Bacterial Chromosome Research Articles

Isolation of Phage WO Particles from Wolbachia-Infected Arthropods.

Nearly all arthropod-associated Wolbachia contain intact and/or genomic remnants of phage WO, temperate bacteriophages that facilitate horizontal gene transfer, genomic rearrangement of the bacterial chromosome, and symbiotic interactions between Wolbachia and their arthropod hosts. Integrated prophage WO genomes produce active, lytic particles; but the lack of a cell-free culturing system for Wolbachia render them difficult to purify and study. This chapter describes polyethylene glycol (PEG) precipitation of phage particles from Wolbachia-infected arthropods, followed by confirmation of phage WO isolation and purification using electron microscopy and PCR.

Mec Genes in Methicillin Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) manifests antibiotic resistance, especially for the β-lactams antibiotic group. MRSA bacteria are a common cause of infection in humans. The antibiotic resistance characteristic comes from the mecA, mecB, and mecC genes in the bacterial chromosome. mecA is the most common gene found in MRSA. Therefore, it is essential to know the role of the mecA gene in antibiotic resistance. This paper searched the literature about MRSA bacteria, the mec gene, and their relationship to cause resistance. The results showed that the mec gene found in MRSA bacteria causes antibiotic resistance in penicillin groups. Methicillin-resistant Staphylococcus aureus (MRSA) manifests antibiotic resistance, especially for the β-lactams antibiotic group. MRSA bacteria are a common cause of infection in humans. The antibiotic resistance characteristic comes from the mecA, mecB, and mecC genes in the bacterial chromosome. mecA is the most common gene found in MRSA. Therefore, it is essential to know the role of the mecA gene in antibiotic resistance. This paper searched literature about MRSA bacteria, the mec gene, and their relationship to cause resistance. The results showed that the mec gene found in MRSA bacteria causes antibiotic resistance in penicillin groups.

Pathogenic bacteria experience pervasive RNA polymerase backtracking during infection.

Eukaryotic hosts have defense mechanisms that may disrupt molecular transactions along the pathogen's chromosome through excessive DNA damage. Given that DNA damage stalls RNA polymerase (RNAP) thereby increasing mutagenesis, investigating how host defense mechanisms impact the movement of the transcription machinery on the pathogen chromosome is crucial. Using a new methodology we developed, we elucidated the dynamics of RNAP movement and association with the chromosome in the pathogenic bacterium Salmonella enterica during infection. We found that dynamics of RNAP movement on the chromosome change significantly during infection genome-wide, including at regions that encode for key virulence genes. In particular, we found that there is pervasive RNAP backtracking on the bacterial chromosome during infections and that anti-backtracking factors are critical for pathogenesis. Altogether, our results suggest that, interestingly, the host environment can promote the development of antimicrobial resistance and hypervirulence as stalled RNAPs can accelerate evolution through increased mutagenesis.

The bacterial replication origin BUS promotes nucleobase capture

Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin (oriC) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown. Here we show that Bacillus subtilis DnaAATP assembles into a continuous oligomer at the site of DNA melting, extending from a dsDNA anchor to engage a single DNA strand. Within this complex, two nucleobases of each ssDNA binding motif (DnaA-trio) are captured within a dinucleotide binding pocket created by adjacent DnaA proteins. These results provide a molecular basis for DnaA specifically engaging the conserved sequence elements within the bacterial chromosome origin basal unwinding system (BUS).

Molecular characteristic of mcr-1 gene in Escherichia coli from aquatic products in Guangdong, China

Aquatic ecosystems serve as a dissemination pathway and a reservoir of both antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study aimed to determine the prevalence of colistin-resistant mcr-like genes in Enterobacteriales in aquatic products, which may be contribute to the transfer of ARGs in water environments. The mcr-1-positive Escherichia coli were recovered from 123 freshwater fish and 34 cultured crocodile cecum samples from 10 farmers' markets in Guangdong, China. Minimum inhibitory concentration (MIC) was determined using the agar dilution method. Genotyping was performed using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Conjugation assay was carried out to investigate the transferability of mcr-1. Genomic information was obtained by whole genome sequencing (WGS) and bioinformatic analysis. Forty-four mcr-1 positive isolates showed co-resistance to tetracycline, trimethoprim/sulfamethoxazole, and gentamicin, while they were all sensitive to tigecycline, meropenem, and amikacin. They were typed into sixteen PFGE clusters. ST10 and ST117 were the most popular sequence types, followed by ST1114. S1-PFGE verified the presence of the mcr-1 gene on plasmids in sizes of ∼60 kb (n = 1) and ∼240 kb (n = 3). Whole genome sequencing-based analysis identified mcr-1 integrated in IncHI2 plasmid (n = 3), IncI2 plasmid (n = 2), and bacterial chromosome in two copies (n = 1). In addition to mcr-1, they carried several other antibiotic resistance genes, such as blaCTX-M-14, fosA3, and aac(6')-Ib-cr. These data suggest that aquatic products are an important antibiotic resistance reservoir and highlight possible risks regarding food safety.

