Broad-range amplification and sequencing of the rpoB gene: a novel assay for bacterial identification in clinical microbiology.

The rpoB gene has been proposed as a promising phylogenetic marker for bacterial identification, providing theoretically improved species-level resolution compared to the 16S rRNA gene for a range of clinically important taxa. However, its utility in diagnostic microbiology has been limited by the lack of broad-range primers allowing for its amplification from most species with a single PCR assay. Here, we present an assay for broad-range partial amplification and Sanger sequencing of the rpoB gene. To reduce cross-reactivity and allow for rpoB amplification directly from patient samples, primers were based on the dual priming oligonucleotide principle. The resulting amplicon is ~550 base pairs in length and appropriate for species-level identification. Systematic in silico evaluation of a wide selection of taxa demonstrated improved resolution within multiple important genera, including Enterococcus, Fusobacterium, Mycobacterium, Streptococcus, and Staphylococcus species and several genera within the Enterobacteriaceae family. Broad-range rpoB amplification and Sanger sequencing of 115 bacterial isolates provided unambiguous species-level identification for 97 (84%) isolates, as compared to 57 (50%) using a clinical 16S rRNA gene assay. Several unresolved taxonomic matters disguised by the low resolution of the 16S rRNA gene were revealed using the rpoB gene. Using a collection of 33 clinical specimens harboring bacteria and assumed to contain high concentrations of human DNA, the rpoB assay identified the pathogen in 29 specimens (88%). Broad-range rpoB amplification and sequencing provides a promising tool for bacterial identification, improving discrimination between closely related species and making it amenable for use in culture-based and culture-independent diagnostic approaches.

Genomic epidemiology of Streptococcus dysgalactiae subsp. equisimilis strains causing invasive disease in Norway during 2018.

Background: Streptococcus dysgalactiae subspecies equisimilis (SDSE) is an emerging global pathogen, yet the epidemiology and population genetics of SDSE species have not been extensively characterized. Methods: We carried out whole genome sequencing to characterize 274 SDSE isolates causing bloodstream infections obtained through national surveillance program in 2018. We conducted multilocus sequence typing (MLST), emm-typing, core genome phylogeny, as well as investigated key features associated with virulence. Moreover, comparison to SDSE from other geographic regions were performed in order to gain more insight in the evolutionary dynamics in SDSE. Results: The phylogenetic analysis indicated a substantial diversity of emm-types and sequence types (STs). Briefly, 17 emm-types and 58 STs were identified that formed 10 clonal complexes (CCs). The predominant ST-types were ST20 (20%), ST17 (17%), and ST29 (11%). While CC17 and CC29 clades showed a substantial heterogeneity with well-separated emm-associated subclades, the CC20 clade harboring the stG62647 emm-type was more homogenous and the most prevalent in the present study. Moreover, we observed notable differences in the distribution of clades within Norway, as well as several disseminated CCs and also distinct geographic variations when compared to data from other countries. We also revealed extensive intra-species recombination events involving surface exposed virulence factors, including the emm gene important for phylogenetic profiling. Conclusion: Recombination events involving the emm as well as other virulence genes in SDSE, are important mechanisms in shaping the genetic variability in the SDSE population, potentially offering selective advantages to certain lineages. The enhanced phylogenetic resolution offered by whole genome sequencing is necessary to identify and delimitate outbreaks, monitor and properly characterize emerging strains, as well as elucidate bacterial population dynamics.

Streptococcus dysgalactiae Bloodstream Infections, Norway, 1999-2021.

Streptococcus dysgalactiae increasingly is recognized as a pathogen of concern for human health. However, longitudinal surveillance data describing temporal trends of S. dysgalactiae are scarce. We retrospectively identified all ß-hemolytic streptococcal bloodstream infections reported in Bergen, in western Norway, during 1999-2021. To explore S. dysgalactiae disease burden in a broader context, we mapped the incidence of all microbial species causing bloodstream infections during 2012-2021. We found S. dysgalactiae incidence rates substantially increased during the study period; by 2021, S. dysgalactiae was the fifth most common pathogen causing bloodstream infections in our region. We noted genotypic shifts and found that the rising trend was related in part to the introduction and expansion of the stG62647 emm-type. S. dysgalactiae is among the most common causes of bloodstream infections in western Norway, and increased surveillance and unambiguous species identification are needed to monitor the disease burden attributable to this pathogen.

