Conjugative Transfer Research Articles

Real-time visualisation of the intracellular dynamics of conjugative plasmid transfer

Conjugation is a contact-dependent mechanism for the transfer of plasmid DNA between bacterial cells, which contributes to the dissemination of antibiotic resistance. Here, we use live-cell microscopy to visualise the intracellular dynamics of conjugative transfer of F-plasmid in E. coli, in real time. We show that the transfer of plasmid in single-stranded form (ssDNA) and its subsequent conversion into double-stranded DNA (dsDNA) are fast and efficient processes that occur with specific timing and subcellular localisation. Notably, the ssDNA-to-dsDNA conversion determines the timing of plasmid-encoded protein production. The leading region that first enters the recipient cell carries single-stranded promoters that allow the early and transient synthesis of leading proteins immediately upon entry of the ssDNA plasmid. The subsequent conversion into dsDNA turns off leading gene expression, and activates the expression of other plasmid genes under the control of conventional double-stranded promoters. This molecular strategy allows for the timely production of factors sequentially involved in establishing, maintaining and disseminating the plasmid.

Establishment of a visual gene knockout system based on CRISPR/Cas9 for the rare actinomycete Nonomuraea gerenzanensis.

To develop a modified CRISPR/Cas9 system with the β-glucuronidase (GusA) reporter and a dual sgRNA cassette for Nonomuraea gerenzanensis (N. gerenzanensis). With the aid of a visual GusA reporter, the complicated and tedious process of cloning and gene identification could be abandoned entirely in the genetic editing of N. gerenzanensis. Moreover, introducing a dual sgRNA cassette into the CRISPR/Cas9 system significantly improved gene deletion efficiency compared to the single sgRNA element. Furthermore, the length of the homologous flanking sequences set to the lowest value of 500bp in this system could still reach the relatively higher conjugation transfer frequency. The enhanced CRISPR/Cas9 system could efficiently perform genetic manipulation on the rare actinomycete N. gerenzanensis.

Conjugative RP4 Plasmid-Mediated Transfer of Antibiotic Resistance Genes to Commensal and Multidrug-Resistant Enteric Bacteria In Vitro

Many antibiotic-resistant bacteria carry resistance genes on conjugative plasmids that are transferable to commensals and pathogens. We determined the ability of multiple enteric bacteria to acquire and retransfer a broad-host-range plasmid RP4. We used human-derived commensal Escherichia coli LM715-1 carrying a chromosomal red fluorescent protein gene and green fluorescent protein (GFP)-labeled broad-host-range RP4 plasmid with ampR, tetR, and kanR in in vitro matings to rifampicin-resistant recipients, including Escherichia coli MG1655, Dec5α, Vibrio cholerae, Pseudomonas putida, Pseudomonas aeruginosa, Klebsiella pneumoniae, Citrobacter rodentium, and Salmonella Typhimurium. Transconjugants were quantified on selective media and confirmed using fluorescence microscopy and PCR for the GFP gene. The plasmid was transferred from E. coli LM715-1 to all tested recipients except P. aeruginosa. Transfer frequencies differed between specific donor-recipient pairings (10-2 to 10-8). Secondary retransfer of plasmid from transconjugants to E. coli LM715-1 occurred at frequencies from 10-2 to 10-7. A serial passage plasmid persistence assay showed plasmid loss over time in the absence of antibiotics, indicating that the plasmid imposed a fitness cost to its host, although some plasmid-bearing cells persisted for at least ten transfers. Thus, the RP4 plasmid can transfer to multiple clinically relevant bacterial species without antibiotic selection pressure.

Triadimefon Promoted Plasmid-mediated Conjugative Transfer of Multiple Antibiotic Resistance Genes within Bacterial Genus

The rapid spread of antibiotic resistance genes (ARGs) has been a worldwide threat to the public health, especially via horizontal gene transfer (HGT). Conjugation is one of the major pathways of HGT. Triadimefon (TF) is a broad-spectrum antifungal compound used to control rust and powdery mildew on crops. Nevertheless, it is not known whether TF could affect the conjugation of ARGs and potential molecular mechanisms. Here, the effects of TF on conjugative transfer of RP4 plasmid within Escherichia coli were systematically investigated. The results demonstrated that TF increased the transconjugant number by 1.41–2.21 folds and the frequency of conjugative transfer by 1.49–2.22 folds at concentrations of 0.1–10 mg/L. There was no obvious change in the number of donor and recipient strains in the mating system of intra-genus with TF. The main mechanisms include increasing intracellular reactive oxygen species and membrane permeability. Our findings highlight the promotion effect of TF on ARG conjugation, providing evidence of the risk of non-antibiotic agrochemical use in ARG dissemination.

