Genotypic diversity and transmission of livestock-associated MRSA

Infections with the human pathogen Staphylococcus aureus were considered to belong to the past with the introduction of antibiotics. However, S. aureus quickly adapted and became resistant against a variety of these drugs, resulting in the emergence of methicillin-resistant S. aureus (MRSA). In 2005, a new MRSA variant was found in pigs and shown to be transmitted to pig farmers. This MRSA variant turned out to be present worldwide and carriage was found in many other livestock animals and designated as livestock-associated MRSA (LA-MRSA). In the Netherlands, the proportion of LA-MRSA isolated from humans rapidly increased and currently around 25% of the Dutch MRSA surveillance isolates are LA-MRSA. Molecular characterization of LA-MRSA has been a challenge since regular typing methods cannot differentiate LA-MRSA strains, hampering studies on transmissions and origin of LA-MRSA. In this thesis, we describe the development and application of new molecular methods to characterize LA-MRSA strains and study their role in human infections. The results showed that LA-MRSA are genetically diverse and that there are three independently evolving genetic groups of LA-MRSA in the Netherlands. Utilizing the new technologies, we could confirm human-to-human transmission of LA-MRSA in Dutch healthcare facilities and showed that prolonged persistence of LA-MRSA in humans occurs. Furthermore, we showed that the number of infections caused by LA-MRSA increases and that there is an increased number people that are colonized or infected with LA-MRSA without having contact with livestock, suggesting LA-MRSA is changing resulting in transmission between humans rather than between animal and human

Swarming behaviour, catchment area and seasonal movement patterns of the Bechstein's bats : implications for conservation

Bats display marked seasonality throughout the temperate zone and use different habitats during different parts of the year. Unfortunately, detailed information regarding seasonal distribution and movements is often lacking, thereby hampering the development of adequate conservation measures. In this study we used radio telemetry to track females of the endangered Bechstein’s bat (Myotis bechsteinii) from autumn swarming sites to their summer maternity colony ranges. We were able to tag 22 individuals, 18 of which were subsequently recovered at nine roost sites up to 20.6 km away. Females from multiple colonies visited the same swarming site on a single night. Concurrently, we recovered females from a single maternity colony at different swarming sites on the same night. The catchment area of the investigated swarming sites measured 27.1 km2, and was skewed to the northwest. Tagged bats were recovered in forest fragments ranging in size from 5.42 to 128.98 ha. Notably, all but one of the recovered roosts were found in forests that have been continuously wooded since at least 1775. Surveys during the summer at these sites confirmed the presence of maternity colonies at six out of seven locations that could be investigated. Our study contributes to our understanding of swarming behavior and seasonal movement patterns, and exemplifies how these can be used to complete the year-round habitat use of bat species

External validation of WGS-based antimicrobial susceptibility prediction tools, KOVER-AMR and ResFinder 4.1, for Escherichia coli clinical isolates

Objective: To externally validate whole genome sequence-antimicrobial susceptibility testing phenotype prediction tools KOVER-AMR and ResFinder 4.1 for Escherichia coli clinical isolates from Dutch routine care. Methods: A random sample of 234 E. coli and 283 third generation cephalosporin-resistant E. coli isolates from urine and blood were collected (2014–2017). Culture-antimicrobial susceptibility testing was performed using VITEK 2 and BD Phoenix. Sequences were used as input for KOVER-AMR-SCM, KOVER-AMR-CART, and ResFinder 4.1. The concordance, major error rate (MER), and very major error rate (VMER) were calculated, with subsequent comparison to U.S. Food and Drug Administration (FDA) criteria (MER ≤3% and VMER with a 95% confidence interval ≤1.5–≤7.5%). Results: ResFinder 4.1 performed better than KOVER-AMR-models; however, neither tool achieved overall (V)MERs below FDA criteria. KOVER-AMR-SCM, KOVER-AMR-CART, and ResFinder 4.1, MER (cumulative all antimicrobials) were: 5.1% (4.4–5.9), 4.3% (3.6–5.0), and 5.1% (4.5–5.8), respectively. MERs ≤3% were achieved for 6 (SCM) and 5 (CART) of the 11 tested antimicrobials for KOVER-AMR-models and for 9/13 antimicrobials tested with ResFinder 4.1. KOVER-AMR-SCM, KOVER-AMR-CART, and ResFinder 4.1 cumulative VMERs were: 26% (24–28), 29% (27–31), and 11% (9.2–12). VMERs with a 95% CI ≤ 1.5–≤7.5 were only achieved for 4/13 tested antimicrobials with ResFinder 4.1. Discussion: In this study, whole genome sequence-antimicrobial susceptibility testing phenotype prediction tools KOVER-AMR and ResFinder 4.1 did not meet the FDA criteria needed for clinical diagnostic use in 517 E. coli clinical isolates from Dutch routine care. The tested tools should be further improved before they can be used for clinical decision making

PFGE diversity within the methicillin-resistant Staphylococcus aureus clonal lineage ST398

