Complex typing of Salmonella typhimurium hospital strains

A salmonellosis outbreak, caused by S. typhimurium, was investigated with the use of some microbiological and molecular-biological methods of typing. This investigation revealed that the outbreak was caused by the "outbreak" electrotype of the multi-resistant variant of the infective agent, found to have several plasmidovars. The possibilities and limitations of typing by sensitivity to antibiotics and plasmid DNA profile were shown. These methods of intraspecific typing were regarded as methods making it possible to establish the heterogeneity of S. typhimurium with the use of intraclonal markers.     

Automated ribotyping and random amplified polymorphic DNA analysis for molecular typing of Salmonella enteritidis and Salmonella typhimurium strains isolated in Italy

AIMS: The ability of automated ribotyping and random amplified polymorphic DNA (RAPD) analysis to differentiate Salmonella enteritidis and Salmonella typhimurium isolates in relation to their origin was evaluated. METHODS AND RESULTS: The restriction enzymes EcoRI, PvuII and PstI, and the random primers OPB17 and P1254, were tested for ribotyping and RAPD analysis, respectively. Seventeen subtypes were identified among the isolates of the two pathogenic Salmonella serovars using the RiboPrinter, and 25 subtypes using RAPD. CONCLUSIONS: The greatest degree of genetic diversity was observed among Salm. typhimurium isolates using both automated ribotyping (Simpson's index of discrimination 0878) and RAPD (Simpson's index of discrimination 0886). SIGNIFICANCE AND IMPACT OF THE STUDY: According to the results of this research, automated ribotyping and RAPD are two useful genotyping techniques for identifying unique and common subtypes associated with a specific source and location, and provide powerful tools for epidemiological investigations.     

Chilling invokes different morphologies in two Salmonella enteritidis PT4 strains

The reduction in chemical preservatives in food processing has resulted in more refrigerated (chilled) products. However, the effect of chilling on Salmonella enteritidis PT4 isolates has received relatively little attention. This study investigates the effect of chilling on two Salm. enteritidis PT4 isolates, denoted E and I. These isolates differ in their tolerance to heat, acidification, survival on surfaces, and behaviour in animal models. E routinely shows greater tolerance and pathogenicity than I. Chilling invokes profound cell elongation and heterogeneity in E which corresponded to a 90% sublethal injury; neither such substantial cell elongation nor significant injury was seen in I. The ability to recover resistance to desoxycholate coincided with a reduction to normal cell size. Incomplete cell division and failure of the septum to form is a likely hypothesis for cell elongation although outer membrane changes could be responsible. Possible links are suggested between cell elongation of the heat- and acid-tolerant strain and pathogenicity.     

Sub-classification of phage types of Salmonella weltevreden and typing of strains by an extended phage typing scheme

By the use of two adapted phage preparations of Typing phage II the S. weltevreden phage types 4 and 5 could be classified into two sub-types each and phage types 9 and 10 into three sub-types each. The 1094 strains of S. weltevreden could be classified into a total of sixteen phage types including the sub-types.The host range mutants of Typing phage II were distinct from the parent strain. After adaptation to two insensitive strains, one of the new preparations, IIA lost its affinity to some strains which were lysed by the parent phage strain but gained lytic affinity for a few others that were originally insensitive. The second preparation IIB showed an increase in lytic range as expected. Antigenically these preparations were shown to be related but not identical. The possible reasons for serological non-identity of host range mutants with the parent strain have been discussed.     

Major secondary metabolites produced by two commercial Trichoderma strains active against different phytopathogens

AIMS: Trichoderma harzianum strains T22 and T39 are two micro-organisms used as active agents in a variety of commercial biopesticides and biofertilizers and widely applied amongst field and greenhouse crops. The production, isolation, biological and chemical characterization of the main secondary metabolites produced by these strains are investigated. METHODS AND RESULTS: Of the three major compounds produced by strain T22, one is a new azaphilone that shows marked in vitro inhibition of Rhizoctonia solani, Pythium ultimum and Gaeumannomyces graminis var. tritici. In turn, filtrates from strain T39 were demonstrated to contain two compounds previously isolated from other T. harzianum strains and a new butenolide. The production of the isolated metabolites was also monitored by liquid chromatography/mass spectrometry during in vitro interaction with R. solani. CONCLUSIONS: This paper reports the isolation and characterization of the main secondary metabolites obtained from culture filtrates of two T. harzianum strains and their production during antagonistic interaction with the pathogen R. solani. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work on secondary metabolites produced by the commercially applied strains T22 and T39. Our results provide a better understanding of the metabolism of these fungi, which are both widely used as biopesticides and/or biofertilizers in biocontrol.     

