Genotypic analyses of IncHI2 plasmids from enteric bacteria.

Incompatibility (Inc) HI2 plasmids are large (typically > 200 kb), transmissible plasmids that encode antimicrobial resistance (AMR), heavy metal resistance (HMR) and disinfectants/biocide resistance (DBR). To better understand the distribution and diversity of resistance-encoding genes among IncHI2 plasmids, computational approaches were used to evaluate resistance and transfer-associated genes among the plasmids. Complete IncHI2 plasmid (N = 667) sequences were extracted from GenBank and analyzed using AMRFinderPlus, IntegronFinder and Plasmid Transfer Factor database. The most common IncHI2-carrying genera included Enterobacter (N = 209), Escherichia (N = 208), and Salmonella (N = 204). Resistance genes distribution was diverse, with plasmids from Escherichia and Salmonella showing general similarity in comparison to Enterobacter and other taxa, which grouped together. Plasmids from Enterobacter and other taxa had a higher prevalence of multiple mercury resistance genes and arsenic resistance gene, arsC, compared to Escherichia and Salmonella. For sulfonamide resistance, sul1 was more common among Enterobacter and other taxa, compared to sul2 and sul3 for Escherichia and Salmonella. Similar gene diversity trends were also observed for tetracyclines, quinolones, ß-lactams, and colistin. Over 99% of plasmids carried at least 25 IncHI2-associated conjugal transfer genes. These findings highlight the diversity and dissemination potential for resistance across different enteric bacteria and value of computational-based approaches for the resistance-gene assessment.

Versatile allelic replacement and self-excising integrative vectors for plasmid genome mutation and complementation.

IMPORTANCE: In spite of the dissemination of multidrug-resistant plasmids among Gram-negative pathogens, including those carrying virulence genes, vector tools for studying plasmid-born genes are lacking. The allelic replacement vectors can be used to generate plasmid or chromosomal mutations including markless point mutations. This is the first report describing a self-excising integrative vector that can be used as a stable single-copy complementing tool to study medically important pathogens including in vivo studies without the need for antibiotic selection. Overall, our newly developed vectors can be applied for the assessment of the function of plasmid-encoded genes by specifically creating mutations, moving large operons between plasmids and to/from the chromosome, and complementing phenotypes associated with gene mutation. Furthermore, the vectors express chromophores for the detection of target gene modification or colony isolation, avoiding time-consuming screening procedures.

Development of an antimicrobial resistance plasmid transfer gene database for enteric bacteria.

Introduction: Type IV secretion systems (T4SSs) are integral parts of the conjugation process in enteric bacteria. These secretion systems are encoded within the transfer (tra) regions of plasmids, including those that harbor antimicrobial resistance (AMR) genes. The conjugal transfer of resistance plasmids can lead to the dissemination of AMR among bacterial populations. Methods: To facilitate the analyses of the conjugation-associated genes, transfer related genes associated with key groups of AMR plasmids were identified, extracted from GenBank and used to generate a plasmid transfer gene dataset that is part of the Virulence and Plasmid Transfer Factor Database at FDA, serving as the foundation for computational tools for the comparison of the conjugal transfer genes. To assess the genetic feature of the transfer gene database, genes/proteins of the same name (e.g., traI/TraI) or predicted function (VirD4 ATPase homologs) were compared across the different plasmid types to assess sequence diversity. Two analyses tools, the Plasmid Transfer Factor Profile Assessment and Plasmid Transfer Factor Comparison tools, were developed to evaluate the transfer genes located on plasmids and to facilitate the comparison of plasmids from multiple sequence files. To assess the database and associated tools, plasmid, and whole genome sequencing (WGS) data were extracted from GenBank and previous WGS experiments in our lab and assessed using the analysis tools. Results: Overall, the plasmid transfer database and associated tools proved to be very useful for evaluating the different plasmid types, their association with T4SSs, and increased our understanding how conjugative plasmids contribute to the dissemination of AMR genes.

Biochemical and Structural Characterization of CRH-1, a Carbapenemase from Chromobacterium haemolyticum Related to KPC ß-Lactamases.

