Influence of volatile metabolites of saprophytic soil microflora on the propagation of pathogenic bacteria

The influence of volatile metabolites of saprophytic soil microflora on the propagation of Listeria monocytogenes and Yersinia pseudotuberculosis is shown. Different character of interspecific relationships between bacteria, influencing their propagation, can be observed on the metabolic level. Volatile compounds produced by microorganisms are capable to act as both intra- and interspecific regulators of microbial communities. In this connection the propagation of pathogenic bacteria inhabiting soil may be stimulated or inhibited by the metabolic products of soil microorganisms. Methanol released by saprophytic bacteria into the environment play an important role in this process.     

Digoxigenin-labelled inv- and ail-probes for the detection and identification of pathogenic Yersinia enterocolitica in clinical specimens and naturally contaminated pig samples

A non-radioactive colony hybridization method was developed for the rapid detection of Yersinia enterocolitica in primary isolates and for differentiation between pathogenic and non-pathogenic strains. The method is based on, respectively, the presence of the inv -locus in all Yersinia spp. and the presence of the ail -gene in pathogenic Y. enterocolitica only. Hybridization results with ail -probes of 132 strains of Y. enterocolitica were in good agreement with pathogenicity phenotypes as indicated by a tissue culture invasion (TCI) assay and by serotyping. All TCI⁺ strains and only two TCI^- strains were positive by hybridization with ail. Hybridization results with inv - or ail -probes of 150 primary isolates of human, animal or slaughterhouse origin were compared with those of conventional methods to detect and identify Y. enterocolitica. All samples that were positive for Yersinia spp. by cultivation (four of 66) or were positive for pathogenic Y. enterocolitica by cultivation and serotyping (six of 84) were also positive by hybridization with, respectively, the inv - or ail -probe. In three slaughterhouse swab samples, in which Yersinia spp. were not detected by cultivation (2%), strong positive hybridization signals were obtained with the inv - and/or ail -probe. Four other swab samples which were negative by cultivation produced weak positive signals by hybridization with inv - and/or ail - probes. These results indicate that the method can be used for (1) the identification of pathogenic Y. enterocolitica isolates and (2) the detection of Yersinia spp. in primary isolates of naturally contaminated samples.     

Patterns of infection by Salmonella and Yersinia spp. in commensal house mouse (Mus musculus domesticus) populations

AIMS: This study sought to examine the risk posed by house mice transmitting pathogens to livestock on typical mixed-agriculture farms in the UK. METHODS AND RESULTS: In a 10-month longitudinal study at one farm, 222 faecal samples were taken from mice and 57 swabs from the farm environment; 3.2% and 15.8%, respectively, were positive for Yersinia. Seventy-five intestinal samples were taken from house mice from three other farms and 9.3% were positive for Yersinia. The commonest species was Y. enterocolitica (of a wide range of serotypes); all isolates were non-pathogenic, except one of Y. pseudotuberculosis. Salmonella was not isolated from any sample. CONCLUSION: This study provides additional evidence that house mice are generally not significant vectors of either pathogenic Yersinia strains or Salmonella species. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first longitudinal study of Yersinia in any small mammal population, and shows infection to be a dynamic series of generally non-pathogenic, transient infections.     

Detection of pathogenic Yersinia enterocolitica using a digoxigenin labelled probe targeting the yst gene

A 145 base pair digoxigenin-d-UTP-labelled probe, specific for pathogenic Yersinia enterocolitica heat-stable enterotoxin yst gene, was prepared by PCR. The probe was used in DNA-DNA colony hybridization and dot-blot hybridization assays. The specificity of the probe was confirmed using 52 strains representing all Yersinia spp., except Y. pestis . Out of a total of 25 Y. enterocolitica strains screened, the probe correctly identified all 18 pathogenic strains. Among the other Yersinia spp. screened, only one strain of Y. kristensenii was positively detected by the yst probe but could be differentiated by its weak signal response as compared with that obtained by pathogenic strains of Y. enterocolitica .     

