Identification of lactobacilli residing in chicken ceca with antagonism against Campylobacter

Bacteriocins produced by Lactobacillus salivarius have been recently recognized as a natural means to control Campylobacter and Salmonella in live poultry. This finding is of relevance since Campylobacter jejuni and Campylobacter coli are the predominant species isolated from poultry that are associated with human campylobacteriosis. In the present work, lactic acid bacteria (LAB) isolated from the cecum of twenty Tunisian chickens were identified and those isolates with antagonism against Campylobacter were further characterized. Following their preliminary confirmation as LAB, 150 strains were identified by combining morphological criteria, biochemical tests, and molecular methods, the latter inluding intergenic 16S- 23S PCR, specific lactobacilli PCR, and a biphasic approach. Most of the LAB isolated belonged to the genus Lactobacillus, among them Lb. sakei (33.3%), Lb. salivarius (19.4%), Lb. reuteri (8.6%), and Lb. curvatus (8.6%). The other LAB strains included those of the genus Weissella (16.7%), Enterococcus faecalis (5.3%), Leuconostoc mesenteroides (2.7%), Lactococcus graviae (2.7%), and Streptococcus sp. (2.7%). The Lactobacilli strains were tested for their antagonism against C. jejuni and C. coli. The activity of three of them, Lb. salivarius SMXD51, Lb. salivarius MMS122, and Lb. salivarius MMS151, against the aforementioned target strains could be ascribed to the production of bacteriocins.     

Characterization of bacteriocin-like inhibitory substances (BLIS) from sourdough lactic acid bacteria and evaluation of their in vitro and in situ activity

AIMS: To identify and characterize bacteriocion-producing lactic acid bacteria (LAB) in sourdoughs and to compare in vitro and in situ bacteriocin activity of sourdough- and nonsourdough LAB. METHODS AND RESULTS: Production of antimicrobial compounds by 437 Lactobacillus strains isolated from 70 sourdoughs was investigated. Five strains (Lactobacillus pentosus 2MF8 and 8CF, Lb. plantarum 4DE and 3DM and Lactobacillus spp. CS1) were found to produce distinct bacteriocin-like inhibitory substances (BLIS). BLIS-producing Lactococcus lactis isolated from raw barley showed a wider inhibitory spectrum than sourdough LAB, but they did not inhibit all strains of the key sourdough bacterium Lb. sanfranciscensis. Antimicrobial production by Lb. pentosus 2MF8 and Lc. lactis M30 was also demonstrated in situ. CONCLUSIONS: BLIS production by sourdough LAB appears to occur at a low frequency, showing limited inhibitory spectrum when compared with BLIS-producing Lc. lactis. Nevertheless, they are active BLIS producers under sourdough and bread-making conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The activity of BLIS has been demonstrated in situ. It may influence the complex sourdough microflora and support the implantation and stability of selected insensitive bacteria, such as Lb. sanfranciscensis, useful to confer good characteristics to the dough.     

Carbon and nitrogen mineralization in soil amended with phenanthrene,anthracene and irradiated sewage sludge

Lactic acid production by Lactobacillus brevis and Lactobacillus pentosus on a hemicellulose hydrolysate (HH) of wet-oxidized wheat straw was evaluated. The potential of 11-12 g/l fermentable sugars was released from the HH through either enzymatic or acidic pretreatment. Fermentation of added xylose in untreated HH after wet-oxidation, showed no inhibition on the lactic acid production by either Lb. pentosus or Lb. brevis. Lb. pentosus produced lactate corresponding to 88% of the theoretical maximum yield regardless of the hydrolysis method, whereas Lb. brevis produced 51% and 61% of the theoretical maximum yield after enzymatic, or acid treatment of HH, respectively. Individually, neither of the two strains were able to fully utilize the relatively broad spectra of sugars released by the acid and enzyme treatments; however, lactic acid production increased to 95% of the theoretical maximum yield by co-inoculation of both strains. Xylulose was the main sugar released after enzymatic treatment of HH with Celluclast. Lb. brevis was able to degrade xylobiose, but was unable to assimilate xylulose, whereas Lb. pentosus was able to assimilate xylulose but unable to degrade xylobiose.     

