Unique Clindamycin-Resistant Clostridioides difficile Strain Related to Fluoroquinolone-Resistant Epidemic BI/RT027 Strain.

During a surveillance study of patients in a long-term care facility and the affiliated acute care hospital in the United States, we identified a Clostridioides difficile strain related to the epidemic PCR ribotype (RT) 027 strain associated with hospital outbreaks of severe disease. Fifteen patients were infected with this strain, characterized as restriction endonuclease analysis group DQ and RT591. Like RT027, DQ/RT591 contained genes for toxin B and binary toxin CDT and a tcdC gene of identical sequence. Whole-genome sequencing and multilocus sequence typing showed that DQ/RT591 is a member of the same multilocus sequence typing clade 2 as RT027 but in a separate cluster. DQ/RT591 produced a similar cytopathic effect as RT027 but showed delayed toxin production in vitro. DQ/RT591 was susceptible to moxifloxacin but highly resistant to clindamycin. Continued surveillance is warranted for this clindamycin-resistant strain that is related to the fluoroquinolone-resistant epidemic RT027 strain.

Association between Clostridioides difficile ribotypes, restriction endonuclease analysis types, and toxin gene expression.

OBJECTIVES: Clostridioides difficile strains cause severe infection. Previous studies suggested that the virulence of C. difficile is dependent on ribotype; however, this hypothesis is still controversial. We aim to investigate the relationship between ribotypes, restriction endonuclease analysis (REA) types, and toxin gene expression in C. difficile strains. METHODS: We utilized 53 clinical C. difficile strains. All strains were assigned a molecular strain type using PCR ribotyping and REA typing and classified into 17 ribotypes and six REA types. The expression of toxin genes (tcdA, tcdB, and cdtB) in C. difficile strains were quantified by real-time PCR using each specific primer set, and expression was normalized to that of the housekeeping gene rpoA. RESULTS: All 53 strains expressed tcdB and four strains expressed cdtB. Five strains did not express tcdA. Most ribotype and REA type strains expressed tcdA and tcdB similar to the BAA-1870 strain. In cdtB-positive strains, the cdtB expression levels were similar to those in the BAA-1870 strain. tcdA and tcdB expression levels were similar in the cdtB-positive and cdtB-negative strains. CONCLUSION: Toxin gene expression was not associated with the ribotype. Production of binary toxin C. difficile transferase was not related to tcdA and tcdB expression levels.

Correlation between restriction endonuclease analysis and PCR ribotyping for the identification of Clostridioides (Clostridium) difficile clinical strains.

Restriction endonuclease analysis (REA) and PCR ribotyping are two typing systems that have been frequently utilized for molecular epidemiologic characterization of Clostridioides (Clostridium) difficile. To correlate typing data obtained from each method, we performed both REA and PCR ribotyping on a large and diverse set of historical and contemporary C. difficile infection clinical isolates. Eighty isolates were selected from each reference laboratory in the United States (Microbiology Reference Laboratory, Hines VA Medical Center) and United Kingdom (Clostridium difficile Network for England and Northern Ireland laboratory, University of Leeds). The 160 isolates were assigned to 82 unique ribotypes and 51 unique REA groups (116 unique REA types). In general, concordance between typing methods was good. Dendrogram analysis of PCR ribotype band patterns demonstrated close genetic relationships among strain types with discordant REA and ribotype assignments. While REA typing was more discriminatory, several REA types in this study were further discriminated by PCR ribotyping, indicating that discriminatory value of these typing methods may be strain dependent. These data will assist with molecular epidemiologic surveillance of strains identified by these two commonly used C. difficile typing systems.

A web-based restriction endonuclease tool for mycobacteriophage cluster prediction.

A recent explosion in the amount of genomic data has revealed a large genetic diversity in the bacteriophages that infect Mycobacterium smegmatis. In an effort to assess the novelty of newly described mycobacteriophage isolates and provide a preliminary determination of their probable cluster assignment prior to full genome sequencing, we have developed a systematic approach that relies on restriction endonuclease analysis. We demonstrate that a web-based tool, the Phage Enzyme Tool (or PET), is capable of rapidly facilitating this analysis and exhibits reliability in the putative placement of mycobacteriophages into specific clusters of previously sequenced phages. We propose that this tool represents a useful analytical step in the initial study of phage genomes and that this tool will increase the efficiency of phage genome characterization and enhance the educational activities involving mycobacteriophage discovery.

