Antibiotic resistance and Caco-2 cell invasion of Pseudomonas aeruginosa isolates from farm environments and retail products

The potential pathogenicity of Pseudomonas aeruginosa isolates from food animals, retail meat products, and food processing environments was evaluated by determining their antibiotic resistance profiles and invasiveness into human intestinal Caco-2 cell. In general, the genomically diversified isolates of P. aeruginosa were resistant to beta-lactams (ampicillin, amoxicillin-clavulanic acid, cefoxitin, ceftiofur, and cephalothin), chloramphenicol, tetracycline, kanamycin, nalidixic acid, and sulfamethoxazole-trimethoprim. Acquisition of any other antibiotic resistance genes, such as class 1 integrons and other beta-lactamase genes, was not found in the tested isolates. The expression of OprM membrane protein, which is associated with a multidrug efflux system, played a major role in their antibiotic resistance. Single mutation in the GyrA to confer resistance to nalidixic acids was also found in the tested isolates, indicating that these factors could synergistically affect the resistance of the P. aeruginosa isolates. The number of bacteria invading into the Caco-2 cells was 2.5 log(10) CFU/ml on average. Therefore, the public health concern of P. aeruginosa could be relevant since its occurrence in food animals could cross contaminate the retail meat products during food handling and processing.     

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.     

Virulence profiles of Klebsiella pneumoniae isolated from 2 large dairy farms in China

We recently reported on the diversity of Klebsiella pneumoniae isolated from dairy herds in China. In our previous work, isolates from subclinical mastitis (SCM) had lower indices of diversity when compared with bacteria from other sources, possibly due to a contagious-like spread of udder adapted strains. Here we explored the virulence profile and capsular types of K. pneumoniae isolated from different sources on 2 dairy farms in China. Our overarching goal was to gain insights on the role of virulence genes toward the severity of mastitis caused by K. pneumoniae. A total of 1,484 samples were collected from clinical mastitis (CM; n = 355), SCM (n = 561), bulk tank milk (BTM; n = 130), and environmental and extramammary (EE) sites (n = 438). From those, 431 K. pneumoniae isolates were obtained, including 129, 77, 66, and 159 isolates from CM, SCM, BTM, and EE samples, respectively. Polymerase chain reactions were used to determine the capsular types and to detect potential virulence genes in all isolates. No significant farm effects were observed when comparing the distribution of most virulence genes in K. pneumoniae isolated from each source. K57 was the most prevalent capsular type in K. pneumoniae from all sources, but with increased detection rate in isolates from CM. entB, kfu, fimH1, mrkD, and β-d-lacZ were frequently detected in K. pneumoniae from all sources. β-d-lacZ, entB, and ituA were more prevalent in isolates from CM, whereas kfu, allS, and nif were more frequently detected in isolates from SCM. ybtS, aerobactin, and rpmA had increased prevalence in K. pneumoniae from BTM when compared with bacteria from other sources. No association was detected between virulence genes and the severity of CM. K57 and the nif gene had the highest discriminatory power to classify isolates from CM and SCM, respectively. Based on our findings, it is likely that K57 is the dominant capsular type in K. pneumoniae causing CM in large Chinese dairy herds. Likewise, we demonstrated that β-d-lacZ is disseminated in K. pneumoniae isolated from large Chinese dairy farms, irrespectively of the source of bacteria. Our results also suggest a low contribution of the virulence profile of K. pneumoniae toward CM severity. Finally, the role of nif in increasing the adaptability to the udder and promoting a contagious-like spread of K. pneumoniae warrants further investigation.     

Occurrence and genetic diversity of ESBL producing Klebsiella species isolated from livestock and livestock products

Klebsiella species have been at the center of attention over the recent years due to its role in the evolution of antimicrobial resistance and is not only associated with nosocomial but also with food related infections worldwide. In this study, out of 336 samples of animal intestinal and foods of animal origin screened, 99 samples were found to harbor Klebsiella spp. Out of 99 isolates, 89 were Klebsiella pneumoniae and 10 were found to be Klebsiella oxytoca by mPCR. The isolates were subjected to antibiotic sensitivity test including ESBL detection by genotypic (PCR) and phenotypic (disc diffusion) methods. β-Lactamase genes were identified in 32 isolates of K. pneumoniae and an isolate of K. oxytoca, blaSHV being the predominant gene detected (31) followed by blaTEM, blaCTX-M1, and blaCTX-M9 (one each), whereas single K. oxytoca isolate harbored blaCTX-M2 gene. Genetic fingerprinting of β-lactamase producing K. pneumoniae using ERIC-PCR and REP-PCR differentiated all the 32 strains with discriminatory power of 1.     

