Magnus Gottfredsson

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.

Risk Factors for Acute Cellulitis of the Lower Limb: A Prospective Case-Control Study

BACKGROUND Acute bacterial cellulitis is a potentially serious infection that commonly recurs. The identification of preventable risk factors could reduce infection-related morbidity and cost and improve patient management. The aim of this study was to identify the risk factors associated with lower-limb cellulitis, including both analysis of risk factors associated with cellulitis in either limb and risk factors in a single limb associated with cellulitis in the same limb. We placed particular emphasis on dermatophytic infections of the foot and bacterial infection and colonization of the toe webs. METHODS We conducted a prospective case-control study of 100 subjects with cellulitis and 200 control subjects, matched for age and sex, who were admitted to a university hospital during the period October 2000-February 2004. Data were obtained with a questionnaire and from examination of lower limbs and microbiological analyses of samples from the feet. RESULTS The median age of the participants was 66.5 years (interquartile range, 48.8-77.0). The following risk factors were strongly and independently associated with cellulitis: previous history of cellulitis (OR, 31.04; 95% CI, 4.15-232.20), the presence of Staphylococcus aureus and/or beta -hemolytic streptococci in the toe webs (OR, 28.97; 95% CI, 5.47-153.48), presence of leg erosions or ulcers (OR, 11.80; 95% CI, 2.47-56.33), and prior saphenectomy (OR, 8.49; 95% CI, 1.62-44.52). Tinea pedis interdigitalis was associated with cellulitis only when toe web bacteria were excluded from the analysis (OR, 3.86; 95% CI, 1.32-11.27). CONCLUSIONS Risk factors for acute bacterial cellulitis in hospitalized patients include predisposing factors and the presence of sites of pathogen entry on legs and toe webs. These findings indicate that improved awareness and management of toe web intertrigo, which may harbor bacterial pathogens, and other skin lesions might reduce the incidence of cellulitis.

Molecular epidemiology of candidemia: evidence of clusters of smoldering nosocomial infections

BACKGROUND Invasive fungal infections pose a serious threat to hospitalized patients worldwide. In particular, the prevalence of clusters of nosocomial infection among patients with candidemia remains unknown. The aim of this study was to investigate the molecular epidemiology of candidemia in a nationwide setting in Iceland during a 16-year period. METHODS The genotypes of all available fungal bloodstream isolates during 1991-2006 (n = 219) were determined by polymerase chain reaction fingerprinting with use of 4 separate primers. Clusters were defined as isolation of > or =2 strains with genotypes that had > or =90% relatedness in the same hospital within a period of 90 days. RESULTS Candida albicans represented 61.6% of isolates, followed by Candida glabrata (13.7%), Candida tropicalis (9.1%), and Candida parapsilosis (8.7%). Polymerase chain reaction fingerprinting revealed 35 clones of C. albicans, 10 clones of C. glabrata, 7 clones of C. tropicalis, 4 clones of C. parapsilosis, and 5 clones of Candida dubliniensis. Overall, 18.7%-39.9% of all infections were part of nosocomial clusters, most commonly caused by C. albicans, C. parapsilosis, and C. tropicalis. Most clusters involved 2 cases and disproportionately affected patients in adult and neonatal intensive care units (P = .045). The 7-day (16%) and 30-day (32%) case-fatality rates among cluster-associated cases did not differ statistically significantly from those for sporadic nosocomial infections. None of the clusters were identified by the hospital surveillance team. CONCLUSIONS In an unselected patient population, as many as one-third of all cases of candidemia may be attributable to nosocomial clusters. The risk is dependent on hospital wards and patient populations; it is highest in intensive care units. Small clusters are not identified by routine hospital surveillance.

Deep sternal wound infections following open heart surgery in Iceland. A population-based study

Objective. The aim of this nationwide case-control study was to study the epidemiology and identify risk factors of deep sternal wound infections (DSWI) in Iceland. Patients and methods. Between 1997–2004, 1 650 adults underwent open cardiac surgery in Iceland. For every infected patient four control subjects were chosen (n =163), matched for time of operation. The groups were compared by multivariable logistic regression analysis. Results. Forty one patients (2.5%) developed DSWI, most often following CABG (76%). The most common pathogens were Staphylococcus aureus (39%) and coagulase-negative staphylococci (24%). All except two patients underwent debridement and rewiring of the sternum. Length of hospital stay was significantly longer in the DSWI group with a trend for increased hospital mortality and significantly greater 1-year mortality (17% vs. 5%, p =0.02). History of stroke (OR 5.12), peripheral arterial disease (OR 5), corticosteroid use (OR 4.25), smoking (OR 3.66) and re-operation for bleeding (OR 4.66) were the strongest independent predictors for DSWI. Conclusion. Incidence of DSWI in Iceland (2.5%) is comparable to other recently published studies, with similar risk factors and significantly reduced survival at one year following the infection.