Microbial analysis of Malaysian tempeh, and characterization of two bacteriocins produced by isolates of Enterococcus faecium

AIMS: Isolation of bacteriocinogenic lactic acid bacteria (LAB) from the Malaysian mould-fermented product tempeh and characterization of the produced bacteriocin(s). METHODS AND RESULTS: LAB were present in high numbers in final products as well as during processing. Isolates, Enterococcus faecium B1 and E. faecium B2 (E. faecium LMG 19827 and E. faecium LMG 19828, respectively) inhibited Gram-positive indicators, including Listeria monocytogenes. Partially purified bacteriocins showed a proteinaceous nature. Activity was stable after heat-treatment except at alkaline pH values. Both strains displayed a bacteriostatic mode of action. Bacteriocin production was associated with late exponential/early stationary growth. Molecular mass, calculated by SDS-PAGE, was 3.4 kDa for B1 bacteriocin, and 3.4 kDa and 5.8 kDa for B2 bacteriocins. PCR screening of enterocin-coding genes revealed three amplified fragments in total genomic DNA that may correspond with PCR signals for enterocin P, enterocin L50A and enterocin L50B. Both B1 and B2 contained a 42-kb plasmid. No differences in bacteriocinogenic capacity were found between wild type strains and plasmid-cured strains. CONCLUSIONS: It was possible to isolate bacteriocinogenic E. faecium active against various Gram-positive bacteria from final products of tempeh. SIGNIFICANCE AND IMPACT OF THE STUDY: A first step in applying biopreservation to fermented South-east Asian foods is to obtain bacteriocinogenic LAB from this source. Such isolates may also be used for biopreservation of mould-fermented foods in general, including various types of mould-ripened cheese.     

Cloning and heterologous production of Hiracin JM79, a Sec-dependent bacteriocin produced by Enterococcus hirae DCH5, in lactic acid bacteria and Pichia pastoris

Hiracin JM79 (HirJM79), a Sec-dependent bacteriocin produced by Enterococcus hirae DCH5, was cloned and produced in Lactococcus lactis, Lactobacillus sakei, Enterococcus faecium, Enterococcus faecalis, and Pichia pastoris. For heterologous production of HirJM79 in lactic acid bacteria (LAB), the HirJM79 structural gene (hirJM79), with or without the HirJM79 immunity gene (hiriJM79), was cloned into the plasmid pMG36c under the control of the constitutive promoter P(32) and into the plasmid pNZ8048 under the control of the inducible P(NisA) promoter. For the production of HirJM79 in P. pastoris, the gene encoding the mature HirJM79 protein was cloned into the pPICZalphaA expression vector. The recombinant plasmids permitted the production of biologically active HirJM79 in the supernatants of L. lactis IL1403, L. lactis NZ9000, L. sakei Lb790, E. faecalis JH2-2, and P. pastoris X-33, the coproduction of HirJM79 and nisin A in L. lactis DPC5598, and the coproduction of HirJM79 and enterocin P in E. faecium L50/14-2. All recombinant LAB produced larger quantities of HirJM79 than E. hirae DCH5, although the antimicrobial activities of most transformants were lower than that predicted from their production of HirJM79. The synthesis, processing, and secretion of HirJM79 proceed efficiently in recombinant LAB strains and P. pastoris.     

Anti‐listerial activity of bacteriocin‐producing Lactobacillus curvatus CWBI‐B28 and Lactobacillus sakei CWBI‐B1365 on raw beef and poultry meat