KPC-2 is one of the most relevant serine-carbapenemases among the carbapenem-resistant Enterobacterales. We previously isolated from the environmental species Chromobacterium haemolyticum a class A CRH-1 ß-lactamase displaying 69% amino acid sequence identity with KPC-2. The objective of this study was to analyze the kinetic behavior and crystallographic structure of this ß-lactamase. Our results showed that CRH-1 can hydrolyze penicillins, cephalosporins (except ceftazidime), and carbapenems with similar efficacy compared to KPC-2. Inhibition kinetics showed that CRH-1 is not well inhibited by clavulanic acid, in contrast to efficient inhibition by avibactam (AVI). The high-resolution crystal of the apoenzyme showed that CRH-1 has a similar folding compared to other class A ß-lactamases. The CRH-1/AVI complex showed that AVI adopts a chair conformation, stabilized by hydrogen bonds to Ser70, Ser237, Asn132, and Thr235. Our findings highlight the biochemical and structural similarities of CRH-1 and KPC-2 and the potential clinical impact of this carbapenemase in the event of recruitment by pathogenic bacterial species.

Whole-Genome Sequences of Nine Hospital-Associated Methicillin-Susceptible Staphylococcus aureus Strains.

We present the draft genome sequences of nine hospital-associated methicillin-susceptible Staphylococcus aureus (HA-MSSA) strains. All strains were from Minnesota (eight from blood and one from bone), harbored various virulence genes, and showed diverse multilocus sequence typing and spa types.

Draft Genome Sequences of 27 Hospital-Associated Methicillin-Resistant Staphylococcus aureus Strains Isolated in Minnesota.

Infections caused by hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) strains have higher morbidity and mortality rates and require longer hospital stays than do those caused by hospital-associated methicillin-sensitive Staphylococcus aureus strains. To gain insight into their genomic makeup, antimicrobial resistance, biofilm formation, and virulence potentials, here we present the draft whole-genome sequences of 27 HA-MRSA strains isolated in Minnesota.

Draft Genome Sequences of 12 Panton-Valentine Leucocidin-Positive and Multidrug-Resistant Methicillin-Resistant Staphylococcus aureus Strains Isolated from an Intensive Care Unit in Pakistan.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium responsible for difficult-to-treat staphylococcal infections due to multidrug resistance. Twelve Panton-Valentine leucocidin (PVL)-positive and multidrug-resistant clinical MRSA isolates from hospitals in Pakistan were sequenced and annotated to investigate genetic markers associated with antimicrobial resistance, virulence, and biofilm formation.

In silico analyses of diversity and dissemination of antimicrobial resistance genes and mobile genetics elements, for plasmids of enteric pathogens.

Introduction: The antimicrobial resistance (AMR) mobilome plays a key role in the dissemination of resistance genes encoded by mobile genetics elements (MGEs) including plasmids, transposons (Tns), and insertion sequences (ISs). These MGEs contribute to the dissemination of multidrug resistance (MDR) in enteric bacterial pathogens which have been considered as a global public health risk. Methods: To further understand the diversity and distribution of AMR genes and MGEs across different plasmid types, we utilized multiple sequence-based computational approaches to evaluate AMR-associated plasmid genetics. A collection of 1,309 complete plasmid sequences from Gammaproteobacterial species, including 100 plasmids from each of the following 14 incompatibility (Inc) types: A/C, BO, FIA, FIB, FIC, FIIA, HI1, HI2, I1, K, M, N, P except W, where only 9 sequences were available, was extracted from the National Center for Biotechnology Information (NCBI) GenBank database using BLAST tools. The extracted FASTA files were analyzed using the AMRFinderPlus web-based tools to detect antimicrobial, disinfectant, biocide, and heavy metal resistance genes and ISFinder to identify IS/Tn MGEs within the plasmid sequences. Results and Discussion: In silico prediction based on plasmid replicon types showed that the resistance genes were diverse among plasmids, yet multiple genes were widely distributed across the plasmids from enteric bacterial species. These findings provide insights into the diversity of resistance genes and that MGEs mediate potential transmission of these genes across multiple plasmid replicon types. This notion was supported by the observation that many IS/Tn MGEs and resistance genes known to be associated with them were common across multiple different plasmid types. Our results provide critical insights about how the diverse population of resistance genes that are carried by the different plasmid types can allow for the dissemination of AMR across enteric bacteria. The results also highlight the value of computational-based approaches and in silico analyses for the assessment of AMR and MGEs, which are important elements of molecular epidemiology and public health outcomes.

Draft Genome Sequences of Eight Community-Associated Methicillin-Resistant Staphylococcus aureus Strains.

Here, we report the draft genome sequences of eight community-associated methicillin-resistant Staphylococcus aureus strains that were resistant to cefoxitin, ampicillin, and erythromycin. Three isolates, i.e., CAR1, CAR2, and CAR8, were sequence type 8 (ST8) with staphylococcal cassette chromosome mec (SCCmec) type IVa and were Panton-Valentine leukocidin (PVL) positive, which has been known as a predominant clone in the United States.

