Antimicrobial resistance of Campylobacter coli isolates from swine

Eighty-three swine isolates of Campylobacter coli were tested for mechanisms underlying resistance to nalidixic acid, ciprofloxacin, erythromycin and tetracycline. Four isolates harbored class 1 integrons but none carried class 2 and 3 integrons. Most of the tetracycline-resistant isolates (97%) possessed tet(O). A Thr-86-Ile substitution in GyrA and an A-2230-G mutation in 23S rRNA were the main resistance mechanisms for quinolones and erythromycin, respectively.     

Genotypic and phenotypic heterogeneity in Enterococcus isolates from Batzos, a raw goat milk cheese

This study investigated the genotypic and phenotypic diversity in 34 isolates of enterococci obtained during ripening of Batzos cheese from raw goat milk and characterized phenotypically as Enterococcus durans. RAPD-PCR, plasmid profiling and PFGE were used to study the genetic variability and distinguish closely related isolates. Species recognition by means of RAPD-PCR was in agreement with the phenotypic identification for 29 strains. One strain was characterized as Lactococcus lactis subsp. lactis by RAPD-PCR and four strains were grouped with the Enterococcus faecium reference strain. All strains were vancomycin sensitive, while 10 strains showed beta-haemolytic reaction on human blood and the majority of them (88.9%) showed decarboxylase activity on tyramine. All strains exhibited antagonistic activity against Bacillus cereus, Staphylococcus aureus, Escherichia coli and Listeria monocytogenes and the majority inhibited Enterococcus faecalis. Isolates displayed weak acidifying ability and low proteolytic activities when grown in milk for 24h. However, their caseinolytic activity after growth in milk for seven days was significant with preference for alphas-casein degradation.     

Heat resistance kinetics variation among various isolates of Escherichia coli

This paper reports an investigation of serotype-specific differences in heat resistance kinetics of clinical and food isolates of Escherichia coli. Heat resistance kinetics for 5 serotypes of E. coli at 60 °C were estimated in beef gravy using a submerged coil heating apparatus. The observed survival curves were sigmoidal and there were significant differences (p=0.05) of the survival curves among the serotypes. Consequently, a model was developed that accounted for the sigmoidal shape of the survival curves and the serotype effects. Specifically, variance components for serotypes and replicates within serotypes were estimated using mixed effect nonlinear modeling. If it is assumed that the studied serotypes represent a random sample from a population of E. coli strains or serotypes, then, from the derived estimates, probability intervals of the expected lethality for random selected serotypes can be computed. For example, expected serotype-specific lethalities at 60 °C for 10 min are estimated to range between 5 and 9 log₁₀ with 95% probability. On the other hand, to obtain a 6-log₁₀ lethality, the expected minutes range, with 95% probability, from 6 to 12 min. The results from this study show that serotypes of E. coli display a wide range of heat resistance with nonlinear survival curves.Industrial relevanceThis paper is of high current interest since it deals with the ongoing international debate on log linear vs. non-log linear microbial inactivation curves observed during thermal and non-thermal processing. The data on 5 serotypes of E. coli indicate a clear need for further studies with more strains to fully characterize the heat resistance kinetics for E. coli.     

Prevalence of Campylobacter within a swine slaughter and processing facility

In this work, the occurrence of Campylobacter in a swine slaughter and processing facility was studied. Thirty composite carcass samples, representing 360 swine carcasses, were taken immediately after exsanguination, immediately after polishing, after the final wash, and after overnight chilling at 2 degrees C. Thirty matching composite rectal samples were also taken immediately after exsanguination, and 60 nonmatching individual colon samples were collected from the same lot of swine during evisceration. Also, 72 environmental samples were collected from equipment used in the slaughter operation (42 samples) and the processing operation (30 samples). Campylobacter was isolated by direct plating on Campy-Line agar (CLA) or Campy-Cefex agar (CCA), as well as by Bolton broth enrichment and subsequent inoculation onto CLA or CCA. For all four recovery methods combined, Campylobacter was detected on 33% (10 of 30) of the composite carcasses immediately after exsanguination, 0% (0 of 30) after polishing, 7% (2 of 30) immediately before chilling, and 0% (0 of 30) after overnight chilling. The pathogen was recovered from 100% (30 of 30) of the composite rectal samples and 80% (48 of 60) of the individual colon samples. Campylobacter was detected in 4.8% (2 of 42) and 3.3% (1 of 30) of the slaughter and processing equipment samples, respectively. The recovery rate achieved with direct plating on CLA was significantly higher (P < 0.05) than those achieved with the other three recovery methods. For the 202 isolates recovered from all of the various samples tested, Campylobacter coli was the predominant species (75%) and was followed by Campylobacter spp. (24%) and Campylobacter jejuni (1%). These results indicate that although Campylobacter is highly prevalent in the intestinal tracts of swine arriving at the slaughter facility, this microorganism does not progress through the slaughtering operation and is not detectable on carcasses after overnight chilling.     

