Antibiotic resistances of intestinal lactobacilli isolated from wild boars

Acquired antibiotic resistances have been reported in lactobacilli of various animal and food sources, but there are no data from wild boar. The objective was a preliminary examination of the antibiotic resistance prevalence of intrinsically vancomycin-resistant lactobacilli isolated from wild boar intestines and analysis of the genetic determinants implicated. Out of three wild boars, 121 lactobacilli were recovered and grouped according to their whole cell protein patterns. Initial phenotypic screening revealed that all were susceptible to erythromycin (2 μg/ml), but 30 were resistant to tetracycline (32 μg/ml). Based on Randomly Amplified Polymorphic DNA-PCR clustering, 64 strains were selected as representative genotypes for identification and minimum inhibitory concentration (MIC) determination. Partial 16S rRNA gene sequencing identified four species: (i) L. mucosae (n = 57), (ii) L. reuteri (n = 47), (iii) L. fermentum (n = 12), and (iv) L. murinus (n = 5). Most heterofermentative strains displayed low MICs for ampicillin (AMP), chloramphenicol (CHL), streptomycin (STR), kanamycin (KAN), gentamicin (GEN), erythromycin (ERY), quinupristin/dalfopristin (Q/D), and clindamycin (CLI). Atypical MICs were found mainly in L. mucosae and L. reuteri for TET, KAN, STR, AMP and CHL, but except the TET MICs of L. mucosae mostly at low level. L. murinus strains revealed atypical MICs for aminoglycosides, and/or CHL, AMP, CLI. PCR screening detected tet(W) in 12 and tet(M) in one of heterofermentative strains, as well as the aph(3′)-III kanamycin gene in L. murinus. This is the first report showing acquired antibiotic resistance determinants in intestinal lactobacilli of wild boar origin.     

海波罗肉用仔鸡在几内亚若干种质特性的研究

选用同日出壳公母健雏各222只，在相同饲养管理条件下，对海波罗肉鸡早期生长发育等有关指标进行观测，结果表明，公鸡早期生长速度较母鸡快，平均日增重比母鸡高4．53g，公母鸡7周龄体重分别为2197．06g和1976．47g；累计耗料量公鸡比母鸡多16．33g；公母鸡料肉比分别为2．10和2．33；经7周龄饲养公母鸡成活率均达92％，抗逆性强，是值得推广的优质鸡种。     

猪源乳酸杆菌对永生化猪小肠上皮细胞的黏附性能

以永生化猪小肠上皮细胞ZYM—SIEC02为体外模型,通过革兰染色法、平板计数法等检测发酵乳杆菌Y091231（Lactobacillus fermentation）、乳酸乳杆菌Y091221（Lactobacillus lactis）,嗜酸乳杆菌Y224031（Lactoba-cillusacidophilus）的黏附性能及对大肠杆菌C83921、致病性沙门菌、金黄色葡萄球菌CVCC2258的黏附抑制作用。结果显示,3株菌对永生化猪小肠上皮细胞的黏附性均较强,黏附指数平均为：Y091231（2903．25±83．33）个／100cells、Y091221（2320．37±81．02）个／100ceils、Y224031（I965．43±37．72）个／100cells,菌株之间差异不显著（P〉0．05）。黏附抑制试验结果显示,Y091231对大肠埃希菌C83921和金黄色葡萄球菌CVCC2258的黏附抑菌能力最强（（99．4±3．17）％和（98．0±2．31）％）,Y091221对沙门菌的抑菌能力最强（（97．1±2．04）％）,Y224031对大肠埃希菌C83921比对另外2种致病菌的黏附抑制能力高,但又显著低于Y091231和Y091221（P〈0．05）,表明不同种乳酸菌对致病菌的黏附抑制作用具有菌种特异性。     

Screening of Lactobacillus isolates from gastrointestinal tract of guinea fowl for probiotic qualities using in vitro tests to select species-specific probiotic candidates

1. A total of 32 Lactobacillus isolates, 8 each from the crop (LGFCP1–LGFCP8), proventriculus (LGFP9–LGFP16), ileum (LGFI17–LGFI24) and caeca (LGFCM25–LGFCM32) were isolated from 25 adult guinea fowl (Pearl variety), 22–28 weeks of age, and characterised morphologically, physiologically, biochemically and by molecular methods.

