Evolution and Population Dynamics of Clonal Complex 152 Community-Associated Methicillin-Resistant Staphylococcus aureus

Since the late 1990s, changes in the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) were recognized with the emergence of community-associated MRSA (CA-MRSA). CA-MRSA belonging to clonal complex 152 (CC152), carrying the small staphylococcal cassette chromosome mec (SCCmec) type V and encoding the Panton-Valentine leukocidin (PVL), has been observed in Europe. The aim of this study was to investigate its origin, evolution, and dissemination. Whole-genome sequencing was performed on a global collection of 149 CC152 isolates spanning 20 years (93 methicillin-susceptible S. aureus [MSSA] and 56 MRSA isolates). Core genome phylogeny, Bayesian inference, in silico resistance analyses, and genomic characterization were applied. Phylogenetic analysis revealed two major distinct clades, one dominated by MSSA and the other populated only by MRSA. The MSSA isolates were predominately from sub-Saharan Africa, whereas MRSA was almost exclusively from Europe. The European MRSA isolates all harbored an SCCmec type V (5C2&5) element, whereas other SCCmec elements were sporadically detected in MRSA from the otherwise MSSA-dominated clade, including SCCmec types IV (2B), V (5C2), and XIII (9A). In total, 93% of the studied CC152 isolates were PVL positive. Bayesian coalescent inference suggests an emergence of the European CC152-MRSA in the 1990s, while the CC152 lineage dates back to the 1970s. The CA-MRSA CC152 clone mimics the European CC80 CA-MRSA lineage by its emergence from a PVL-positive MSSA ancestor from North Africa or Europe. The CC152 lineage has acquired SCCmec several times, but acquisition of SCCmec type V (5C2&5) seems associated with expansion of MRSA CC152 in Europe. IMPORTANCE Understanding the evolution of CA-MRSA is important in light of the increasing importance of this reservoir in the dissemination of MRSA. Here, we highlight the story of the CA-MRSA CC152 lineage using whole-genome sequencing on an international collection of CC152. We show that the evolution of this lineage is novel and that antibiotic usage may have the potential to select for the phage-encoded Panton-Valentine leukocidin. The diversity of the strains correlated highly to geography, with higher level of resistance observed among the European MRSA isolates. The mobility of the SCCmec element is mandatory for the emergence of novel MRSA lineages, and we show here distinct acquisitions, one of which is linked to the successful clone found throughout Europe today

Infection control of Staphylococcus aureus : spa typing to elucidate transmission

Staphylococcus aureus is a commensal of the human flora, primarily colonizing the anterior nares and throat, but it may also cause infections ranging from mild skin and soft tissue infections to severe diseases such as endocarditis and septicemia. S. aureus is also a major nosocomial problem increasing with the worldwide dissemination of methicillin-resistant S. aureus (MRSA). The main vector for bacterial cross-transmission in healthcare settings is the hands of healthcare workers (HCWs). No S. aureus was detected in the air in this thesis demonstrating that transmission through air is not important. Despite the fact that good compliance with hand hygiene is essential to prevent cross-transmission the compliance is generally less than 50 %. Gold standard to track bacterial transmission in healthcare settings has for long been pulsed-field gel electrophoresis (PFGE), a method that is labor- intensive, lacks consensus protocol and relies on semi-subjective analysis. Molecular typing by sequencing of the hypervariable part of the S. aureus protein A gene (spa typing) has overcome these problems and has shown promising results in epidemiological investigations. The aims of this thesis were to study bacterial transmission with S. aureus colonization of newborn infants as a model and to evaluate spa typing as a molecular tool. Additionally, the influence of compliance with hygiene guidelines on S. aureus transmission was assessed. Analysis of 280 MRSA isolates by spa typing revealed excellent typeability and epidemiological concordance and satisfactory discriminatory power. Additionally, spa typing was considered superior to PFGE thanks to its accessibility, ease of use and rapidity. Also, spa typing results are registered in a global database, facilitating inter-laboratory comparison. The prevalence of S. aureus ranged from 41 % to 66 % in the populations studied and males had the highest colonization rate. Throat was the premier colonization site for adults and transmission from individuals colonized in the throat only was documented, suggesting that throat cultures should be included in S. aureus screening programs. The umbilicus was the premier colonization site for newborn infants. Incubating the swabs in enrichment broth prior to plating increased the prevalence of S. aureus positive samples by 46 %, resulting in prevalence ranging from 51 % to 70 % in the populations studied. Thus enrichment prior to plating is necessary to determine more truthful S. aureus colonization rates. There were no indications of an institutional flora, as the colonization rates, spa type distribution and antibiotic resistance prevalence were similar among parents and HCWs. Direct observations and self-reporting by HCWs were both validated as tools for monitoring compliance with hygiene guidelines. The compliance with hygiene guidelines was significantly higher following a 10-point hygiene intervention as compared to baseline. The compliance was also higher three years after the intervention in three of four participating departments. These data show that it is possible to markedly improve the compliance with hygiene guidelines, but to achieve a long-term effect, continuous and varied reminders seems necessary. Both at baseline and following the intervention almost 60 % of the colonized infants were colonized with an S. aureus of the same spa type as isolated from their own family. At baseline approximately 25 % of the colonized infants received their S. aureus from non-family individuals, indicating transmission directly or indirectly from HCWs. Despite the improvement in compliance with barrier precautions from 41 % at baseline to 86 % following the hygiene intervention, the transmission from non-family did not decrease. This indicates that other factors may have a prominent impact on bacterial transmission. One factor might be the quality of hand hygiene technique which therefore needs to be studied further. However, to ensure patient safety it is still recommended that all HCWs comply with hygiene guidelines at all time