Azithromycin Maintains Airway Epithelial Integrity during Pseudomonas aeruginosa Infection

Tight junctions (TJs) play a key role in maintaining bronchial epithelial integrity, including apical-basolateral polarity and paracellular trafficking. Patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) often suffer from chronic infections by the opportunistic Gram-negative bacterium Pseudomonas aeruginosa, which produces multiple virulence factors, including rhamnolipids. The macrolide antibiotic azithromycin (azm) has been shown to improve lung function in patients with CF without reducing the bacterial count within the lung. However, the mechanism of this effect is still debated. It has previously been shown that azm increased transepithelial electrical resistance (TER) in a bronchial epithelial cell line. In this study we used an air-liquid interface model of human airway epithelia and measured TER, changes in TJ expression and architecture after exposure to live P. aeruginosa PAO1, and PAO1-Deltarhl which is a PAO1 mutant lacking rhlA and rhlB, which encode key enzymes for rhamnolipid production. In addition, the cells were challenged with bacterial culture medium conditioned by these strains, purified rhamnolipids, or synthetic 3O-C(12)-HSL. Virulence factors secreted by P. aeruginosa reduced TER and caused TJ rearrangement in the bronchial epithelium, exposing the epithelium to further bacterial infiltration. Pretreatment of the bronchial epithelium with azm attenuated this effect and facilitated epithelial recovery. These data suggest that azm protects the bronchial epithelium during P. aeruginosa infection independent of antimicrobial activity, and could explain in part the beneficial results seen in clinical trials of patients with CF.

Transcriptome Remodeling Contributes to Epidemic Disease Caused by the Human Pathogen Streptococcus pyogenes

ABSTRACT For over a century, a fundamental objective in infection biology research has been to understand the molecular processes contributing to the origin and perpetuation of epidemics. Divergent hypotheses have emerged concerning the extent to which environmental events or pathogen evolution dominates in these processes. Remarkably few studies bear on this important issue. Based on population pathogenomic analysis of 1,200 Streptococcus pyogenes type emm89 infection isolates, we report that a series of horizontal gene transfer events produced a new pathogenic genotype with increased ability to cause infection, leading to an epidemic wave of disease on at least two continents. In the aggregate, these and other genetic changes substantially remodeled the transcriptomes of the evolved progeny, causing extensive differential expression of virulence genes and altered pathogen-host interaction, including enhanced immune evasion. Our findings delineate the precise molecular genetic changes that occurred and enhance our understanding of the evolutionary processes that contribute to the emergence and persistence of epidemically successful pathogen clones. The data have significant implications for understanding bacterial epidemics and for translational research efforts to blunt their detrimental effects. IMPORTANCE The confluence of studies of molecular events underlying pathogen strain emergence, evolutionary genetic processes mediating altered virulence, and epidemics is in its infancy. Although understanding these events is necessary to develop new or improved strategies to protect health, surprisingly few studies have addressed this issue, in particular, at the comprehensive population genomic level. Herein we establish that substantial remodeling of the transcriptome of the human-specific pathogen Streptococcus pyogenes by horizontal gene flow and other evolutionary genetic changes is a central factor in precipitating and perpetuating epidemic disease. The data unambiguously show that the key outcome of these molecular events is evolution of a new, more virulent pathogenic genotype. Our findings provide new understanding of epidemic disease. The confluence of studies of molecular events underlying pathogen strain emergence, evolutionary genetic processes mediating altered virulence, and epidemics is in its infancy. Although understanding these events is necessary to develop new or improved strategies to protect health, surprisingly few studies have addressed this issue, in particular, at the comprehensive population genomic level. Herein we establish that substantial remodeling of the transcriptome of the human-specific pathogen Streptococcus pyogenes by horizontal gene flow and other evolutionary genetic changes is a central factor in precipitating and perpetuating epidemic disease. The data unambiguously show that the key outcome of these molecular events is evolution of a new, more virulent pathogenic genotype. Our findings provide new understanding of epidemic disease.

Negative-pressure wound therapy for deep sternal wound infections reduces the rate of surgical interventions for early re-infections

OBJECTIVES To evaluate the outcome of treatment for deep sternal wound infection (DSWI) in a nationwide patient cohort, before and after the introduction of negative-pressure wound therapy (NPWT). METHODS This was a population-based cohort of all patients treated for DSWI in Iceland out of 2446 open heart operations performed between 2000 and 2010. Length of hospital stay, survival and reoperations were compared in (i) 23 patients treated with open and/or closed irrigation before August 2005 (conventional treatment, CvT group) and in (ii) 20 patients treated after this time with NPWT as a first-line therapy (NPWT group). RESULTS The DSWI rate was 1.8% and did not change during the study period. Demographics were similar for both groups, except for peripheral arterial disease which was less common in the NPWT group. Coagulase-negative staphylococci were also more common (as the only pathogen identified) in the NPWT group (70% vs 30%, P = 0.01). The median length of hospital stay was 43 days in both groups and the sternum could be closed with delayed primary closure in all except 2 patients, one in each group. Eight patients in the CvT group required surgical revision for re-infections, including debridement and rewiring, when compared with 1 patient in the NPWT group (P = 0.02). Furthermore, 6 patients in the CvT group developed late chronic infections of the sternum requiring surgical revision, compared with one in the NPWT group (P = 0.10). The 30-day mortality was not significantly different between groups (4% vs 0%, P > 0.1) and the same was true for 1-year mortality (17% vs 0%, P = 0.11). CONCLUSIONS NPWT significantly reduces the risk of early re-infections in patients with DSWI. There was a lower rate of late chronic sternal infections and lower mortality in the NPWT group, but the difference was not statistically significant. We conclude that NPWT should be considered as a first-line treatment for most DSWIs.

