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.     

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.     

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.     

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.     

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-Resistant Enterococcus faecalis in Abattoir Pigs and Plasmid Colocalization and Cotransfer of tet(M) and erm(B) Genes

This study was conducted to determine plasmid colocalization and transferability of both erm(B) and tet(M) genes in Enterococcus faecalis isolates from abattoir pigs in Canada. A total of 124 E. faecalis isolates from cecal contents of abattoir pigs were examined for antibiotic susceptibility. High percentages of resistance to macrolides and tetracyclines were found. Two predominant multiresistance patterns of E. faecalis were examined by PCR and sequencing for the presence of genes encoding antibiotic resistance. Various combinations of antibiotic resistance genes were detected; erm(B) and tet(M) were the most common genes. Plasmid profiling and hybridization revealed that both genes were colocated on a ～9-kb transferable plasmid in six strains with the two predominant multiresistant patterns. Plasmid colocalization and cotransfer of tet(M) and erm(B) genes in porcine E. faecalis isolates indicates that antibiotic coselection and transferability could occur via this single genetic element. To our knowledge, this is the first report on plasmid colocalization and transferability of erm(B) and tet(M) genes in E. faecalis on a mobile genetic element of ～9 kb. Physical linkage between important antibiotic resistance determinants in enterococci is of interest for predicting potential transfer to other bacterial genera.     

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。     

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.     

Isolation of halotolerant Lactococcus lactis subsp. lactis from intestinal tract of coastal fish

We isolated lactic acid bacteria from the intestinal tract of the pufferfish Takifugu niphobles caught in Shimoda, Shizuoka, Japan by using MRS broth prepared with 50% seawater. Additional screening was carried out using phenotypic tests such as Gram staining, cell morphology, catalase, oxidase and fermentation of glucose. Subsequently 227 isolates screened by the phenotypic tests were subjected to species-specific PCR for Lactococcus lactis, resulting in four positive isolates. The 16S rRNA gene sequences from three isolates were highly similar to that of L. lactis subsp. lactis (DNA database accession number M58837), while that of one isolate was identical to that of Leuconostoc mesenteroides (AB023246). These isolates were characterized by API 50 CH for carbohydrate fermentation and other phenotypic criteria for salt tolerance, and the characteristics were compared with those of L. lactis subsp. lactis from a cheese starter culture. The carbohydrate fermentation profiles of these isolates were characteristic of L. lactis subsp. lactis strains, whereas the tolerance of these isolates to salt was higher than that of L. lactis subsp. lactis from the cheese starter culture: the new L. lactis isolates showed high salt tolerance in MRS-agar plates containing 200% seawater or 6% sodium chloride. This is the first report of the isolation of halotolerant strains of L. lactis subsp. lactis from a marine environment.     

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。     

Antibiotic survey of Lactococcus lactis strains to six antibiotics by Etest, and establishment of new susceptibility-resistance cut-off values

In order to establish cut-off values for Lactococcus lactis to six antibiotics to distinguish susceptible and intrinsically resistant strains from those having acquired resistances, the minimum inhibitory concentration (MIC) of tetracycline, erythromycin, clindamycin, streptomycin, chloramphenicol and vancomycin was determined in 93 different Lc. lactis strains using the Etest. These bacterial strains were originally isolated from dairy and animal sources in widely separated geographical locations. Cut-offs were defined on the basis of the distribution of the MICs frequency of the studied antibiotics, which in the absence of acquired determinants should approach to a normal statistical distribution. In general, the new cut-off values proposed in this study are higher than previously defined (European Commission, 2005. The EFSA Journal 223, 1-12). Based on these new values, all the strains tested were susceptible to erythromycin, chloramphenicol and vancomycin, and 79 susceptible to all six antibiotics. However, 11 strains (around 12%) were considered resistant to tetracycline (six of which had been identified after screening of a large collection of lactococci strains for tetracycline resistance) and five (5.4%) resistant to streptomycin. Of these, two fish isolates proved to be resistance to both tetracycline and streptomycin. From the tetracycline resistant strains, tet(M) and mosaic tet(L/S) genes were amplified by PCR, demonstrating they harboured acquired antibiotic resistance determinants.     

