Emergence of Klebsiella pneumoniae ST14 co-harboring blaNDM-1, blaOXA-232, mcr-1.1, and a novel IncI1 tet(X4) plasmid, with evidence of ColKP3 mobilization under antibiotic pressure

ABSTRACTProphages are often involved in host survival strategies and contribute toward increasing the genetic diversity of the host genome. Prophages also drive horizontal propagation of various genes as vehicles. However, there are few retrospective studies contributing to the propagation of antimicrobial resistance (AMR) and virulence factor (VF) genes by prophage. We extracted the complete genome sequences of seven pathogens, including ESKAPE bacteria and Escherichia coli from a public database, and examined the distribution of both the AMR and VF genes in prophage-like regions. We found that the ratios of AMR and VF genes greatly varied among the seven species. More than 70% of Enterobacter cloacae strains had VF genes, but only 1.2% of Klebsiella pneumoniae strains had VF genes from prophages. AMR and VF genes are unlikely to exist together in the same prophage region except in E. coli and Staphylococcus aureus, and the distribution patterns of prophage types containing AMR genes are distinct from those of VF gene-carrying prophage types. AMR genes in the prophage were located near transposase and/or integrase. The prophage containing class 1 integrase possessed a significantly greater number of AMR genes than did prophages with no class 1 integrase. The results of this study present a comprehensive picture of AMR and VF genes present within, or close to, prophage-like elements and different prophage patterns between AMR- or VF-encoding prophage-like elements.IMPORTANCE Although we believe phages play an important role in horizontal gene transfer in exchanging genetic material, we do not know the distribution of the antimicrobial resistance (AMR) and/or virulence factor (VF) genes in prophages. We collected different prophage elements from the complete genome sequences of seven species—Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, and Escherichia coli—and characterized the distribution of antimicrobial resistance and virulence genes located in the prophage region. While virulence genes in prophage were species specific, antimicrobial resistance genes in prophages were highly conserved in various species. An integron structure was detected within specific prophage regions such as P1-like prophage element. Maximum of 10 antimicrobial resistance genes were found in a single prophage region, suggesting that prophages act as a reservoir for antimicrobial resistance genes. The results of this study show the different characteristic structures between AMR- or VF-encoding prophages.

Antimicrobial agents are widely employed in swine and cattle production systems to treat and prevent diseases. The overall benefits include increased animal productivity, improved animal health, and reduction in gut prevalence and fecal shedding of foodborne pathogens such as Shiga-toxin producing Escherichia coli, Salmonella and Campylobacter. The use of in-feed antimicrobials is of concern because of the emergence and dissemination of antimicrobial resistance (AMR) bacteria within the gut microbial ecosystem. These resistant bacteria, or the genetic elements they carry, can be transmitted to humans through the food chain, direct animal contact, or environmental pathways, ultimately posing a threat to public health by limiting treatment options for bacterial infections. The emergence and persistence of AMR in food-producing animals are driven by a complex interplay of microbial, host, and environmental factors. This complexity highlights the urgent need to better understand the ecological and evolutionary consequences of antimicrobial use particularly its impact on microbial community structure, resistance gene reservoirs, and the horizontal gene transfer dynamics within the gastrointestinal tract. Insights into these processes are critical for developing effective strategies to limit the spread of resistance without compromising animal health or productivity. A potential approach to AMR mitigation is to minimize or eliminate exposure of gut bacteria to antimicrobials by minimizing oral administration of antimicrobials. This approach aligns with global efforts to implement antimicrobial stewardship practices in animal agriculture and encourages the exploration of alternative interventions that can maintain animal performance while mitigating the selection pressure for resistance. Because of public health concern associated with AMR, antimicrobial alternatives, such as heavy metals, mainly copper and zinc, probiotics and essential oils, are being used to achieve health and performance benefits similar to antimicrobials. The alternatives are sustainable because performance benefits are attained with a concurrent reduction in the overall antimicrobial use. Additionally, the changes in the gut microbial community composition induced by antimicrobial alternatives could have favorable effects in mitigating AMR in gut bacteria. However, antimicrobial alternatives, particularly copper, zinc and certain probiotics, have the potential to contribute to AMR in gut bacteria. Copper and zinc, particularly at elevated levels in the feed, induce metal resistance in bacteria and genes that confer metal resistance have been shown to be genetically linked to other AMR genes, suggesting potential co-selection of AMR in the absence of antibiotic selection pressure. Similarly, probiotics, particularly Enterococcus faecium, which is one of the commonly used probiotic species, can carry virulence genes and AMR genes to medically-important antibiotics and have the potential to horizontally transfer the genes to other bacteria, including pathogens in the gut. In conclusion, antimicrobial alternatives used in livestock production must be critically evaluated not only for their benefits but also for their potential to contribute to AMR. Any intervention that alters the microbial community has the potential to select for resistance. Therefore, coordinated, interdisciplinary efforts involving human and animal health sectors are essential to guide research and ensure the safe and effective use of antibiotic alternatives in food animal production systems.

