Isolation and molecular characterization of antibiotic-resistant lactic acid bacteria from poultry and swine meat products

The transfer via the food chain from animals to humans of microbes that are resistant to antimicrobial agents is of increasing concern. To determine the contributions of nonpathogenic microflora to the occurrence and spread of antibiotic resistance (AR) genes in the food chain, 123 lactic acid bacteria were isolated from 29 samples of raw and processed pork and chicken meat products that had previously tested positive for one or more AR genes that encode clinically relevant ARs: tet(M), tet(O), tet(K), erm(A), erm(B), erm(C), aac (6')-Ie aph (2")-Ia, mecA, and blaZ. All of the isolates were initially tested for their AR gene profiles by PCR. The 59 isolates carrying a tet, erm, or blaZ gene were taken through molecular identification, analyzed by determination of the MIC, and subjected to genetic fingerprinting. Lactococcus garvieae was the predominant species (28 isolates), followed by Lactobacillus plantarum (11 isolates) and L. salivarius (6 isolates), whereas Lactococcus lactis subsp. lactis, Lactobacillus johnsonii, L. reuteri, L. crispatus, and L. brevis were identified at lower frequencies. The tet(M) and erm(B) genes were the most frequently detected. Assessment of multiple resistances in 18 tet positive (tet+) isolates revealed that tet(M) plus erm(B) and tet(K) plus erm(B) were the most frequent AR gene patterns. Partial sequencing of the tet(M) open reading frame of three selected strains showed high sequence similarities (> 99%) with tet(M) genes previously found in human pathogens (Listeria monocytogenes and Neisseria meningitidis). Southern hybridization with plasmid profiles revealed these strains contained tet(M)-carrying plasmids.     

Multiple-antibiotic resistance of Enterococcus faecalis and Enterococcus faecium from cecal contents in broiler chicken and turkey flocks slaughtered in Canada and plasmid colocalization of tetO and ermB genes

This study was conducted to characterize the antimicrobial resistance determinants and investigate plasmid colocalization of tetracycline and macrolide genes in Enterococcus faecalis and Enterococcus faecium from broiler chicken and turkey flocks in Canada. A total of 387 E. faecalis and E. faecium isolates were recovered from poultry cecal contents from five processing plants. The percentages of resistant E. faecalis and E. faecium isolates, respectively, were 88.1 and 94% to bacitracin, 0 and 0.9% to chloramphenicol, 0.7 and 14.5% to ciprofloxacin, 72.6 and 80.3% to erythromycin, 3.7 and 41% to flavomycin, 9.6 and 4.3% (high-level resistance) to gentamicin, 25.2 and 17.1% (high-level resistance) to kanamycin, 100 and 94% to lincomycin, 0 and 0% to linezolid, 2.6 and 20.5% to nitrofurantoin, 3 and 27.4% to penicillin, 98.5 and 89.7% to quinupristin-dalfopristin, 7 and 12.8% to salinomycin, 46.7 and 38.5% (high-level resistance) to streptomycin, 95.6 and 89.7% to tetracycline, 73 and 75.2% to tylosin, and 0 and 0% to vancomycin. One predominant multidrug-resistant phenotypic pattern was identified in both E. faecalis and E. faecium (bacitracin, erythromycin, lincomycin, quinupristin-dalfopristin, tetracycline, and tylosin). These isolates were further examined by PCR and sequencing for the genes encoding their antimicrobial resistance. Various combinations of vatD, vatE, bcrR, bcrA, bcrB, bcrD, ermB, msrC, linB, tetM, and tetO genes were detected, and ermB, tetM, and bcrB were the most common antimicrobial resistance genes identified. For the first time, plasmid extraction and hybridization revealed colocalization of tetO and ermB genes on a ca. 11-kb plasmid in E. faecalis isolates, and filter mating experiments demonstrated its transferability. Results indicate that the intestinal enterococci of healthy poultry, which can contaminate poultry meat at slaughter, could be a reservoir for quinupristin-dalfopristin, bacitracin, tetracycline, and macrolide resistance genes.     