Genotypic and phenotypic characteristics of Yersinia spp. isolates from food and man

Yersinia strains collected by the Unit of Enteric Pathogens of the Istituto Superiore di Sanità were analysed for bioserogroup, virulence-related characteristics, DNA plasmid profile and biotype. The isolates were from clinical and food samples, 26 were Y. enterocolitica, four Y. intermedia and four Y. frederiksenii. Seventeen were isolated in Northern Italy, 11 in Southern Italy, five in Central Italy and one from a sample of mussels imported from Greece. Virulence-related characteristics were expressed only by four Y. enterocolitica, bioserogroup 4:O3 associated with the presence of a plasmid weighing 76·8 Kbp. The DNA digested by Spe I and Xba I and analysed by PFGE generated restriction patterns of 12–23 bands, ranging between 400 and 20 Kbp. The similarities of the DNA fingerprintings ofY. enterocolitica strains generated using Spe I and Xba I and calculated by Dice's index were not closely related to bioserogroup.     

Genotypic and phenotypic diversity of Lactococcus lactis isolates from Batzos, a Greek PDO raw goat milk cheese

The genotypic and phenotypic variability of 40 Lactococcus lactis isolates obtained from three cheese-making trials of Batzos cheese made one in each, winter, spring and summer was investigated. RAPD-PCR, plasmid profiling and PFGE were used to study the genetic variability and distinguish closely related isolates. Results showed a high degree of heterogeneity among strains. According to PFGE data, all strains except one were clustered together (at a similarity level of approximately 50%) with the L. lactis subsp. lactis reference strain and eleven groups of isolates consisting of 2-8 strains each were distinguished. Plasmid profiling results revealed that there were eight isolates lacking plasmids and nine having unique plasmids. Twenty-three isolates were allocated into six groups. There was an interesting similarity between the plasmid profiling groups and those formed according to PFGE. Clustering of strains according to RAPD-PCR was in agreement with results obtained by both plasmid profiling and PFGE for the majority of the strains. In addition, results obtained by molecular methods indicate a grouping of most of the strains according to the season of cheese production. All strains inhibited the growth of Escherichia coli O157:H7. Their ability to affect the growth of Yersinia enterocolitica, Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes and Enterococcus faecalis was strain dependent. In 42.5% of the isolates high acidifying ability in milk after 24 h was recorded and these were isolates, mainly, from fresh cheese. The 75% of the isolates from winter cheese exhibited higher Lys- than Leu-aminopeptidase activity while the approximately 67% of the isolates from summer cheese showed higher Leu- than Lys-aminopeptidase activity. Their caseinolytic activity after growth in milk for 24 h was significant with preference for alpha(s)-casein degradation. The majority (90%) of the strains formed methanethiol from methionine and this ability was strain dependent. These results suggest that among the wild lactococcal population from Batzos cheese there are interesting strains appropriate to be used as starters for the dairy industry.     