Escherichia marmotae-a Human Pathogen Easily Misidentified as Escherichia coli.

We hereby present the first descriptions of human-invasive infections caused by Escherichia marmotae, a recently described species that encompasses the former "Escherichia cryptic clade V." We describe four cases, one acute sepsis of unknown origin, one postoperative sepsis after cholecystectomy, one spondylodiscitis, and one upper urinary tract infection. Cases were identified through unsystematic queries in a single clinical lab over 6 months. Through genome sequencing of the causative strains combined with available genomes from elsewhere, we demonstrate Es. marmotae to be a likely ubiquitous species containing genotypic virulence traits associated with Escherichia pathogenicity. The invasive isolates were scattered among isolates from a range of nonhuman sources in the phylogenetic analyses, thus indicating inherent virulence in multiple lineages. Pan genome analyses indicate that Es. marmotae has a large accessory genome and is likely to obtain ecologically advantageous traits, such as genes encoding antimicrobial resistance. Reliable identification might be possible by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), but relevant spectra are missing in commercial databases. It can be identified through 16S rRNA gene sequencing. Escherichia marmotae could represent a relatively common human pathogen, and improved diagnostics will provide a better understanding of its clinical importance. IMPORTANCE Escherichia coli is the most common pathogen found in blood cultures and urine and among the most important pathogenic species in the realm of human health. The notion that some of these isolates are not Es. coli but rather another species within the same genus may have implications for what Es. coli constitutes. We only recently have obtained methods to separate the two species, which means that possible differences in important clinical aspects, such as antimicrobial resistance rates, virulence, and phylogenetic structure, may exist. We believe that Es. marmotae as a common pathogen is new merely because we have not looked or bothered to distinguish between the thousands of invasive Escherichia passing through microbiological laboratories each day.

Alternative vanHAX promoters and increased vanA-plasmid copy number resurrect silenced glycopeptide resistance in Enterococcus faecium.

BACKGROUND: Vancomycin variable enterococci (VVE) are van-positive isolates with a susceptible phenotype that can convert to a resistant phenotype during vancomycin selection. OBJECTIVES: To describe a vancomycin-susceptible vanA-PCR positive ST203 VVE Enterococcus faecium isolate (VVESwe-S) from a liver transplantation patient in Sweden which reverted to resistant (VVESwe-R) during in vitro vancomycin exposure. METHODS: WGS analysis revealed the genetic differences between the isolates. Expression of the van-operon was investigated by qPCR. Fitness and stability of the revertant were investigated by growth measurements, competition and serial transfer. RESULTS: The VVESwe-R isolate gained high-level vancomycin (MIC >256 mg/L) and teicoplanin resistance (MIC = 8 mg/L). VVESwe-S has a 5'-truncated vanR activator sequence and the VVESwe-R has in addition acquired a 44 bp deletion upstream of vanHAX in a region containing alternative putative constitutive promoters. In VVESwe-R the vanHAX-operon is constitutively expressed at a level comparable to the non-induced prototype E. faecium BM4147 strain. The vanHAX operon of VVESwe is located on an Inc18-like plasmid, which has a 3-4-fold higher copy number in VVESwe-R compared with VVESwe-S. Resistance has a low fitness cost and the vancomycin MIC of VVESwe-R decreased during in vitro serial culture without selection. The reduction in MIC was associated with a decreased vanA-plasmid copy number. CONCLUSIONS: Our data support a mechanism by which vancomycin-susceptible VVE strains may revert to a resistant phenotype through the use of an alternative, constitutive, vanR-activator-independent promoter and a vanA-plasmid copy number increase.

Role of Horizontal Gene Transfer in the Development of Multidrug Resistance in Haemophilus influenzae.