Aim: The study aimed to evaluate the effect of the bacteriocins produced by Lactobacillus sakei CWBI‐B1365 and Lactobacillus curvatus CWBI‐B28 on the growth and survival of Listeria monocytogenes in raw beef and poultry meat. Methods and Results: The sakacin P and sakacin G structural genes were identified in Lact. curvatus CWBI‐B28 and Lact. sakei CWBI‐B1365 using PCR amplification, respectively. The effect of the two bacteriocinogenic strains either alone or together, and that of the nonbacteriocin‐producing strain Lact. sakei LMG17302, on the growth of L. monocytogenes was evaluated in beef and poultry meat. In raw beef, the pathogenic bacteria were inhibited by the bacteriocinogenic strains. The bacteriocinogenic strains had no activity in raw chicken meat when inoculated separately, while they showed a clear anti‐Listeria effect when applied together. Conclusion: Sakacin G producing Lact. sakei and sakacin P producing Lact. curvatus may be applied in raw beef to inhibit L. monocytogenes. In poultry meat, the inhibition of L. monocytogenes could only be achieved by a combined application of these bacteriocin‐producing strains. Significance and Impact of the Study: In some meat products, the combined application of different class IIa bacteriocin producing lactic acid bacterium can enhance the anti‐listerial activity.     

Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolates from broiler chickens isolated at an Irish poultry processing plant

AIMS: The antibiotic susceptibility of Campylobacter jejuni and Campylobacter coli isolates from broiler chickens were determined in order to evaluate the level of antibiotic resistance of Campylobacter species in the Irish poultry industry. METHODS AND RESULTS: Seventy-eight Camp. jejuni and 22 Camp. coli strains were examined for susceptibility to eight antibiotics using the disc diffusion assay. The highest level of resistance of the Camp. jejuni isolates was recorded to ampicillin (35.9%), followed by 20.5% to tetracycline, 20.5% to naladixic acid, 17.9% to ciprofloxacin, 10.2% to erythromycin, 2.5% to streptomycin and 1.2% to kanamycin. Multidrug resistance to two or more antibiotics was seen for 30.7% of Camp. jejuni strains. Resistance of the Camp. coli isolates was shown to ampicillin (9%) and tetracycline (18.2%). CONCLUSIONS: The majority of Camp. jejuni strains were susceptible to antibiotics commonly used for human therapy. Camp. coli strains showed very low resistance levels and were susceptible to six of the eight antimicrobial agents studied. SIGNIFICANCE AND IMPACT OF THE STUDY: Levels of Camp. jejuni and Camp. coli antimicrobial resistance in Irish poultry production was assessed to determine the current situation in Ireland. The prevalence of antibiotic resistance of Campylobacter strains isolated from broiler chickens was low.     

High-resolution genotyping of Campylobacter strains isolated from poultry and humans with amplified fragment length polymorphism fingerprinting.

For epidemiological studies of Campylobacter infections, molecular typing methods that can differentiate campylobacters at the strain level are needed. In this study we used a recently developed genotyping method, amplified fragment length polymorphism (AFLP), which is based on selective amplification of restriction fragments of chromosomal DNA, for genetic typing of Campylobacter jejuni and Campylobacter coli strains derived from humans and poultry. We developed an automated AFLP fingerprinting method in which restriction endonucleases HindIII and HhaI were used in combination with one set of selective PCR primers. This method resulted in evenly distributed band patterns for amplified fragments ranging from 50 to 500 bp long. The discriminatory power of AFLP was assessed with a C. jejuni strain, an isogenic flagellin mutant, and distinct C. jejuni strains having known pulsed-field gel electrophoresis and fla PCR-restriction fragment length polymorphism genotypes. Unrelated C. jejuni strains produced heterogeneous patterns, whereas genetically related strains produced similar AFLP patterns. Twenty-five Campylobacter strains obtained from poultry farms in The Netherlands grouped in three C. jejuni clusters that were separate from a C. coli cluster. The band patterns of 10 C. jejuni strains isolated from humans were heterogeneous, and most of these strains grouped with poultry strains. Our results show that AFLP analysis can distinguish genetically unrelated strains from genetically related strains of Campylobacter species. However, desirable genetically related strains can be differentiated by using other genotyping methods. We concluded that automated AFLP analysis is an attractive tool which can be used as a primary method for subtyping large numbers of Campylobacter strains and is extremely useful for epidemiological investigations.     

