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.     

Escherichia coli of poultry food origin as reservoir of sulphonamide resistance genes and integrons

The antimicrobial resistance phenotype and genotype, the flanking regions of sulphonamide resistance genes and the integrons were analyzed in 166 Escherichia coli isolates recovered from poultry meat in Tunisia. High percentages of resistance were detected to ampicillin, streptomycin, nalidixic acid, sulphonamide and tetracycline (66-95%), and lower percentages to gentamicin, amoxicillin-clavulanic acid and cefoxitin (1-4%). The bla_TEM, tet(A)/tet(B), aph(3′)-Ia, aac(6′)-Ib-cr, aac(3)-II and cmlA genes were identified in 92, 82, 29, 2, 2 and 7 isolates, respectively. Class 1 and/or class 2 integrons were detected in 52% of E. coli isolates and five different gene cassette arrangements were identified in the variable regions of class 1 integrons, which included antimicrobial resistance determinants. Sixty-eight isolates contained the sul1 gene and 37 of them presented this gene into a class 1 integron structure. The sul3 gene was detected associated with non-classic class 1 integrons in 4 out of 46 sul3-positive isolates. The sul2 gene was detected in 66 isolates, 51 of them were linked to strA/B genes in seven different genetic structures. Seventy-three-per-cent of integron-positive isolates presented resistance to at least five different antimicrobial families versus 38.7% of integron-negative isolates. Our study highlights the role of commensal E. coli isolates from poultry meat as an important reservoir for sulphonamide resistance genes and integrons carrying antimicrobial resistance genes.     

Prevalence,antimicrobial susceptibility,and molecular characterization of Staphylococcus aureus isolated from dairy herds in northern China

Staphylococcus aureus is one of the main pathogens involved in dairy cow mastitis. Monitoring of antibiotic use would prove useful to assess the risk of Staph. aureus in raw milk. The objective of this work was to investigate the prevalence of Staph. aureus strais isolated from raw milk in northern China, and to characterize antimicrobial susceptibility of these strains and their key virulence genes. In total, 195 raw milk samples were collected from 195 dairy farms located in 4 cities of northern China from May to September 2015. Out of 195 samples, 54 (27.7%) were positive for Staph. aureus. Among these 54 samples, 54 strains of Staph. aureus were isolated, and 16 strains were identified as methicillin-resistant Staph. aureus. The strains exhibited high percentages of resistance to penicillin G (85.2%), ampicillin (79.6%), and erythromycin (46.3%). Moreover, 72% of the strains showed resistance to more than one antimicrobial agent. Overall, 63% of penicillin-resistant strains possessed the blaZ gene, and 60% of the erythromycin-resistant strains possessed erm(A), erm(B), erm(C), msr(A), or msr(B) genes with 8 different gene patterns. All isolates resistant to gentamicin, kanamycin, and oxacillin carried the aac6'-aph2”, ant(4')-Ia, and mecA genes, respectively. Two tet(M)-positive isolates carried specific genes of the Tn916-Tn1545 transposon. The most predominant virulence genes were sec, sea, and pvl, which encode staphylococcal enterotoxins (sec and sea) and Panton-Valentine leukocidin, respectively. Thirty-two isolates (59.2%) harbored one or more virulence genes. The majority of Staph. aureus strains were multidrug resistant and carried multiple virulence genes, which may pose a risk to public health. Our data indicated that antimicrobial resistance of Staph. aureus was prevalent in dairy herds in northern China, and that antibiotics, especially penicillin G and ampicillin, to treat mastitis caused by Staph. aureus should be used with caution in northern China.     

