[Show abstract][Hide abstract] ABSTRACT: Campylobacter jejuni, one of the most common causes of gastroenteritis worldwide, is transmitted to humans through poultry. We previously reported that Lactobacillus gasseri SBT2055 (LG2055) reduced C. jejuni infection in human epithelial cells in vitro and inhibited pathogen colonization of chickens in vivo. This suggested that the LG2055 adhesion and/or co-aggregation phenotype mediated by cell-surface aggregation-promoting factors (APFs) may be important for the competitive exclusion of C. jejuni. Here, we show that cell surface-associated APF1 promoted LG2055 self-aggregation and adhesion to human epithelial cells, and exhibited high affinity for the extracellular matrix component fibronectin. These effects were absent in the apf1 knockout mutant, indicating the role of APF1 in LG2055-mediated inhibition of C. jejuni in epithelial cells and chicken colonization. Similar to APF1, APF2 promoted the co-aggregation of LG2055 and C. jejuni, but did not inhibit C. jejuni infection. Our data suggest a pivotal role for APF1 in mediating the interaction of LG2055 with human intestinal cells and in inhibiting C. jejuni colonization of the gastrointestinal tract. We thus provide new insight into the health-promoting effects of probiotics and mechanisms of competitive exclusion in poultry. Further research is needed to determine whether the probiotic strains reach the epithelial surface.
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[Show abstract][Hide abstract] ABSTRACT: The virulence plasmid profile of R. equi strains isolated from Suidae and humans is similar. Recent evidence suggests that the consumption of pork products contaminated with feces might be a potential source of R. equi infections in humans, mainly to patients with rhodococcosis without history of contact with pigs or pig farms. The present study investigated the virulence-associated genes (vapA and vapB) and plasmid profiles of R. equi among the 150 samples of small intestinal content obtained from slaughtered pigs. In addition, all samples were subjected to microbiological culture in conventional sheep blood agar and CAZ-NB, TCP and TVP selective media. A total of 40 (26.7%) of the samples recovered R. equi, with two samples recovering isolates harboring the VapB type 8 plasmid. Among the 150 pigs sampled herein, CAZ-NB was considered the best selective medium for the isolation of R. equi from feces. Our results provide evidence that the contamination of slaughtered pig carcasses with pathogenic R. equi might occur through feces, representing a public health concern. Furthermore, the present study is the first description of R. equi strains carrying the VapB plasmid in the gut of pigs. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: A virulence plasmid of Rhodococcus equi harbors the vap mutigene family. Here we show that transcription of vap gene family members other than vapA (vapD, vapE and vapG) was regulated by temperature and pH and was abolished when either virS or virR was deleted. Expression of VirS in the absence of functional VirR increased the transcriptional levels of vap genes to those expressed in the presence of VirR. These findings suggest that the transcription of vap genes is regulated by VirS and that VirR is involved in the mechanism of transcriptional responses to temperature and pH.
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Microbiology and Immunology 06/2015; 59(8):n/a-n/a. DOI:10.1111/1348-0421.12277 · 1.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background Rhodococcus equi is an important pulmonary pathogen in foals and in immunocompromised individuals. Virulent R. equi strains carry an 80-90 kb virulence plasmid that expresses the virulence-associated protein A (VapA). VapA expression is regulated by temperature and pH. The LysR-type transcriptional regulator, VirR, is involved in the regulation of the vapA gene. To examine the mechanism underlying transcriptional regulation of vapA, we characterized an R. equi mutant in which another putative transcriptional regulator encoded on the virulence plasmid, VirS, was deleted.ResultsDeletion of virS reduced vapA promoter activity to non-inducible levels. Complementary expression of VirS in the virS deletion mutant restored transcription at the P vapA promoter, even under non-inducing conditions (30°C and pH 8.0). In addition, VirS expression increased P vapA promoter activity in the absence of functional VirR. Further, transcription of the icgA operon containing virS was regulated by pH and temperature in the same manner as vapA.Conclusions
This study suggests that VirS is required for VapA expression and that regulation of P vapA -promoter activity may be achieved by controlling VirS expression levels.