Antibacterial activity, strain identification and DNA transfer by bacterial conjugation in Streptomyces sp. SS200

In the finding of new antimicrobial agents to respond to drug-resistant bacterial pathogens, the genus Streptomyces is still an important source of secondary metabolites associated with antibacterial activity. Strikingly, two-thirds of antibiotics used in human medicine are secondary metabolites from Streptomyces bacteria, among which the biosynthetic pathway of several antibiotics was studied. We report here the isolation of an endophytic bacterial strain from Bougainvillea glabra collected in Vung Tau City, Vietnam. This bacterial strain showed strong antibacterial activity against multiple pathogenic bacteria, including those belonging to the ESKAPE pathogens group. Cultural and molecular characterization identified the isolated strain as Streptomyces sp. SS200. A DNA transfer system by bacterial conjugation was established in Streptomyces sp. SS200 to pave the way for following studies on biosynthetic pathways of secondary metabolites having antibacterial activity in this strain. The key conditions in the conjugal transfer were investigated, including the recipient’s cellular forms, donor Escherichia coli strains, effects of cation2+, conjugation media, and antibiotics overlaying time. The mycelial form of Streptomyces sp. SS200 gave a 5-fold higher conjugation frequency than the spore form. ET12567 strain carrying the mobilizing plasmid pUZ8002 had the highest frequency of conjugation among two E. coli donors, specifically at a double frequency than SM10pir strain. Ca2+ gave more exconjugants than Mg2+, and the highest frequency of conjugation was obtained at 10 mM of this cation. Of four conjugation media, the exconjugants between E. coli and Streptomyces were only found on the Soya Flour Mannitol medium. The overlaying time of antibiotics was optimal at 24 hours of conjugation. These conjugal conditions will be used for the following experiments to find gene candidates involved in the biosynthesis of secondary metabolites with antibacterial activity in Streptomyces sp. SS200.

Nanoplastics promote the dissemination of antibiotic resistance through conjugative gene transfer: implications from oxidative stress and gene expression

S-PSNPs facilitated conjugative transfer frequency of ARGs between E. coli strains than L-PSNPs, and the mechanisms include SOS response, membrane permeability and altered related gene expression.

Dihydroartemisinin inhibits plasmid transfer in drug-resistant Escherichia coli via limiting energy supply.

Conjugative transfer of antibiotic resistance genes (ARGs) by plasmids is an important route for ARG dissemination. An increasing number of antibiotic and nonantibiotic compounds have been reported to aid the spread of ARGs, highlighting potential challenges for controlling this type of horizontal transfer. Development of conjugation inhibitors that block or delay the transfer of ARG-bearing plasmids is a promising strategy to control the propagation of antibiotic resistance. Although such inhibitors are rare, they typically exhibit relatively high toxicity and low efficacy in vivo and their mechanisms of action are inadequately understood. Here, we studied the effects of dihydroartemisinin (DHA), an artemisinin derivative used to treat malaria, on conjugation. DHA inhibited the conjugation of the IncI2 and IncX4 plasmids carrying the mobile colistin resistance gene ( mcr-1) by more than 160-fold in vitro in Escherichia coli, and more than two-fold (IncI2 plasmid) in vivo in a mouse model. It also suppressed the transfer of the IncX3 plasmid carrying the carbapenem resistance gene bla NDM-5 by more than two-fold in vitro. Detection of intracellular adenosine triphosphate (ATP) and proton motive force (PMF), in combination with transcriptomic and metabolomic analyses, revealed that DHA impaired the function of the electron transport chain (ETC) by inhibiting the tricarboxylic acid (TCA) cycle pathway, thereby disrupting PMF and limiting the availability of intracellular ATP for plasmid conjugative transfer. Furthermore, expression levels of genes related to conjugation and pilus generation were significantly down-regulated during DHA exposure, indicating that the transfer apparatus for conjugation may be inhibited. Our findings provide new insights into the control of antibiotic resistance and the potential use of DHA.