克雷伯杆菌甘油脱氢酶的分离纯化及性质

在有氧条件下,利用Q Sepharose Fast Flow离子交换层析和Blue Sepharose CL 6B亲和层析提纯克雷伯杆菌胞内甘油脱氢酶.酶的纯化倍数和回收率分别为 32.61 倍和 5.83%.通过SDS PAGE电泳测得该酶亚基的相对分子质量约为 34 000.该酶最适表观反应温度和最适反应pH值分别为60 ℃和11.在30 ℃以下及pH值10~12时,该酶具有良好的稳定性.在45 ℃和pH值11条件下,该酶以甘油和NAD 为底物的米氏常数Km分别为 0.75 mmol/L和 0.12 mmol/L.甘油脱氢酶对甘油的生理反应活性最大,对其它醇类如 1,2 丙二醇、乙二醇也有氧化能力.NH4 和Na 对酶有显著激活作用.巯基保护剂可明显地提高酶的活力.     

A review of the incidence and transmission of Listeria monocytogenes in ready-to-eat products in retail and food service environments

Contamination of ready-to-eat products with Listeria monocytogenes may occur at several stages before consumption. Accessibility to the public and relatively limited control interventions at retail and food service establishments (compared with the processing sector of the food industry) and the lack of a specific regulatory framework increase the likelihood of introduction of this pathogen into some foods in these establishments. This review is a compilation of available information on the incidence and transmission of L. monocytogenes through ready-to-eat products at the retail and food service level. The potential transmission of L. monocytogenes within retail and food service operations has been indicated in epidemiological investigations and by survey data. Potential sources of the organism in these operations include the environment, food handlers, and incoming raw ingredients or processed products that have become contaminated after the lethality treatment at the manufacturing facility. L. monocytogenes may be present at retail and food service establishments in various ready-to-eat products, both prepackaged and those packaged in the store, and occasionally at high concentrations. This issue dictates the need for development and application of effective control measures, and potential control approaches are discussed here. Good manufacturing practices, appropriate cleaning, sanitation and hygiene programs, and temperature control required for prevention or inhibition of growth of the pathogen to high levels are critical for control of L. monocytogenes in the retail and food service sector. A comprehensive food safety system designed to be functional in retail and food service operations and based on the philosophy of hazard analysis and critical control point systems and a series of sound prerequisite programs can provide effective control of L. monocytogenes in these environments. However, competent delivery of food safety education and training to retail and food service managers and food handlers must be in place for successful implementation of such a system.     

Characterization of Listeria monocytogenes isolated from retail foods

Listeria monocytogenes isolates recovered from retail ready-to-eat (RTE) meats, raw chickens and fresh produce were characterized by serogroup identification using PCR, genotyping using pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. Five L. monocytogenes serogroups were identified. Of the 167 isolates 68 (41%) belonged to serogroup 1/2b, 3b; 53 (32%) belonged to serogroup 4b, 4d, 4e; 43 (26%) belonged to serogroup 1/2a, 3a; 2 (1.2%) belonged to serogroup 1/2c, 3c; and 1 (0.6%) belonged to serogroup 4a, 4c. PFGE generated 120 patterns which correlated well with PCR serogrouping. Most L. monocytogenes isolates were resistant to sulfonamide (73%) and some were resistant to tetracycline (8.4%) and ciprofloxacin (1.8%). Tetracycline resistance was conjugatively transferable and the tet(M) gene was identified in 14 tetracycline-resistant isolates as well as their transconjugants. These findings indicate that L. monocytogenes present in food were diverse, and that resistance to one or more antibiotics among these isolates was common. In addition, the presence of potential serotype 4b in all food categories is of public health concern, as serotype 4b has been the serotype most frequently associated with human listeriosis.     

