Identification of tet(M) in two Lactococcus lactis strains isolated from a Spanish traditional starter-free cheese made of raw milk and conjugative transfer of tetracycline resistance to lactococci and enterococci

Specific PCR and sequencing showed that a tet(M) gene was present in two tetracycline-resistant Lactococcus lactis strains isolated from a raw milk, starter-free cheese. Hybridisation experiments using as a probe an internal segment of the gene obtained by PCR associated tet(M) with plasmids of around the same size (30 kbp) in both strains. Molecular analysis of the tetracycline resistance loci, including the upstream and downstream regions of the genes, showed them to be identical to one other and to the tet(M) encoded by the conjugative transposon Tn916. Amplification of Tn916-derived segments suggested the transposon was complete in the two L. lactis strains. Further, curing of the tetracycline resistance was accompanied by a reduction in size of the plasmids comparable to that expected for Tn916. Tetracycline resistance could be transferred by conjugation to plasmid-free Lactococcus and Enterococcus strains. However, no plasmid DNA was detected among the transconjugants while both tet(M) and transposon-related sequences were amplified by PCR. This suggested that only the transposon was mobilized.     

Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins

The inhibitory activity of 122 out of 426 Enterococcus strains of geographically widespread origin and from different sources (food and feed, animal isolates, clinical and nonclinical human isolates) was tested against a wide range of indicator bacteria. Seventy-two strains, mainly belonging to the species Enterococcus faecium and Enterococcus faecalis were bacteriocinogenic. A remarkable variation of inhibitory spectra occurred among the strains tested, including inhibition of, for instance, only closely related enterococci, other lactic acid bacteria (LAB), food spoilage and pathogenic bacteria. No correlation could be found between the origin of the strains and the type of inhibitory spectrum, although a clustering of human isolates from both fecal and clinical origin was observed in the group of strains inhibiting lactic acid bacteria, Listeria, and either Staphylococcus or Clostridium. No relationship could be established between the presence of enterocin structural genes and the origin of the strain either, and hence no correlation seemed to exist between the presence of known enterocin genes and the activity spectra of these enterococci. The structural gene of enterocin A was widely distributed among E. faecium strains, whereas that of enterocin B only occurred in the presence of enterocin A. The vancomycin resistance phenotype as well as the presence of vancomycin resistance genes was also investigated. The vanA gene only occurred among E. faecium strains. The incidence of beta-hemolysis was not restricted to E. faecalis strains, but among the E. faecium strains the structural genes of cytolysin were not detected. beta-Hemolysis occurred in strains both from food and nonfood origin. It has been concluded that bacteriocin-producing E. faecium strains lacking hemolytic activity and not carrying cytolysin nor vancomycin resistance genes may be useful as starter cultures, cocultures, or probiotics.     

Assessment of tetracycline and erythromycin resistance transfer during sausage fermentation by culture-dependent and -independent methods

The food chain is considered one of the main routes of antibiotic resistance diffusion between animal and human population. The resistance to antimicrobial agents among enterococci could be related to the efficient exchange of transferable genetic elements. In this study a sausage model was used to evaluate the persistence of antibiotic resistant enterococci during meat fermentation and to assess horizontal gene transfer among bacteria involved in meat fermentation. Enterococcus faecalis OG1rf harbouring either pCF10 or pAMβ1 plasmid was used as donor strain. The analysis of population dynamics during fermentation confirmed that the human isolate E. faecalis OG1rf was able to colonize the meat ecosystem with similar growth kinetics to that of food origin enterococci and to transfer the mobile genetic elements coding for tetracycline and erythromycin resistances. Transconjugant strains were detected after only two days of fermentation and increased their numbers during ripening even in the absence of selective antibiotic pressure. By means of culture-dependent and -independent molecular techniques, transconjugant strains carrying both tetracycline and erythromycin resistance genes were identified in enterococci, pediococci, lactobacilli and staphylococci groups. Our results suggest that the sausage model provides a suitable environment for horizontal transfer of conjugative plasmids and antibiotic resistance genes among food microbiota.     

