are a major reservoir of pathogenic Y. enterocolitica. However, no clear evidence shows that such a transmission route exists. Using PCR, the detection rate of pathogenic Y. enterocolitica in raw pork products is high, which reinforces the assumption that these products are a transmission link between pigs and humans. Several different DNA-based methods have been used to characterize Y. enterocolitica strains. However, the high genetic similarity between strains and the predominating genotypes within the bio- and serotype have limited the benefit of these methods in epidemiological studies. Similar DNA patterns have been obtained among human and pig strains of pathogenic Y. enterocolitica, corroborating the view that pigs are an important source of human yersiniosis. Indistinguishable genotypes have also been found between human strains and dog, cat, sheep and wild rodent strains, indicating that these animals are other possible infection sources for humans.

Sporadic human Yersinia enterocolitica infections caused by bioserotype 4/O : 3 originate mainly from pigs.

Yersinia enterocolitica 4/O : 3 is the most frequent cause of sporadic human yersiniosis in Finland and Germany. To investigate the possible link between pigs and humans, 282 human and 534 porcine strains from Finland and Germany were characterized with PFGE using NotI, ApaI and XhoI enzymes. Most of the human strains (>80 %) were indistinguishable from the porcine strains in both countries and most of the genotypes (178/182) were different in Finland and Germany. The indistinguishable genotypes among human and porcine strains together with different genotypes in Finland and Germany indicate that pigs are an important source of sporadic yersiniosis in both countries.

Substituted indanylacetic acids as PPAR-alpha-gamma activators.

A series of oxazole-substituted indanylacetic acids were prepared which show a spectrum of activity as ligands for PPAR nuclear receptor subtypes.

Intramolecular Pauson-Khand reactions of methylenecyclopropane and bicyclopropylidene derivatives as an approach to spiro(cyclopropanebicyclo[n.3.0]alkenones).

The trimethylsilyl-protected enynes 9a-c and 14a,b with alkynyl substituents on the three-membered ring or on the double bond of a methylenecyclopropane or a bicyclopropylidene moiety were prepared in two steps from the alcohols 6a-c and 12a,b, respectively, by conversion to the iodides and their coupling with lithium (trimethylsilyl)acetylide (8) in 38-73% overall yields. The bicyclopropylidene derivative 9d was synthesized in 49% yield directly from bicyclopropylidene (3) by lithiation followed by coupling with (5-iodopent-1-ynyl)trimethylsilane (11). Enynes 9b-d were protiodesilylated by treatment with K2CO3 in methanol to give the corresponding unprotected enynes 10b-d in 53, 74 and 94% yield, respectively. Enynes 17a-c with a carbonyl group adjacent to the acetylenic moiety were synthesized from oxo derivatives 15a-c by Wittig olefination followed by coupling with 8 in 47, 18 and 12% overall yield, respectively. Pauson-Khand reactions of the methylenecyclopropane derivatives with a substituent on the ring (9a,b and 10a) as well as on the double bond (14a,b and their in situ prepared protiodesilylated analogues) proceeded smoothly by stirring of the corresponding enyne with [Co2(CO)8] in dichloromethane at ambient temperature followed by treatment of the formed complexes with trimethylamine N-oxide under an oxygen atmosphere at -78 degrees C to give tricyclic or spirocyclopropanated bicyclic enones 18a,b, 19a, 20a,b, 21a,b in good yields. Alkynylbicyclopropylidene derivatives 9c,d and 10c,d formed the corresponding cobalt complexes at -78 to -20 degrees C. Treatment of the latter with N-methylmorpholine N-oxide under an argon atmosphere at -20 degrees C gave the spirocyclopropanated tricyclic enones 18c, 19c and 18d in 31-45% yields. The structure of 19c was proved by X-ray crystal structure analysis. The cyclization of enynones 17a-c in MeCN at 80 degrees C gave the spirocyclopropanated bicyclic diketones 22a-c in 38-65% yields. Intramolecular PKRs of the enynes 25a,d with a chiral auxiliary adjacent to the triple bond gave the corresponding products 26a,d in 70 and 79% yield, respectively, as 5:1 and 8:1 mixtures of diastereomers, respectively. Addition of lithium dimethylcuprate or higher order cuprates to the double bond of the former furnished bridgehead-substituted bicyclo[3.3.0]octanones 27a-c in 57-86% yields. Protiodesilylation of 27a followed by acetal cleavage gave the enantiomerically pure spirocyclopropanated bicyclo[3.3.0]octanedione (1R,5R)- 29a with [alpha]D(20)=-148 (c=1.0 in CHCl3) in 55% overall yield.

Versatile Synthesis of Enantiomerically Pure 2-Alkoxy-1-Ethynylcyclopropanes and their Application in the Synthesis of Enantiomerically Pure Bicyclo-[3.3.0]oct-1-en-3-ones.

A variety of chiral, nonracemic 2-alkoxy-1-alkynylcyclopropanes 7 were synthesized in good to very good yields from enantiomerically pure glycidol derivatives (glycidol tosylate, epichlorohydrin) by boron trifluoride promoted addition of lithium trimethylsilylacetylide followed by protection of the secondary hydroxyl group and finally a diastereoselective γ-elimination. The 2-ethoxy derivative (S,R)-7 b was deprotonated with n-butyllithium, and the resulting 1-lithio-2-ethoxy derivative (S,R)-20 functionalized by treatment with oxygen followed by tosyl chloride. Protodesilylation and catalytic hydrogenation smoothly furnished 1-ethenylcyclopropyl sulfonates, which underwent a clean Pd0 -catalyzed SN 2'-type substitution with dimethyl propargylsodiummalonate to give the (E)-configurated enyne (R,E)-26 with a methylenecyclopropane end group. A diastereoselective Pauson-Khand reaction completed the sequence to give the enantiomerically pure spirocyclopropaneannelated bicyclo[3.3.0]octane derivative 31.