Charged nanoplastics differentially affect the conjugative transfer of antibiotic resistance genes

The transfer of antibiotic resistance genes was influenced by the concentration and surface charge of nanoplastics. This was due to a combination of the promotion effect of ROS and the inhibition caused by nanoplastic agglomeration.

Prevalence, antimicrobial susceptibility, and antibiotic resistance gene transfer of Bacillus strains isolated from pasteurized milk.

Pasteurization is carried out in dairy industries to kill harmful bacteria present in raw milk. However, endospore-forming bacteria, such as Bacillus, cannot be completely eliminated by pasteurization. In this study, a total of 114 Bacillus strains were isolated from 133 pasteurized milk samples. Antibiotic susceptibility tests showed that the percentage of Bacillus with intrinsic resistance to ampicillin and penicillin were 80 and 86%, respectively. Meanwhile, some Bacillus isolates had acquired resistance, including trimethoprim-sulfamethoxazole resistance (10 isolates), clindamycin resistance (8 isolates), erythromycin resistance (2 isolates), and tetracycline resistance (1 isolate). To further locate these acquired resistance genes, the plasmids were investigated in these 16 Bacillus strains. The plasmid profile indicated that Bacillus cereus BA008, BA117, and BA119 harbored plasmids, respectively. Subsequently, the Illumina Novaseq PE150 was applied for the genomic and plasmid DNA sequencing. Notably, the gene tetL encoding tetracycline efflux protein was found to be located on plasmid pBC46-TL of B. cereus BA117. In vitro conjugative transfer indicated that pBC46-TL can be transferred into Bacillus invictae BA142, Bacillus safensis BA143, and Bacillus licheniformis BA130. The frequencies were of 1.5 × 10-7 to 1.7 × 10-5 transconjugants per donor cells. Therefore, Bacillus strains with acquired antibiotic resistance may represent a potential risk for the spread of antibiotic resistance between Bacillus and other clinical pathogens via horizontal gene transfer.

Conjugation-mediated transfer of antibiotic resistance genes influenced by primary soil components and underlying mechanisms

Soil is the main natural reservoir of antibiotic resistant bacteria and antibiotic resistance genes (ARGs). Their dissemination and proliferation were largely motivated by conjugative transfer, while the influence of soil components on bacterial conjugative transfer and the underlying mechanisms remain poorly understood. In the present study, two Escherichia coli strains were exposed to soil minerals (quartz, kaolinite and montmorillonite) and organic matters (humic acid, biochar and soot) respectively to investigate their impact on ARGs conjugation. The results showed that quartz had no significant effect on conjugation; montmorillonite promoted the growth of the donor, but inhibited the recipient and conjugant; kaolinite and three organic matters significantly promoted the production of conjugant, while biochar promoted and then inhibited it with time prolong. Within the range of bacterial concentration involved in this study, the concentration of conjugant increased with the ratio of the concentration of donor and recipient (RD/R), indicating that the variation of conjugant production was mainly mediated by changing RD/R. Further observation of biochar treatment group showed that the bacterial responses such as cell membrane permeability, cell surface hydrophobicity and biofilm formation ability shifted with the exposure time, which might be a potential factor affecting conjugative transfer. Collectively, our findings suggest that the type and exposure time of soil components jointly affected conjugation, while the change of RD/R and related bacterial responses are the main underlying mechanisms.

Hormetic Effect of Pyroligneous Acids on Conjugative Transfer of Plasmid-mediated Multi-antibiotic Resistance Genes within Bacterial Genus.