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 Aerobic Bacteria Carrying Tetracycline and Sulfonamide Resistance Genes Obtained from a Meat Processing Plant

Microbial contamination in food‐processing plants can play a fundamental role in food quality and safety. The purpose of this study was to investigate aerobic bacteria carrying tetracycline and sulfonamide resistance genes from a meat processing plant as possible sources of meat contamination. One hundred swab samples from surfaces of conveyor belts, meat slicers, meat knives, benches, plastic trays, gloves, and aprons were analyzed. A total of 168 isolates belonging to 10 genera were obtained, including Pseudomonas sp. (n = 35), Acinetobacter sp. (n = 30), Aeromonas sp. (n = 20), Myroides sp. (n = 15), Serratia sp. (n = 15), Staphylococcus sp. (n = 14), Enterobacter sp. (n = 11), Escherichia coli (n = 10), Lactococcus sp. (n = 10), and Klebsiella sp. (n = 8). Of the 168 isolates investigated, 60.7% showed resistance to tetracycline and 57.7% to trimethoprim/sulfamethoxazole. The tetracycline resistance genes tetL, tetA, tetB, tetC, tetE, tetM, tetS, tetK, and tetX were found in the frequency of 7.7%, 6.0%, 4.8%, 4.8%, 3.6%, 3.6%, 3.6%, 1.2%, and 0.6%, respectively. Sulfonamide resistance genes sul1 and sul2 were observed in the frequency of 17.9% and 38.1%, respectively. The tetracycline resistance genes tetX was first found in Myroides sp. This investigation demonstrated that food contact surfaces in a meat processing plant may be sources of contamination of aerobic bacteria carrying tetracycline and sulfonamide antibiotic resistance genes.     

Prevalence and characterization of antimicrobial resistance of foodborne Listeria monocytogenes isolates in Hebei province of Northern China, 2005-2007

A total of 2177 food samples collected from nine cities in northern China during 2005 to 2007 were screened for the presence of Listeria monocytogenes. All L. monocytogenes isolates were subjected to serotyping, antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), as well as PCR screening to identify genes responsible for tetracycline resistance [tet(L), tet(M), tet(K), tet(S) and tet(B)], transposon Tn916, and class 1 integron. Contamination with L. monocytogenes was detected in 4.13% (90/2177) of the total samples representing various food products. The pathogen was mainly isolated from frozen food made of wheat flour or rice products (26/252, 10.32%) and raw meat products (46/733, 6.28%). Besides, 3.31% (10/302) of cooked meat, 1.17% (4/343) of seafood, 0.98% (2/204) of non-fermented bean products and 0.62% (2/323) of vegetables samples were contaminated by this bacterium. The L. monocytogenes isolates belonged to five serotypes (1/2a, 1/2b, 1/2c, 4b, and 3a), with serotype 1/2a being dominant (48.88%). Antimicrobial resistance was most frequently observed for ciprofloxacin (17.8%), tetracycline (15.6%) and streptomycin (12.2%). Overall, resistance was observed against 14 out of 18 antimicrobials tested while multiple resistances occurred among 18.9% (17/90) isolates. Interestingly, two isolates were resistant to more than five antimicrobials. Among 14 tetracycline-resistant isolates, 13 carried tet(M) gene including nine possessing Tn916, and one harbored tet(S) gene. PFGE analysis revealed genetic heterogeneity among individual serotypes as well as scattered occurrence of some genotypes without any clear-cut correlation to source or food type. The widespread distribution of epidemiologically important serotypes (1/2a, 1/2b and 4b) of L. monocytogenes, and their resistance to commonly used antibiotics indicate a potential public health risk. Our data also indicate that L. monocytogenes could act as a reservoir of mobile tet genes along the food chain.     

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.     

Characterization of antibiotic resistant enterococci isolated from untreated waters for human consumption in Portugal

Untreated drinking water is frequently overlooked as a source of antibiotic resistance in developed countries. To gain further insight on this topic, we isolated the indicator bacteria Enterococcus spp. from water samples collected in wells, fountains and natural springs supplying different communities across Portugal, and characterized their antibiotic resistance profile with both phenotypic and genetic approaches. We found various rates of resistance to seven antibiotic families. Over 50% of the isolates were resistant to at least ciprofloxacin, tetracyclines or quinupristin-dalfopristin and 57% were multidrug resistant to ≥ 3 antibiotics from different families. Multiple enterococcal species (E. faecalis, E. faecium, E. hirae, E. casseliflavus and other Enterococcus spp) from different water samples harbored genes encoding resistance to tetracyclines, erythromycin or gentamicin [tet(M)-46%, tet(L)-14%, tet(S)-5%, erm(B)-22%, aac(6´)-Ie-aph(2″)-12%] and putative virulence factors [gel-28%, asa1-16%]. The present study positions untreated drinking water within the spectrum of ecological niches that may be reservoirs of or vehicles for antibiotic resistant enterococci/genes. These findings are worthy of attention as spread of antibiotic resistant enterococci to humans and animals through water ingestion cannot be dismissed.     

