Antibiotic Resistance Plasmids Research Articles

Effect of non-antibiotic factors on conjugative transfer of antibiotic resistance genes in aquaculture water

Aquaculture water with antibiotic resistance genes (ARGs) is escalating due to the horizontal gene transfer. Non-antibiotic stressors specifically found, including those from fishery feed and disinfectants, are potential co-selectors. However, the mechanisms underlying this process remains unclear. Intragenus and intergenus conjugative transfer systems of the antibiotic-resistant plasmid RP4 were established to examine conjugative transfer frequency under exposure to five widely used non-antibiotic factors in aquaculture water: iodine, oxolinic acid, NO2-N, NO3-N and H2O2 and four different recipient bacteria: E. coli HB101, Citrobacter portucalensis SG1, Vibrio harveyi and Vibrio alginolyticus. The study found that low concentrations of non-antibiotic factors significantly promoted conjugative transfer, whereas high concentrations inhibited it. Moreover, the conjugation transfer efficiencies were significantly different with different bacterial species within (E. coli HB101 ∼ 10-3 %) or cross genera (C. portucalensis SG1 ∼10-5 %, V. harveyi ∼1 %). Besides, excessive exposure concentrations inhibited the expression of related genes and the generation of reactive oxygen species (ROS). Regulation of multiple related genes and ROS-induced SOS responses are common primary mechanisms. However, the mechanisms of non-antibiotic factors differ from those of standard antibiotics, with direct changes in cell membrane permeability potentially playing a dominant role. Additionally, variations among non-antibiotic factors and the specific characteristics of bacterial species contribute to differences in conjugation mechanisms. Notably, this study found that non-antibiotic factors could increase the frequency of intergeneric conjugation beyond that of intrageneric conjugation. Furthermore, non-antibiotic factors influenced by multiple transport systems may raise the risk of unintended cross-resistance, significantly amplifying the potential for resistance gene spread. This study underscores the significance of non-antibiotic factors in the propagation of ARGs, highlighting their role in advancing aquaculture development and protecting human health.

Multidrug-resistant ESBL-producing Klebsiella pneumoniae complex in Czech hospitals, wastewaters and surface waters

BackgroundMultidrug-resistant (MDR) bacteria pose a significant challenge to the treatment of infectious diseases. Of particular concern are members of the Klebsiella pneumoniae species complex (KpSC), which are frequently associated with hospital-acquired infections and have the potential to spread outside hospitals via wastewaters. In this study, we aimed to investigate the occurrence and phylogenetic relatedness of MDR KpSC from patients with urinary tract infections (UTIs), hospital sewage, municipal wastewater treatment plants (mWWTPs) and surface waters and to evaluate the clinical relevance of the KpSC subspecies.MethodsA total of 372 KpSC isolates resistant to third-generation cephalosporins and/or meropenem were collected from patients (n = 130), hospital sewage (n = 95), inflow (n = 54) and outflow from the mWWTPs (n = 63), river upstream (n = 13) and downstream mWWTPs (n = 17) from three cities in the Czech Republic. The isolates were characterized by antimicrobial susceptibility testing and whole-genome sequencing (Illumina). The presence of antibiotic resistance genes, plasmid replicons and virulence-associated factors was determined. A phylogenetic tree and single nucleotide polymorphism matrix were created to reveal the relatedness between isolates.ResultsThe presence of MDR KpSC isolates (95%) was identified in all water sources and locations. Most isolates (99.7%) produced extended-spectrum beta-lactamases encoded by blaCTX−M−15. Resistance to carbapenems (5%) was observed mostly in wastewaters, but carbapenemase genes, such as blaGES−51 (n = 10), blaOXA−48 (n = 4), blaNDM−1 (n = 4) and blaKPC−3 (n = 1), were found in isolates from all tested locations and different sources except rivers. Among the 73 different sequence types (STs), phylogenetically related isolates were observed only among the ST307 lineage. Phylogenetic analysis revealed the transmission of this lineage from patients to the mWWTP and from the mWWTP to the adjacent river and the presence of the ST307 clone in the mWWTP over eight months. We confirmed the frequent abundance of K. pneumoniae (K. pneumoniae sensu stricto and K. pneumoniae subsp. ozaenae) in patients suffering from UTIs. K. variicola isolates formed only a minor proportion of UTIs, and K. quasipneumoniae was not found among UTIs isolates; however, these subspecies were frequently observed in hospital sewage communities during the first sampling period.ConclusionThis study provides evidence of the transmission and persistence of the ST307 lineage from UTIs isolates via mWWTPs to surface waters. Isolates from UTIs consisted mostly of K. pneumoniae. Other isolates of KpSC were observed in hospital wastewaters, which implies the impact of sources other than UTIs. This study highlights the influence of urban wastewaters on the spread of MDR KpSC to receiving environments.