Survival of Yersinia enterocolitica in the environment

When Yersinia enterocolitica was introduced into soils (or physiological saline), very little decrease in the population was observed throughout the test period. If the soil was allowed to air dry slowly, only 0.1% (2.8 x 10(3) colony forming units/g of soil) of the original population added still remained viable by day 10. On the other hand, the introduced organisms disappeared rapidly in river water but their longevities could be extended significantly if a eucaryote inhibitor was added to the river water or the river water was passed through a 0.8-micron membrane filter to remove eucaryotic predators. Furthermore, the rapid decrease of the Yersinia population coincided with an increase in numbers of protozoans. However, when Yersinia was added to filter-sterilized river water or when small numbers of the organism, below the threshold level believed necessary for active predation to occur, were added to the river water, no response in predators was observed; nevertheless, the population of Yersinia still showed a continued decline. When the organism was introduced into sephadex-treated river water or groundwater, its survival improved significantly compared with its survival in nontreated water samples. Low ambient temperature dramatically increased its ability to survive in the aquatic environment. It is concluded that, in addition to the temperature factor, the longevity of Y. enterocolitica in river water is chiefly regulated by predators and toxin producers.     

Pasteurized milk as a factor in the transmission of the causative agents of yersiniosis]

From 43 (35.8%) out of 120 samples of pasteurized milk used by volunteers 182 cultures of five Yersinia species--Y. enterocolitica, biovar 1 (70.3%), Y. intermedia (18.1%), Y. frederiksenii (7.7%), Y. kristensenii (3.3%) and Y. aldovae (0.5%)--were isolated in the course of bacteriological studies carried out in 1987-1990. The isolated Yersinia cultures belonged to nonpathogenic pyraminidaze-positive serovars. Yersinia pathogenic bio/serovars (4/O3, 2/O9, 1B/O8) were not detected. No deviations from the normal state were registered in the volunteers drinking pasteurized milk, and Yersinia could be isolated from their feces only during the first 3 days following milk consumption. In 390 patients examined for the presence of Yersinia infections the isolation rate of Yersinia of biovar 1 (99.6% of the cultures) varied between 4.4% and 21.3% and correlated with the increase of the isolation rate from milk (13.6% to 53.9%). The results of the study suggest that unboiled pasteurized milk contributes to the spread of Yersinia carriership among the population.     

Identification and characterization of pathogenic Yersinia enterocolitica isolates by PCR and pulsed-field gel electrophoresis

Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n=48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.     

Effects of the pesticides atrazine and lindane and of manganese ions on cellular immunity of carp, Cyprinus carpio

Pesticides and metals in the aquatic environment can peturb the fish immune system and, consequently, increase their susceptibility to pathogenic agents. Lindane, an insecticide, was tested for its effects on skin graft rejection after daily administration in the food for 1 month (1000 mg lindane kg⁻¹ food). No difference was observed from the control for the first set of graft rejection times (8 days) or for the second set of graft rejection times (5 days), even if the lymphoid organs were highly contaminated with lindane. Atrazine, lindane and manganese were tested for their effect on in vitro phagocytosis of Yersinia ruckeri and zymosan (yeast extract) by pronephric and splenic macrophages. Neither pesticide had any effect on macrophage phagocytosis at concentrations up to their limit of solubility in water. Manganese ions, in the range 6.25–50 ppm, had a strong enhancing effect on phagocytosis of Y. ruckeri , as did zymosan, but only at a concentration of less than 6.25 ppm.     

Hydrobionts as reservoir hosts for infectious agents of bacterial sapronoses

Data on parasitism of the infectious agents of sapronoses in hydrobionts (protozoans, crustaceans, worms, mollusks, echinoderms, and fishes) are considered from the population-ecological viewpoint. The symbiotic relationships between populations of pathogenic bacteria and protozoans are of the host-parasite type. An ultrastructural analysis demonstrates that phagocytosis is incomplete both in planktonic forms and in biofilms formed by bacteria and protozoa. This provides for long-term survival of infectious agents in the environment. The migration of Yersinia pseudotuberculosis along trophic chains from the lowest to the highest level has been simulated experimentally. The long-term survival of pathogenic bacteria in aquatic animals and the ability of bacteria to migrate along trophic chains, reaching soil animals and humans, provide evidence that comprehensive studies on the routes of circulation of pathogens in natural ecosystems are highly relevant from the ecological and epidemiological viewpoints.     

