Steen Hoffmann

Evolutionary pathway to increased virulence and epidemic group A Streptococcus disease derived from 3,615 genome sequences

Significance Epidemics of microbial infections are a considerable threat to human and animal health. Analysis of 3,615 genome sequences, coupled with virulence studies in animals, permitted us to delineate the nature and timing of molecular events that contributed to an ongoing global human epidemic of infections caused by group A Streptococcus, the “flesh-eating” pathogen. We clarified decades-long uncertainty about the timing and sequence of genomic alterations that underpinned the global epidemic. Analyses of this type are crucial for developing better strategies to predict and monitor strain emergence and epidemics, formulate effective protective public health maneuvers, and develop or modify vaccines. We sequenced the genomes of 3,615 strains of serotype Emm protein 1 (M1) group A Streptococcus to unravel the nature and timing of molecular events contributing to the emergence, dissemination, and genetic diversification of an unusually virulent clone that now causes epidemic human infections worldwide. We discovered that the contemporary epidemic clone emerged in stepwise fashion from a precursor cell that first contained the phage encoding an extracellular DNase virulence factor (streptococcal DNase D2, SdaD2) and subsequently acquired the phage encoding the SpeA1 variant of the streptococcal pyrogenic exotoxin A superantigen. The SpeA2 toxin variant evolved from SpeA1 by a single-nucleotide change in the M1 progenitor strain before acquisition by horizontal gene transfer of a large chromosomal region encoding secreted toxins NAD+-glycohydrolase and streptolysin O. Acquisition of this 36-kb region in the early 1980s into just one cell containing the phage-encoded sdaD2 and speA2 genes was the final major molecular event preceding the emergence and rapid intercontinental spread of the contemporary epidemic clone. Thus, we resolve a decades-old controversy about the type and sequence of genomic alterations that produced this explosive epidemic. Analysis of comprehensive, population-based contemporary invasive strains from seven countries identified strong patterns of temporal population structure. Compared with a preepidemic reference strain, the contemporary clone is significantly more virulent in nonhuman primate models of pharyngitis and necrotizing fasciitis. A key finding is that the molecular evolutionary events transpiring in just one bacterial cell ultimately have produced millions of human infections worldwide.

Target Gene Sequencing To Characterize the Penicillin G Susceptibility of Neisseria meningitidis

ABSTRACT Clinical isolates of Neisseria meningitidis with reduced susceptibility to penicillin G (intermediate isolates, PenI) harbor alterations in the penA gene encoding the penicillin binding protein 2 (PBP2). A 402-bp DNA fragment in the 3′ half of penA was sequenced from a collection of 1,670 meningococcal clinical isolates from 22 countries that spanned 60 years. Phenotyping, genotyping, and the determination of MICs of penicillin G were also performed. A total of 139 different penA alleles were detected with 38 alleles that were highly related, clustered together in maximum-likelihood analysis and corresponded to the penicillin G-susceptible isolates. The remaining 101 penA alleles were highly diverse, corresponded to different genotypes or phenotypes, and accounted for 38% of isolates, but no clonal expansion was detected. Analysis of the altered alleles that were represented by at least five isolates showed high correlation with the PenI phenotype. The deduced amino acid sequence of the corresponding PBP2 comprised five amino acid residues that were always altered. This correlation was not complete for rare alleles, suggesting that other mechanisms may also be involved in conferring reduced susceptibility to penicillin. Evidence of mosaic structures through events of interspecies recombination was also detected in altered alleles. A new website was created based on the data from this work (http://neisseria.org/nm/typing/penA ). These data argue for the use of penA sequencing to identify isolates with reduced susceptibility to penicillin G and as a tool to improve typing of meningococcal isolates, as well as to analyze DNA exchange among Neisseria species.

Variations in emm type among group A streptococcal isolates causing invasive or noninvasive infections in a nationwide study.

ABSTRACT Since the late 1980s several studies have described the increased incidence and severity of invasive group A streptococcal (GAS) infections. However, most studies on GAS pathogenesis have focused on information obtained during outbreaks. We analyzed isolate distribution and host susceptibility as part of a nationwide prospective surveillance study performed between January 2001 and August 2002. GAS isolates collected from 201 patients with invasive infections, 335 patients with noninvasive infections, and 17 asymptomatic carriers were characterized with respect to their emm types and superantigen genotypes. The superantigen-neutralizing capacity and levels of antibodies against streptolysin O and DNAse B were determined for isolates from the sera from 36 invasive cases and 91 noninvasive cases. emm type 1 (emm-1) isolates were significantly more common among invasive cases, whereas emm-4, emm-6, and emm-12 dominated among the noninvasive cases. The distributions of the phage-associated superantigen genes (speA, speC, speH, speI, ssa) differed among invasive and noninvasive isolates, mainly due to their linkage to certain emm types. No significant differences in serum superantigen-neutralizing capacities were observed. The levels of anti-streptolysin O and anti-DNAse B antibodies were highest in the sera from invasive cases. Our study emphasizes the importance of obtaining data during years with stable incidences, which will enable evaluation of future outbreak data.