Distribution and transferability of tetracycline resistance determinants in Escherichia coli isolated from meat and meat products

Escherichia coli is used to assess the hygienic quality of food products and the dissemination of antimicrobial resistance. In particular, tetracycline-resistant E. coli can be chosen as an indicator of antibiotic resistant bacteria because it has a high frequency of occurrence. The purpose of this study was to investigate the distribution and transfer of tetracycline resistance determinants in meatborne E. coli. A total of 121 tetracycline-resistant E. coli isolates were collected from meat and meat products (raw meat, fish, and processed foods) from 2004 to 2006 in Korea. Among these isolates, tet(A) (52.4%) was the most frequent tetracycline resistance determinant, followed by tet(B) (41.3%), whereas tet(C) (1.7%) and tet(D) (0.8%) were less frequently identified. Two isolates (1.6%) contained two tet genes simultaneously, tet(A) and tet(B). Minimal inhibitory concentrations (MICs) to tetracycline family antibiotics, such as tetracycline, minocycline, doxycycline, oxytetracycline, and chlortetracycline were higher for isolates carrying the tet(B) gene compared to isolates carrying tet(A) (P<0.0001). Conjugation experiments were performed by the broth mating method; 119 isolates (98.3%) containing at least one of the tet genes were shown to transfer tetracycline resistance to recipient E. coli J53. Also, we observed high diversity of tetracycline-resistant E. coli isolates in meat and meat products in Korea by using XbaI pulsed-field gel electrophoresis (PFGE) typing. This study suggests that the high prevalence of tetracycline-resistant E. coli in meat may be due to the high transferability of tet determinants.     

Contribution of enterococci to the spread of antibiotic resistance in the production chain of swine meat commodities

Thirty-six samples, including fecal specimens, dry feedstuffs, raw and processed pork meat products, and dry fermented sausages, were collected from two production chains of swine meat commodities and analyzed for the presence of 11 antibiotic resistance (AR) genes. Specific PCR assays carried out on DNA extracted directly from the samples revealed a high incidence of the genes tet(K) (80.5%), ermB (66.7%), and tet(M) (66.7%). Feces and feedstuffs gave the largest number of positive amplifications. To elucidate the contribution of enterococci to the occurrence and spread of AR, 146 resistant enterococci were isolated, and their identity, genetic fingerprints, and AR gene profiles were determined by means of molecular techniques. Enterococcus faecalis and Enterococcus faecium were the predominant isolated species (43.8 and 38.4%, respectively); Other Enterococcus species identified were E. durans (8.9%), E. hirae (2.7%), E. gallinarum (2.1%), E. mundtii (2.1%), and E. casseliflavus (2.1%). A number of isolates displayed a complex AR gene profile comprising up to four different resistance determinants. The genes tet(M) and ermB were highly diffused, being present in 86.9 and 84.9%, respectively, of the isolates. The application of amplified fragment length polymorphism fingerprinting was particularly valuable to monitor the resistant enterococcal isolates along the production chain and to individuate steps in which contamination might occur. In fact, isolates of E. faecalis and E. faecium showing the same amplified fragment length polymorphism profile and AR gene pattern were detected in samples taken at different steps of the food chain suggesting three cases of bacterial clonal spread.     

四川泡菜发酵原料-灯笼辣椒附生乳酸菌的抗生素耐药性评估与耐药基因分析

以四川泡菜蔬菜原料——新鲜灯笼辣椒为对象,分析其表面附生乳酸菌Enterococcus mundtii（5 株）、Enterococcus faecalis（2 株）、Enterococcus hirae（5 株）、Lactococcus lactis（7 株）、Leuconostocmesenteroides（2 株）、Leuconostoc holzapfelii（3 株）和Weissella cibaria（79 株）对青霉素（penicillin,PEN）、红霉素（erythromycin,ERY）、四环素（tetracycline,TET）、链霉素（streptomycin,STR）和氯霉素（chloramphenicol,CHL）的抗生素耐药性和耐药基因分布,为制定合理的食品安全防控措施提供科学依据。研究表明：所有分离菌株均无PEN和ERY耐药性,其他种属部分菌株对TET、STR和CHL表现出单一、二重或三重耐药性。除E. hirae、E. faecalis和L. holzapfelii部分菌株对STR表现出单一耐药性外,所有L. mesenteroide菌株只表现出了STR单一耐药性;STR和TET、STR和CHL二重耐药菌株在E. faecalis、E. hirae、L. lactis和W. cibaria分离菌株中都有发现,但是STR、TET、CHL三重耐药菌株仅在W. cibaria中发现。聚合酶链式反应检测发现：除基因norA、sepA、tet(A)、tet(O)和aac(6’)-aph(2’)未被检出外,其他耐药菌株都有相应1 个或多个耐药基因被检出。多重耐药外排泵基因efrA、tolC、norC、sugE和mdfA较核糖体蛋白质保护和酶修饰基因检出率高,分别达到了49%、41%、48%、41%和47%。虽然辣椒表面附生乳酸菌的抗生素耐药基因在四川泡菜发酵过程中的扩散行为需要进一步研究,但根据食品加工过程安全规范标准,也应关注其表面附生的乳酸菌抗生素耐药性存在的潜在食品安全问题。     

