An application of monoclonal antibodies to identification of leptospires of serogroup icterohaemorrhagiae found in China

For the purpose of improving the procedures of identification of leptospires, a set of 5 monoclonal antibodies with different serological reactivity against serovars of Leptospira interrogans Icterohaemorrhagiae serogroup isolated in China was developed. One hundred and eight strains isolated from epidemic fields in 5 provinces in southern China were distinctly identified into 4 serovars of Icterohaemorrhagiae serogroup by the monoclonal antibody procedure, i.e., 98 isolates were identified as serovar lai, 7 as icterohaemorrhagiae, 2 as copenhageni, and 1 as H2. Factor antiserum procedure was used at the same time as control for typing these strains and an identical result was obtained.     

Comparison of 40 Salmonella enterica serovars injured by thermal, high-pressure and irradiation stress

AIM: To investigate and compare the inherent resistance of 40 Salmonella serovars to heat, irradiation and high-pressure stress. METHODS AND RESULTS: D10 values for each of the three stresses were calculated for four serovars, chosen as representatives from a catalogue of 40. Based on these results, conditions for each stress were defined, which produced, on average, a three-log reduction in viability. Heat stress (57 degrees C for 13 min), high-pressure stress (350 MPa for 10 min at 20 degrees C) and irradiation stress (1.5 kGy at 20 degrees C) were applied to all 40 serovars in the collection. Injury and loss of viability for all serovars were determined. CONCLUSIONS: Cluster analysis identified five groupings of isolates in terms of resistance to the applied stresses. The independent response of each isolate to all three stresses suggests that there is no relationship between resistances. SIGNIFICANCE AND IMPACT OF THE STUDY: Each serovar is inherently different. For modelling of real-life food preservation processing the most resistant isolates for that process should be chosen. The results also emphasize the importance of including multiple stress resistant strains when food preservation systems apply multiple stresses.     

Phenotypic characterization of Bacillus thuringiensis and Bacillus cereus

One hundred and thirty-seven strains of Bacillus thuringiensis and 35 strains of Bacillus cereus were tested for the presence or absence of 99 traits. An analysis of these data indicated that strains of B. thuringiensis were indistinguishable from B. cereus, except for their ability to produce parasporal crystals. This conclusion was based on a comparison of the phenotypic properties of B. thuringiensis and B. cereus, as well as on the results of numerical analyses of the data which grouped strains into clusters on the basis of phenotypic similarity. In the resulting dendrograms, strains of B. thuringiensis and B. cereus were interspersed, exhibiting no tendency to segregate. In addition, with the exception of serovar israelensis, strains on B. thuringiensis belonging to the same flagellar serovar showed little or no tendency to group in different clusters. A comparison of the phenotypic differences between serovars indicated that the greater the number of strains in the serovars, the fewer, if any, phenotypic traits separating them. This suggests that the properties reported to differentiate serovars can be attributed to the internal phenotypic diversity of the species. Characterization of 10 mosquitocidal strains of Bacillus sphaericus indicated that the traits employed in this study readily distinguished these highly related organisms from strains of B. thuringiensis and B. cereus.     

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.     

Differentiation of Listeria monocytogenes serovars by using artificial neural network analysis of Fourier-transformed infrared spectra

A classification system based on Fourier transform infrared (FTIR) spectroscopy combined with artificial neural network analysis was designed to differentiate 12 serovars of Listeria monocytogenes using a reference database of 106 well-defined strains. External validation was performed using a test set of another 166 L. monocytogenes strains. The O antigens (serogroup) of 164 strains (98.8%) could be identified correctly, and H antigens were correctly determined in 152 (91.6%) of the test strains. Importantly, 40 out of 41 potentially epidemic serovar 4b strains were unambiguously identified. FTIR analysis is superior to PCR-based systems for serovar differentiation and has potential for the rapid, simultaneous identification of both species and serovar of an unknown Listeria isolate by simply measuring a whole-cell infrared spectrum.     

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.     

Effect of osmotic stress on plant growth promoting Pseudomonas spp.

