High diversity in the regulatory region of Stx-converting bacteriophage genomes

Shiga toxin (Stx) is the major virulence factor of enterohemorrhagic Escherichia coli (EHEC), and the stx genes are carried by temperate bacteriophages (Stx phages). The switch between lysogenic and lytic life cycle of the phage, which is crucial for Stx production and for severity of the disease, is regulated by the CI repressor. CI maintain latency by preventing transcription of the replication proteins. Three EHEC phage replication units (Eru1-3) in addition to the classical lambdoid replication region have been described previously, and Stx phages carrying the Eru1 replication region were associated with highly virulent EHEC strains. In this study, we have classified the Eru replication region of 419 Stx phages. In addition to the lambdoid replication region and the three already described Erus, ten novel Erus (named Eru4 to Eru13) were detected. The lambdoid type, Eru1, Eru4 and Eru7 seem to be widely distributed in Western Europe. Notably, EHEC strains involved in severe outbreaks in England and Norway carry Stx phages with Eru1, Eru2, Eru5 and Eru7 replication regions. Phylogenetic analysis of CI repressors from Stx phages revealed eight major clades that largely separate according to Eru type. The classification of replication regions and CI proteins of Stx phages provides an important platform for further studies aimed to assess how characteristics of the replication region influence the regulation of phage life cycle and, consequently, the virulence potential of the host EHEC strain. IMPORTANCE: EHEC is an emerging health challenge worldwide and outbreaks caused by this pathogen tend to be more frequent and severe. Increased knowledge on how characteristics of the replication region influence the virulence of E. coli may be used for more precise identification of high-risk EHEC strains.

Replication Region Analysis Reveals Non-lambdoid Shiga Toxin Converting Bacteriophages

Shiga toxin is the major virulence factor of enterohemorrhagic Escherichia coli (EHEC), and the gene encoding it is carried within the genome of Shiga toxin-converting phages (Stx phages). Numerous Stx phages have been sequenced to gain a better understanding of their contribution to the virulence potential of EHEC. The Stx phages are classified into the lambdoid phage family based on similarities in lifestyle, gene arrangement, and nucleotide sequence to the lambda phages. This study explores the replication regions of non-lambdoid Stx phages that completely lack the O and P genes encoding the proteins involved in initiating replication in the lambdoid phage genome. Instead, they carry sequences encoding replication proteins that have not been described earlier, here referred to as eru genes (after EHEC phage replication unit genes). This study identified three different types of Eru-phages, where the Eru1-type is carried by the highly pathogenic EHEC strains that caused the Norwegian O103:H25 outbreak in 2006 and the O104:H4 strain that caused the large outbreak in Europe in 2011. We show that Eru1-phages exhibit a less stable lysogenic state than the classical lambdoid Stx phages. As production of phage particles is accompanied by production of Stx toxin, the Eru1-phage could be associated with a high-virulence phenotype of the host EHEC strain. This finding emphasizes the importance of classifying Stx phages according to their replication regions in addition to their Stx-type and could be used to develop a novel strategy to identify highly virulent EHEC strains for improved risk assessment and management.

A Possible Role of Insertion Sequence IS1216V in Dissemination of Multidrug-Resistant Elements MESPM1 and MES6272-2 between Enterococcus and ST59 Staphylococcus aureus

