Serovars of nonagglutinating vibrios isolated on the territory of the Uzbek SSR and their role in the development of acute intestinal diseases

As the result of the serotyping of 1,223 NAG vibrio strains isolated in the Uzbek SSR from the environment and 659 strains isolated from gastroenteritis patients and from vibriocarriers, the serovars of 563 strains (46%) and 420 strains (63%), respectively, were determined. The prevailing serovars were 08, 06, 09, 014, 05, 039, 041, 013, 019 among the strains isolated from the environment and 08, 041, 037, 05, 039, 024, 06 among the strains isolated from humans. The strains belonging to serovars 05, 06, 08, 024, 037, 039, 041 were isolated both from the environment and from humans. The possible role of these strains in the development of acute gastroenteritis and in the formation of vibriocarriership in humans is pointed out.     

Cholera and NAG vibrio utilization of different C-containing substrates in Hiss media and a medium with a limited mineral content

A total of 55 V. cholerae strains and 175 NAG vibrio strains were studied with a view to establish their capacity for utilizing citrate in Simmons citrate agar or for growing in it in the absence of the source of carbon. The strains were divided into 3 groups, each containing approximately an equal number of cholera and NAG vibrios irrespective of their origin or serovars. None of 50 signs used in this investigation permitted the reliable differentiation of the cholera and NAG vibrio groups due to considerable differences between the strains within each group. The use of Hiss medium with starch instead of Kodam medium is proposed for the determination of the diastatic activity of cholera and NAG vibrios.     

Novel virulence gene and clustered regularly interspaced short palindromic repeat (CRISPR) multilocus sequence typing scheme for subtyping of the major serovars of Salmonella enterica subsp. enterica

Salmonella enterica subsp. enterica is the leading cause of bacterial food-borne disease in the United States. Molecular subtyping methods are powerful tools for tracking the farm-to-fork spread of food-borne pathogens during outbreaks. In order to develop a novel multilocus sequence typing (MLST) scheme for subtyping the major serovars of S. enterica subsp. enterica, the virulence genes sseL and fimH and clustered regularly interspaced short palindromic repeat (CRISPR) loci were sequenced from 171 clinical isolates from nine Salmonella serovars, Salmonella serovars Typhimurium, Enteritidis, Newport, Heidelberg, Javiana, I 4,[5],12:i:-, Montevideo, Muenchen, and Saintpaul. The MLST scheme using only virulence genes was congruent with serotyping and identified epidemic clones but could not differentiate outbreaks. The addition of CRISPR sequences dramatically improved discriminatory power by differentiating individual outbreak strains/clones. Of particular note, the present MLST scheme provided better discrimination of Salmonella serovar Enteritidis strains than pulsed-field gel electrophoresis (PFGE). This method showed high epidemiologic concordance for all serovars screened except for Salmonella serovar Muenchen. In conclusion, the novel MLST scheme described in the present study accurately differentiated outbreak strains/clones of the major serovars of Salmonella, and therefore, it shows promise for subtyping this important food-borne pathogen during investigations of outbreaks.     

Discrimination of Escherichia coli O157, O26 and O111 from Other Serovars by MALDI-TOF MS Based on the S10-GERMS Method

Enterohemorrhagic Escherichia coli (EHEC), causes a potentially life-threatening infection in humans worldwide. Serovar O157:H7, and to a lesser extent serovars O26 and O111, are the most commonly reported EHEC serovars responsible for a large number of outbreaks. We have established a rapid discrimination method for E. coli serovars O157, O26 and O111 from other E. coli serovars, based on the pattern matching of mass spectrometry (MS) differences and the presence/absence of biomarker proteins detected in matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF MS). Three biomarkers, ribosomal proteins S15 and L25, and acid stress chaperone HdeB, with MS m/z peaks at 10138.6/10166.6, 10676.4/10694.4 and 9066.2, respectively, were identified as effective biomarkers for O157 discrimination. To distinguish serovars O26 and O111 from the others, DNA-binding protein H-NS, with an MS peak at m/z 15409.4/15425.4 was identified. Sequence analysis of the O157 biomarkers revealed that amino acid changes: Q80R in S15, M50I in L25 and one mutation within the start codon ATG to ATA in the encoded HdeB protein, contributed to the specific peak pattern in O157. We demonstrated semi-automated pattern matching using these biomarkers and successfully discriminated total 57 O157 strains, 20 O26 strains and 6 O111 strains with 100% reliability by conventional MALDI-TOF MS analysis, regardless of the sample conditions. Our simple strategy, based on the S10-spc-alpha operon gene-encoded ribosomal protein mass spectrum (S10-GERMS) method, therefore allows for the rapid and reliable detection of this pathogen and may prove to be an invaluable tool both clinically and in the food industry.     