Interrelation Between Pathoadaptability Factors and Crispr-Element Patterns in the Genomes of Escherichia coli Isolates Collected from Healthy Puerperant Women in Ural Region, Russia.

Escherichia coli is a commensal and opportunistic bacterium widely distributed around the world in different niches including intestinal of humans and animals, and its extraordinary genome plasticity led to the emergence of pathogenic strains causing a wide range of diseases. E. coli is one of the monitored species in maternity hospitals, being the main etiological agent of urogenital infections, endometriosis, puerperal sepsis, and neonatal diseases. This study presents a comprehensive analysis of E. coli isolates obtained from the maternal birth canal of healthy puerperant women 3-4 days after labor. According to whole genome sequencing data, 31 sequence types and six phylogenetic groups characterized the collection containing 53 isolates. The majority of the isolates belonged to the B2 phylogroup. The data also includes phenotypic and genotypic antibiotic resistance profiles, virulence factors, and plasmid replicons. Phenotypic and genotypic antibiotic resistance testing did not demonstrate extensive drug resistance traits except for two multidrug-resistant E. coli isolates. The pathogenic factors revealed in silico were assessed with respect to CRISPR-element patterns. Multiparametric and correlation analyses were conducted to study the interrelation of different pathoadaptability factors, including antimicrobial resistance and virulence genomic determinants carried by the isolates under investigation. The data presented will serve as a valuable addition to further scientific investigations in the field of bacterial pathoadaptability, especially in studying the role of CRISPR/Cas systems in the E. coli genome plasticity and evolution.

Altered genomic methylation promotes Staphylococcus aureus persistence in hospital environment

Staphylococcus aureus can cause outbreaks and becomes multi-drug resistant through gene mutations and acquiring resistance genes. However, why S. aureus easily adapts to hospital environments, promoting resistance and recurrent infections, remains unknown. Here we show that a specific S. aureus lineage evolved from a clone that expresses the accessory gene regulator (Agr) system to subclones that reversibly suppressed Agr and caused an outbreak in the hospital setting. S. aureus with flexible Agr regulation shows increased ability to acquire antibiotic-resistant plasmids, escape host immunity, and colonize mice. Bacteria with flexible Agr regulation shows altered cytosine genomic methylation, including the decreased 5mC methylation in transcriptional regulator genes (pcrA and rpsD), compared to strains with normal Agr expression patterns. In this work, we discover how altered genomic methylation promotes flexible Agr regulation which is associated with persistent pathogen colonization in the hospital environment.

Antibiotic resistance plasmids in Enterobacteriaceae isolated from fresh produce in northern Germany.

This study showed that Enterobacteriaceae from raw vegetables carried plasmids ranging in size from 2,715 to 326,286 bp, of which about less than one-third carried antibiotic resistance genes encoding resistance toward antibiotics such as tetracyclines, aminoglycosides, fosfomycins, sulfonamides, quinolones, and β-lactam antibiotics. Some strains encoded multiple resistances, and some encoded extended-spectrum β-lactamases. The study highlights the potential of produce, which may be eaten raw, as a potential vehicle for the transfer of antibiotic-resistant bacteria.

Co-Occurrence of Two Plasmids Encoding Transferable blaNDM-1 and tet(Y) Genes in Carbapenem-Resistant Acinetobacter bereziniae.