ArdA genes from pKM101 and from B. bifidum chromosome have a different range of regulated genes

The ardA genes are present in a wide variety of conjugative plasmids and play an important role in overcoming the restriction barrier. To date, there is no information on the chromosomal ardA genes. It is still unclear whether they keep their antirestriction activity and why bacterial chromosomes contain these genes. In the present study, we confirmed the antirestriction function of the ardA gene from the Bifidobacterium bifidum chromosome. Transcriptome analysis in Escherichia coli showed that the range of regulated genes varies significantly for ardA from conjugative plasmid pKM101 and from the B. bifidum chromosome. Moreover, if the targets for both ardA genes match, they often show an opposite effect on regulated gene expression. The results obtained indicate two seemingly mutually exclusive conclusions. On the one hand, the pleiotropic effect of ardA genes was shown not only on restriction-modification system, but also on expression of a number of other genes. On the other hand, the range of affected genes varies significally for ardA genes from different sources, which indicates the specificity of ardA to inhibited targets.Author Summary. Conjugative plasmids, bacteriophages, as well as transposons, are capable to transfer various genes, including antibiotic resistance genes, among bacterial cells. However, many of those genes pose a threat to the bacterial cells, therefore bacterial cells have special restriction systems that limit such transfer.Antirestriction genes have previously been described as a part of conjugative plasmids, and bacteriophages and transposons. Those plasmids are able to overcome bacterial cell protection in the presence of antirestriction genes, which inhibit bacterial restriction systems.This work unveils the antirestriction mechanisms, which play an important role in the bacterial life cycle. Here, we clearly show that antirestriction genes, which are able to inhibit cell protection, exist not only in plasmids but also in the bacterial chromosomes themselves.Moreover, antirestrictases have not only an inhibitory function but also participate in the regulation of other bacterial genes. The regulatory function of plasmid antirestriction genes also helps them to overcome the bacterial cell protection against gene transfer, whereas the regulatory function of genomic antirestrictases has no such effect.

Mid-cell migration of the chromosomal terminus is coupled to origin segregation in Escherichia coli

Bacterial chromosomes are dynamically and spatially organised within cells. In slow-growing Escherichia coli, the chromosomal terminus is initially located at the new pole and must therefore migrate to midcell during replication to reproduce the same pattern in the daughter cells. Here, we use high-throughput time-lapse microscopy to quantify this transition, its timing and its relationship to chromosome segregation. We find that terminus centralisation is a rapid discrete event that occurs ~25 min after initial separation of duplicated origins and ~50 min before the onset of bulk nucleoid segregation but with substantial variation between cells. Despite this variation, its movement is tightly coincident with the completion of origin segregation, even in the absence of its linkage to the divisome, suggesting a coupling between these two events. Indeed, we find that terminus centralisation does not occur if origin segregation away from mid-cell is disrupted, which results in daughter cells having an inverted chromosome organisation. Overall, our study quantifies the choreography of origin-terminus positioning and identifies an unexplored connection between these loci, furthering our understanding of chromosome segregation in this bacterium.

The environmentally-regulated interplay between local three-dimensional chromatin organisation and transcription of proVWX in E. coli

