Perceived utility and feasibility of pathogen genomics for public health practice: a survey among public health professionals working in the field of infectious diseases, Belgium, 2019.

BACKGROUND: Pathogen genomics is increasingly being translated from the research setting into the activities of public health professionals operating at different levels. This survey aims to appraise the literacy level and gather the opinions of public health experts and allied professionals working in the field of infectious diseases in Belgium concerning the implementation of next-generation sequencing (NGS) in public health practice. METHODS: In May 2019, Belgian public health and healthcare professionals were invited to complete an online survey containing eight main topics including background questions, general attitude towards pathogen genomics for public health practice and main concerns, genomic literacy, current and planned NGS activities, place of NGS in diagnostic microbiology pathways, data sharing obstacles, end-user requirements, and key drivers for the implementation of NGS. Descriptive statistics were used to report on the frequency distribution of multiple choice responses whereas thematic analysis was used to analyze free text responses. A multivariable logistic regression model was constructed to identify important predictors for a positive attitude towards the implementation of pathogen genomics in public health practice. RESULTS: 146 out of the 753 invited public health professionals completed the survey. 63% of respondents indicated that public health agencies should be using genomics to understand and control infectious diseases. Having a high level of expertise in the field of pathogen genomics was the strongest predictor of a positive attitude (OR = 4.04, 95% CI = 1.11 - 17.23). A significantly higher proportion of data providers indicated to have followed training in the field of pathogen genomics compared to data end-users (p < 0.001). Overall, 79% of participants expressed interest in receiving further training. Main concerns were related to the cost of sequencing technologies, data sharing, data integration, interdisciplinary working, and bioinformatics expertise. CONCLUSIONS: Belgian health professionals expressed favorable views about implementation of pathogen genomics in their work activities related to infectious disease surveillance and control. They expressed the need for suitable training initiatives to strengthen their competences in the field. Their perception of the utility and feasibility of pathogen genomics for public health purposes will be a key driver for its further implementation.

Widespread implementation of EUCAST breakpoints for antibacterial susceptibility testing in Europe.

The European Committee on Antimicrobial Susceptibility Testing (EUCAST) was established to harmonise clinical antimicrobial breakpoints and to define breakpoints for new agents in Europe. Data from the European Antimicrobial Resistance Surveillance Network (EARS-Net) external quality assessment (EQA) exercises from 2009 to 2012, from the United Kingdom External Quality Assessment Scheme (UK NEQAS) from November 2009 to March 2013 and data collected by EUCAST through a questionnaire in the first quarter of 2013 were analysed to investigate implementation of EUCAST guidelines in Europe. A rapid change to use of EUCAST breakpoints was observed over time. Figures for implementation of EUCAST breakpoints at the end of the studied period were 61.2% from EARSNet data and 73.2% from UK NEQAS data. Responses to the EUCAST questionnaire indicated that EUCAST breakpoints were used by over 50% of laboratories in 18 countries, by 10 to 50% of laboratories in eight countries and by less than 10% in seven countries. The EUCAST disk diffusion method was used by more than 50% of laboratories in 12 countries, by 10 to 50% of laboratories in ten countries and by less than 10% in eleven countries. EUCAST guidelines implementation is essential to ensure consistent clinical reporting of antimicrobial susceptibility results and antimicrobial resistance surveillance.

Laboratory capability and surveillance testing for Middle East respiratory syndrome coronavirus infection in the WHO European Region, June 2013.

Since September 2012, over 90 cases of respiratory disease caused by a novel coronavirus, now named Middle East respiratory syndrome coronavirus (MERSCoV), have been reported in the Middle East and Europe. To ascertain the capabilities and testing experience of national reference laboratories across the World Health Organization (WHO) European Region to detect this virus, the European Centre for Disease Prevention and Control (ECDC) and the WHO Regional Office for Europe conducted a joint survey in November 2012 and a follow-up survey in June 2013. In 2013, 29 of 52 responding WHO European Region countries and 24 of 31 countries of the European Union/European Economic Area (EU/EEA) had laboratory capabilities to detect and confirm MERS-CoV cases, compared with 22 of 46 and 18 of 30 countries, respectively, in 2012. By June 2013, more than 2,300 patients had been tested in 23 countries in the WHO European Region with nine laboratory-confirmed MERS-CoV cases. These data indicate that the Region has developed significant capability to detect this emerging virus in accordance with WHO and ECDC guidance. However, not all countries had developed capabilities, and the needs to do so should be addressed. This includes enhancing collaborations between countries to ensure diagnostic capabilities for surveillance of MERS-CoV infections across the European Region.