Acinetobacter bereziniae has emerged as a significant human pathogen, acquiring multiple antibiotic resistance genes, including carbapenemases. This study focuses on characterizing the plasmids harboring the blaNDM-1 and tet(Y) genes in two carbapenem-resistant A. bereziniae isolates (UCO-553 and UCO-554) obtained in Chile during the COVID-19 pandemic. Methods: Antibiotic susceptibility testing was conducted on UCO-553 and UCO-554. Both isolates underwent whole-genome sequencing to ascertain their sequence type (ST), core genome multilocus sequence-typing (cgMLST) profile, antibiotic resistance genes, plasmids, and mobile genetic elements. Conjugation experiments were performed for both isolates. Results: Both isolates exhibited broad resistance, including resistance to carbapenems, third-generation cephalosporins, fluoroquinolones, tetracycline, cotrimoxazole, and aminoglycosides. Both isolates belong to sequence type STPAS1761, with a difference of 17 out of 2984 alleles. Each isolate carried a 47,274 bp plasmid with blaNDM-1 and aph(3')-VI genes and two highly similar plasmids: a 35,184 bp plasmid with tet(Y), sul2, aph(6)-Id, and aph(3″)-Ib genes, and a 6078 bp plasmid containing the ant(2″)-Ia gene. Quinolone-resistance mutations were identified in the gyrA and parC genes of both isolates. Importantly, blaNDM-1 was located within a Tn125 transposon, and tet(Y) was embedded in a Tn5393 transposon. Conjugation experiments successfully transferred blaNDM-1 and tet(Y) into the A. baumannii ATCC 19606 strain, indicating the potential for horizontal gene transfer. Conclusions: This study highlights the critical role of plasmids in disseminating resistance genes in A. bereziniae and underscores the need for the continued genomic surveillance of this emerging pathogen. The findings emphasize the importance of monitoring A. bereziniae for its potential to cause difficult-to-treat infections and its capacity to spread resistance determinants against clinically significant antibiotics.

Comparison of Plasmid Curing Efficiency across Five Lactic Acid Bacterial Species.

With the recent stringent criteria for antibiotic susceptibility in probiotics, the presence of antibiotic resistance genes and plasmids associated with their transfer has become a limiting factor in the approval of probiotics. The need to remove genes related to antibiotic resistance and virulence through plasmid curing for the authorization of probiotics is increasing. In this study, we investigated the curing efficiency of ethidium bromide, acridine orange, and novobiocin at different concentrations and durations in five strains of plasmid-bearing lactic acid bacteria and examined the curing characteristics in each strain. Limosibacillus reuteri and Lacticaseibacillus paracasei exhibited curing efficiencies ranging from 5% to 44% following treatment with ethidium bromide (10-50 μg/ml) for 24-72 h, while Lactobacillus gasseri showed the highest efficiency at 14% following treatment with 10 μg/ml novobiocin for 24 h. Lactiplantibacillus plantarum, which harbors two or more plasmids, demonstrated curing efficiencies ranging from 1% to 8% after an additional 72-h treatment of partially cured strains with 10 μg/ml novobiocin. Plasmid curing in strains with larger plasmids exhibited lower efficiencies and required longer durations. In strains harboring two or more plasmids, a relatively low curing efficiency with a single treatment and a high frequency of false positives, wherein recovery occurred after curing, were observed. Although certain strains exhibited altered susceptibilities to specific antibiotics after curing, these outcomes could not be attributed to the loss of antibiotic resistance genes. Furthermore, the genomic data from the cured strains revealed minimal changes throughout the genome that did not lead to gene mutations.

Loss of Antibiotic Resistance Transposons if grown in the absence of Antibiotics

In a recent article I have proven F is not a single replicon but co-integrate of two complete replicons – F and R. These two replicons are not allowed to function simultaneously but their individual functional states are stringently controlled by the transposon Tn 1000(5.7 Kb). Besides these two replicons we have previously mentioned the Ent plasmid P307. In 1977 we characterized the antibiotic resistance plasmid pCG86 in ETEC isolated from the stool of a piglet with diarrhoeal disease. The genes for heat-labile enterotoxin and heat-stable enterotoxin were also carried by the same plasmid pCG86. In the year 1975 we received this same bacterial isolate of Carlton Gyles (Department of Veterinary Microbiology and Immunology, University of Guelph, Ontario Canada) from Professor S. Falkow (University of California) who saved the stab at room temperature but without adding antibiotics. The same strain can’t grow in the presence of antibiotics. After the discovery of transposons, I felt the re-interpretation of this heteroduplex molecule formed between pCG86 and Ent P307 is absolutely necessary.