Nucleoid associated proteins (NAPs) maintain the architecture of bacterial chromosomes and regulate gene expression. Thus, their role as transcription factors may involve three-dimensional chromosome re-organisation. While this model is supported by in vitro studies, direct in vivo evidence is lacking. Here, we use RT-qPCR and 3C-qPCR to study the transcriptional and architectural profiles of the H-NS (histone-like nucleoid structuring protein)-regulated, osmoresponsive proVWX operon of Escherichia coli at different osmolarities and provide in vivo evidence for transcription regulation by NAP-mediated chromosome re-modelling in bacteria. By consolidating our in vivo investigations with earlier in vitro and in silico studies that provide mechanistic details of how H-NS re-models DNA in response to osmolarity, we report that activation of proVWX in response to a hyperosmotic shock involves the destabilization of H-NS-mediated bridges anchored between the proVWX downstream and upstream regulatory elements (DRE and URE), and between the DRE and ygaY that lies immediately downstream of proVWX. The re-establishment of these bridges upon adaptation to hyperosmolarity represses the operon. Our results also reveal additional structural features associated with changes in proVWX transcript levels such as the decompaction of local chromatin upstream of the operon, highlighting that further complexity underlies the regulation of this model operon. H-NS and H-NS-like proteins are wide-spread amongst bacteria, suggesting that chromosome re-modelling may be a typical feature of transcriptional control in bacteria.

Massive integration of large gene libraries in the chromosome of Escherichia coli.

Large gene libraries are frequently created in Escherichia coli plasmids, which can induce cell toxicity and expression instability due to the high gene dosage. To address these limitations, gene libraries can be integrated in a single copy into the bacterial chromosome. Here, we describe an efficient system for the massive integration (MAIN) of large gene libraries in the E. coli chromosome that generates in-frame gene fusions that are expressed stably. MAIN uses a thermosensitive integrative plasmid that is linearized invivo to promote extensive integration of the gene library via homologous recombination. Positive and negative selections efficiently remove bacteria lacking gene integration in the target site. We tested MAIN with a library of 107 VHH genes that encode nanobodies (Nbs). The integration of VHH genes into a custom target locus of the E. coli chromosome enabled stable expression and surface display of the Nbs. Next-generation DNA sequencing confirmed that MAIN preserved the diversity of the gene library after integration. Finally, we screened the integrated library to select Nbs that bind a specific antigen using magnetic and fluorescence-activated cell sorting. This allowed us to identify Nbs binding the epidermal growth factor receptor that were not previously isolated in a similar screening of a multicopy plasmid library. Our results demonstrate that MAIN enables large gene library integration into the E. coli chromosome, creating stably expressed in-frame fusions for functional screening.

Genomic insights into the adaptation of Acinetobacter johnsonii RB2-047 to the heavy metal-contaminated subsurface mine environment

The subsurface mine environments characterized by high levels of toxic metals and low nutrient availability represent an extreme threat to bacterial persistence. In recent study, the genomic analysis of the Acinetobacter johnsonii strain RB2-047 isolated from the Rozália Gold Mine in Slovakia was performed. As expected, the studied isolate showed a high level of heavy metal tolerance (minimum inhibitory concentrations were 500 mg/L for copper and nickel, 1,500 mg/L for lead, and 250 mg/L for zinc). The RB2-047 strain also showed noticeable resistance to several antibiotics (ampicillin, kanamycin, chloramphenicol, tetracycline and ciprofloxacin). The genomic composition analysis demonstrated a low number of antibiotic and metal resistance coding genes, but a high occurrence of efflux transporter genes located on the bacterial chromosome. The experimental inhibition of efflux pumps resulted in decreased tolerance to Zn and Ni (but not to Cu and Pb) and to all antibiotics tested. In addition, the H33342 dye-accumulation assay confirmed the high efflux activity in the RB2-047 isolate. These findings showed the important role of efflux pumps in the adaptation of Acinetobacter johsonii strain RB2-047 to metal polluted mine environment as well as in development of multi-antibiotic resistance.

Genomic characterization of extended spectrum beta lactamases producing multidrug-resistant Escherichia coli clinically isolated harboring chromosomally mediated CTX-M-15 from Alkharj, KSA

