The skin microbiota of atopic dermatitis (AD) patients is characterized by increased
colonization, which exacerbates disease symptoms and has been linked to reduced bacterial diversity. Skin ...bacterial communities in AD patients have mostly been described at family and genus levels, while species-level characterization has been limited. In this study, we investigated the role of the bacteria belonging to the
genus using targeted sequencing of the
gene with genus-specific primers. We compared staphylococcal communities on lesional and non-lesional skin of AD patients, as well as AD patients with healthy controls, and determined the absolute abundance of bacteria present at each site. We observed that the staphylococcal community, bacterial alpha diversity, and bacterial densities were similar on lesional and non-lesional skin, whereas AD severity was associated with significant changes in staphylococcal composition. Increased
,
, and
abundances were correlated with increased severity. Conversely,
abundance was negatively correlated with severity. Furthermore,
relative abundance was reduced on AD skin compared to healthy skin. In conclusion, various staphylococcal species appear to be important for skin health.
Clostridium perfringens causes gastrointestinal diseases in both humans and domestic animals. Type A strains expressing the NetB toxin are the main cause of necrotic enteritis (NE) in chickens, which ...has remarkable impact on animal welfare and production economy in the international poultry industry. Three pathogenicity loci NELoc-1, -2 and -3 and a collagen adhesion gene cnaA have been found to be associated with NE in chickens, whereas the presence of these has not been investigated in diseased turkeys. The purpose was to investigate the virulence associated genome content and the genetic relationship among 30 C. perfringens isolates from both healthy and NE infected chickens and turkeys, applying whole-genome sequencing.
NELoc-1, -3, netB and cnaA were significantly associated with NE isolates from chickens, whereas only NELoc-2 was commonly observed in both diseased turkeys and chickens. A putative collagen adhesion gene that encodes a von Willebrand Factor (vWF) domain was identified in all diseased turkeys and designated as cnaD. The phylogenetic analysis based on single nucleotide polymorphisms showed that the isolates generally were not closely related. These results indicate that virulence factors and pathogenicity loci associated with NE in chickens are not important to the same extent in diseased turkeys except for NELoc-2. A putative collagen adhesion gene which potentially could be of importance in regard to the NE pathogenesis in turkeys was identified and need to be further investigated. Thus, the pathogenesis of NE in turkeys appears to be different from that of broiler chickens.
A proportion of classical Hodgkin lymphoma (HL) is believed to be causally related to infection with the ubiquitous lymphotropic EBV. The determining factors for development of EBV-related HL remain ...poorly understood, but likely involve immunological control of the viral infection. Accordingly, markers of the HLA class I region have been associated with risk of EBV-related HL. To study the host genetic component of EBV-related HL further, we investigated the lymphoma's association with HLA-A*01 and HLA-A*02 simultaneously in the setting of infectious mononucleosis (IM), a risk factor for EBV-related HL, in a case-series analysis including 278 EBV-related and 656 EBV-unrelated cases of HL. By logistic regression, HLA-A*01 alleles odds ratio (OR) per allele, 2.15; 95% CI, 1.60-2.88 were associated with increased and HLA-A*02 alleles (OR per allele, 0.70; 95% CI, 0.51-0.97) with decreased risk of EBV-related HL. These allele-specific associations corresponded to nearly 10-fold variation in risk of EBV-related HL between HLA-A*01 and HLA-A*02 homozygotes. History of IM was also associated with risk of EBV-related HL (OR, 3.40; 95% CI, 1.74-6.66). The association between history of IM and EBV-related HL was not seen in the presence of HLA-A*02 because this allele appeared to neutralize the effect of IM on EBV-related HL risk. Our findings suggest that HLA class I-restricted EBV-specific cytotoxic T-cell responses and events in the early immune response to EBV infection in IM play critical roles in the pathogenesis of EBV-related HL.
The aim of the study was to characterize clinical and environmental Staphylococcus pettenkoferi isolates with regard to genomic diversity and antibiotic susceptibility pattern. ...Repetitive-sequence-based PCR and core genome phylogenetic analysis of whole-genome sequencing (WGS) data verified the presence of distinct clades comprising closely related S. pettenkoferi isolates from different geographical locations and origins.
Phylogenetic relationships between 25 S. pettenkoferi isolates collected from blood cultures and intra-operative air sampling were determined by repetitive-sequence-based PCR typing and analysis of ~157 000 SNPs identified in the core genome after WGS. Antibiotic susceptibility testing and tests for biofilm production (microtitre plate assay) were performed.