Impact of aeration rate on the transfer range of antibiotic-resistant plasmids during manure composting

Conjugative plasmids are important vectors of mobile antibiotic resvistance genes (ARGs), facilitating their horizontal transfer within the environment. While composting is recognized as an effective method to reduce antibiotics and ARGs in animal manure, its impact on the bacterial host communities containing antibiotic-resistant plasmids remains unclear. In this study, we investigated the permissiveness of bacterial community during composting when challenged with multidrug-resistant conjugative RP4 plasmids, employing Pseudomonas putida as the donor strain. Ultimately, this represents the first exploration of the effects of aeration rates on the range of RP4 plasmid transfer hosts. Transconjugants were analyzed through fluorescent reporter gene–based fluorescence-activated cell sorting and Illumina sequencing. Overall, aeration rates were found to influence various physicochemical parameters of compost, including temperature, pH, total organic matter, total nitrogen, and potassium. Regarding RP4 plasmid host bacteria, the dominant phylum was determined to shift from Bacteroidetes in the raw material to Proteobacteria in the compost. Notably, a moderate-intensity aeration rate (0.05 L/min/L) was found to be more effective in reducing the diversity and richness of the RP4 plasmid host bacterial community. Following composting, the total percentage of dominant transconjugant-related genera decreased by 66.15–76.62%. Ultimately, this study determined that the aeration rate negatively impacts RP4 plasmid host abundance primarily through alterations to the environmental factors during composting. In summary, these findings enhance our understanding of plasmid host bacterial communities under varying composting aeration rates and offer novel insights into preventing the dissemination of ARGs from animal manure to farmland.

CRISPR-Cas inhibits plasmid transfer and immunizes bacteria against antibiotic resistance acquisition in manure.

The horizontal transfer of antibiotic resistance genes among bacteria is a pressing global issue. The bacterial defense system clustered regularly interspaced short palindromic repeats (CRISPR)-Cas acts as a barrier to the spread of antibiotic resistance plasmids, and CRISPR-Cas-based antimicrobials can be effective to selectively deplete antibiotic-resistant bacteria. While significant surveillance efforts monitor the spread of antibiotic-resistant bacteria in the clinical context, a major, often overlooked aspect of the issue is resistance emergence in agriculture. Farm animals are commonly treated with antibiotics, and antibiotic resistance in agriculture is on the rise. Yet, CRISPR-Cas efficacy has not been investigated in this setting. Here, we evaluate the prevalence of CRISPR-Cas in agricultural Enterococcus faecalis strains and its antiplasmid efficacy in an agricultural niche: manure. Analyzing 1,986 E. faecalis genomes from human and animal hosts, we show that the prevalence of CRISPR-Cas subtypes is similar between clinical and agricultural E. faecalis strains. Using plasmid conjugation assays, we found that CRISPR-Cas is a significant barrier against resistance plasmid transfer in manure. Finally, we used a CRISPR-based antimicrobial approach to cure resistant E. faecalis of erythromycin resistance, but this was limited by delivery efficiency of the CRISPR antimicrobial in manure. However, immunization of bacteria against resistance gene acquisition in manure was highly effective. Together, our results show that E. faecalis CRISPR-Cas is prevalent and effective in an agricultural setting and has the potential to be utilized for depleting antibiotic-resistant populations. Our work has broad implications for tackling antibiotic resistance in the increasingly relevant agricultural setting, in line with a One Health approach.IMPORTANCEAntibiotic resistance is a growing global health crisis in human and veterinary medicine. Previous work has shown technologies based on CRISPR-Cas-a bacterial defense system-to be effective in tackling antibiotic resistance. Here we test if CRISPR-Cas is present and effective in agricultural niches, specifically in the ubiquitously present bacterium, Enterococcus faecalis. We show that CRISPR-Cas is both prevalent and functional in manure and has the potential to be used to specifically kill bacteria carrying antibiotic resistance genes. This study demonstrates the utility of CRISPR-Cas-based strategies for control of antibiotic resistance in agricultural settings.