Extended Spectrum Beta Lactamases (ESBLs) are the most prevalent enzymes conferring resistance to beta-lactams encoded on plasmids and rarely in chromosomes. This genomic study aims to characterize Escherichia coli to identify antimicrobial resistance genes (ARG), virulence factors, and phylogenetic relationships among ESBL-positive and negative isolates of E. coli obtained from Al-Kharj, Riyadh region, Saudi Arabia. Three clinical isolates from urine and vaginal swabs were obtained and subjected to whole genome sequencing, minimum inhibitory concentration, and antibiotic sensitivity tests. The pathogenicity and ARG were discovered, and the raw genomic sequences were assembled and annotated. Two isolates (E5 and E15) were MDR and ESBLs producers; the sequence type (ST) for E5 was 58, while those for E15 and E21 were 106. Most of the virulence genes were detected as plasmid-mediated; E21 was identified with a hyper-virulent plasmid (pH 2332–166) carrying different virulence factors (TraJ, traT, iss, etsC, hlyF, and iron acquisition associated proteins), plasmids (IncFII, IncFIB, and IncFIA), and insertion sequences (ISEc31). While most of the antimicrobial resistance genes were chromosomally mediated, a rare chromosome insertion of qnrS1 and blaCTX−M−15 with co-occurrence of Tn2 and ISKpn19 was identified in the E5 isolate. The consistent preservation of these genetic elements on bacterial chromosomes and plasmids could enhance the spread of Multidrug-Resistant (MDR) strains across various Enterobacteriaceae Species. This poses a significant threat to the effectiveness of existing antimicrobial treatments.

Bacteria can maintain rRNA operons solely on plasmids for hundreds of millions of years

It is generally assumed that all bacteria must have at least one rRNA operon (rrn operon) on the chromosome, but some strains of the genera Aureimonas and Oecophyllibacter carry their sole rrn operon on a plasmid. However, other related strains and species have chromosomal rrn loci, suggesting that the exclusive presence of rrn operons on a plasmid is rare and unlikely to be stably maintained over long evolutionary periods. Here, we report the results of a systematic search for additional bacteria without chromosomal rrn operons. We find that at least four bacterial clades in the phyla Bacteroidota, Spirochaetota, and Pseudomonadota (Proteobacteria) lost chromosomal rrn operons independently. Remarkably, Persicobacteraceae have apparently maintained this peculiar genome organization for hundreds of millions of years. In our study, all the rrn-carrying plasmids in bacteria lacking chromosomal rrn loci possess replication initiator genes of the Rep_3 family. Furthermore, the lack of chromosomal rrn operons is associated with differences in copy numbers of rrn operons, plasmids, and chromosomal tRNA genes. Thus, our findings indicate that the absence of rrn loci in bacterial chromosomes can be stably maintained over long evolutionary periods.

Analysis of Pseudomonas aeruginosa Isolates from Patients with Cystic Fibrosis Revealed Novel Groups of Filamentous Bacteriophages.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause infections in humans, especially in hospital patients with compromised host defence mechanisms, including patients with cystic fibrosis. Filamentous bacteriophages represent a group of single-stranded DNA viruses infecting different bacteria, including P. aeruginosa and other human and animal pathogens; many of them can replicate when integrated into the bacterial chromosome. Filamentous bacteriophages can contribute to the virulence of P. aeruginosa and influence the course of the disease. There are just a few isolated and officially classified filamentous bacteriophages infecting P. aeruginosa, but genomic studies indicated the frequent occurrence of integrated prophages in many P. aeruginosa genomes. An analysis of sequenced genomes of P. aeruginosa isolated from upper respiratory tract (throat and nasal swabs) and sputum specimens collected from Russian patients with cystic fibrosis indicated a higher diversity of filamentous bacteriophages than first thought. A detailed analysis of predicted bacterial proteins revealed prophage regions representing the filamentous phages known to be quite distantly related to known phages. Genomic comparisons and phylogenetic studies enabled the proposal of several new taxonomic groups of filamentous bacteriophages.

Alicyclobacillus sp. SO9, a novel halophilic acidophilic iron-oxidizing bacterium isolated from a tailings-contaminated beach, and its effect on copper extraction from chalcopyrite in the presence of high chloride concentration

Many acidophilic iron-oxidizing bacteria used in the mining industry for the bioleaching of sulfidic minerals are intolerant to high chloride concentrations, resulting in problems where chloride occurs in the deposit at high concentrations or only seawater is available. In search for strains tolerating such conditions a tetrathionate- and iron-oxidizing bacterium was isolated from a tailings-contaminated beach sample at Portman Bay, Cartagena-La Union mining district, Spain, in the presence of 20 g l−1 (0.34 M) sodium chloride. The isolate was able to form spores, did not grow in the absence of NaCl, and oxidized ferrous iron in the presence of up to 1.5 M (∼87 g l−1) NaCl. Genome sequencing based on a combination of Illumina and PacBio reads revealed two contigs, a circular bacterial chromosome of 5.2 Mbp and a plasmid of 90 kbp, respectively. The chromosome comprised seven different 16S rRNA genes. Submission of the chromosome to the Type (Strain) Genome Server (TYGS) without preselection of similar sequences revealed exclusively type strains of the genus Alicyclobacillus. In the TYGS analyses the respective most similar species were dependent on whether the final tree was derived from just 16S rRNA, from the genomes, or from the proteomes. Thus, TYGS analysis clearly showed that isolate SO9 represents a novel species of the genus Alicyclobacillus. In the presence of artificial seawater with almost 0.6 M chloride, the addition of Alicyclobacillus sp. SO9 improved copper dissolution from chalcopyrite (CuFeS2) compared to abiotic leaching without bacteria. The new isolate SO9, therefore, has potential for bioleaching at elevated chloride concentrations.

