Conjugation Frequency Research Articles

Residual concentrations of antimicrobial growth promoters in poultry litter favour plasmid conjugation among Escherichia coli.

Considering that plasmid conjugation is a major driver for the dissemination of antimicrobial resistance in bacteria, this study aimed to investigate the effects of residual concentrations of antimicrobial growth promoters (AGPs) in poultry litter on the frequencies of IncFII-FIB plasmid conjugation among Escherichia coli organisms. A 2 × 5 factorial trial was performed invitro, using two types of litter materials (sugarcane bagasse and wood shavings) and five treatments of litter: non-treated (CON), herbal alkaloid sanguinarine (SANG), AGPs monensin (MON), lincomycin (LCM) and virginiamycin (VIR). E.coli H2332 and E.coli J62 were used as donor and recipient strains, respectively. The presence of residues of monensin, lincomycin and virginiamycin increased the frequency of plasmid conjugation among E.coli in both types of litter materials. On the contrary, sanguinarine significantly reduced the frequency of conjugation among E.coli in sugarcane bagasse litter. The conjugation frequencies were significantly higher in wood shavings compared with sugarcane bagasse only in the presence of AGPs. Considering that the presence of AGPs in the litter can increase the conjugation of IncFII-FIB plasmids carrying antimicrobial resistance genes, the real impact of this phenomenon on the dissemination of antimicrobial resistant bacteria in the poultry production chain must be investigated.

Environmentally relevant concentrations of triclosan exposure promote the horizontal transfer of antibiotic resistance genes mediated by Edwardsiella piscicida.

Aquaculture pathogen and antibiotic resistance genes (ARGs) co-occur in the aquatic environment. Accumulated evidence suggests that aquaculture pathogens can facilitate the horizontal transfer of plasmid-mediated ARGs. However, the role of Edwardsiella piscicida (E. piscicida) in ARG dissemination is still not fully understood. In addition, the potential impact of triclosan (TCS) on the spread of ARGs mediated by E. piscicida is still unknown, so a mating model system was established to investigate the transfer process of ARGs. The results showed that E. piscicida disseminated ARGs on RP4 by horizontal gene transfer (HGT). Furthermore, TCS exposure promoted this process. The conjugative transfer frequencies were enhanced approximately 1.2–1.4-fold by TCS at concentrations from 2 to 20 μg/L, when compared with the control. TCS promoted the HGT of ARGs by stimulating reactive oxygen species (ROS) production, increasing cell membrane permeability, and altering expressions of conjugative transfer–associated genes. Together, the results suggested that aquaculture pathogens spread ARGs and that the emerging contaminant TCS enhanced the transfer of ARGs between bacteria.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11356-022-20082-8.

Conjugative transfer of mcr-1-bearing plasmid from Salmonella to Escherichia coli in vitro on chicken meat and in mouse gut