In this study we isolated and screened drought tolerant Pseudomonas isolates from arid and semi arid crop production systems of India. Five isolates could tolerate osmotic stress up to −0.73 MPa and possessed multiple PGP properties such as P-solubilization, production of phytohormones (IAA, GA and cytokinin), siderophores, ammonia and HCN however under osmotic stress expression of PGP traits was low compared to non-stressed conditions. The strains were identified as Pseudomonas entomophila, Pseudomonas stutzeri, Pseudomonas putida, Pseudomonas syringae and Pseudomonas monteilli respectively on the basis of 16S rRNA gene sequence analysis. Osmotic stress affected growth pattern of all the isolates as indicated by increased mean generation time. An increase level of intracellular free amino acids, proline, total soluble sugars and exopolysaccharides was observed under osmotic stress suggesting bacterial response to applied stress. Further, strains GAP-P45 and GRFHYTP52 showing higher levels of EPS and osmolytes (amino acids and proline) accumulation under stress as compared to non-stress conditions, also exhibited higher expression of PGP traits under stress indicating a relationship between stress response and expression of PGP traits. We conclude that isolation and screening of indigenous, stress adaptable strains possessing PGP traits can be a method for selection of efficient stress tolerant PGPR strains.     

Emergence of new variants of antibiotic resistance genomic islands among multidrug-resistant Salmonella enterica in poultry

Non-typhoidal Salmonella enterica (NTS) are diverse and important bacterial pathogens consisting of more than 2600 different serovars, with varying host-specificity. Here, we characterized the poultry-associated serovars in Israel, analysed their resistome and illuminated the molecular mechanisms underlying common multidrug resistance (MDR) patterns. We show that at least four serovars including Infantis, Muenchen, Newport and Virchow present a strong epidemiological association between their temporal trends in poultry and humans. Worrisomely, 60% from all of the poultry isolates tested (n = 188) were multidrug resistant, mediated by chromosomal SNPs and different mobile genetics elements. A novel streptomycin-azithromycin resistance island and previously uncharacterized versions of the mobilized Salmonella genomic island 1 (SGI1) were identified and characterized in S. Blockley and S. Kentucky isolates respectively. Moreover, we demonstrate that the acquisition of SGI1 does not impose fitness cost during growth under nutrient-limited conditions or in the context of Salmonella infection in the mouse model. Overall, our data emphasize the role of the poultry production as a pool of specific epidemic MDR strains and autonomous genetic elements, which confer resistance to heavy metals and medically relevant antibiotics. These are likely to disseminate to humans via the food chain and fuel the increasing global antibiotic resistance crisis.     

Serovars and biovars of Campylobacter strains isolated from humans and slaughterhouse animals in northern Germany

A total of 318 Campylobacter strains from sporadic cases of human enteritis (109 strains) and healthy slaughterhouse animals in northern Germany (209 strains) were bio- and serotyped according to the Lior typing schemes. Three hundred strains were typable (94.3%) and 38 serovars were identified. Among human strains 28 serovars were identified with 30% of them belonging to serovar 4. Strains from pigs were associated with 25 serovars, the most frequent being serovar 20 (21.2%). Fourteen serovars were identified in the ovine strains of which 31.1% were of serovar 49, and 22.2% of serovar 4. All of the strains from one chicken farm were of serovar 11, whereas in those from another serovar 1 was predominant (85.4%). Twenty-five of the 38 serovars identified were associated with at least two different biovars. Campylobacter jejuni biovar I was predominant in humans, sheep and chickens and Campylobacter coli biovar I in pigs. The results suggest that the combined use of bio- and serotyping according to the Lior typing schemes would be of use in studies on the epidemiology of human campylobacteriosis in Germany.     