市售鸡肉中沙门菌分离株多重耐药谱测定

为了解我国市售鸡肉中沙门菌多重耐药状况及耐药程度，对国家食源性疾病监测网从市售鸡肉中分离到的51株沙门菌进行耐药检测，并对多重耐药谱进行分析。利用NccLs(National Committee of Clinical Labaratory Standard)推荐的纸片法进行耐药检测，51株分离菌株全部对至少3种以上的抗生素耐药，属多重耐药株，耐5种抗生素的9株(17.6％)，耐6～9种抗生素的12株(23．5％)，耐10种以上抗生素的9株(17．6％)。51株沙门菌对万古霉素、红霉素、苯唑西林都具有耐药性(100％)，对其它抗生素的耐药情况是：萘啶酮酸(52.9％)、磺胺(35．3％)、链霉素(33．3％)、四环素(29.4％)、氨苄青霉素(23．5％)、羧苄西林(21.6％)、阿莫西林(19．6％)、吡拉西林(19．6％)、美唑西林(17．6％)、强力霉素(17．6％)、氯霉素(13．7)和头孢噻吩(9．8％)。有3株菌对庆大霉素耐药，有2株菌对丁胺卡那霉素和甲氧苄胺嘧啶耐药，有1株菌对复方新诺明耐药。分离出1株耐15种抗生素的鼠伤寒沙门菌，其耐药谱与超级耐药鼠伤寒沙门菌DTl04的耐药谱类似。从8省市市售鸡肉中分离的51株沙门菌均为多重耐药株，提示我国畜牧养殖业在使用抗生素方面存在严重问题，应加强对饲料添加剂使用的安全性管理，合理使用抗生素。     

Butyltin compounds in retail mollusc products

Butyltin was found in 62 of 74 mollusc products purchased from major supermarket outlets in seven cities across Canada. Tribuyltin was detected in 41 samples, with levels up to 233 ng g^-1. Dibutyltin and monobutyltin levels ranged up to 88 and 53 ng g^-1, respectively. Products originating from East and South East Asia generally contained the highest levels. Gastropods contained lower levels than other molluscs. Enzymatically hydrolysed samples were extracted with 0.05% tropolone in hexane-diethyl ether (1 : 1). Ethyl derivatives were made by Grignard reaction and analysed by gas chromatography coupled to an atomic emission detector. Gas chromatograph-mass spectrometry confirmed the presence of butyltins in the mollusc samples.     

Prevalence and characterization of foodborne pathogens from Australian dairy farm environments

The ability of foodborne pathogens to gain entry into food supply systems remains an ongoing concern. In dairy products, raw milk acts as a major vehicle for this transfer; however, the sources of pathogenic bacteria that contaminate raw milk are often not clear, and environmental sources of contamination or the animals themselves may contribute to the transfer. This survey examined the occurrence of 9 foodborne pathogens in raw milk and environments of 7 dairy farms (3 bovine, 3 caprine, and 1 ovine farm) in summer and autumn, in Victoria, Australia. A total of 120 samples were taken from sampling points common to dairy farms, including pasture, soil, feed, water sources, animal feces, raw milk, and milk filters. The prevalence of the Bacillus cereus group, Campylobacter, Clostridium perfringens, Cronobacter, Shiga-toxigenic Escherichia coli, Listeria, Salmonella, coagulase-positive staphylococci (CPS), and Yersinia enterocolitica across the farms was investigated. The 2 most prevalent bacteria, which were detected on all farms, were the B. cereus group, isolated from 41% of samples, followed by Cl. perfringens, which was isolated from 38% of samples. The highest occurrence of any pathogen was the B. cereus group in soil, present in 93% of samples tested. Fecal samples showed the highest diversity of pathogens, containing 7 of the 9 pathogens tested. Salmonella was isolated from 1 bovine farm, although it was found in multiple samples on both visits. Out of the 14 occurrences where any pathogen was detected in milk filters, only 5 (36%) of the corresponding raw milk samples collected at the same time were positive for the same pathogen. All of the CPS were Staphylococcus aureus, and were found in raw milk or milk filter samples from 6 of the 7 farms, but not in other sample types. Pathogenic Listeria species were detected on 3 of the 7 farms, and included 4 L. ivanovii-positive samples, and 1 L. monocytogenes-positive water sample. Shiga-toxigenic Escherichia coli were identified in fecal samples from 3 of the 7 farms and in a single raw milk sample. Cronobacter species were identified on 4 of the 7 farms, predominantly in feed samples. No Y. enterocolitica was detected. Results of this study demonstrate high standards of pathogen safety across the 7 farms, with a low incidence of pathogens detected in raw milk samples. Monitoring feed contamination levels may help control the spread of bacterial species such as Cl. perfringens and B. cereus through the farm environment, which is a natural reservoir for these organisms.     