Characterization of a Clostridioides difficile outbreak caused by PCR ribotype 046, associated with increased mortality

This study describes a large nosocomial outbreak of Clostridioides difficile infections (CDI) dominated by ribotype (RT) 046 in a Swedish hospital. The present study aimed to examine the pathogenicity of this RT, explore epidemiological links by whole genome sequencing (WGS), and evaluate different interventions implemented to stop the outbreak. Clinical isolates (n = 366) collected during and after the outbreak were ribotyped and 246 isolates were subjected to WGS. Medical records of patients infected with the seven most common RTs were evaluated. RT046 was spread effectively throughout the hospital and was the most common among the 44 different RTs found (114/366 isolates). Infection with RT046 was associated with higher mortality compared to other strains (20.2% to 7.8%), although there were no differences in concomitant disease, age or antibiotic treatment. To control the outbreak, several measures were successfully implemented.Funding: Academy for Health and Care, Region Jonkoping County, Sweden</p

Molecular epidemiology of community- and hospital-associated Clostridioides difficile infections in Jönköping, Sweden, October 2017-March 2018

Clostridioides difficile infections (CDIs) in Sweden are mostly hospital-associated (HA) with limited knowledge regarding community-associated (CA) infections. Here, we investigated the molecular epidemiology of clinical isolates of CA-CDI and HA-CDI in a Swedish county. Data and isolates (n = 156) of CDI patients (n = 122) from Jonkoping county, October 2017-March 2018, were collected and classified as CA (without previous hospital care or onset &amp;lt;= 2 days after admission or &amp;gt;12 weeks after discharge from hospital) or HA (onset &amp;gt;3 days after hospital admission or within 4 weeks after discharge). Molecular characterization of isolates included PCR ribotyping (n = 156 isolates) and whole genome sequencing with single nucleotide polymorphisms (SNP) analysis (n = 53 isolates). We classified 47 patients (39%) as CA-CDI and 75 (61%) as HA-CDI. Between CA-CDI and HA-CDI patients, we observed no statistically significant differences regarding gender, age, 30-day mortality or recurrence. Ribotype 005 (RR 3.1; 95% CI: 1.79-5.24) and 020 (RR 2.5; 95% CI: 1.31-4.63) were significantly associated with CA-CDI. SNP analysis identified seven clusters (0-2 SNP difference) involving 17/53 isolates of both CA-CDI and HA-CDI. Molecular epidemiology differed between CA-CDI and HA-CDI and WGS analysis suggests transmission of CDI within and between hospitals and communities

Analyzing Multiclonality of Staphylococcus aureus in Clinical Diagnostics Using spa-Based Denaturing Gradient Gel Electrophoresis

We present a novel denaturing gradient gel electrophoresis (DGGE) method which characterizes multiclonal communities of Staphylococcus aureus. The spa PCR-based DGGE method simultaneously separates strains that differ in only one base, thereby revealing multiclonal colonization and infections.Funding Agencies|Swedish Society of Medicine||Futurum||Research Council of South-East Sweden (FORSS)|

Genome-wide association study of hemolytic uremic syndrome causing Shiga toxin-producing Escherichia coli from Sweden, 1994-2018

Shiga toxin-producing Escherichia coli (STEC) infection can cause clinical manifestations ranging from diarrhea to potentially fatal hemolytic uremic syndrome (HUS). This study is aimed at identifying STEC genetic factors associated with the development of HUS in Sweden. A total of 238 STEC genomes from STEC-infected patients with and without HUS between 1994 and 2018 in Sweden were included in this study. Serotypes, Shiga toxin gene (stx) subtypes, and virulence genes were characterized in correlation to clinical symptoms (HUS and non-HUS), and pan-genome wide association study was performed. Sixty-five strains belonged to O157:H7, and 173 belonged to non-O157 serotypes. Our study revealed that strains of O157:H7 serotype especially clade 8 were most commonly found in patients with HUS in Sweden. stx2a and stx2a + stx2c subtypes were significantly associated with HUS. Other virulence factors associated with HUS mainly included intimin (eae) and its receptor (tir), adhesion factors, toxins, and secretion system proteins. Pangenome wide-association study identified numbers of accessory genes significantly overrepresented in HUS-STEC strains, including genes encoding outer membrane proteins, transcriptional regulators, phage-related proteins, and numerous genes related to hypothetical proteins. Whole-genome phylogeny and multiple correspondence analysis of pangenomes could not differentiate HUS-STEC from non-HUS-STEC strains. In O157:H7 cluster, strains from HUS patients clustered closely; however, no significant difference in virulence genes was found in O157 strains from patients with and without HUS. These results suggest that STEC strains from different phylogenetic backgrounds may independently acquire genes determining their pathogenicity and confirm that other non-bacterial factors and/or bacteria-host interaction may affect STEC pathogenesis

