Dissemination of Enterococcus faecalis and Enterococcus faecium in a Ricotta Processing Plant and Evaluation of Pathogenic and Antibiotic Resistance Profiles

In this work, the sources of contamination by Enterococcus spp. in a ricotta processing line were evaluated. The isolated strains were tested for virulence genes (gelE, cylA,B, M, esp, agg, ace, efaA, vanB), expression of virulence factors (hemolysin and gelatinase), and the resistance to 10 different antibiotics. Enterococcus faecium and Enterococcus faecalis were subjected to discriminatory identification by intergenic spacer region (ITS)‐polymerase chain reaction and sequencing of the ITS region. The results showed that Enterococcus spp. was detected in the raw materials, environment samples and the final product. None of the 107 Enterococcus isolates were completely free from all virulence genes considered. A fraction of 21.5% of the isolates containing all of the genes of the cylA, B, M operon also expressed β‐hemolysis. Most of the isolates showed the gelE gene, but only 9.3% were able to hydrolyze gelatin. In addition, 23.5% of the observed Enterococcus isolates had the vanB gene but were susceptible to vancomycin in vitro. The dissemination of antibiotic‐resistant enterococci was revealed in this study: 19.3% of the E. faecium samples and 78.0% of the E. faecalis samples were resistant to at least one of the antibiotics tested. Sequencing of region discriminated 5 and 7 distinct groups among E. faecalis and E. faecium, respectively. Although some similarity was observed among some of the isolates, all E. faecalis and E. faecium isolates had genetic differences both in the ITS region and in the virulence profile, which makes them different from each other.     

Diversity of Antibiotic Resistance Genes in Enterococcus Strains Isolated from Ready‐to‐Eat Meat Products

The objective of the study was to answer the question of whether the ready‐to‐eat meat products can pose indirect hazard for consumer health serving as reservoir of Enterococcus strains harboring tetracyclines, aminoglycosides, and macrolides resistance genes. A total of 390 samples of ready‐to‐eat meat products were investigated. Enterococcus strains were found in 74.1% of the samples. A total of 302 strains were classified as: Enterococcus faecalis (48.7%), Enterococcus faecium (39.7%), Enterococcus casseliflavus (4.3%), Enterococcus durans (3.0%), Enterococcus hirae (2.6%), and other Enterococcus spp. (1.7%). A high percentage of isolates were resistant to streptomycin high level (45%) followed by erythromycin (42.7%), fosfomycin (27.2%), rifampicin (19.2%), tetracycline (36.4%), tigecycline (19.9%). The ant(6′)‐Ia gene was the most frequently found gene (79.6%). Among the other genes that encode aminoglycosides‐modifying enzymes, the highest portion of the strains had the aac(6′)‐Ie‐aph(2′′)‐Ia (18.5%) and aph(3′′)‐IIIa (16.6%), but resistance of isolates from food is also an effect of the presence of aph(2′′)‐Ib, aph(2′′)‐Ic, aph(2′′)‐Id genes. Resistance to tetracyclines was associated with the presence of tetM (43.7%), tetL (32.1%), tetK (14.6%), tetW (0.7%), and tetO (0.3%) genes. The ermB and ermA genes were found in 33.8% and 18.9% of isolates, respectively. Nearly half of the isolates contained a conjugative transposon of the Tn916/Tn1545 family. Enterococci are widely present in retail ready‐to‐eat meat products. Many isolated strains (including such species as E. casseliflavus, E. durans, E. hirae, and Enterococcus gallinarum) are antibiotic resistant and carry transferable resistance genes.     

Short communication: Antimicrobial resistance and virulence genes of Enterococcus faecalis isolated from subclinical bovine mastitis cases in China

This study aimed to investigate the antimicrobial resistance and virulence genes of Enterococcus faecalis isolated from subclinical bovine mastitis cases in China. Enterococcus faecalis isolates were identified by 16S rRNA amplification and sequencing. Antimicrobial susceptibility was determined by the disc diffusion method. Antimicrobial resistance and virulence genes were tested by PCR. Overall, E. faecalis was recovered from 81 of 1,787 (4.5%) mastitic milk samples. The isolates showed high resistance against tetracycline (87.7%) and erythromycin (79.0%). The most prevalent resistance genes found in the E. faecalis were tetK (96.3%), tetL (79.0%), and tetM (87.7%) for tetracycline and ermC (97.5%) for erythromycin. Moreover, gelE (70.4%), esp (85.2%), efaA (91.4%) were the most common virulence genes. This is the first report to characterize E. faecalis recovered from subclinical bovine mastitis cases in China.     

