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.     

The leg strength of two commercial strains of meat chicken subjected to different incubation profiles

Lower egg shell temperatures (EST) during the first 2 weeks of incubation, notionally known as Slow start incubation, extended the standing time of a 5-week-old fast feathering meat chicken parent line. This study was designed to evaluate the effect of Slow start incubation on the standing ability of commercial meat chickens. Eggs from two strains of meat chickens, Strains 1 and 2, were incubated using either the Slow start incubation, (the initial EST was 36.75°C followed by a gradual increase in EST, reaching 37.8°C at day 16 of incubation), or Control incubation (EST 37.75°C to 38°C from the start of incubation until day 18 of incubation). Eggs were observed every 6 h from 468 h until 516 h of incubation to identify chick hatch window. At 516 h of incubation all chicks were taken out of the incubator (take-off). Chicks from each Strain and incubation treatment were randomly selected for assessment of chick weight, chick length, yolk sac weight, serum Ca and P, and femoral bone ash (BA). All unhatched eggs were inspected to determine the stage of embryo failure. Remaining chicks were grown for 5 weeks in floorpens. Weekly feed intake (FI), chick weight and feed conversion ratio (FCR) were determined. At 35 days of age the standing ability of visibly male birds was assessed in a latency-to-lie test. Compared to the Control, Slow start incubation delayed the average hatch time of both strains by ∼13 h, and reduced hatchability with 4.6% live but unhatched chicks, which was most evident in Strain 2. Significant differences due to main effects only were observed at take-off. Strain 1 chicks were significantly heavier and longer with higher serum Ca but significantly lower BA and serum P than Strain 2. Slow start incubation generated significantly heavier chicks that were shorter, but had significantly heavier yolk sacs, lower serum Ca but higher serum P than Control incubated chicks. During the 1^st week post hatch Strain 1 Control incubated chicks had significantly higher FI and higher FCR than all other Strain and incubation treatments. At 35 days of age Slow start incubated birds of both Strains stood significantly longer than those from the Control incubation. This experiment clearly demonstrated the ability of Slow start incubation of commercial meat chickens to improve their leg strength.     

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.     

Multilocus sequence typing of Salmonella strains by high-throughput sequencing of selectively amplified target genes

Rapid development of next generation sequencing (NGS) technologies in recent years has made whole genome sequencing of bacterial genomes widely accessible. However, it is often unnecessary or not feasible to sequence the whole genome for most applications of genetic analyses in bacteria. Selectively capturing defined genomic regions followed by NGS analysis could be a promising approach for high-resolution molecular typing of a large set of strains. In this study, we describe a novel and straightforward PCR-based target-capturing method, hairpin-primed multiplex amplification (HPMA), which allows for simultaneous amplification of numerous target genes. To test the feasibility of NGS-based strain typing using HPMA, 20 target gene sequences were simultaneously amplified with barcode tagging in each of 41 Salmonella strains. The amplicons were then pooled and analyzed by 454 pyrosequencing. Analysis of the sequence data, as an extension of multilocus sequence typing (MLST), demonstrated the utility and potential of this novel typing method, MLST-seq, as a high-resolution strain typing method. With the rapidly increasing sequencing capacity of NGS, MLST-seq or its variations using different target enrichment methods can be expected to become a high-resolution typing method in the near future for high-throughput analysis of a large collection of bacterial strains.     

2014年大连市伤寒沙门菌耐药及 分子分型研究

摘要：目的 了解大连市伤寒沙门菌耐药性及分子分型特点,建立沙门菌分子特征本底信息,为今后防治工作提供科学依据。方法 采用微量肉汤稀释法对46株伤寒沙门菌进行8种抗生素敏感试验;运用脉冲场凝胶电泳（PFGE）方法对46株伤寒沙门菌进行分子分型及聚类分析。结果 46株伤寒沙门菌对萘啶酸（NAL）100%敏感,对氯霉素（CHL）和甲氧苄啶/磺胺甲噁唑（TMP/SMZ）耐药率为4.35%,对庆大霉素（GEN）耐药率为47.83%,发现多重耐药株1株;BioNumerics分析结果显示,46株伤寒沙门菌共产生30种PFGE带型,有7株表现为同一PFGE型别。结论 大连地区存在耐庆大霉素的伤寒沙门菌;PFGE结果表明这些菌株存在遗传多态性,并有优势菌株的存在。