Lack of a cytolethal distending toxin among Arcobacter isolates from various sources

Arcobacter has been shown to be present in numerous different sources, including poultry, water, and humans exhibiting gastroenteritis. The production of a cytolethal distending toxin (CDT) has been documented in Campylobacter, Helicobacter, and other species. The polymerase chain reaction was used to screen Arcobacter isolates from poultry, cattle, irrigation water, and human diarrhea for the presence of CDT genes. Cell filtrates and sonic extracts were also tested for CDT-like activity on Chinese Hamster Ovary, HeLa, and Intestinal 407 (INT407) cells in culture. No CDT amplimers were observed in any of the Arcobacter isolates investigated. However, toxicity to HeLa and INT407 cells was observed and was subsequently analyzed for cell cycle arrest in the presence of the Arcobacter extracts with flow cytometry. Cells treated with Arcobacter sonic extracts and filtrates exhibited normal cell cycles, suggesting that CDT is not expressed by Arcobacter. Thus, Arcobacter was shown to produce an entity that was toxic to some cells in culture, but this entity was toxic in a manner different from that of Campylobacter CDT.     

A multiplex polymerase chain reaction for the differentiation of Campylobacter jejuni and Campylobacter coli from a swine processing facility and characterization of isolates by pulsed-field gel electrophoresis and antibiotic resistance profiles

A multiplex polymerase chain reaction (PCR) was developed for the detection and speciation of 60 Campylobacter strains isolated from porcine rectal swabs and from different areas in a pork processing plant. The PCR assay was based on primers specific for the cadF gene of pathogenic Campylobacter species, a specific but undefined gene of Campylobacter jejuni, and the ceuE gene of Campylobacter coli. Further characterization of these isolates was established by pulsed-field gel electrophoresis (PFGE) analyses with the restriction endonuclease SmaI. In addition to molecular discrimination, the antibiotic resistance profiles of the isolates were examined by the Kirby Bauer disc diffusion method with 22 antibiotics. Differentiation of isolates by multiplex PCR identified 86.9% (52 of 60) as C. coli and 13.1% (8 of 60) as C. jejuni. Using the Molecular Analyst software, 60 PFGE types were identified. The percentages of relatedness among C. jejuni strains with PFGE ranged from 25 to 86%, while those among C. coli strains ranged from 34 to 99%. Among the 60 PFGE types, each of 12 C. coli isolates showed > or =90% similarity to one other isolate. The antibiotic resistance profiles of all 60 isolates were distinct. Analyses of antibiotic resistance profiles showed that all isolates were resistant to five or more antibiotics. Twenty-five percent (2 of 8) of C. jejuni isolates and 15% (8 of 52) of C. coli isolates were resistant to at least one of the three fluoroquinolones tested, antibiotics that are commonly used in the treatment of human Campylobacter infections. Three percent (2 of 60) of Campylobacter isolates examined were resistant to all three fluoroquinolones. On the basis of the PFGE and antibiotic resistance profiles, each of the 60 isolates was distinct, suggesting that C. jejuni and C. coli strains originating from diverse sources were present in porcine samples and in the pork processing plant.     

Persistent Listeria monocytogenes subtypes isolated from a smoked fish processing facility included both phage susceptible and resistant isolates

Contamination of Ready-To-Eat foods with Listeria monocytogenes can typically be traced back to post-processing contamination from environmental sources; contamination is often linked to subtypes that persist in food associated environments. Although phage-based biocontrol strategies have been proposed for controlling this pathogen, information on the efficacy of phage treatment against diverse L. monocytogenes subtypes from food associated environments is still limited. We identified subtypes that were repeatedly found (“persistent”) in a smoked fish processing facility by using EcoRI ribotyping data for isolates obtained in 1998–2009. PFGE analysis of 141 isolates (9 ribotypes) supported persistence for up to 11 years. Characterization of selected isolates, representing persistent subtypes, against a panel of 28 listeriaphages showed a wide range of likelihood of phage susceptibility, ranging from 4.6% (for 7 ribotype DUP-1043A isolates) to 95.4% (for 7 ribotype DUP-1044A isolates). In challenge studies with 10⁵ and 10⁶ CFU/ml L. monocytogenes, using phage cocktails and a commercial phage product at different phage-host ratios, one isolate (ribotype DUP-1043A) was not affected by any treatment. A reduction in L. monocytogenes counts of up to 4 log units was observed, after 8 h of treatment, in isolates of two ribotypes, but subsequent re-growth occurred. Survivor isolates obtained after 24 h of treatment showed decreased susceptibility to individual phages included in the phage cocktail, suggesting rapid emergence of resistant subtypes.     