The occurrence of virulence traits among high-level aminoglycosides resistant Enterococcus isolates obtained from feces of humans, animals, and birds in South Korea

Enterococcus isolates (1500) obtained from the feces of 48 humans, 209 domesticated food animals, and 155 wild geese in South Korea were characterized with respect to species status by PCR analyses and resistance to antibiotics. Of the 1500 strains examined, the majority (n = 577) were Enterococcus faecalis from 224 (54.4%) of the samples feces, while 299 were of E. faecium from 125 of the samples (30.3%), 224 were E. hirae from 101 (24.5%) of the samples, 94 were E. casseliflavus from 43 (10.4%) of the samples, and one was E. gallinarum. While 305 isolated from 125 (30.3%) of the samples were unidentified species. Approximately 15, 60, 50, 55, 3, and 40% of samples obtained from beef cattle, chickens, ducks, swine, wild geese, and humans, respectively, yielded Enterococcus isolates that were resistant to high-levels of aminoglycosides (i.e., of gentamicin, kanamycin, and streptomycin, minimum inhibitory concentrations were > 1000 mg/l). The 180 Enterococcus isolates that showed high levels of resistance to aminoglycoside antibiotics (HLAR) were screened for virulence genes encoding for aggregation substance (agg), cytolysin activator (cylA), gelatinase (gelE) and surface protein (esp). Of those, the gelE gene was found most frequently in chickens and ducks of the HLAR isolates, while 56 E. faecalis and 13 E. faecium HLAR were gelatinase positive and showed hemolysin activity. Multiple antibiotic resistant Enterococcus isolates carrying virulence genes were most frequently isolated from poultry and swine, and were mostly E. faecalis or E. faecium. These findings suggest that restriction of the use of antibiotics in food animal operations in South Korea, especially those involved in poultry and swine production would be desirable.     

Speciation and frequency of virulence genes of Enterococcus spp. isolated from rainwater tank samples in Southeast Queensland, Australia

In this study, 212 Enterococcus isolates from 23 rainwater tank samples in Southeast Queensland (SEQ), Australia were identified to the species level. The isolates were also tested for the presence of 6 virulence genes associated with Enterococcus related infections. Among the 23 rainwater tank samples, 20 (90%), 10 (44%), 7 (30%), 5 (22%), 4 (17%), 2 (9%), and 1 (4%) samples yielded E. faecalis, E. mundtii, E. casseliflavus, E. faecium, E. hirae, E. avium, and E. durans, respectively. Among the 6 virulence genes tested, gelE and efaA were most prevalent, detected in 19 (83%) and 18 (78%) of 23 rainwater tank samples, respectively. Virulence gene ace was also detected in 14 (61%) rainwater tank samples followed by AS, esp (E. faecalis variant), and cylA genes which were detected in 3 (13%), 2 (9%), and 1 (4%) samples, respectively. In all, 120 (57%) Enterococcus isolates from 20 rainwater tank samples harbored virulence genes. Among these tank water samples, Enterococcus spp. from 5 (25%) samples harbored a single virulence gene and 15 (75%) samples were harboring two or more virulence genes. The significance of these strains in terms of health implications remains to be assessed. The potential sources of these strains need to be identified for the improved management of captured rainwater quality. Finally, it is recommended that Enterococcus spp. should be used as an additional fecal indicator bacterium in conjunction with E. coli for the microbiological assessment of rainwater tanks.     