不同来源屎肠球菌的全基因组序列分析

目的 了解不同分离来源的屎肠球菌标准菌株的全基因组特征。     

Enterococcus populations in Pecorino Abruzzese cheese: biodiversity and safety aspects

The presence of enterococci in Pecorino Abruzzese cheese during ripening was evaluated. Counts were high, especially in fully ripened summer batches. Seventy strains were isolated and identified based on phenotypical and genotypical features as Enterococcus faecium (48.5%), Enterococcus faecalis (40%), and Enterococcus durans (11.5%), with the first species predominant in spring batches and the second predominant in summer batches. High biodiversity was revealed by random amplification of polymorphic DNA and a PCR assay, suggesting the presence of autochthonous strains. E. faecium isolates were the most resistant to the tested antibiotics, especially to erythromycin, chloramphenicol, and penicillin, but all strains were susceptible to vancomycin, as confirmed by the absence of vanA and vanB genes. The presence of some virulence determinants was investigated, revealing the diffusion of aggregation substance (asal) and gelatinase (gelE) genes in 37.5% of E. faecalis strains. However, none of the isolates produced gelatinase in vitro, suggesting the presence of silent genes. The virulence genes were absent in E. durans. Among E. faecium strains, only Lab 41/1 possessed gelE and asal, whose presence previously has been reported only in E. faecalis. Decarboxylating activity was revealed for phenylalanine (27% of the strains) and tyrosine (96%) but not histidine. The presence of a tyrosine decarboxylase-encoding gene was observed for all strains. A comparison of these results with those of previous studies of clinical and food isolates indicates that enterococci from Pecorino Abruzzese cheese have low pathogenic potential.     

Comparative analysis of genetic diversity and incidence of virulence factors and antibiotic resistance among enterococcal populations from raw fruit and vegetable foods, water and soil, and clinical samples

A comparative study was carried out among enterococci isolated from fruits and vegetable foods, water and soil, and clinical samples. Results indicate strong differences in the numbers of enterococcal species found in different environments as well as their abundance. While Enterococcus faecalis was clearly the predominant species in clinical samples, Enterococcus faecium predominated in vegetables, and it slightly outnumbered E. faecalis in water samples. Other species (Enterococcus hirae, Enterococcus mundtii, Enterococcus durans, Enterococcus gallinarum and Enterococcus casseliflavus) were found more frequently in vegetables, water, and specially in soil. Isolates from vegetable foods showed a lower incidence of antibiotic resistance compared to clinical isolates for most antimicrobials tested, especially erythromycin, tetracycline, chloramphenicol, ciprofloxacin, levofloxacin, gentamicin and streptomycin for E. faecalis, and quinupristin/dalfopristin, ampicillin, penicillin, ciprofloxacin, levofloxacin, rifampicin, choramphenicol, gentamicin and nitrofurantoin for E. faecium. E. faecium isolates from vegetable foods and water showed an average lower number of antibiotic resistance traits (2.95 and 3.09 traits for vegetable and water isolates, respectively) compared to clinical samples (7.5 traits). Multi-resistant strains were also frequent among clinical E. faecalis isolates (5.46 traits on average). None of E. faecalis or E. faecium isolates from vegetable foods, water and soil showed beta-haemolytic activity, while 25.64% of clinical E. faecalis did. A 51.28% of E. faecalis clinical isolates tested positive for the cylA, cylB, cylM set of genes, while some or all of these genes were missing in the rest of isolates. In clinical E. faecalis and E. faecium isolates, the genetic determinants for the enterococcal surface protein gene (esp), the collagen adhesin gene (ace) and the sex pheromone gene ccf (as well as cob in E. faecalis) showed a clearly higher incidence compared to isolates from other sources. E. faecalis isolates from vegetable foods and water had much lower average numbers of virulence genetic determinants per strain (4.23 and 4.0, respectively) compared to clinical isolates (8.71). Similarly, among E. faecium the lowest average number of traits per strain occurred in vegetable food isolates (1.72) followed by water (3.9) and clinical isolates (4.73). Length heterogeneity (LH)-PCR typing with espF-aceF-ccfF and espF-ccfF primers revealed genomic groups that clearly differentiated clinical isolates from those of vegetable foods, water and soil (except for two clinical isolates). The large differences found in the incidence of antibiotic resistance and virulence factors and in the genetic fingerprints determined by LH-PCR suggest a clear separation of hospital-adapted populations of enterococci from those found in open environments.     