Quantity of the tetracycline resistance gene tet(M) differs substantially between meat at slaughterhouses and at retail

Abstract: Concentrations of the tetracycline resistance gene tet(M) per square centimeter were assessed in meat from the slaughterhouse (n = 100) and from retail (n = 100) by real‐time quantitative PCR. The study revealed a substantial contamination of retail meat with the tetracycline resistance gene tet(M), with a mean of 4.34 log copies per cm² fasces in chicken and 5.58 log copies per cm² fasces in pork. Quantitative resistance gene analysis provides an interesting tool for risk assessment and is becoming increasingly important. For both chicken and pork, tet(M) concentrations were significantly higher in meat at retail, compared to meat at slaughter. Cultural investigations revealed substantial differences in the prevalence of listeria and enterococci, and of E. coli and coliforms, between meat at slaughter (n = 500) and at retail (n = 500). However, the differences in the prevalence of 2 investigated groups of potential tet(M)‐carriers (enterococci, listeria) could not sufficiently explain the differences in tet(M) concentrations, since increasing concentrations of tet(M) were accompanied by decreasing prevalences of these potential tet(M)‐carriers. The percentage of tetracycline susceptible indicator bacteria (E. faecalis, E. coli) did not differ between meat at slaughter and meat at retail. Higher concentrations of tet(M) at retail might correlate with the proliferation of other genera than enterococci and listeria, but there is also a reason to discuss whether secondary contaminants might carry tet(M) more often than the primary flora of meat. Practical Application: We successfully applied the direct quantitative monitoring of resistance genes in meat, which generally might aid as a useful and rapid additional tool for risk assessment. We know that bacteria provide a large pool of resistance genes, which are widely shared between each other—the larger the pool is, the more genes might be exchanged. Thus, in terms of resistance gene monitoring, we should sometimes overcome the restricted view on single bacteria and look at the gene pool, instead.     

Characteristics of Escherichia coli from raw vegetables at a retail market in the Czech Republic

A large epidemic caused by shigatoxigenic Escherichia coli (E. coli) in spring 2011 in Germany resulted in reduction of trust in the health safety of raw vegetables and sprouted seeds. This study focused on the detection and characterization of E. coli in raw vegetables and sprouted seeds sold in the Czech Republic. Out of 91 samples, 24 (26.4%) were positive for the presence of E. coli. Resistance to antimicrobial agents was determined by the disk diffusion method and E-test. Polymerase chain reaction was used for the detection of selected genes encoding virulence — eaeA, hly, stx₁, and stx₂ and genes encoding resistance to tetracycline — tet(A), tet(B), tet(C), and tet(G) and to β-lactams — bla_TEM, bla_SHV, and bla_CTX. The bla_TEM gene was detected in two isolates, the tet(B) gene in three and tet(A) in one isolate. No hly, stx₁, or stx₂ genes were present, but the eaeA gene was found in three (11.1%) isolates from imported vegetables. These isolates can be considered as potentially enteropathogenic. Results of this study show that raw vegetables and sprouted seeds sold in the retail market can represent a potential risk for consumers.     

Identification and functional traits of lactic acid bacteria isolated from Ciauscolo salami produced in Central Italy

Lactic acid bacteria (LAB) from Ciauscolo salami produced in Marche Region of Central Italy, and LAB strains belonging to our laboratory collection were examined for their capability to survive at low pH and bile, to adhere to Caco-2 cells, and for antibiotic resistance. LAB from Ciauscolo were identified by ARDRA and RAPD-PCR. Our study showed that all LAB strains had good adaptation to gastric juice and moderate tolerance to bile. The adhesiveness was variable among strains but significantly lower in LAB from food. Antibiotic resistance was broadly spread among food strains, with level of resistance exceeding 15% for all the antibiotics tested. The resistance determinants erm(B) and tet(M) were found in nine strains of food origin (21.4%) while tet(L) in one strain of our collection (5%). Our work suggests that fermented foods are valuable sources of bacterial strains with functional traits of intestinal lactobacilli. These bacteria may be further studied for their use in probiotic applications.     