The prevalence of campylobacters and arcobacters in broiler chickens

Chicken carcasses from a supermarket and from a poultry abattoir were examined using methods designed to isolate as many strains of campylobacters and related organisms as possible. Strains of arcobacter, but no campylobacters, were isolated from every carcass after enrichment. Campylobacter jejuni subsp. jejuni was isolated from all carcasses examined by direct plating and other Campylobacter -like strains were isolated from nine out of 15 abattoir carcasses by direct plating but not after enrichment. Only the Camp. jejuni subsp. jejuni strains could be identified to species level using a readily available identification scheme and/or a commercial identification kit. Examination of caecal contents from the 15 abattoir poultry yielded Camp. jejuni subsp. jejuni and Campylobacter -like strains from 15 and eight by direct plating, and from six and nine after enrichment, respectively. Four sites in the intestine of the abattoir birds (60 samples) were examined for arcobacters and only one strain was isolated. This indicates that arcobacters are probably not normal inhabitants of the poultry intestine. Poultry is a rich source of other campylobacteria besides the thermophilic Campylobacter spp.     

139株空肠弯曲菌耐药性分析

空肠弯曲菌(Campylobacter jejuni)是最常见的食源性病原菌之一。本研究采用微量肉汤稀释法对分离得到的139株空肠弯曲菌(117株为禽源样本分离株,22株为人源样本分离株)进行耐药性检测。通过对最小抑菌浓度(MIC)的判定结果得出:120株(86. 33%)空肠弯曲菌分离株对6类9组临床常用的抗生素表现出不同程度的耐药,其中禽源空肠弯曲菌耐药率为83. 76%,22株人源空肠弯曲菌均表现出耐药性。对喹诺酮类抗生素表现出高度耐药(环丙沙星80. 58%,萘啶酸77. 70%);对四环素类表现为中等耐药(四环素53. 24%);对部分大环内酯类、氨基糖苷类、林可酰胺类表现为低耐药(庆大霉素7. 19%,阿奇霉素5. 76%,克林霉素6. 47%);对酰胺醇类、部分大环内酯类表现为敏感(氟苯尼考0%,红霉素0%、泰利霉素0%)。139株空肠弯曲菌共产生14种耐药谱型,以TET-CIP-NAL谱型最多,占比38. 13%,耐三重及以上抗生素的多重耐药菌株占比53. 24%。禽源菌株中多重耐药占比46. 15%,人源菌株中多重耐药占比90. 91%。研究结果显示空肠弯曲菌耐药现状不容乐观,尤其对喹诺酮类与四环素类抗生素耐药性较为突出,且过半数菌株为多重耐药。本研究为食源性空肠弯曲菌的防控及临床用药提供参考。     

Serotypes, antimicrobial susceptibility, and gyr A gene mutation of Campylobacter jejuni isolates from humans and chickens in Thailand

In Thailand, 51% (36/70) Campylobacter jejuni isolates from humans and 68% (47/69) isolates from poultry were classified into 10 Penner serotypes (serotype B, C, R, E, G, A, K, D, I, and L) and 9 serotypes (serotype A, C, I, K, B, E, S, D, and L), respectively. The rate of antimicrobial drug resistance to nalidixic acid, ciprofloxacin, ampicillin, tetracycline, and erythromycin shown by human isolates were 96%, 96%, 29%, 57%, and 14%, while that shown by poultry isolates were 77%, 77%, 22%, 26%, and 17%, respectively. All quinolone-resistant strains contained a mutation in the gyrA gene (T(86)-->I(86)), suggesting that the strains were already widespread in Thailand.     

Improved method for the isolation of Campylobacter jejuni and Campylobacter coli bacteriophages

A centrifugation and filtration method of isolating Campylobacter phages has been developed. Forty-nine Campylobacter phages were isolated from 272 effluent samples of which 42 produced lysis with Campylobacter jejuni strains and seven with C. coli strains. Phages were recovered from pig manure, abattoir effluents, human faeces, sewage and poultry manure. Phages were not isolated from water samples, cattle and sheep faeces or farm pasture soil.     

