Diversification of plasmids in a genus of pathogenic and nitrogen-fixing bacteria

Members of the agrobacteria–rhizobia complex (ARC) have multiple and diverse plasmids. The extent to which these plasmids are shared and the consequences of their interactions are not well understood. We extracted over 4000 plasmid sequences from 1251 genome sequences and constructed a network to reveal interactions that have shaped the evolutionary histories of oncogenic virulence plasmids. One newly discovered type of oncogenic plasmid is a mosaic with three incomplete, but complementary and partially redundant virulence loci. Some types of oncogenic plasmids recombined with accessory plasmids or acquired large regions not known to be associated with pathogenicity. We also identified two classes of partial virulence plasmids. One class is potentially capable of transforming plants, but not inciting disease symptoms. Another class is inferred to be incomplete and non-functional but can be found as coresidents of the same strain and together are predicted to confer pathogenicity. The modularity and capacity for some plasmids to be transmitted broadly allow them to diversify, convergently evolve adaptive plasmids and shape the evolution of genomes across much of the ARC. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

Reoccurring Bovine Anthrax in Germany on the Same Pasture after 12 Years

The zoonotic disease anthrax caused by the endospore-forming bacterium Bacillus anthracis is very rare in Germany. In the state of Bavaria, the last case occurred in July of 2009 resulting in four dead cows. In August of 2021, the disease reemerged after heavy rains, killing one gestating cow. Notably, both outbreaks affected the same pasture, suggesting a close epidemiological connection. B. anthracis could be grown from blood culture and the presence of both virulence plasmids (pXO1 and pXO2) were confirmed by PCR. Also, recently developed diagnostic tools enabled rapid detection of B. anthracis cells and nucleic acids directly in clinical samples. The complete genome of the strain isolated from blood, designated BF-5, was DNA-sequenced and phylogenetically grouped within the B.Br.CNEVA clade that is typical for European B. anthracis strains. The genome was almost identical to BF-1, the isolate of 2009, separated only by three single nucleotide polymorphisms on the chromosome, one on plasmid pXO2 and three indel-regions. Further, B. anthracis DNA was detected by PCR from soil-samples taken from spots, where the cow had fallen onto the pasture. New tools based on phage receptor binding proteins enabled the microscopic detection and isolation of B. anthracis directly from soil-samples. These environmental isolates were genotyped and found to be SNP-identical to BF-1. Therefore, it seems that the BF-5 genotype is currently the prevalent one at the affected premises. The contaminated area was subsequently disinfected with formaldehyde.

Long-Read Sequencing Reveals Evolution and Acquisition of Antimicrobial Resistance and Virulence Genes in Salmonella enterica

Salmonella enterica is a significant and phylogenetically diverse zoonotic pathogen. To understand its genomic heterogeneity and antimicrobial resistance, we performed long-read sequencing on Salmonella isolated from retail meats and food animals. A collection of 134 multidrug-resistant isolates belonging to 33 serotypes were subjected to PacBio sequencing. One major locus of diversity among these isolates was the presence and orientation of Salmonella pathogenic islands (SPI), which varied across different serotypes but were largely conserved within individual serotypes. We also identified insertion of an IncQ resistance plasmid into the chromosome of fourteen strains of serotype I 4,[5],12:i:– and the Salmonella genomic island 1 (SGI-1) in five serotypes. The presence of various SPIs, SGI-1 and integrated plasmids contributed significantly to the genomic variability and resulted in chromosomal resistance in 55.2% (74/134) of the study isolates. A total of 93.3% (125/134) of isolates carried at least one plasmid, with isolates carrying up to seven plasmids. We closed 233 plasmid sequences of thirteen replicon types, along with twelve hybrid plasmids. Some associations between Salmonella isolate source, serotype, and plasmid type were seen. For instance, IncX plasmids were more common in serotype Kentucky from retail chicken. Plasmids IncC and IncHI had on average more than five antimicrobial resistance genes, whereas in IncX, it was less than one per plasmid. Overall, 60% of multidrug resistance (MDR) strains that carried >3 AMR genes also carried >3 heavy metal resistance genes, raising the possibility of co-selection of antimicrobial resistance in the presence of heavy metals. We also found nine isolates representing four serotypes that carried virulence plasmids with the spv operon. Together, these data demonstrate the power of long-read sequencing to reveal genomic arrangements and integrated plasmids with a high level of resolution for tracking and comparing resistant strains from different sources. Additionally, the findings from this study will help expand the reference set of closed Salmonella genomes that can be used to improve genome assembly from short-read data commonly used in One Health antimicrobial resistance surveillance.