Species distribution,antibiotic resistance and virulence traits in enterococci from meat in Tunisia

Antimicrobial resistance and the mechanisms implicated were studied in 119 enterococci from 105 meat samples from Tunisian markets. Almost 24.5% of recovered enterococci showed resistance against four or more antimicrobial agents and these isolates were identified to the species level. Enterococcus faecalis was the most prevalent species (41%). High percentages of erythromycin and tetracycline resistances were found among our isolates, and lower percentages were identified to aminoglycosides, ciprofloxacin and chloramphenicol. All tetracycline-resistant isolates carried the tet(M) and/or tet(L) genes. The erm(B) gene was detected in 78.5% of erythromycin-resistant isolates, ant(6)-Ia gene in 58.8% of streptomycin-resistant isolates, and cat(A) gene in one chloramphenicol-resistant isolate. Forty-eight isolates carried the gelE gene and exhibited gelatinase activity. The hyl and esp genes were detected in one and three Enterococcus faecium isolates, respectively. Streptomycin-resistant isolates showed a high genetic diversity by PFGE and MLST. Meat might play a role in the spread through the food chain of enterococci with these virulence and resistance characteristics to humans.     

Prevalence,characterization and antibiotic resistance of enterococci from traditional cheeses in Turkey

The aim of the present work is to provide information about Enterococcus strains isolated from traditional Turkish cheese samples in Ankara (Turkey), focusing on their prevalence, phenotypic and genotypic characteristics, and antibiotic resistance. A total of 213 probable enterococcal isolates isolated from 215 samples were identified by phenotypic and genotypic methods. As a result of 16S rDNA sequence analysis, 88 of the 213 enterococci strains were identified as Enterococcus faecium and 125 as Enterococcus faecalis. The E. faecalis strains (58.7%) were identified as the dominant species isolated from cheese samples in Turkey. The 213 Enterococcus strains were tested for susceptibility to 12 different antimicrobial agents. The resistance phenotype were as follow: nalidixic acid (100%), kanamycin (98.6%), rifampicin (78.4%), ampicillin (48.8%), ciprofloxacin (45.5%), erythromycin (18.8%), tetracycline (11.7%), penicillin G (5.6%), chloramphenicol (4.2%), gentamycin (3.8%) and streptomycin (1.4%). None of the strains was resistant to vancomycin. E. faecium strains showed more resistant phenotypes than E. faecalis strains as shown by the antibiotic resistance levels. It was also observed that the resistance of E. faecium and E. faecalis strains against the antibiotics was statistically significant (p ? 0.05). In total, 100% of E. faecium and 88.8% of E. faecalis strains were resistant to multiple drugs.     

Prevalence of Salmonella serotypes S. Enteritidis and S. Typhimurium in poultry and poultry products

Salmonella continues to be a major food safety and public health threat. In the present study, Salmonella enterica subsp. enterica serotypes Enteritidis (SE) and Typhimurium (ST) were isolated from poultry and characterized for virulence, antimicrobial susceptibility, and biofilm formation. Prevalence of Salmonella serotypes in poultry was 3.35%; predominant serotypes isolated were S. Enteritidis (68.1%) and S. Typhimurium (31.8%). Source-wise, Salmonella were isolated from retail market chicken meat (4.8%), live chicken at farm (2.5%), and table eggs (2.1%). Salmonella isolates produced invA gene of 284 bp (100%), spvR gene of 310 bp (77.27%), spvC gene of 571 bp (22.72%), and stn gene of 260 bp (100%) as virulence/ pathogenicity determinants. Salmonella isolates exhibited resistance to common antimicrobials; 72.7% isolates showed multiple resistance (≥3 antimicrobial class), highest resistance was observed for polymyxin-B (81.8%) followed by nalidixic acid (72.7%), colistin (59.1%), ampicillin/tetracyline (45.5%), ampicillin + sulbactam (40.9%), cefodroxil (18.2%), streptomycin (9.1%), and cefazidine/ceftriaxone-tazobactam (4.5%). Multiple antimicrobial resistance (MAR) index of poultry Salmonella isolates ranged from 0.11 to 0.35; wherein, 59.1% isolates showed MAR of >0.2. About 81.8% Salmonella isolates produced biofilm and were categorized as strong (13.6%), moderate (45.4%), and weak (22.7%) biofilm producers. Occurrence of antimicrobial resistant virulent Salmonella strains in poultry requires implementation of suitable strategies so as to protect the public health.     