The Effect of Lactic Acid Sprays on Campylobacter jejuni Inoculated onto Poultry Carcasses

Spraying poultry carcasses with 1 % lactic acid 10 min after inoculation with Campylobacter jejuni, resulted in a significant reduction in the number of the bacteria after 4 h at 4°C. Some of the inoculated cells, however, survived for at least 144 h. Spraying 10 min after inoculation with 2% lactic acid, totally eliminated all inoculated C. jejuni within 24 h. On the other hand, spraying 24 h after inoculation, with either 1 % or 2 % lactic acid did not eliminate all the bacteria. Inoculated C. jejuni on poultry carcasses not sprayed with lactic acid, survived at 4°C throughout the sampling period (up to 144 h) and showed little tendency to decrease in number even when the carcasses started to deteriorate. Resident Campylobacters on poultry carcasses were significantly reduced by the lactic acid treatment. Frozen and thawed chickens appeared to show a graying of the skins immediately after spraying with lactic acid, slightly stronger with 2 % lactic acid, but the colour reverted to normal after 24 h. We were not able to observe any colour change on the fresh broiler chickens after lactic acid treatment. Our results indicated that lactic acid had a significant bactericidal effect on C. jejuni on both naturally and artificially contaminated poultry carcasses. This effect, however, became manifest only several hours after acid treatment.     

Probiotic properties of Lactobacillus strains with high cinnamoyl esterase activity isolated from jeot-gal,a high-salt fermented seafood

Cinnamoyl esterases (CEs) improve the bioavailability of caffeic acid, a potent antioxidant with beneficial health effects. This study aimed to characterize the probiotic properties of 14 strains of CE-producing lactic acid bacteria (LAB) isolated from jeot-gal, a high-salt fermented seafood. We evaluated properties of the probiotic LAB with high CE activity, including tolerance to low pH and bile salts, antimicrobial activity, surface hydrophobicity, adhesion, and immunomodulatory effects, in vitro. All LAB tested tolerated pH 2.0 and 3% Oxgall, i.e., conditions comparable with those in the gastrointestinal environment. Three isolates, Lactobacillus paracasei JBCC10650, Lactobacillus pentosus JBCC10659, and Lactobacillus plantarum JBCC10543, showed stronger adherence to epithelial cells (12.3, 9.6, and 9.4%) than a commercial probiotic Lactobacillus rhamnosus GG (9.1%; p < 0.05), and exhibited broad antibacterial activity against putative pathogens. Most of the 14 LAB strains were able to regulate mRNA expression of pro- and anti-inflammatory cytokines in macrophages, indicating their potential immunomodulatory effects. Our findings suggest that the newly isolated CE-producing probiotics may show beneficial health effects by supporting the host immune system.     

Study on the epidemiology and control of Campylobacter jejuni in poultry broiler flocks.

Broiler flocks are frequently infected with Campylobacter jejuni. The origin of the infection is still unclear. The question of whether colonization of flocks results from transmission of C. jejuni from breeder flocks to progeny (vertical transmission) or from environmental sources (horizontal transmission) remains to be answered. Therefore, in this study samples were taken from successive broiler flocks in two broiler houses (house A on farm A and house B1 on farm B) as well as from the environment of the houses. All C. jejuni isolates were typed by using the Penner serotyping system, and part of the isolates from farm B were typed by using a randomly amplified polymorphic DNA-typing system. In poultry house A, C. jejuni was isolated from the first flock but not from subsequent flocks. In poultry house B1, C. jejuni strains of the same Penner serotypes and exhibiting identical DNA profiles were isolated from successive flocks. Infection of the flocks from a common source via horizontal pathways is suspected, while a vertical route of infection is not likely to exist. Application of measures to control horizontal transmission of C. jejuni on farm B was successful.     

Identification and characterization of Lactic Acid Bacteria isolated from Tibetan Qula cheese

Fourteen strains of Lactic Acid Bacteria (LAB) isolated from Qula, a Tibetan traditional yak cheese, were divided into four groups (A-D) according to morphological and biochemical characteristics. On the basis of the 16S rRNA gene sequence analysis, group A and group B strains were placed in the cluster making up the genus Leuconostoc, which together with Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides, formed a distinct cluster. The group C strain was clearly identified as Enterococcus faecium by forming a very well defined cluster with this species. The group D strains were placed in the lactobacilli cluster with Lactobacillus plantarum and Lactobacillus pentosus being the closely related species. On the basis of DNA-DNA hybridization, strains in the groups A, B, C and D were identified as Leuconostoc mesenteroides subsp. dextranicum, Leuconostoc pseudomesenteroides, Enterococcus faecium and Lactobacillus plantarum, respectively. Leuconostoc mesenteroides subsp. dextranicum was the dominate member of the population.     