[Show abstract][Hide abstract] ABSTRACT: Campylobacter is a normal inhabitant of the chicken gut. Pathogenic infection with this organism in humans is accompanied by severe inflammation of the intestinal mucosal surface. The aim of this study was to evaluate the ability of Lactobacillus gasseri SBT2055 (LG2055) to inhibit the adhesion and invasion of Campylobacter jejuni in vitro and to suppress C. jejuni colonization of chicks in vivo. Pretreatment with LG2055 significantly reduced adhesion to and invasion of a human epithelial cell line, Intestine 407, by C. jejuni 81-176. Methanol (MeOH)-fixed LG2055 also reduced infection by C. jejuni 81-176. However, proteinase K (ProK)-treated LG2055 eliminated the inhibitory effects. Moreover, LG2055 co-aggregated with C. jejuni 81-176. ProK treatment prevented this co-aggregation, indicating that the co-aggregation phenotype mediated by the proteinaceous cell-surface components of LG2055 is important for reducing C. jejuni 81-176 adhesion and invasion. In an in vivo assay, oral doses of LG2055 were administered to chicks daily for 14 days after oral inoculation with C. jejuni 81-176. At 14 days post-inoculation, chicks treated with LG2055 had significantly reduced cecum colonization by C. jejuni. Reduction in the number of C. jejuni 81-176 cells adhering to and internalized by human epithelial cells demonstrated that LG2055 is an organism that effectively and competitively excludes C. jejuni 81-176. In addition, the results of the chick colonization assay suggest that treatment with LG2055 could be useful in suppressing C. jejuni colonization of the chicks at early growth stages.
PLoS ONE 09/2014; 9(9):e108827. DOI:10.1371/journal.pone.0108827 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rhodococcus equi was isolated from the submaxillary lymph nodes of wild boars (Sus scrofa) in Wakayama and Hyogo, Japan, with a high prevalence. Rhodococcus equi isolation rates between locations, sexes, or body weights were not different, except in the prevalence of vapB-positive R. equi between locations.
[Show abstract][Hide abstract] ABSTRACT: Acute hypotonic stress becomes a threat to the survival of bacteria in the environment. Mechanosensitive channels play an essential role in the maintenance of bacterial cell integrity during hypoosmotic shock. A database search suggested that Campylobacter jejuni, a major worldwide cause of bacterial gastroenteritis in humans, possesses two putative mechanosensitive channels, designated Cjj0263 and Cjj1025, in C. jejuni strain 81-176. Osmotic downshock experiments demonstrated that a mutant lacking Cjj0263 showed a severe defect in survival of hypoosmotic shock, while a mutant lacking Cjj1025 exhibited the same survival capacity as the wild type. We further examined the colonization ability of each mutant using the one-day old chick model. Cjj0263 or Cjj1025 mutants were able to colonize chick ceca at the same level as the wild type, but a Cjj0263 Cjj1025 double mutant revealed significantly reduced ability to colonize chick ceca. To examine whether C. jejuni that have grown in the digestive tract of chicks are protected against acute hypotonic stress, bacteria in ceca were directly exposed to water. The wild type was able to survive acute osmotic downshift, but the Cjj0263 mutant suffered a substantial loss of viability when subjected to a rapid osmotic downshock. Immunoblot analysis suggested that both Cjj0263 and Cjj1025 were glycosylated via the N-linked protein glycosylation pathway, but glycan modification of these proteins was unlikely to have a major effect on their function and stability. Our data suggest that Cjj0263, a mechanosensitive channel, has a pivotal role in protection against hypoosmotic stress experienced during environmental transmission.