Since mcr-1 was first discovered in 2015, this gene has shown excellent transmission ability and evolutionary characteristics worldwide, leading to major public health and food safety concerns. In this study, chicken meat was used as a food vehicle for the conjugation of mcr-1-bearing Salmonella at different storage temperatures (4 °C, 25 °C, and 37 °C) to simulate mcr-1 transmission during food transportation and storage and determine its efficiency and mechanism. In addition, conjugation experiments were performed in mouse gut to further confirm that mcr-1 is horizontally transferred in vivo during food consumption. 16S rDNA sequencing of mouse stool samples was performed to understand the effect of horizontal transfer of mcr-1 on mouse gut bacteria. mcr-1-bearing plasmids were characterized using pulsed-field gel electrophoresis (PFGE) and S1 nuclease-PFGE and sequenced by Illumina sequencing. Our results showed that mcr-1-bearing plasmids in donors are successfully transferred to recipients on chicken meat at not only 25 °C and 37 °C but also 4 °C with conjugation frequencies between 1.32 × 10-6 and 3.85 × 10-4 per recipient cell. In mouse gut, mcr-1 was transferred not only to the recipient bacteria introduced by intragastric administration but also to the intestinal bacteria (E. coli strain named as E6353). Horizontal transfer of mcr-1-bearing plasmid in mouse gut negatively affected the mouse intestinal microbiota. In a constant conjugative environment, plasmid replicon type is the most decisive factor affecting the conjugation frequency. The peak number of transconjugants in group D6-E. coli C600 with an IncHI2-type mcr-1-bearing plasmid (1.43 × 102 colony-forming units [CFU]/g feces) was significantly higher than that of transconjugants in group D7-E. coli C600 with an IncX4-type mcr-1-bearing plasmid (0.3 × 102 CFU/g feces). The upstream and downstream genetic environment of mcr-1 in different plasmid replicon types in Salmonella varied during conjugation in different horizontal transfer environments. An IncI2 plasmid (p25-D4R7S1_mcr-1) lost the insertion sequence ISApl1, which originally existed upstream of mcr-1, when this plasmid transferred from donor to recipient cells on chicken meat at 25 °C. An IncHI2 plasmid was more active than IncI2 and IncX4 plasmids during bacterial reproduction and evolution; an IncFIB–IncHI2 hybrid plasmid (p6176253_mcr-1) was formed in mouse gut during conjugation from pD6_tet(M) and pD6_mcr-1. mcr-1 is captured by mobile genetic elements IS26 in IncX4 plasmids and ISApl1 in IncI2, IncHI2, and IncFIB–IncHI2 hybrid plasmids and is disseminated among bacteria.

Antimicrobial activity and safety features assessment of Weissella spp. from environmental sources.

Weissella strains have been reported to be useful in biotechnological and probiotic determinations, and some of them are considered opportunistic pathogens. Given the widespread interest about antimicrobial susceptibilities, transmission of resistances, and virulence factors, there is little research available on such topics for Weissella. The aim of this study was to assess the safety aspects and antimicrobial potential of 54 Weissella spp. strains from different environmental sources. Antibiotic susceptibility, hemolytic activity, horizontal transfer, and antibacterial activity were studied, as well as the detection of biogenic amine BA production on decarboxylase medium and PCR was performed. All the strains were nonhemolytic and sensitive to chloramphenicol and ampicillin. Several strains were classified as resistant to fusidic acid, and very low resistance rates were detected to ciprofloxacin, tetracycline, streptomycin, lincomycin, erythromycin, and rifampicin, although all strains had intrinsic resistance to vancomycin, nalidixic acid, kanamycin, and teicoplanin. Two BA‐producing strains (W. halotolerans FAS30 and FAS29) exhibited tyrosine decarboxylase activity, and just one W. confusa FS077 produced both tyramine and histamine, and their genetic determinants were identified. Ornithine decarboxylase/odc gene was found in 16 of the Weissella strains, although 3 of them synthesize putrescine. Interestingly, eight strains with good properties displayed antibacterial activity. Conjugation frequencies of erythromycin from Bacillus to Weissella spp. varied in the average of 3 × 10−9 transconjugants/recipient. However, no tetracycline‐resistant transconjugant was obtained with Enterococcus faecalis JH2‐2 as recipient. The obtained results support the safe status of Weissella strains, derived from environmental sources, when used as probiotics in animal feed.

Response mechanisms of different antibiotic-resistant bacteria with different resistance action targets to the stress from photocatalytic oxidation

The stress response of antibiotic-resistant bacteria (ARB) and the spread of antibiotic resistance genes (ARGs) pose a serious threat to the aquatic environment and human beings. This study mainly explored the effect of the heterogeneous photocatalytic oxidation (UVA-TiO2 system) on the stress response mechanism of ARB with different antibiotic resistance action targets, including the cell wall, proteins, DNA, RNA, folate and the cell membrane. Results indicate that the stress response mechanism of tetracycline- and sulfamethoxazole-resistant E. coli DH5α, which targets the synthesis of protein and folate, could rapidly induce global regulators by the overexpression of relative antibiotic resistance action target genes. Different stress response systems were mediated via cross-protection mechanism, causing stronger tolerance to an adverse environment than other ARB. Moreover, the photocatalytic inactivation mechanism of bacterial cells and a graded response of cellular stress mechanism caused differences in the intensity of the stress mechanism of antibiotic resistance action targets. E. coli DH5α resistant to cefotaxime and polymyxin, targeting synthesis of the cell wall and cell membrane, respectively, could confer greater advantages to bacterial survival and higher conjugative transfer frequency than E. coli DH5α resistant to nalidixic acid and rifampicin, which target the synthesis of DNA and RNA, respectively. This new perspective provides detailed information on the practical application of photocatalytic oxidation for inactivating ARB and hampering the spreading of ARGs in the aquatic environment.