The fatty acid composition of the total lipids in Vibrio cholerae]

The fatty acid composition of total cell lipids of V. albensis typing strains, serovars 01-012, isolated from patients and environmental objects, as well as V. eltor and NAG vibrios, has been studied. Fatty acids contain 14-18 carbon atoms and are mainly represented by unsaturated monoacids. Palmitoleic and oleic acids constitute the greater part of unsaturated acids (their total content is 55.2-71.0%). The level of saturated fatty acids in considerably lower (27.0-43.1%), and these acids are mainly represented by palmitic acid, its maximum level being 28.4%. The similarity of the fatty acid profiles of the lipids common to the strains under study may indicate that these strains are phylogenetically related.     

On the microbiological diagnostics of Campylobacter jejuni

Investigation of campylobacteriosis cases in 1983-1989 resulted in the isolation of a total of 245 antigenically identified and 23 unidentified strains from humans, animals and foods. A commonly accepted method developed in 1985 using our own experience was used for strain isolation and culturing. A variety of nutrient media in combination with different supplementary substances (antibiotics, growth factors) and additives, such as horse serum, were verified as well as filtration and Fortner's procedures. The best results were obtained when material was stored in thioglycolate transport medium accompanied by cold enrichment (24 h at 4 degrees C) and repeated inoculation into appropriate solid nutrient medium. Owing to the simple culturing of C. jejuni, the number of not elucidated diarrheas was reduced and the incidence of campylobacteriosis (approximately 12 %) is higher than that of salmonellosis and shigellosis. A total of 245 C. jejuni strains was classified using Kahlich's antigenic scheme. The incidence of serovars 1 and 2 was greater than 10 %. Five serovars (13, 17, 25, 26 and 27) were represented by only one strain. The study of campylobacteriosis also revealed the long-term excretion of C. jejuni by convalescents (71 days at most) as well as the occurrence of family outbreaks. Procedures were developed to ensure short-term and long-term (freeze-drying) preservation of isolated strains. The number of cases reported by microbiological laboratories in the framework of the Hygienic Service throughout Czechoslovakia suggest an increase in positive findings with C. jejuni as the etiological agent.     

Biological characteristics of Streptococcus pneumoniae strains isolated from children with pneumococcal diseases

The biological properties of 97 S. pneumoniae strains isolated from 92 children with purulent meningitides, meningoencephalitides, pneumonia and otitis, hospitalized at the Leningrad Research Institute of Childhood Infections, were studied. The data obtained in this investigation were indicative of the formation of atypical forms of pneumococci (R-forms, unbalanced growth forms and L-forms) in all clinical forms of pneumococcal infection in children. In purulent meningitides and meningoencephalitides serovars 1 and 6, in pneumonia serovars 1, 3 and 6 and in otitis serovars 6 and 19 played the leading role. The determination of sensitivity to antibiotics showed that the forms under study retained high sensitivity to a number of antibiotics. The appearance of strains resistant to benzylopenicillin was registered. (10%). The isolated strains either possessed low virulence or were avirulent in bioassay on white mice.     