Salmonella on pig carcasses: positive pigs and cross contamination in the slaughterhouse

AIMS: The purpose of this study was to investigate the prevalence of Salmonella in pigs at the moment of slaughter and in the slaughterhouse environment. METHODS AND RESULTS: In total, five different commercial slaughterhouses were sampled during eight slaughterhouse visits. Carcass swabs, colon content and mesenteric lymph nodes were taken to reflect the animal status and from the slaughterhouse environmental samples were taken. Salmonella was isolated from 37% of the carcass samples as a mean value. High variations were noticed between different slaughterhouses (between 0 and 70%) and sampling days in the same abattoir (between 3 and 52%). A correlation was found between the carcass contamination and the status of the delivered animals (P=0.01675). Cross contamination was estimated to account for 29% of the positive carcasses. The slaughterhouse environment was highly contaminated; before starting the slaughtering activities 25% of the samples were positive on average. The most prevalent serotypes isolated at the slaughterhouse environment and from the colon content were S. Typhimurium, S. Livingstone and S. Derby. On carcasses S. Typhimurium was predominately isolated (71%). The biggest variability of serotypes was found in the mesenteric lymph nodes. Serologically 56.3% of the pigs were found positive for Salmonella using a cut-off level of the optical density percentage higher than 10 (O.D.% > or = 10). While on individual pig level the correlation between the bacteriological and serological data was poor, because of recent Salmonella infections, a better correlation was found at the herd level on the moment of slaughtering. CONCLUSION: A high degree of carcass contamination is noticed after slaughtering. This contamination resulted from the delivery of Salmonella-positive pigs and cross-contamination from the slaughterhouse environment. SIGNIFICANCE AND IMPACT OF THE STUDY: In pigs, Salmonella carriage is high, but it is obvious that slaughterhouse hygiene is a determinative factor for managing carcass contamination.     

辽宁省耐药沙门菌PFGE分子分型研究

目的对辽宁省2011-2012年间鸡肉中检测出的沙门菌进行耐药谱分析和PFGE分子分型分析,为沙门菌的监测、暴发预警提供依据。方法 Phoenix-100全自动微生物鉴定/药敏系统做耐药性分析;PFGE分型依据国际实验室分子分型监测网络PulseNet中沙门菌PFGE分型标准化方案进行。结果鸡肉中38株沙门菌均多重耐药,PFGE指纹图谱与血清具有一定的相关性,相同血清型的菌株聚类后,都能分到同一群。结论同一地区的沙门菌具有很高的相似带型,为以实验室为基础的食源性疾病暴发的发现及疫情控制提供依据。     

Development of a sequence typing scheme for differentiation of Salmonella Enteritidis strains

A DNA sequence typing scheme based on the caiC and SEN0629 loci was developed for differentiation of Salmonella Enteritidis strains and validated using a diverse collection of 102 isolates representing 38 phage types from different sources, year of isolation, geographical locations and epidemiological backgrounds. caiC encodes a probable crotonobetaine/carnitine-CoA ligase, and SEN0629 is a pseudogene. Our system allowed for discrimination of 16 sequence types (STs) among the 102 isolates analysed and intraphage type differentiation. Our findings also suggested that the stability of phage typing may be adversely affected by the occurrence of phage type conversion events. During a confirmatory phage typing analysis performed by a reference laboratory, 13 of 31 S. Enteritidis strains representing nine phage types were assigned phage types that differed from the ones originally determined by the same reference laboratory. It is possible that this phenomenon passes largely unrecognized in reference laboratories performing routine phage typing analyses. Our results demonstrate that phage typing is an unstable system displaying limited reproducibility and that the two-loci sequence typing scheme is highly discriminatory, stable, truly portable and has the potential to become the new gold standard for epidemiological typing of S .?Enteritidis strains.     