Isolation and characterization of Carnobacterium, Lactococcus, and Enterococcus spp. from cooked, modified atmosphere packaged, refrigerated, poultry meat

The microbiota of commercially produced, cooked and modified atmosphere packaged poultry meat was followed during storage at 3.5 degrees C for up to 7 weeks. The dominant microbiota consisted of Lactococcus raffinolactis (117 isolates), Carnobacterium divergens (61 isolates), Carnobacterium piscicola (11 isolates), Lactococcus garvieae (four isolates), Lactococcus lactis (one isolate) and Enterococcus faecalis (three isolates). All isolates were screened for production of bacteriocins. Only C. piscicola isolates produced an inhibitory substance active against other lactic acid bacteria and against several Listeria spp. Species-specific polymerase chain reaction (PCR) primers were used for the differentiation of Carnobacterium, L. raffinolactis, L. lactis, and L. garvieae strains associated with the modified atmosphere packaged poultry products. No false PCR products were observed with other closely related bacterial species.     

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.     

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.     

Tetracycline resistance associated with commensal bacteria from representative ready-to-consume deli and restaurant foods

Proper knowledge of antibiotic resistance (AR) dissemination is essential for effective mitigation. This study examined the profiles of tetracycline-resistant (Tetr) commensal bacteria from representative ready-to-consume food samples from salad bars at local grocery stores and restaurants. Out of 900 Tetr isolates examined, 158 (17.6%) carried one or more of tetM, tetL, tetS, and tetK genes by conventional PCR, 28 harbored more than one Tetr determinants. The most prevalent genotype was tetM, which was detected in 70.9% of the AR gene carriers, followed by tetL (31.6%), tetS (13.9%), and tetK (2.5%). Identified AR gene carriers included Enterococcus, Lactococcus, Staphylococcus, Brochothrix, Carnobacterium, Stenotrophomonas, Pseudomonas, and Sphingobacterium, by 16S rRNA gene sequence analysis. AR determinants were successfully transmitted, and led to resistance in Streptococcus mutans via natural gene transformation and Enterococcus faecalis via electroporation, suggesting the functionality and mobility of the AR genes from the food commensal bacteria. In addition, the AR traits in many isolates are quite stable, even in the absence of the selective pressure. The identification of new commensal carriers for representative AR genes revealed the involvement of a broad spectrum of bacteria in the horizontal transmission of AR genes. Meanwhile, the spectrum of the antibiotic-resistant bacteria differed from the spectrum of the total bacteria (by denaturing gradient gel electrophoresis) associated with the food items. Our data revealed a common avenue in AR exposure and will assist in proper risk assessment and the development of comprehensive mitigation strategies to effectively combat AR.     

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.     

Novel conjugative plasmids from the natural isolate Lactococcus lactis subspecies cremoris DPC3758: a repository of genes for the potential improvement of dairy starters

A collection of 17 natural lactococcal isolates from raw milk cheeses were studied in terms of their plasmid distribution, content, and diversity. All strains in the collection harbored an abundance of plasmids, including Lactococcus lactis ssp. cremoris DPC3758, whose 8-plasmid complement was selected for sequencing. The complete sequences of pAF22 (22,388 kb), pAF14 (14,419 kb), pAF12 (12,067 kb), pAF07 (7,435 kb), and pAF04 (3,801 kb) were obtained, whereas gene functions of technological interest were mapped to pAF65 (65 kb) and pAF45 (45 kb) by PCR. The plasmids of L. lactis DPC3758 were found to encode many genes with the potential to improve the technological properties of dairy starters. These included 3 anti-phage restriction/modification (R/M) systems (1 of type I and 2 of type II) and genes for immunity/resistance to nisin, lacticin 481, cadmium, and copper. Regions encoding conjugative/mobilization functions were present in 6 of the 8 plasmids, including those containing the R/M systems, thus enabling the food-grade transfer of these mechanisms to industrial strains. Using cadmium selection, the sequential stacking of the R/M plasmids into a plasmid-free host provided the recipient with increased protection against 936- and c2-type phages. The association of food-grade selectable markers and mobilization functions on L. lactis DPC3758 plasmids will facilitate their exploitation to obtain industrial strains with enhanced phage protection and robustness. These natural plasmids also provide another example of the major role of plasmids in contributing to host fitness and preservation within its ecological niche.     