SHEDDING LIGHT ON GENETIC RELATIONSHIPS, PLASMID PROFILES AND ANTIBIOTIC RESISTANCE PATTERNS OF CLINICAL ESCHERICHIA COLI STRAINS: PULSED-FIELD GEL ELECTROPHORESIS PERSPECTIVE

In the present work, 47 Escherichia coli isolates collected from various clinical samples were elucidated with regard to genetic relationship, antibiotic resistance and plasmid profile. The genetic diversity of the isolates was defined by the pulsed field gel electrophoresis (PFGE) method. With a degree of similarity of 80%, the results from PFGE separated the E. coli strains into 39 different groups representing four subtypes. Antibiotic resistance patterns and extended spectrum of beta lactamase (ESBL) producing properties of the strains were determined by using a disk diffusion and double disc synergy method, respectively. According to the susceptibility test results, 36 distinct resistance profiles (resisto type) were observed among clinical strains. The strains were mostly resistant to ampicillin (100%) and this has been followed by cephalothin and ticarcillin/clavulanic acid with the ratio of 89.36%, cefuroxime with the ratio of 87.24%, tetracycline and cefotaxime with the ratio of 80.85%. It was observed that 24 of the strains (51.06%) were defined as ESBL positive. When E. coli strains were evaluated according to plasmid size, it was determined that 27 of 47 strains (57.44%) carried plasmids and the sizes of the determined plasmids were ranging between 77.1 kb and 1.6 kb. It was concluded that plasmids of E. coli strains are randomly distributed and no significant correlation was found between antibiotic resistance patterns and plasmids.

Investigation of the impact of widely used pesticides on conjugative transfer of multidrug resistance plasmids

Plasmid-mediated conjugative transfer has emerged as a major driver accounting for the dissemination of antibiotic resistance genes (ARGs). In addition to the use of antimicrobial agents, there is growing evidence that non-antibiotic factors also play an important role. Pesticides are widely used to protect crops against vectors of diseases, and are indispensable agents in agricultural production, whereas the impact of pesticide pollution on the transmission of antimicrobial resistance remains poorly understood. Here we reveal that the pesticides at environmentally relevant concentrations, especially cyromazine (Cyr) and kresoxim-methyl (Kre), greatly facilitate the conjugative transfer of antibiotic-resistance plasmids carrying clinically important ARGs. Mechanistic studies indicate that Cyr and Kre treatments trigger reactive oxygen species (ROS) production and SOS response, increase membrane permeability, upregulate bacterial proton motive force (PMF) and promote ATP supply. Further non-targeted metabolomics and biochemical analysis demonstrate that the addition of Cyr and Kre accelerates tricarboxylic acid (TCA) cycle and electron transport chain (ETC), thereby activating bacterial energy metabolism. In the constructed soil model, we prove that two pesticides contribute to the dissemination of resistance plasmids in the soil microbiota. 16S rRNA sequencing analyses indicate that pesticides alter transconjugant microbial communities, and enable more opportunistic pathogens, such as Pseudomonas and Enterobacter, to acquire the multidrug resistance plasmids. Collectively, our work indicates the potential risk in accelerating the spread of antimicrobial resistance owing to pesticide pollution, highlighting the importance of continuous surveillance of pesticide residues in complex environmental settings.

Genome analysis of multidrug resistant Enterococcus faecium and Enterococcus faecalis circulating among hospitalized patients in uMgungundlovu District, KwaZulu-Natal, South Africa