禽致病性大肠杆菌（CVCC1565）中耶尔森菌强毒力岛核心区的检测

采用PCR法,检测了大肠杆菌（CVCC1565）中耶尔森菌强毒力岛（high pathogenicity island,HPI）核心区的irp1、irp2、irp3、irp4、irp5及fyuA基因片段,并与小肠结肠炎耶尔森菌毒力岛的类似基因进行同源性比较。结果显示,E.coliCVCC1565菌株irp1、irp2、irp3、irp4、irp5及fyuA基因大小分别为799bp、414bp、798bp、504bp、758bp、948bp,与GenBank中公布的小肠结肠炎耶尔森菌（Yersinia enterocoliticaO：8 WA）HPI的irp1、irp2、irp3、irp4、irp5及fyuA基因同源性分别达到98%、98%、98%、95%、98%、98%。研究结果表明禽致病性大肠杆菌标准株（CVCC1565）携带耶尔森菌强毒力岛基因,这几个毒力岛基因在小肠结肠炎耶尔森菌和禽致病性大肠杆菌之间可能存在水平性转移。     

云南省新平县家鼠鼠疫疫原地小肠结肠炎耶尔森菌的调查分析

目的了解新平县家鼠鼠疫疫源地小肠结肠炎耶尔森菌的分布及病原学特征。方法采集家鼠盲肠、舌头和猪粪便、咽喉粘液以及腹泻患者粪便标本进行小肠结肠炎耶尔森菌的检测与分析。结果检测家鼠盲肠、鼠舌头、猪粪便、猪咽喉粘液物、腹泻患者粪便的标本数分别为722、722、467、237和107份,共分离到61株小肠结肠炎耶尔森菌,总检出率为2. 71%,5种标本的检出率分别为2. 63%、1. 39%、3. 85%、2.53%和7. 48%,差异有统计学意义（x^2= 16. 422,P = 0. 003）;分离株包括致病株10株、非致病株51株,有1A、2、3三种生物型和0：3、0：5、0：8等多种血清型,以及六种毒力基因型。猪、鼠、腹泻患者标本检出致病菌株数分别为9、1、0株。结论新平县家鼠鼠疫自然疫源地猪、鼠、腹泻患者是小肠结肠炎耶尔森菌的重要宿主,分离菌株具有遗传多样性,猪、鼠是小肠结肠炎耶尔森菌病的主要传染源。     

Seasonal fluctuations and long-term persistence of pathogenic populations of Agrobacterium spp. in soils

Short- and long-term persistence of pathogenic (i.e., tumor forming) agrobacteria in soil was investigated in six nursery plots with a history of high crown gall incidence. No pathogenic Agrobacterium strains were isolated in soil samples taken in fall and winter in any plots, but such strains were isolated from both bulk soils and weed rhizospheres (over 0.5 x 10(5) pathogenic CFU/g of bulk soil or rhizosphere) in three out of six plots in spring and summer. PCR amplifications of a vir sequence from DNA extracted from soil confirmed the presence of Ti plasmids in summer and their absence in fall and winter. The results indicate that strains that harbor a Ti plasmid had an unforeseen positive fitness versus Ti plasmid-free strains in soil and rhizosphere in spring and summer in spite of the apparent absence of tumor, and hence of opines. The gain of fitness occurred during a bloom of all cultivable agrobacteria observed only in conducive soils. An evolution of the pathogenic population was recorded during a 4-year period in one particularly conducive soil. In 1990, the pathogenic population in this soil consisted of only biovar 1 strains harboring both octopine- and nopaline-type Ti plasmids. In 1994, it consisted of only nopaline-type Ti plasmids equally distributed among biovar 1 and 2 strains. These results suggest that nopaline-type Ti plasmids conferred a better survival ability than octopine-type Ti plasmids to biovar 2 agrobacteria under the present field conditions.     