克雷伯杆菌利用生物柴油副产物甘油生产氢气和1,3-丙二醇的研究

以Klebsiella pneumoniaeDSM2026为出发菌株,通过紫外线诱变,选育得到能耐较高浓度生物柴油副产物甘油生产H2和1,3-丙二醇(1,3-PD)的菌株21株,命名为Kp1~Kp21。通过比较,Kp8菌株产量最高,1,3-PD和H2产量分别达到0.36 g/50 mL和0.99 mmol/50 mL,比出发菌株分别提高了3.5倍和4.2倍。对Kp8菌株发酵条件进行优化,得到最佳培养条件为pH 7.0,培养温度37℃,接种量10%(v/v),废甘油浓度为30 g/L。在该条件下H2产量为1.0 mmoL/50 mL,1,3-PD产量为7.5 g/L,甘油转化率为83.3%。     

Purification and characterization of phytase from Klebsiella pneumoniae 9-3B

Phytase, an enzyme that catalyzes the hydrolysis of phytate, was purified from Klebsiella pneumoniae 9-3B. The isolate was preferentially selected in a medium which contains phytate as a sole carbon and phosphate source. Phytic acid was utilized for growth and consequently stimulated phytase production. Phytase production was detected throughout growth and the highest phytase production was observed at the onset of stationary phase. The purification scheme including ion exchange chromatography and gel filtration resulted in a 240 and 2077 fold purification of the enzyme with 2% and 15% recovery of the total activity for liberation of inorganic phosphate and inositol, respectively. The purified phytase was a monomeric protein with an estimated molecular weight of 45kDa based on size exclusion chromatography and SDS-PAGE analyses. The phytase has an optimum pH of 4.0 and optimum temperature of 50°C. The phytase activity was slightly stimulated by Ca(2+) and EDTA and inhibited by Zn(2+) and Fe(2+). The phytase exhibited broad substrate specificity and the K(m) value for phytate was 0.04mM. The enzyme completely hydrolyzed myo-inositol hexakisphosphate (phytate) to myo-inositol and inorganic phosphate. The properties of the enzyme prove that it is a good candidate for the hydrolysis of phytate for industrial applications.     

Antimicrobial susceptibility of vancomycin-susceptible and -resistant enterococci isolated in Italy from raw meat products, farm animals, and human infections

The susceptibility of vancomycin-resistant (VRE) and vancomycin-susceptible (VSE) enterococci to 10 antimicrobial agents was evaluated. The strains, belonging to different species, were isolated in Italy from raw meat products, farm animals, and human clinical infections in the years 1997-2000. High frequency of resistance to tetracycline and erythromycin was observed in all the groups of strains. On the contrary, chloramphenicol was the only drug that showed a relatively low rate of resistance in all the groups examined. In general, the resistance rates observed for VSE did not differ from those observed for VRE of the same species and origin. Some differences could be noticed among the different enterococcal species, with Enterococcus faecium strains being usually more resistant to beta-lactams, and Enterococcus faecalis strains more resistant to gentamicin. However, the strongest differences were observed when the strains were compared according to their source, the human isolates being usually more resistant than the isolates of animal origin. No significant difference was observed between isolates of swine and poultry origin. Among VRE E. faecium, multiple resistance was much more frequent among the human strains (90%) than among poultry (48.9%) and swine (26.5%) strains. These results show that in Italy VRE isolates from human clinical infections are usually more resistant than isolates from meat products and farm animals, and possess different antimicrobial resistance profiles.     

Antimicrobial resistance and virulence of Enterococcus faecalis isolated from retail food

Although enterococci are considered opportunistic nosocomial pathogens, their contribution to foodborne illnesses via dissemination through retail food remains undefined. In this study, prevalence and association of antimicrobial resistance and virulence factors of 80 Enterococcus faecalis isolates from retail food items were investigated. The highest rates of resistance were observed for lincomycin (73 of 80 isolates, 91%) and bacitracin (57 of 80 isolates, 71%), and lower rates of resistance (< or =40%) were found for chloramphenicol, ciprofloxacin, erythromycin, flavomycin, gentamicin, kanamycin, nitrofurantoin, penicillin, and tylosin. Overall resistance to antimicrobials was low for most isolates tested. Of the virulence factors tested, the majority of isolates were positive for ccf (78 of 80 isolates, 98%), efaAfs (77 of 80, 96%), and cpd (74 of 80, 93%). Isolates also commonly contained cob (72 of 80 isolates, 90%) and gelE (68 of 80, 85%). Very few isolates contained cylMBA (12 of 80 isolates [15%] for cylM and 9 of 80 isolates [11%] for both cylB and cylA) and efaAfm (2 of 80 isolates, 3%). Positive statistical associations (significance level of 0.05) were found between agg and tetracycline resistance, cylM and erythromycin resistance, and gelE and efaAfs and lincomycin resistance. The presence of the cylB and cylA alleles also was positively correlated with bacitracin and tetracycline resistance. Negative correlations were observed between many of the virulence attributes and resistance to ciprofloxacin, erythromycin, flavomycin, gentamicin, kanamycin, and tylosin. These data suggest that both positive and negative associations exist between antimicrobial resistance genes and virulence factors in E. faecalis isolates from foods commonly purchased from grocery stores.     