Effect of various sludge digestion conditions on sulfonamide, macrolide, and tetracycline resistance genes and class I integrons

Wastewater treatment processes are of growing interest as a potential means to limit the dissemination of antibiotic resistance. This study examines the response of nine representative antibiotic resistance genes (ARGs) encoding resistance to sulfonamide (sulI, sulII), erythromycin (erm(B), erm(F)), and tetracycline (tet(O), tet(W), tet(C), tet(G), tet(X)) to various laboratory-scale sludge digestion processes. The class I integron gene (intI1) was also monitored as an indicator of horizontal gene transfer potential and multiple antibiotic resistance. Mesophilic anaerobic digestion at both 10 and 20 day solids retention times (SRTs) significantly reduced sulI, suII, tet(C), tet(G), and tet(X) with longer SRT exhibiting a greater extent of removal; however, tet(W), erm(B) and erm(F) genes increased relative to the feed. Thermophilic anaerobic digesters operating at 47 °C, 52 °C, and 59 °C performed similarly to each other and provided more effective reduction of erm(B), erm(F), tet(O), and tet(W) compared to mesophilic digestion. However, thermophilic digestion resulted in similar or poorer removal of all other ARGs and intI1. Thermal hydrolysis pretreatment drastically reduced all ARGs, but they generally rebounded during subsequent anaerobic and aerobic digestion treatments. To gain insight into potential mechanisms driving ARG behavior in the digesters, the dominant bacterial communities were compared by denaturing gradient gel electrophoresis. The overall results suggest that bacterial community composition of the sludge digestion process, as controlled by the physical operating characteristics, drives the distribution of ARGs present in the produced biosolids, more so than the influent ARG composition.     

Antibiotic resistance and virulence factors among Enterococci isolated from chouriço, a traditional Portuguese dry fermented sausage

Enterococci are ubiquitous microorganisms, found as part of the normal intestinal microbiota of many animals. They can be present in food products, for example, the Portuguese dry fermented sausage chouri?o. Twenty enterococci were isolated from chouri?o in two processing units; after identification and typification by conventional-molecular methods, the isolates were screened for virulence factors and antibiotic resistance. Identification allocated all enterococci to the species Enterococcus faecalis, and PCR fingerprinting demonstrated that each isolate was specific to the processing unit and chouri?o from which it was recovered. Regarding the screening for virulence factors, 1 strain produced cytolysin and 4 were gelatinase positive, but none produced lipase. The ace gene was detected in 1 enterococci, ebpABC and efaA(fs) in 16 isolates each, esp in 3, fsrB in 5, gelE in 7, and cylA in 1. A multiresistant phenotype was observed in 8 isolates, 6 belonging to factory A. The antibiotic resistance gene ere(B) was detected in 9 enterococci, whereas the genes tet(M), aac(6')-Ie-aph(2'), and vanA were detected in 8 isolates each. As some of the E. faecalis chouri?o isolates present a multiresistant profile and harbor virulence and/or resistance genes, to assess further the safety of Portuguese dry sausages, a larger number of products and processing units must by analyzed.     

Antibiotic resistance and virulence traits of enterococci isolated from Baylough, an Irish artisanal cheese

Eight representative Enterococcus strains from a collection of over 600 previously isolated from an Irish artisanal cheese were subjected to phenotypic and genotypic analysis of antibiotic resistance and virulence properties. Genes encoding resistance to tetracycline (tet(M) and tet(L)) and/or erythromycin (erm(B)) were detected in five strains. In addition, all strains contained two or more of the virulence genes tested (agg, gel, cyl, esp, ace, efaAfs, and efaAfm). Further investigation into the transferability and environmental dissemination of these resistance and virulence traits will allow risk assessment and safety evaluation of artisanal cheeses.     