Genetic variation of Listeria monocytogenes isolates from domestic and imported foods in Japan

Phylogenetic analyses were carried out on a total of 118 Listeria monocytogenes isolates from foods or food processing environments, and 7 isolates from listeriosis patients in Japan to evaluate the genetic variation in the pathogen in this country. Isolates of serotypes 1/2a, 1/2b and 4b were mainly examined to assess the risk of exposure of humans to L. monocytogenes from foods in Japan. The nucleotide sequences of the part of the iap gene that contains the region encoding the threonine-asparagine repeat units were determined in order to construct phylogenetic trees of the isolates investigated. A phylogram showed high genetic diversity among lineage 2 isolates, while the lineage 1 isolates showed clonal characteristics. The results of the genetic analyses suggested the presence of rare putative lineage 3 isolates and epidemic clone I (ECI) isolates in foods in Japan. The results showed that ECI was also isolated from listeriosis patients. The genetic variation in L. monocytogenes in Japan reported here suggests the necessity of monitoring the pathogen in foods and environments in addition to surveillance of listeriosis patients.     

Prevalence, distribution, and molecular characterization of Salmonella recovered from swine finishing herds and a slaughter facility in Santa Catarina, Brazil

Swine can carry Salmonella strains that may be transmitted to humans by pork products. This investigation determined the distribution and types of Salmonella in 12 swine finishing herds and a slaughter facility in Santa Catarina, Brazil. A total of 1258 samples, consisting of environmental, feed, carcass, lymph node, and fecal material were collected and submitted to bacteriological isolation of Salmonella. From 487 positive samples, 1255 isolates were recovered and confirmed to be Salmonella. The distribution of positive samples was as follows: finishing pen floors 26% (16/61); feed 29% (42/143); feces 44% (52/119); pooled feces 59% (35/59); slaughter holding pens 90% (36/40); lymph nodes 46% (220/478); pre-chilled carcass surfaces 24% (24/98); and post-chilled carcass surfaces 24% (62/260). The most prevalent serovars were Typhimurium, Panama, Senftenberg, Derby, and Mbandaka. By pulsed-field gel electrophoresis, 1071 isolates were subtyped using XbaI, and duplicate isolates were removed. From the remaining 747 isolates, 163 macrorestriction profiles (pulsotypes) were identified. Six pulsotypes were considered very frequent, occurring in 33 isolates or more. The multiple correspondence analyses showed correlations between pulsotypes from shedding pigs (feces), herd environment (pen floors), and subiliac and prescapular lymph nodes and between lairage and carcass surface samples before and after chilling. All sources of Salmonella investigated contributed to the carrier state; however, pre-slaughter contamination at lairage was the variable most strongly associated with carcass contamination. A total of 59 different antimicrobial resistance profiles were observed in 572 Salmonella isolates. From these isolates, 17% (97/572) were susceptible to all 15 antibiotics tested, 83% (475/572) were resistant to at least one, and 43% (246/572) were resistant to four or more antibiotics (multi-resistant). The AmpGenKanTet profile was the most prevalent in carcass isolates and was associated with farm origin.     

Application of standardized quality control procedures to open-path Fourier transform infrared data collected at a concentrated swine production facility

Open-path Fourier transform infrared (OP/FT-IR) spectrometry was used to measure the concentrations of ammonia, methane, and other atmospheric gases at a concentrated swine production facility. A total of 2200 OP/FT-IR spectra were acquired along nine different monitoring paths during an 8-day period between January 11 and 22, 1999. Standardized quality control (QC) procedures were applied to the archived OP/FT-IR spectra to verify that the instrument was set up and operating properly during the field study and to identify outliers in the concentration data. These QC procedures included measuring the random baseline noise, the signal strength, and the relative single-beam intensity in selected wavenumber regions; inspecting the archived spectra for wavenumber shifts, changes in resolution, and evidence of detector saturation; and examining time series plots of the target gas concentrations and the uncertainty values reported by the classical least-squares (CLS) analysis. Application of these QC procedures to the archived spectra identified 252 potential outliers. After a careful review of the original spectra, 41 of the 252 suspected outliers were designated as actual outliers. Of the QC criteria used during this study, the uncertainty values reported by the CLS analysis were the most reliable indicators of actual outliers.     