Evolving epidemiology of invasive Haemophilus infections in the post‐vaccination era: results from a long‐term population‐based study

Historically, Haemophilus influenzae (Hi) serotype b (Hib) caused most invasive Haemophilus infections worldwide, mainly in children. In 1989 routine childhood vaccination against Hib was initiated in Iceland. We conducted a population-based study of all patients in the country with Haemophilus spp. isolated from sterile sites (n = 202), from 1983 to 2008. Epidemiology, clinical characteristics of the infections and serotypes of the isolates were compared during the pre-vaccination (1983-1989) and post-vaccination era (1990-2008). Following the vaccination, the overall incidence of Hib decreased from 6.4 to 0.3/100,000 per year (p <0.05) whereas the incidence did not change significantly for infections caused by Haemophilus sensu lato not serotype b, hereafter referred to as non-type b Hi (0.9 vs 1.2, respectively). The most frequent diagnosis prior to 1990 was meningitis caused by Hib, which was subsequently replaced by pneumonia and bacteraemia caused by non-type b Hi. Most commonly, non-type b Hi were non-typeable (NTHi; 40/59), followed by Hi serotype f (14/59) and Hi serotype a (3/59). Pregnancy was associated with a markedly increased susceptibility to invasive Haemophilus infections (RR 25.7; 95% CI 8.0-95.9, p <0.0001) compared with non-pregnant women. The case fatality rate for Hib was 2.4% but 14% for non-type b Hi, highest at the extremes of age. Hib vaccination gives young children excellent protection and decreases incidence in the elderly due to herd effect in the community. Replacement with other species or serotypes has not been noted. Pregnant women are an overlooked risk group.

The importance of strain variation in virulence of Candida dubliniensis and Candida albicans: results of a blinded histopathological study of invasive candidiasis

The pathogenic yeast Candida dubliniensis is increasingly reported as a cause of systemic fungal infections. We compared the virulence of 9 clinical bloodstream isolates of C. dubliniensis with 3 C. albicans isolates in a murine model of invasive candidiasis. Quantification of organisms and inflammatory changes in kidneys of infected animals were evaluated in a blinded, systematic manner. Average 7-day mortality among animals infected with C. dubliniensis was 21.0% (33/157 animals; range for strains: 0-57.1%); and with C. albicans 23.2%, (23/99 animals; range for strains: 6.7-85.0%) (p 0.65). Greater strain variation was noted within species than between the two species. Both species comprised strains of either high or low virulence, and six of the nine C. dubliniensis strains showed negligible virulence. Colony counts determined on samples from liver and kidneys did not differ between species. According to histopathological analysis, C. dubliniensis produced significantly lower levels of hyphae than C. albicans (p <0.001). Candida albicans caused a greater inflammatory response in kidneys (p <0.001) and was more commonly associated with granulomatous inflammation (p 0.003) and greater mononuclear infiltrate (p <0.001). According to multivariate analysis, increasing tissue burden of both hyphal forms (p 0.032) and yeasts (p 0.016) was independently associated with death, whereas higher levels of mononuclear cells were protective (p <0.001). The results suggest a great overlap between the virulence properties of C. dubliniensis and C. albicans. Both yeast and hyphal forms are independently associated with mortality, suggesting similar virulence for both. The source of the fungal isolates may be a neglected confounding factor in virulence studies in animal models.

S100B Profiles and Cognitive Function at High Altitude

Exposure to high altitude can lead to acute mountain sickness (AMS) and high altitude cerebral edema (HACE). In this study we investigated the effect of high altitude on neurocognitive function and S100B release. Increased S100B release has been hypothesized to signify a loss of integrity in the blood-brain barrier (BBB). Seven healthy volunteers trekked to Capanna Regina Margherita (4554 m above sea level) in the Monte Rosa massif. During ascent and descent, five test events were undertaken; participants underwent neurocognitive testing, Lake Louise scoring (LLS), and blood sampling to measure levels of S100B. The blood tests revealed that S100B levels increased 42% to 122% from baseline, and mean LLS increased from 0.57 to 2.57. A significant correlation was observed between both S100B levels and LLS and S100B and some neurocognitive scores. The study indicates that S100B can be released by a mild hypoxia during AMS. Moreover, an observed correlation between S100B and a lower score on neurocognitive tests suggests that the pathogenetic mechanisms may be linked. The study indicates that a decline in cognitive function is associated with symptoms of AMS.