Listeria monocytogenes and Salmonella spp. in raw milk produced in Brazil: occurrence and interference of indigenous microbiota in their isolation and development

This study aimed to verify the occurrence of Listeria monocytogenes and Salmonella spp. in raw milk produced in Brazil. On account of the poor microbiological quality of this product, possible interference from the indigenous microbiota in these pathogens was also evaluated. Two-hundred and ten raw milk samples were collected in four important milk-producing areas in Brazil, tested for L. monocytogenes and Salmonella spp. presence, and for enumeration of indicator microorganisms: mesophilic aerobes, total coliforms and Escherichia coli. The interference of the indigenous microbiota in the isolation procedures was also tested, as well the frequency of naturally occurring raw milk strains with antagonistic activity against both pathogens. The pathogens were not isolated in any raw milk sample, but poor microbiological quality was confirmed by the high levels of indicator microorganisms. When present at high levels, the indigenous microbiota generated an evident interference in the methodologies of L. monocytogenes and Salmonella spp. isolation, mainly when the pathogens appeared at low levels. Three-hundred and sixty raw milk strains were tested for antagonistic activity against both pathogens, and 91 (25.3%) showed inhibitory activity against L. monocytogenes and 33 (9.2%) against Salmonella spp. The majority of the antagonistic strains were identified as Lactic Acid Bacteria species, mainly Lactococcus lactis subsp. lactis and Enterococcus faecium, known by antimicrobial substance production.     

Influence of intensive and extensive breeding on lactic acid bacteria isolated from Gallus gallus domesticus ceca

In the present study, lactic acid bacteria (LAB) from the cecum of chickens bred either under intensive (commercial broilers) or extensive (free-range) conditions were isolated, identified and some of their probiotic characteristics determined. The LAB identified by 16S-23S rRNA PCR-ARDRA were mainly of Lactobacillus species and to a lesser extent of Enterococcus spp. for all animals. Free-range chickens showed a higher presence of Lactobacillus acidophilus while Lactobacillus reuteri and Lactobacillus johnsonii were more frequently recovered from commercial broilers. Lactobacillus crispatus was found only in commercial broilers, Lactobacillus vaginalis and Lactobacillus agilis only in free-range chickens and Lactobacillus salivarius in both types. Enterococcus isolates from ceca of commercial broilers showed a higher resistance to antimicrobial drugs. Lactobacillus isolates from free-range chickens presented a higher frequency of in vitro antagonistic activity against selected pathogens than from commercial broilers. All LAB isolates had predominantly non-hydrophobic surfaces, but with variations depending on age of the chickens and breeding conditions. Animal breeding caused variation on composition, antimicrobial susceptibility, antagonistic activity and surface hydrophobicity of LAB from chicken cecum. LAB isolates from ceca of free-range chickens have potential as probiotic agents, which may be used in the future as replacing the use of antimicrobials as growth promoters.     

木霉菌对草坪草病原菌的抑菌效果及其机理初步研究

木霉菌是自然界中普遍存在的拮抗微生物,多种木霉菌对植物病原菌具有重要的生防价值。为挖掘出防治草坪草病害的生防菌剂,采用对峙培养法测定了4株木霉菌株对14种草坪草病原菌的拮抗作用,通过显微镜观察、玻璃纸法及粗提液抑菌试验研究了其抑菌机理。研究结果表明,供试木霉菌对所有供试病原菌均有拮抗作用,其中菌株SQ-1Q-18抑菌效果最佳,对玉蜀黍丝核菌和地衣状伏革菌的拮抗等级为I级,抑制率高达100%,对灰葡萄孢菌、地毯草炭疽菌和立枯丝核菌的拮抗等级为Ⅱ级,抑制率大于80%,且对供试病原菌的平均抑制率高达72.8%。进一步研究菌株SQ-1Q-18的抑菌机理发现,该菌株在培养基上产生抑菌圈侵占病原菌的生长空间,覆盖和深入病原菌内部并与其相互缠绕使得病原菌菌丝变细、缢缩甚至断裂,或直接穿入病原菌菌丝内部吸取营养致使菌丝细胞溶解,也可产生拮抗物质,其中玻璃纸法中对草坪草币斑病菌、球黑孢霉及地衣状伏革菌的相对抑制率分别高达91.77%、89.80%和79.59%;粗提取液对草坪草币斑病菌和佩立金平脐蠕孢的抑制率分别为43.60%和42.91%。经鉴定,SQ-1Q-18为哈茨木霉。研究结果为高效防治草坪草真菌病害的生防菌剂的研制、开发提供了科学资料。     