Possible gene interchange between plasmid and chromosome in yeast

Genomic DNAs isolated from 420 yeast strains stocked in the Department of Fermentation Technology, Hiroshima University (HUT) were screened for the presence of a plasmid sequence both as plasmid or in the chromosome. Five DNA samples gave rise to a positive hybridization signal when 32P-labelled Zygosaccharomyces plasmid pSR1 was used as a probe. Two among these contain hybridizing sequences as plasmids while the other three apparently were chromosomal. Two chromosomal DNA segments of HUT 7195 (Zygosaccharomyces spp.) which hybridized with pSR1 probe were cloned and sequenced. Both DNAs hybridized with a plasmid sequence covering the P gene of pSR1. One of the two segments contains a large open reading frame which can encode 410 amino acid residues. The deduced amino acid sequence is closely related with that of the P gene of pSR1. The present finding suggests that there was an interchange(s) of a gene between yeast plasmid(s) and chromosomes.     

Technological characterization of the natural lactic acid bacteria of artisanal Turkish White Pickled cheese

The aim of this study was to characterize the lactic acid bacteria (LAB) isolated from White Pickled cheeses produced with traditional methods; and to improve the quality of cheesemaking with a selection of bacterial cultures from artisanal White cheeses. LAB were isolated and identified from 30 White Pickled cheese samples collected from various cities in Turkey. Also, the numbers of several microbial groups (total aerobic mesophilic bacteria, LAB, enterococci, coliforms, moulds and yeasts) of cheese samples were enumerated. Lactobacilli, lactococci and enterococci were the most abundant microbial groups. The numbers of Enterococcus and Lactobacillus isolates were higher than those of the other LAB. Enterococcus faecalis (24.43%), Enterococcus faecium (17.61%) and Lactobacillus fermentum (19.88%) isolates were the most frequently isolated species. Lactococcus strains showed the highest acidifying activity, followed by Enterococcus and Lactobacillus strains. Proteolytic activity of Enterococcus faecalis strains was higher than that of the other enterococci species, except Enterococcus avium strains. Within lactobacilli strains, the highest mean proteolytic activity was that of Lactobacillus bifermentans, Lactobacillus brevis and Lactobacillus casei strains.     

食品中副溶血性弧菌toxR和tdh基因双色荧光PCR检测方法的建立

目的 建立对副溶血性弧菌（Vibrio parahaemolyticus）特异性检测toxR（跨膜转录激活蛋白）基因和tdh（热稳定性直接溶血素）毒力基因的Taqman探针双色荧光PCR检测方法。方法 根据副溶血性弧菌toxR基因和tdh基因,分别设计引物和探针,建立Taqman探针双色荧光PCR扩增体系,进行特异性、灵敏度试验;对副溶血性弧菌分离菌株实施检测,了解其tdh基因和tdh基因分布情况。结果 结果表明,副溶血性弧菌标准菌株和3株从食物中毒患者中分离获得的分离株均出现toxR基因和tdh扩增曲线,而溶藻弧菌、单增李斯特菌等31株弧菌属其他菌株和肠杆菌科的菌株未见扩增曲线。从食品中分离的37株副溶血性弧菌分离株均未携带tdh毒力基因。副溶血性弧菌检测灵敏度可达到3.6×102 cfu/mL。结论 该方法可用于同时检测食品中副溶血性弧菌的特异性和毒力基因。     

Resistance to gentamicin and vancomycin in enterococcal strains isolated from retail broiler chickens in Japan

A total of 137 Enterococcus strains isolated from chicken meat were subjected to antimicrobial susceptibility tests. Strains with the vanCl gene were isolated from seven of nine samples of chicken meat processed in Japan and from all chickens from China and Brazil between July 2001 and April 2002. The pulsed-field gel electrophoresis (PFGE) patterns of the isolates were distinguishable from each other, suggesting that VanCl-type vancomycin-resistant Enterococcus is preferentially colonized in broiler chickens in these countries. The incidence of high-level gentamicin resistant (HLGR) enterococci that harbored the aac(6')-le-aph(2")-la or aph(2')-Id gene varied among the countries from which the chickens originated (Japan, 2 of 65; China, 11 of 43; Brazil, 6 of 29). Moreover, the PFGE patterns of the HLGR strains were distinguishable from each other, except for two strains obtained from chickens from Brazil. The results suggest that HLGR Enterococcus is highly prevalent in broiler chickens.     