Spread of antibiotic resistance genes (ARGs) by conjugation poses great challenges to public health. Application of pyroligneous acids (PA) as soil amendments has been evidenced as a practical strategy to remediate pollution of ARGs in soils. However, little is known about PA effects on horizontal gene transfer (HGT) of ARGs by conjugation. This study investigated the effects of a woody waste-derived PA prepared at 450°C and its three distillation components (F1, F2, and F3) at different temperatures (98, 130, and 220°C) on conjugative transfer of plasmid RP4 within Escherichia coli. PA at relatively high amount (40-100 μL) in a 30-mL mating system inhibited conjugation by 74-85%, following an order of PA > F3 ≈ F2 ≈ F1, proving the hypothesis that PA amendments may mitigate soil ARG pollution by inhibiting HGT. The bacteriostasis caused by antibacterial components of PA, including acids, phenols, and alcohols, as well as its acidity (pH 2.81) contributed to the inhibited conjugation. However, a relatively low amount (10-20 μL) of PA in the same mating system enhanced ARG transfer by 26-47%, following an order of PA > F3 ≈ F2 > F1. The opposite effect at low amount is mainly attributed to the increased intracellular reactive oxygen species production, enhanced cell membrane permeability, increased extracellular polymeric substance contents, and reduced cell surface charge. Our findings highlight the hormesis (low-amount promotion and high-amount inhibition) of PA amendments on ARG conjugation and provide evidence for selecting an appropriate amount of PA amendment to control the dissemination of soil ARGs. Moreover, the promoted conjugation also triggers questions regarding the potential risks of soil amendments (e.g., PA) in the spread of ARGs via HGT.

Comparative genomic analysis of plasmids harboring bla OXA-48-like genes in Klebsiella pneumoniae.

The emergence and spread of carbapenem-resistant Klebsiella pneumoniae (CRKP) is a serious medical problem worldwide. Acquired OXA-48-like carbapenemases encoded by plasmids are important causes of carbapenem resistance in K. pneumoniae. To explore the links between plasmids and bla OXA-48-like genes in K. pneumoniae, we systematically analyzed the variants of bla OXA-48-like plasmid replicon types, phylogenetic patterns, geographic distribution, conjugative transfer regions, and the genetic environments surrounding bla OXA-48-like of 191 bla OXA-48-like-harboring plasmids, which were identified from 4451 plasmids of K. pneumoniae downloaded from GenBank. Our results showed that seven different variants of bla OXA-48-like genes were identified from the 191 bla OXA-48-like-harboring plasmids in K. pneumoniae, with bla OXA-48, bla OXA-232, and bla OXA-181 being highly prevalent. In K. pneumoniae, bla OXA-48 was mainly carried by the composite transposon Tn1999.2 located on IncL/M-type conjugative plasmids, which were mainly geographically distributed in Switzerland, Germany, and China. In K. pneumoniae, the blaOXA-232 gene was mainly carried by 6.1-kb ColKP3-type mobilizable plasmids, which were mainly isolated in India. In K. pneumoniae, bla OXA-181 was mainly carried by a group of 50-kb ColKP3-IncX3 hybrid conjugative plasmids and a group of small ColKP3-type mobilizable plasmids with lengths of 5.9-9.3 kb, the former was sporadically discovered in China, South Korea, India, and Czech Republic, while the latter was almost all isolated in India. In addition, five bla OXA-245-harboring 65.9-kb IncL plasmids of K. pneumoniae isolated in Spain were found to have the genetic context of bla OXA-245 more complicated than that of bla OXA-48-harboring IncL/M-type plasmids, with two copies of IS1R inserted both upstream and downstream of bla OXA-245-lysR. These findings enhance our understanding of the genetic diversity of bla OXA-48-like-harboring plasmids in K. pneumoniae.

The Emerging Nosocomial Pathogen Klebsiella michiganensis: Genetic Analysis of a KPC-3 Producing Strain Isolated from Venus Clam.