Repetitive-sequence-based PCR as well as WGS data demonstrated the close relatedness of clinically significant blood culture isolates to probable contaminants, as well as to environmental isolates. Antibiotic-susceptibility testing demonstrated a low level of antimicrobial resistance. The mecA gene was present in two cefoxitin-resistant isolates. No isolates were found to produce biofilm.
Close genomic relatedness of S. pettenkoferi isolates from different geographical locations and origins were found within clades, but with substantial genomic difference between the two major clades. The ecological niche of S. pettenkoferi remains unconfirmed, but the presence of S. pettenkoferi in the air of the operating field favours the suggestion of a role in skin flora. Identification of S. pettenkoferi in clinical samples should, in a majority of cases, most likely be regarded as a probable contamination, and its role as a possible pathogen in immunocompromised hosts remains to be clarified.
Small selected cohort studies suggest that mutations in the cardiac myosin binding protein-C (MYBPC3) gene cause late-onset, clinically benign hypertrophic cardiomyopathy (HCM). The aim of this study ...was to test this hypothesis in a large series of families with HCM associated with MYBPC3 mutations.
The initial study population comprised 57 probands with 42 mutations (26 61.9% novel) in MYBPC3. Missense mutations (15, 45.6%) were the most frequent, and multiple mutations occurred in 4 (7.0%) probands. Another 110 mutation carriers were identified during familial evaluation; 38 were clinically affected with left ventricular hypertrophy ≥13 mm. Disease penetrance was, therefore, incomplete (56.9% in all mutation carriers, 34.5% in relatives), related to age (38.4% <40 versus 68.6% ≥40 years, P<0.001), and was greater in males than females (65.1% versus 48.1%, P=0.03). In 9 families (25 individuals) with the R502W mutation, there was marked heterogeneity in age at diagnosis (5 to 80 years), pattern of hypertrophy (11 none, 9 asymmetrical, 3 concentric, 1 apical, 1 eccentric), and prognosis (premature sudden death in 2 individuals compared with survival to advanced age in 6 individuals). During follow up of 7.9+/-4.5 years, in 82 clinically affected individuals the annual risk of sudden death and all cause mortality was 0.46% and 0.93% per year, respectively.
Disease expression in families with HCM related to MYBPC3 mutations shows marked heterogeneity with incomplete, age-related, and gender specific penetrance. Importantly, complex genetic status is observed and should be considered when mutation analysis and cascade screening is used in the evaluation of at risk family members.
CRISPR-Cas is an adaptive immune system that allows bacteria to inactivate mobile genetic elements. Approximately 50% of bacteria harbor CRISPR-Cas; however, in the human pathogen
Staphylococcus ...aureus
, CRISPR-Cas loci are less common and often studied in heterologous systems. We analyzed the prevalence of CRISPR-Cas in genomes of methicillin-resistant
Staphylococcus aureus
(MRSA) strains isolated in Denmark. Only 2.9% of the strains carried CRISPR-Cas systems, but for strains of sequence type ST630, over half were positive. All CRISPR-Cas loci were type III-A and located within the staphylococcal cassette chromosome
mec
(SCC
mec
) type V(5C2&5), conferring β-lactam resistance. Curiously, only 23 different CRISPR spacers were identified in 69 CRISPR-Cas positive strains, and almost identical SCC
mec
cassettes, CRISPR arrays, and
cas
genes are present in staphylococcal species other than
S. aureus
, suggesting that these were transferred horizontally. For the ST630 strain 110900, we demonstrate that the SCC
mec
cassette containing CRISPR-Cas is excised from the chromosome at high frequency. However, the cassette was not transferable under the conditions investigated. One of the CRISPR spacers targets a late gene in the lytic bacteriophage phiIPLA-RODI, and we show that the system protects against phage infection by reducing phage burst size. However, CRISPR-Cas can be overloaded or circumvented by CRISPR escape mutants. Our results imply that the endogenous type III-A CRISPR-Cas system in
S. aureus
is active against targeted phages, albeit with low efficacy. This suggests that native
S. aureus
CRISPR-Cas offers only partial immunity and in nature may work in tandem with other defense systems.
IMPORTANCE
CRISPR-Cas is an adaptive immune system protecting bacteria and archaea against mobile genetic elements such as phages. In strains of
Staphylococcus aureus
, CRISPR-Cas is rare, but when present, it is located within the SCC
mec
element, which encodes resistance to methicillin and other β-lactam antibiotics. We show that the element is excisable, suggesting that the CRISPR-Cas locus is transferable. In support of this, we found almost identical CRISPR-Cas-carrying SCC
mec
elements in different species of non-
S. aureus
staphylococci, indicating that the system is mobile but only rarely acquires new spacers in
S. aureus
. Additionally, we show that in its endogenous form, the
S. aureus
CRISPR-Cas is active but inefficient against lytic phages that can overload the system or form escape mutants. Thus, we propose that CRISPR-Cas in
S. aureus
offers only partial immunity in native systems and so may work with other defense systems to prevent phage-mediated killing.