Systematic literature analysis and review of targeted preventive measures to limit healthcare-associated infections by meticillin-resistant Staphylococcus aureus.

Meticillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare-associated infections in Europe. Many examples have demonstrated that the spread of MRSA within healthcare settings can be reduced by targeted infection control measures. The aim of this systematic literature analysis and review was to summarise the evidence for the use of bacterial cultures for active surveillance the benefit of rapid screening tests, as well as the use of decolonisation therapies and different types of isolation measures. We included 83 studies published between 2000 and 2012. Although the studies reported good evidence supporting the role of active surveillance followed by decolonisation therapy, the effectiveness of single-room isolation was mostly shown in non-controlled studies, which should inspire further research regarding this issue. Overall, this review highlighted that when planning the implementation of preventive interventions, there is a need to consider the prevalence of MRSA, the incidence of infections, the competing effect of standard control measures (e.g. hand hygiene) and the likelihood of transmission in the respective settings of implementation.

Development and validation of potential structure indicators for evaluating antimicrobial stewardship programmes in European hospitals.

This study describes the development of structure indicators for hospital antimicrobial stewardship programmes and pilot validation across European hospitals. A multi-disciplinary panel from four European countries developed structure indicators in three steps: identification and listing of indicators, remote ranking of indicators using multi-criteria scoring, selection of indicators in a face-to-face consensus meeting. Additionally, the top-ten indicators were identified as a minimal set of key indicators. A survey was sent to the directors of antimicrobial stewardship programmes in European hospitals. The yes/no answers for the indicators were transformed into numbers in order to calculate the total scores. A list of 58 indicators was selected and categorised into the following topics: antimicrobial stewardship services (12 items), tools (16 items), human resources and mandate (6 items), health care personnel development (4 items), basic diagnostic capabilities (6 items), microbiological rapid tests (2 items), evaluation of microbiological drug resistance data (3 items), antibiotic consumption control (5 items) and drug use monitoring (4 items). The indicator scores, reported by 11 pilot hospitals from five European countries, ranged from 32 to 50 (maximum score = 58) and from 5 to 10 points (maximum score = 10) for, respectively, the complete and the top-ten list. An international panel selected 58 potential structure indicators, among which was a minimal set of ten key structure indicators, that could be useful for assessment of the comprehensiveness and resource-intensity of antimicrobial stewardship programmes. There was significant heterogeneity among participating centres with regard to their score for structural components of effective antimicrobial stewardship.

Community-acquired methicillin-resistant Staphylococcus aureus clones circulating in Belgium from 2005 to 2009: changing epidemiology.

The present study reports the evolution of the demographic characteristics and the molecular epidemiology of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) in Belgium from 2005 to 2009. Four hundred and ten CA-MRSA isolates were prospectively collected and screened for the presence of Panton-Valentin leucocidin (PVL) and toxic shock syndrome toxin 1 (TSST-1) encoding genes, while clinical information were recorded. PVL- and TSST-1-positive isolates were genotyped by pulsed-field gel electrophoresis (PFGE). Staphylococcal cassette chromosome mec (SCCmec) type, spa type and multilocus sequence type (MLST) were determined on representative isolates. One hundred and fifty-nine (39 %) isolates were PVL-positive. PVL-positive isolates were significantly more frequently isolated from skin or soft tissue than PVL-negative isolates, causing mainly subcutaneous abscesses and furuncles. Patients with PVL-positive CA-MRSA were significantly younger than patients with PVL-negative CA-MRSA. Eighty-seven percent of the PVL-positive isolates belonged to a limited number (n = 7) of PFGE types belonging to sequence types (ST) ST80, ST8, ST30, ST5, ST152, ST338 and a new ST, a single-locus variant of ST1. A temporal evolution of the distribution of these PFGE types was observed, characterised by (1) the dissemination of the ST8-SCCmecIV arcA-positive (USA300) genotype and (2) a genetic diversification. Forty-seven (11 %) strains were TSST-1-positive, of which 65 % clustered into four PFGE types, all belonging to ST5. The epidemiology of CA-MRSA in Belgium is changing, as the rapid diffusion of the USA300 clone seems to occur, together with a clonal diversification.