2. Isolates were screened for their probiotic quality using range of in vitro tests: aggregation test, cell surface hydrophobicity, resistance to bile salts and acidic conditions, enzymatic tests and coaggregation and antagonistic test.

3. Based on in vitro test results and a novel scoring method, the two best isolates were selected and partial 16S rRNA sequencing was done. BLAST (Basic Local Alignment Search Tool) analysis of sequence of isolate LGFCP4 showed 99% genetic identity with Lactobacillus plantarum and LGFP16 with Lactobacillus reuteri.

4. The study shows that these two microbial agents may be suitable as potential probiotic candidates in guinea fowl, as well as in a feed supplement for other poultry species.

     

Bacteriocin production of probiotic Lactobacillus gasseri LA39 isolated from human feces in milk-based media

The use of bacteriocins from Lactobacillus gasseri , a probiotic lactic acid bacterium, as bio-preservatives in the food industry and animal formulations has been limited because few strains of Lb. gasseri are cultivated and produce a bacteriocin in natural media such as milk and milk-based media. By the determination of the growth-supplements to milk among the 47 nutrients, Lb. gasseri JCM1131^T, LA39 and LA158 isolated from human feces were successfully cultured in reconstituted skim milk and cheese whey using proteose peptone as a nutrient supplement, where Lb. gasseri LA39 produced a useful bacteriocin, gassericin A, with effective growth-inhibiting activity against Gram-positive food-borne pathogens. The data suggest these developed low-cost safe media supporting enough production of bacteriocins by the probiotic Lb. gasseri LA39 could be used to improve the safe bio-preservation of foods and therapy of bovine mastitis, and extra cheese whey produced by cheese making industry is reused in the cultivation for probiotics effectively.     

Evaluation in vitro of the antagonistic substances produced by Lactobacillus spp. isolated from chickens

To determine the inhibitory capacity of lactic acid bacteria due to the action of antagonistic substances, we tested 474 isolates of Lactobacillus from the crop and cecum of chickens against gram-positive and gram-negative indicator microorganisms by the spot-on-the-lawn and well-diffusion antagonism methods. Of the 474 isolates, 265 demonstrated antimicrobial activity against the indicator microorganisms. Isolates identified as L. reuteri, L. salivarius, or Lactobacillus spp. inhibited Enterococcus faecalis, E. faecium, Listeria monocytogenes, and Salmonella spp. but not L. casei, L. delbrueckii, L. fermentum, or L. helveticus by the well-diffusion simultaneous antagonism method under anaerobic incubation conditions. The antagonistic substances produced by some of the Lactobacillus isolates were inactivated after treatment by proteolytic enzymes, which suggested that the substances could be antimicrobial peptides or bacteriocins.     

In vitro probiotic selection and characterization of Lactobacillus spp. isolated from healthy domesticated Turkeys