Interlaboratory comparison of agar dilution and etest methods for determining the MICs of antibiotics used in management of Neisseria meningitidis infections

ABSTRACT Previous studies have shown that there is considerable variation in the methods and media used to determine the susceptibility of Neisseria meningitidis to antimicrobial agents in different countries. In this study, national and regional reference laboratories used a standardized methodology to determine the MICs of antibiotics used in the management of meningococcal infection. Fourteen laboratories participated in the study, determining the susceptibility to penicillin G, rifampin, cefotaxime, ceftriaxone, ciprofloxacin, and ofloxacin of a collection of 17 meningococci, of which 11 strains were previously defined as having intermediate resistance to penicillin (PenI) by sequencing and restriction fragment length polymorphism analysis of the penA gene. The MIC was determined by agar dilution and Etest with Mueller-Hinton agar (MH), MH supplemented with sheep blood (MH+B), and MH supplemented with heated (chocolated) blood. Several laboratories encountered problems obtaining confluent growth with unsupplemented MH. MH+B was considered to give the most congruent and reproducible results among the study laboratories. The modal MIC for MH+B for each antibiotic and method was calculated to define the MIC consensus, allowing assessment of each individual laboratorys data in relation to the others. The agreement in each antibiotic/method/medium combination was defined as the percentage of laboratories with a result within one dilution of the modal result. For the whole study, an agreement of 90.6% was observed between agar dilution and Etest methods. The agreement in each laboratory/antibiotic/method combination ranged from 98.2% to 69.7%, with six laboratories demonstrating agreement higher than 90% and 11 more than 80%. The ability of the laboratories to detect the PenI isolates ranged from 18.2% to 100%. The apparent difficulty in interpreting susceptibility to rifampin, particularly with the Etest method, is very interesting.

Impact of pneumococcal vaccination in Denmark during the first 3 years after PCV introduction in the childhood immunization programme.

BACKGROUND AND AIMS The 7-valent pneumococcal conjugate vaccine (PCV7) was introduced in Denmark in October 2007 in a 2+1 schedule with a catch-up programme for children up to 17 months of age. To assess the impact of PCV we evaluated on the whole population: (1) direct and indirect effects on incidence of invasive pneumococcal disease (IPD), (2) changes in pneumococcal serotype distribution and (3) IPD related mortality. METHODS We compared disease incidence in pre-PCV (years 2000-2007) and PCV periods (years 2008-2010) based on national surveillance data. RESULTS In children aged 0-5 years the overall incidence of IPD decreased from 26.7 to 16.3 cases per 100,000 (IRR 0.58; 95% Confidence Interval (CI) [0.48-0.69]) and case fatality declined from 1.8% (12 deaths) in the eight-year pre-PCV period to 0% (no deaths) in the three-year PCV period. In the whole population the overall incidence of IPD and of IPD caused by vaccine serotypes declined significantly from 19.5 to 17.7 and from 7.7 to 3.8 cases per 100,000 persons comparing the two periods. The incidence of IPD due to non-vaccine serotypes (NVT-IPD) increased significantly from 11.8 to 13.9 cases per 100,000 in the whole population (incidence rate ratio 1.18; 95% CI [1.12-1.24]) with predominance of the serotypes 1.7F and 19A. CONCLUSIONS We report a marked decline in incidence in IPD in both vaccinated and non-vaccinated age groups and a minor but statistically significant increase in incidence of IPD due to NVTs in both vaccinated and non-vaccinated groups with predominance of serotypes covered by higher valence pneumococcal conjugate vaccines.

An evaluation of gentamicin susceptibility of Neisseria gonorrhoeae isolates in Europe

OBJECTIVES The emergence of decreased susceptibility to third-generation, extended-spectrum cephalosporins in Neisseria gonorrhoeae and associated treatment failures highlights the need to consider alternatives for future therapeutic use, such as gentamicin. METHODS The three laboratories surveying gonococcal antimicrobial susceptibility as part of the European Network for Sexually Transmitted Infections Surveillance compared agar dilution and Etest to determine gentamicin MICs and performed the first survey of gentamicin susceptibility on 1366 gonococcal isolates from 17 European Union/European Economic Area (EU/EAA) countries in 2009. RESULTS Sentinel surveillance of gentamicin susceptibility showed that 95% of European isolates were within a narrow MIC range (4-8 mg/L), with 79% showing an MIC of 8 mg/L. Most countries showed little variation, but wider MIC ranges were observed in Greece (1-16 mg/L) and France, Norway and Sweden (2-16 mg/L). While MICs for both methods generally differed by just one doubling dilution, they were lower by Etest. CONCLUSIONS This is the first reported evidence that the European gonococcal population susceptibility to gentamicin is similar to that reported in other world regions. Clinical trials to evaluate the therapeutic efficacy of gentamicin may be warranted.