Genomic Insights Into Clinical Shiga Toxin-Producing Escherichia coli Strains: A 15-Year Period Survey in Jonkoping, Sweden

Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens that can cause human infections ranging from asymptomatic carriage to bloody diarrhea (BD) and fatal hemolytic uremic syndrome (HUS). However, the molecular mechanism of STEC pathogenesis is not entirely known. Here, we demonstrated a large scale of molecular epidemiology and in-depth genomic study of clinical STEC isolates utilizing clinical and epidemiological data collected in Region Jonkoping County, Sweden, over a 15-year period. Out of 184 STEC isolates recovered from distinct patients, 55 were from patients with BD, and 129 were from individuals with non-bloody stools (NBS). Five individuals developed HUS. Adults were more associated with BD. Serotypes O157:H7, O26:H11, O103:H2, O121:H19, and O104:H4 were more often associated with BD. The presence of Shiga toxin-encoding gene subtypes stx(2a), stx(2a) + stx(2c), and stx(1a) + stx(2c) was associated with BD, while stx(1)(a) was associated with milder disease. Multiplex virulence and accessory genes were correlated with BD; these genes encode toxins, adhesion, autotransporters, invasion, and secretion system. A number of antimicrobial resistance (AMR) genes, such as aminoglycoside, aminocoumarin, macrolide, and fluoroquinolone resistance genes, were prevalent among clinical STEC isolates. Whole-genome phylogeny revealed that O157 and non-O157 STEC isolates evolved from distinct lineages with a few exceptions. Isolates from BD showed more tendency to cluster closely. In conclusion, this study unravels molecular trait of clinical STEC strains and identifies genetic factors associated with severe clinical outcomes, which could contribute to management of STEC infections and disease progression if confirmed by further functional validation.Funding Agencies|Scandinavian Society for Antimicrobial Chemotherapy Foundation [SLS884041]; National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [81701977]</p

Table_1_Shiga Toxin-Producing Escherichia coli Infection in Jönköping County, Sweden: Occurrence and Molecular Characteristics in Correlation With Clinical Symptoms and Duration of stx Shedding.DOCX

<p>Shiga toxin–producing Escherichia coli (STEC) cause bloody diarrhea (BD), hemorrhagic colitis (HC), and even hemolytic uremic syndrome (HUS). In Nordic countries, STEC are widely spread and usually associated with gastrointestinal symptoms and HUS. The objective of this study was to investigate the occurrence of STEC in Swedish patients over 10 years of age from 2003 through 2015, and to analyze the correlation of critical STEC virulence factors with clinical symptoms and duration of stx shedding. Diarrheal stool samples were screened for presence of stx by real-time PCR. All STEC isolates were characterized by DNA microarray assay and PCR to determine serogenotypes, stx subtypes, and presence of intimin gene eae and enterohaemolysin gene ehxA. Multilocus sequencing typing (MLST) was used to assess phylogenetic relationships. Clinical features were collected and analyzed using data from the routine infection control measures in the county. A total of 14,550 samples were enrolled in this 12-years period study, and 175 (1.2%) stools were stx positive by real-time PCR. The overall incidence of STEC infection was 4.9 cases per 100,000 person-years during the project period. Seventy-five isolates, with one isolate per sample were recovered, among which 43 were from non-bloody stools, 32 from BD, and 3 out of the 75 STEC positive patients developed HUS. The presence of stx2 in both stools and isolates were associated with BD (p = 0.008, p = 0.05), and the presence of eae in isolates was related to BD (p = 0.008). The predominant serogenotypes associated with BD were O157:H7, O26:H11, O121:H19, and O103:H2. Isolates from HUS were O104:H4 and O98: H21 serotypes. Phylogenetic analysis revealed our strains were highly diverse, and showed close relatedness to HUS-associated STEC collection strains. In conclusion, the presence of stx2 in stool was related to BD already at the initial diagnostic procedure, thus could be used as risk predictor at an early stage. STEC isolates with stx2 and eae were significantly associated with BD. The predominant serotypes associated with BD were O157:H7, O26:H11, O121:H19, and O103:H2. Nevertheless, the pathogenic potential of other serotypes and genotypes should not be neglected.</p