The ClpX protease is essential for inactivating the CI master repressor and completing prophage induction in Staphylococcus aureus

Bacteriophages (phages) are the most abundant biological entities on Earth, exerting a significant influence on the dissemination of bacterial virulence, pathogenicity, and antimicrobial resistance. Temperate phages integrate into the bacterial chromosome in a dormant state through intricate regulatory mechanisms. These mechanisms repress lytic genes while facilitating the expression of integrase and the CI master repressor. Upon bacterial SOS response activation, the CI repressor undergoes auto-cleavage, producing two fragments with the N-terminal domain (NTD) retaining significant DNA-binding ability. The process of relieving CI NTD repression, essential for prophage induction, remains unknown. Here we show a specific interaction between the ClpX protease and CI NTD repressor fragment of phages Ф11 and 80α in Staphylococcus aureus. This interaction is necessary and sufficient for prophage activation after SOS-mediated CI auto-cleavage, defining the final stage in the prophage induction cascade. Our findings unveil unexpected roles of bacterial protease ClpX in phage biology.

Dynamic ParB-DNA interactions initiate and maintain a partition condensate for bacterial chromosome segregation.

In most bacteria, chromosome segregation is driven by the ParABS system where the CTPase protein ParB loads at the parS site to trigger the formation of a large partition complex. Here, we present in vitro studies of the partition complex for Bacillus subtilis ParB, using single-molecule fluorescence microscopy and AFM imaging to show that transient ParB-ParB bridges are essential for forming DNA condensates. Molecular Dynamics simulations confirm that condensation occurs abruptly at a critical concentration of ParB and show that multimerization is a prerequisite for forming the partition complex. Magnetic tweezer force spectroscopy on mutant ParB proteins demonstrates that CTP hydrolysis at the N-terminal domain is essential for DNA condensation. Finally, we show that transcribing RNA polymerases can steadily traverse the ParB-DNA partition complex. These findings uncover how ParB forms a stable yet dynamic partition complex for chromosome segregation that induces DNA condensation and segregation while enabling replication and transcription.

Functional diversification despite structural congruence in the HipBST toxin-antitoxin system of Legionella pneumophila.

Toxin-antitoxin (TA) systems are parasitic genetic elements found in almost all bacterial genomes. They are exchanged horizontally between cells and are typically poorly conserved across closely related strains and species. Here, we report the characterization of a tripartite TA system in the bacterial pathogen Legionella pneumophila that is highly conserved across Legionella species genomes. This system (denoted HipBSTLp) is a distant homolog of the recently discovered split-HipA system in Escherichia coli (HipBSTEc). We present bioinformatic, molecular, and structural analyses of the divergence between these two systems and the functionality of this newly described TA system family. Furthermore, we provide evidence to refute previous claims that the toxin in this system (HipTLp) possesses bifunctionality as an L. pneumophila virulence protein. Overall, this work expands our understanding of the split-HipA system architecture and illustrates the potential for undiscovered biology in these abundant genetic elements.

Pseudobulbiferamides: Plasmid-Encoded Ureidopeptide Natural Products with Biosynthetic Gene Clusters Shared Among Marine Bacteria of Different Genera.

Ureidopeptidic natural products possess a wide variety of favorable pharmacological properties. In addition, they have been shown to mediate core physiological functions in producer bacteria. Here, we report that similar ureidopeptidic natural products with conserved biosynthetic gene clusters are produced by different bacterial genera that coinhabit marine invertebrate microbiomes. We demonstrate that a Microbulbifer strain isolated from a marine sponge can produce two different classes of ureidopeptide natural products encoded by two different biosynthetic gene clusters that are positioned on the bacterial chromosome and on a plasmid. The plasmid encoded ureidopeptide natural products, which we term the pseudobulbiferamides (5-8), resemble the ureidopeptide natural products produced by Pseudovibrio, a different marine bacterial genus that is likewise present in marine sponge commensal microbiomes. Using imaging mass spectrometry, we find that the two classes of Microbulbifer-derived ureidopeptides occupy different physical spaces relative to the bacterial colony, perhaps implying different roles for these two compound classes in Microbulbifer physiology and environmental interactions.

