Evaluation of within-host evolution of methicillin-resistant Staphylococcus aureus (MRSA) by comparing cgMLST and SNP analysis approaches.

Whole genome sequencing (WGS) of methicillin-resistant Staphylococcus aureus (MRSA) provides high-resolution typing, facilitating surveillance and outbreak investigations. The aim of this study was to evaluate the genomic variation rate in MRSA, by comparing commonly used core genome multilocus sequencing (cgMLST) against single nucleotide polymorphism (SNP) analyses. WGS was performed on 95 MRSA isolates, collected from 20 carriers during years 2003-2019. To assess variation and methodological-related differences, two different cgMLST schemes were obtained using Ridom SeqSphere+ and the cloud-based 1928 platform. In addition, two SNP methods, 1928 platform and Northern Arizona SNP Pipeline (NASP) were used. The cgMLST using Ridom SeqSphere+ and 1928 showed a median of 5.0 and 2.0 allele variants/year, respectively. In the SNP analysis, performed with two reference genomes COL and Newman, 1928 showed a median of 13 and 24 SNPs (including presumed recombination) and 3.8 respectively 4.0 SNPs (without recombination) per individual/year. Accordantly, NASP showed a median of 5.5 and 5.8 SNPs per individual/year. In conclusion, an estimated genomic variation rate of 2.0-5.8 genetic events per year (without recombination), is suggested as a general guideline to be used at clinical laboratories for surveillance and outbreak investigations independently of analysis approach used.

Genotypic Characterization of Clinical Klebsiella spp. Isolates Collected From Patients With Suspected Community-Onset Sepsis, Sweden.

Klebsiella is a genus of Gram-negative bacteria known to be opportunistic pathogens that may cause a variety of infections in humans. Highly drug-resistant Klebsiella species, especially K. pneumoniae, have emerged rapidly and are becoming a major concern in clinical management. Although K. pneumoniae is considered the most important pathogen within the genus, the true clinical significance of the other species is likely underrecognized due to the inability of conventional microbiological methods to distinguish between the species leading to high rates of misidentification. Bacterial whole-genome sequencing (WGS) enables precise species identification and characterization that other technologies do not allow. Herein, we have characterized the diversity and traits of Klebsiella spp. in community-onset infections by WGS of clinical isolates (n = 105) collected during a prospective sepsis study in Sweden. The sequencing revealed that 32 of the 82 isolates (39.0%) initially identified as K. pneumoniae with routine microbiological methods based on cultures followed by matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) had been misidentified. Of these, 23 were identified as Klebsiella variicola and nine as other members of the K. pneumoniae complex. Comparisons of the number of resistance genes showed that significantly fewer resistance genes were detected in Klebsiella oxytoca compared to K. pneumoniae and K. variicola (both values of p < 0.001). Moreover, a high proportion of the isolates within the K. pneumoniae complex were predicted to be genotypically multidrug-resistant (MDR; 79/84, 94.0%) in contrast to K. oxytoca (3/16, 18.8%) and Klebsiella michiganensis (0/4, 0.0%). All isolates predicted as genotypically MDR were found to harbor the combination of ß-lactam, fosfomycin, and quinolone resistance markers. Multi-locus sequence typing (MLST) revealed a high diversity of sequence types among the Klebsiella spp. with ST14 (10.0%) and ST5429 (10.0%) as the most prevalent ones for K. pneumoniae, ST146 for K. variicola (12.0%), and ST176 for K. oxytoca (25.0%). In conclusion, the results from this study highlight the importance of using high-resolution genotypic methods for identification and characterization of clinical Klebsiella spp. isolates. Our findings indicate that infections caused by other members of the K. pneumoniae complex than K. pneumoniae are a more common clinical problem than previously described, mainly due to high rates of misidentifications.

Epidemiological typing of ST80 vancomycin-resistant Enterococcus faecium: core genome multilocus sequence typing versus single nucleotide polymorphism-based typing.

OBJECTIVES: Vancomycin-resistant Enterococcus faecium (VRE) belonging to sequence type 80 (ST80) have been successfully disseminating in hospital settings. This study aimed to explore whole-genome sequencing (WGS)-based approaches in epidemiological typing of VRE by investigating a collection of ST80 strains. METHODS: In total, 41 VRE ST80 isolates in three suspected transmission chains were subjected to WGS. The genetic relationship of the isolates was analysed by core genome multilocus sequence typing (cgMLST) and single nucleotide polymorphism (SNP)-based typing. The published genome sequence of a predominant ST80 clone in Copenhagen was also included in the analyses. RESULTS: Group 1 isolates (n = 10) were recovered in the same geographic area, including two isolates from an outbreak of vanA genotype. Group 2 isolates (n = 4) were detected from another outbreak. Group 3 consisted of 3 non-outbreak isolates and 24 representative isolates from an outbreak of vanB genotype. Our findings indicated ≥24 allelic difference and >16 SNPs as the cut-off for excluding VRE strains from an outbreak. cgMLST identified >200 differing alleles between genomes of the Copenhagen clone and outbreak strains in this study. Our findings also demonstrated that SNP analysis was not suitable for typing samples from different groups, even with the same ST, owing to lack of an optimal reference genome. CONCLUSION: cgMLST and SNPs provided comparable epidemiological discrimination for isolates with a suspected transmission chain. For diverse isolates, SNP analysis could be suboptimal. An approach applying cgMLST as the first-line typing method and SNPs as a complementary tool is suggested.

Whole genome sequencing of Clostridioides difficile PCR ribotype 046 suggests transmission between pigs and humans.

BACKGROUND: A zoonotic association has been suggested for several PCR ribotypes (RTs) of Clostridioides difficile. In central parts of Sweden, RT046 was found dominant in neonatal pigs at the same time as a RT046 hospital C. difficile infection (CDI) outbreak occurred in the southern parts of the country. OBJECTIVE: To detect possible transmission of RT046 between pig farms and human CDI cases in Sweden and investigate the diversity of RT046 in the pig population using whole genome sequencing (WGS). METHODS: WGS was performed on 47 C. difficile isolates from pigs (n = 22), the farm environment (n = 7) and human cases of CDI (n = 18). Two different core genome multilocus sequencing typing (cgMLST) schemes were used together with a single nucleotide polymorphisms (SNP) analysis and the results were related to time and location of isolation of the isolates. RESULTS: The pig isolates were closely related (≤6 cgMLST alleles differing in both cgMLST schemes) and conserved over time and were clearly separated from isolates from the human hospital outbreak (≥76 and ≥90 cgMLST alleles differing in the two cgMLST schemes). However, two human isolates were closely related to the pig isolates, suggesting possible transmission. The SNP analysis was not more discriminate than cgMLST. CONCLUSION: No general pattern suggesting zoonotic transmission was apparent between pigs and humans, although contrasting results from two isolates still make transmission possible. Our results support the need for high resolution WGS typing when investigating hospital and environmental transmission of C. difficile.