[Show abstract][Hide abstract] ABSTRACT: Campylobacter jejuni is a leading cause of bacterial gastroenteritis in humans and a commensal bacterium of the intestinal tracts of animals, especially poultry. Chemotaxis is an important determinant for chicken colonization of C. jejuni. Adaptation has a crucial role in the gradient-sensing mechanism that underlies chemotaxis. The genome sequence of C. jejuni reveals the presence of genes encoding putative adaptation proteins, CheB and CheR. In-frame deletions of cheB, cheR and cheBR were constructed and the chemosensory behaviour of the resultant mutants was examined on swarm plates. CheB and CheR proteins significantly influence chemotaxis but are not essential for this behaviour to occur. Increased mobility of two methyl-accepting chemotaxis proteins (MCPs), DocC and Tlp1, during SDS-PAGE was detected in the mutants lacking functional CheB in the presence of CheR, presumably resulting from stable methylation of receptors. In vitro studies using tissue culture revealed that deletion of cheR resulted in hyperadherent and hyperinvasive phenotypes, while deletion of cheB resulted in nonadherent, noninvasive phenotypes. Furthermore, the ΔcheBR mutant showed significantly reduced ability to colonize chick caeca. Our data suggest that modification of chemoreceptors by the CheBR system is involved in regulation of chemotaxis in C. jejuni although CheB is apparently not controlled by phosphorylation.
[Show abstract][Hide abstract] ABSTRACT: Campylobacter jejuni reportedly exhibits chemotactic behavior towards fucose, several amino acids and organic acids, mucin and bile. The chemotaxis of C. jejuni has mainly been studied using the chemical-in-plug chemotaxis assay. In this study, a nonchemotactic mutant (cheY mutant) and nonmotile mutant (flhA mutant) were constructed and used as negative controls in an assay. Apparent zones of accumulation around test plugs containing several amino acids and organic acids were observed with both of the mutants. Our results suggest that positive responses of C. jejuni in the chemical-in-plug assay are not always indicative of chemotaxis.
Journal of Veterinary Medical Science 10/2010; 73(3):389-91. DOI:10.1292/jvms.10-0396 · 0.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Campylobacter jejuni infection is a leading cause of bacterial gastroenteritis in the United States and is acquired primarily through the ingestion
of contaminated poultry products. Here, we describe the C. jejuni orthologue of ZnuA in other gram-negative bacteria. ZnuA (Cj0143c) is the periplasmic component of a putative zinc ABC transport
system and is encoded on a zinc-dependent operon with Cj0142c and Cj0141c, which encode the other two likely components of
the transport system of C. jejuni. Transcription of these genes is zinc dependent. A mutant lacking Cj0143c is growth deficient in zinc-limiting media, as
well as in the chick gastrointestinal tract. The protein is glycosylated at asparagine 28, but this modification is dispensable
for zinc-limited growth and chick colonization. Affinity-purified FLAG-tagged Cj0143c binds zinc in vitro. Based on our findings
and on its homology to E. coli ZnuA, we conclude that Cj0143c encodes the C. jejuni orthologue of ZnuA.
Journal of bacteriology 03/2009; 191(5):1631-40. DOI:10.1128/JB.01394-08 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The presence of large plasmids in 30 Rhodococcus equi strains isolated from pig lymph nodes with granulomatous changes was investigated. Plasmid DNAs were isolated and digested with the restriction endonu- cleases BamHI, EcoRI, EcoT22I and HindIII for detailed comparison and estimation of plasmid sizes. A total of nine isolates were identified as intermediately virulent (VapB-positive), harbouring large plasmids of type 5 (n = 5) and four new variants that we tentatively designated as type 19 (n = 1), 20 (n = 1), 21 (n = 1) and 24 (n = 1). All isolates were subjected to genotyping with pulsed-field gel electrophoresis (PFGE). High genetic diversity was observed: 21 distinct genotypes were detected; five were found in multiple isolates and the others were unique. Isolates of the same plasmid type exhibited different PFGE profiles and vice versa. In a few cases, multiple strains from certain farms were analysed, the majority of which exhibited diverse PFGE profiles. Our findings demonstrate the presence of a wide variety of R. equi strains even in small confined environments such as farms. This is the first molecular epidemiology study of intermediately virulent R. equi isolates from Slovenian pigs.