Species Diversity and Pheno‐ and Genotypic Antibiotic Resistance Patterns of Staphylococci Isolated from Retail Ground Meats

The presence and species diversity of staphylococci in 250 ground beef and lamb meat samples obtained from Diyarbakir, Turkey were investigated. The presence of the 16S rRNA gene, mecA, nuc, pvl, and femA was analyzed by multiplex PCR. Pheno‐ and genotypic antibiotic resistance profiles of 208 staphylococci isolates were established. Of the ground beef and ground lamb samples, 86.4% and 62.4% were positive for staphylococci, respectively. Staphylococcus aureus, S. saprophyticus, S. hominis, S. lentus, S. pasteuri, S. warneri, S. intermedius, and S. vitulinus made up 40.8%, 28.8%, 11%, 3.8%, 3.8%, 2.4%, 2.4%, and 2.4% of isolates, respectively. Of the 85 S. aureus isolates, 40%, 47%, and 5.8% carried femA, mecA, and pvl, respectively, whereas the corresponding rates for the 118 coagulase‐negative staphylococci (CoNS) were 0%, 10.1%, and 0%, respectively. We determined from the 208 isolates, the highest antibiotic resistances were to tetracycline and oxytetracycline (85.5%), followed by penicillin (51.4%), novobiocin (45.6%), ampicillin (39.9%), and doxycycline (31.7%), using the Clinical and Laboratory Standards Inst. (CLSI) method. All isolates were sensitive to gentamycin, ofloxacin, and tobramycin, but 2.3% of the S. aureus isolates had resistance to vancomycin. The staphylococci isolates carried tet(K), blaZ, tet(L), tet(W), cat, tet(S), tet(M), ermB, ermA, and ermC antibiotic resistance genes at rates of 59%, 51.7%, 36.9%, 31.8%, 27.2%, 27.2%, 24.4%, 18.1%, 7.9%, and 3.9%, respectively.     

Molecular characterization of antimicrobial resistance in Klebsiella pneumoniae isolated from Brazilian dairy herds

In this observational study, phenotypic and genotypic patterns of antimicrobial resistance (AMR) in Klebsiella pneumoniae isolated from intramammary infections, clinical mastitis, fresh feces, rectal swabs, animal hindlimbs, and bulk tank milk samples from Brazilian dairy herds were investigated. In addition, we identified specific genetic variants present among extended-spectrum β-lactamase (ESBL) producers. We obtained 169 isolates of K. pneumoniae from 2009 to 2011 on 24 Brazilian dairy farms located in 4 Brazilian states. The AMR profile of all isolates was determined using disk-diffusion assays. The antimicrobial panel included drugs commonly used as mastitis treatment in Brazilian dairy herds (gentamicin, cephalosporins, sulfamethoxazole-trimethoprim, tetracycline) as well as antimicrobials of critical importance for human health (meropenem, ceftazidime, fluoroquinolones). The K. pneumoniae isolates resistant to tetracycline, fluoroquinolones, sulfamethoxazole-trimethoprim, or chloramphenicol were screened for presence of drug-specific AMR genes [tet, qnr, aac(6')-Ib, floR, catA2, cm1A, dfr, sul] using PCR. In addition, we identified ESBL genes present among ESBL-producers by using whole genome sequencing. Genomes were assembled and annotated, and patterns of AMR genes were investigated. Resistance was commonly detected against tetracycline (22.5% of all isolates), streptomycin (20.7%), and sulfamethoxazole-trimethoprim (9.5%). Antimicrobial resistance rates were higher in K. pneumoniae isolated from intramammary infections in comparison with isolates from feces (19.2 and 0% of multidrug resistance in intramammary and fecal isolates, respectively). In contrast, no difference in AMR rates was observed when contrasting hind limbs and isolates from intramammary infections. The genes tetA, sul2, and floR were the most frequently observed AMR genes in K. pneumoniae resistant to tetracycline, sulfamethoxazole-trimethoprim, and chloramphenicol, respectively. The tetA gene was present exclusively in isolates from milk. The genes bla_CTX-M8 and bla_SHV-108 were present in 3 ESBL-producing K. pneumoniae, including an isolate from bulk tank milk. The 3 isolates were of sequence type 281 and had similar mobile genetic elements and virulence genes. Our study reinforced the epidemiological importance and dissemination of bla_CTX-M-8 pST114 plasmid in food-producing animals in Brazil.     