The recovery and characterization of a multidrug-resistant, KPC-3-producing Klebsiella michiganensis that was obtained from Venus clam samples is reported in this study. A whole-genome sequencing (WGS) analysis using Illumina and Nanopore technologies of the K. michiganensis 23999A2 isolate revealed that the strain belonged to the new sequence type 382 (ST382) and carried seven plasmid replicon sequences, including four IncF type plasmids (FII, FIIY, FIIk, and FIB), one IncHI1 plasmid, and two Col plasmids. The FIB and FIIk plasmids showed high homology to each other and to multireplicon pKpQIL-like plasmids that are found in epidemic KPC-K. pneumoniae clones worldwide. The strain carried multiple β-lactamase genes on the IncF plasmids: blaOXA-9 and blaTEM-1A on FIB, blaKPC-3 inserted in a Tn4401a on FIIK, and blaSHV-12 on FIIY. The IncHI1-ST11 harbored no resistance gene. The curing of the strain caused the loss of all of the bla genes and a rearrangement of the IncF plasmids. Conjugal transfer of the blaOXA-9, blaTEM-1A and blaKPC-3 genes occurred at a frequency of 5 × 10-7, using K. quasipneumoniae as a recipient, and all of the bla genes were transferred through a pKpQIL that originated from the recombination of the FIB and FIIk plasmids of the donor. A comparison with 31 K. michiganensis genomes that are available in the NCBI database showed that the closest phylogenetic relatives of K. michiganensis 23999A2 are an environmental isolate from soil in South Korea and a clinical isolate from human sputum in Japan. Finally, a pan-genome analysis showed a large accessory genome of the strain as well as the great genomic plasticity of the K. michiganensis species. IMPORTANCE Klebsiella michiganensis is an emerging nosocomial pathogen, and, so far, few studies describe isolates of clinical origin in the environment. This study contributes to the understanding of how the dissemination of carbapenem-resistance outside the hospital setting may be related to the circulation of pKpQIL-like plasmids that are derived from epidemic Klebsiella pneumoniae strains. The recovery of a carbapenem-resistant isolate in clams is of great concern, as bivalves could represent vehicles of transmission of pathogens and resistance genes to humans via the food chain. The study demonstrates the plasticity of K. michiganensis genome, which is probably useful to multiple environment adaptation and to the evolution of the species.

Non-caloric artificial sweeteners modulate conjugative transfer of multi-drug resistance plasmid in the gut microbiota

ABSTRACT Non-caloric artificial sweeteners have been widely permitted as table sugar substitutes with high intensities of sweetness. They can pass through the intestinal tract without significant metabolization and frequently encounter the gut microbiome, which is composed of diverse bacterial species and is a pool of antibiotic resistance genes (ARGs). However, little is known about whether these sweeteners could accelerate the spread of ARGs in the gut microbiome. Here, we established an in vitro conjugation model by using Escherichia coli that carries chromosome-inserted Tn7 lacIq-pLpp-mCherry and plasmid-encoded gfpmut3b gene as the donor and murine fecal bacteria as the recipient. We found that four commonly used artificial sweeteners (saccharin, sucralose, aspartame, and acesulfame potassium) can increase reactive oxygen species (ROS) production and promote plasmid-mediated conjugative transfer to the gut microbiome. Cell sorting and 16S rRNA gene amplicon sequencing analysis of fecal samples reveal that the tested sweeteners can promote the broad-host-range plasmid permissiveness to both Gram-negative and Gram-positive gut bacteria. The increased plasmid permissiveness was also validated with a human pathogen Klebsiella pneumoniae. Collectively, our study demonstrates that non-caloric artificial sweeteners can induce oxidative stress and boost the plasmid-mediated conjugative transfer of ARGs among the gut microbiota and a human pathogen. Considering the soaring consumption of these sweeteners and the abundance of mobile ARGs in the human gut, our results highlight the necessity of performing a thorough risk assessment of antibiotic resistance associated with the usage of artificial sweeteners as food additives.

Cotransfer of resistance to cephalosporins, colistin, and fosfomycin mediated by an IncHI2/pSH16G4928-like plasmid in ESBL-producing monophasic Salmonella Typhimurium strains of pig origin.

To assess the co-transmission risk of phenotypic and genetic resistance to cephalosporins, colistin, and fosfomycin in Salmonella strains collected along the whole pork production chain. From a total of 107 Salmonella isolates from samples collected in pig slaughterhouses and markets, 15 ESBL-producing Salmonella strains resistant to cefotaxime were identified by broth microdilution method and clavulanic acid inhibition test, including 14 monophasic Salmonella Typhimurium strains and one Salmonella Derby strain. Whole genome sequence analysis showed that nine monophasic S. Typhimurium strains coresistant to colistin and fosfomycin carried the resistance genes blaCTX-M-14, mcr-1, and fosA3. Conjugational transfer tests demonstrated that the phenotypic and genetic resistance to cephalosporins, colistin, and fosfomycin could cotransfer back and forth between Salmonella and Escherichia coli via an IncHI2/pSH16G4928-like plasmid. This study reports the cotransmission of phenotypic and genetic resistance to cephalosporins, colistin, and fosfomycin via an IncHI2/pSH16G4928-like plasmid in Salmonella strains of animal origin, giving an alarm for the prevention of the development and spread of bacterial multidrug resistance.

Multiple relaxases contribute to the horizontal transfer of the virulence plasmids from the tumorigenic bacterium Pseudomonas syringae pv. savastanoi NCPPB 3335.