Acquisition of the Conjugative Virulence Plasmid From a CG23 Hypervirulent Klebsiella pneumoniae Strain Enhances Bacterial Virulence

The emergence of hypervirulent and carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has become a hot topic and confounding problem for clinicians and researchers alike. Conjugative virulence plasmids have the potential to cause more threatening dissemination of hv-CRKP strains. We previously identified K2606, a CG23 clinical hypervirulent strain of Klebsiella pneumoniae harboring a conjugative virulence plasmid designated pK2606. In this study we examined hypervirulence levels using assays of biofilm formation, serum resistance, and wax larvae and mouse in vivo infection models. Moreover, to define the transfer ability of pK2606 and whether this confers hypervirulence to other strains we performed plasmid transconjugation experiments between K2606 and the ST11 CRKP strain HS11286 along with E. coli J53. We found that although biofilm formation and serum resistance were not significantly increased, the transconjugants acquired the ability of produce high level of siderophores and also caused high mortality of wax larvae and mice. Furthermore, we identified pK2606-like conjugative virulence plasmids in GenBank, providing evidence that such plasmids may have begun to spread throughout China. These findings provide an evidence base for the possible mechanisms of the emergence of hv-CRKP strains and highlight the potential of pK2606-like conjugative virulence plasmids to spread worldwide.

Infection of Cronobacter sakazakii ST1 Producing SHV-12 in a Premature Infant Born from Triplet Pregnancy

Cronobacter sakazakii can cause severe life-threatening invasive infections in neonates, with a high mortality rate mostly associated with powdered infant formula consumption. The study describes a fatal C. sakazakii infection in premature infant fed only with expressed human milk. Despite the identification of etiological factor from patient’s blood, the epidemiological investigation, including mother’s skin, hospital surfaces, milk expressing devices, and milk samples, did not show bacterial contamination. The infection was caused by C. sakazakii ST1, being one of the leading genotypes reported in invasive infections. The phylogenetic analysis of the international collection of the ST1 organisms allowed us to identify the isolate as a member of the main cluster. The pathogenic potential of the isolate was augmented by the presence of IncFIB-like molecule representing virulence plasmids of pESA-3 family. Isolate presented ESBL phenotype associated with blaSHV-12 gene harboured by IncX3 plasmid. The described case gave valuable information to genetics of Cronobacter, and also urges the need of wider whole-genome sequencing implementation as a part of diagnostic procedure.

Mobilization of the nonconjugative virulence plasmid from hypervirulent Klebsiella pneumoniae

Background Klebsiella pneumoniae, as a global priority pathogen, is well known for its capability of acquiring mobile genetic elements that carry resistance and/or virulence genes. Its virulence plasmid, previously deemed nonconjugative and restricted within hypervirulent K. pneumoniae (hvKP), has disseminated into classic K. pneumoniae (cKP), particularly carbapenem-resistant K. pneumoniae (CRKP), which poses alarming challenges to public health. However, the mechanism underlying its transfer from hvKP to CRKP is unclear. Methods A total of 28 sequence type (ST) 11 bloodstream infection-causing CRKP strains were collected from Ruijin Hospital in Shanghai, China, and used as recipients in conjugation assays. Transconjugants obtained from conjugation assays were confirmed by XbaI and S1 nuclease pulsed-field gel electrophoresis, PCR detection and/or whole-genome sequencing. The plasmid stability of the transconjugants was evaluated by serial culture. Genetically modified strains and constructed mimic virulence plasmids were employed to investigate the mechanisms underlying mobilization. The level of extracellular polysaccharides was measured by mucoviscosity assays and uronic acid quantification. An in silico analysis of 2608 plasmids derived from 814 completely sequenced K. pneumoniae strains available in GenBank was performed to investigate the distribution of putative helper plasmids and mobilizable virulence plasmids. Results A nonconjugative virulence plasmid was mobilized by the conjugative plasmid belonging to incompatibility group F (IncF) from the hvKP strain into ST11 CRKP strains under low extracellular polysaccharide-producing conditions or by employing intermediate E. coli strains. The virulence plasmid was mobilized via four modes: transfer alone, cotransfer with the conjugative IncF plasmid, hybrid plasmid formation due to two rounds of single-strand exchanges at specific 28-bp fusion sites or homologous recombination. According to the in silico analysis, 31.8% (242) of the putative helper plasmids and 98.8% (84/85) of the virulence plasmids carry the 28-bp fusion site. All virulence plasmids carry the origin of the transfer site. Conclusions The nonconjugative virulence plasmid in ST11 CRKP strains is putatively mobilized from hvKP or E. coli intermediates with the help of conjugative IncF plasmids. Our findings emphasize the importance of raising public awareness of the rapid dissemination of virulence plasmids and the consistent emergence of hypervirulent carbapenem-resistant K. pneumoniae (hv-CRKP) strains.