[Show abstract][Hide abstract] ABSTRACT: Rhodococcus equi has been isolated from the submaxillary lymph nodes of domesticated pigs, but little is known about the presence of R. equi in wild boars. The aim of the study was the evaluation of the incidence of R. equi in wild boars and the characterisation of them. Of 482 submaxillary lymph nodes of wild boars shot in 39 settlements throughout Hungary, R. equi was isolated from 60 specimens, and plasmid types of 82 isolates were examined. The isolates were tested for the presence of 15-17-kDa (VapA) and 20-kDa virulence-associated protein antigen (VapB) genes by polymerase chain reaction (PCR). Plasmid DNAs were isolated and analysed by digestion with restriction endonucleases to estimate size and compare their polymorphisms. None of the 82 isolates contained vapA gene but 21 isolates (25.6%) were positive for vapB gene showing 827bp product of the expected size in the PCR amplification. Sixty-one strains (74.4%) did not contain plasmid. The 21 isolates of intermediate virulence contained virulence plasmids that were identified as types 1 (1 isolate), 5 (16 isolates), 21 (1 isolate), and three new distinct plasmid variants (1-1-1 isolate), respectively. On the basis of restriction digestion patterns of plasmid DNAs, we tentatively designated the new variants as types 25-27, respectively. The prevalence of R. equi strains of intermediate virulence among the isolates originated from the submaxillary lymph nodes of wild boars (25.6%) is very similar to those of domestic pigs (26.8%) in Hungary, and plasmid type 5 is the predominating one in both groups. This is the first report of isolation of VapB-positive R. equi from wild boars in the world.
[Show abstract][Hide abstract] ABSTRACT: Pulsotypes of VapA positive Rhodococcus equi isolated from foals and soil on a farm in Germany were characterized on the basis of nasal and tracheal samples simultaneously collected in 2003 from 217 foals with sonographic evidence of pneumonia or pulmonary abscesses. Of the 217 double samples, R. equi was isolated in 118 (54%) of the tracheal samples and in 52 of the nasal swab samples (24%) (P<0.001). Furthermore, 37 and 55 isolates were also randomly selected from nasal swabs and the tracheal samples, respectively, and further processed to determine the presence of VapA by colony blot enzyme-linked immunosorbent assay. This method showed that 26 (68%) of the nasal swabs and 40 (73%) of the tracheal samples were VapA-positive. R. equi was isolated from 56 (87%) of the 64 soil samples taken from the paddocks and stables in March and from 17 (68%) of the 25 samples taken in July of 2003. Three (21%) of these randomly selected 14 isolates from March and 13 (81%) of the 16 from July were VapA-positive. The VapA positive isolates from foals and soil were genotyped by plasmid profiling, restriction fragment length polymorphism analysis and pulsed-field gel electrophoresis. Of the 83 isolates, 80 contained an 85-kb type I plasmid and three contained an 87-kb type I plasmid. Pulsed-field gel electrophoresis yielded four distinct VspI profiles dividing 83 isolates into three major (A1, 51; D, 14; and 11 isolates) and three minor (C, 4; A3, 2; and A2, 1 isolates) groups. These results suggest that the majority of foals were exposed to and infected with three pulsotypes of VapA positive R. equi containing an 85-kb type I plasmid.