Study on the epidemiology and control of Campylobacter jejuni in poultry broiler flocks.

Broiler flocks are frequently infected with Campylobacter jejuni. The origin of the infection is still unclear. The question of whether colonization of flocks results from transmission of C. jejuni from breeder flocks to progeny (vertical transmission) or from environmental sources (horizontal transmission) remains to be answered. Therefore, in this study samples were taken from successive broiler flocks in two broiler houses (house A on farm A and house B1 on farm B) as well as from the environment of the houses. All C. jejuni isolates were typed by using the Penner serotyping system, and part of the isolates from farm B were typed by using a randomly amplified polymorphic DNA-typing system. In poultry house A, C. jejuni was isolated from the first flock but not from subsequent flocks. In poultry house B1, C. jejuni strains of the same Penner serotypes and exhibiting identical DNA profiles were isolated from successive flocks. Infection of the flocks from a common source via horizontal pathways is suspected, while a vertical route of infection is not likely to exist. Application of measures to control horizontal transmission of C. jejuni on farm B was successful.     

Evaluation of new broth media for microdilution antibiotic susceptibility testing of Lactobacilli, Pediococci, Lactococci, and Bifidobacteria

Nine pure or mixed broth media were evaluated for their suitabilities to determine MICs in a microdilution test of 19 antibacterial agents for lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Lactococcus, and Bifidobacterium. A mixed formulation of Iso-Sensitest broth (90%) and deMan-Rogosa-Sharpe broth (10%) with or without supplementation with L-cysteine, referred to as the LAB susceptibility test medium, provided the most optimal medium basis in terms of growth support of nonenterococcal LAB and correct indication of MICs of international control strains.     

Expression of Campylobacter genes for proline biosynthesis in Escherichia coli

Cloned DNA from Campylobacter jejuni was found to complement auxotrophic defects in proline metabolism in several strains of Escherichia coli. A 4.4-kilobase fragment of Campylobacter DNA encodes the genes analogous to the proA and B genes of E. coli and Salmonella typhimurium.     

Characteristics and identification of enterocins produced by Enterococcus faecium JCM 5804T

AIMS: To screen bacteriocin-producing lactic acid bacteria (LAB) in 52 type and reference strains, which have not previously been studied, with respect to bacteriocins, and to characterize the presence of bacteriocins. METHODS AND RESULTS: Only Enterococcus faecium JCM 5804T showed bacteriocin-like activity. It inhibited the growth of Lactobacillus spp., Enterococcus spp., Clostridium spp., Listeria monocytogenes, and vancomycin resistant Enterococcus (VRE). However, it was not effective against Gram-negative strains, Weisella spp., Leuconostoc spp., Lactococcus spp., or methicillin resistant Staphylococcus aureus (MRSA). The inhibitory activity of Ent. faecium JCM 5804T was inactivated by proteinase K, trypsin, alpha-chymotrypsin, and papain, but not by lysozyme, lipase, catalase, or beta-glucosidase. The inhibitory activity was stable at 100 degrees C for 30 min, and had a pH range from 2 to 10. The molecular weight of the partially purified bacteriocin(s) was approx. 4.5 kDa, according to tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Polymerase chain reaction and direct sequencing methods identified three different types of bacteriocins produced by Ent. faecium JCM 5804T, enterocin A, enterocin B, and enterocin P-like bacteriocin. CONCLUSION: Enterococcus faecium JCM 5804T produced three different types of bacteriocins, and they inhibited LAB and pathogens. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report of enterocin A, enterocin B, and enterocin P-like bacteriocin, detected in Ent. faecium JCM 5804T among LAB type and reference strains.