Evaluation of a Single Procedure Allowing the Isolation of Enteropathogenic Yersinia along with Other Bacterial Enteropathogens from Human Stools

Enteropathogenic Yersinia are among the most frequent agents of human diarrhea in temperate and cold countries. However, the incidence of yersiniosis is largely underestimated because of the peculiar growth characteristics of pathogenic Yersinia, which make their isolation from poly-contaminated samples difficult. The use of specific procedures for Yersinia isolation is required, but is expensive and time consuming, and therefore is not systematically performed in clinical pathology laboratories. A means to circumvent this problem would be to use a single procedure for the isolation of all bacterial enteropathogens. Since the Statens Serum Institut enteric medium (SSI) has been reported to allow the growth at 37°C of most Gram-negative bacteria, including Yersinia, our study aimed at evaluating its performances for Yersinia isolation, as compared to the commonly used Yersinia-specific semi-selective Cefsulodin-Irgasan-Novobiocin medium (CIN) incubated at 28°C. Our results show that Yersinia pseudotuberculosis growth was strongly inhibited on SSI at 37°C, and therefore that this medium is not suitable for the isolation of this species. All Yersinia enterocolitica strains tested grew on SSI, while some non-pathogenic Yersinia species were inhibited. The morphology of Y. enterocolitica colonies on SSI allowed their differentiation from various other Gram-negative bacteria commonly isolated from stool samples. However, in artificially contaminated human stools, the recovery of Y. enterocolitica colonies on SSI at 37°C was difficult and was 3 logs less sensitive than on CIN at 28°C. Therefore, despite its limitations, the use of a specific procedure (CIN incubated at 28°C) is still required for an efficient isolation of enteropathogenic Yersinia from stools.     

Prevalence of Yersinia enterocolitica in pigs slaughtered in Chinese abattoirs

The distribution of Yersinia enterocolitica in slaughtered pigs in China was studied. A total of 8,773 samples were collected and examined from different pig abattoirs in 11 provinces from 2009 to 2011. Of these, 4,495 were oral-pharyngeal swab (tonsils) samples from pigs, 1,239 were from intestinal contents, and 3,039 were feces samples from abattoirs or local pigpens. The data showed that 1,132 strains were obtained, from which the isolation rate for Yersinia enterocolitica was 19.53% (878/4,495) from the tonsil samples, 7.51% (93/1,239) from intestinal contents, and 5.30% (161/3,039) from feces. Of the 850 pathogenic Yersinia strains, except for three of bioserotype 2/O:9 and three of bioserotype 4/O:3, most (844/850) were of bioserotype 3/O:3. Interestingly, pathogenic Y. enterocolitica accounted for the majority of the isolated strains from most provinces (85.17% to 100%), whereas from Heilongjiang, 96.52% (111/115) were classified as nonpathogenic biotype 1A with various serotypes, and only 3.48% of the strains (4/115) were pathogenic 3/O:3. All of the pathogenic strains were analyzed using pulsed-field gel electrophoresis (PFGE), and 49 patterns were obtained for the O:3 pathogenic strains; most of them were K6GN11C30021 (53.13%: 450/847) and K6GN11C30012 (21.37%: 181/847). Several strains from diarrhea patient samples revealed PFGE patterns identical to that from samples of local pigs, suggesting a possible link between porcine isolates and human infection. The results above suggested that Yersinia enterocolitica in slaughtered pigs from Chinese abattoirs was characterized by region-specific PFGE patterns and confirmed that strains isolated from pigs are closely related to those from human infections.     

The Yersinia Yops inhibit invasion of Listeria, Shigella and Edwardsiella but not Salmonella into epithelial cells