Prevalence,Antimicrobial Resistance,and Virulence Characteristics of mecA‐Encoding Coagulase‐Negative Staphylococci Isolated from Soft Cheese in Brazil

Coagulase‐negative staphylococci (CoNS), which are generally neglected as foodborne bacteria, are emerging as significant opportunistic pathogens that may be highly resistant to available antimicrobial drugs. In this study, antimicrobial susceptibility patterns, mecA gene occurrence, and virulence‐associated characteristics were evaluated in CoNS isolated from soft cheese in Brazil. A total of 227 bacterial isolates were recovered from 35 cheese samples belonging to 5 batches with 7 different trademarks. The CoNS counts ranged from 10⁶ to 10⁷ CFU/g. High antimicrobial resistance percentages were observed for oxacillin (76.2%), penicillin (78.5%), erythromycin (67.8%), gentamicin (47.2%), clindamycin (35.7%), rifampicin (26.8%), azithromycin (14.7%), tetracycline (14.7%), levofloxacin (14.2%), and sulfamethoxazole‐trimethoprim (11.9%). A low antimicrobial resistance percentage was observed for chloramphenicol (2.3%), and all of the tested bacteria were susceptible to vancomycin and linezolid. In total, a multiple antibiotic resistance (MAR) index of >0.2 was observed for 80.6% of the isolated CoNS. However, the MAR index ranged from 50% to 92.6% when only bacterial cheese isolates belonging to the same trademark were considered. Regarding to the prevalence of CoNS carrying mecA gene, 81.5% of the isolated strains were mecA⁺, and 76.2% of these were phenotypically resistant to oxacillin. Three isolates carried the enterotoxin A gene (sea), 29.5% produced biofilm in a laboratory test, and α‐ or ß‐hemolysis were observed for 3% and 5.2%, respectively. This study highlights the extent of the antimicrobial resistance phenomenon in neglected foodborne microorganisms and the potential public health risks that are related to the consumption of CoNS‐contaminated soft cheese.     

A survey of the occurrence and properties of methicillin‐resistant Staphylococcus aureus and methicillin‐resistant Staphylococcus intermedius in water buffalo milk and dairy products in Turkey

Antimicrobial resistance, β‐lactamase activity and mecA gene of Staphylococcus aureus and Staphylococcus intermedius isolated from raw water buffalo milk and dairy products in Turkey were determined. All strains showed resistance to at least one antibiotic but none was resistant to vancomycin. Of the 97 S. aureus and 35 S. intermedius strains, 9 (9.2%) and 2 (5.7%) were resistant to oxacillin and harboured mecA gene. β‐lactamase activity of 13.4% and 5.7% of S. aureus and S. intermedius strains was positive, respectively. Overall, 2.5% and 0.55% of the samples were contaminated with methicillin‐resistant S. aureus and S. intermedius, respectively.     

Determination of Enterotoxigenic and Methicillin Resistant Staphylococcus aureus in Ice Cream

The aim of this study was to determine the prevalence of enterotoxigenic and methicillin‐resistant Staphylococcus aureus in ice creams. After culture‐based identification of isolates, the presence of 16S rRNA and nuc was confirmed by mPCR. S. aureus was identified in 18 of 56 fruity (32.1%), 4 of 32 vanilla (12.5%), and 1 of 12 chocolate (8.3%) ice creams. S. aureus was identified as 38 isolates in 23 ice cream samples by culture‐based techniques, but only 35 isolates were confirmed by PCR as S. aureus. To determine the enterotoxigenic properties of PCR‐confirmed S. aureus isolates, a toxin detection kit was used (SET RPLA®). Of the 12 enterotoxigenic S. aureus isolates, 9 SEB (75%), 1 SED (8.3%), 1 SEB+SED (8.3%), and 1 SEA+SEB+SED (8.3%) expressing isolates were found. The presence of enterotoxin genes (sea, seb, sed) was identified in 13 (37.1%) out of 35 isolates by the mPCR technique. In the ice cream isolates, the sea, seb, and sed genes were detected: 1 sea (7.6%), 9 seb (69.2%), 1 sed (7.6%), 1 seb+sed (7.6%), and 1 sea+seb+sed (7.6%), respectively. The sec gene was not detected in any of these isolates. One of the 35 (2.8%) S. aureus strain was mecA positive.     