Detection of plasmids in Salmonella from poultry and investigating the potential horizontal transfer of antimicrobial resistance and virulence genes: PLASMID TRANSFER OF RESISTANCE AND VIRULENCE.

Although most Brazilian dairy products meet high technological standards, there are quality issues regarding milk production, which may reduce the final product quality. Several microbial species may contaminate milk during manufacture and handling. If antimicrobial usage remains uncontrolled in dairy cattle, the horizontal transfer of antimicrobial resistance genes in foodstuffs may be of particular concern for both food producers and dairy industry. This study focused on the evaluation of putative Gram positive cocci in Minas cheese and of antimicrobial and biocide resistance genes among the isolated bacteria. Representative samples of 7 different industrially trademarked Minas cheeses (n = 35) were processed for selective culture and isolation of Gram positive cocci. All isolated bacteria were identified by DNA sequencing of the 16S rRNA gene. Antimicrobial resistance genes were screened by PCR. Overall, 208 strains were isolated and identified as follows: Enterococcus faecalis (47.6%), Macrococcus caseolyticus (18.3%), Enterococcus faecium (11.5%), Enterococcus caseliflavus (7.7%), Staphylococcus haemolyticus (7.2%), Staphylococcus aureus (4.3%), Staphylococcus epidermidis (2.9%), and Enterococcus hirae (0.5%). The genetic markers mecA (78.0%) and smr (71.4%) were the most prevalent, but others were also detected, such as blaZ (65.2%), msrA (60.9%), msrB (46.6%), linA (54.7%), and aacA-aphD (47.6%). The occurrence of opportunist pathogenic bacteria harboring antimicrobial resistance markers in the cheese samples are of special concern, since these bacteria are not considered harmful contaminating agents according to the Brazilian sanitary regulations. However, they are potentially pathogenic bacteria and the cheese may be considered a reservoir for antimicrobial resistance genes available for horizontal transfer through the food chain, manufacturing personnel and consumers.

IntroductionIn the past decades, extended-spectrum beta-lactamase (ESBL)-producing and carbapenem-resistant (CR) Escherichia coli isolates have been detected in Vietnamese hospitals. The transfer of antimicrobial resistance (AMR) genes carried on plasmids is mainly responsible for the emergence of multidrug-resistant E. coli strains and the spread of AMR genes through horizontal gene transfer. Therefore, it is important to thoroughly study the characteristics of AMR gene-harboring plasmids in clinical multidrug-resistant bacterial isolates.MethodsThe profiles of plasmid assemblies were determined by analyzing previously published whole-genome sequencing data of 751 multidrug-resistant E. coli isolates from Vietnamese hospitals in order to identify the risk of AMR gene horizontal transfer and dissemination.ResultsThe number of putative plasmids in isolates was independent of the sequencing coverage. These putative plasmids originated from various bacterial species, but mostly from the Escherichia genus, particularly E. coli species. Many different AMR genes were detected in plasmid contigs of the studied isolates, and their number was higher in CR isolates than in ESBL-producing isolates. Similarly, the blaKPC-2, blaNDM-5, blaOXA-1, blaOXA-48, and blaOXA-181 β-lactamase genes, associated with resistance to carbapenems, were more frequent in CR strains. Sequence similarity network and genome annotation analyses revealed high conservation of the β-lactamase gene clusters in plasmid contigs that carried the same AMR genes.DiscussionOur study provides evidence of horizontal gene transfer in multidrug-resistant E. coli isolates via conjugative plasmids, thus rapidly accelerating the emergence of resistant bacteria. Besides reducing antibiotic misuse, prevention of plasmid transmission also is essential to limit antibiotic resistance.