Genotypic variation of glucosinolates in broccoli (Brassica oleracea var. italica) florets from China

Five main cultivars from China and 143 parent materials grown in a greenhouse were used to investigate the glucosinolates in broccoli florets. Eight aliphatic glucosinolates, four indole glucosinolates, and one aromatic glucosinolate were identified and quantified using high-performance liquid chromatography (HPLC). The results showed that glucoraphanin, glucobrassicin, 4-methoxyglucobrassicin and neoglucobrassicin were present in all samples. However, the predominant type of glucosinolate was different among pure lines. The anti-cancer glucoraphanin concentration ranged from 0.06 to 24.17 μmol/g in pure lines and from 1.57 to 5.95 μmol/g in commercial cultivars. The progoitrin concentration in commercial cultivars varied from 1.77 to 6.07 μmol/g with a mean value of 3.20 μmol/g. Significant variations were observed in the concentration of individual glucosinolates and in each class of glucosinolates among broccoli populations. Dozens of specific lines with altered glucosinolate profiles, as well as ten good candidates for breeding high-chemoprotective glucosinolate cultivars, were obtained according to the putative glucosinolate pathway in broccoli.     

Genotypic and environmental variation in the total phenol and flavanol contents of barley grain

Flavanol and total phenol contents have been determined in samples of barley grown in Britain under a wide range of environmental conditions. An assessment has also been made of the genetic variation in flavanol and total phenol contents among exotic genotypes and current UK varieties. Total phenol content decreased in the first 3 months immediately after harvest but no indication was observed that prolonged storage significantly reduced the levels of flavanols and, after the initial decline, no systematic decreases were found in total phenol content. Significant differences were found between trial sites for both constituents but these could not be related to geographical locations. An examination of inter-varietal variation revealed significant differences among both the current UK varieties (flavanols 0.11–0.17%, total phenols 0.43–0.53%) and 85 diverse genotypes (flavanols 0.08–0.15%, total phenols 0.37–0.54%). The levels of both constituents appeared to be independent of grain size, malting quality, oil content and protein content and no significant differences were found between either two and six-rowed varieties or husked and naked grain types.     

Morphological and physiological variants among isolates of saccharomyces cerevisiae from palmwine and other sources

Using 34 carbon and four nitrogen compounds, and 12 standard yeast identification tests, eight isolates of Saccharomyces cerevisiae Hansen from Raphia Beaur (Palmae) palmwine and other sources were evaluated comparatively for morphological and physiological characteristics. Differences among the isolates were found mainly in their formation of pseudomycelium and blastospores, and in the fermentative utilisation of galactose, maltose, melibiose, raffinose, trehalose and α-methyl-D -galactoside. The wine isolates varied primarily in their assimilatory capabilities for lactic acid and maltose. Observed fermentative and assimilative differences among the wine isolates confirmed the presence of physiological races of S cerevisiae in Raphia palmwine, and suggest that physiological races of S cerevisiae in palmwine may influence the wine's organoleptic and aromatic qualities.     

Determination of MICs of streptomycin for resistant Salmonella isolates in swine and poultry using a micro-broth dilution system

The MICs of streptomycin for Salmonella isolates from swine and poultry were determined by a micro-broth dilution technique. The Salmonella isolates were recovered from the lymph nodes and cecal contents of market-age swine and from the cecal contents of poultry at the time of slaughter and were found by disk diffusion to be resistant to 10 microg of streptomycin. MIC testing was carried out with the Sensititre susceptibility system for streptomycin, which uses a microwell concentration gradient of 16 to 800 microg/ml. Results indicated that >80% of the swine isolates had MICs of < or = 64 microg/ml, while 51% of poultry isolates exhibited MICs of > or = 128 microg/ml. The highest MICs observed in swine and poultry were 256 and 800 microg/ml, respectively. Replicate tests performed on 12 of the isolates chosen at random indicated a 100% correlation between runs. Advantages of this system include easily read results and precoated wells. Disadvantages include the cost and the inability to test concentrations of streptomycin other than those in the wells. We found this micro-broth dilution commercial test kit to provide a relatively quick and easy testing procedure for the determination of streptomycin resistance in Salmonella.     