Prevalence and characterization of antibiotic resistant Enterococcus faecalis in French cheeses

Prevalence of enterococci and antibiotic resistance profiles of Enterococcus faecalis was analyzed in 126 French cheeses from retail stores. Forty-four percent of pasteurized or thermised-milk cheeses, and up to 92% of raw-milk cheeses contained detectable enterococci. A total of 337 antibiotic resistant enterococci were isolated in 29% and 60% of pasteurized-milk and raw-milk cheeses, respectively. E. faecalis was the predominant antibiotic resistant species recovered (81%), followed by Enterococcus faecium (13%), and Enterococcus durans (6%). The most prevalent antibiotic resistances were tetracycline (Tet) and minocycline (Min), followed by erythromycin (Ery), kanamycin (Kan) and chloramphenicol (Cm). The most common multiple antibiotic resistance phenotype was Cm Ery Kan Min Tet. The occurrence of antibiotic genes, as searched by PCR, was 100 % for aph3′IIIa, 96 % for ermB, 90 % for tetM and 80 % for catA in isolates resistant to Kan, Ery, Tet or Cm, respectively. MLST analysis of 30 multidrug resistant E. faecalis revealed that ST19, CC21, CC25 and CC55 isolates were the most common in cheeses. In conclusion, as in many other European countries, French cheeses do contain enterococci with multiple antibiotics resistances. However, low occurrence of high-level gentamicin resistant or sulfamethoxazole/trimethoprim-resistant enterococci and absence of vancomycin- or ampicillin- resistant enterococci indicate that cheeses cannot be considered as a major reservoir for nosocomial multi-drug resistant enterococci.     

Identification of virulence factors in contact lens associated bacteria: A physiological approach

PurposeWearing contact lens requires awareness about possible contaminants, the causative agents of multiple complications. The present study focused on identification of potential pathogens and presence of virulence associated markers in contact lens associated bacteria.MethodsBacterial contaminants were isolated from contact lenses or cleaning solutions collected from University students. Isolates were identified using conventional methods followed by 16S rRNA gene sequencing and screened for the presence of virulence factors which included capsular presence, adhesion, serum resistance, iron chelation, haemagglutination and hemolysis. Moreover, antibiotic resistance profile was also monitored.ResultsContamination was observed in 79% (45 of 57) of lenses. Based on 16S rRNA sequencing Bacillus sp. was found to be most abundant (26%). The presence of at least three pathogenic characteristics was recorded in 75.8% isolates. Among the pathogenic characteristics, capsule presence was found to be the most prevalent character (73%) followed by hemolysin production (65%), serum resistance (61%), haemagglutination (56%), iron chelation (50%) and polystyrene adherence (42%). Multiple antibiotic resistance was recorded in 66.13% isolates. Cluster analysis on the basis of virulence markers separated all isolates in two groups. Potential pathogens and non-pathogens were found to be equally frequent among contaminants of contact lens cases.ConclusionThe present work provides evidence that pathogenic bacteria can adhere and survive in contact lens or lens solution. It highlights the need for the development of new methods to protect contact lenses and lens care accessories. Drugs targeting capsule formation may offer a good option for treatment or use in cleaning solution.     

Monitoring of radical scavenging compounds from oxidized lipids (RSOL) by 2,4-dinitrophenylhydrazine (DNPH) derivatization in thermally oxidized linoleic acid and lard systems

From February to October 2011, 209 samples of retail raw meat and fishery products were randomly obtained from 41 grocery stores in Korea and cultured for the presence of Enterococcus spp. Ninety-six enterococcal isolates were recovered from 76 samples, with contamination rates ranging from 18.5% in fishery product samples to 43.9% in chicken samples. Antimicrobial disk susceptibility testing was conducted according to the Clinical and Laboratory Standards Institute protocol. The antibiotic resistance rates of the 96 enterococci isolates were as follows: tetracycline (TE) 77.1%; erythromycin (E) 50%; rifampin (RD) 44.8%; and vancomycin (VAN) 9.4%. Disk diffusion showed that 9 isolates were resistant to vancomycin. The minimum inhibitory concentration (MIC) was ≥32 μg/mL for all 9 isolates, and all were resistant to vancomycin. Among the 9 vancomycin-resistant enterococci (VRE) identified, the vanA gene was carried by 1 Enterococcus durans strain and the vanB gene was carried by 2 Enterococcus faecium, and 1 Enterococcus hirae strains. Further genotyping of the VRE isolates using pulsed-field gel electrophoresis (PFGE) revealed extensive heterogeneity. The VRE were recovered from various animal sources with a particularly high prevalence of E. faecium found in fishery products and chicken.     