草坪镰刀枯萎病菌拮抗木霉菌的筛选及拮抗机制的研究

通过木霉属5个菌株与大连高尔夫球场上草坪镰刀枯萎病菌的对峙培养试验,结果表明:哈茨木霉1、哈茨木霉2、钩状木霉及桔绿木霉的抑菌效果很好,可以作为草坪镰刀枯萎病菌的生物防治拮抗菌加以利用,在这5株木霉中以哈茨木霉2的拮抗作用最强.观察结果表明,木霉对镰刀枯萎病菌的拮抗机制主要表现为生长竞争、重寄生及产生抗菌物质使病菌菌丝消解,而木霉菌生长迅速,可附着或缠绕于病菌菌丝上,可产生吸器侵入菌丝.另外,木霉菌还能分泌使病原菌菌丝原生质浓缩、断裂、消解的物质.     

乳酸杆菌抑制致病菌及生物保鲜发酵剂特性评价

以15株乳酸杆菌为研究对象,动物双歧杆菌乳亚种BB-12作为对照,研究它们对肠道致病菌的抑菌特性.结果表明:9株菌对5株指示菌均有较好抑制作用,2株菌对金黄色葡萄球菌、肠炎沙门氏菌、大肠杆菌和蜡样芽孢杆菌均有抑制作用;以抑菌效果、凝乳状态、产酸性能为指标,筛选出4株菌,与嗜热链球菌ST-21组合制备生物保鲜发酵剂;通过...     

In vitro competitive adhesion and production of antagonistic compounds by lactic acid bacteria against fish pathogens

The present study describes the screening of five lactic acid bacteria (LAB) for use as probiotics based on their competitive adhesion and production of antagonistic substances against some fish pathogens. A reduction of adhesion of all pathogenic strains tested was obtained with three of the LAB strains (Lactococcus lactis subsp. lactis CLFP100, Lactococcus lactis subsp. cremoris CLFP102 and Lactobacillus curvatus CLFP150). With the exception of fish pathogens Flavobacterium psychrophilum and Renibacterium salmoninarum that were not inhibited by LAB strains, production of antagonistic compounds by all tested LAB was observed against at least one of the indicator strains. Based on mucus adhesion, competitive exclusion, and suppression of fish pathogen growth, the selected LAB strains can be considered for future challenge experiments in fish as a very promising alternative to the use of chemotherapeutic agents.     

抑制奶牛乳房炎源金黄色葡萄球菌的乳酸菌的筛选

为获得抑制奶牛乳房炎源金黄色葡萄球菌(S. aureus)的乳酸菌(LAB),本研究从新疆巴音布鲁克牧区鲜牛乳和哈萨克族乳制品奶疙瘩样品中分离培养LAB,通过传统的分离鉴定与16S rDNA基因序列测序相结合的方法鉴定LAB种类,同时以临床奶牛乳房炎源金黄色葡萄球菌S. aureus N2为指示菌,采用双层琼脂扩散法检测分离株的抑菌能力。通过测定生长曲线确定分离株的生长稳定期,进而利用硫酸铵沉淀法透析提取稳定期内分离株的细菌素,并检测其细菌素抑菌效价。结果显示:从样品中筛选获得5株能够抑制指示菌生长的LAB,分别为希氏乳杆菌、干酪乳杆菌、粪肠球菌、戊糖片球菌和乳酸乳球菌亚种。生长规律曲线表明20 h^30 h为5株LAB的稳定期,此期培养液pH值维持在3.8~4.5。从培养20 h的5株LAB上清液中提取到了细菌素,经检测其具有抑菌活性,抑菌效价分别为457 IU/mL、1 023 IU/mL、676 IU/mL、1 862 IU/mL和1 023 IU/mL。本研究结果表明5株LAB通过在生长稳定期内维持较低酸性环境(pH<4.5),代谢产生细菌素对乳房炎源S. aureus发挥抑制生长作用。本研究为S. aureus性奶牛乳房炎的生物防治提供了实验依据。     