Haemophilus influenzae colonizes the respiratory tract in humans and causes both invasive and noninvasive infections. Resistance to extended-spectrum cephalosporins in H. influenzae is rare in Europe. In this study, we defined acquired resistance gene loci and ftsI mutations in multidrug-resistant (MDR) and/or PBP3-mediated beta-lactam-resistant (rPBP3) H. influenzae strains, intending to understand the mode of spread of antibiotic resistance determinants in this species. Horizontal transfer of mobile genetic elements and transformation with resistance-conferring ftsI alleles were contributory. We found one small plasmid and three novel integrative conjugative elements (ICEs) which carry different combinations of resistance genes. Demonstration of transfer and/or ICE circular forms showed that the ICEs are functional. Two extensively MDR genetically unrelated H. influenzae strains (F and G) from the same geographical region shared an identical novel MDR ICE (Tn6686) harboring bla TEM-1, catA2-like, and tet(B). The first Nordic case of MDR H. influenzae septicemia, strain 0, originating from the same geographical area as these strains, had a similar resistance pattern but contained another ICE [Tn6687 with bla TEM-1, catP and tet(B)] with an overall structure quite similar to that of Tn6686. Comparison of the complete ftsI genes among rPBP3 strains revealed that the entire gene or certain regions of it are identical in genetically unrelated strains, indicating horizontal gene transfer. Our findings illustrate that H. influenzae is capable of acquiring resistance against a wide range of commonly used antibiotics through horizontal gene transfer, in terms of conjugative transfer of ICEs and transformation of chromosomal genes.IMPORTANCE Haemophilus influenzae colonizes the respiratory tract in humans and causes both invasive and noninvasive infections. As a threat to treatment, resistance against critically important antibiotics is on the rise in H. influenzae Identifying mechanisms for horizontal acquisition of resistance genes is important to understand how multidrug resistance develops. The present study explores the antimicrobial resistance genes and their context in beta-lactam-resistant H. influenzae with coresistance to up to four non-beta-lactam groups. The results reveal that this organism is capable of acquiring resistance to a wide range of commonly used antibiotics through conjugative transfer of mobile genetic elements and transformation of chromosomal genes, resulting in mosaic genes with a broader resistance spectrum. Strains with chromosomally mediated resistance to extended-spectrum cephalosporins, co-trimoxazole, and quinolones combined with mobile genetic elements carrying genes mediating resistance to ampicillin, tetracyclines, and chloramphenicol have been reported, and further dissemination of such strains represents a particular concern.

Spread of Plasmid-Encoded NDM-1 and GES-5 Carbapenemases among Extensively Drug-Resistant and Pandrug-Resistant Clinical Enterobacteriaceae in Durban, South Africa.

Whole-genome sequence analyses revealed the presence of blaNDM-1 (n = 31), blaGES-5 (n = 8), blaOXA-232 (n = 1), or blaNDM-5 (n = 1) in extensively drug-resistant and pandrug-resistant Enterobacteriaceae organisms isolated from in-patients in 10 private hospitals (2012 to 2013) in Durban, South Africa. Two novel NDM-1-encoding plasmids from Klebsiella pneumoniae were circularized by PacBio sequencing. In p19-10_01 [IncFIB(K); 223.434 bp], blaNDM-1 was part of a Tn1548-like structure (16.276 bp) delineated by IS26 The multireplicon plasmid p18-43_01 [IncR_1/IncFIB(pB171)/IncFII(Yp); 212.326 bp] shared an 80-kb region with p19-10_01, not including the blaNDM-1-containing region. The two plasmids were used as references for tracing NDM-1-encoding plasmids in the other genome assemblies. The p19-10_01 sequence was detected in K. pneumoniae (n = 7) only, whereas p18-43_01 was tracked to K. pneumoniae (n = 4), Klebsiella michiganensis (n = 1), Serratia marcescens (n = 11), Enterobacter spp. (n = 7), and Citrobacter freundii (n = 1), revealing horizontal spread of this blaNDM-1-bearing plasmid structure. Global phylogeny showed clustering of the K. pneumoniae (18/20) isolates together with closely related carbapenemase-negative ST101 isolates from other geographical origins. The South African isolates were divided into three phylogenetic subbranches, where each group had distinct resistance and replicon profiles, carrying either p19-10_01, p18-10_01, or pCHE-A1 (8,201 bp). The latter plasmid carried blaGES-5 and aacA4 within an integron mobilization unit. Our findings imply independent plasmid acquisition followed by local dissemination. Additionally, we detected blaOXA-232 carried by pPKPN4 in K. pneumoniae (ST14) and blaNDM-5 contained by a pNDM-MGR194-like genetic structure in Escherichia coli (ST167), adding even more complexity to the multilayer molecular mechanisms behind nosocomial spread of carbapenem-resistant Enterobacteriaceae in Durban, South Africa.