Detection of virulence determinants by real time PCR in E. coli isolated from mutton

Ninety three Escherichia coli isolates belonging to 35 serotypes isolated from market mutton were tested to find out the prevalence of virulence determinants, Verotoxin 1 (VT1), Verotoxin 2 (VT2), Intimin (eae) genes and enterohemolysin production. Real Time PCR based detection was carried out for virulence genes using SYBR green format and amplicons were confirmed by melt curve analysis. Prevalence of VT1 gene in these isolates was much higher (38.70%) on the other hand, that of VT2 gene was nil (0%) while that of eae was very low (3.22%). Enterohemolysin production was found in 31.18% isolates when tested on washed sheep blood agar supplemented with CaCl₂. All enterohemolysin producing isolates were also positive for the VT1 gene.     

Characteristics of virulent vibrio parahaemolyticus isolated from Oregon and Washington

Thirty-four virulent strains of Vibrio parahaemolyticus containing tdh and/or trh genes isolated from Oregon and Washington coastal water were analyzed for O-group antigens and urease activity, and by pulsed-field gel electrophoresis. Six O serotypes (O1, O3, O4, O5, O10, and O11) were identified among the isolates, with the O5 group (19 isolates) being the most prevalent, followed by the 01 group (9 isolates). Nearly all (33 of 34) isolates were capable of producing urease, which reaffirmed the correlation between urease production and virulence factors of V. parahaemolyticus strains isolated from the Pacific Northwest. Pulsed-field gel electrophoresis analysis with NotI and SfiI digestions of the 34 V. parahaemolyticus isolates plus five clinical strains revealed 22 patterns (NlS1 to N20S22), with NIS1 (25.6%) being the most common, followed by N2S2 (10.3%). Nine Oregon isolates were grouped with a 1997 Oregon outbreak strain (027-1C1) with the same serotype (O5), virulence factors (tdh+ and trh+), and genotype (N S 1). Three Washington isolates were found to share the same serotype (O1), virulence factors (tdh' and trh'), and genotype (N2S2) with a 1997 Washington outbreak strain (10293). The repetitive isolation of virulent strains of V. parahaemolyticus identical to clinical strains involved in previous outbreaks indicates potential hazards associated with oyster consumption. These data may be useful in risk assessment of V. parahaemolyticus infections associated with raw oyster consumption in Oregon and Washington.     

Biodiversity of Bacteriocin-Producing Lactic Acid Bacteria from Mexican Regional Cheeses and their Contribution to Milk Fermentation

The aim of this work was to examine the biodiversity of bacteriocin-producing lactic acid bacteria from homemade cheeses produced in Veracruz (México) and assess their contribution as adjunct cultures in dairy products. Ninety-three presumptive bacteriocinogenic strains were detected by direct antagonism assays and 29 of them were active against Enterococcus faecalis NRRL-B537, Listeria innocua 062 AST, or Listeria monocytogenes ATCC19115 by the well diffusion test using cell-free supernatants, adjusted to pH 6.0 to exclude inhibition by organic acids. Positive isolates were identified by partial sequencing of the 16s rDNA as Pediococcus acidilactici (four isolates), Enterococcus faecium (17 isolates), Lactobacillus plantarum (six isolates) and Lactobacillus fermentum (two isolates). RAPD-PCR discriminated seven groups with a 50% similarity and revealed the presence of the same isolates. The coding genes for the synthesis of plantaricin EF, plantaricin JK, plantaricin N, plantaricin NC8 and the inducing peptide plantaricin A were detected by PCR in L. plantarum. Similarly, enterocin P and pediocin PA-1 genes were amplified from Enterococcus and Pediococcus genomic DNA, respectively. Overall, co-culturing of bacteriocin producing Lactobacillus and Pediococcus strains with the dairy starter Lactococcus lactis IPLA947 did not interfere with milk acidification. Lactose consumption, acidification rate and production of lactic acid were unchanged. Nonetheless, higher levels of acetic acid, ethanol and succinic acid were detected depending on the strain. Our results demonstrate the diversity of bacteriocinogenic species in homemade Mexican cheeses which may be used as adjunct cultures to enhancing safety of this well-appreciated cheese while providing a richer range of metabolites.     