Cladal relatedness among Aspergillus oryzae isolates and Aspergillus flavus S and L morphotype isolates

Aspergillus flavus is the main etiological agent for aflatoxin contamination of crops. Its close relative, A. oryzae, does not produce aflatoxins and has been widely used to produce fermented foods. We compared the phylogeny of A. oryzae isolates and L- and S-type sclerotial isolates of A. flavus using single nucleotide polymorphisms in the omtA gene in the aflatoxin biosynthesis gene cluster and deletions in and distal to the norB-cypA intergenic region as phylogenetic signals. Aflatoxin-producing ability and sclerotial size also were weighted in the analysis. Like A. flavus, the A. oryzae isolates form a polyphyletic assemblage. A. oryzae isolates in one clade strikingly resemble an A. flavus subgroup of atoxigenic L-type isolates. All toxigenic S-type isolates closely resemble another subgroup of atoxigenic L-type isolates. Because atoxigenic S-type isolates are extremely rare, we hypothesize that loss of aflatoxin production in S-type isolates may occur concomitantly with a change to L-type sclerotia. All toxigenic L-type isolates, unlike A. oryzae, have a 1.0 kb deletion in the norB-cypA region. Although A. oryzae isolates, like S-type, have a 1.5 kb deletion in the norB-cypA region, none were cladally related to S-type A. flavus isolates. Our results show that A. flavus populations are genetically diverse. A. oryzae isolates may descend from certain atoxigenic L-type A. flavus isolates.     

大豆种子贮藏蛋白亚基特异种质的蛋白功能性评价

以4个蛋白亚基变异类型大豆品种(系)制备的分离蛋白和南农大黄豆粗7S蛋白为材料,以亚基正常品种南农大黄豆制备的分离蛋白为对照,对其溶解性、凝胶质构特性、乳化性、乳化稳定性以及DSC特性进行了测定.结果表明,11S组分单个亚基缺失对大豆蛋白的溶解性、乳化性、乳化稳定性和热稳定性影响不显著;11S组分含量显著降低或缺失能提高大豆蛋白的乳化性和乳化稳定性,但降低大豆蛋白变性温度和变性焓;在凝胶质构特性方面,7S组分含量与凝胶弹性呈显著正相关,11S组分含量与凝胶内聚性呈极显著正相关,与凝胶硬度、胶黏性和破裂强度呈显著正相关,与凝胶弹性呈极显著负相关.     

Molecular characterization of Listeria monocytogenes isolated from a poultry further processing facility and from fully cooked product

This study was undertaken to explore environmental sources of Listeria monocytogenes in a commercial chicken further processing facility and to compare the isolates obtained from this facility with others obtained from fully cooked product. In a survey conducted at the processing facility, 40 environmental sites (encompassing two production lines and representing areas in which raw and cooked products are processed) were cultured for L. monocytogenes. The resulting isolates were subjected to molecular subtyping by ribotyping, and these isolates were compared with 25 isolates collected by plant personnel from product contact surfaces and from fully cooked product. Eighty-nine environmental and product isolates were divided into 15 distinct ribogroups. Two ribogroups included isolates from fully cooked product; the members of these two ribogroups were subjected to further analysis by pulsed-field gel electrophoresis, resulting in four clusters. L. monocytogenes isolates from fully cooked product produced on line 1 were found to be indistinguishable from isolates collected from (i) drains on the raw-product side of line 1 and (ii) the floor surface in the cooked-product area of line 1. L. monocytogenes isolates from fully cooked product from line 2 were found to be indistinguishable from isolates collected from (i) the spiral freezer exit conveyor on line 2, (ii) raw product contact surfaces on line 1, and (iii) drains in the cooked-product area of line 1. These data suggest that L. monocytogenes can colonize a poultry further processing facility and eventually be transferred to fully cooked product.