Phenotypic and genetic characterisation of bacteriocin-producing strains of Staphylococcus aureus involved in bovine mastitis

Fifty strains of Staphylococcus spp. isolated from bovine mastitis cases in several herds from different Argentinian provinces were screened for antimicrobial substances. Twelve strains exhibited a high antagonistic activity against the indicator strain (Corynebacterium fimi) and were chosen for further characterisation. The antimicrobial substances were sensitive to proteolytic enzymes suggesting that they might be bacteriocins (Bac). These strains were identified as S. aureus by the amplification of the femA gene. Plasmid profile analysis of these strains revealed the presence of at least one plasmid. Eleven strains carried a plasmid with a size similar to that of pRJ6 (8.0kb), which encodes aureocin A70, a bacteriocin produced by the Brazilian S. aureus strain A70 isolated from commercial milk. The other strain harboured a much larger plasmid. PCR experiments, using specific primers for amplification of the bacteriocin operon found in pRJ6, showed that all strains had the expected 525bp amplicon, suggesting that the bacteriocin produced may be related to aureocin A70. The genomic DNA of all Bac(+) strains was then analysed by pulsed-field gel electrophoresis (PFGE) in order to investigate clonal relationships amongst strains. Based on the results of PFGE experiments, 10 out of the 12 Bac(+) strains belonged to the same clone. The remaining two strains are possibly related to the prevalent clone. The aureocin A70 producer-strain belonged to a distinct clone.     

枯草芽孢杆菌MA139类细菌素抑菌活性的研究

为测定枯草芽孢杆菌MA139产生的类细菌素的抑菌活性。本试验采用管碟法对枯草芽孢杆菌MA139产生的类细菌素对多株革兰氏阳性菌、革兰氏阴性菌和真菌的抑菌活性进行测定。结果表明:类细菌素不但对芽孢杆菌属的其他菌种有抗菌活性,对病原菌和霉菌也有拮抗作用;不同指示菌对类细菌素的敏感性不一样,其中金黄色葡萄球菌对其最敏感。该类细菌素具有广谱抗菌作用,有作为畜禽饲料添加剂的潜力。     

Influence of a xylanase feed additive on Lactobacillus species in the jejunum of piglets

rRNA extracts from the jejunum of rearing piglets fed a wheat/rye based diet with or without a xylanase feed additive were hybridized against eight Lactobacillus spp. specific digoxigenin labelled oligonucleotide probes. Known concentrations of sample- or culture RNA extracts were used to calibrate and quantify hybridization signals from different sample extracts and hybridizations. The xylanase feed additive tended to increase total eubacterial rRNA in the terminal jejunum, probably a result of increased Lactobacillus spp. rRNA in the central and terminal jejunum. However, individual animal variations were high. L. reuteri was the major Lactobacillus species followed by L. acidophilus and L. mucosae. L. amylophilus and L. crispatus were not detected in all RNA extracts.The results of this study show that the xylanase feed additive modifies Lactobacillus spp. populations on the species level in piglets in addition to their well known effects on digesta viscosity and nutrient digestibility.The implications of these effects for the intestinal microbiota require further analysis.     

