Treponema pallidum PCR testing for diagnosis of mucocutaneous ulcers suspicious for syphilis.

BACKGROUND: Primary syphilis is characterised by the appearance of an ulcerated lesion (chancre) on the anogenital or oral mucosa from which Treponema pallidum DNA may be detectable by PCR. Serological tests for syphilis may be non-reactive in early infection, even after the appearance of a chancre. We reviewed the use of a multiplex-PCR (M-PCR) test to determine the added value of T. pallidum DNA detection in the management of individuals presenting with mucocutaneous ulceration at a sexual health service in central London. METHODS: We performed a cross-sectional analysis of all individuals with detectable T. pallidum DNA from September 2019 to April 2020. Electronic patient records were reviewed and concomitant results for treponemal serology and/or rapid plasma reagin (RPR) extracted, along with demographic data, history of syphilis and indices of sexual behaviour including number of sexual partners contacted. Any subsequent treponemal serology and RPR results were also reviewed. RESULTS: M-PCR swab specimens were performed in 450 individuals, of whom 63 (14%) had detectable T. pallidum DNA; 60 of 63 (95%) were gay or bisexual men and 11 of 63 (17%) were living with HIV. A history of treated syphilis was present in 17 of 63 (27%). Same-day treponemal serology/RPR testing was performed in 58 of 63 (92%) patients. Of the 58 who had same-day syphilis serology/RPR, 9 (16%) had their syphilis infection confirmed by treponemal DNA PCR alone. A total of 165 partners were traced as contacts of infection, of whom 25 (15%) were contacts of individuals diagnosed by M-PCR testing alone. CONCLUSION: In individuals with T. pallidum PCR-positive lesions, around one in six in our cohort were negative on standard diagnostic serological tests for syphilis. Treponemal DNA testing is an important addition to serological assays in individuals with mucocutaneous ulceration who are at risk of recent syphilis infection and facilitates early diagnosis and contact tracing.

Characterization of the impact of rpoB mutations on the in vitro and in vivo competitive fitness of Clostridium difficile and susceptibility to fidaxomicin.

Objectives: To establish the role of specific, non-synonymous SNPs in the RNA polymerase ß subunit (rpoB) gene in reducing the susceptibility of Clostridium difficile to fidaxomicin and to explore the potential in vivo significance of rpoB mutant strains. Methods: Allelic exchange was used to introduce three different SNPs into the rpoB gene of an erythromycin-resistant derivative (CRG20291) of C. difficile R20291. The genome sequences of the created mutants were determined and each mutant analysed with respect to growth and sporulation rates, toxin A/B production and cytotoxicity against Vero cells, and in competition assays. Their comparative virulence and colonization ability was also assessed in a hamster infection model. Results: The MIC of fidaxomicin displayed by three mutants CRG20291-TA, CRG20291-TG and CRG20291-GT was substantially increased (>32, 8 and 2 mg/L, respectively) relative to that of the parent strain (0.25 mg/L). Genome sequencing established that the intended mutagenic substitutions in rpoB were the only changes present. Relative to CRG20291, all mutants had attenuated growth, were outcompeted by the parental strain, had lower sporulation and toxin A/B production capacities, and displayed diminished cytotoxicity. In a hamster model, virulence of all three mutants was significantly reduced compared with the progenitor strain, whereas the degree of caecum colonization was unaltered. Conclusions: Our study demonstrates that particular SNPs in rpoB lead to reduced fidaxomicin susceptibility. These mutations were associated with a fitness cost in vitro and reduced virulence in vivo.

Comparison of the first whole genome sequence of 'Haemophilus quentini' with two new strains of 'Haemophilus quentini' and other species of Haemophilus.

Comparison of the genome of the Gram negative human pathogen Haemophilus quentini MP1 with other species of Haemophilus revealed that, although it is more closely related to Haemophilus haemolyticus than Haemophilus influenzae, the pathogen is in fact genetically distinct, a finding confirmed by phylogenetic analysis using the H. influenzae multilocus sequence typing genes. Further comparison with two other H. quentini strains recently identified in Canada revealed that these three genomes are more closely related than any other species of Haemophilus; however, there is still some sequence variation. There was no evidence of acquired antimicrobial resistance within the H. quentini MP1 genome nor any mutations within the DNA gyrase or topoisomerase IV genes known to confer resistance to fluoroquinolones, which has been previously identified in other H. quentini isolates. We hope by presenting the annotation and genetic comparison of the H. quentini MP1 genome it will aid the future molecular detection of this potentially emerging pathogen via the identification of unique genes that differentiate it from other species of Haemophilus.

Improving the reproducibility of the NAP1/B1/027 epidemic strain R20291 in the hamster model of infection.

Comparative analysis of the Clostridium difficile BI/NAP1/027 strain R20291 and ClosTron-derived ermB mutants in the hamster infection model are compromised by the clindamycin susceptibility of the parent. Mutants can appear more virulent. We have rectified this anomaly by genome engineering. The variant created (CRG20291) represents an ideal control strain for virulence assays of ClosTron mutants.