Laboratory capability for molecular detection and confirmation of novel coronavirus in Europe, November 2012.

A rapid survey by the European Centre for Disease Prevention and Control (ECDC) and the World Health Organization (WHO) Regional Office for Europe ascertained the availability of national reference laboratory testing for a recently detected novel coronavirus as of 28 November 2012. Screening by internal quality controlled upE-RT-PCR assay was available in 23/46 of responding countries in the WHO European Region, of which 19/30 in European Union (EU) and European Economic Area (EEA) countries. Confirmation of positive screened samples by either ORF1b - RT-PCR, or other target RT-PCR assays with sequence analysis or whole-genome sequence analysis was available in 22/46 responding countries of which 18/30 in EU/EEA countries.

Prospective multicentre feasibility study of a quality of care indicator for intravenous to oral switch therapy with highly bioavailable antibiotics.

BACKGROUND: Enhanced oral (po) bioavailability of antimicrobial drugs allows conversion to po therapy once a patient meets defined clinical criteria. This can reduce length of hospital stay, healthcare costs and risk of complications related to intravenous (iv) access. We developed a quality indicator for assessing the appropriate iv-to-po switch of bioavailable antibiotics and evaluated its feasibility and clinical relevance across acute healthcare systems. METHODS: The study was designed as a multicentre, multinational observational audit. The indicator was the proportion of inappropriate iv treatments at any point in time in adult patients treated with fluoroquinolones, clindamycin, linezolid or metronidazole. Treatments were prospectively evaluated by a trained physician or clinical pharmacist using predefined clinical criteria. The feasibility of the indicator was evaluated by measuring data availability, data collection workload and sensitivity to improvement RESULTS: Data were collected over a 3 month period in five university hospitals in Austria, Belgium and Germany and iv treatment was assessed in 211 patients. The indicator was measurable in 99.1% of cases. By intention-to-treat analysis, 37.0% (95% CI 30.5-43.9) of treatments were inappropriate, ranging from 17.5% to 53.8% across hospitals. The median time needed for case assessment and documentation was 29 min. CONCLUSIONS: This quality indicator was found to be generally feasible in hospitals across three European countries, and informative about the local need for clinical quality improvement.

Previous healthcare exposure is the main antecedent for methicillin-resistant Staphylococcus aureus carriage on hospital admission in Belgium.

This study aimed to estimate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) carriage upon hospital admission and to study the molecular epidemiology of MRSA in order to assess the proportion of Panton-Valentine leukocidin (PVL)-positive community-associated (CA) and livestock-associated (LA) MRSA strains. Epidemiological data on MRSA carriage upon hospital admission (2006-2009) were collected in a compulsory, continuous, national MRSA surveillance in Belgian acute-care hospitals. Additionally, 328 MRSA strains in 2005 and 314 strains in 2008 were collected in a separate, multicenter microbiological survey. Spa-typing, SCCmec-typing and MLST were performed; toxin genes were detected by PCR. The overall prevalence of MRSA carriage upon hospital admission was 8.9 cases/1,000 admissions between 2006 and 2009. Of MRSA carriers, 37.5% had a known MRSA history, 39.4% had stayed in a care facility, 12.2% reported no contact with healthcare. Over 90% of MRSA belonged to five healthcare-associated clones. Of these, MRSA spa-CC038-ST45-IV was in decline, mainly in favor of spa-CC008-ST8-IV. MRSA spa-CC002-ST5-IV, spa-CC002-ST5-II and spa-CC032-ST22-IV remained relatively stable. The proportion of PVL-positive CA-MRSA and LA-MRSA ST398 was below 2% of all MRSA. The extra-hospital MRSA reservoir in Belgium mainly consists of persons with previous healthcare exposure. PVL-positive CA-MRSA and LA-MRSA strains remained infrequent among hospitalized patients.

Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance.

Many different definitions for multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR) bacteria are being used in the medical literature to characterize the different patterns of resistance found in healthcare-associated, antimicrobial-resistant bacteria. A group of international experts came together through a joint initiative by the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC), to create a standardized international terminology with which to describe acquired resistance profiles in Staphylococcus aureus, Enterococcus spp., Enterobacteriaceae (other than Salmonella and Shigella), Pseudomonas aeruginosa and Acinetobacter spp., all bacteria often responsible for healthcare-associated infections and prone to multidrug resistance. Epidemiologically significant antimicrobial categories were constructed for each bacterium. Lists of antimicrobial categories proposed for antimicrobial susceptibility testing were created using documents and breakpoints from the Clinical Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the United States Food and Drug Administration (FDA). MDR was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories, XDR was defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e. bacterial isolates remain susceptible to only one or two categories) and PDR was defined as non-susceptibility to all agents in all antimicrobial categories. To ensure correct application of these definitions, bacterial isolates should be tested against all or nearly all of the antimicrobial agents within the antimicrobial categories and selective reporting and suppression of results should be avoided.

Positive predictive value of the Xpert MRSA assay diagnostic for universal patient screening at hospital admission: influence of the local ecology.

The purpose of this study was to assess the accuracy of the Xpert MRSA assay (XP) for the detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage upon hospital admission. Nasal swabs were prospectively collected for MRSA screening from 1,891 patients admitted to a teaching hospital. XP results were compared to chromogenic agar culture results. MRSA was cultured in 61 specimens (3%). Compared with culture, XP had a sensitivity, specificity, positive, and negative predictive value of 60.7, 97.3, 37.8, and 98.9%, respectively. The median turnaround time (TAT) for the results was 3 h. Of 24 MRSA isolated from XP-negative samples, three harbored composite SCCmec. Among 61 samples with culture-negative but XP-positive results, 15 methicillin-susceptible S. aureus (MSSA) isolates tested positive by XP on pure colony lysates. These MSSA included: (i) strains with SCCmec deletion encompassing mecA and (ii) multilocus sequence typing (MLST) clonal complex (CC) 1 strains harboring a chromosomal sequence homologous to one of the orfX-SCCmec junction sequences targeted by XP. On account of the low sensitivity and positive predictive value in a hospital patient population with moderate prevalence of MRSA, culture still appears to be necessary in order to confirm polymerase chain reaction (PCR) results. The emergence of new SCCmec variants and the presence of MSSA harboring cross-reactive SCCmec-like elements may challenge the successful implementation of such detection systems.

Diversity of accessory genome of human and livestock-associated ST398 methicillin resistant Staphylococcus aureus strains.

BACKGROUND: Molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) has documented the diversity of the genetic background of strains associated with healthcare (HA-MRSA), community (CA-MRSA) and livestock (LA-MRSA). The accessory and core-variable genomes of those strains however remain largely unknown. OBJECTIVE: To compare the genetic background and accessory and core variable gene content of ST398 LA-MRSA strains with those of HA-and CA-MRSA strains from the same region. METHODS: Representative strains of HA- (n=21), CA- (n=13) and ST398 LA-MRSA (n=18) were selected from Belgian National Reference Laboratory collections. The accessory and core-variable genomes of these strains were characterized by a DNA-microarray composed of oligonucleotide probes targeting ~400 resistance, adhesion and virulence associated genes. RESULTS: ST398 strains displayed very homogenous hybridization profiles irrespective of their host origin. This ST398 genomic profile was moderately related to that of certain human HA- or CA-lineages but distinctively lacked several virulence- and colonization-associated genes implicated in carriage in humans, such as proteases and adhesins. No enterotoxin gene was found among ST398 strains. Differences were observed in the mobile resistance gene content of ST398 strains, including antibiotic resistance determinants. CONCLUSION: LA-MRSA strains represent a homogenous lineage distinct from co-local HA- and CA-MRSA strains, especially in its accessory genome content characterized by a lack of human-associated virulence and adhesion determinants. The absence of detectable enterotoxin gene among ST398 LA-MRSA strains from a wide host range is reassuring regarding their foodborne pathogenic potential.