Acinetobacter baumannii contributes significantly to the global issue of multidrug-resistant (MDR) nosocomial infections. Often, these strains demonstrate resistance to carbapenems (MDR-CRAB), the first-line treatment for infections instigated by MDR A. baumannii. Our study focused on the antimicrobial susceptibility and genomic sequences related to plasmids from 12 clinical isolates of A. baumannii that carry both the blaOXA-58 and bla NDM-1 carbapenemase genes. Whole-genome sequencing with long-read technology was employed for the characterization of an A. baumannii plasmid that harbors the bla OXA-58 and blaNDM-1 genes. The location of the bla OXA-58 and bla NDM-1 genes was confirmed through Southern blot hybridization assays. Antimicrobial susceptibility tests were conducted, and molecular characterization was performed using PCR and PFGE. Multilocus Sequence Typing analysis revealed considerable genetic diversity among bla OXA-58 and bla NDM-1 positive strains in Brazil. It was confirmed that these genes were located on a plasmid larger than 300 kb in isolates from the same hospital, which also carry other antimicrobial resistance genes. Different genetic contexts were observed for the co-occurrence of these carbapenemase-encoding genes in Brazilian strains. The propagation of bla OXA-58 and bla NDM-1 genes on the same plasmid, which also carries other resistance determinants, could potentially lead to the emergence of bacterial strains resistant to multiple classes of antimicrobials. Therefore, the characterization of these strains is of paramount importance for monitoring resistance evolution, curbing their rapid global dissemination, averting outbreaks, and optimizing therapy.

In the environment, unicellular organisms such as prokaryotes are exposed to direct invasion of viruses and its consequent transduction. In addition, some of the prokaryotic species can uptake naked DNA molecules outside or transfer their own DNA to other species through conjugative plasmids. Hence, prokaryotic genomes could be often mosaic: they may have the extrinsic genes which are not vertically transmitted from the ancestor but horizontally transferred from other organisms. Such a phenomenon, namely, “horizontal (or lateral) gene transfer,” is the main issue of this chapter. Horizontal gene transfer can rapidly cause genotypic/phenotypic changes in the recipient organisms, apparently beyond the theory of traditional population genetics based on mutation. Thus, it has been considered that horizontal gene transfer has influenced very much on the evolution of prokaryotes. In response to the accumulation of genomic data, the amount of horizontally transferred genes has been estimated at the large scale, but the significance of horizontal gene transfer in real environment has not been fully assessed. How often does horizontal gene transfer occur among taxa? How much does it affect the gene pool in environment? The challenging studies have just started. Metagenomic approaches have a great potential for this purpose, but many methodological limitations for treating the data remains unsolved. In this chapter, traditional genomics methods for estimating horizontally transferred genes are first reviewed. In the latter part, technical perspectives on prediction of horizontal gene transfer from the metagenomics data are discussed.