Importance of toxin A, toxin B, and CDT in virulence of an epidemic Clostridium difficile strain.

Clostridium difficile infection is the main cause of healthcare-acquired diarrhea in the developed world. In addition to the main virulence factors toxin A and B, epidemic, PCR Ribotype 027 strains, such as R20291, produce a third toxin, CDT. To develop effective medical countermeasures, it is important to understand the importance of each toxin. Accordingly, we created all possible combinations of isogenic toxin mutants of R20291 and assessed their virulence. We demonstrated that either toxin A or toxin B alone can cause fulminant disease in the hamster infection model and present tantalizing data that C. difficile toxin may also contribute to virulence.

Associations between enterotoxin gene cluster types egc1, egc2 and egc3, agr types, enterotoxin and enterotoxin-like gene profiles, and molecular typing characteristics of human nasal carriage and animal isolates of Staphylococcus aureus.

Twenty genes encoding enterotoxin and enterotoxin-like proteins have been described in Staphylococcus aureus strains. Five of these occur commonly in the enterotoxin gene cluster (egc: selo, selm, sei, seln and seg). In the sei-seln intergenic region, two pseudogenes, psient1 and psient2, can be present or an additional gene designated selu or a variant selu(v). Whilst frequencies of loci bearing pseudogenes (egc1) or the selu gene (egc2) have been reported, the distinction between selu-bearing and selu(v)-bearing (egc3) loci has rarely been made. A PCR-RFLP procedure involving cleavage of the sei-seln intergenic region by restriction endonuclease BbvI or TseI was developed that allowed differentiation of selu(+) and selu(v)(+) loci. In addition, PCR primers were designed to yield a 203 bp amplimer for sequencing of a selu or selu(v) intragenic region, which encompassed ten signature nucleotide differences. A total of 43 egc(+) human nasal isolates and 53 egc(+) bovine, ovine, caprine, leporine and gallinaceous isolates were egc typed and agr typed. None of the animal isolates was of agr type III. A total of 12 out of 17 egc3(+) human nasal isolates were of agr type III, the other 5 being agr type I. On the basis of representative multilocus sequence typing, agr type III/egc3(+) strains belonged to CC30. Human nasal isolates bearing an egc1 locus were distributed evenly across agr types I, II and III. Only two nasal isolates had an egc2 locus. All 14 agr type IV isolates, only 1 of which was of human origin, possessed an egc2 locus. The agr IV nasal isolate was fusidic acid sensitive and was found to be ST123 (CC121). There were strong associations between bovine, leporine and gallinaceous S. aureus clonal types and egc locus types. The PCR-RFLP procedure was used to screen an additional 45 S. aureus isolates from dogs, cats, rats, pigs and horses for egc locus types. Of these, 33 were egc(-). Six equine isolates were selu(+). One canine and three porcine isolates possessed pseudogenes psient1 and psient2. One porcine and one canine isolate each had the selu(v) gene. Putative relationships between disease-causing propensity and egc type need (re-)evaluation.

Molecular typing of nasal carriage isolates of Staphylococcus aureus from an Irish university student population based on toxin gene PCR, agr locus types and multiple locus, variable number tandem repeat analysis.

Forty-eight Staphylococcus aureus isolates collected from a young, healthy, Irish university student population from 1995 to 2004 were screened for 16 enterotoxin (SE) and enterotoxin-like (SEl) genes (sea-see, seg-sei, selj-selo, selq, selu), and for the toxic shock toxin syndrome toxin-1 gene, tst. All of the isolates harboured at least one SE or SEl gene and 66.7 % possessed a classical SE gene (sea, seb, sec), the commonest being the seb gene. Most of the isolates (85.4 %) had a complete egc locus (selo, selm, sei, seln, seg). The intergenic sei-seln region of the egc locus was typed by PCR-RFLP in 34 isolates, 15 possessing pseudogenes psient1 and psient2 and 19 having the selu gene. The seh and sell genes, the selk-selq gene combination, and the tst gene were each found in <15 % of isolates. The agr genotype distribution was agr type III, 37.5 %; agr type I, 35.4 %; agr type II, 25 %; and agr type IV, 2.1 %. There was no association between SE-SEl genotype and agr type. All tst gene-positive isolates were of agr type III and harboured a classical SE gene. Multiple locus, variable number tandem repeat analysis (MLVA) produced 47 different patterns. While the sdr locus was present in all isolates, half of them lacked one or two of the sdr gene amplimers. Twenty isolates harboured the bbp gene, its presence being associated with agr type III, but not with the SE-SEl gene profile. The agr types of isolates were associated with MLVA subclusters. Selective MLST analysis revealed seven novel sequence types and a new aroE allele. Five clonal clusters (CCs), including CCs comprising major pandemic clones CC30, CC5 and CC22 and minor lineages CC6 and CC9, and three singletons were identified.

Rapid differentiation of Staphylococcus aureus isolates harbouring egc loci with pseudogenes psient1 and psient2 and the selu or seluv gene using PCR-RFLP.