Research in Veterinary Science 12/2007; 83(3):311-7. DOI:10.1016/j.rvsc.2007.01.009 · 1.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The complete nucleotide sequence of a plasmid, pMBO-1, from Moraxella bovis strain Epp63 was determined. We identified 30 open reading frames (ORFs) encoded by the 44,215bp molecule. Two large ORFs, flpA and flpB, encoding proteins with similarity to Bordetella pertussis filamentous haemagglutinin (FHA), were identified on the same plasmid. The gene for a specific accessory protein (Fap), which may play a role in the secretion of Flp protein, was also identified. Reverse transcriptase PCR analysis of total RNA isolated from M. bovis Epp63 indicated that the flpA, flpB, and fap genes are all transcribed. Southern blot analysis indicated that the flp and fap genes are present in other clinical isolates of geographically diverse M. bovis.
[Show abstract][Hide abstract] ABSTRACT: Campylobacter jejuni has an N-linked protein glycosylation pathway that is required for efficient cell invasion and chick gastrointestinal colonization
by the microbe. In this study, we constructed insertion mutants of 22 putative glycoprotein genes and examined the ability
of each to invade the human intestinal epithelial cell line INT-407. Among the mutants tested, one carrying an insertion in
Cj1496c was defective for invasion into INT-407 cells; this defect was also observed in an in-frame deletion mutant of Cj1496c
(ΔCj1496c). The ΔCj1496c mutant C. jejuni also showed a reduced ability to colonize chick ceca. Site-specific mutagenesis combined with Western blot analysis suggested
that the Cj1496c protein is glycosylated at N73 and N169. However, the ΔCj1496c mutant expressing a nonglycosylated form of
Cj1496c exhibited levels of invasion and colonization equivalent to those of the parent strain, suggesting that glycans are
not directly involved in the function of Cj1496c.
Infection and Immunity 09/2006; 74(8):4715-23. DOI:10.1128/IAI.00033-06 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Little is known about the distribution of Rhodococcus equi in the soil environment of native horses in China. One hundred and eight soil samples were collected from native-horse farms in the Hulun Beier grasslands of eastern Mongolia, the Xilin Goler grasslands of southern Mongolia, and Tongliao City in Inner Mongolia, China. The isolation rates of R. equi from soil samples from the Hulun Beier and Xilin Goler grasslands ranged from 25.9% to 30.0%. In contrast, isolation rates from soil samples from Tongliao City were as high as 82.3% and the mean number of R. equi in soil samples from Tongliao City was 10 times more than those of samples from the grasslands. The 488 isolates were examined using PCR for the presence of genes that encode virulence-associated 15-17 kDa antigen protein (VapA) and the 20 kDa antigen protein (VapB). All isolates were negative for virulence-associated proteins. Plasmid profiles of these avirulent isolates showed that cryptic plasmids of various sizes were present with an incidence of 13.3% to 21.5%. The results of the present study contrast with those of our recent study (J. Vet. Med. Sci. 67:611-613, 2005), in which we reported that R. equi was absent from Mongolian horses in Ulaanbaatar, Mongolia. It is suggested that the difference between the results of these two studies is due to the mobile pasturing system in Mongolia and nonmobile pasturing system in Inner Mongolia.
Journal of Veterinary Medical Science 08/2006; 68(7):739-42. DOI:10.1292/jvms.68.739 · 0.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The plasmid profiles of virulent Rhodococcus equi strains isolated on three horse-breeding farms located in different parts of Hungary were investigated. From 49 soil samples collected on the three farms, 490 R. equi isolates (10 from each sample) were obtained and tested for the presence of 15- to 17-kDa antigens (VapA) by immunoblotting and PCR. Ninety-eight VapA-positive isolates were detected from 30 of the 49 culture-positive samples with a prevalence ranging from 13.1% to 23.2%. Of the 98 virulent isolates, 70 contained an 85-kb type I plasmid, 13 contained an 87-kb type I plasmid, and 15 contained an 85-kb type III plasmid which had been uniquely isolated from soil isolates in the United States. This study demonstrates that the virulent form of R. equi is very widespread in the soil environment of these stud farms in Hungary and the plasmid pattern is different from farm to farm.