BackgroundVancomycin-resistant enterococci (VRE) are important pathogens categorized as high-priority bacteria in the Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics published by the World Health Organization. The aim of this study was to determine the risk factors, resistance, virulence, mobilomes associated with multidrug-resistant and clonal lineages of Enterococcus faecium and faecalis circulating among hospitalized patients following the health system in South Africa, using whole genome sequencing (WGS).MethodsA cross-sectional study was conducted during a two-month periods among hospitalized patients in 2017. Rectal swabs were collected from patients admitted to medical and surgical wards in an urban tertiary hospital, and a rural district hospital in uMgungundlovu district, South Africa. Enterococci were screened for vancomycin resistance on bile esculin azide agar supplemented with 6 mg/L of vancomycin and confirmation of VRE was done using ROSCO kits. Conventional and real-time PCR methods were used to ascertain the presence of VanA, VanB, VanC-2/3 and VanC-1 genes. All six multidrug-resistant Enterococcus faecalis and faecium selected were identified using multiplexed paired-end libraries (2 × 300 bp) with the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA) and genome sequencing was done using Illumina MiSeq instrument with 100× coverage at the National Institute of Communicable Diseases Sequencing Core Facility, South Africa. Antibiotic resistance genes, virulence factors, plasmids, integrons and CRISPR were characterized using RAST, ResFinder, VirulenceFinder, PlasmidFinder, PHAST and ISFinder respectively.ResultsSequencing analysis revealed that these strains harbouring numerous resistance genes to glycopeptides (vanC[100%], vex3[100%], vex2[83,33%] and vanG[16,66%]), macrolides, lincosamides, sterptogramine B (ermB[33,32%], Isa[16,66%], emeA[16,66%]) and tetracyclines (tetM[33,32%]) in both district and tertiary hospitals. Multidrug efflux pumps including MATE, MFS and pmrA conferring resistance to several classes of antibiotics were also identified. The main transposable elements observed were in the Tn3 family, specifically Tn1546. Four single sequence types (STs) were identified among E. faecium in the district hospital, namely ST822, ST636, ST97 along with a novel ST assigned ST1386, while one lineage, ST29 was detected in the tertiary hospital.ConclusionThe study reveals the genetic diversity and high pathogenicity of multidrug-resistant Enterococcus faecalis and faecium circulating among hospitalized patients. It underlines the necessity to implement routine screening of admitted patients coupled with infection control procedures, antimicrobial stewardship and awareness should be strengthened to prevent and/or contain the carriage and spread of multidrug resistant E. faecium and E. faecalis in hospitals and communities in South Africa.

β-lactamase expression induces collateral sensitivity in Escherichia coli

Major antibiotic groups are losing effectiveness due to the uncontrollable spread of antimicrobial resistance (AMR) genes. Among these, β-lactam resistance genes –encoding β-lactamases– stand as the most common resistance mechanism in Enterobacterales due to their frequent association with mobile genetic elements. In this context, novel approaches that counter mobile AMR are urgently needed. Collateral sensitivity (CS) occurs when the acquisition of resistance to one antibiotic increases susceptibility to another antibiotic and can be exploited to eliminate AMR selectively. However, most CS networks described so far emerge as a consequence of chromosomal mutations and cannot be leveraged to tackle mobile AMR. Here, we dissect the CS response elicited by the acquisition of a prevalent antibiotic resistance plasmid to reveal that the expression of the β-lactamase gene blaOXA-48 induces CS to colistin and azithromycin. We next show that other clinically relevant mobile β-lactamases produce similar CS responses in multiple, phylogenetically unrelated E. coli strains. Finally, by combining experiments with surveillance data comprising thousands of antibiotic susceptibility tests, we show that β-lactamase-induced CS is pervasive within Enterobacterales. These results highlight that the physiological side-effects of β-lactamases can be leveraged therapeutically, paving the way for the rational design of specific therapies to block mobile AMR or at least counteract their effects.

Molecular Characteristics of Salmonella Spp. Responsible for Bloodstream Infections in a Tertiary Hospital in Nanjing, China, 2019-2021.