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.     

STUDIES ON OCCURRENCE AND CHARACTERIZATION OF CLOSTRIDIUM PERFRINGENS FROM SELECT MEATS

A study was undertaken to assess the prevalence of Clostridium perfringens in meat and to characterize the isolates obtained in the study for virulence factors. A total of 211 meat samples of different animals (70 each of buffalo and goat and 71 of poultry) were screened and the highest occurrence of C. perfringens was observed in goat (91.4%) followed by poultry (70.4%) and buffalo (65.7%). Among the 116 isolates (buffalo‐32, goat‐37 and poultry‐45) of C. perfringens screened for the presence of enterotoxin gene by PCR, 9.3, 32.4 and 15.5% isolates of buffalo, goat and poultry, respectively, were found to possess enterotoxin gene. Screening of 15 enterotoxin gene possessing isolates for verocytotoxicity revealed that 12 isolates exhibited cytopathic effect while 3 isolates did not show any cytopathic effect in spite of the presence of enterotoxin gene. A total of 115 C. perfringens isolates were screened for other virulence markers, i.e., lecithinase and hemolysin. The results revealed that the majority of the isolates expressed these activities. Antibiogram studies of C. perfringens isolates using 16 antibiotics displayed multidrug resistance. The isolates showed resistance to streptomycin, ceftazidime, colistin sulfate, cephalothin, ampicillin and gentamicin. Whereas 100% sensitivity to ciprofloxacin, ofloxacin and nitrofurantoin was seen, moderate sensitivity was observed with tetracycline and sulfatriad.     

Microbiological and physicochemical properties of farm bulk tank milk and antimicrobial resistance of its dominant bacteria

This study determined the antibiotic resistance of the dominant bacteria in the 85 farm BTMs according to the guidelines recommended by the epidemiological cutoff values in the EUCAST. In addition, some physicochemical and microbiological properties of farm BTMs were investigated. The milk samples were divided into two groups according to their SCC values. The milk samples with higher SCC than 400,000 cells mL⁻¹ were further examined bacteriologically, and the antibiotic resistance of isolates was determined. The average TAMB value was 6.34 log CFU/mL in farm BTM. It was found that high-SCC values did not affect other physicochemical properties of BTM samples, such as fat, protein and total solids, except for lactose content. Seventy-two strains were isolated from 45 bulk milk samples. The most prevalent bacteria were Enterococcus spp. (23.61%). The other isolates were Citrobacter spp. (12.5%), Staphylococcus spp. (12.51%), Serratia spp. (11.12%), Klebsiella spp. (9.72%), Bacillus spp. (9.72%), and Enterobacter spp. (8.33%). In antibiotic resistance analysis, 52.6% of Enterobacterales isolates showed cefoxitin resistance, and nine Enterobacterales isolates were determined as the presumptive ESBL producers. None of them was confirmed as ESBL producers. Moreover, MDR was detected in 83.3% of Enterobacter spp. isolates and all Bacillus spp. isolates. The over and inappropriate use of antibiotics in mastitis treatment may cause antibiotic-resistant microorganisms in milk. It was found that 52.7% of the isolated bacteria were MDR, which could pose a risk to public health and food safety, with the consumer's increasing interest in consuming raw milk.     