A new molecular method for the rapid subtyping of bovine herpesvirus 1 field isolates.

Bovine herpesvirus 1 (BoHV-1) causes several clinical syndromes in cattle worldwide. There are 3 subtypes of BoHV-1: 1.1, 1.2a, and 1.2b. Several molecular methods are commonly used in the detection and characterization of BoHV-1. Among them, restriction endonuclease analysis (REA) and single-nucleotide polymorphism (SNP) analysis of the complete viral genome allow classification of BoHV-1 into different subtypes. However, developing countries need simpler and cheaper screening assays for routine testing. We designed a standard multiplex PCR followed by a REA assay allowing straightforward subclassification of all BoHV-1 isolates tested into 1.1, 1.2a, and 1.2b subtypes based on the analysis of fragment length polymorphism. Our standard multiplex PCR-REA was used to analyze 33 field strains of BoHV-1 isolated from various tissues. The assay confirmed the subtype identified previously by REA. In addition, non-polymorphic or undigested fragments were sequenced in order to confirm the mutation affecting the RE HindIII site. Our PCR-REA method is an affordable and rapid test that will subtype all BoHV-1 strains.

The Relative Role of Toxins A and B in the Virulence of Clotridioides difficile.

Most pathogenic strains of C. difficile possess two large molecular weight single unit toxins with four similar functional domains. The toxins disrupt the actin cytoskeleton of intestinal epithelial cells leading to loss of tight junctions, which ultimately manifests as diarrhea in the host. While initial studies of purified toxins in animal models pointed to toxin A (TcdA) as the main virulence factor, animal studies using isogenic mutants demonstrated that toxin B (TcdB) alone was sufficient to cause disease. In addition, the natural occurrence of TcdA-/TcdB+ (TcdA-/B+)mutant strains was shown to be responsible for cases of C. difficile infection (CDI) with symptoms identical to CDI caused by fully toxigenic (A+/B+) strains. Identification of these cases was delayed during the period when clinical laboratories were using immunoassays that only detected TcdA (toxA EIA). Our hospital laboratory at the time performed culture as well as toxA EIA on patient stool samples. A total of 1.6% (23/1436) of all clinical isolates recovered over a 2.5-year period were TcdA-/B+ variants, the majority of which belonged to the restriction endonuclease analysis (REA) group CF and toxinotype VIII. Despite reports of serious disease due to TcdA-/B+ CF strains, these infections were typically mild, often not requiring specific treatment. While TcdB alone may be sufficient to cause disease, clinical evidence suggests that both toxins have a role in disease.

Comparison of whole genome sequencing to restriction endonuclease analysis and gel diffusion precipitin-based serotyping of Pasteurella multocida.

The gel diffusion precipitin test (GDPT) and restriction endonuclease analysis (REA) have commonly been used in the serotyping and genotyping of Pasteurella multocida. Whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis has become the gold standard for other organisms, offering higher resolution than previously available methods. We compared WGS to REA and GDPT on 163 isolates of P. multocida to determine if WGS produced more precise results. The isolates used represented the 16 reference serovars, isolates with REA profiles matching an attenuated fowl cholera vaccine strain, and isolates from 10 different animal species. Isolates originated from across the United States and from Chile. Identical REA profiles clustered together in the phylogenetic tree. REA profiles that differed by only a few bands had fewer SNP differences than REA profiles with more differences, as expected. The GDPT results were diverse but it was common to see a single serovar show up repeatedly within clusters. Several errors were found when examining the REA profiles. WGS was able to confirm these errors and compensate for the subjectivity in analysis of REA. Also, results of WGS and SNP analysis correlated more closely with the epidemiologic data than GDPT. In silico results were also compared to a lipopolysaccharide rapid multiplex PCR test. From the data produced in our study, WGS and SNP analysis was superior to REA and GDPT and highlighted some of the issues with the older tests.

Genomic diversity of human adenovirus type 3 isolated in Fukui, Japan over a 24-year period.

