Undetectable Production of the VIM-1 Carbapenemase in an Atlantibacter hermannii Clinical Isolate

The differential expression of VIM-1 in Atlantibacter hermannii WEB-2 and Enterobacter hormaechei ssp. hoffmannii WEB-1 clinical isolates from a rectal swab of a hospitalized patient in France was investigated. A. hermannii WEB-2 was resistant to all β-lactams except carbapenems. It produced ESBL SHV-12, but the Carba NP test failed to detect any carbapenemase activity despite the production of VIM-1. Conversely, E. hormaechei WEB-1, previously recovered from the same patient, was positive for the detection of carbapenemase activity. The blaVIM–1 gene was located on a plasmid and embedded within class 1 integron. Both plasmids were of the same IncA incompatibility group and conferred the same resistance pattern when electroporated in Escherichia coli TOP10 or Enterobacter cloacae CIP7933. Quantitative RT-PCR experiments indicated a weaker replication of pWEB-2 in A. hermannii as compared to E. hormaechei. An isogenic mutant of A. hermannii WEB-2 selected after sequential passages with increased concentrations of imipenem possessed higher MICs for carbapenems and cephalosporins including cefiderocol, higher levels of the blaVIM–1 gene transcripts, and detectable carbapenemase activity using the Carba NP test. Assessment of read coverage demonstrated that a duplication of the region surrounding blaVIM–1 gene occurred in the A. hermannii mutant with detectable carbapenemase activity. The lack of detection of the VIM-1 carbapenemase activity in A. hermannii WEB-2 isolate was likely due to a weak replication of the IncA plasmid harboring the blaVIM–1 gene. Imipenem as selective pressure led to a duplication of this gene on the plasmid and to the restoration of a significant carbapenem-hydrolyzing phenotype.

The higBA- Type Toxin-Antitoxin System in IncC Plasmids Is a Mobilizable Ciprofloxacin-Inducible System

Toxin-antitoxin (TA) systems play vital roles in maintaining plasmids in bacteria. Plasmids with incompatibility group C are large plasmids that disseminate via conjugation and carry high-profile antibiotic resistance genes.

Incompatibility Group I1 (IncI1) Plasmids: Their Genetics, Biology, and Public Health Relevance

SUMMARY Bacterial plasmids are extrachromosomal genetic elements that often carry antimicrobial resistance (AMR) genes and genes encoding increased virulence and can be transmissible among bacteria by conjugation. One key group of plasmids is the incompatibility group I1 (IncI1) plasmids, which have been isolated from multiple Enterobacteriaceae of food animal origin and clinically ill human patients. The IncI group of plasmids were initially characterized due to their sensitivity to the filamentous bacteriophage If1. Two prototypical IncI1 plasmids, R64 and pColIb-P9, have been extensively studied, and the plasmids consist of unique regions associated with plasmid replication, plasmid stability/maintenance, transfer machinery apparatus, single-stranded DNA transfer, and antimicrobial resistance. IncI1 plasmids are somewhat unique in that they encode two types of sex pili, a thick, rigid pilus necessary for mating and a thin, flexible pilus that helps stabilize bacteria for plasmid transfer in liquid environments. A key public health concern with IncI1 plasmids is their ability to carry antimicrobial resistance genes, including those associated with critically important antimicrobials used to treat severe cases of enteric infections, including the third-generation cephalosporins. Because of the potential importance of these plasmids, this review focuses on the distribution of the plasmids, their phenotypic characteristics associated with antimicrobial resistance and virulence, and their replication, maintenance, and transfer.

pCTX-M3—Structure, Function, and Evolution of a Multi-Resistance Conjugative Plasmid of a Broad Recipient Range

pCTX-M3 is the archetypic member of the IncM incompatibility group of conjugative plasmids (recently referred to as IncM2). It is responsible for the worldwide dissemination of numerous antibiotic resistance genes, including those coding for extended-spectrum β-lactamases and conferring resistance to aminoglycosides. The IncM plasmids acquired during evolution diverse mobile genetic elements found in one or two multiple resistance regions, MRR(s), grouping antibiotic resistance genes as well as mobile genetic elements or their remnants. The IncM plasmids can be found in bacteria inhabiting various environments. The information on the structure and biology of pCTX-M3 is integrated in this review. It focuses on the functional modules of pCTX-M3 responsible for its replication, stable maintenance, and conjugative transfer, indicating that the host range of the pCTX-M3 replicon is limited to representatives of the family Enterobacteriaceae (Enterobacterales ord. nov.), while the range of recipients of its conjugation system is wide, comprising Alpha-, Beta-, and Gammaproteobacteria, and also Firmicutes.