Pathogenicity of Salmonella Strains Isolated from Egg Shells and the Layer Farm Environment in Australia

In Australia, the egg industry is periodically implicated during outbreaks of Salmonella food poisoning. Salmonella enterica serovar Typhimurium and other nontyphoidal Salmonella spp., in particular, are a major concern for Australian public health. Several definitive types of Salmonella Typhimurium strains, but primarily Salmonella Typhimurium definitive type 9 (DT9), have been frequently reported during egg-related food poisoning outbreaks in Australia. The aim of the present study was to generate a pathogenicity profile of nontyphoidal Salmonella isolates obtained from Australian egg farms. To achieve this, we assessed the capacity of Salmonella isolates to cause gastrointestinal disease using both in vitro and in vivo model systems. Data from in vitro experiments demonstrated that the invasion capacity of Salmonella serovars cultured to stationary phase (liquid phase) in LB medium was between 90- and 300-fold higher than bacterial suspensions in normal saline (cultured in solid phase). During the in vivo infection trial, clinical signs of infection and mortality were observed only for mice infected with either 10³ or 10⁵ CFU of S. Typhimurium DT9. No mortality was observed for mice infected with Salmonella serovars with medium or low invasive capacity in Caco-2 cells. Pathogenicity gene profiles were also generated for all serovars included in this study. The majority of serovars tested were positive for selected virulence genes. No relationship between the presence or absence of virulence genes by PCR and either in vitro invasive capacity or in vivo pathogenicity was detected. Our data expand the knowledge of strain-to-strain variation in the pathogenicity of Australian egg industry-related Salmonella spp.     

The agglutination reactions of cholera vibrios.

The present study with 11 strains of vibrio using single-dose and hyperimmune antisera confirmed earlier observations on the cross-reactivity of the flagellar (H) agglutinating antigens of cholera and NAG vibrios. The effect of several variables on the agglutinating sensitivity of cell suspensions was determined by measuring the reaction rate in the presence of constant O- or H-antibody. The variables investigated were culture conditions, antigen dilution, reaction temperature, formalin fixation and heat-treatment; all were found to affect cholera and NAG serovars similarly. The optimal conditions for the O- and H-tests were markedly different. Dilute, young living broth cultures were highly sensitive to O- but not H-antibody. Conditions favoring the H-reaction were 48-hr culture on firm dry agar, high suspension opacity, a reaction temperature of 45 C and formalin fixation. The inverse relationship of O- and H-sensitivity under these conditions indicated that the flagellar antigen in the growing vibrio is masked by an O-sensitive layer. The temperature of denaturation of the unfixed H-antigen before or after reaction with antiserum was 64 +/- 0.5 C and could be used as a criterion of the H-reaction.     

Amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes

AFLP analysis using four selective primers was performed on a set of 33 Listeria monocytogenes including strains from patients and foods implicated in outbreaks, human sporadic cases or foods. Strains were tested belonging to serovars 1/2a, 1/2b, 1/2c, 3b, and 4b. Using one of the primers, the AFLP technique generated 20 different sized DNA fragments. The 33 cultures segregated into 14 different patterns, each comprising 7-12 different fragments. Although the method was not sufficiently discriminatory for epidemiological typing, AFLP analysis reconfirmed the observation that L. monocytogenes comprises two major genetic groups: group 1 includes strains of serovars 1/2a and 1/2c, while group 2 serovars 1/2b, 3b and 4b.     

Serovars of Vibrio parahaemolyticus

Seashore water samples collected along the coastline in Bulgaria and Rumania contained in large numbers OK serovars of V. parahaemolyticus; some of these had been isolated repeatedly over an extended time period: 01 K32, 03 K30, 03 K48, 04 K37, 04 K53, 05 K17, 05 K30. The serovar 05 K17 was virtually present in all water samples and was also isolated from a case of purulent ear infection in a child from Burgas. In contrast, strains recovered from Asian and African coastal water had different K antigens and were never identified in Europe. Two strains of V. parahaemolyticus (serovars 05 K15 and 07 K10) had positive swarming growth resembling that of V. alginolyticus. The first of these was Kanagawa-positive and was isolated from a case of severe diarrhea in Brazzaville. Vibrio parahaemolyticus isolates came from marine or brackish water specimens collected on sand banks, 3 strains were recovered from marine or brackish water in Africa. Vibrio harveyi, a sucrose-negative species important from differential diagnostic aspects, has been isolated from seashore water samples collected on coarse-sand or pebbly beaches.     