The egc locus of Staphylococus aureus harbours two enterotoxin genes (seg and sei) and three enterotoxin-like genes (selm, seln and selo). Between the sei and seln genes are located two pseudogenes, psient1 and psient2, or the selu or seluv gene. While these two alternative sei-seln intergenic regions can be distinguished by PCR, to date, DNA sequencing has been the only confirmatory option because of the very high degree of sequence similarity between egc loci bearing the pseudogenes and the selu or seluv gene. In silico restriction enzyme digestion of genomic regions encompassing the egc locus from the 3' end of the sei gene through the 5' first quarter of the seln gene allowed pseudogene- and selu- or seluv-bearing egc loci to be distinguished by PCR-RFLP. Experimental application of these findings demonstrated that endonuclease HindIII cleaved PCR amplimers bearing pseudogenes but not those with a selu or seluv gene, while selu- or seluv-bearing amplimers were susceptible to cleavage by endonuclease HphI, but not by endonuclease HindIII. The restriction enzyme BccI cleaved selu- or seluv-harbouring amplimers at a unique restriction site created by their signature 15 bp insertion compared with pseudogene-bearing amplimers, thereby allowing distinction of these egc loci. PCR-RFLP analysis using these restriction enzymes provides a rapid, easy to interpret alternative to DNA sequencing for verification of PCR findings on the nature of an egc locus type, and can also be used for the primary identification of the intergenic sei-seln egc locus type.

What's a SNP between friends: The influence of single nucleotide polymorphisms on virulence and phenotypes of Clostridium difficile strain 630 and derivatives.

Clostridium difficile is a major cause of antibiotic induced diarrhea worldwide, responsible for significant annual mortalities and represents a considerable economic burden on healthcare systems. The two main C. difficile virulence factors are toxins A and B. Isogenic toxin B mutants of 2 independently isolated erythromycin-sensitive derivatives (630E and 630Δerm) of strain 630 were previously shown to exhibit substantively different phenotypes. Compared to 630, strain 630E and its progeny grow slower, achieve lower final cell densities, exhibit a reduced capacity for spore-formation, produce lower levels of toxin and are less virulent in the hamster infection model. By the same measures, strain 630Δerm and its derivatives more closely mirror the behavior of 630. Genome sequencing revealed that 630Δerm had acquired 7 unique Single Nucleotide Polymorphisms (SNPs) compared to 630 and 630E, while 630E had 9 SNPs and a DNA inversion not found in the other 2 strains. The relatively large number of mutations meant that the identification of those responsible for the altered properties of 630E was not possible, despite the restoration of 3 mutations to wildtype by allelic exchange and comparative RNAseq analysis of all 3 strains. The latter analysis revealed large differences in gene expression between the 3 strains, explaining in part why no single SNP could restore the phenotypic differences. Our findings suggest that strain 630Δerm should be favored over 630E as a surrogate for 630 in genetic-based studies. They also underline the importance of effective strain curation and the need to genome re-sequence master seed banks wherever possible.

Draft Whole-Genome Sequence of a Haemophilus quentini Strain Isolated from an Infant in the United Kingdom.

Haemophilus quentini is a rare and distinct genospecies of Haemophilus that has been suggested as a cause of neonatal bacteremia and urinary tract infections in men. We present the draft whole-genome sequence of H. quentini MP1 isolated from an infant in the United Kingdom, aiding future identification and detection of this pathogen.

Expanding the repertoire of gene tools for precise manipulation of the Clostridium difficile genome: allelic exchange using pyrE alleles.

Sophisticated genetic tools to modify essential biological processes at the molecular level are pivotal in elucidating the molecular pathogenesis of Clostridium difficile, a major cause of healthcare associated disease. Here we have developed an efficient procedure for making precise alterations to the C. difficile genome by pyrE-based allelic exchange. The robustness and reliability of the method was demonstrated through the creation of in-frame deletions in three genes (spo0A, cwp84, and mtlD) in the non-epidemic strain 630Δerm and two genes (spo0A and cwp84) in the epidemic PCR Ribotype 027 strain, R20291. The system is reliant on the initial creation of a pyrE deletion mutant, using Allele Coupled Exchange (ACE), that is auxotrophic for uracil and resistant to fluoroorotic acid (FOA). This enables the subsequent modification of target genes by allelic exchange using a heterologous pyrE allele from Clostridium sporogenes as a counter-/negative-selection marker in the presence of FOA. Following modification of the target gene, the strain created is rapidly returned to uracil prototrophy using ACE, allowing mutant phenotypes to be characterised in a PyrE proficient background. Crucially, wild-type copies of the inactivated gene may be introduced into the genome using ACE concomitant with correction of the pyrE allele. This allows complementation studies to be undertaken at an appropriate gene dosage, as opposed to the use of multicopy autonomous plasmids. The rapidity of the 'correction' method (5-7 days) makes pyrE(-) strains attractive hosts for mutagenesis studies.