Analysis of the complete genome sequences of Clostridium perfringens strains harbouring the binary enterotoxin BEC gene and comparative genomics of pCP13-like family plasmids

BackgroundBEC-producing Clostridium perfringens is a causative agent of foodborne gastroenteritis. It was first reported in 2014, and since then, several isolates have been identified in Japan and the United Kingdom. The novel binary ADP-ribosylating toxin BEC, which consists of two components (BECa and BECb), is encoded on a plasmid that is similar to pCP13 and harbours a conjugation locus, called Pcp, encoding homologous proteins of the type 4 secretion system. Despite the high in vitro conjugation frequency of pCP13, its dissemination and that of related plasmids, including bec-harbouring plasmids, in the natural environment have not been characterised. This lack of knowledge has limited our understanding of the genomic epidemiology of bec-harbouring C. perfringens strains.ResultsIn this study, we determined the complete genome sequences of five bec-harbouring C. perfringens strains isolated from 2009 to 2019. Each isolate contains a ~ 3.36 Mbp chromosome and 1–3 plasmids of either the pCW3-like family, pCP13-like family, or an unknown family, and the bec-encoding region in all five isolates was located on a ~ 54 kbp pCP13-like plasmid. Phylogenetic and SNP analyses of these complete genome sequences and the 211 assembled C. perfringens genomes in GenBank showed that although these bec-harbouring strains were split into two phylogenetic clades, the sequences of the bec-encoding plasmids were nearly identical (>99.81%), with a significantly smaller SNP accumulation rate than that of their chromosomes. Given that the Pcp locus is conserved in these pCP13-like plasmids, we propose a mechanism in which the plasmids were disseminated by horizontal gene transfer. Data mining showed that strains carrying pCP13-like family plasmids were unexpectedly common (58/216 strains) and widely disseminated among the various C. perfringens clades. Although these plasmids possess a conserved Pcp locus, their ‘accessory regions’ can accommodate a wide variety of genes, including virulence-associated genes, such as becA/becB and cbp2. These results suggest that this family of plasmids can integrate various foreign genes and is transmissible among C. perfringens strains.ConclusionThis study demonstrates the potential significance of pCP13-like plasmids, including bec-encoding plasmids, for the characterisation and monitoring of the dissemination of pathogenic C. perfringens strains.

Effects of hematite on the dissemination of antibiotic resistance in pathogens and underlying mechanisms

The dissemination of antibiotic resistance genes (ARGs) in pathogens is becoming a pervasive global health threat, to which the importance of the environment attracts more and more attention. However, how natural minerals affect ARGs transfer in pathogens is still unclear. In this study, the concentration and size effects of hematite on the ARGs conjugative transfer to a common zoonotic pathogen Escherichia coli O157:H7 and underlying mechanisms were explored. Results revealed that bulk hematite reduced the conjugation of resistant plasmids by inhibiting cell growth at any concentration (1–100 mg/L), different from nano-hematite. Low concentrations of nano-hematite (≤ 25 mg/L) induced significant increases in conjugative transfer frequency of 1.83–4.49 folds, while its high concentrations (50 and 100 mg/L) showed no impact, compared with the control group. This low-concentration effect was likely attributed to the increased intracellular ROS level, the reduced intercellular repulsion by increasing the extracellular polymeric substances production and cell surface hydrophobicity, the formation of transfer channels and the increased membrane permeability evidenced by significant changes in gene expression level, and the increased proton motive force by increasing the transmembrane potential of recipients. These findings shed light on potential health risks caused by nano minerals-mediated ARGs dissemination in pathogens in the environment.

Occurrence and Characteristics of Mcrs among Gram-Negative Bacteria Causing Bloodstream Infections of Infant Inpatients between 2006 and 2019 in China.