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.     

Resistance of potential probiotic lactic acid bacteria and bifidobacteria of African and European origin to antimicrobials: determination and transferability of the resistance genes to other bacteria

Probiotic bacteria and starter cultures of Lactobacillus, Weissella and Bifidobacterium of African and European origins were studied and compared for their susceptibility to antimicrobials. The study included, for all isolates, determination of MICs (Minimal Inhibitory Concentration) for 24 antimicrobials, detection of resistance genes by PCR reactions using specific primers and sequencing of positive amplicons. The ability of Lb. reuteri from Africa to transfer the erythromycin resistance gene erm(B) to closely related bacteria was investigated by conjugation. Variations were observed and high levels of intrinsic resistance were found among the tested species. Positive amplicons were obtained for resistance genes encoding aminoglycoside (aph(3')-III, aadA, aadE) and tetracycline (tet(S)) from isolates from Europe and macrolide (erm(B)) from an isolate from Africa. However, only the erm(B) gene found in Lb. reuteri L4: 12002 from Africa contained a homologous sequence to previously published sequences. This gene could be transferred in vitro to enterococci. Higher prevalence of phenotypic resistance for aminoglycoside was found in isolates from Europe.     

Phenotyping and genotyping of antibiotic-resistant Escherichia coli isolated from a natural river basin

Scientists have become increasingly concerned about the occurrence of antibacterial resistance in the environment. In this study, Escherichia coli resistant to one or more antibiotics among nine antibiotics was screened from Wenyu River Basin in Beijing, China, with mean frequency of 48.7 +/- 8.7% of 388 isolates in summer and 47 +/- 6% of 236 isolates in winter. The mean multiantibiotic resistance (MAR) index in summer was 0.11 +/- 0.03, slightly lower than that (0.14 +/- 0.04) in winter. Most frequent resistance appeared for sulfonamides, tetracycline, and ampicillin. The distribution of 20 tetracycline, three sulfonamide, and three beta-lactam resistance genes was assessed in the resistant isolates. While 97% of the ampicillin (AMP) resistant mechanism could be explained by the resistance gene TEM, 90% of the tetracycline (TC) and 96% of the sulfonamide (SXT) resistances could be explained by tet(A), tet(B), tet(M), and their combinations and sul(I), sul(II), sul(III), and their combinations, respectively. tet(M), a tetracycline-resistant gene originally detected in Gram-positive bacteria, and its combinations with tet(A) or tet(B) were first detected in E. coli isolated from a natural river basin, suggesting that tet(M) in E. coli might have been transferred from other bacterial species through horizontal gene transfer, which was supported by the fact that no tet(M) was detected in the isolates of human and chicken sources, except for only one isolate from swine. The source of sulfonamide-resistant E. coli in the river was supposed to be mainly from humans, based on a comparison of the sulfonamide resistance genotypes in animals and humans.     

Direct detection of antibiotic resistance genes in specimens of chicken and pork meat

Antibiotic resistance (AR) in bacteria, a major threat to human health, has emerged in the last few decades as a consequence of the selective pressure exerted by the widespread use of antibiotics in medicine, agriculture and veterinary practice and as growth promoters in animal husbandry. The frequency of 11 genes [tet(M), tet(O), tet(K), erm(A), erm(B), erm(C), vanA, vanB, aac (6')-Ie aph (2')-Ia, mecA, blaZ] encoding resistance to some antibiotics widely used in clinical practice was analysed in raw pork and chicken meat and in fermented sausages as well as in faecal samples from the relevant farm animals using a molecular approach based on PCR amplification of bacterial DNA directly extracted from specimens. Some of the 11 AR genes were highly prevalent, the largest number being detected in chicken meat and pig faeces. The genes found most frequently in meat were tet(K) and erm(B); vanB and mecA were the least represented. All 11 determinants were detected in faecal samples except mecA, which was found only in chicken faeces. erm(B) and erm(C) were detected in all faecal samples. The frequency of AR genes was not appreciably different in meat compared to faecal specimens of the relevant animal except for vanB, which was more prevalent in faeces. Our findings suggest that AR genes are highly prevalent in food-associated bacteria and that AR contamination is likely related to breeding rather than processing techniques. Finally, the cultivation-independent molecular method used in this work to determine the prevalence of AR genes in foods proved to be a rapid and reliable alternative to traditional tools.     