Antimicrobial susceptibilities of Listeria monocytogenes strains isolated from food and food processing environment in Poland

A total of 471 Listeria monocytogenes isolates from different types of food and food-related sources in Poland during 2004-2010 were examined. This number includes 200 isolates from fish, 144 from fresh and frozen vegetables, 43 ready-to-eat products (deli foods, cold cuts), 13 from dairy products, 16 from raw meats, 15 from confectionery products and 40 directly from processing plants. All isolates were subjected to serotyping and lineage assays using PCR, and antimicrobial susceptibility using E-test and a broth microdilution method. Of all isolates, 256 (54.4%), 120 (25.5%), 59 (12.5%), 36 (7.6%) were identified as serotypes 1/2a (or 3a), 1/2c (or 3c), 1/2b (or 3b or 7), and 4b (or 4d or 4e), respectively. A direct correlation between the most common serotypes and three L. monocytogenes lineages was also observed. All L. monocytogenes isolates belonged to lineages I (20.2%) and II (79.8%). All strains were sensitive to ampicillin, amoxicillin, gentamicin, erythromycin, trimethoprim, rifampicin, vancomycin, chloramphenicol and sulfamethoxazol. Two of the L. monocytogenes strains (0.42%) showed phenotypic resistance. One strain was resistant to tetracycline and minocycline due to the presence of tet(M). It did not carry gene int, which may indicate that the tet(M) gene in this strain was not integrated in the transposon Tn916-Tn1545 family. The resistance of the second strain to ciprofloxacin and norfloxacin was attributed to active efflux associated with overexpression of gene lde. Our data indicate the low prevalence of antimicrobial resistance among L. monocytogenes isolates from food and food-related sources in Poland.     

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.     

Characterization of the lactic acid bacteria in ewe's milk and cheese from northwest Argentina

Indigenous lactic acid bacteria in ewe's milk and artisanal cheese were studied in four samples of fresh raw milk and four 1-month-old cheeses from the provinces of northwest Argentina. Mean growth counts on M17, MRS, and MSE agar media did not show significant differences (P < 0.05) in raw milk and cheeses. Isolates of lactic acid bacteria from milk were identified as Enterococcus (48%), lactococci (14%), leuconostocs (8%), and lactobacilli (30%). All lactococci were identified as Lactococcus lactis (subsp. lactis and subsp. cremoris). Lactobacilli were identified as Lactobacillus plantarum (92%) and Lactobacillus acidophilus (8%). Enterococci (59%) and lactobacilli (41%) were isolated from cheeses. L. plantarum (93%), L. acidophilus (5%), and Lactobacillus casei (2%) were most frequently isolated. L. lactis subsp. lactis biovar diacetylactis strains were considered as fast acid producers. L. lactis subsp. cremoris strains were slow acid producers. L. plantarum and L. casei strains identified from the cheeses showed slow acid production. The majority of the lactobacilli and Lactococcus lactis strains utilized citrate and produced diacetyl and acetoin in milk. Enzyme activities (API-ZYM tests) of lactococci were low, but activities of L. plantarum strains were considerably higher. The predominance of L. plantarum in artisanal cheese is probably important in the ripening of these cheeses due to their physiological and biochemical characteristics.