Integrons in the development of antimicrobial resistance: critical review and perspectives.

Antibiotic resistance development and pathogen cross-dissemination are both considered essential risks to human health on a worldwide scale. Antimicrobial resistance genes (AMRs) are acquired, expressed, disseminated, and traded mainly through integrons, the key players capable of transferring genes from bacterial chromosomes to plasmids and their integration by integrase to the target pathogenic host. Moreover, integrons play a central role in disseminating and assembling genes connected with antibiotic resistance in pathogenic and commensal bacterial species. They exhibit a large and concealed diversity in the natural environment, raising concerns about their potential for comprehensive application in bacterial adaptation. They should be viewed as a dangerous pool of resistance determinants from the "One Health approach." Among the three documented classes of integrons reported viz., class-1, 2, and 3, class 1 has been found frequently associated with AMRs in humans and is a critical genetic element to serve as a target for therapeutics to AMRs through gene silencing or combinatorial therapies. The direct method of screening gene cassettes linked to pathogenesis and resistance harbored by integrons is a novel way to assess human health. In the last decade, they have witnessed surveying the integron-associated gene cassettes associated with increased drug tolerance and rising pathogenicity of human pathogenic microbes. Consequently, we aimed to unravel the structure and functions of integrons and their integration mechanism by understanding horizontal gene transfer from one trophic group to another. Many updates for the gene cassettes harbored by integrons related to resistance and pathogenicity are extensively explored. Additionally, an updated account of the assessment of AMRs and prevailing antibiotic resistance by integrons in humans is grossly detailed-lastly, the estimation of AMR dissemination by employing integrons as potential biomarkers are also highlighted. The current review on integrons will pave the way to clinical understanding for devising a roadmap solution to AMR and pathogenicity. Graphical AbstractThe graphical abstract displays how integron-aided AMRs to humans: Transposons capture integron gene cassettes to yield high mobility integrons that target res sites of plasmids. These plasmids, in turn, promote the mobility of acquired integrons into diverse bacterial species. The acquisitions of resistant genes are transferred to humans through horizontal gene transfer.

Emergence of Raoultella ornithinolytica in human infections from different hospitals in Ecuador with OXA-48-producing resistance.

The purpose of this study was to highlight the clinical and molecular features of 13 Raoultella ornithinolytica strains isolated from clinical environments in Ecuador, and to perform comparative genomics with previously published genomes of Raoultella spp. As Raoultella is primarily found in environmental, clinical settings, we focused our work on identifying mechanisms of resistance that can provide this bacterium an advantage to establish and persist in hospital environments. We analyzed 13 strains of Raoultella ornithinolytica isolated from patients with healthcare associated infections (HAI) in three hospitals in Quito and one in Santo Domingo de Los Tsáchilas, Ecuador, between November 2017 and April 2018. These isolates were subjected to phenotypic antimicrobial susceptibility testing, end-point polymerase chain reaction (PCR) to detect the presence of carbapenemases and whole-genome sequencing. Polymerase chain reaction revealed that seven isolates were positive isolates for blaOXA-48 and one for blaKPC-2 gene. Of the seven strains that presented the blaOXA-48 gene, six harbored it on an IncFII plasmid, one was inserted into the bacterial chromosome. The blaKPC gene was detected in an IncM2/IncR plasmid. From the bioinformatics analysis, nine genomes had the gene blaOXA-48, originating from Ecuador. Moreover, all R. ornithinolytica strains contained the ORN-1 gene, which confers resistance for β-lactams, such as penicillins and cephalosporins. Comparative genome analysis of the strains showed that the pangenome of R. ornithinolytica is considered an open pangenome, with 27.77% of core genes, which could be explained by the fact that the antibiotic resistance genes in the ancestral reconstruction are relatively new, suggesting that this genome is constantly incorporating new genes. These results reveal the genome plasticity of R. ornithinolytica, particularly in acquiring antibiotic-resistance genes. The genomic surveillance and infectious control of these uncommon species are important since they may contribute to the burden of antimicrobial resistance and human health.