Prevalence and Relatedness of mcr-1-Mediated Colistin-Resistant Escherichia coli Isolated From Livestock and Farmers in Japan

Colistin is used to treat infectious diseases in humans and livestock; it has also been used as a feed additive for livestock for approximately 50 years. Since the mcr-1 plasmid-mediated colistin resistance gene was discovered in China in 2015, it has been detected worldwide, mainly in livestock. In this study, we investigated the prevalence and characteristics of mcr-mediated colistin-resistant Escherichia coli in livestock and farmers in Japan. We collected fecal samples from 295 healthy livestock (202 cattle and 93 swine) and 62 healthy farmers from 72 livestock farms (58 cattle farms and 14 swine farms) between 2013 and 2015. Twenty-eight mcr-1-harboring E. coli strains were isolated from 25 livestock (six cattle and 19 swine) and three farmers (two cattle farmers and one swine farmer). The prevalence rates of mcr-1-harboring E. coli in livestock and farmers were 8.47 and 4.84%, respectively. Of the 28 strains, the resistance genes of three were transferable via the mcr-1-coding plasmids to E. coli J53 at low frequencies (10−7–10−8). Six strains coharbored mcr-1 with CTX-M β-lactamases (CTX-M-14, CTX-M-27, or CTX-M-156). Of the isolates obtained from livestock and farmers in four farms (farms C, I, N, and P), nine strains had the same genotypical characteristics (sequence types and pulsed-field gel electrophoresis band patterns), plasmid characteristics (incompatibility group and plasmid transferability), and minimum inhibitory concentrations. Thus, the findings suggested that clonal strains could spread among livestock and farmers within farms. To our knowledge, this is the first study to detect clonal relatedness of mcr-1-mediated colistin-resistant E. coli in livestock and farmers. It is suggested that farmers are at a higher risk of acquiring mcr-1-harboring strains, calling for our attention based on the One Health concept.

Genotypic and Phenotypic Characterization of Incompatibility Group FIB Positive Salmonella enterica Serovar Typhimurium Isolates from Food Animal Sources

Salmonella enterica is one of the most common bacterial foodborne pathogens in the United States, causing illnesses that range from self-limiting gastroenteritis to more severe, life threatening invasive disease. Many Salmonella strains contain plasmids that carry virulence, antimicrobial resistance, and/or transfer genes which allow them to adapt to diverse environments, and these can include incompatibility group (Inc) FIB plasmids. This study was undertaken to evaluate the genomic and phenotypic characteristics of IncFIB-positive Salmonella enterica serovar Typhimurium isolates from food animal sources, to identify their plasmid content, assess antimicrobial resistance and virulence properties, and compare their genotypic isolates with more recently isolated S. Typhimurium isolates from food animal sources. Methods: We identified 71 S. Typhimurium isolates that carried IncFIB plasmids. These isolates were subjected to whole genome sequencing and evaluated for bacteriocin production, antimicrobial susceptibility, the ability to transfer resistance plasmids, and a subset was evaluated for their ability to invade and persist in intestinal human epithelial cells. Results: Approximately 30% of isolates (n = 21) displayed bacteriocin inhibition of Escherichia coli strain J53. Bioinformatic analyses using PlasmidFinder software confirmed that all isolates contained IncFIB plasmids along with multiple other plasmid replicon types. Comparative analyses showed that all strains carried multiple antimicrobial resistance genes and virulence factors including iron acquisition genes, such as iucABCD (75%), iutA (94%), sitABCD (76%) and sitAB (100%). In 17 cases (71%), IncFIB plasmids, along with other plasmid replicon types, were able to conjugally transfer antimicrobial resistance and virulence genes to the susceptible recipient strain. For ten strains, persistence cell counts (27%) were noted to be significantly higher than invasion bacterial cell counts. When the genome sequences of the study isolates collected from 1998–2003 were compared to those published from subsequent years (2005–2018), overlapping genotypes were found, indicating the perseverance of IncFIB positive strains in food animal populations. This study confirms that IncFIB plasmids can play a potential role in disseminating antimicrobial resistance and virulence genes amongst bacteria from several food animal species.