ABSTRACTThe aim of this study was to determine the occurrence of mobilized colistin resistance (mcr) genes in Gram-negative bacteria causing bloodstream infections of child inpatients in China. Bacteria were collected between 2006 and 2019 in a maternal and child health hospital, and mcr genes were screened by PCR. Five of 252 isolates were mcr-positive, including one mcr-1-positive colistin-resistant Escherichia coli isolate, two mcr-9-positive colistin-susceptible Salmonella enterica isolates, and two mcr-9-positive colistin-susceptible Enterobacter hormaechei isolates. These were obtained from two neonate and three infant patients admitted between 2009 and 2018. The E. coli isolate was obtained from a neonate aged 20 min, suggestive of a possible mother-to-neonate transmission. The five mcr-positive isolates were multidrug resistant, and two S. enterica and one E. hormaechei isolate showed a hypervirulent phenotype compared to a hypervirulent Klebsiella pneumoniae type strain in a Galleria mellonella infection model. The mcr-1 gene was carried by an IncX4-type pA1-like epidemic plasmid, and the mcr-9 gene was detected on IncHI2/2A-type novel plasmids co-carrying multiple resistance genes. The four IncHI2/2A-type plasmids shared a backbone and a high similarity (≥77% coverage and ≥ 90% nucleotide identity), suggesting that they were derived from a common ancestor with cross-species transmission and have circulated locally over a long period. The conjugation assay showed that the mcr-1-encoding plasmid and one mcr-9-encoding plasmid were self-transmissible to E. coli with high conjugation frequencies. Our findings demonstrate that mcr genes have disseminated in the community and/or hospitals, mediated by epidemic/endemic plasmids over a long period. The study shows that continuous monitoring of mcr genes is imperative for understanding and tackling their dissemination.IMPORTANCE Antimicrobial resistance, especially the spread of carbapenemase-producing Enterobacteriaceae (CPE), represents one of the largest challenges to One Health coverage of environmental, animal, and human sectors. Colistin is one of the last-line antibiotics for clinical treatment of CPE. However, the emergence of the mobilized colistin resistance (mcr) gene largely threatens the usage of colistin in the clinical setting. In this study, we investigated the existence of mcr genes in 252 Gram-negative bacteria collected between 2006 and 2019 which caused bloodstream infections of child inpatients in China. We found a high prevalence of mcr carriage among children inpatients in the absence of professional exposure, and mcr might have widely disseminated in the community via different routes. This study emphasizes the importance of rational use of colistin in the One Health frame, and highlights both the urgent need for understanding the prevalence and dissemination of mcr genes in different populations and the importance of effective measures to control their spread.

Mechanism and potential risk of antibiotic resistant bacteria carrying last resort antibiotic resistance genes under electrochemical treatment

The significant rise in the number of antibiotic resistance genes (ARGs) that resulted from our abuse of antibiotics could do severe harm to public health as well as to the environment. We investigated removal efficiency and removal mechanism of electrochemical (EC) treatment based on 6 different bacteria isolated from hospital wastewater carrying 3 last resort ARGs including NDM-1, mcr-1 and tetX respectively. We found that the removal efficiency of ARGs increased with the increase of both voltage and electrolysis time while the maximum removal efficiency can reach 90%. The optimal treatment voltage and treatment time were 3 V and 120 min, respectively. Temperature, pH and other factors had little influence on the EC treatment process. The mechanism of EC treatment was explored from the macroscopic and microscopic levels by scanning electron microscopy (SEM) and flow cytometry. Our results showed that EC treatment significantly changed the permeability of cell membrane and caused cells successively experience early cell apoptosis, late cell apoptosis and cell necrosis. Moreover, compared with traditional disinfection methods, EC treatment had less potential risks. The conjugative transfer frequencies of cells were significantly reduced after treatment. Less than 1% of bacteria entered the viable but nonculturable (VBNC) state and less than 5% of intracellular ARGs (iARGs) turned into extracellular ARGs (eARGs). Our findings provide new insights into as well as important reference for future electrochemical treatment in removing ARB from hospital wastewater.