干酪中肠球菌种群的遗传差异及万古霉素抗性基因分析

通过种、属引物特异性扩增、重复序列PCR（rep-PCR）技术和万古霉素抗性基因检测对新疆北疆地区干酪样品中球菌的遗传结构差异进行分析。结果表明,15份样品共分离52株肠球菌,包括31株耐久肠球菌(Enterococcus durans)、18株粪肠球菌(Enterococcus faecalis)和3株屎肠球菌(Enterococcus faecium)。依据rep-PCR遗传指纹带谱分析,52株菌可以聚类成7个群,其中4个由E. durans构成。24株肠球菌检测到了万古霉素抗性基因,17株E. durans为VanC2/C3型,4株E. faecalis为VanC1型,2株E. faecium为VanB型,只有1株E. faecium为VanA型。新疆北疆地区干酪中肠球菌种群分布较为广泛,地域之间优势种群和基因型不同,同种肠球菌菌株之间存在广泛的遗传差异。     

Antimicrobial and safety aspects, and biotechnological potential of bacteriocinogenic enterococci isolated from mallard ducks (Anas platyrhynchos)

Samples from the intestinal content and carcasses of mallard ducks (Anas platyrhynchos) were evaluated for enterococci with antimicrobial activity, presence of genes coding bacteriocins and their expression, and potential virulence factors. Enterococcus faecalis comprised the largest enterococcal species with antagonistic activity followed by E. faecium, E. hirae, Enterococcus spp., and the non-enterococci. Although all E. faecalis isolates manifested a potent direct antimicrobial activity, no activity was detected in supernatants of most producer cultures. However, all E. faecium isolates showed antimicrobial activity in their supernatants and encoded bacteriocins, although the occurrence in the isolates of several enterocin genes did not always correlate with a higher antagonistic activity in supernatants. The efaAfm determinant was the only virulence gene detected in E. faecium, while E. faecalis showed a larger number of virulence determinants, and E. hirae did not carry any of the virulence genes examined. The rapid identification of genes coding described bacteriocins permits recognition of isolates that are potentially producers of novel bacteriocins. Purification of the antimicrobial activity of E. hirae DCH5 and Lactococcus garvieae DCC43 revealed unique chromatographic fragments after MALDI-TOF mass spectrometry analysis, suggesting the antagonistic peptides were purified to homogeneity. Bacteriocinogenic E. faecium and E. hirae isolates may be considered hygienic for production of bacteriocins, and potentially safe due to their low incidence of potential virulence genes and susceptibility to most clinically relevant antibiotics. However, the presence among the enterococci of E. faecalis strains with a potent antagonistic activity and multiple virulence factors, raises concerns regarding their potential pathogenicity to consumers.     

Technological performance of several Lactococcus and Enterococcus strains of dairy origin in milk

Thirty-nine strains (29 Lactococcus strains and 10 Enterococcus strains) isolated from five different artisanal cheeses were subjected to technological characterization. Several strains of lactococci and enterococci produced lactic acid at a rate and final concentration suitable for large-scale cheesemaking. However, extensive phenotypic differences between strains were encountered. Proteolytic activity correlated quite well with acidification for all strains, with the more proteolytic strains being the best acidifiers. The strains were also assayed for the production of organic acids and volatile components in milk. With few exceptions, enterococcus isolates produced more formic acid and acetic acid than did lactococcus isolates. The volatile-compound profiles obtained were rather simple. The main volatile component produced by most strains was ethanol. Since the inclusion of enterococcus strains in food systems is controversial, tests were also performed to detect recognized determinants of virulence (namely, aggregation, gelatinase and hemolysin production, and antibiotic resistance). Aggregation in both liquid and solid media was observed only for two Enterococcus durans isolates. None of the strains studied produced gelatinase under the conditions of the assay. Beta-hemolysin activity was clearly detected in two Enterococcus faecalis strains, which also produced the biogenic amine tyramine from tyrosine in a laboratory medium. In general, the enterococcus strains were more resistant to the antibiotics assayed than were the lactococcus strains. Both the minimum inhibitory concentration (MIC) modes and the highest MIC values were consistently higher for the enterococci. Nevertheless, particular strains of lactococci were resistant to antibiotics such as bacitracin, cephalothin, clindamycin, streptomycin, and tetracycline.     