Yersinia virulence is dependent on the expression of plasmid-encoded secreted proteins called Yops. After bacterial adherence to receptors on the mammalian cell membrane, several Yops are transported by a type III secretion pathway into the host cell cytoplasm. Two Yops, YopH and YopE, prevent macrophages from phagocytosing Yersinia by disrupting the host cell cytoskeleton and signal transduction pathways. In contrast to this active inhibition of phagocytosis by Yersinia , other pathogens such as Salmonella , Shigella , Listeria and Edwardsiella actively promote their entry into mammalian cells by binding to specific host surface receptors and exploiting existing cell cytoskeletal and signalling pathways. We have tested whether Yersinia Yops can prevent the uptake of these diverse invasive pathogens. We first infected epithelial cells with Yersinia to permit delivery of Yops and subsequently with an invasive pathogen. We then measured the level of bacterial invasion. Preinfection with Yersinia inhibited invasion of Edwardsiella , Shigella and Listeria , but not Salmonella . Furthermore, we found that either YopE or YopH prevented Listeria invasion, whereas only YopE prevented Edwardsiella and Shigella invasion. We correlated the inhibitory effect of the Yops with the inhibitory action of the cell-signalling inhibitors Wortmannin, LY294002 and NDGA, and concluded that the four invasive pathogenic species enter epithelial cells using at least three distinct host cell pathways. We also speculate that YopE affects the rho pathway.     

耶尔森菌强毒力岛*

近年来,人们发现了耶尔森菌强毒力岛,并对其结构与功能进行了大量研究。同时,在一些研究中,发现它也广泛存在于其它几种肠道致病菌中。就耶尔森菌毒力岛HPI结构与功能及它在其它肠道致病菌分布的研究现状作一综述。     

Detection of virulence markers in clinical strains of Yersinia]

The dispersion of plasmid pYV associated virulence markers in 474 Yersinia strains isolated from people has been studied. The ability to autoagglutination, calcium dependence of growth and the specific antigens were identified in 157 strains of traditionally pathogenic Yersinia enterocolitica serovars 03, 09, Yersinia pseudotuberculosis serovar I. They were not found in 223 strains of other 12 serovars of Yersinia enterocolitica, in 40 strains of Yersinia frederiksenii, Yersinia kristensenii, Yersinia intermedia. The proportion of virulent clones in the population of Yersinia is noted to depend on the conditions of its existence in vivo or in vitro. Identification of virulence markers is acknowledged to be expedient in epidemiological and ethiological estimation of the role of isolated Yersinia strains.     

virF-positive Yersinia pseudotuberculosis and Yersinia enterocolitica found in migratory birds in Sweden

During spring and autumn migrations, 468 fecal samples from 57 different species of migratory birds were collected in Sweden. In total, Yersinia spp. were isolated from 12.8% of collected samples. The most commonly found species was Yersinia enterocolitica, which was isolated from 5.6% of all collected samples, followed by Y. intermedia (3.8%), Y. frederiksenii (3.0%), Y. kristensenii (0.9%), Y. pseudotuberculosis (0.6%), and Y. rohdei (0.4%). The pathogenic, virF-positive Y. pseudotuberculosis strains were recovered from three thrushes. These strains belonged to the same bioserotype, 1/O:2, but had two different profiles as determined by pulsed-field gel electrophoresis with NotI and SpeI enzymes. In addition, 10 Y. enterocolitica strains, all from barnacle geese, belonged to bioserotype 3/O:3, which is associated with human disease. Two of the strains were pathogenic, carrying the virF gene on their plasmids. All pathogenic Y. pseudotuberculosis and Y. enterocolitica strains were recovered during the spring, and as the birds were caught during active migration they likely became infected at an earlier stage of the migration, thus potentially transporting these bacterial pathogens over long geographical distances.     

The multiplication dynamics of pathogenic bacteria in relation to the trophic and temperature cultivation conditions

The comparative study of the dynamics of multiplication of Listeria monocytogenes and Yersinia pseudotuberculosis in organic and synthetic media and in distilled water at temperatures of 37 degrees C and 6 degrees C was carried out. This study revealed that in organic media the multiplication of bacteria was good at 37 degrees C and 6 degrees C. In mineral media and distilled water their multiplication was observed only at 6 degrees C. Moreover, conditions necessary for the multiplication of pathogenic bacteria in distilled water were shown; these conditions depended on the inoculated dose, the number of autolyzed microbial cells and the state of the culture. Proofs of the multiplication of the bacteria under the conditions of minimum nutrition and low temperature were obtained with the use of labeled 3H-thymidine.