Phenotypic typing, technological properties and safety aspects of Lactococcus garvieae strains from dairy environments

AIMS: To characterize Lactococcus garvieae strains of dairy origin and to determine their technological properties and safety for their possible use in starter culture preparation. METHODS AND RESULTS: Forty-seven L. garvieae isolates, recovered from two artisanal Italian cheeses were studied, in comparison with 12 fish isolates and the type strain of the species. Phenotypic typing revealed that the strains could be differentiated on the basis of their ecological niche of origin in lactose positive strains (all isolated from dairy sources) and lactose negative strains (all isolated from fish). Furthermore, the strains exhibited a high degree of physiological variability, showing the presence of 26 different biotypes. The strains possessed moderate acidifying and proteolytic activities and did not produce bacteriocins. A safety investigation revealed that all strains were sensitive to vancomycin and moderately resistant to kanamycin; some biotypes were tetracycline resistant. Production of biogenic amines or presence of genes encoding virulence determinants occurred in some isolates. CONCLUSIONS: The prevalence of L. garvieae in some artisanal Italian cheeses can be linked to the typicity of the products. Although in a few cases an antimicrobial resistance or a presence of virulence determinants may imply a potential hygienic risk, most of the strains showed positive properties for their possible adjunction in a starter culture preparation, to preserve the natural bacterial population responsible for the typical sensorial characteristics of the traditional raw milk cheeses. SIGNIFICANCE AND IMPACT OF THE STUDY: L. garvieae strains can be considered an important part of the microbial population associated with the natural fermentation of artisanal Italian cheeses. A deepened characterization of the strains may aid in understanding the functional and ecological significance of their presence in dairy products and in selecting new strains for the dairy industry.     

Dissimilar properties of two recombinant Lactobacillus acidophilus strains displaying Salmonella FliC with different anchoring motifs

Display of heterologous antigens on the cell surface is considered a useful technique for vaccine delivery by recombinant lactobacilli. In this study, two recombinant Lactobacillus acidophilus derivatives displaying Salmonella flagellin (FliC) were constructed using different anchor motifs. In one instance, the FliC protein was fused to the C-terminal region of a cell envelope proteinase (PrtP) and was bound to the cell wall by electrostatic bonds. In the other case, the same antigen was conjugated to the anchor region of mucus binding protein (Mub) and was covalently associated with the cell wall by an LPXTG motif. These two recombinant L. acidophilus cell surface displays resulted in dissimilar maturation and cytokine production by human myeloid dendritic cells. The surface-associated antigen was highly sensitive to simulated gastric and small intestinal juices. By supplementation with bicarbonate buffer and soybean trypsin inhibitor, the cell surface antigen was protected from proteolytic enzymes during gastric challenge in vitro. The protective reagents also increased the viability of the L. acidophilus cells upon challenge with simulated digestive juices. These results demonstrate the importance of protecting cells and their surface-associated antigens during oral immunization.     

Phenotypic and molecular typing of Vibrio harveyi isolates and their pathogenicity to tiger shrimp larvae

AIMS: The objective of the present study was to identify the biotype(s) and molecular type(s) of Vibrio harveyi associated with pathogenicity in tiger shrimp (Penaeus monodon) larvae. METHODS AND RESULTS: Five luminescent and four nonluminescent V. harveyi isolates were subjected to phenotyping and random amplified polymorphic DNA (RAPD) fingerprinting, and pathogenicity testing to P. monodon mysis. Four isolates induced 34-41% mortality of P. monodon mysis when challenged at the rate of 10(6) CFU ml(-1) within 60 h. Sucrose-fermenting biotypes of V. harveyi appeared to be associated with pathogenicity to larval shrimp. Higher temperature and salinity appeared to play a role on the onset of vibriosis and mortality in the challenged larval shrimp. Pathogenic isolates of V. harveyi could be demarcated as revealed by their clustering in the dendrogram constructed based on the RAPD fingerprints. CONCLUSIONS: Nonluminescent V. harveyi also appear to be important aetiological agents of vibriosis of shrimp larvae. Sucrose-fermenting biotypes are likely to be pathogenic. High temperature may trigger onset of vibriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Biotyping of V. harveyi isolates and looking for traits, such as ability to ferment sucrose may be helpful in identifying the pathogenic forms, and such approach requires to be investigated further with larger number of isolates.     

Survey of molecular methods for the typing of wine yeast strains

A survey of the genetic polymorphisms produced by distinct methods was performed in 23 commercial winery yeast strains. Microsatellite typing, using six different loci, an optimized interdelta sequence analysis and restriction fragment length polymorphism of mitochondrial DNA generated by the enzyme HinfI had the same discriminatory power: among the 23 commercial yeast strains, 21 distinct patterns were obtained. Karyotype analysis gave 22 patterns, thereby allowing the discrimination of one of the three strains that were not distinguished by the other methods. Due to the equivalence of the results obtained in this survey, any of the methods can be applied at the industrial scale.     