Vancomycin-resistant enterococci from animal sources in Korea

Enterococci for which the minimum inhibitory concentration (MIC) of vancomycin was >/=8 mg/l were isolated from meat, feces, and raw milk samples collected in Korea from March to November 2003. Among the 243 vancomycin-resistant enterococci (VRE) that were identified the vanA vancomycin resistance gene was carried by 51 Enterococcus faecium and one Enterococcus sp., vanC1 was carried by 151 Enterococcus gallinarum, vanC2 was carried by 39 Enterococcus casseliflavus, and one Enterococcus sp. carried no van genes. Of the isolated enterococci carrying vanA, 4% were found to be highly resistant to gentamicin and 11% were resistant to ampicillin. Further genotyping of the E. faecium isolates carrying vanA using pulsed-field gel electrophoresis (PFGE) revealed extensive heterogeneity. The vancomycin resistance transferability test revealed that only two of the 52 enterococci carrying the vanA gene were able to transfer vancomycin resistance to other enterococci. The VRE were recovered from various animal sources with a particularly high prevalence of E. faecium carrying the vanA gene being found in poultry meat.     

Duplication of the beta-galactosidase gene in some Lactobacillus plantarum strains.

Curing of a plasmid that encoded a beta-galactosidase gene (beta-gal) from the Lactobacillus plantarum strain of dairy origin LL441 was not accompanied by complete loss of the lactose utilization phenotype. DNA-DNA hybridization, using an internal fragment of the beta-gal gene as a probe, revealed a second determinant located on the chromosome of the cured derivatives. The chromosomal copy was present in all of a series of beta-Gal+ L. plantarum and Lactobacillus pentosus strains from different origins. In addition, four other L. plantarum strains harboured plasmid encoded beta-gal genes as well. Since both sequences cross-hybridized and present a similar genetic organization, it is postulated that the plasmid copy was generated through gene duplication and, probably, selected by growth of the strains in lactose rich environments.     

Gene transfer of vancomycin and tetracycline resistances among Enterococcus faecalis during cheese and sausage fermentations

This study assessed the frequency of transfer of two mobile genetic elements coding for virulence determinants and antibiotic resistance factors, into food associated enterococci during fermentation processes. First, the transfer of the pheromone-inducible pCF10 plasmid, carrying tetracycline resistance and aggregation substance (AS) as virulence factor, between clinical and food strains of Enterococcus faecalis, was investigated in models of cheese and fermented sausage. The experiments demonstrated that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from E. faecalis OG1rf cells to food strain E. faecalis BF3098c and that the plasmid subsequently persisted in these environments. Very high frequency of transfer was observed in sausage (10(-3)/recipient) if compared to cheese (10(-6)) and plate mating (10(-4)). Transconjugants were subsequently verified by PCR. The second transmissible element was the plasmid harbouring the vancomycin resistance (VanA phenotype) from E. faecalis A256. The transfer of this antibiotic resistance to a food strain of E. faecalis was studied in vitro and in food models. Although the transfer of vancomycin resistance was achieved in all the environments, the highest conjugation frequencies were observed during the ripening of fermented sausages, reaching 10(-3) transconjugants/recipient cell. PCR confirmed the transfer of the VanA genotype into a food associated Enterococcus strain. This study showed that even in the absence of selective pressure, mobile genetic elements carrying antibiotic resistance and virulence determinants can be transferred at high frequency to food associated enterococci during cheese and sausage fermentation.     