Serovars and biovars of Campylobacter strains isolated from humans and slaughterhouse animals in northern Germany

A total of 318 Campylobacter strains from sporadic cases of human enteritis (109 strains) and healthy slaughterhouse animals in northern Germany (209 strains) were bio- and serotyped according to the Lior typing schemes. Three hundred strains were typable (94.3%) and 38 serovars were identified. Among human strains 28 serovars were identified with 30% of them belonging to serovar 4. Strains from pigs were associated with 25 serovars, the most frequent being serovar 20 (21.2%). Fourteen serovars were identified in the ovine strains of which 31.1% were of serovar 49, and 22.2% of serovar 4. All of the strains from one chicken farm were of serovar 11, whereas in those from another serovar 1 was predominant (85.4%). Twenty-five of the 38 serovars identified were associated with at least two different biovars. Campylobacter jejuni biovar I was predominant in humans, sheep and chickens and Campylobacter coli biovar I in pigs. The results suggest that the combined use of bio- and serotyping according to the Lior typing schemes would be of use in studies on the epidemiology of human campylobacteriosis in Germany.     

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.     

Pyrosequencing as a method for grouping of Listeria monocytogenes strains on the basis of single-nucleotide polymorphisms in the inlB gene.

By using pyrosequencing (i.e., sequencing by synthesis) 106 strains of different serovars of Listeria monocytogenes were rapidly grouped into four categories based on nucleotide variations at positions 1575 and 1578 of the inlB gene. Strains of serovars 1/2a and 1/2c constituted one group, and strains of serovars 1/2b and 3b constituted another group, whereas serovar 4b strains were separated into two groups.     

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.     

Dynamics of the etiological structure of salmonellosis and of the biological properties of the Salmonellae isolated in a selected territory of the Moldavian SSR 1982-1985

The etiological structure of Salmonella infections and the biological properties of salmonellae, isolated in one of the regions of the Moldavian SSR where the epidemic process of Salmonella infections reflected the regularities observed on the whole territory of this republic, were studied. Changes in the predominant serovars at the period of 1982-1985 in comparison with the preceding years were shown. Salmonellae belonging to the dominating serovars were characterized by more pronounced drug resistance in comparison with other salmonellae. Some parameters of the epidemic process of Salmonella infections were found to be related to the biological properties of the causative agents of these diseases.     

Occurrence of sopE gene and its phenotypic expression among different serovars of Salmonella enterica isolated from man and animals

Salmonella pathogenesis is a complex phenomenon and a Type III secretion system plays a central role in the development of Salmonella-induced enteritis. One such Type III secretion protein is Salmonella outer protein E (SopE). Prevalence of sopE gene and its phenotypic expression (SopE protein) among different serovars of Salmonella enterica isolated from man and animals were investigated. Of 305 strains of S. enterica belonging to 11 serovars tested for the presence of sopE, 130 strains belonging to three serovars viz., Enteritidis, Gallinarum and Virchow were found to carry sopE gene irrespective of their source of isolation when tested by PCR amplification technique using its specific primers. Of these 130 strains, 112 strains were found to express SopE protein phenotypically as detected by Dot-ELISA using SopE antibody. Among the different serovars tested only serovars Gallinarum, Enteritidis and Virchow expressed SopE protein phenotypically in vitro. Role of SopE protein in pathogenesis of salmonellosis has been discussed.     

Pyrosequencing as a Method for Grouping of Listeria monocytogenes Strains on the Basis of Single-Nucleotide Polymorphisms in the inlB Gene

By using pyrosequencing (i.e., sequencing by synthesis) 106 strains of different serovars of Listeria monocytogenes were rapidly grouped into four categories based on nucleotide variations at positions 1575 and 1578 of the inlB gene. Strains of serovars 1/2a and 1/2c constituted one group, and strains of serovars 1/2b and 3b constituted another group, whereas serovar 4b strains were separated into two groups.     