Incidence of Vibrio cholerae and related vibrios in a coastal lagoon and seawater influenced by lake discharges along an annual cycle

Most probable numbers of Vibrio cholerae and related vibrios were determined in Albufera Lake, Valencia, Spain, and in coastal waters under the influence of the lake discharges over the course of an annual cycle. The influence of temperature, kind of water, and characteristics of the different sampling sites on the numbers of vibrios recovered was evaluated. Maximum recovery of vibrios reached 10(3)/ml in both types of waters analyzed. V. cholerae numbers reached 10(3)/ml in the lake and 10(2) in one of the coastal sites. Frequently during the warm season, all vibrios isolated were identified as V. cholerae. Occasionally, no V. cholerae was recovered. The recovery of vibrios was significantly influenced by the temperature of the water and the type of water analyzed. Most of the V. cholerae isolates were included in Heiberg groups I and II, and nearly 50% of the strains used chitin as sole carbon source. Indole was not produced by 100% of the strains. All strains tested were non-O1 serovars.     

Incidence of Vibrio cholerae and related vibrios in a coastal lagoon and seawater influenced by lake discharges along an annual cycle.

Most probable numbers of Vibrio cholerae and related vibrios were determined in Albufera Lake, Valencia, Spain, and in coastal waters under the influence of the lake discharges over the course of an annual cycle. The influence of temperature, kind of water, and characteristics of the different sampling sites on the numbers of vibrios recovered was evaluated. Maximum recovery of vibrios reached 10(3)/ml in both types of waters analyzed. V. cholerae numbers reached 10(3)/ml in the lake and 10(2) in one of the coastal sites. Frequently during the warm season, all vibrios isolated were identified as V. cholerae. Occasionally, no V. cholerae was recovered. The recovery of vibrios was significantly influenced by the temperature of the water and the type of water analyzed. Most of the V. cholerae isolates were included in Heiberg groups I and II, and nearly 50% of the strains used chitin as sole carbon source. Indole was not produced by 100% of the strains. All strains tested were non-O1 serovars.     

胸膜肺炎放线杆菌基因分型方法的建立及其临床应用

根据胸膜肺炎放线杆菌各血清型之间外毒素(Apx),外膜脂蛋白(OmlA),转铁蛋白B(TbpB)的基因差异,分别对各血清型进行PCR扩增,得到不同的特异性片段,可区分开生物Ⅰ型13个标准菌株血清型中的8个血清型。临床检测结果与血清学分型一致,将此分型系统用于临床送检的126份肺脏和42份扁桃体的病原学检测,可直接检测出胸膜肺炎放线杆菌感染。此方法还可以将一些尚未定型的菌株进行归类,弥补了血清学方法的不足,为细菌的流行病学调查提供了可靠的技术手段。     

Ribotypes and plasmid contents of Vibrio anguillarum strains in relation to serovar.

Eighty-six strains of the 10 major agglutination types of Vibrio anguillarum (serovars O1 to O10) and 6 nontypeable strains of V. anguillarum have been characterized by ribotyping with a probe complementary to 16S and 23S rRNA of Escherichia coli and by plasmid profile analysis. Forty-four different ribotypes were observed with the restriction enzyme HindIII. Ribotype similarity was compared by using the Dice coefficient (Sd), and three significantly different levels of homogeneity within the V. anguillarum serovars were observed (serovars O1, O3A, O7, and O9, Sds of > 90%; serovars O2B, O4, and O10, Sds of 80 to 90%; serovars O2A, O3B, O5, and O8, Sds between 46 and 70%). None of the ribotype patterns of V. anguillarum strains were observed among 20 other Vibrio strains typed for comparison. By cluster analysis, the V. anguillarum strains were divided into a main cluster containing 83 strains, while all strains of serovar O3B, one strain (each) of serovars O2A, O5, and O8, and a nontypeable strain were separated from this cluster by at least 15% difference in similarity coefficients. Plasmids were demonstrated in only six strains other than serovar O1. In serovar O1, a 67- to 70-kilobase-pair (kb) plasmid molecule was present in 17 of 19 strains tested; of the two remaining strains, one strain harbored two plasmids (45 and 6.5 kb) and one strain had no plasmids.     