The health of poultry is closely related to how the animals are raised, and the gut microbiota plays a key role in the fulfillment of their productive potential. Lactobacillus spp. are bacteria present in the natural microbiota of poultry that, when employed as probiotic agents, should present several characteristics. This study aims at performing the in vitro probiotic isolation and characterization of Lactobacillus spp. Isolates were obtained from ceca content of healthy turkeys through the isolation and identification of morphological, molecular, and physiologic characteristics. They were identified through Gram staining, catalase and hydrogen peroxide production tests, gas production tests during glucose fermentation, and hydrogen sulfide production during the triple sugar iron test. The samples were identified molecularly through polymerase chain reaction tests and were subjected to genetic sequencing. The assessment of the probiotic potential was conducted through artificial gastric juice tolerance and bile salt tolerance tests. A hydrophobicity test was used as an indirect method of assessing an isolate’s probable ability to adhere to the intestinal mucosa. In addition, other analyses were performed, such as multiplication potential tests, Salmonella Heildelberg antagonism assays, hydrogen peroxide production tests, and antibiograms, as well as an assessment of the genes responsible for resistance to antimicrobial agents in Integron C. In conclusion, we identified, through morphologic, physiologic, pathogen antagonism, and antimicrobial resistance tests, 11 strains of Lactobacillus spp. belonging to species L. reuteri (9), L. johnsonii (1), and L. frumenti (1) that showed potential as probiotic candidates for in vivo application.     

Cytokine expression in an ex vivo culture system of duodenal samples from dogs with chronic enteropathies: modulation by probiotic bacteria

There is evidence that probiotics have immune-modulating effects on intestinal inflammation during chronic enteropathies (CE). In an ex vivo culture system we investigated the influence of probiotics on mRNA and protein expression levels of cytokines in intestinal samples from dogs suffering from CE. Duodenal samples of client-owned dogs with CE (group CE; n = 12) were collected during diagnostic endoscopy. Additional duodenal samples of gastrointestinally healthy dogs (group C; n = 4) from an unrelated study were available. Based on histopathological analyses, no pathological changes or only mild to moderate eosinophilic and/or lymphoplasmacytic duodenitis were diagnosed. Tissue samples were cultured: (1) with cell culture medium alone (negative control), (2) with a probiotic cocktail (PC), constituted of three Lactobacilli spp. from healthy canine fecal isolates, (3) with the individual strains of PC, and (4) with a placebo powder. Viability of intestinal tissue and probiotic bacteria before and after culture was evaluated. The mRNA abundance of interleukin (IL)-10, IL-12p40, interferon (IFN)-γ, tumor necrosis factor (TNF)-, and transforming growth factor (TGF)-β1 was analyzed by real-time polymerase chain reaction (RT-PCR). Protein concentrations of IFN-γ and IL-10 were measured in culture supernatant by ELISA. Results of RT-PCR were expressed as 2^{(−2ΔCrossing Point)} × 100 after normalization with β-actin. There was a loss of about 1 log CFU/mL of probiotic bacteria during the incubation period. Viability of tissue was maintained as confirmed by non-significant release of lactate dehydrogenase. In C, addition of PC increased IL-10 mRNA levels (P < 0.1). In CE, PC increased mRNA and protein levels of IL-10 (P < 0.05). On the mRNA level, the ratio of TNF-/IL-10, IFN-γ/IL-10, and IL-12p40/IL-10 decreased after addition of PC (P < 0.05). The results demonstrate favorable effects of PC on regulatory cytokines relative to inflammatory cytokines that might contribute to reduction of intestinal inflammation.     

禽类采食量生理调节因子的研究进展

采食量是影响禽类生长最重要的因素之一。认识采食量的调节规律和机制,旨在更有效地调节家禽的采食量,为生产实践提供重要指导。禽类采食量的生理调节机制十分复杂,涉及许多神经通路和体液因子的调节。本文将重点介绍采食量生理调节因子。     

Relation between cell‐bound exopolysaccharide production via plasmid‐encoded genes and rugose colony morphology in the probiotic Lactobacillus brevis KB290