The Co-occurrence of NDM-5, MCR-1, and FosA3-Encoding Plasmids Contributed to the Generation of Extensively Drug-Resistant Klebsiella pneumoniae.

The rise and global dissemination of extensively drug-resistant (XDR) bacteria are often related to plasmid-borne mobile antimicrobial resistance genes. Notably, isolates having multiple plasmids are often highly resistant to almost all the antibiotics available. In this study, we characterized an extensively drug-resistant Klebsiella pneumoniae 1678, which exhibited high-level resistance to almost all the available antibiotics. Through whole-genome sequencing (WGS), more than 20 resistant elements and 5 resistant plasmids were observed. Notably, the tigecycline resistance of K. pneumoniae 1678 was not related to the plasmid-borne tetA gene but associated with the overexpression of AcrAB and OqxAB efflux pumps, according to the susceptibility results of tetA-transformant and the related mRNA quantification of RND efflux pumps. Except for tigecycline resistance, three plasmids, mediating resistance to colistin, Fosfomycin, and ceftazidime–avibactam, respectively, were focused. Detailed comparative genetic analysis showed that all these plasmids belonged to dominated epidemic plasmids, and harbored completed conjugation systems. Results of conjugation assay indicated that these three plasmids not only could transfer to E. coli J53 with high conjugation frequencies, respectively, but also could co-transfer to E. coli J53 effectively, which was additionally confirmed by the S1-PFGE plasmids profile. Moreover, multiple insertion sequences (IS) and transposons (Tn) were also found surrounding the vital resistant genes, which may form several novel mechanisms involved in the resistant determinants’ mobilization. Overall, we characterized and reported the uncommon co-existence and co-transferring of FosA3-, NDM-5, and MCR-1-encoding plasmids in a K. pneumoniae isolate, which may increase the risk of spread of these resistant phenotypes and needing great concern.

Differences in recipient ability of uropathogenic Escherichia coli strains in relation with their pathogenic potential.

Conjugation is recognized as a mechanism driving dissemination of antibacterial resistances and virulence factors among bacteria. In the presented work conjugative transfer frequency into clinical uropathogenic Escherichia coli strains (UPEC) isolated from patients with symptomatic urinary tract infections was investigated. From 93 obtained UPEC strains only 29 were suitable for conjugation experiments with the plasmid pOX38, a well-known F-plasmid derivative. The study was focused on comparison of conjugation frequencies in plankton and biofilm, including comparison of conjugation frequencies in high and low biofilm biomass with their virulence potential. It was shown that the conjugation frequency depended on the biofilm biomass and was significantly higher in thin (OD580<0.3) than in thick biofilm (OD580≥0.3). Nonmetric multidimensional scaling analysis revealed that higher conjugation frequencies in plankton and biofilm were directly positively correlated with the sum of virulence-associated genes of the recipient strain and presence of multidrug antibiotic resistances. On the other hand, the sum of insensitivities to different bacteriocins was negatively correlated with an increase in the conjugative transfer level. Our results obtained hence indicate that the evolution of potentially more pathogenic strains via conjugation is depended on the strains' ability to be a "good" recipient in the conjugative transfer, possibly due to the ability to form thinner biofilms.

A simple judgment method for joint action of antibacterial agents on bacterial resistance

The severe pollution of bacterial resistance induced by the wide and even indiscriminate use of antibacterial agents has posed serious threats to human health and ecological safety. Furthermore, the combined effects of antibacterial agents have a closer relationship with the pollution of bacterial resistance than single antibacterial agent. However, there is little information regarding how multiple antibacterial agents interplay to induce bacterial resistance. Here, we developed a simple judgment method with five basic procedures for the joint action of antibacterial agents on bacterial resistance, involving toxicity determination, mutation frequency determination, conjugative transfer frequency determination, dose-response relationship fitting, key parameters obtaining, and joint resistance action judgment. This proposed approach was validated through investigating the joint resistance action between silver nanoparticle (AgNP) and 1-pyrrolidino-1-cyclohexene (1P1C, a kind of quorum sensing inhibitors). According to the procedures, the mutation unit and conjugative transfer unit for the AgNP-1P1C mixture were calculated to be 64.27 and 5.10, respectively, indicating the antagonism for their joint resistance action. This method can not only benefit the mechanistic explanation for how mixed antibacterial agents stimulate the bacterial resistance, but also guide the environmental risk assessment and clinical use of combined antibacterial agents in the related fields.• We present a novel method to judge the joint resistance action of antibacterial agents, taking the emergence and dissemination of antibiotic resistance genes into account.• Toxicity determination can help to design the mixtures of antibacterial agents and confirm the appropriate test concentration range of antibacterial agents used in mutation frequency and conjugative transfer frequency determination.• The mutation unit and conjugative transfer unit were proposed according to the toxic unit in the judgment of joint toxic action.