To investigate the clinical and molecular characteristics of Salmonella spp. causing bloodstream infections (BSIs) in our hospital. We studied 22 clinical Salmonella isolates from BSIs and 16 from non-BSIs, performing antimicrobial susceptibility testing (AST) and whole genome sequencing (WGS). The analysis included serovars, antibiotic resistance genes (ARGs), virulence factors (VFs), sequence types (STs), plasmid replicons, and genetic relationships. We also assessed pathogenicity of the isolates causing BSIs through growth, biofilm formation, and anti-serum killing assays. WGS analysis identified 13 Salmonella serovars, with four responsible for BSIs. S. Enteritidis was the most prevalent serovar, involved in 19 (50.0%) cases. BSIs were caused by 17S. Enteritidis, two S. Typhimurium, two S. Munster and one S. Diguel. Of the 38 isolates, 27 (71.1%) exhibited high resistance to ampicillin, and 24 (63.2%) to ampicillin/sulbactam. Thirty-six types of ARGs were identified, with blaTEM-1B (n = 25, 65.8%) being the most frequent. Ten plasmid replicons were found; the combination of IncFIB(S)-IncFII(S)-IncX1 was the most common in S. Enteritidis (94.7%). Fifteen STs were identified, among which ST11 was the most prevalent and clonally disseminated, primarily responsible for BSIs. A total of 333 different VFs were detected, 177 of which were common across all strains. No significant differences were observed between the BSI and non-BSI isolates in terms of resistance rates, ARGs, plasmid replicons, and VFs, except for seven VFs. No strong pathogenicity was observed in the BSI-causing isolates. BSIs were predominantly caused by clonally disseminated S. Enteritidis ST11, the majority of which carried multiple ARGs, VFs and plasmid replicons. This study provides the first data on clonally disseminated S. Enteritidis ST11 causing BSIs, highlighting the urgent need for enhanced infection control measures.

Genome sequence, antibiotic resistance genes, and plasmids in a monophasic variant of Salmonella typhimurium isolated from retail pork.

A Salmonella isolate from retail pork was whole genome sequenced using Illumina NovaSeq6000, with a 5,320,119 bp genome and 51.06% GC content. Several antibiotic resistance genes and plasmids, including blaTEM-1, aac(6')-IIc, IncHI2, and p0111 were obtained from subsequent analysis. These findings provide vital insights into generic determinants of antimicrobial resistance (AMR) in this foodborne pathogen.

Type I BREX system defends against antibiotic-resistant plasmids in Escherichia coli.

The Bacteriophage Exclusion (BREX) system is a novel antiphage defense system identified in Bacillus cereus in 2015. The purpose of this study was to investigate the presence of the BREX system defenses against antibiotic-resistant plasmids such as blaKPC and blaNDM invasion in Escherichia coli. The BREX system was present in 5.4% (23/424) of E. coli clinical isolates and 6.5% (84/1283) of E. coli strains with completely sequenced genomes in the GenBank database. All 23 BREX-positive E. coli clinical isolates were susceptible to carbapenems, while all five isolates carrying blaKPC and 11 carrying blaNDM were BREX-negative. For E. coli strains in the GenBank database, 37 of 38 strains carrying blaKPC and 109 of 111 strains carrying blaNDM were BREX negative. The recognition site sequence of methyltransferase PglX in a clinical E. coli 3756 was 5'-CANCATC-3' using PacBio single-molecular real-time sequencing. The transformation efficiency of plasmid psgRNA-ColAori-target with the PglX recognition site was reduced by 100% compared with the plasmid without the recognition site in E. coli DH5α-pHSG398-BREX. The BREX showed lower defense efficacy against plasmid psgRNA-15Aori-target which had the same plasmid backbone but different surrounding sequences of recognition sites with psgRNA-ColAori-target. The conjugation frequency of the KPC-2 plasmid and NDM-5 plasmid in E. coli 3756-ΔBREX was higher than that in E. coli 3756 clinical isolate (1.0 × 10-6 vs 1.3 × 10-7 and 5.5 × 10-7 vs 1.7 × 10-8, respectively). This study demonstrated that the type I BREX system defends against antibiotic-resistant plasmids in E. coli.

PlasmidEC and gplas2: an optimized short-read approach to predict and reconstruct antibiotic resistance plasmids in Escherichia coli.