食源性单核细胞增生李斯特菌四环素、红霉素耐药基因研究

研究了食源性单核细胞增生李斯特菌四环素、红霉素耐药基因的分布状况及和耐药表型的关系。采用微量肉汤稀释法对2005~2007年河北省疾病预防控制中心分离到的食源性单核细胞增生李斯特菌株进行四环素、红霉素药敏实验;应用PCR方法对实验菌株进行四环素耐药基因tet(M)、tet(S)、tet(L)、tet(K)、tet(B)、及与tet(M)基因关系密切的转座子Tn916、红霉素核糖体甲基化酶基因ermB、ermC、及与ermB基因关系密切的转座子Tn917检测,对阳性样本序列进行鉴定分析;应用血清学分型、脉冲场凝胶电泳(PFGE)、及脂肪酸聚类分析方法分析四环素耐药菌株之间的相关性,确定基因型和多态性。结果表明,91株单核细胞增生李斯特菌四环素敏感77株、耐药14株;红霉素敏感89株、耐药2株,其中包含1株菌同时交叉耐药四环素和红霉素;14株四环素耐药株中含tet(M)基因的13株,在13株tet(M)基因阳性菌中,tet(M)位于Tn916转座子上的9株;1株同时交叉耐药四环素、红霉素菌同时携带tet(S)、ermB基因;ermC基因、转座子Tn917均为阴性;四环素、红霉素敏感株中未检测到上述任何耐药基因。14株四环素耐药菌株血清型分布以1/2a型为主(n=12),部分菌株PFGE、脂肪酸分型完全一致。食源性单核细胞增生李斯特菌获得tet(M)基因是耐四环素的主要机制之一,具有水平传播耐药基因能力的接合型转座子Tn916与该菌四环素耐药播散有直接关系;ermB基因介导的核糖体靶位点改变存在食源性单核细胞增生李斯特菌红霉素耐药株中;PFGE基因型结合脂肪酸聚类分析能够用来分析菌株之间的相关性。     

Phenotypic and molecular assessment of antimicrobial resistance in Lactobacillus paracasei strains of food origin

Antimicrobial resistance data in food-associated lactic acid bacteria (LAB) such as lactobacilli are mostly based on nonstandardized methodologies and/or have been obtained for only a limited number of strains. This susceptibility study included a diverse collection of 115 isolates mainly of food origin originally identified as Lactobacillus paracasei or Lactobacillus casei. Upon reidentification and removal of potential replicate isolates using repetitive DNA element PCR fingerprinting, 65 genotypically unique L. paracasei strains and the L. casei type strain were selected for broth microdilution and Etest assays using the LAB susceptibility test medium. In both methodologies, strains appeared uniformly susceptible to ampicillin and clindamycin but exhibited natural resistance to streptomycin and gentamicin. Three L. paracasei strains from cheese displayed acquired resistance to tetracycline (MIC > or = 32 microg/ml) and/or to erythromycin (MIC >16 microg/ml), which was linked to the presence of a tet(M) or tet(W) gene and/or an erm(B) gene, respectively. Partial sequencing revealed that the tet(M) genes found in two of these strains belonged to two tet(M) sequence homology groups previously found in enterococci. Collectively, phenotypic and genotypic data allowed us to propose tentative epidemiological cutoffs for L. paracasei and L. casei for differentiating susceptible strains from those strains harboring one or more acquired resistance factors.     