Environmental free radicals efficiently inhibit the conjugative transfer of antibiotic resistance by altering cellular metabolism and plasmid transfer

Spread of antibiotic-resistant genes (ARGs) is a global public safety issue and inhibition their transfer is imperative. In this study, a novel strategy using environmental free radical exposure was developed to inhibit conjugative transfer of ARGs (RP4 plasmid) in aqueous solutions. Long-time free radical (·OH, 1O2, and O2·−) exposure significantly suppressed the conjugative transfer frequency of ARGs between Escherichia coli (E. coli) strains, and ·OH was more likely to attack ARG, thereby inhibiting the conjugate transfer frequency, compared to 1O2 and O2·−. Compared with the control, the conjugative transfer frequency significantly decreased from 4.08 × 10−5 to 1.2 × 10−8 after 10 min free radical exposure, confirming that the transfer and proliferation of ARGs were well inhibited. Correspondingly, the number of transconjugant significantly decreased by 61.7% after 10 min free radical exposure. Significant reductions in reactive oxygen species levels (ROS content and enzyme levels) and DNA damage-induced responses in the donor strains were observed after 10 min free radical exposure. Concurrently, intercellular contact was also weakened via inhibiting the synthesis of polysaccharides in extracellular polymeric substances. Moreover, the expressions of plasmid transfer genes were down-regulated after 10 min exposure due to the shortage of adenosine-triphosphate supply. This study firstly disclosed the underneath mechanisms for depressing ARGs transfer and dissemination via environmental free radical exposure.

Melatonin prevents conjugative transfer of plasmid-mediated antibiotic resistance genes by disrupting proton motive force

The widespread dissemination of antibiotic resistance genes (ARGs) is a serious problem and constitutes a threat for public health. Plasmid-mediated conjugative transfer of ARGs is recognized as one of the most important pathways accounting for this global crisis. Inhibiting the conjugative transfer of resistant gene-bearing plasmids provides a feasible strategy to prevent the spread of antibiotic resistance. Here we found that melatonin, a neurohormone secreted from pineal gland, substantially inhibited the horizontal transfer of RP4–7 plasmid in a dose-dependent manner. Furthermore, melatonin could also suppress the conjugal frequency of different types of clinical plasmids that carrying colistin resistance gene mcr-1 rather than blaNDM or tet(X) genes. Next, we investigated the mechanisms underlying the inhibitory effect of melatonin on conjugation. As a result, we showed that the addition of melatonin markedly reduced bacterial membrane permeability and inhibited the oxidative stress. In line with these observations, the conjugative transfer-related genes were regulated accordingly. Most importantly, we uncovered that melatonin disrupted bacterial proton motive force (PMF), which is an essential bacterial energy metabolism substance and is important for conjugative process. Collectively, these results provide implications that some non-antibiotics such as melatonin are effective inhibitors of transmission of ARGs and raise a promising strategy to confront the increasing resistant infections.