MgrA Negatively Impacts Staphylococcus aureus Invasion by Regulating Capsule and FnbA.

Virulence genes are regulated by a complex regulatory network in Staphylococcus aureus Some of the regulators are global in nature and affect many downstream genes. MgrA is a multiple-gene regulator that has been shown to activate genes involved in capsule biosynthesis and repress surface protein genes. The goal of this study was to demonstrate the biological significance of MgrA regulation of capsule and surface proteins. We found that strain Becker possessed one fibronectin-binding protein, FnbA, and that FnbA was the predominant protein involved in invasion of nonphagocytic HeLa cells. By genetic analysis of strains with different amounts of capsule, we demonstrated that capsule impeded invasion of HeLa cells by masking the bacterial cell wall-anchored protein FnbA. Using variants with different levels of mgrA transcription, we further demonstrated that MgrA negatively impacted invasion by activating the cap genes involved in capsule biosynthesis and repressing the fnbA gene. Thus, we conclude that MgrA negatively impacts cell invasion of S. aureus Becker by promoting capsule and repressing FnbA.

Contribution of hla Regulation by SaeR to Staphylococcus aureus USA300 Pathogenesis.

The SaeRS two-component system in Staphylococcus aureus is critical for regulation of many virulence genes, including hla, which encodes alpha-toxin. However, the impact of regulation of alpha-toxin by Sae on S. aureus pathogenesis has not been directly addressed. Here, we mutated the SaeR-binding sequences in the hla regulatory region and determined the contribution of this mutation to hla expression and pathogenesis in strain USA300 JE2. Western blot analyses revealed drastic reduction of alpha-toxin levels in the culture supernatants of SaeR-binding mutant in contrast to the marked alpha-toxin production in the wild type. The SaeR-binding mutation had no significant effect on alpha-toxin regulation by Agr, MgrA, and CcpA. In animal studies, we found that the SaeR-binding mutation did not contribute to USA300 JE2 pathogenesis using a rat infective endocarditis model. However, in a rat skin and soft tissue infection model, the abscesses on rats infected with the mutant were significantly smaller than the abscesses on those infected with the wild type but similar to the abscesses on those infected with a saeR mutant. These studies indicated that there is a direct effect of hla regulation by SaeR on pathogenesis but that the effect depends on the animal model used.

Expanding the Repertoire of Carbapenem-Hydrolyzing Metallo-ß-Lactamases by Functional Metagenomic Analysis of Soil Microbiota.

Carbapenemases are bacterial enzymes that hydrolyze carbapenems, a group of last-resort ß-lactam antibiotics used for treatment of severe bacterial infections. They belong to three ß-lactamase classes based amino acid sequence (A, B, and D). The aim of this study was to elucidate occurrence, diversity and functionality of carbapenemase-encoding genes in soil microbiota by functional metagenomics. Ten plasmid libraries were generated by cloning metagenomic DNA from agricultural (n = 6) and grassland (n = 4) soil into Escherichia coli. The libraries were cultured on amoxicillin-containing agar and up to 100 colonies per library were screened for carbapenemase production by CarbaNP test. Presumptive carbapenemases were characterized with regard to DNA sequence, minimum inhibitory concentration (MIC) of ß-lactams, and imipenem hydrolysis. Nine distinct class B carbapenemases, also known as metallo-beta-lactamases (MBLs), were identified in six soil samples, including two subclass B1 (GRD23-1 and SPN79-1) and seven subclass B3 (CRD3-1, PEDO-1, GRD33-1, ESP-2, ALG6-1, ALG11-1, and DHT2-1). Except PEDO-1 and ESP-2, these enzymes were distantly related to any previously described MBLs (33 to 59% identity). RAIphy analysis indicated that six enzymes (CRD3-1, GRD23-1, DHT2-1, SPN79-1, ALG6-1, and ALG11-1) originated from Proteobacteria, two (PEDO-1 and ESP-2) from Bacteroidetes and one (GRD33-1) from Gemmatimonadetes. All MBLs detected in soil microbiota were functional when expressed in E. coli, resulting in detectable imipenem-hydrolyzing activity and significantly increased MICs of clinically relevant ß-lactams. Interestingly, the MBLs yielded by functional metagenomics generally differed from those detected in the same soil samples by antibiotic selective culture, showing that the two approaches targeted different subpopulations in soil microbiota.