Sequence type 59 (ST59) is the dominant type of community-associated methicillin-resistant Staphylococcus aureus (MRSA) in Taiwan. Previously, we reported that ST59 MRSA harbors enterococcal IS1216V-mediated multidrug-resistant composite transposons MESPM1 or MES6272-2. The MES were found to have a mosaic structure, largely originating in enterococci and partly native to S. aureus. The current study aimed to track the origin of the MES and how they disseminated from enterococci to ST59 S. aureus. A total of 270 enterococcal isolates were analyzed, showing that two ST64 Enterococcus faecalis isolated in 1992 and 11 clonal complex 17 Enterococcus faecium harbored MESPM1-like and MES6272-2-like structures, respectively. Sequence analysis revealed that ST64 E. faecalis strain N48 acquired the MESPM1-like structure on the plasmid pEflis48. The pEflis48 harbored the enterococci-originated region (erythromycin, kanamycin, and streptomycin resistances) and the S.aureus-originated region (chloramphenicol resistance) of MESPM1 but was separated by the replication region of the plasmid. Homologous recombination between the two direct repeats of IS1216V resulted in excision of the replication region of the plasmid to regenerate MESPM1. The p4780-1 and pV19 of E. faecium carried MES6272-2-like structures with IS1216V, albeit with multiple insertions by other insertion sequences. The findings show that IS1216V plays important roles in bidirectional gene transfer of multidrug resistance between enterococci and S. aureus.

Discovery and comparative genomic analysis of elk circovirus (ElkCV), a novel circovirus species and the first reported from a cervid host

The complete genome sequence of a novel circovirus (elk circovirus (ElkCV) Banff/2019) was determined via high throughput sequencing of liver tissue from a euthanized Rocky Mountain elk (Cervus canadensis nelsoni) from Alberta, Canada. The genome is circular and 1,787 nucleotides long, with two major ORFs encoding predicted proteins. Comparative genomic analysis to 4,164 publicly available complete and near complete circovirus genomes showed that ElkCV shares approximately 65% pairwise genome-wide nucleotide identity with the most closely related circovirus species, porcine circoviruses (PCV) 1 and 2 and bat-associated circovirus (BatACV) 11. ElkCV features a stem-loop within the origin of replication region characteristic of circoviruses. However, it differs from those found in PCV1, PCV2 and BatACV11 since it has a longer stem and contains hexamer repeats that overlap the stem in opposing orientations. Interestingly, stem-loop structures of similar length featuring repeats in a similar position and orientation are also seen in some avian circoviruses. Based on the demarcation threshold established by the International Committee on Taxonomy of Viruses (ICTV) for members of Circoviridae (80% pairwise genome-wide nucleotide identity), ElkCV represents a novel species and is the first complete circovirus genome reported from a cervid host.

RocS drives chromosome segregation and nucleoid occlusion in Streptococcus pneumoniae

Segregation of replicated chromosomes in bacteria is poorly understood outside some prominent model strains and even less is known about how it is coordinated with other cellular processes. Here we report that RocS is crucial for chromosome segregation in the opportunistic human pathogen Streptococcus pneumoniae. RocS is membrane-bound and interacts both with DNA and the chromosome partitioning protein ParB to properly segregate the origin of replication region to new daughter cells. In addition, we show that RocS interacts with the tyrosine-autokinase CpsD required for polysaccharide capsule biogenesis, which is crucial for S. pneumoniae’s ability to prevent host immune detection. Altering the RocS-CpsD interaction drastically hinders chromosome partitioning and cell division. Altogether, this work reveals that RocS is the cornerstone of an atypical nucleoid occlusion system ensuring proper cell division in coordination with the biogenesis of a protective capsular layer.

Characterization of pKP-M1144, a Novel ColE1-Like Plasmid Encoding IMP-8, GES-5, and BEL-1 β-Lactamases, from a Klebsiella pneumoniae Sequence Type 252 Isolate

ABSTRACTIMP-8 metallo-β-lactamase was identified inKlebsiella pneumoniaesequence type 252 (ST252), isolated in a Portuguese hospital in 2009.blaIMP-8was the first gene cassette of a novel class 3 integron, In1144, also carrying theblaGES-5,blaBEL-1, andaacA4cassettes. In1144 was located on a ColE1-like plasmid, pKP-M1144 (12,029 bp), with a replication region of limited nucleotide similarity to those of other RNA-priming plasmids, such as pJHCMW1. In1144 and pKP-M1144 represent an interesting case of evolution of resistance determinants in Gram-negative bacteria.