The probiotic Lactobacillus brevis KB290 is a natural producer of cell‐bound exopolysaccharide (EPS), and the plasmid‐encoded glycosyltransferase genes are responsible for this EPS production. KB290 forms unique rugose colonies inside an agar medium; this characteristic is useful for detecting and enumerating KB290 in the gut or feces. However, the genetic elements associated with this morphology remain unclear. Here, we aimed to investigate the relation between the plasmid eps genes and rugose colony morphology in KB290. The plasmid‐cured mutants formed smooth colonies, and the rugose colony morphology was restored after complementation with the eps genes. The eps genes were successfully cloned and expressed in other L. brevis and L. plantarum strains. In these transformant strains, the presence of the EPS, consisting of glucose and N‐acetylglucosamine, correlated with rugose colonies, indicating that EPS is responsible for rugose colony formation. To the best of our knowledge, this is the first report identifying the genetic factors influencing rugose colonies in Lactobacillus strains. This rugose colony formation may serve as a useful selective marker for KB290 in routine laboratory and research settings and can be used to detect the spontaneous loss of plasmids in this strain.     

益生菌对致病性大肠杆菌在雏鸡消化道内的作用效果

1日龄雏鸡120只,随机分为6组,用不同的方法处理后,对各组雏鸡7个部位（嗉囔、腺胃、十二指肠、空肠、回肠、盲肠、直肠）消化道黏膜黏附的O78进行分离计数,研究禽致病性大肠杆菌在雏鸡消化道的分布,比较益生菌对禽致病性大肠杆菌O78在不同雏鸡动物模型中的抑制作用。结果表明：禽致病性大肠杆菌O78主要定植在雏鸡的嗉囔、盲肠和直肠;抗生素处理组和非处理组在灌服益生菌后24、48、72、96h与对照纽相比,各消化道部位定植的O78均有不同程度的降低,说明益生菌对禽致病性大肠杆菌O78。具有较强的抑制能力;未处理组的雏鸡消化道内黏附的O78菌数低于抗生素处理组,说明益生菌在抑制病原菌同时可促进消化道有益菌群的生长,从而防止致病菌的感染。     

Starch digestion rates in multiple samples of commonly used feed grains in diets for broiler chickens

In this study the starch digestion rates in broiler chickens from 18 samples of 5 commonly used feed grains (sorghum, wheat, maize, barley, triticale) were determined. The methodology to determine starch digestion rates in poultry is detailed herein. Starch digestion rates were not significantly different (P = 0.128) across the 18 feed grains, which reflects the wide variations that were observed within a given feedstuff. Nevertheless, starch digestion rates in broiler chickens offered wheat-based diets were significantly more rapid by 56.0% (0.117 versus 0.075 min⁻¹; P = 0.012) than their sorghum-based counterparts on the basis of a pair-wise comparison. In descending order, the following starch digestion rates were observed: wheat (0.117 min⁻¹), barley (0.104 min⁻¹), triticale (0.093 min⁻¹), maize (0.086 min⁻¹), sorghum (0.075 min⁻¹). The implications of these findings are discussed as they almost certainly have implications for poultry nutrition and the development of reduced crude protein diets for broiler chickens.     

Screening of two probiotic products for use in fermented liquid feed

In this trial two commercial probiotic products (Bactocell® and Adjulact® Pro) were investigated in vitro for their use as microbial inoculum for the production of fermented liquid feed (FLF) for pigs. Bactocell® was applied at a dose of 9 and 10 log₁₀ CFU/kg and Adjulact® Pro at a level of 9 log₁₀ CFU/kg. The FLF (control and treatments) was prepared with a water to feed ratio of 4:1 and run in batch for 72 h at 30 °C. The microbial population was followed with plate countings and the lactic acid, acetic acid and ethanol concentration was determined at different time points in the FLF. After 24 h, significant differences (P < 0.05) were found between the control and the Adjulact® Pro FLF for pH (4.7 vs 4.3), lactic acid (57.9 vs 91.5 mmol/L), acetic acid (23.1 vs 6.8 mmol/L), ethanol (24.5 vs 1.1 mmol/L), coliforms (7.2 vs 4.3 log₁₀ CFU/mL) and E. coli (6.2 vs 4.4 log₁₀ CFU/mL). Bactocell® addition did not alter the fermentation characteristics compared to the control FLF. After 72 h no significant differences between treatments were noted, except for the yeast count which was higher in the FLF inoculated with Adjulact® Pro.     