肺炎克雷伯氏杆菌二羟丙酮激酶基因(dhaKL)克隆与表达

以肺炎克雷伯氏菌As1.1736的基因组为模板,通过PCR技术成功地扩增了dhaKL基因,并构建了pET-28a(+)/dhaKL表达载体。DNA序列分析的结果表明:dhaKL基因由2个开放阅读框架组成:ORF1全长为1017bp,编码356个氨基酸;ORF2全长633bp,编码210个氨基酸。SDS-PAGE电泳结果表明:dhaKL基因获得了有效表达,上清液中的酶活性为15.6U/mL。     

肺炎克雷伯氏杆菌二羟丙酮激酶基因(dhaKL)

以肺炎克雷伯氏菌Asl.1736的基因组为模板,通过PCR技术成功地扩增了dhaKL基因,并构建了pET-28a(+)/dhaKL表达载体.DNA序列分析的结果表明:dhaKL基因由2个开放阅读框架组成:ORF1全长为1017bp,编码356个氨基酸;ORF2全长633bp,编码210个氨基酸.SDS-PAGE电泳结果表明:dhaKL基因获得了有效表达,上清液中的酶活性为15.6U/mL.     

Production of 2,3-butylene glycol from whey by Klebsiella pneumoniae and Enterobacter aerogenes

Production of 2,3-butylene glycol from whey with Klebsiella pneumoniae and Enterobacter aerogenes was studied. Sterilization of the whey was unnecessary. Acid whey required neutralization, but sweet whey did not. Butylene glycol production was most efficient at 33 degrees C for Klebsiella pneumoniae and at 37 degrees C for Enterobacter aerogenes. Aeration significantly improved yields. Klebsiella pneumoniae produced more butylene glycol than did Enterobacter aerogenes in unsupplemented whey. The addition of 50 mM sodium acetate to whey increased the production of butylene glycol and acetoin by Enterobacter aerogenes; it also increased the production of glycol by Klebsiella pneumoniae, but the increase in this case was offset by a decrease of production of acetoin. Maximal yields of the glycol plus acetoin in whey were obtained in 48 to 64 h, but Enterobacter aerogenes required about 160 h for complete utilization of the lactose. Highest yields were about .3 M butylene glycol plus acetoin, which corresponds to the production of about 10 kg of glycol from 380 liters of whey.     

Molecular characterization of multidrug-resistant Proteus mirabilis isolates from retail meat products

Sixty-four multidrug-resistant isolates of Proteus mirabilis were collected from retail meat products in Oklahoma. The isolates showed four different patterns of antibiotic resistance based on their resistant phenotype and genotypes. Most of these isolates were resistant to ampicillin, tetracycline, gentamycin, and kanamycin. Class 1 integrons were detected as a common carrier of the antibiotic-resistant genes, such as aadA1, aadB, and aadA2. A few isolates (9%) contained class 2 integrons with three gene cassettes included: dhfr1, sat1, and aadA1. These isolates were even resistant to nalidixic acid due to mutations in gyrA and parC. All ampicillin-resistant isolates contained blaTEM-1. Plasmids that contained class 1 or 2 integrons and blaTEM-1 were able to be transferred from P. mirabilis isolates into Escherichia coli by conjugation, indicating that conjugal transfer could contribute to the dissemination of antibiotic resistance genes between the Enterobacteriaceae species.     

木糖发酵高产2,3-丁二醇菌株的选育

以肺炎克雷伯氏菌(Klebsiella pneumoniae)CICC10011为出发菌株,进行紫外诱变选育。通过2,3-丁二醇抗性和产酸能力对诱变菌株进行初筛,发酵后23,-丁二醇产量检测进行复筛,获得6株高产2,3-丁二醇的肺炎克雷伯氏菌,其中产量最高的一株诱变菌U3-17,与出发菌株相比,产量提高了21.5%。在摇瓶批式补料发酵中,诱变株U3-17的23,-丁二醇产量达49.3 g/L。