Emergence and genomic diversification of a virulent serogroup W:ST-2881(CC175) Neisseria meningitidis clone in the African meningitis belt.

Countries of the African 'meningitis belt' are susceptible to meningococcal meningitis outbreaks. While in the past major epidemics have been primarily caused by serogroup A meningococci, W strains are currently responsible for most of the cases. After an epidemic in Mecca in 2000, W:ST-11 strains have caused many outbreaks worldwide. An unrelated W:ST-2881 clone was described for the first time in 2002, with the first meningitis cases caused by these bacteria reported in 2003. Here we describe results of a comparative whole-genome analysis of 74 W:ST-2881 strains isolated within the framework of two longitudinal colonization and disease studies conducted in Ghana and Burkina Faso. Genomic data indicate that the W:ST-2881 clone has emerged from Y:ST-175(CC175) bacteria by capsule switching. The circulating W:ST-2881 populations were composed of a variety of closely related but distinct genomic variants with no systematic differences between colonization and disease isolates. Two distinct and geographically clustered phylogenetic clonal variants were identified in Burkina Faso and a third in Ghana. On the basis of the presence or absence of 17 recombination fragments, the Ghanaian variant could be differentiated into five clusters. All 25 Ghanaian disease isolates clustered together with 23 out of 40 Ghanaian isolates associated with carriage within one cluster, indicating that W:ST-2881 clusters differ in virulence. More than half of the genes affected by horizontal gene transfer encoded proteins of the 'cell envelope' and the 'transport/binding protein' categories, which indicates that exchange of non-capsular antigens plays an important role in immune evasion

DNA methylation assessed by SMRT sequencing is linked to mutations in Neisseria meningitidis isolates

The Gram-negative bacterium Neisseria meningitidis features extensive genetic variability. To present, proposed virulence genotypes are also detected in isolates from asymptomatic carriers, indicating more complex mechanisms underlying variable colonization modes of N. meningitidis. We applied the Single Molecule, Real-Time (SMRT) sequencing method from Pacific Biosciences to assess the genome-wide DNA modification profiles of two genetically related N. meningitidis strains, both of serogroup A. The resulting DNA methylomes revealed clear divergences, represented by the detection of shared and of strain-specific DNA methylation target motifs. The positional distribution of these methylated target sites within the genomic sequences displayed clear biases, which suggest a functional role of DNA methylation related to the regulation of genes. DNA methylation in N. meningitidis has a likely underestimated potential for variability, as evidenced by a careful analysis of the ORF status of a panel of confirmed and predicted DNA methyltransferase genes in an extended collection of N. meningitidis strains of serogroup A. Based on high coverage short sequence reads, we find phase variability as a major contributor to the variability in DNA methylation. Taking into account the phase variable loci, the inferred functional status of DNA methyltransferase genes matched the observed methylation profiles. Towards an elucidation of presently incompletely characterized functional consequences of DNA methylation in N. meningitidis, we reveal a prominent colocalization of methylated bases with Single Nucleotide Polymorphisms (SNPs) detected within our genomic sequence collection. As a novel observation we report increased mutability also at 6mA methylated nucleotides, complementing mutational hotspots previously described at 5mC methylated nucleotides. These findings suggest a more diverse role of DNA methylation and Restriction-Modification (RM) systems in the evolution of prokaryotic genomes

Azithromycin Resistance through Interspecific Acquisition of an Epistasis-Dependent Efflux Pump Component and Transcriptional Regulator in Neisseria gonorrhoeae

Mosaic interspecifically acquired alleles of the multiple transferable resistance (mtr) efflux pump operon correlate with increased resistance to azithromycin in Neisseria gonorrhoeae in epidemiological studies. However, whether and how these alleles cause resistance is unclear. Here, we use population genomics, transformations, and transcriptional analyses to dissect the relationship between variant mtr alleles and azithromycin resistance. We find that the locus encompassing the mtrR transcriptional repressor and the mtrCDE pump is a hot spot of interspecific recombination introducing alleles from Neisseria meningitidis and Neisseria lactamica into N. gonorrhoeae, with multiple rare haplotypes in linkage disequilibrium at mtrD and the mtr promoter region. Transformations demonstrate that resistance to azithromycin, as well as to other antimicrobial compounds such as polymyxin B and crystal violet, is mediated through epistasis between these two loci and that the full-length mosaic mtrD allele is required. Gene expression profiling reveals the mechanism of resistance in mosaics couples novel mtrD alleles with promoter mutations that increase expression of the pump. Overall, our results demonstrate that epistatic interactions at mtr gained from multiple neisserial species has contributed to increased gonococcal resistance to diverse antimicrobial agents.Neisseria gonorrhoeae is the sexually transmitted bacterial pathogen responsible for more than 100 million cases of gonorrhea worldwide each year. The incidence of resistance to the macrolide azithromycin has increased in the past decade; however, a large proportion of the genetic basis of resistance remains unexplained. This study is the first to conclusively demonstrate the acquisition of macrolide resistance through mtr alleles from other Neisseria species, demonstrating that commensal Neisseria bacteria are a reservoir for antibiotic resistance to macrolides, extending the role of interspecies mosaicism in resistance beyond what has been previously described for cephalosporins. Ultimately, our results emphasize that future fine-mapping of genome-wide interspecies mosaicism may be valuable in understanding the pathways to antimicrobial resistance. Our results also have implications for diagnostics and public health surveillance and control, as they can be used to inform the development of sequence-based tools to monitor and control the spread of antibiotic-resistant gonorrhea