Modulation of AggR levels reveals features of virulence regulation in enteroaggregative E. coli

Enteroaggregative Escherichia coli (EAEC) strains are one of the diarrheagenic pathotypes. EAEC strains harbor a virulence plasmid (pAA2) that encodes, among other virulence determinants, the aggR gene. The expression of the AggR protein leads to the expression of several virulence determinants in both plasmids and chromosomes. In this work, we describe a novel mechanism that influences AggR expression. Because of the absence of a Rho-independent terminator in the 3′UTR, aggR transcripts extend far beyond the aggR ORF. These transcripts are prone to PNPase-mediated degradation. Structural alterations in the 3′UTR result in increased aggR transcript stability, leading to increased AggR levels. We therefore investigated the effect of increased AggR levels on EAEC virulence. Upon finding the previously described AggR-dependent virulence factors, we detected novel AggR-regulated genes that may play relevant roles in EAEC virulence. Mutants exhibiting high AggR levels because of structural alterations in the aggR 3′UTR show increased mobility and increased pAA2 conjugation frequency. Furthermore, among the genes exhibiting increased fold change values, we could identify those of metabolic pathways that promote increased degradation of arginine, fatty acids and gamma-aminobutyric acid (GABA), respectively. In this paper, we discuss how the AggR-dependent increase in specific metabolic pathways activity may contribute to EAEC virulence.

Effects of voltage on the emergence and spread of antibiotic resistance genes in microbial electrolysis cells: From mutation to horizontal gene transfer

Microbial electrolysis cells (MECs) are widely considered as promising alternatives for degrading antibiotics. As one of the major operating parameters in MECs, voltage might affect the spread of antibiotic resistance genes (ARGs) given it can affect the physiological characteristics of bacteria. However, little is known about the impacts of voltage on the acceleration of bacterial mutation and the promotion of ARG dissemination via horizontal transfer in MECs. In this study, two voltages (0.9 V and 1.5 V) were applied to identify if electrical stimulation could increase bacterial mutation frequency. Three voltages (0.9 V, 1.5 V, and 2.5 V) were used to evaluate the conjugative transfer frequency of plasmid-encoded the ARGs from the donor (E. coli K-12) to the recipient (E. coli HB101) in MECs. After repeating subculture in MECs for 10 days, the mutation frequency of E. coli K-12 was promoted, consequently, the generated mutants became more resistant against tetracycline. When the voltage was higher than 0.9 V, conjugative ARG transfer frequency was significantly increased in the anode chamber (p < 0.05). The over-production of reactive oxygen species (ROS) (voltage >0.9 V) and cell membrane permeability (voltage >1.5 V) were significantly enhanced under electrical stimulations (p < 0.05). Genome-wide RNA sequencing indicated that the expressions of genes related to oxidative stress and cell membrane were upregulated with exposure to electrical stimulation. Electrical stimulations induced oxidative reactions, which triggered ROS over-production, SOS response, and enhancement of cell membrane permeability for both donor and recipient in the MECs. These findings provide insights into the potential role of voltage in the generation and spread of ARGs in MECs.

Transfer of plasmids harbouring blaOXA-48-like carbapenemase genes in biofilm-growing Klebsiella pneumoniae: Effect of biocide exposure

The spread of OXA-48-encoding plasmids from Klebsiella pneumoniae (OXA-48-Kpn), especially successful high-risk (HR) clones, is a growing concern. Biofilm formation can contribute to the dissemination of OXA-48-Kpn. It is not known whether biocides can affect the transfer of OXA-48-Kpn in biofilm. The aim of this study was to evaluate the effect of biocides on the conjugation frequency (CF) of OXA-48-Kpn in both biofilm and planktonic cultures. For that, seven OXA-48-Kpn isolates (4 belonging to HR clones and 3 to non-HR clones) were selected as donors. Each isolate was mixed (1:1) with Escherichia coli J53 (recipient) and grown on polystyrene microplates without biocides (control) and with 0.25x MIC of triclosan (TRI), chlorhexidine digluconate (CHX), povidone-iodine (POV), sodium hypochlorite (SOD) or ethanol (ETH). The CF was calculated as the number of transconjugants/number of E. coli J53. The results showed that for isolates growing in the absence of biocide, the mean fold change in the CF in biofilm with respect to that determined in planktonic cells (CF-BF/CF-PK) was 0.2 in non-HR isolates and ranged from 2.0 to 14.7 in HR isolates. In HR isolates grown in the presence of biocide, especially CHX, TRI, and ETH, the fold changes in CF-BF/CF-PK decreased, whereas in non-HR isolates the fold changes were similar or increased slightly with CHX, ETH, SOD and POV. In conclusion, the fold changes in the CF-BF/CF-PK are higher in HR isolates comparing to non-HR isolates in abscence of biocides. The fold changes in CF-BF/CF-PK of the HR isolates in the presence of biocides varied with the type of biocides, whereas in non-HR isolates, biocides have no significant effect, or produce only a slight increase in the fold change of CF-BF/CF-PK.