Aims
Fabry disease, an X‐linked storage disorder caused by defective lysosomal enzyme alpha‐galactosidase A activity, may resemble sarcomere‐gene‐associated hypertrophic cardiomyopathy (HCM). The ...‘cardiac variant’ of Fabry disease which only affects the heart may be missed unless specifically tested for.
Methods and results
We evaluated 90 consecutively recruited HCM probands and their relatives. Probands without sarcomere‐gene mutations were tested for alpha‐galactosidase A gene (GLA) mutations. Of the 90 families, 31 (34%) had sarcomere gene mutations and were therefore excluded. In the remaining 59 probands, 3 (5%) had GLA mutations as follows. The first proband, a female with asymmetric septal hypertrophy (ASH), a significant left ventricular outflow tract gradient, and chronic obstructive pulmonary disease, was heterozygous for a novel missense mutation (p.N139S). The second proband, a male with ASH and multiple episodes of ventricular tachycardia, was hemizygous for a missense mutation (p.A156T). His daughter was heterozygous, but had normal enzyme activity. The third proband was a female with ASH, and no other indices of Fabry disease. She was heterozygous for a GLA missense mutation (p.G271S). She had one affected daughter but her two other children were unaffected. The affected daughter had three children, of whom two were also affected—a boy aged 8 and a daughter aged 10 years.
Conclusion
This is the first report of systematic mutation screening of GLA in HCM patients without sarcomere gene mutations. GLA mutations were found in 3/90 (3%) of HCM families and in 2/20 (10%) of females without sarcomere‐gene mutations. None of the probands presented other indices of Fabry disease. This, in combination with putative reversibility of cardiac changes by enzyme replacement therapy, supports systematic testing for Fabry disease. Enzyme measurements are sufficient in men, but genetic testing is needed in women.
There have been conflicting reports in the literature on association of gene copy number with disease, including CCL3L1 and HIV susceptibility, and β-defensins and Crohn's disease. Quantification of ...precise gene copy numbers is important in order to define any association of gene copy number with disease. At present, real-time quantitative PCR (QPCR) is the most commonly used method to determine gene copy number, however the Paralogue Ratio Test (PRT) is being used in more and more laboratories.
In this study we compare a Pyrosequencing-based Paralogue Ratio Test (PPRT) for determining beta-defensin gene copy number with two currently used methods for gene copy number determination, QPCR and triplex PRT by typing five different cohorts (UK, Danish, Portuguese, Ghanaian and Czech) of DNA from a total of 576 healthy individuals. We found a systematic measurement bias between DNA cohorts revealed by QPCR, but not by the PRT-based methods. Using PRT, copy number ranged from 2 to 9 copies, with a modal copy number of 4 in all populations.
QPCR is very sensitive to quality of the template DNA, generating systematic biases that could produce false-positive or negative disease associations. Both triplex PRT and PPRT do not show this systematic bias, and type copy number within the correct range, although triplex PRT appears to be a more precise and accurate method to type beta-defensin copy number.
The aim was to study alterations of bacterial communities in patients undergoing hip or knee arthroplasty to assess the impact of chlorhexidine gluconate soap decolonisation and systemic antibiotic ...prophylaxis. A Swedish multicentre, prospective collection of samples obtained from elective arthroplasty patients (n = 83) by swabbing anterior nares, skin sites in the groin and the site of planned surgery, before and after arthroplasty surgery, was analysed by 16S rRNA (V3-V4) gene sequencing and a complementary targeted
gene sequencing approach to comprehensively characterise alterations in staphylococcal communities. Significant reductions in alpha diversity was detected for both bacterial (
= 0.04) and staphylococcal (
= 0.03) groin communities after arthroplasty surgery with significant reductions in relative
(
= 0.001) abundance and
(
0.01) relative staphylococcal abundance. In nares, significant reductions occurred for
(
= 0.02),
(
= 0.02)
and
(
= 0.003) relative to other staphylococci.
colonised 35% of anterior nares before and 26% after arthroplasty surgery.
was the most abundant staphylococcal species at all sampling sites. No bacterial genus or staphylococcal species increased significantly after arthroplasty surgery. Application of a targeted
gene sequencing approach provided auxiliary staphylococcal community profiles and allowed species-level characterisation directly from low biomass clinical samples.
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