<p>Abstract</p> <p>Background</p> <p>Livestock has recently been identified as a new reservoir of methicillin-resistant <it>Staphylococcus aureus </it>(MRSA). Most isolates belong to ST398 and are non-typeable with PFGE using <it>Sma</it>I, making it difficult to study transmission and outbreaks. Therefore, a new PFGE using <it>Cfr</it>9I, a neoschizomer of <it>Sma</it>I was optimized and evaluated to investigate ST398 isolates.</p> <p>Results</p> <p>After optimizing and evaluating the <it>Cfr</it>9I PFGE, clear and reproducible banding patterns were obtained from all previously non-typeable MRSA (NT_{<it>Sma</it>I}-MRSA) isolates. The PFGE patterns of ST398 isolates showed more diversity than with <it>spa</it>-typing and/or MLST. The PFGE results showed diversity within and between the two most prevalent <it>spa</it>-types of NT_{<it>Sma</it>I}-MRSA (t011 and t108). No match was found, when comparing banding patterns of the NT_{<it>Sma</it>I}-MRSA with 700 different PFGE types, obtained with <it>Sma</it>I digestion, in our database of more than 4000 strains. Furthermore, possible transmission among veterinarians and their family members was investigated and an outbreak of ST398 MRSA in a residential care facility was confirmed with the <it>Cfr</it>9I PFGE.</p> <p>Conclusions</p> <p>The adjusted PFGE can be used as a method for selecting important and distinct ST398 isolates for further research. The adjustments in the PFGE protocol using <it>Cfr</it>9I are easy to implement to study the ST398 clonal lineage in laboratories which already have a PFGE facility.</p

Molecular characterization of MRSA collected during national surveillance between 2008 and 2019 in the Netherlands

BACKGROUND: Although the Netherlands is a country with a low endemic level, methicillin-resistant Staphylococcus aureus (MRSA) poses a significant health care problem. Therefore, high coverage national MRSA surveillance has been in place since 1989. To monitor possible changes in the type-distribution and emergence of resistance and virulence, MRSA isolates are molecularly characterized.METHODS: All 43,321 isolates from 36,520 persons, collected 2008-2019, were typed by multiple-locus variable number tandem repeats analysis (MLVA) with simultaneous PCR detection of the mecA, mecC and lukF-PV genes, indicative for PVL. Next-generation sequencing data of 4991 isolates from 4798 persons were used for whole genome multi-locus sequence typing (wgMLST) and identification of resistance and virulence genes.RESULTS: We show temporal change in the molecular characteristics of the MRSA population with the proportion of PVL-positive isolates increasing from 15% in 2008-2010 to 25% in 2017-2019. In livestock-associated MRSA obtained from humans, PVL-positivity increases to 6% in 2017-2019 with isolates predominantly from regions with few pig farms. wgMLST reveals the presence of 35 genogroups with distinct resistance, virulence gene profiles and specimen origin. Typing shows prolonged persistent MRSA carriage with a mean carriage period of 407 days. There is a clear spatial and a weak temporal relationship between isolates that clustered in wgMLST, indicative for regional spread of MRSA strains.CONCLUSIONS: Using molecular characterization, this exceptionally large study shows genomic changes in the MRSA population at the national level. It reveals waxing and waning of types and genogroups and an increasing proportion of PVL-positive MRSA.</p

Left ventricular strain-volume loops in bicuspid aortic valve disease:new insights in cardiomechanics

AIMS: By combining temporal changes in left ventricular (LV) global longitudinal strain (GLS) with LV volume, LV strain-volume loops can assess cardiac function across the cardiac cycle. This study compared LV strain-volume loops between bicuspid aortic valve (BAV) patients and controls, and investigated the loop’s prognostic value for clinical events.METHODS AND RESULTS: From a prospective cohort of congenital heart disease patients, BAV patients were selected and compared with healthy volunteers, who were matched for age and sex at group level. GLS7 analysis from apical views were used to construct strain-volume loops. Associations with clinical events, i.e. a composite of all-cause mortality, heart failure, arrhythmias and aortic valve replacement, were assessed by Cox regression. 113 BAV patients were included (median age 32 years, 40% female). BAV patients demonstrated lower Sslope (0.21%/mL, [Q1-Q3: 0.17-0.28] vs. 0.27%/mL [0.24-0.34], p&lt;0.001) and ESslope (0.19%/mL [0.12-0.25] vs. 0.29%/mL [0.21-0.43], p&lt;0.001) compared to controls, but also greater uncoupling during early (0.48±1.29 vs. 0.06±1.2, p=0.018) and late diastole (0.66±1.01 vs -0.06±1.09, p&lt;0.001). Median follow-up duration was 9.9 [9.3-10.4] years. Peak aortic jet velocity (HR 1.22, p=0.03), enlarged left atrium (HR 3.16, p=0.003), E/e’ ratio (HR 1.17, p=0.002), GLS (HR 1.16, p=0.008) and ESslope (HR 0.66, p=0.04) were associated with the occurrence of clinical events.CONCLUSION: Greater uncoupling and lower systolic and diastolic slopes were observed in BAV patients compared to healthy controls, suggesting presence of altered LV cardiomechanics. Moreover, lower ESslope was associated with clinical events, highlighting the strain-volume loop’s potential as prognostic marker