Horizontal gene transfer (HGT) is a driving force for the dissemination of antimicrobial resistance (AMR) genes among Campylobacter jejuni organisms, a leading cause of foodborne gastroenteritis worldwide. Although HGT is well documented for C. jejuni planktonic cells, the role of C. jejuni biofilms in AMR spread that likely occurs in the environment is poorly understood. Here, we developed a cocultivation model to investigate the HGT of chromosomally encoded AMR genes between two C. jejuni F38011 AMR mutants in biofilms. Compared to planktonic cells, C. jejuni biofilms significantly promoted HGT (P < 0.05), resulting in an increase of HGT frequencies by up to 17.5-fold. Dynamic study revealed that HGT in biofilms increased at the early stage (i.e., from 24 h to 48 h) and remained stable during 48 to 72 h. Biofilms continuously released the HGT mutants into supernatant culture, indicating spontaneous dissemination of AMR to broader niches. DNase I treatment confirmed the role of natural transformation in genetic exchange. HGT was not associated with biofilm biomass, cell density, or bacterial metabolic activity, whereas the presence of extracellular DNA was negatively correlated with the altered HGT frequencies. HGT in biofilms also had a strain-to-strain variation. A synergistic HGT effect was observed between C. jejuni with different genomic backgrounds (i.e., C. jejuni NCTC 11168 chloramphenicol-resistant strain and F38011 kanamycin-resistant strain). C. jejuni performed HGT at the frequency of 10-7 in Escherichia coli-C. jejuni biofilms, while HGT was not detectable in Salmonella enterica-C. jejuni biofilms. IMPORTANCE Antimicrobial-resistant C. jejuni has been listed as a high priority of public health concern worldwide. To tackle the rapid evolution of AMR in C. jejuni, it is of great importance to understand the extent and characteristics of HGT in C. jejuni biofilms, which serve as the main survival strategy of this microbe in the farm-to-table continuum. In this study, we demonstrated that biofilms significantly enhanced HGT compared to the planktonic state (P < 0.05). Biofilm cultivation time and extracellular DNA (eDNA) amount were related to varied HGT frequencies. C. jejuni could spread AMR genes in both monospecies and dual-species biofilms, mimicking the survival mode of C. jejuni in food chains. These findings indicated that the risk and extent of AMR transmission among C. jejuni organisms have been underestimated, as previous HGT studies mainly focused on the planktonic state. Future AMR controlling measures can target biofilms and their main component eDNA.

BackgroundHorizontal gene transfer, i.e. the acquisition of genetic material from nonparent organism, is considered an important force driving species evolution. Many cases of horizontal gene transfer from prokaryotes to eukaryotes have been registered, but no transfer mechanism has been deciphered so far, although viruses were proposed as possible vectors in several studies. In agreement with this idea, in our previous study we discovered that in two eukaryotic proteins bacteriophage recombination site (AttP) was adjacent to the regions originating via horizontal gene transfer. In one of those cases AttP site was present inside the introns of cysteine-rich repeats. In the present study we aimed to apply computational tools for finding multiple horizontal gene transfer events in large genome databases. For that purpose we used a sequence of cysteine-rich repeats to identify genes potentially acquired through horizontal transfer.ResultsHMMER remote similarity search significantly detected 382 proteins containing cysteine-rich repeats. All of them, except 8 sequences, belong to eukaryotes. In 124 proteins the presence of conserved structural domains was predicted. In spite of the fact that cysteine-rich repeats are found almost exclusively in eukaryotic proteins, many predicted domains are most common for prokaryotes or bacteriophages. Ninety-eight proteins out of 124 contain typical prokaryotic domains. In those cases proteins were considered as potentially originating via horizontal transfer. In addition, HHblits search revealed that two domains of the same fungal protein, Glycoside hydrolase and Peptidase M15, have high similarity with proteins of two different prokaryotic species, hinting at independent horizontal gene transfer events.ConclusionsCysteine-rich repeats in eukaryotic proteins are usually accompanied by conserved domains typical for prokaryotes or bacteriophages. These proteins, containing both cysteine-rich repeats, and characteristic prokaryotic domains, might represent multiple independent horizontal gene transfer events from prokaryotes to eukaryotes. We believe that the presence of bacteriophage recombination site inside cysteine-rich repeat coding sequence may facilitate horizontal genes transfer. Thus computational approach, described in the present study, can help finding multiple sequences originated from horizontal transfer in eukaryotic genomes.