Trends in Antibiotic Susceptibility in Staphylococcus aureus in Boston, Massachusetts, from 2000 to 2014

ABSTRACT The rate of infection by methicillin-resistant Staphylococcus aureus (MRSA) has declined over the past decade, but it is unclear whether this represents a decline in S. aureus infections overall. To evaluate the trends in the annual rates of infection by S. aureus subtypes and mean antibiotic resistance, we conducted a 15-year retrospective observational study at two tertiary care institutions in Boston, MA, of 31,753 adult inpatients with S. aureus isolated from clinical specimens. We inferred the gain and loss of methicillin resistance through genome sequencing of 180 isolates from 2016. The annual rates of infection by S. aureus declined from 2003 to 2014 by 4.2% (2.7% to 5.6%), attributable to an annual decline in MRSA of 10.9% (9.3% to 12.6%). Penicillin-susceptible S. aureus (PSSA) increased by 6.1% (4.2% to 8.1%) annually, and rates of methicillin-susceptible penicillin-resistant S. aureus (MSSA) did not change. Resistance in S. aureus decreased from 2000 to 2014 by 0.8 antibiotics (0.7 to 0.8). Within common MRSA clonal complexes, 3/14 MSSA and 2/21 PSSA isolates arose from the loss of resistance-conferring genes. Overall, in two tertiary care institutions in Boston, MA, a decline in S. aureus infections has been accompanied by a shift toward increased antibiotic susceptibility. The rise in PSSA makes penicillin an increasingly viable treatment option

ELF-MF exposure affects the robustness of epigenetic programming during granulopoiesis

Extremely-low-frequency magnetic fields (ELF-MF) have been classified as "possibly carcinogenic" to humans on the grounds of an epidemiological association of ELF-MF exposure with an increased risk of childhood leukaemia. Yet, underlying mechanisms have remained obscure. Genome instability seems an unlikely reason as the energy transmitted by ELF-MF is too low to damage DNA and induce cancer-promoting mutations. ELF-MF, however, may perturb the epigenetic code of genomes, which is well-known to be sensitive to environmental conditions and generally deranged in cancers, including leukaemia. We examined the potential of ELF-MF to influence key epigenetic modifications in leukaemic Jurkat cells and in human CD34+ haematopoietic stem cells undergoing in vitro differentiation into the neutrophilic lineage. During granulopoiesis, sensitive genome-wide profiling of multiple replicate experiments did not reveal any statistically significant, ELF-MF-dependent alterations in the patterns of active (H3K4me2) and repressive (H3K27me3) histone marks nor in DNA methylation. However, ELF-MF exposure showed consistent effects on the reproducibility of these histone and DNA modification profiles (replicate variability), which appear to be of a stochastic nature but show preferences for the genomic context. The data indicate that ELF-MF exposure stabilizes active chromatin, particularly during the transition from a repressive to an active state during cell differentiation

Bayesian modeling of the impact of antibiotic resistance on the efficiency of MRSA decolonization.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality. Colonization by MRSA increases the risk of infection and transmission, underscoring the importance of decolonization efforts. However, success of these decolonization protocols varies, raising the possibility that some MRSA strains may be more persistent than others. Here, we studied how the persistence of MRSA colonization correlates with genomic presence of antibiotic resistance genes. Our analysis using a Bayesian mixed effects survival model found that genetic determinants of high-level resistance to mupirocin was strongly associated with failure of the decolonization protocol. However, we did not see a similar effect with genetic resistance to chlorhexidine or other antibiotics. Including strain-specific random effects improved the predictive performance, indicating that some strain characteristics other than resistance also contributed to persistence. Study subject-specific random effects did not improve the model. Our results highlight the need to consider the properties of the colonizing MRSA strain when deciding which treatments to include in the decolonization protocol

Two N. meningitidis serogroup A strains Z2491 and NM1264 display divergent DNA modifications.

<p>DNA modification scores are plotted against the coverage in SMRT sequencing of Tet1 converted samples. Each dot represents a position on either strand with a modification score larger than 20, the color specifying the nucleotide base, on which the modification was detected. Modified adenosines (red dots) are predominantly detected in strain NM1264. The horizontal line indicates the threshold score 50 applied for subsequent motif finding.</p

Variability at DNA methyltransferase loci.

<p>101 N. meningitidis isolates clustered according to SNP distance, yielding in two sequence type (ST) groups. Each column represents an isolate and rows specify the ORF status of 13 DNA methyltransferases (Rebase geneIDs of Z2491 reference strain). Bars in grey at the bottom represent the number of repeat units determining ON/OFF status of the phase-variable modA12 (M.NmeAORF1589P)</p