[Show abstract][Hide abstract] ABSTRACT: We recently demonstrated the presence of virulence-associated protein antigen (VapA)-positive Rhodococcus equi in Jeju Island, Korea. These bacteria contained one of two distinct plasmid types, a 90-kb type II plasmid, which has been found in isolates from the native Kiso horses of Japan, and a new variant, a 90-kb type V plasmid. However, the genotypic characters of the VapA-positive R. equi from Jeju native horses and their environments are poorly understood. Ninety-eight isolates from soil samples and 89 isolates from fecal samples were collected from five farms that breed or have bred Jeju native horses, and were tested for the presence of VapA by immunoblotting and PCR. Of the 98 soil isolates and 89 fecal isolates, seven and 13 were VapA-positive R. equi, respectively. In 2003, two Jeju foals died suddenly and were brought to the Faculty of Veterinary Medicine, Cheju National University, for postmortem examination. Pure cultures of R. equi were isolated from the lung lesions of both foals. Of the 16 clinical isolates, 14 were VapA-positive R. equi. Of the 34 VapA-positive clinical and environmental isolates, 16 contained the 90-kb type II plasmid and 18 contained a 90-kb type V plasmid. Pulsed-field gel electrophoresis (PFGE) patterns of the VapA-positive isolates from Jeju horses and Kiso horses, containing the 90-kb type II plasmid, were compared and formed two distinct groups. Furthermore, 18 virulent isolates containing the 90-kb type V plasmid formed two distinct PFGE groups (of 16 and two isolates). These results demonstrate that two virulence plasmid types are widespread in R. equi in Jeju native horses. However, there is little diversity in the PFGE patterns of virulent isolates, suggesting the clonal spread of virulent R. equi. The PFGE pattern of the virulent R. equi isolates from Jeju native horses in Korea is not identical to those of Kiso native horses in Japan.
Journal of Veterinary Medical Science 04/2006; 68(3):249-53. DOI:10.1292/jvms.68.249 · 0.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To develop a method for typing Streptococcus equi on the basis of the DNA sequence of the genes that produce an M-like protein and to compare isolates among the United States, Japan, and other countries.
S equi strains CF32, Hidaka/95/2, and NCTC9682 as well as 82 other isolates from the United States, Japan, and other countries obtained during 1975 to 2001.
DNA sequences of the structural genes ( SeM and SzPSe) that produce M-like proteins were determined for 3 representative strains to find a variable region. Variability in this region of SeM was then determined for the other isolates. Amino acid sequences were deduced and analyzed phylogenetically by use of the neighbor-joining method.
Sequence diversity was detected in the N-terminal region of SeM but not in SzPSe of the 3 representative strains. Base substitutions in the variable region of SeM varied in a nonsynonymous manner, resulting in variation in the amino acid sequence. Eighty-five isolates were categorized as 32 types of SeM on the basis of differences in the deduced amino acid sequences.
This study documented a region in the N-terminal portion of SeM that varies in a nonsynonymous manner. This information should be useful in molecular epidemiologic studies of S equi.
American Journal of Veterinary Research 01/2006; 66(12):2167-71. DOI:10.2460/ajvr.2005.66.2167 · 1.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In native Mongolian horses, the incidence and distribution of Rhodococcus equi are poorly understood. One hundred and fourteen equine fecal samples and 71 soil samples were collected from the camp sites of 26 nomadic families located in three areas less than 100 km from Ulaanbaatar, Mongolia. Five fecal samples were also collected from foals of Przewalski's Horses introduced into the Hustai National Park, Mongolia. No R. equi was isolated from the Mongolian horses or the soil samples. However, three colonies of R. equi were isolated from two fecal samples collected from foals of Przewalski's Horses. These isolates were avirulent, with neither 15- to 17-kDa antigens (VapA) nor a 20-kDa antigen (VapB) genes being detected. We concluded that native Mongolian horses and their environment appear free from contamination with R. equi.
Journal of Veterinary Medical Science 07/2005; 67(6):611-3. DOI:10.1292/jvms.67.611 · 0.78 Impact Factor