A Silenced vanA Gene Cluster on a Transferable Plasmid Caused an Outbreak of Vancomycin-Variable Enterococci.

We report an outbreak of vancomycin-variable vanA(+) enterococci (VVE) able to escape phenotypic detection by current guidelines and demonstrate the molecular mechanisms for in vivo switching into vancomycin resistance and horizontal spread of the vanA cluster. Forty-eight vanA(+) Enterococcus faecium isolates and one Enterococcus faecalis isolate were analyzed for clonality with pulsed-field gel electrophoresis (PFGE), and their vanA gene cluster compositions were assessed by PCR and whole-genome sequencing of six isolates. The susceptible VVE strains were cultivated in brain heart infusion broth containing vancomycin at 8 µg/ml for in vitro development of resistant VVE. The transcription profiles of susceptible VVE and their resistant revertants were assessed using quantitative reverse transcription-PCR. Plasmid content was analyzed with S1 nuclease PFGE and hybridizations. Conjugative transfer of vanA was assessed by filter mating. The only genetic difference between the vanA clusters of susceptible and resistant VVE was an ISL3-family element upstream of vanHAX, which silenced vanHAX gene transcription in susceptible VVE. Furthermore, the VVE had an insertion of IS1542 between orf2 and vanR that attenuated the expression of vanHAX Growth of susceptible VVE occurred after 24 to 72 h of exposure to vancomycin due to excision of the ISL3-family element. The vanA gene cluster was located on a transferable broad-host-range plasmid also detected in outbreak isolates with different pulsotypes, including one E. faecalis isolate. Horizontally transferable silenced vanA able to escape detection and revert into resistance during vancomycin therapy represents a new challenge in the clinic. Genotypic testing of invasive vancomycin-susceptible enterococci by vanA-PCR is advised.

A multicentre hospital outbreak in Sweden caused by introduction of a vanB2 transposon into a stably maintained pRUM-plasmid in an Enterococcus faecium ST192 clone.

The clonal dissemination of VanB-type vancomycin-resistant Enterococcus faecium (VREfm) strains in three Swedish hospitals between 2007 and 2011 prompted further analysis to reveal the possible origin and molecular characteristics of the outbreak strain. A representative subset of VREfm isolates (n = 18) and vancomycin-susceptible E. faecium (VSEfm, n = 2) reflecting the spread in time and location was approached by an array of methods including: selective whole genome sequencing (WGS; n = 3), multi locus sequence typing (MLST), antimicrobial susceptibility testing, virulence gene profiling, identification of mobile genetic elements conferring glycopeptide resistance and their ability to support glycopeptide resistance transfer. In addition, a single VREfm strain with an unrelated PFGE pattern collected prior to the outbreak was examined by WGS. MLST revealed a predominance of ST192, belonging to a hospital adapted high-risk lineage harbouring several known virulence determinants (n≥10). The VREfm outbreak strain was resistant to ampicillin, gentamicin, ciprofloxacin and vancomycin, and susceptible to teicoplanin. Consistently, a vanB2-subtype as part of Tn1549/Tn5382 with a unique genetic signature was identified in the VREfm outbreak strains. Moreover, Southern blot hybridisation analyses of PFGE separated S1 nuclease-restricted total DNAs and filter mating experiments showed that vanB2-Tn1549/Tn5382 was located in a 70-kb sized rep17/pRUM plasmid readily transferable between E. faecium. This plasmid contained an axe-txe toxin-antitoxin module associated with stable maintenance. The two clonally related VSEfm harboured a 40 kb rep17/pRUM plasmid absent of the 30 kb vanB2-Tn1549/Tn5382 gene complex. Otherwise, these two isolates were similar to the VREfm outbreak strain in virulence- and resistance profile. In conclusion, our observations support that the origin of the multicentre outbreak was caused by an introduction of vanB2-Tn1549/Tn5382 into a rep17/pRUM plasmid harboured in a pre-existing high-risk E. faecium ST192 clone. The subsequent dissemination of VREfm to other centres was primarily caused by clonal spread rather than plasmid transfer to pre-existing high-risk clones.