Intracerebral pathogenicity for chickens of avian influenza viruses isolated from free-living waterfowl in Japan

The pathogenicity for chickens of 91 strains of avian influenza A virus isolated from such free-living waterfowl as whistling swan, pintail, tufted duck, mallard and black-tailed gull in Japan was tested. The majority of the virus strains infected and were pathogenic for the chickens. The virulence of these viruses seemed not to be as high as that of fowl plague virus. There were no significant differences in the intracerebral index score among the viruses belonging to the same subtype, irrespective of year of isolation or host.     

Study and use of the probiotic Lactobacillus reuteri in pigs: a review

Probiotics are living microorganisms that provide a wide variety of health benefits to the host when ingested in adequate amounts. The bacterial strains most frequently used as probiotic agents are lactic acid bacteria, such as Lactobacillus reuteri, which is one of the few endogenous Lactobacillus species found in the gastrointestinal tract of vertebrates, including humans, rats, pigs and chickens. L. reuteri is one of the most well documented probiotic species and has been widely utilized as a probiotic in humans and animals for many years. Initially, L. reuteri was used in humans to reduce the incidence and the severity of diarrhea, prevent colic and necrotic enterocolitis, and maintain a functional mucosal barrier. As interest in alternatives to in-feed antibiotics has grown in recent years, some evidence has emerged that probiotics may promote growth, improve the efficiency of feed utilization, prevent diarrhea, and regulate the immune system in pigs. In this review, the characteristics of L. reuteri are described, in order to update the evidence on the efficacy of using L. reuteri in pigs.     

Pathogenicity for chickens of a paramyxovirus type 1, isolated from racing pigeons in Sweden

Strains of paramyxovirus type 1 (PMV-1) have been isolated from diseased racing pigeons in Sweden. One of these isolates was selected for studies of the pathogenicity and contagiousness in chickens.The same isolate was previously found to have a high intravenous pathogenicity index (IVPI) in 6 weeks old chickens.In three experiments it was found that the PMV-1 isolate was very pathogenic for 1 week old chickens but not pathogenic for 120 day old pullets inoculated intranasally and ocularly.Symptoms in the young chickens were similar to those seen in the neurotropic form of Newcastle disease. The mortality was high and the incubation period 5–11 days.The disease easily spread to young chickens kept in contact with diseased birds.The microscopic examination revealed an interstitial nonpurulent pneumonia and a nonpurulent encephalitis in the young chickens. In the pullets the only finding was a mild encephalitis.PMV-1 was recovered from all young chickens but not from the pullets.Both the chickens and the inoculated pullets developed antibodies to PMV-1.     

Selection of useful probiotic lactic acid bacteria from the Lactobacillus acidophilus group and their applications to functional foods

In the present review, a new mass screening system for selecting probiotic strains from Lactobacillus (L) acidophilus group lactic acid bacteria (LAB) with strong adhesion to the human intestinal tract is described. Characteristics of antimicrobial peptides (bacteriocin), lactose‐hydrolyzing enzymes and immunostimulative oligo DNA motifs in L. gasseri strains are also described. Finally, the use of L. acidophilus LAB, selected by our screening method, that have strong adhesion to the human colonic mucosa in functional yogurt products is described. Adhesiveness to the human intestine is one of the most important characteristics of probiotic LAB. A new screening system that involves a combination of three methods is proposed: rat colonic mucin (RCM)‐micro plate assay, Carnoy's histochemical staining method and carbohydrate probe binding assay. By using an RCM‐coated poly‐vinylidene‐diflouride membrane that mimics the human colonic mucous layer, a new lectin was isolated and its structure was clarified by gene cloning. Furthermore, the structures and functions of a new cyclic bacteriocin (gassericin A), new lactose‐hydrolyzing enzymes, and new immunostimulating oligo DNA motifs from Lactobacillus gasseri (B₁ subgroup) were clarified. A new functional yogurt ‘Fit down’ is proposed, that is fermented by an adhesive strain of L. acidophilus LA67 selected by our screening and contains antihypertensive peptides derived from whey proteins by protease digestion. In the future, superior functional foods containing more effective probiotic LAB are expected to be developed by the use of the proposed mass screening system.     