Unique Properties of the Alpha-Helical DNA-Binding Protein KfrA Encoded by the IncU Incompatibility Group Plasmid RA3 and Its Host-Dependent Role in Plasmid Maintenance

ABSTRACT KfrA, encoded on the broad-host-range RA3 plasmid, is an alpha-helical DNA-binding protein that acts as a transcriptional autoregulator. The KfrARA3 operator site overlaps the kfrA promoter and is composed of five 9-bp direct repeats (DRs). Here, the biological properties of KfrA were studied using both in vivo and in vitro approaches. Localization of the DNA-binding helix-turn-helix motif (HTH) was mapped to the N29-R52 region by protein structure modeling and confirmed by alanine scanning. KfrA repressor ability depended on the number and orientation of DRs in the operator, as well as the ability of the protein to oligomerize. The long alpha-helical tail from residues 54 to 355 was shown to be involved in self-interactions, whereas the region from residue 54 to 177 was involved in heterodimerization with KfrC, another RA3-encoded alpha-helical protein. KfrA also interacted with the segrosome proteins IncC (ParA) and KorB (ParB), representatives of the class Ia active partition systems. Deletion of the kfr genes from the RA3 stability module decreased the plasmid retention in diverse hosts in a species-dependent manner. The specific interactions of KfrA with DNA are essential not only for the transcriptional regulatory function but also for the accessory role of KfrA in stable plasmid maintenance. IMPORTANCE Alpha-helical coiled-coil KfrA-type proteins are encoded by various broad-host-range low-copy-number conjugative plasmids. The DNA-binding protein KfrA encoded on the RA3 plasmid, a member of the IncU incompatibility group, oligomerizes, forms a complex with another plasmid-encoded, alpha-helical protein, KfrC, and interacts with the segrosome proteins IncC and KorB. The unique mode of KfrA dimer binding to the repetitive operator is required for a KfrA role in the stable maintenance of RA3 plasmid in distinct hosts.

OmpK36 and TraN facilitate conjugal transfer of the Klebsiella pneumoniae carbapenem resistance plasmid pKpQIL

We investigated the mechanism of conjugal transfer of the endemic Klebsiella pneumoniae carbapenem resistance plasmid, pKpQIL. Transfer efficiency of this plasmid was found to be dependent on the expression of the major outer membrane porin, OmpK36, in recipient cells. We also found that conjugal uptake is reduced in recipients expressing an OmpK36 isoform associated with the globally pervasive K. pneumoniae ST258 clade (OmpK36ST258). This reduction was attributed to a glycine-aspartate insertion in loop 3 of OmpK36ST258, which constricts the pore by 26%. Deletion of finO, which encodes an RNA-binding protein, derepressed transfer of pKpQIL and enabled visualisation of the conjugation pilus and OmpK36-dependent conjugation in real time. While deletion of traN abolished pKpQIL conjugation, substituting traN in pKpQIL with its homologue from R100-1 circumvented OmpK36 dependency. These results suggest that OmpK36 in recipient K. pneumoniae and the pKpQIL-encoded TraN in donor bacteria cooperate to facilitate plasmid transfer. This is the first report since 1998 to suggest a novel recipient cell receptor for IncF plasmid transfer and supports the idea that TraN mediates receptor specificity for plasmids belonging to this incompatibility group.

Detailed characterization of an IncFII plasmid carrying blaOXA-48 from Lebanon

Background The spread of carbapenem-resistant Enterobacteriaceae is an important challenge and an increasing healthcare problem. OXA-48 is a class D carbapenemase that is usually localized on a conjugative plasmid belonging to the IncL incompatibility group. Methods In this study, we used a combination of short- and long-read WGS approaches and molecular typing techniques to characterize the genetic environment of the smallest reported 27 029 bp IncFII plasmid carrying blaOXA-48 (pLAU-OXA48). Results The plasmid recovered from a clinical Escherichia coli isolate was positive for blaOXA-48, which was located within the Tn6237 composite transposon. Primers targeting junctions between the IncF fragment and Tn6237 for the rapid identification of pLAU-OXA48-like plasmids were designed. Conclusions To our knowledge, this is the first report showing the complete sequence of an IncFII plasmid carrying blaOXA-48 within Tn6237 using hybrid assembly of long- and short-read sequencing.