Antibiotic susceptibility patterns of Enterococcus strains isolated from Turkish Tulum cheese

The aim of this study was to detect the plasmid profiles, haemolytic activity and antibiotic susceptibility patterns of 47 Enterococcus strains isolated from Turkish Tulum cheese. Enterococcus strains were found to comprise 1–9 plasmids with molecular weight from 2.0 to 47.6 kb. None of the Enterococcus strains displayed haemolytic activity. The Enterococcus strains were found mostly resistant to tetracycline (30 μg) followed by high‐level streptomycin (300 μg). The Enterococcus faecalis strains were found to be highly resistant to antibiotics than Enterococcus faecium and Enterococcus durans strains. In total, 4.3% of the strains exhibited multiple antibiotic resistance patterns.     

Enterococci from Tolminc cheese: population structure, antibiotic susceptibility and incidence of virulence determinants

Microbiological analysis of ripened artisanal Tolminc cheese revealed the presence of an enterococcal population in numbers of up to 10(6) per g. All colonies, isolated from the citrate azide tween carbonate (CATC) enterococcal selective medium were Gram positive and coccal-shaped and were analysed with PhenePlate FS system. This system discriminated 10 PhP clusters among the 90 enterococcal isolates. From each cluster the most representative isolate for that particular type was selected for further study. The 10 representative enterococci were catalase negative and grew in the presence of NaCl (2%, 4% and 6.5%) and bile salts (0.06%). Genus specific primers confirmed all 10 enterococcal representatives as Enterococcus members, while species specific primers determined them further as strains of Enterococcus faecalis species. PCR for vanA and vanB genes detection, respectively, amplified no PCR products. The absence of van genes was confirmed with both disc and E-test, as isolates were susceptible to vancomycin according to the National Committee for Clinical Laboratory Standards (NCCLS). The results of disc tests with other antimicrobial agents (ampicillin, vancomycin, kanamycin, penicillin, erythromycin, neomycin, chloramphenicol, clindamycin, rifampin) did not differ much among the tested enterococci: they were all very resistant to clindamycin only. The incidence of enterococcus virulence determinants was as expected: all of the 10 E. faecalis strains tested possessed multiple determinants (between 7 and 11).     

Species distribution and antibiotic resistance patterns of enterococci isolated from food of animal origin in Germany

Presently, enterococci take the third place of bacterial pathogens associated with nosocomial infections, after staphylococci and Escherichia coli. Especially, the resistances of enterococci to several available antibiotics are threatening. We attempted to determine which species of enterococci could be found in food of animal origin and their significance according to their antibiotic resistances for human beings. From November 2000 to May 2002 we investigated 155 samples of food of animal origin bought in retail outlets in Germany: 27 samples of sausages, 19 of ham, 83 of minced meat, 26 of cheese. From these food samples we isolated 416 enterococcal strains. The most frequent species was Enterococcus faecalis (299 strains); furthermore, we found Enterococcus faecium (54 strains), Enterococcus durans together with Enterococcus hirae (24 strains), Enterococcus casseliflavus (22 strains), Enterococcus avium (9 strains) and Enterococcus gallinarum (8 strains). We focused on the resistance patterns of 118 selected E. faecium and E. faecalis strains to 13 antimicrobial active agents (ampicillin, amoxicillin/clavulanic acid, avilamycin, chloramphenicol, enrofloxacin, erythromycin, flavomycin, gentamicin, penicillin, quinupristin/dalfopristin, teicoplanin, tetracycline and vancomycin). From the clinical point of view, the situation of antibiotic resistance to the examined antimicrobial agents seemed to be favourable. The investigated strains were sensitive to ampicillin and amoxicillin/clavulanic acid. These antibiotics are, in combination with an aminoglycoside, for example gentamicin, agents of choice for the treatment of enterococcal infections in human medicine. Only one E. faecium strain was resistant to penicillin, while all strains were sensitive to the glycopeptide antibiotics, vancomycin and teicoplanin. Resistances found against the antibiotics, tetracycline, quinupristin/dalfopristin and erythromycin, are causes for concern.