BackgroundMultidrug resistant (MDR) bacteria which resist at least three different antibiotic classes are serious threat to public health and patient treatment. Antimicrobial resistant (AMR) genes are often mediated through plasmids due to its horizontal transferability from bacteria to bacteria. Here, we assessed the prevalence of AMR genes in the USA by analyzing longitudinal K. pneumoniae plasmid genome data obtained from PATRIC (Pathosystems Resources Integration Center) database.MethodsThe PATRIC database have 176 K. pneumoniae plasmid genome data collected in 9 states of US from 2004 to 2019. The isolation source are various patient samples including urine, blood, etc. The sequence information was downloaded from GeneBank. The AMR genes on plasmids of K. pneumoniae were identified using open-source AMR database, Resfinder which search AMR genes for 16 types of antibiotic classes.ResultsMultidrug resistance was spread all over the US as most of isolates have AMR genes against more than one antibiotic classes, and some isolates contain against up to 8 antibiotic classes AMR genes. Most common AMR genes are against 9 classes including Aminoglycosides, beta-lactamase (carbapenem), Chloramphenicol, Macrolide, Quinolone, Rifampicin, Sulfonamide, Tetracycline, and Trimethoprim. Aminoglycosides and beta-lactamase including carbapenem resistance genes showed higher frequency than other classes of antibiotics. Carbapenem resistance genes, especially blaKPC showed higher frequency in east region of US including NY, PA, and VA (Fig 1A). While some resistances reduced over time, Aminoglycosides and beta-lactamase resistance genes are sharply increased (Fig 1B). Many isolates contain 3∼4 plasmids and plasmid amplicon types were various by isolates but no co-relation between plasmid types and AMR genes was observed.Figure 1A.The number of antibiotics resistance genes over the geographical region by the isolates. The states that have more than 3 isolates were included in the analysis.Figure IB.Changes in the number of antibiotics resistance genes over the years. Only time points that have more than 3 isolates been included in the analysis.ConclusionDatabase analysis of K. pneumoniae isolates from different regions and time provides insight of the prevalence of AMR genes. The prevalence of the multidrug resistance obtained from databases analysis showed the abundance of AMR genes regardless of the region or time, especially for Aminoglycoside and beta-lactamase. Bacterial sequence database such as PATRIC is a useful tool for tracking the existence and emergence of AMR genes.DisclosuresChetan Jinadatha, MD, MPH, AHRQ R01 Grant-5R01HS025598: Grant/Research Support|EOS Surfaces: Copper Coupons and materials for testing.

Simple SummaryWorldwide, antimicrobial resistance (AMR) is of major concern for human and animal health since infections with multidrug-resistant bacteria are often more challenging and costly. In the family Staphyloccocaceae, the species Staphylococcusaureus in particular was reported to cause severe infections. Although most of the other Staphylococcaceae members were not shown to cause severe illnesses, the transmission of AMR genes to harmful species might take place. Therefore, the monitoring of AMR potential in different environments is of high relevance. Mammaliicocci on dairy farms might represent such an AMR gene reservoir. Thus, in this study, the AMR potential of mammaliicocci isolates from German dairy farms was investigated. Whole-genome sequencing (WGS) of the isolates was conducted to evaluate the phylogenetic relationship of the isolates and analyze AMR genes. In addition, antimicrobial susceptibility testing was performed to compare the AMR genotype with the phenotype. It turned out that mammaliicocci may harbor large numbers of different AMR genes and exhibit phenotypic resistance to various antibiotics. Since some AMR genes are likely located on mobile genetic elements, such as plasmids, AMR gene transmission between members of the Staphylococcaceae family might occur.Mammaliicocci might play a major role in antimicrobial resistance (AMR) gene transmission between organisms of the family Staphylococcaceae, such as the potentially pathogenic species Staphylococcus aureus. The interest of this study was to analyze AMR profiles of mammaliicocci from German dairy farms to evaluate the AMR transmission potential. In total, 65 mammaliicocci isolates from 17 dairy farms with a history of MRSA detection were analyzed for AMR genotypes and phenotypes using whole genome sequencing and antimicrobial susceptibility testing against 19 antibiotics. The various genotypic and phenotypic AMR profiles of mammaliicocci from German dairy farms indicated the simultaneous occurrence of several different strains on the farms. The isolates exhibited a non-wildtype phenotype to penicillin (58/64), cefoxitin (25/64), chloramphenicol (26/64), ciprofloxacin (25/64), clindamycin (49/64), erythromycin (17/64), fusidic acid (61/64), gentamicin (8/64), kanamycin (9/64), linezolid (1/64), mupirocin (4/64), rifampicin (1/64), sulfamethoxazol (1/64), streptomycin (20/64), quinupristin/dalfopristin (26/64), tetracycline (37/64), tiamulin (59/64), and trimethoprim (30/64). Corresponding AMR genes against several antimicrobial classes were detected. Linezolid resistance was associated with the cfr gene in the respective isolate. However, discrepancies between genotypic prediction and phenotypic resistance profiles, such as for fusidic acid and tiamulin, were also observed. In conclusion, mammaliicocci from dairy farms may carry a broad variety of antimicrobial resistance genes and exhibit non-wildtype phenotypes to several antimicrobial classes; therefore, they may represent an important source for horizontal gene transfer of AMR genes to pathogenic Staphylococcaceae.