Emergence of Hybrid Resistance and Virulence Plasmids Harboring New Delhi Metallo-β-Lactamase in Klebsiella pneumoniae in Russia

The emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) is a new threat to healthcare. In this study, we analyzed nine CR-hvKp isolates of different sequence-types (ST) recovered from patients with nosocomial infections in two hospitals in Saint Petersburg. Whole-genome sequencing showed that eight of them harbored large mosaic plasmids carrying resistance to carbapenems and hypervirulence simultaneously, and four different types of hybrid plasmids were identified. BLAST analysis showed a high identity with two hybrid plasmids originating in the UK and Czech Republic. We demonstrated that hybrid plasmids emerged due to the acquisition of resistance genes by virulent plasmids. Moreover, one of the hybrid plasmids carried a novel New Delhi metallo-beta-lactamase (NDM) variant, differing from NDM-1 by one amino acid substitution (D130N), which did not provide significant evolutionary advantages compared to NDM-1. The discovery of structurally similar plasmids in geographically distant regions suggests that the actual distribution of hybrid plasmids carrying virulence and resistance genes is much wider than expected.

Identification of a novel hybrid plasmid encoding KPC-2 and virulence factors in Klebsiella pneumoniae sequence type 11

Recent emergence of carbapenem-resistant K. pneumoniae (CRKP) co-harbouring blaKPC-2 and pLVPK-like virulence plasmids represented a novel clinical challenge. In the present study, we characterized a blaKPC-2 and virulence hybrid plasmid, designated as pCRHV-C2244, from a clinical ST11-K64 CRKP strain. pCRHV-C2244 was non-self-transmissible due to the incomplete conjugative elements, but mobilizable together with a conjugative helper. Enhanced virulence and stable maintenance without significant fitness loss in its original host were confirmed in vitro and in vivo.

Virulence Plasmids of Rhodococcus equi Isolates From Cuban Patients With AIDS

Rhodococcus equi is an animal pathogen and zoonotic human opportunistic pathogen associated with immunosuppressive conditions. The pathogenicity of R. equi is linked to three animal host-associated virulence plasmids encoding a family of “Virulence Associated Proteins” (VAPs). Here, the PCR-based TRAVAP molecular typing system for the R. equi virulence plasmids was applied to 26 R. equi strains isolated between 2010 and 2016 at the Institute of Tropical Medicine “Pedro Kourí,” Cuba, from individuals living with HIV/AIDS. TRAVAP detects 4 gene markers, traA common to the three virulence plasmids, and vapA, vapB, and vapN specific to each of the host-associated plasmid types (equine pVAPA, porcine pVAPB, and ruminant pVAPN). Of the 26 isolates, six were positive to the vapB (porcine-type) marker, 4 (15.4%) to the vapA (equine-type) marker, and 1 (3.8%) to the vapN (ruminant-type) marker. Most of the isolates 14 (53.8%) were negative to all TRAVAP markers, suggesting they lacked a virulence plasmid. To our knowledge, this work is the first to report the molecular characterization of R. equi isolates from Cuba. Our findings provide insight into the zoonotic origin of R. equi infections in people and the potential dispensability of the virulence plasmid in immunosuppressed patients.