Pseudomonas syringae pv. savastanoi NCPPB 3335 is the causal agent of olive knot disease and contains three virulence plasmids: pPsv48A (pA), 80 kb; pPsv48B (pB), 45 kb, and pPsv48C (pC), 42 kb. Here we show that pB contains a complete MPFT (previously type IVA secretion system) and a functional origin of conjugational transfer adjacent to a relaxase of the MOBP family; pC also contains a functional oriT-MOBP array, whereas pA contains an incomplete MPFI (previously type IVB secretion system), but not a recognizable oriT. Plasmid transfer occurred on solid and in liquid media, and on leaf surfaces of a non-host plant (Phaseolus vulgaris) with high (pB) or moderate frequency (pC); pA was transferred only occasionally after cointegration with pB. We found three plasmid-borne and three chromosomal relaxase genes, although the chromosomal relaxases did not contribute to plasmid dissemination. The MOBP relaxase genes of pB and pC were functionally interchangeable, although with differing efficiencies. We also identified a functional MOBQ mobilization region in pC, which could only mobilize this plasmid. Plasmid pB could be efficiently transferred to strains of six phylogroups of P. syringae sensu lato, whereas pC could only be mobilized to two strains of phylogroup 3 (genomospecies 2). In two of the recipient strains, pB was stably maintained after 21 subcultures in liquid medium. The carriage of several relaxases by the native plasmids of P. syringae impacts their transfer frequency and, by providing functional diversity and redundancy, adds robustness to the conjugation system.

Conjugative IncC Plasmid Entry Triggers the SOS Response and Promotes Effective Transfer of the Integrative Antibiotic Resistance Element SGI1.

The broad-host-range IncC plasmid family and the integrative mobilizable Salmonella genomic island 1 (SGI1) and its derivatives enable the spread of medically important antibiotic resistance genes among Gram-negative pathogens. Although several aspects of the complex functional interactions between IncC plasmids and SGI1 have been recently deciphered regarding their conjugative transfer and incompatibility, the biological signal resulting in the hijacking of the conjugative plasmid by the integrative mobilizable element remains unknown. Here, we demonstrate that the conjugative entry of IncC/IncA plasmids is detected at an early stage by SGI1 through the transient activation of the SOS response, which induces the expression of the SGI1 master activators SgaDC, shown to play a crucial role in the complex biology between SGI1 and IncC plasmids. Besides, we developed an original tripartite conjugation approach to directly monitor SGI1 mobilization in a time-dependent manner following conjugative entry of IncC plasmids. Finally, we propose an updated biological model of the conjugative mobilization of the chromosomal resistance element SGI1 by IncC plasmids. IMPORTANCE Antimicrobial resistance has become a major public health issue, particularly with the increase of multidrug resistance (MDR) in both animal and human pathogenic bacteria and with the emergence of resistance to medically important antibiotics. The spread between bacteria of successful mobile genetic elements, such as conjugative plasmids and integrative elements conferring multidrug resistance, is the main driving force in the dissemination of acquired antibiotic resistances among Gram-negative bacteria. Broad-host-range IncC plasmids and their integrative mobilizable SGI1 counterparts contribute to the spread of critically important resistance genes (e.g., extended-spectrum β-lactamases [ESBLs] and carbapenemases). A better knowledge of the complex biology of these broad-host-range mobile elements will help us to understand the dissemination of antimicrobial resistance genes that occurred across Gammaproteobacteria borders.

Structural effects of sulfonamides on the proliferation dynamics of sulfonamide resistance genes in the sequencing batch reactors and the mechanism