New Delhi metallo-beta-lactamase 1-producing Enterobacteriaceae: emergence and response in Europe.

Acquired carbapenemases confer extensive antibiotic resistance to Enterobacteriaceae and represent a public health threat. A novel acquired carbapenemase, New Delhi metallo-beta-lactamase 1 (NDM-1), has recently been described in the United Kingdom and Sweden, mostly in patients who had received care on the Indian subcontinent. We conducted a survey among 29 European countries (the European Union Member States, Iceland and Norway) to gather information on the spread of NDM-1-producing Enterobacteriaceae in Europe, on public health responses and on available national guidance on detection, surveillance and control. A total of 77 cases were reported from 13 countries from 2008 to 2010. Klebsiella pneumoniae was the most frequently reported species with 54%. Among 55 cases with recorded travel history, 31 had previously travelled or been admitted to a hospital in India or Pakistan and five had been hospitalised in the Balkan region. Possible nosocomial acquisition accounted for 13 of 77 cases. National guidance on NDM-1 detection was available in 14 countries and on NDM-1 control in 11 countries. In conclusion, NDM-1 is spreading across Europe, where it is frequently linked to a history of healthcare abroad, but also to emerging nosocomial transmission. National guidance in response to the threat of carbapenemase-producing Enterobacteriaceae is available in approximately half of the surveyed European countries. Surveillance of carbapenemase- producing Enterobacteriaceae must be enhanced in Europe and effective control measures identified and implemented.

Methicillin-resistant Staphylococcus aureus (MRSA): burden of disease and control challenges in Europe.

Methicillin-resistant Staphylococcus aureus (MRSA) isa major cause of healthcare- and community-associated infections worldwide. Within the healthcare setting alone, MRSA infections are estimated to affect more than 150,000 patients annually in the European Union (EU), resulting in attributable extra in-hospital costs of EUR 380 million for EU healthcare systems. Pan-European surveillance data on bloodstream infections show marked variability among EU Member States in the proportion of S. aureus that are methicillin-resistant, ranging from less than 1% to more than 50%. In the past five years, the MRSA bacteraemia rates have decreased significantly in 10 EU countries with higher endemic rates of MRSA infections. In addition to healthcare-associated infections, new MRSA strains have recently emerged as community and livestock-associated human pathogens in most EU Member States. The prevention and control of MRSA have therefore been identified as public health priorities in the EU. In this review, we describe the current burden of MRSA infections in healthcare and community settings across Europe and outline the main threats caused by recent changes in the epidemiology of MRSA. Thereby, we aim at identifying unmet needs of surveillance, prevention and control of MRSA in Europe.

Evaluation of the Xpert MRSA assay for rapid detection of methicillin-resistant Staphylococcus aureus from nares swabs of geriatric hospitalized patients and failure to detect a specific SCCmec type IV variant.

Rapid and reliable detection of methicillin-resistant Staphylococcus aureus (MRSA) carriers is crucial for control of MRSA nosocomial transmission. We aimed to evaluate the performance of the GeneXpert real-time PCR system using the Xpert MRSA assay on a collection of 40 representative Belgian MRSA strains and for MRSA screening of geriatric inpatients. Double nasal swabs were used: the first swab for the Xpert MRSA assay and the second for culture onto chromogenic selective medium and enrichment broth. All but 1 of the 40 collection strains were recognized as MRSA by the Xpert MRSA assay. Nares swabs were prospectively collected from 246 inpatients including 25 nasal MRSA carriers. Compared with enriched cultures, the sensitivity, the specificity, and the positive and negative predictive values of the Xpert MRSA assay were 69.2%, 97.7%, 78.3%, and 96.3% respectively. The 7 evaluable false-negative results according to the assay were due to its possible lack of sensitivity (n = 3) and to the occurrence of a Belgian MRSA clone carrying a particular staphylococcal chromosomal cassette mec (SCCmec) type IV variant (n = 4) not targeted by the current Xpert MRSA assay. Because of the evolution of SCCmec in MRSA, new primers should be designed and further studies are warranted to ensure continuous monitoring of this assay.