Screening of the enterocin genes and antimicrobial activity against pathogenic bacteria in Enterococcus strains obtained from different origins

Antimicrobial activities of 139 Enterococcus isolates (48 E. faecium and 91 E. faecalis) obtained from canine feces, boiler meat samples, swine feces, wild waterfowl feces, and human feces were examined against respective bacteria, including Streptococcus pyogenes, Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes, Salmonella Enteritidis, and Escherichia coli. Bacteriocin (BAC) production assay revealed that the antimicrobial activity against at least one of 6 indicator strains (BAC+ phenotype) was found in 51 (37%) isolates (29 E. faecium and 22 E. faecalis). Twenty-four of 46 isolates positive for at least one of the enterocin structural genes (entA, entB, entL50AB, and cylL) showed a BAC+ phenotype. The existence of other enterocins or nonenterocin factors was implied because the BAC+ phenotype was detected in a total of 27 Enterococcus isolates that had none of the enterocin genes tested. The antimicrobial activity against Gram-negative strains (Salmonella Enteritidis and E. coli) was detected in the 6 Enterococcus isolates that had either the entA, entB, entL50AB or cylL genes. Moreover, the proportion of the antimicrobial activity against L. monocytogenes among the cylL-positive E. faecalis isolates showing beta-hemolysis (10/16) was significantly (p<0.01) higher than among those lacking beta-hemolysis (2/15). The results suggested that certain characteristics are likely to be associated with the antimicrobial activity against specific organisms.     

The In Vitro Activity of 15 Antimicrobial Agents against Bacterial Isolates from Dogs

The in vitro activity of 15 antimicrobial agents against clinical isolates of Staphylococcus pseudintermedius, Staphylococcus aureus, Escherichia coli, Pasteurella spp. and Streptococcus canis from dogs was investigated. For Staphylococcus spp., the highest frequency of resistance was observed for penicillin, followed by ampicillin, tetracycline and chloramphenicol. The highest frequency of resistance in E. coli isolates was recorded for tetracycline and streptomycin. Pasteurella spp. and S. canis had the highest resistance rate for tetracycline and chloramphenicol. Most isolates showed full susceptibility to low-level resistance to colistin, florfenicol and fluoroquinolones. Further studies using larger number of isolates from both healthy and diseased dogs would provide a broader picture of antimicrobial resistance at a national level and promote prudent use of antimicrobial agents in companion animals.     

旗草内生真菌与旗草抗病性研究

旗草内生真菌交织顶孢霉的7个不同分离物对旗草主要病源真菌德氏霉和立枯丝核菌在PDA培养基上的体外对峙试验表明,大多数内生真菌分离物对这2个病原菌都有不同程度的抑制作用,不同分离物对同一病原菌的抑制作用不同,同一分离物对不同病原菌的抑制作用也不同,EB6780.501和EH32a对德氏霉和立枯丝核菌的抑制作用最强。旗草植株抗病试验表明,内生真菌感染的植株对叶枯病具有明显的抗性,但这种抗性随着病原菌立枯丝核菌入侵时间的延长而减弱。在体外抑菌试验中,对病原菌生长抑制能力强的内生真菌分离物,在旗草体内也表现出较强的抗病性,说明抗病内生真菌的体外抑菌筛选是有效的。     

Antimicrobial susceptibility of Klebsiella spp. and Proteus spp. from various organ systems of horses, dogs and cats as determined in the BfT-GermVet monitoring program 2004-2006

A total of 120 isolates of Klebsiella spp. and Proteus spp. collected from horses and small animals (dogs and cats) were screened for their susceptibility to 24 different antimicrobial agents. Klebsiella spp. were included from infections of the genital tract (GT) of horses (36 isolates) and the urinary/genital tract (UGT) from dogs and cats (17 isolates), while Proteus spp. were from small animal (dogs and cats) infections of the UGT (37 strains) and the skin (incl. ear/mouth) (30 isolates). In Klebsiella spp. resistance appeared most frequently to ampicillin (53-67%), sulfamethoxazole (19-29%) and potentiated sulfonamides (trimethoprim/sulfamethoxazole 1/19 combination) (19-24%). A further 29% of enrofloxacin resistant Klebsiella isolates were observed for the UGT of small animals. From the GT of horses for this antimicrobial agent there was no isolate detected with a comparably high minimum inhibitory concentration (MIC) value. In Proteus spp. highest percentages of resistance occurred against tetracycline (90-92%). Due to drug efflux proteins, high MIC values against this antimicrobial agent have been frequently reported in literature. In Proteus spp. relevant resistance percentages also occurred for potentiated sulfonamides (27-37%), sulfamethoxazole (24-37%) and chloramphenicol (24-37%).     