Typing of lymphogranuloma venereum Chlamydia trachomatis strains

We analyzed by multilocus sequence typing 77 lymphogranuloma venereum Chlamydia trachomatis strains from men who have sex with men in Europe and the United States. Specimens from an outbreak in 2003 in Europe were monoclonal. In contrast, several strains were in the United States in the 1980s, including a variant from Europe.

Coexistence of Urogenital Schistosomiasis and Sexually Transmitted Infection in Women and Men Living in an Area Where Schistosoma haematobium is Endemic

BACKGROUND In settings in which adequate laboratory service is lacking, the coexistence of urogenital schistosomiasis and sexually transmitted infections (STIs) poses a diagnostic challenge for health care providers in the management of patients with urogenital complaints. METHODS Symptoms were recorded with use of a semistructured questionnaire at baseline and in follow-up surveys after STI and Schistosoma haematobium infection had been assessed and systematically treated as part of a community-based study of 253 women and 236 men, aged 15-49 years, living in an area of Madagascar where S. haematobium is endemic. RESULTS Of those infected with S. haematobium, 35% of the women had concordant STI (e.g., infection with Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, and/or Trichomonas vaginalis), compared with 17% of the men. Both S. haematobium infection and STI were significantly more common among younger individuals, aged 15-24 years, than among older individuals. A broad spectrum of urogenital symptoms was reported. However, one-half of the women and men who had positive test results for an STI or for S. haematobium infection were asymptomatic. Gross hematuria and dysuria were, in multivariate analysis, associated with S. haematobium infection, as were genitopelvic discomfort in women and ejaculation pain in men. The association became stronger with higher intensity of infection. In bivariate analysis but not in the multiregression model, STI was associated with vaginal discharge and genitopelvic discomfort in women and was associated with urethral discharge in men. CONCLUSIONS The rationale for empirical antischistosoma treatment of adolescents and younger adults in areas where S. haematobium is endemic, with praziquantel alone or in combination with existing anti-STI regimens, is discussed.

The epidemiology of invasive meningococcal disease in EU/EEA countries, 2004–2014

BACKGROUND Invasive meningococcal disease (IMD) is a major cause of bacterial meningitis and septicaemia although infection by some serogroups may be prevented through vaccination. We aimed to describe the epidemiology of IMD in EU/EEA countries during 2004-2014 to monitor serogroup- and age-specific trends, and compare country trends by the period of meningococcal C conjugate (MCC) vaccine introduction. METHODS We analysed IMD surveillance data by age, gender, serogroup, country and outcome. We estimated the percentage change in annual notification rate (NR), using linear regression analysis of the log of the annual NR. We grouped countries by the year they introduced MCC vaccination into their routine immunisation programmes. RESULTS The overall NR was 0.9/100 000 population, and decreased 6.6% (95%CI: -8.0%;-5.1%) annually. Infants had the highest NR (16.0/100 000), and there were decreasing trends in all age groups <50years. Serogroup B (SgB) caused 74% of all cases, and the majority of cases in all age groups. There were decreasing trends in SgB and serogroup C (SgC) and an increasing trend in serogroup Y. Countries that introduced MCC vaccination before, and between 2004 and 2014, had decreasing trends in NR of SgC, but not countries without routine MCC vaccination. CONCLUSIONS Our findings support evidence that routine MCC vaccination was the driving force behind the decreasing SgC trend. Vaccinating against SgB in the first year of life could help reduce the burden of IMD due to this serogroup. Changing serogroup-specific NR trends highlight the need for high-quality surveillance data to accurately assess the changing epidemiology of IMD, the effectiveness and impact of implemented vaccines, and the need for future vaccines.

Emerging cephalosporin and multidrug-resistant gonorrhoea in Europe

Neisseria gonorrhoeae has consistently developed resistance to antimicrobials used therapeutically for gonorrhoea and few antimicrobials remain for effective empiric first-line therapy. Since 2009 the European gonococcal antimicrobial surveillance programme (Euro-GASP) has been running as a sentinel surveillance system across Member States of the European Union (EU) and European Economic Area (EEA) to monitor antimicrobial susceptibility in N. gonorrhoeae. During 2011, N. gonorrhoeae isolates were collected from 21 participating countries, and 7.6% and 0.5% of the examined gonococcal isolates had in vitro resistance to cefixime and ceftriaxone, respectively. The rate of ciprofloxacin and azithromycin resistance was 48.7% and 5.3%, respectively. Two (0.1%) isolates displayed high-level resistance to azithromycin, i.e. a minimum inhibitory concentration (MIC) ≥256 mg/L. The current report further highlights the public health need to implement the European response plan, including further strengthening of Euro-GASP, to control and manage the threat of multidrug resistant N. gonorrhoeae.