Molecular characterization of AmpC-producing Escherichia coli clinical isolates recovered at two Belgian hospitals.

OBJECTIVE: To characterize the genetic determinants associated with an AmpC phenotype in clinical Escherichia coli isolates. METHODS: E. coli strains recovered at two Belgian hospitals between 2004 and 2006 were selected on the basis of an AmpC-producing phenotype. Plasmid-mediated cephalosporinases coding genes and the sequence of chromosomal ampC genes were identified by PCR/sequencing. The isolates were submitted to phylotyping and genotyping analysis using rep-PCR (Diversilab) and PFGE. A novel chromosomal ampC gene was cloned. RESULTS: Eighty-three out of 6850 E. coli isolates were selected. Seventy-two isolates were found to overexpress their chromosomal cephalosporinases while 11 contained plasmid-mediated cephalosporinases. Among chromosomal AmpC overproducers, 12 were extended-spectrum AmpC (ESAC) expressing isolates which all displayed reduced susceptibility to cefepime. Cloning of a new ESAC allele suggested that L293P mutation was responsible of the extension of the hydrolysis spectrum to cefepime. AmpC overproducers, including ESAC producers, predominantly belonged to phylogenetic group A and B1, while plasmid-mediated AmpC-producing isolates preferentially belong to phylogroup B2 and D. According to rep-PCR, the majority of the E. coli isolates belonging to phylogroup A were clonally related which was further confirmed by PFGE for the 11 ESAC expressing isolates. CONCLUSIONS: Chromosomal AmpC overproduction was the most common resistance mechanism, and the occurrence of ESAC was found to be as frequent as plasmid-mediated cephalosporinases. The detection of a new ESAC allele, of an ESAC producing strain belonging to phylogroup D and the existence of a clonal relationship between ESAC producing strains underline the need for study of the clinical relevance of this mechanism of resistance.

First case of bacteremia caused by Moellerella wisconsensis: case report and a review of the literature.

Moellerella wisconsensis, a member of the Enterobacteriaceae family, is rarely isolated in clinical specimens. We report here a case of M. wisconsensis infection in a 46-year-old cirrhotic patient with acute cholecystitis. This is the first reported case of a M. wisconsensis infection in Belgium and the first reported case of human bacteremia caused by this bacterium. Our case report is followed by a review of the literature.

StaphVar-DNA microarray analysis of accessory genome elements of community-acquired methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Approximately 75% of the genome of Staphylococcus aureus (the 'core' genome) is highly conserved between strains, whereas the remaining 25% (the 'accessory' genome) is composed of mobile genetic elements (MGEs), containing virulence and resistance genes. We developed a composite microarray focused on resistance and virulence genes located on the accessory or core-variable genome to characterize a collection of Belgian community-acquired methicillin-resistant S. aureus (CA-MRSA) strains. METHODS: Oligonucleotide probes targeting 403 genes encoding antimicrobial resistance (35%), virulence (28%) and adhesion (31%) factors were designed among eight S. aureus sequenced genomes. The StaphVar Array was validated by testing five of the strains used for the design and utilized to characterize 13 CA-MRSA strains representative of the multilocus sequence typing (MLST) sequence types circulating in Belgium. RESULTS: Analysis of the gene content of the five reference strains by the StaphVar Array matched 90% to 97% of the theoretical results. Analysis of CA-MRSA strains showed that 54.4% of the genes tested were strain-dependent. Strains presented specific exotoxin, enterotoxin, cytolysin and adhesin gene profiles by MLST lineage. One exception to these 'lineage-specific' profiles was the variable presence of the arginine catabolic mobile element (characteristic of the USA300 clone) within ST8 strains. CONCLUSIONS: The StaphVar Array enables the characterization of approximately 400 variable resistance and virulence determinants in S. aureus. CA-MRSA strains displayed extensive diversity in virulence and resistance profiles. The presence of the USA300 clone in Belgium was confirmed. Although mainly located on MGEs, associations of virulence genes were highly conserved within strains of the same MLST lineage.