Antibiotic resistance genes (ARGs) can be easily promoted by antibiotics, however, the structural effects of antibiotics on the proliferation of ARGs dynamic and the associated mechanisms remain obscure in, especially, activated sludge sequencing batch reactors. In the present study, the effects of 9 sulfonamides (SAs) with different structures on the proliferation dynamic of sulfonamide resistance genes (Suls) in the activated sludge sequencing batch reactors and the corresponding mechanisms were determined (30 days), and the results showed that the largest proliferation value (∆AR) of Suls dynamic for SAs (sulfachloropyridazine) was approximately 2.9 times than that of the smallest one (sulfadiazine). The proliferation of Suls was significantly related to the structural features (minHBint6, SssNH, SHBd and SpMax2_Bhm) that represent the biological activity of SAs. To interpret the phenomenon, a mechanistic model was developed and the results indicated that the biodegradation of SAs (T1/2) rather than conjugative transfer frequency or mutation frequency tends to be the key process for affecting Suls proliferation. T1/2 was proved to be dependent on the interactions between SAs and receptors (Ebinding), the cleavage mode (bond dissociation energy), and the site of nucleophilic assault. Besides, the metagenomic analysis showed that SAs posed significant effect on antibiotic resistome and Tnp31 played a vital role in the proliferation of Suls. Overall, our findings provide important insight into a theoretical basis for understanding the structural effects of SAs on the proliferation of ARGs in SBR systems.

Genetic heterogeneity and predominance of blaCTX-M -15 in cefotaxime-resistant Enterobacteriaceae isolates colonizing hospitalized children in Tunisia.

Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae have emerged as important nosocomial pathogens. Community infections by these organisms have been also reported and were associated with previous intestinal colonization. We aimed to characterize cefotaxime-resistant Enterobacteriaceae (CTX-R-En) isolated from hospitalized children in a Tunisian paediatric ward. Seventy CTX-R-En isolates were collected from 227 rectal swabs from hospitalized children in a paediatric ward. Antimicrobial susceptibility testing was determined according to the EUCAST guidelines. Isolates were characterized by polymerase chain reaction (PCR, genes encoding: ESBLs, pAmpC, carbapenemases, plasmid-mediated quinolone resistance, virulence factors in Escherichia coli and Klebsiella pneumoniae isolates, occurrence of classes 1 and 2 integrons, phylogenetic groups of E.coli isolates, ERIC-PCR and PCR-based replicon typing) and conjugal transfer experiments. In total, 65 out of 227 (28·6%) hospitalized children were colonized with CTX-M-R-En, and 70 isolates were identified. Isolates were 59 ESBL-, 7 plasmidic-AmpC (pAmpC)-, 3 ESBL+pAmpC-, and one ESBL+carbapenemase producers. The following bla genes were identified: blaCTX-M-15 (n = 54), blaCTX-M-1 (n = 5), blaCTX-M-9 (n = 2), blaCTX-M-13 (n = 1) and blaCTX-M-14 (n = 1), blaCMY-2 (n = 5), blaCMY-4 (n = 4), blaACC-1 (n = 1) and blaOXA-48 (n = 1). Our results showed that hospitalized children were colonized with various CTX-R-En-producing several beta-lactamase enzymes.

Dose-dependent joint resistance action of antibacterial mixtures in their hormetic effects on bacterial resistance based on concentration addition model

The judgment of joint resistance action is significant for evaluating the resistance risk of antibacterial mixture. Using bacterial mutation frequency (MF) and conjugative transfer frequency (CTF) to respectively characterize the bacterial endogenous and exogenous resistance, mutation unit and conjugative transfer unit have been proposed to judge the joint resistance action of antibacterial mixture at a certain dose. However, these methods could not evaluate the antibacterial mixture's joint resistance action at a larger concentration-range. In this study, the concentration addition for bacterial resistance (CA-BR) approach was used to judge the joint resistance actions between kanamycin sulfate (KAN) and some other typical antibacterial agents, including sulfonamides (SAs), sulfonamide potentiators (SAPs), and silver antibacterial compounds (SACs). Through comparing the hormetic dose-response curves of the binary mixtures on the MF (or CTF) in Escherichia coli (E. coli) and the corresponding CA-BR curves calculated from the hormetic dose-responses of the single agents, the joint resistance actions between KAN and other agents were judged to exhibit dose-dependent feature: with the increase of mixture concentration, the joint mutation actions between KAN and SAs (or SAPs) were fixed at synergism, and the joint mutation actions between KAN and SACs varied from antagonism to synergism; the joint conjugative transfer actions between KAN and other agents changed from antagonism to synergism. Mechanistic explanation suggested that the heterogeneous pattern of joint resistance action had a close relationship with the interplays among the agents' modes of action, and meanwhile was significantly influenced by their joint survival pressure on E. coli. This study reveals the dose-dependent feature for the joint resistance action of antibacterial mixture and highlights the importance of exposure concentration, which will benefit clarifying the resistance risk of antibacterial mixture in the environment.