Accurate reconstruction of Escherichia coli antibiotic resistance gene (ARG) plasmids from Illumina sequencing data has proven to be a challenge with current bioinformatic tools. In this work, we present an improved method to reconstruct E. coli plasmids using short reads. We developed plasmidEC, an ensemble classifier that identifies plasmid-derived contigs by combining the output of three different binary classification tools. We showed that plasmidEC is especially suited to classify contigs derived from ARG plasmids with a high recall of 0.941. Additionally, we optimized gplas, a graph-based tool that bins plasmid-predicted contigs into distinct plasmid predictions. Gplas2 is more effective at recovering plasmids with large sequencing coverage variations and can be combined with the output of any binary classifier. The combination of plasmidEC with gplas2 showed a high completeness (median=0.818) and F1-Score (median=0.812) when reconstructing ARG plasmids and exceeded the binning capacity of the reference-based method MOB-suite. In the absence of long-read data, our method offers an excellent alternative to reconstruct ARG plasmids in E. coli.

Dissemination of antibiotic resistance genes is regulated by iron oxides: Insight into the influence on bacterial transformation

The transportation of antibiotic resistance genes (ARGs) in manure-soil-plant continuums poses risks to human health. Horizontal gene transfer, particularly for bacterial transformation, is an important way for ARG dissemination. As crucial components in soils, iron oxides impacted the fates of various abiotic and biotic contaminants due to their active properties. However, whether they can influence the transformation of ARGs is unknown, which waits to be figured out to boost the assessment and control of ARG spread risks. In this study, we have investigated the effects of goethite, hematite, and magnetite (0–250 mg/L, with sizes < 100 nm and > 100 nm) on the transfer of ampicillin resistance genes to Escherichia coli cells. At lower iron oxide concentrations, the transformation of ARGs was first facilitated (transformation frequency reached up to 3.38-fold higher), but the facilitating effects gradually weakened and eventually disappeared as concentrations further increased. Particle size and iron oxide type were not the universal determinants controlling the transformation. At lower concentrations, iron oxides interacted with proteins and phospholipids in E. coli envelope structures, and induced the overgeneration of intracellular reactive oxygen species. Consequently, they led to pore formation and permeability enhancement on the cell membrane, thus promoting the transformation. The facilitation was also associated with the carrier-like effect of iron oxides for antibiotic resistance plasmids. At higher concentrations, the weakened facilitations were attributed to the aggregation of iron oxides. In this study, we highlight the crucial roles of the concentrations (contents) of iron oxides on the dissemination of ARGs in soils; this study may serve as a reference for ARG pollution control in future agricultural production.

Photoaged microplastics enhanced the antibiotic resistance dissemination in WWTPs by altering the adsorption behavior of antibiotic resistance plasmids

Growing concerns have raised about the microplastic eco-coronas in the ultraviolet (UV) disinfection wastewater, which accelerated the pollution of antibiotic resistance genes (ARGs) in the aquatic environment. As the hotspot of gene exchange, microplastics (MPs), especially for the UV-aged MPs, could alter the spread of ARGs in the eco-coronas and affect the resistance of the environment through adsorbing antibiotic resistant plasmids (ARPs). However, the relationship between the MP adsorption for ARPs and ARG spreading characteristics in MP eco-corona remain unclear. Herein, this study explored the distribution of ARGs in the MP eco-corona through in situ investigations of the discharged wastewater, and the adsorption behaviors of MPs for ARPs by in vitro adsorption experiments and in silico calculations. Results showed that the adsorption capacity of MPs for ARPs was enhanced by 42.7–48.0 % and the adsorption behavior changed from monolayer to multilayer adsorption after UV-aging. It was related to the increased surface roughness and oxygen-containing functional groups of MPs under UV treatment. Moreover, the abundance of ARGs in MP eco-corona of UV-treated wastewater was 1.33–1.55 folds higher than that without UV treatment, promoting the proliferation of drug resistance. DFT and DLVO theoretical calculations indicated that the MP-ARP interactions were dominated by electrostatic physical adsorption, endowing the aged MPs with low potential oxygen-containing groups to increase the electrostatic interaction with ARPs. Besides, due to the desorption of ARPs on MPs driven by the electrostatic repulsion, the bioavailability of ARGs in the MP eco-coronas was increased with pH and decreased with salinity after the wastewater discharge. Overall, this study advanced the understanding of the adsorption behavior of MPs for ARPs and provided inspirations for the evaluation of the resistance spread in the aquatic environment mediated by MP eco-coronas.