自贡市肉及其制品中粪肠球菌耐药性毒力基因和多位点序列分析

目的了解四川省自贡市肉及其制品分离粪肠球菌的抗生素耐药性和携带毒力基因情况,以及多重耐药菌株的序列分型(ST)。方法 2013年4月17~21日对147份肉及其制品样品污染的粪肠球菌进行分离鉴定,使用全自动微生物鉴定仪对分离菌株的耐药性进行检验,应用聚合酶链式反应(PCR)方法检测分离到的粪肠球菌携带4种常见毒力基因的情况,并对多重耐药菌株进行多位点序列分型(MLST)研究。结果从147份肉及其制品样品中共分离到65株粪肠球菌,其中耐药菌株比例为58.5%(38/65);分离菌株对四环素的耐药率最高,为41.5%(27/65),对利福平、氯霉素和红霉素也均有较高的耐药率;分离菌株对高浓度链霉素和高浓度庆大霉素的耐药率分别达到了15.4%(10/65)和12.3%(8/65);未分离到对青霉素类(青霉素和氨苄西林)、糖肽类(万古霉素)和脂肽类(达托霉素)抗生素耐药的菌株。4种常见毒力基因(gel E、asa1、esp、cyl A)在65株分离株中均有携带,阳性率分别为56.9%(37/65)、21.5%(14/65)、9.2%(6/65)和7.7%(5/65)。14株多重耐药菌株共有9个MLST型别,包括4株ST16、2株ST81和2株ST480菌株,且相同ST型别的粪肠球菌有相似的耐药谱和毒力基因携带情况。结论四川省自贡市肉及其制品中相同ST型别的粪肠球菌有相似的耐药谱和毒力基因携带情况,应重视肉及其制品中耐药粪肠球菌对公众健康的潜在威胁。     

Antimicrobial‐Resistance Genetic Markers in Potentially Pathogenic Gram Positive Cocci Isolated from Brazilian Soft Cheese

Although most Brazilian dairy products meet high technological standards, there are quality issues regarding milk production, which may reduce the final product quality. Several microbial species may contaminate milk during manufacture and handling. If antimicrobial usage remains uncontrolled in dairy cattle, the horizontal transfer of antimicrobial resistance genes in foodstuffs may be of particular concern for both food producers and dairy industry. This study focused on the evaluation of putative Gram positive cocci in Minas cheese and of antimicrobial and biocide resistance genes among the isolated bacteria. Representative samples of 7 different industrially trademarked Minas cheeses (n = 35) were processed for selective culture and isolation of Gram positive cocci. All isolated bacteria were identified by DNA sequencing of the 16S rRNA gene. Antimicrobial resistance genes were screened by PCR. Overall, 208 strains were isolated and identified as follows: Enterococcus faecalis (47.6%), Macrococcus caseolyticus (18.3%), Enterococcus faecium (11.5%), Enterococcus caseliflavus (7.7%), Staphylococcus haemolyticus (7.2%), Staphylococcus aureus (4.3%), Staphylococcus epidermidis (2.9%), and Enterococcus hirae (0.5%). The genetic markers mecA (78.0%) and smr (71.4%) were the most prevalent, but others were also detected, such as blaZ (65.2%), msrA (60.9%), msrB (46.6%), linA (54.7%), and aacA‐aphD (47.6%). The occurrence of opportunist pathogenic bacteria harboring antimicrobial resistance markers in the cheese samples are of special concern, since these bacteria are not considered harmful contaminating agents according to the Brazilian sanitary regulations. However, they are potentially pathogenic bacteria and the cheese may be considered a reservoir for antimicrobial resistance genes available for horizontal transfer through the food chain, manufacturing personnel and consumers.     

Isolation and Identification of Lactic Acid Bacteria from Fermented Red Dragon Fruit Juices

Abstract: Red dragon fruit or red pitaya is rich in potassium, fiber, and antioxidants. Its nutritional properties and unique flesh color have made it an attractive raw material of various types of food products and beverages including fermented beverages or enzyme drinks. In this study, phenotypic and genotypic methods were used to confirm the identity of lactic acid bacteria (LAB) appeared in fermented red dragon fruit (Hylocereus polyrhizus) beverages. A total of 21 isolates of LAB were isolated and characterized. They belonged to the genus of Enterococcus based on their biochemical characteristics. The isolates can be clustered into two groups by using the randomly amplified polymorphic DNA method. Nucleotide sequencing and restriction fragment length polymorphism of the 16S rRNA region suggested that they were either Enterococcus faecalis or Enterococcus durans. Practical Application: Current research revealed the use of biochemical analyses and molecular approaches to identify the microbial population particularly lactic acid bacteria from fermented red dragon fruit juices.