Bacterial isolates from blood and their susceptibility patterns in critically ill foals: 543 cases (1991-1998)

OBJECTIVE: To assess microorganisms isolated from blood specimens obtained from critically ill neonatal foals and to evaluate their antimicrobial susceptibility patterns. DESIGN: Retrospective study. ANIMALS: 543 neonatal foals. PROCEDURE: Medical records of foals that were < 1 month old and were admitted to a referral neonatal intensive care unit were reviewed for results of bacteriologic culture of blood and antimicrobial susceptibility patterns. RESULTS: At least 1 microorganism was isolated from 155 of 543 (28.5%) foals. Escherichia coli was the most commonly isolated bacterium. A single gram-positive organism was detected in 49 foals. Although 90% of the E coli isolates were susceptible to amikacin, some gram-negative and gram-positive organisms had resistance against multiple antimicrobials. CONCLUSIONS AND CLINICAL RELEVANCE: Gram-negative bacteria remain the most common isolates from blood of neonatal foals; however, gram-positive organisms were also found, and with greater prevalence than reported elsewhere. Susceptibility patterns may vary, and resistance to multiple antimicrobials may develop. This is especially true for organisms such as Enterobacter spp and Enterococcus spp. Prudent empirical treatment for neonatal sepsis should include broad-spectrum antimicrobials.     

Selection and characterization of broad‐spectrum antibacterial substance‐producing Lactobacillus curvatus PA40 as a potential probiotic for feed additives

Lactic acid bacteria were screened for potential probiotics for use as feed additives. We obtained 3,000 isolates from feces of: cattle, dogs, goats, and infants; milk; yogurt; cheese; fermented sausages; Kimchi; and Cheonggukjang and tested their antibacterial activity toward indicator pathogens, including Bacillus cereus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enterica Enteritidis. We further tested their tolerance to artificial gastric juice (1% [w/v] pepsin, pH 2.5) and bile acid (0.1% [w/v] oxgall, pH 6.8). Six isolates exhibited strong antibacterial activity against indicator pathogens. The PA40 isolate from Kimchi exhibited marked resistance to artificial gastric juice and bile acid. The antibacterial substances produced by PA40 were stable to heat, pH, and enzymes. Strain PA40 was identified as a Lactobacillus curvatus strain using chemical tests and 16S rDNA sequencing and produced 248.4 mmol/L lactic acid after 48 hr of fermentative growth. The L. curvatus PA40 strain was also highly tolerant of the artificial gastrointestinal model system. Our results indicate that L. curvatus PA40 could be used as a potential probiotic feed additive.     

Antibiotic resistance among indicator bacteria isolated from healthy pigs in New Zealand