The prevalence of Yersinia and its serologic variants in patients with acute intestinal diseases of undetermined etiology in Leningrad

Among Yersinia enterocolitica strains of 32 serovars, proposed as typing strains, some strains were found to belong to new species. Y. enterocolitica sensu stricto was represented by 21 serovars in the collection of typing strains. The occurrence of different Yersinia serovars in patients with acute enteric diseases of unknown etiology in Leningrad in 1983-1986 was determined with the use of the set of monoreceptor to 21 serovars. Out of 2,947 cultures studied by biochemical and serological methods, 81% were typed. Among them 18 Y. enterocolitica serovars were determined. Their characteristic feature was the prevalence of serovar O3 and an insignificant proportion of serovar O9. More frequently Yersinia were detected in patients with the primary diagnosis of acute enteric diseases (93.5%). The overwhelming majority (two-thirds) of Yersinia strains were isolated from children. A great number of strains detected in this study (70%) was isolated on days 10-15 of the bacteriological examination. In 927 cultures the following biovars were determined: the strains of serovar O3 belonged to biovar 4 and all other strains, to biovar 1.     

Agricultural animals as a source of yersiniosis

The role of agricultural animals as the sources of infection was studied. 8 Yersinia strains were isolated from 83 samples taken from cattle. Yersinia were isolated from feces, udder washings and milk. In the examination of 63 samples obtained from sheep the infective agent was isolated from feces in 6 cases. 6 Yersinia strains were isolated from 92 fecal samples taken from pigs. Y. enterocolitica were isolated from sick animals. The isolated strains were classified with serovars 03, 058, 08 and 09. In the foci of animal infection thus revealed antibodies to various Yersinia strains were detected in the blood sera of the animal-tending personnel, which indicates the professional character of the spread of this infection among humans. The infection is probably transferred by the contact and alimentary routes.     

Leptospire genomic diversity revealed by microarray-based comparative genomic hybridization

Comparative genomic hybridization was used to compare genetic diversity of five strains of Leptospira (Leptospira interrogans serovars Bratislava, Canicola, and Hebdomadis and Leptospira kirschneri serovars Cynopteri and Grippotyphosa). The array was designed based on two available sequenced Leptospira reference genomes, those of L. interrogans serovar Copenhageni and L. interrogans serovar Lai. A comparison of genetic contents showed that L. interrogans serovar Bratislava was closest to the reference genomes while L. kirschneri serovar Grippotyphosa had the least similarity to the reference genomes. Cluster analysis indicated that L. interrogans serovars Bratislava and Hebdomadis clustered together first, followed by L. interrogans serovar Canicola, before the two L. kirschneri strains. Confirmed/potential virulence factors identified in previous research were also detected in the tested strains.     

The gene identification of bacterial species and serovariants by the polymerase chain reaction with universal oligonucleotides: the reidentification of earlier isolated strains of Yersinia pseudotuberculosis]

Genetic analysis of 19 standard strains belonging to 6 Yersinia species (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica, Y. kirstensenii, Y. frederiksenii, Y. intermedia) revealed that gene typing by the method of polymerase chain reaction (PCR) with the use of universal primers permitted the identification of species in bacterial cultures by PCR patterns and the determination of Y. pseudotuberculosis serovars within 4 hours. By this method 23 Y. pseudotuberculosis strains (serovar 1), earlier isolated in different regions of the USSR from humans and rodents, were studied. The study showed that out of 14 strains of human origin only two strains could actually be classified with serovar 1, while the remaining strains were reidentified as belonging to serovar 5. Among 9 strains isolated from rodents those of serovar 1 prevailed (8 strains). The authors suppose that strains of serovar 5 cause outbreaks and sporadic cases of pseudotuberculosis, occurring considerably more often than it is commonly believed in the USSR.     

Protective activity of a cell-free Klebsiella vaccine in relation to different Klebsiella pneumoniae serovars

The capacity of dried Klebsiella cell-free vaccine, obtained from strain No. 204 by the disintegration of microbial cells with hydroxylamine, for protecting mice from Klebsiella septic infection caused by the homologous serovar and 9 heterologous serovars of K. pneumoniae was studied. The newly developed preparation was found capable of stimulating immunity not only to the homologous K. pneumoniae serovar, but also to other K. pneumoniae heterologous serovars: K1, K9, K11, K16, K20, K61. The protective capacity of the preparation with respect to these serovars was not inferior to that of the vaccines prepared by the same method from the corresponding homologous strains. The capacity of the vaccine to protect mice from Klebsiella sepsis was manifested irrespective of the virulence of the strains used for challenge.