上海市闵行地区副溶血性弧菌分子流行病学特征分析

目的 分析上海市闵行地区副溶血性弧菌临床和食源性分离株的分子流行病学特征,了解本地区副溶血性弧菌的流行规律.方法 对2007-2010年临床来源菌株(食物中毒患者和散发腹泻病例)以及食源性样品中分离的184株副溶血性弧菌进行血清分型、耐热直接溶血素基因(tdh)和耐热相关溶血素基因(trh)检测.对102株临床来源O3:K6型菌株及41株食源性样品来源株,应用脉冲场凝胶电泳(PFGE)进行分子分型和溯源分析.结果 在调查的52起副溶血性弧菌引起的食物中毒事件中,tdh阳性、trh阴性的O3:K6血清型引起的有46起(88.5％),tdh阳性、trh阴性的O4:K8血清型引起的有5起(9.6％).在散发腹泻病例中,tdh阳性、trh阴性的O3:K6、O4:K8和O3:KUT菌株分别占60.3％(38/63)、19.0％(12/63)和15.9％(10/63).41株食源性样品株分属9种O血清群,未见O3:K6和O4:K8血清型菌株,而且tdh和trh均为阴性.PFGE聚类分析显示闵行地区食物中毒O3:K6分离株中存在着遗传关系密切相关的优势流行克隆,散发腹泻患者O3:K6分离株中存在着与食物中毒优势株相同谱型,而所有食源性样品分离株与临床患者来源株亲缘关系都较远.结论 临床和食源性样品来源的副溶血性弧菌在血清分型、毒力基因和分子特征等方面存在显著差异,一大群遗传关系密切、tdh阳性、trh阴性的O3:K6血清型菌株在闵行地区呈优势流行.     

First report of methicillin‐resistant Staphylococcus aureus in tank cultured dusky kob (Argyrosomus japonicus), and evaluation of three phenotypic methods in the detection of MRSA

This study was conducted to ascertain the occurrence of methicillin‐resistant Staphylococcus aureus (MRSA) in marine finfish (Argyrosomus japonicus) harvested from the wild and two re‐circulatory aquaculture systems, and evaluating the reliability of three phenotypic methods in the detection of methicillin resistance. A total of 120 dusky kob fish were sampled for S. aureus detection using conventional methods and confirmed by polymerase chain reaction (PCR) targeting the nuc gene. Methicillin resistance was determined by molecular detection of the mecA gene. Using mecA as the defining standard, the specificities and sensitivities of cefoxitin disc diffusion, oxacillin screen agar, and growth of S. aureus on Brilliance MRSA II Agar were evaluated. A total of 321 presumptive S. aureus isolates were recovered by culture, out of which 202 (62.9%) were confirmed by PCR. Of these, 33 (16.3%) strains were mecA positive while 169 (83.7%) were mecA negative. The sensitivities and specificities of MRSA detection was 93.9 and 91.7%, 81.8 and 92.3%, and 87.9 and 94.1% for cefoxitin disc, oxacillin screen agar test, and Brilliance MRSA II agar, respectively. This is so far the first report of MRSA in dusky kob aquaculture in South Africa. In the absence of molecular techniques, cefoxitin disc diffusion test is recommended along with any other phenotypic method to improve MRSA detection from samples of veterinary origin.