AIM: To determine the resistance to antibiotics among the indictor bacteria, Escherichia coli and Enterococcus spp, isolated from the faeces of healthy pigs on three conventional pig farms and one organic farm in the North Island of New Zealand. METHODS: Faecal samples, collected at intervals between March and October 2001, were plated onto MacConkey agar and Slanetz-Bartley agar and examined after 1-3 days incubation for colonies resembling E. coli and Enterococcus spp, respectively. Typical colonies were subcultured for further identification and storage. The isolates were tested for antibiotic resistance, using disc diffusion, to ampicillin, gentamicin, streptomycin, and tetracycline. Escherichia coli isolates were also tested for resistance to ciprofloxacin, cotrimoxazole and neomycin. Enterococcus spp isolates were also tested for resistance to vancomycin, erythromycin and virginiamycin. RESULTS: A total of 296 E. coli and 273 Enterococcus spp isolates were obtained from the three conventional farms, and 79 E. coli and 80 Enterococcus spp isolates were obtained from the organic farm. All the E. coli isolates from both the conventional and organic pig farms were susceptible to ciprofloxacin, and all the Enterococcus spp isolates were susceptible to ampicillin, gentamicin and vancomycin. Isolates of E. coli from conventional pig farms were resistant to gentamicin (0.7%), neomycin (0.7%), ampicillin (2.7%), cotrimoxazole (11%), streptomycin (25%) and tetracycline (60%). Enterococcus spp isolates from the same farms were resistant to erythromycin (68%), tetracycline (66%), streptomycin (54%) and virginiamycin (49%). By contrast, for the organic pig farm 相似文献   

Blood culture isolates and antimicrobial sensitivities from 427 critically ill neonatal foals

OBJECTIVE: To assist correct decision-making about antimicrobial treatment of equine neonates with septicaemia. DESIGN: Retrospective study of microbial blood culture results obtained from foals less than 7 days of age. METHODS: Microbial blood culture results from foals less than 7 days of age admitted to an intensive care unit between July 1999 and December 2004 were reviewed. Antimicrobial sensitivity was assessed by the Kirby Bauer disc diffusion method. Antimicrobials were defined as an effective first-line choice antimicrobial if greater than 70% of isolates were susceptible. Multiple drug resistance (MDR) was defined as resistance to at least three antimicrobials in different chemical classes or with different mechanisms of resistance. RESULTS: Of the 427 Thoroughbred foals included in the study, a positive blood culture was obtained in 110 foals and 124 microorganisms were isolated. Gram-positive isolates, predominantly Streptococcus/Enterococcus spp, were obtained in 41% of foals. Gram-negative isolates were predominantly of the Enterobacteriaceae family, in particular Escherichia coli. The overall antimicrobial sensitivity of the isolates was low. The Gram-positive organisms had unpredictable sensitivity patterns. MDR was recorded in 32% of isolates. In total, 81% of foals were discharged from hospital and 74.5% of foals with a positive blood culture were discharged. CONCLUSION: With the increasing prevalence of Gram-positive microorganisms and their unpredictable sensitivity patterns, blood cultures remain important in the diagnosis and treatment of equine neonatal septicaemia.     

草坪褐斑病菌和镰刀枯萎病菌拮抗木霉菌的筛选及效果研究

通过木霉属5个菌株与草坪禾草褐斑病菌、草坪禾草镰刀枯萎病菌的对峙培养试验,发现哈茨木霉1、哈茨木霉2、深绿木霉、钩状木霉及桔绿木霉的抑菌效果很好,可以作为草坪镰刀枯萎病菌的生物防治拮抗菌加以利用。在这5株木霉中以哈茨木霉对草坪镰刀枯萎病菌的拮抗作用最强。     

Effect of chosen lactic acid bacteria strains on Staphylococcus aureus in vitro as well as in meat and raw sausages

The present study was aimed at determining the influence of 15 strains of lactic acid bacteria on the growth of 2 Staphylococcus aureus strains in vitro as well as in meat and raw sausages. The investigations were performed within the framework of three alternate stages which differed in respect to the products studied, the number of Lactobacillus sp. strains and, partly, methodological approach. The study also considered water activity (a(w)) and pH of the products investigated. The results obtained are demonstrated in 5 diagrams. It was found that among 15 strains of Lactobacillus aureus investigated only one strain, Lactobacillus helveticus T 78, showed antagonistic effect on studied strains of Staphylococcus aureus both in vitro as well as in meat and raw sausages. Five other strains of Lactobacillus spp. displayed the antagonistic effect in vitro only. The temperature and incubation time of sausages, but also the type of sausage stuffing were found to have a distinct or slight influence, respectively, on the antagonistic interaction between the bacteria. However, this phenomenon was affected by neither a(w) nor pH.