Prevalence and characterization of Enterococcus spp. isolated from Brazilian foods

Enterococci can be used in the food industry as starter or probiotic cultures. However, enterococci are also implicated in severe multi-resistant nosocomial infections. In this study, the prevalence of enterococci in selected Brazilian foodstuffs (raw and pasteurized milk, meat products, cheeses and vegetables) was evaluated. Phenotypic and PCR protocols were used for species identification. Tests for production of gelatinase, haemolysin, bacteriocin and bile salt hydrolysis were done with all enterococci isolates, whereas molecular determination of virulence markers (genes esp, gel, ace, as, efaA, hyl and cylA) and antibiotic resistance was checked only for Enterococcus faecium and Enterococcus faecalis isolates. The antibiotic-resistant isolates were assayed for biofilm formation and adhesion to mammalian cells. From the 120 food samples analyzed, 52.5% were positive for enterococci, meat and cheese being the most contaminated. E. faecium was the predominant species, followed by E. faecalis, E. casseliflavus and Enterococcus gallinarum. Phenotypic tests indicated that 67.7% of isolates hydrolyzed bile salts, 15.2% produced bacteriocin, 12.0% were beta-hemolytic and 18.2% produced gelatinase. Antibiotic resistance (gentamicin, tetracycline and erythromycin) and genes encoding for virulence traits were more frequent in E. faecalis than in E. faecium. Three E. faecium isolates were resistant to vancomycin. Among antibiotic-resistant isolates, 72.4% of E. faecalis were able to form biofilm and 13.8% to adhere to Caco-2 cells. Antibiotic-resistant E. faecalis and E. faecium isolates were grouped by RAPD-PCR and a scattered distribution was noted, indicating that resistance was not related to a particular clone. The spread of virulence/resistance traits in isolates of the two species and different RAPD-types suggest the pathogenic potential of both species. By contrast, the recovery of bacteriocinogenic E. faecium isolates with no virulence traits suggests their potential for biotechnological applications. In conclusion, our results showed that enterococci from Brazilian foods present important dualist aspects for food safety.     

食源性乳酸菌安全性的评价

从不同来源的样品中分离得到36 株乳酸菌,对其进行常见抗生素如氯霉素、四环素、红霉素抗药性分析,结果表明,26 株菌具有抗药性,其中4 株携带抗生素抗性基因,并且菌株Enterococcus faecium KN9所携带的四环素抗性基因tet（M）和tet（L）能通过接合作用,转移到受体菌Lactococcus lactis MG1614中。对这些乳酸菌产生有害代谢产物分析结果表明,部分菌株可以产生生物胺,所有的菌株都不产生硝基还原酶和偶氮还原酶。所以,本实验分离到的大部分乳酸菌是安全的,只有携带有抗四环素基因的E. faecium KN9具有潜在的安全隐患。因此,需要在食用前对乳酸菌进行安全性评价,以减少可能的安全隐患。     

Antibiotic resistance genes and identification of staphylococci collected from the production chain of swine meat commodities

Staphylococci harbouring antibiotic resistance (AR) genes may represent a hazard for human health and, as other resistant food-related bacteria, they contribute to the spread of AR. In this study, we isolated resistant staphylococci from an entire swine production chain and investigated the occurrence of 11 genes [aac(6')Ie-aph(2')Ia, blaZ, mecA, vanA, vanB, ermA, ermB, ermC, tet(M), tet(O) and tet(K)] encoding resistance to some antibiotics largely used in clinical practice. The 66 resistant staphylococcal isolates were identified as Staphylococcus epidermidis (27 isolates), Staphylococcus aureus (12), Staphylococcus xylosus (12), Staphylococcus simulans (5), Staphylococcus pasteuri (4), Staphylococcus carnosus (3), Staphylococcus lentus (2) and Staphylococcus sciuri (1). Specific-PCR detection of AR genes showed the prevalence of the tet(K) gene in most of the isolates (89.4%), followed by tet(M) and ermC (about 75%); mecA was detected in more than half of S. aureus and S. epidermidis isolates. The genes vanA and vanB were not retrieved. It was found that a high proportion of coagulase-positive and -negative isolates are multidrug-resistant and some of them carry up to six AR genes. Our findings show that the swine production chain is a source of antibiotic-resistant staphylococci suggesting the importance of resistance surveillance in the food production environment.     