Practical implications

Methicillin‐resistant Staphylococcus aureus currently presents one of the greatest challenges for medical research worldwide, as well as is one of the most important causes of bacteria gastroenteritis due to preformed toxins in foods. There is a dearth of knowledge on marine foods as carriers/sources of MRSA infection. There are also major discrepancies obtained with MRSA detection methods, making effective detection of this pathogen complicated. The results from this study show that healthy aquaculture fish are reservoirs of MRSA, thus it is necessary to regularly monitor marine foods. Cefoxitin disc diffusion test is recommended as the preferred method for detection of MRSA from fish/food samples where molecular methods are lacking.     

Characterization of Antibiotic Resistance Genes from Lactobacillus Isolated from Traditional Dairy Products

Lactobacilli are widely used as starter cultures or probiotics in yoghurt, cheese, beer, wine, pickles, preserved food, and silage. They are generally recognized as safe (GRAS). However, recent studies have shown that some lactic acid bacteria (LAB) strains carry antibiotic resistance genes and are resistant to antibiotics. Some of them may even transfer their intrinsic antibiotic resistance genes to other LAB or pathogens via horizontal gene transfer, thus threatening human health. A total of 33 Lactobacillus strains was isolated from fermented milk collected from different areas of China. We analyzed (1) their levels of antibiotic resistance using a standardized dilution method, (2) their antibiotic resistance gene profiles by polymerase chain reaction (PCR) using gene‐specific primers, and (3) the transferability of some of the detected resistance markers by a filter mating assay. All Lactobacillus strains were found to be resistant to vancomycin, but susceptible to gentamicin, linezolid, neomycin, erythromycin, and clindamycin. Their susceptibilities to tetracycline, kanamycin, ciprofloxacin, streptomycin, quinupristin/dalfopristin, trimethoprim, ampicillin, rifampicin, and chloramphenicol was different. Results from our PCR analysis revealed 19 vancomycin, 10 ciprofloxacin, and 1 tetracycline‐resistant bacteria that carried the van(X), van(E), gyr(A), and tet(M) genes, respectively. Finally, no transferal of the monitored antibiotic resistance genes was observed in the filter mating assay. Taken together, our study generated the antibiotic resistance profiles of some milk‐originated lactobacilli isolates and preliminarily assessed their risk of transferring antibiotic gene to other bacteria. The study may provide important data concerning the safe use of LAB.     

Phenotypic and genetic characterization of antimicrobial resistance in Salmonella serovars isolated from retail meats in Alberta, Canada

This study determined the prevalence of Salmonella serovars, antimicrobial resistance (AMR) and resistance genes in Salmonella isolated from retail meats purchased in Alberta, Canada. Samples were collected during one year period (May 2007–April 2008) on weekly basis from 19 census divisions in Alberta. A total of 564 samples including chicken (n = 206), turkey (n = 91), beef (n = 134) and pork (n = 133) were purchased. Salmonella were recovered from chicken (40%), turkey (27%) and pork (2%) samples and was not found in ground beef. A total of 21, 8, and 3 different serovars were recovered from chicken, turkey and pork meats, respectively. Salmonella Hadar was most common in chicken whereas S. Heidelberg was common in turkey meat. Overall 29% (32/110) of isolates were susceptible to tested antimicrobials and resistance to ciprofloxacin, amikacin and nalidixic acid was not found in any isolate. Multiresistance (≥2 antimicrobials) was found in 56% of isolates. Resistance to amoxicillin–clavulanic acid (AMC), ceftiofur (TIO), and ceftriaxone (CRO) was found in about 21% of chicken and 25% of turkey isolates. Resistance to either of tetracycline (TET), streptomycin (STR) or ampicillin (AMP) was unconditionally associated with S. Hadar but resistance to either of TET, AMP, AMC, TIO, CRO or cefoxitin was associated with S. Heidelberg. The strA/B (42% isolates), tet(A) (28% isolates), bla_CMY-2 (21% isolates) and bla_TEM (17% isolates) were the most common resistance genes found. The bla_CMY-2 and bla_TEM genes were unconditionally associated with S. Heidelberg; tet(A) and strA/B with S. Hadar and tet(B) gene with S. Kentucky. The strA/B genes were not associated with S. Heidelberg. Our data suggests that the prevalence of Salmonella serovars varied by the meat type and that AMR and resistance genes varied by the Salmonella serovars.