Recently, human adenovirus type 3 (HAdV-3) has become the most isolated HAdV worldwide. Restriction endonuclease analysis of globally isolated strains of HAdV-3 has uncovered 51 genome types to date. Information on the genome type is important to the epidemiological study of HAdV-3. In this study, analysis of 75 isolates of HAdV- 3 collected over a 24-year period in Fukui revealed: (1) the emergence of three novel genome types (HAdV-3a52, HAdV-3a53 and HAdV-3a54) and two known genome types (HAdV-3a and HAdV-3a54); (2) the spectrum of diseases caused by individual genome types and their major involvement in the paediatric age population; and (3) the co-circulation and replacement of genome types as a usual phenomenon. The rising number of HAdV-3 genome types indicates that the genetic variation of HAdV-3 is more than other HAdVs. Considering the clinical importance of HAdV-3 infection, its genetic diversity underscores the need for its continuous surveillance and genetic characterization.

Genetic diversity in Echinococcus shiquicus from the plateau pika (Ochotona curzoniae) in Darlag County, Qinghai, China.

The metacestode of Echinococcus shiquicus has been recorded previously in the lung and liver of its intermediate host, the plateau pika (Ochotona curzoniae), but there is limited information regarding other organ sites. There is also limited evidence of intra-specific genetic variation within E. shiquicus. A PCR-amplified mitochondrial (mt) nad1 gene fragment (approximately 1400bp in size), with unique EcoRI and SspI restriction sites, was used to distinguish cysts or cyst-like lesions of E. shiquicus from E. multilocularis. Then, the complete mt nad1 and cox1 genes for the E. shiquicus isolates were amplified and sequenced. Phylogenetic tree and haplotype network analyses for the isolates were then generated based on a concatenated dataset of the nad1 and cox1 genes using the neighbour-joining (NJ) method and TCS1.21 software. Nineteen of eighty trapped pikas were found to harbor cysts (71 in total) when dissected at the survey site. Seventeen animals had cysts (fertile) present only in the lungs, one animal had fertile cysts in the lungs and spleen, and one individual had an infertile kidney cyst. Restriction endonuclease analysis of a fragment of the nad1 gene indicated all the cysts were due to E. shiquicus. Genetic diversity analysis revealed that the nad1 and cox1 genes varied by 0.1-1.2% and 0.1-1.0%, respectively. Haplotype network analysis of the concatenated nad1 and cox1 sequences of the isolates showed they were classified into at least 6 haplotypes, and different haplotype percentages ranged from 4.2% to 29.6%. Although, high haplotype diversity was evident in the study area, the complete nad1 and cox1 gene sequences obtained indicated that all samples represented isolates of E. shiquicus. The study has also provided a new PCR-restriction endonuclease-based method to rapidly distinguish E. shiquicus from E. multilocularis which provides a useful tool for epidemiological investigations where the two species overlap.

Clostridium difficile virulence factors: Insights into an anaerobic spore-forming pathogen.

The worldwide emergence of epidemic strains of Clostridium difficile linked to increased disease severity and mortality has resulted in greater research efforts toward determining the virulence factors and pathogenesis mechanisms used by this organism to cause disease. C. difficile is an opportunist pathogen that employs many factors to infect and damage the host, often with devastating consequences. This review will focus on the role of the 2 major virulence factors, toxin A (TcdA) and toxin B (TcdB), as well as the role of other putative virulence factors, such as binary toxin, in C. difficile-mediated infection. Consideration is given to the importance of spores in both the initiation of disease and disease recurrence and also to the role that surface proteins play in host interactions.

Plasmid and Restriction Endonuclease Analyses of Bacteria Associated with Pneumonia in HIV/AIDS Patients in Ekiti State, Nigeria.

Bacterial infections are an important cause of morbidity and mortality in individuals with the human immunodeficiency virus. The emergence of multiple-drug resistant bacteria has been documented by many researches. This study was therefore carried out to determine whether the resistances of bacterial isolates from HIV positive and HIV negative patients are plasmid mediated or chromosomal mediated. The Plasmid, Post Plasmid-curing Sensitivity and Restriction enzymes endonuclease were done using standard methods. The result of plasmid analysis showed that Plasmid-mediated resistance was observed in both populations and the molecular weight of the plasmid DNA was 1000 base pairs. Plasmid mediated resistance was common, and this was observed in all isolates from HIV/AIDS patients with exceptions of P. aeruginosa in which the resistance was chromosomally mediated. Restriction endonuclease analysis from E. coli revealed 3 distinct clusters. The result of restriction enzymes analysis indicate that the pneumonia infection in HIV/AIDS patients is likely to be hospital acquired in the study location. The study also suggests a common source of infection of the patients.