Antibiotic susceptibility of Bifidobacterium thermophilum and Bifidobacterium pseudolongum isolates from animal sources

The widespread use of antimicrobial substances has led to resistant populations of microorganisms in several ecosystems. In animal husbandry, the application of antibiotics has contributed to resistance development in pathogenic and commensal bacteria. These strains or their resistance genes can be spread along several ecological routes, including the food chain. Antibiotic resistance is important in terms of the safety of industrial strains, such as probiotics for food and feed. Bifidobacterium thermophilum and Bifidobacterium pseudolongum are known to comprise the major part of the bifidobacterial microbiota in the gut and feces of cattle and pigs. In this study, the antimicrobial susceptibility in bifidobacterial isolates of these species was investigated. Isolates from the beef and pork production chain were identified and typed to strain level, and the antimicrobial susceptibility level was tested to a set of antibiotics. Isolates with low susceptibility levels were screened by PCR for already described resistance genes. Strains atypically resistant to clindamycin, erythromycin, and tetracycline were determined. The resistance genes tet(O), tet(W), and erm(X) were detected in the bifidobacterial species that were examined.     

Molecular structure and evolution of the conjugative multiresistance plasmid pRE25 of Enterococcus faecalis isolated from a raw-fermented sausage

Plasmid pRE25 from Enterococcus faecalis transfers resistances against kanamycin, neomycin, streptomycin, clindamycin, lincomycin, azithromycin, clarithromycin, erythromycin, roxithromycin, tylosin, chloramphenicol, and nourseothricin sulfate by conjugation in vitro to E. faecalis JH2-2, Lactococcus lactis Bu2, and Listeria innocua L19. Its nucleotide sequence of 50237 base pairs represents the largest, fully sequenced conjugative multiresistance plasmid of enterococci (Plasmid 46 (2001) 170). The gene for chloramphenicol resistance (cat) was identified as an acetyltransferase identical to the one of plasmid pIP501 of Streptococcus agalactiae. Erythromycin resistance is due to a 23S ribosomal RNA methyl transferase, again as found in pIP501 (ermB). The aminoglycoside resistance genes are packed in tandem as in transposon Tn5405 of Staphylococcus aureus: an aminoglycoside 6-adenyltransferase, a streptothricin acetyl transferase, and an aminoglycoside phosphotransferase.). Identical resistance genes are known from pathogens like Streptococcus pyogenes, S. agalactiae, S. aureus, Campylobacter coli, Clostridium perfringens, and Clostridium difficile. pRE25 is composed of a 30.5-kbp segment almost identical to pIP501. Of the 15 genes involved in conjugative transfer, 10 codes for putative transmembrane proteins (e.g. trsB, traC, trsF, trsJ, and trsL). The enterococcal part is joined into the pIP501 part by insertion elements IS1216V of E. faecium Tn1545 (three copies), and homologs of IS1062 (E. faecalis) and IS1485 (E. faecium). pRE25 demonstrates that enterococci from fermented food do participate in the molecular communication between Gram-positive and Gram-negative bacteria of the human and animal microflora.     

Enterococcus faecalis and Enterococcus faecium isolates from milk,beef, and chicken and their antibiotic resistance

The occurrence and antibiotic resistance of enterococci, especially Enterococcus faecalis and Enterococcus faecium, in milk, beef, and chicken in Gaborone, Botswana, were studied. Enterococci were isolated from these sources with the use of bile esculin agar and identified with API 20 Strep kits. Antibiotic resistance was determined by the disk diffusion method. The antibiotics tested were vancomycin, teicoplanin, ampicillin, tetracycline, and cephalothin. Among the 1,467 enterococci isolated from the samples, E. faecalis (46.1%) and E. faecium (29.0%) were found to be the predominant species. Other enterococcal species made up 25% of the isolates. More than 96 and 97% of the E. faecalis and E. faecium isolates, respectively, were found to be resistant to ampicillin. Almost 34, 27.3, and 22.4% of the E. faecalis isolates from milk, beef, and chicken, respectively, were also resistant to cephalothin. The percentages of E. faecium isolates that were found to be resistant to cephalothin were 32.8, 16.9, and 17.3% for milk, beef, and chicken, respectively. Resistance to vancomycin was widespread. It was found that 18.8, 7.8. and 13.1% of the E. faecalis isolates from milk, beef, and chicken samples, respectively, were resistant to vancomycin. In contrast, 32.8, 24.7, and 30.7% of the E. faecium isolates from milk, beef, and chicken samples, respectively, were resistant to vancomycin. Isolates that were resistant to multiple drugs were found in relatively large numbers.