鸡源性沙门氏菌耐药基因检测与耐药相关性分析

为研究沙门氏菌耐药基因与其耐药性之间的关系,本研究以K-B纸片法对临床分离的29株鸡源性沙门氏菌进行10种抗菌药物敏感性测定,应用PCR技术进行这些抗菌药物相关耐药基因检测.并通过质粒接合试验证明了耐药性及耐药基因的转移.结果显示:29株分离株对氨苄西林耐药率为31.0％,对头孢唑啉耐药率为10.3％,对四环素耐药率为44.8％,对复合磺胺耐药率为41.4％,对氯霉素耐药率为3.4％,对环丙沙星、氧氟沙星、诺氟沙星的耐药率均为37.9％,但对庆大霉素和卡那霉素均敏感.本实验共检测到8种耐药基因,其中blatem-1阳性率为31.0％,tetA和tetB阳性率为44.8％,sull和sul2阳性率为41.3％,aadAl阳性率为3.4％,dfrA1阳性率为10.3％,qnr阳性率为37.9％,而tetG、blaCMY-2和catI均未检出.此外,通过与非耐药菌接合后绝大部分接合子均获得了供体菌质粒编码的相应抗性表型,耐药基因在接合过程也发生了转移,赋予被接合细菌新的耐药性.以上结果表明沙门氏菌的耐药性与其相关耐药基因的检出率基本一致,而且耐药性通过质粒进行转移.     

Early-age feed restriction affects viability and gene expression of satellite cells isolated from the gastrocnemius muscle of broiler chicks

Background: Muscle growth depends on the fusion of proliferate satellite cells to existing myofibers. We reported previously that 0-14 day intermittent feeding led to persistent retardation in myofiber hypertrophy. However, how satellite cells respond to such nutritional insult has not been adequately elucidated. Results: One-day-old broiler chicks were allocated to control (Con, ad libitum feeding), intermittent feeding (IF, feed provided on alternate days) and re-feeding (RF, 2 days ad libitum feeding after 12 days of intermittent feeding) groups. Chickens were killed on Day 15 and satellite cells were isolated. When cultured, satellite cells from the IF group demonstrated significant retardation in proliferation and differentiation potential, while RF partly restored the proliferation rate and differentiation potential of the satellite cells. Significant up-regulation of insulin like growth factor I receptor (IGF-IR) (P<0.05) and thyroid hormone receptor α (TRα) (P<0.05), and down-regulation of growth hormone receptor (GHR) (P<0.01) and IGF-I (P<0.01) mRNA expression was observed in freshly isolated IF satellite cells when compared with Con cells. In RF cells, the mRNA expression of IGF-I was higher (P<0.05) and of TRα was lower (P<0.01) than in IF cells, suggesting that RF restored the mRNA expression of TRα and IGF-I, but not of GHR and IGF-IR. The Bax/Bcl-2 ratio tended to increase in the IF group, which was reversed in the RF group (P<0.05), indicating that RF reduced the pro-apoptotic influence of IF. Moreover, no significant effect of T 3 was detected on cell survival in IF cells compared with Con (P<0.001) or RF (P<0.05) cells. Conclusions: These data suggest that early-age feed restriction inhibits the proliferation and differentiation of satellite cells, induces changes in mRNA expression of the GH/IGF-I and thyroid hormone receptors in satellite cells, as well as blunted sensitivity of satellite cells to T3 , and that RF partially reverses these effects. Thus, a moderate nutritional strategy for feed restriction should be chosen in early chick rearing systems.