Caracterization of Aujeszky's disease virus isolated from South Brazil in the last twenty years by restriction enzyme analysis

Aujeszky's disease virus (ADV) belongs to Herpesviridae family and is an important etiological agent which infects pigs causing economic losses in swine producing countries worldwide and international trade restrictions to products of swine origin. An eradication program for ADV was established in Santa Catarina State since 2001. The last outbreak was reported in July 2004 and since then none has been reported. The disease has been controlled with the use of a genetic modified vaccine and elimination of seropositives. Aiming the characterization of ADV isolated in the South of Brazil in the last twenty years (1983-2003), a retrospective study based on the genomic analysis of the isolates through a digestion of viral genomic DNA with restriction enzyme Bam HI was done. Thirty-seven ADV samples isolated from swine from the States of Santa Catarina, Parana and Rio Grande do Sul were analyzed. These isolates were compared to the reference strains NIA-4, Bartha and Begonia. The most predominant genomic arrangement was type II found in 33 samples isolated in Santa Catarina State and in one isolate from Rio Grande do Sul State. Genomic arrangement type I, characteristic of vaccine strains was identified in 2 isolates from Parana State and in 1 isolate from Rio Grande do Sul State.

O vírus da doença de Aujeszky (VDA) pertencente à família Herpesviridae é um importante agente etiológico que infecta suínos causando perdas na produção de suínos no mundo inteiro e restrições para o comércio internacional de suínos ou de seus subprodutos. No estado de Santa Catarina, Brasil, foi instituído em 2001 um programa de erradicação da doença de Aujeszky (DA). O último surto da DA foi reportado em julho de 2004 e desde então não foram notificados mais casos. A doença tem sido controlada com o uso de uma vacina geneticamente modificada e eliminação de animais soropositivos para o VDA. Visando caracterizar amostras do VDA isoladas nos últimos vinte anos (1983-2003) na região Sul do Brasil, foi realizado um estudo retrospectivo baseado na análise do genoma dos vírus isolados de acordo com os perfís de restrição apresentados após a digestão do DNA genômico viral com a enzima Bam HI. Foram analisadas trinta e sete amostras isoladas de suínos oriundos dos estados de Santa Catarina, Paraná e Rio Grande do Sul. Os isolados foram comparados com as amostras de referência NIA-4, Bartha e Begônia. O arranjo genômico predominante, encontrado em 33 amostras isoladas em Santa Catarina e em uma isolada no Rio Grande do Sul, foi o do tipo II. O arranjo genômico do tipo I, característico de amostras de vírus vacinal foi identificado em duas amostras isoladas no Paraná e em uma amostra isolada no Rio Grande do Sul.

A Case of Nocardia asteroides type I Induced Pneumonia / 대한진단검사의학회지

Nocardia species are opportunistic pathogens that are known to affect mostly the immunocompromised patients. Recently, we experienced a young systemic lupus erythromatosus female patient having infected with Nocardia species, which we were able to isolate from her lung abscess. The patient is twenty-nine years old female who was diagnosed as having systemic lupus erythromatosus two years ago and is currently engaged with ongoing treatment. During her admission, new symptoms of fever and dyspnea along with a lesion in the apical lobe of her left lung found by simple chest X-rays were observed. Under lung biopsy examination, there were seen neutrophilic exudates that were gram-positive, AFB-negative, and modified AFB-positive. By culturing the biopsy material, we found gram-positive, AFB-negative, and modified AFB-positive branching hyphaes that were morphologically matched for Nocardia species. We have analyzed the Nocardia DNA by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and found the organism to be Nocardia asteroides type I. Treatment of patient was done using sulfamethoxazole/trimethoprim and ceftriazone, and her clinical conditions as well as her radiological findings improved.