Determination of molecular phylogenetics of Vibrio parahaemolyticus strains by multilocus sequence typing

Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. There is a growing public health concern due to the emergence of a pandemic strain causing severe outbreaks worldwide. Many questions remain unanswered regarding the evolution and population structure of V. parahaemolyticus. In this work, we describe a multilocus sequence typing (MLST) scheme for V. parahaemolyticus based on the internal fragment sequences of seven housekeeping genes. This MLST scheme was applied to 100 V. parahaemolyticus strains isolated from geographically diverse clinical (n = 37) and environmental (n = 63) sources. The sequences obtained from this work were deposited and are available in a public database (http://pubmlst.org/vparahaemolyticus). Sixty-two unique sequence types were identified, and most (50) were represented by a single isolate, suggesting a high level of genetic diversity. Three major clonal complexes were identified by eBURST analysis. Separate clonal complexes were observed for V. parahaemolyticus isolates originating from the Pacific and Gulf coasts of the United States, while a third clonal complex consisted of strains belonging to the pandemic clonal complex with worldwide distribution. The data reported in this study indicate that V. parahaemolyticus is genetically diverse with a semiclonal population structure and an epidemic structure similar to that of Vibrio cholerae. Genetic diversity in V. parahaemolyticus appears to be driven primarily by frequent recombination rather than mutation, with recombination ratios estimated at 2.5:1 and 8.8:1 by allele and site, respectively. Application of this MLST scheme to more V. parahaemolyticus strains and by different laboratories will facilitate production of a global picture of the epidemiology and evolution of this pathogen.     

Prevalence of Salmonella enterica serovars and genovars from chicken carcasses in slaughterhouses in Spain

AIMS: To determine the prevalence of Salmonella enterica serovars in chicken carcasses in slaughterhouses in Spain and to examine genotypic relations among these serovars. METHODS AND RESULTS: A total of 336 chicken carcasses were collected from six slaughterhouses in Northwestern Spain. Salmonellae were isolated (ISO-6579-1993), serotyped, phage-typed, ribotyped and antibiotyped against 20 antibiotics. Salmonella strains were detected in 60 (17.9%) carcasses. Isolates belonged to nine different serotypes, with Salm. Enteritidis being the most common. Three strains (5%) were resistant to one antibiotic and 24 (40%) were multi-resistant (to more than one antibiotic). The most frequently encountered resistances were to sulphamides, fluoroquinolones and tetracycline. Ribotyping was able to differentiate isolates of the same serotype and phage type. CONCLUSIONS: The Salmonella serotypes and phage types detected are among those most frequently associated with human diseases in Spain. The large percentage of antimicrobial resistant strains is a matter for concern. A high genetic relationship between strains from different slaughterhouses was found. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides detailed information about Salmonella isolates from poultry in Spain. It emphasizes the importance of controlling this pathogen in poultry products, and suggests the need for more prudent use of antibiotics.     

Toward a global database for the molecular typing of Saccharomyces cerevisiae strains

Most of the yeast strains used in fermented beverages and foods are classified as Saccharomyces cerevisiae. However, different strains are suitable for different fermentation processes. The purpose of this work is the proposal of a standardized methodology for the molecular genotyping of S. cerevisiae strains based on polymorphisms at microsatellite loci and/or single nucleotide polymorphisms (SNPs). Single nucleotide variants in the coding region of FLO8, a key regulator of flocculation and pseudohyphae formation, were analyzed in a subset of Uruguayan wine strains. Polymorphism analysis at nine microsatellite loci (selected from 33 loci tested) was performed in a collection of 120 strains, mostly wine strains, from different origins. From a total of 184 different alleles scored, 50 were exclusive alleles that could identify 29 strains. Four selected microsatellite loci are located within or near genes of putative enological interest. The Uruguayan strains are highly diverse and evenly distributed in the phylogenetic reconstructions, suggesting an evolutionary history previous to human use. The Saccharomyces cerevisiae Microsatellites and SNPs Genotyping Database is presented (www.pasteur.edu.uy/yeast). Comparison of standardized results from strains coming from different settings (industrial, clinical, environmental) will provide a reliable and growing source of information on the molecular biodiversity of S. cerevisiae strains.