Characterization of Vibrio anguillarum Strains Isolated from Diseased Fish in Finland

Characterization of V anguillarum strains (n=109) isolated from diseased salmonids was performed. Eight O serovars were found among the strains. Serovar Ol was predominant (90 %), while serovars O2, O3, O5, O8, O9, and a new serovar Va NT2, were represented by 1 or 2 strains. Two strains remained non-typeable. One of these was cross-reactive with several antisera, but had a LPS profile identical to that of serovar O8. All serovars showed specific LPS profiles. All but 1 of the Ol strains had a plasmid comparable in size to the pJMl virulence plasmid, while plasmids of different sizes were found in O2, Va NT2 and the non-typeable strains. Apart from a single strain resistant to tetracycline, all the strains were sensitive to oxolinic acid, tetracycline, and trimethoprim-sulfonamides. By their biochemical and antigenic properties strains causing vibriosis among salmonids in Finland closely resemble Scandinavian strains. Predominance of the serovars Ol and O2 suggests that commercial vaccines containing these serovars should afford sufficient protection against vibriosis in Finland.     

Molecular Typing of Pathogenic Leptospira Serogroup Icterohaemorrhagiae Strains Circulating in China during the Past 50 Years

Background

Leptospirosis is one of the most important neglected tropical infectious diseases worldwide. Icterohaemorrhagiae has been throughout recent history, and still is, the predominant serogroup of this pathogen in China. However, very little in detail is known about the serovars or genotypes of this serogroup.

Methodology/Principal Findings

In this study, 120 epidemic strains from five geographically diverse regions in China collected over a 50 year period (1958~2008), and 8 international reference strains characterized by 16S rRNA sequencing and MLST analysis. 115, 11 and 2 strains were identified as L. interrogans, L. borgpetersenii, and L. kirschneri, respectively. 17 different STs were identified including 69 ST1 strains, 18 ST17, 18 ST128, 9 ST143 and 2 ST209. The remaining 12 strains belonged to 12 different STs. eBURST analysis demonstrated that, among the clonal complexes isolated (CCs), CC1 accounted for 73.3% (88/120) strains representing three STs: ST1, ST128 and ST98. ST1 was the most likely ancestral strain of this CC, followed by singleton CC17 (17/120) and CC143 (11/120). Further analysis of adding 116 serogroup Icterohaemorrhagiae strains in the MLST database and studies previously described using global eBURST analysis and MST dendrogram revealed relatively similar ST clustering patterns with five main CCs and 8 singletons among these 244 strains. CC17 was found to be the most prevalent clone of pathogenic Leptospira circulating worldwide. This is the first time, to our knowledge, that ST1 and ST17 strains were distributed among 4 distinct serovars, indicating a highly complicated relationship between serovars and STs.

Conclusions/Significance

Our studies demonstrated a high level of genetic diversity in the serogroup Icterohaemorrhagiae strains. Distinct from ST17 or ST37 circulating elsewhere, ST1 included in CC1, has over the past 50 years or so, proven to be the most prevalent ST of pathogenic leptospires isolated in China. Moreover, the complicated relationship between STs and serovars indicates an urgent need to develop an improved scheme for Leptospira serotyping.     

Mutant Prevention Concentration (MPC) of Ciprofloxacin Against Salmonella enterica of Epidemic and Poultry Origin

Salmonella isolates resistant or with reduced susceptibility to quinolones increased in recent years. The mutant prevention concentration (MPC) is a new alternative that can prevent the selection and multiplication of resistant Salmonella spp. strains. The MPC of ciprofloxacin (CipMPC) was evaluated for 312 Salmonella enterica strains of epidemic and poultry origin susceptible and resistant to nalidixic acid (NAL). The CipMPC for NAL-susceptible strains were in the range from 0.002 to 4???g/ml and for NAL-resistant strains, it ranged from 0.004 to 16???g/ml. The average MPC/MIC ratio for NAL-resistant strains was higher than NAL susceptible. S. Enteritidis showed the highest CipMPC and the highest MPC/MIC ratio also for NAL-resistant strains and with mutations in gyrA. Serovar Corvallis, a NAL-resistant strain without mutations, and of poultry origin showed the highest CipMPC value. The lowest value was observed for epidemic NAL-susceptible strains serovars Typhimurium and London. The average MPC/MIC ratio for strains with mutations in Aspartate 87 was higher than that mutated in Serine 83. The results show the importance of MPC in determining the correct dosage of Cip for treatment of Salmonella spp.     

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.