In 2004-2005, an outbreak of impetigo occurred at a correctional facility during a sentinel outbreak of methicillin- resistant Staphylococcus aureus (MRSA) in Alberta, Canada. Next-generation sequencing (NGS) was used to characterize the group A Streptococcus (GAS) isolates and evaluate whether genomic biomarkers could distinguish between those recovered alone and those co-isolated with S. aureus. Superficial wound swabs collected from all adults with impetigo during this outbreak were cultured using standard methods. NGS was used to characterize and compare all of the GAS and S. aureus genomes. Fifty-three adults were culture positive for GAS, with a subset of specimens also positive for MRSA (n = 5) or methicillin-sensitive S. aureus (n = 3). Seventeen additional MRSA isolates from this facility from the same time frame (no GAS co-isolates) were also included. All 78 bacterial genomes were analyzed for the presence of known virulence factors, plasmids, and antimicrobial resistance (AMR) genes. Among the GAS isolates were 12 emm types, the most common being 41.2 (n = 27; 51%). GAS genomes were phylogenetically compared with local and public datasets of invasive and non-invasive isolates. GAS genomes had diverse profiles for virulence factors, plasmids, and AMR genes. Pangenome analysis did not identify horizontally transferred genes in the co-infection versus single infections. GAS recovered from invasive and non-invasive sources were not genetically distinguishable. Virulence factors, plasmids, and AMR profiles grouped by emm type, and no genetic changes were identified that predict co-infection or horizontal gene transfer between GAS and S. aureus.

Chapter 25 - Horizontal Gene Transfer and its Role in the Evolution of Prokaryotes

Antimicrobial resistance (AR) is a global problem with serious implications for public health. AR genes are frequently detected on animal farms, but little is known about their origin and distribution patterns. We hypothesized that AR genes can transfer from animal feces to the environment through manure, and to this end, we characterized and compared the resistomes (collections of AR genes) of animal feces, manure, and soil samples collected from five dairy farms using a metagenomics approach. Resistomes constituted only up to 1% of the total gene content, but were variable by sector and also farm. Broadly, the identified AR genes were associated with 18 antibiotic resistances classes across all samples; however, the most abundant genes were classified under multidrug transporters (44.75%), followed by resistance to vancomycin (12.48%), tetracycline (10.52%), bacitracin (10.43%), beta-lactam resistance (7.12%), and MLS efflux pump (6.86%) antimicrobials. The AR gene profiles were variable between farms. Farm 09 was categorized as a high risk farm, as a greater proportion of AR genes were common to at least three sectors, suggesting possible horizontal transfer of AR genes. Taxonomic characterization of AR genes revealed that a majority of AR genes were associated with the phylum Proteobacteria. Nonetheless, there were several members of Bacteroidetes, particularly Bacteroides genus and several lineages from Firmicutes that carried similar AR genes in different sectors, suggesting a strong potential for horizontal transfer of AR genes between unrelated bacterial hosts in different sectors of the farms. Further studies are required to affirm the horizontal gene transfer mechanisms between microbiomes of different sectors in animal agroecosystems.

Effects of selected emerging contaminants found in wastewater on antimicrobial resistance and horizontal gene transfer