Inhibited conjugative transfer of antibiotic resistance genes in antibiotic resistant bacteria by surface plasma

Antibiotic resistant bacteria (ARB) and resistance genes (ARGs) are emerging environmental pollutants with strong pathogenicity. In this study, surface plasma was developed to inactivate the donor ARB with Escherichia coli (AR E. coli) as a model, eliminate ARGs, and inhibit conjugative transfer of ARGs in water, highlighting the influences of concomitant inorganic ions. Surface plasma oxidation significantly inactivated AR E. coli, eliminated ARGs, and inhibited conjugative transfer of ARGs, and the presence of NO3−, Cu2+, and Fe2+ all promoted these processes, and SO42− did not have distinct effect. Approximately 4.5log AR E. coli was inactivated within 10 min treatment, and it increased to 7.4log AR E. coli after adding Fe2+. Integrons intI1 decreased by 3.10log (without Fe2+) and 4.43log (adding Fe2+); the addition of Fe2+ in the surface plasma induced 99.8% decline in the conjugative transfer frequency. The inhibition effects on the conjugative transfer of ARGs were mainly attributed to the reduced reactive oxygen species levels, decreased DNA damage-induced response, decreased intercellular contact, and down-regulated expression of plasmid transfer genes. This study disclosed underlying mechanisms for inhibiting ARGs transfer, and supplied a prospective technique for ARGs control.

Cunning plasmid fusion mediates antibiotic resistance genes represented by ESBLs encoding genes transfer in foodborne Salmonella

Foodborne disease caused by antibiotic resistant Salmonella is quite difficult to deal with. In order to further explore the antibiotic resistance associated with gene transfer among foodborne Salmonella, several wild-type Salmonella strains were used as donors and recipients, respectively, to investigate how extended spectrum β-lactamases (ESBLs) encoding genes co-transfer with transposable elements to transmit antibiotic resistance. Antibiotic susceptibility was determined by agar dilution method, the transposase encoding gene was detected via PCR combined with DNA sequencing, S1 nuclease and pulsed field gel electrophoresis (S1-PFGE), and southern-blot. Illumina HiSeq 4000 platform and Nanopore MinION long-read sequencing technology were used to determine the antibiotic resistance encoding genes (ARGs) and their surrounding gene environment. The results indicated that the conjugation frequency was from ×10−4 to ×10−5 per recipient cell. A 185,608-bp-long DNA fragment and two short backbone protein encoding regions in pG19 in the donor fused with part genes in pS3 in the recipient during conjugation, the size of this fusion plasmid is as same as that of pG19. Cefoxitin resistance of the transconjugant was mediated by a tnpA21-related blaDHA-1 transfer. Resistance of Salmonella to ceftriaxone, cefoperazone and ceftiofur was mediated by a tnpU1548 related blaTEM-1B and blaCTX-M-3 transfer. The study indicated that transposase synergy and plasmid selective fusion act as important roles for foodborne Salmonella gathering ARGs. The consistent size of the plasmid before and after fusion suggested the invisibility and complexity of bacterial conjugation without DNA sequencing, the fact reminded us that the rampant transmission of antibiotic-resistance encoding genes would pose tremendous threat to food safety.

Acquisition of a Stable and Transferable blaNDM-5-Positive Plasmid With Low Fitness Cost Leading to Ceftazidime/Avibactam Resistance in KPC-2-Producing Klebsiella pneumoniae During Treatment.

The emergence and prevalence of carbapenem-resistant Enterobacteriaceae (CRE) have drawn worldwide attention. Ceftazidime/avibactam (CAZ/AVI) gives us a valuable alternative strategy to treat CRE infections. Unfortunately, CAZ/AVI resistance could occur during CAZ/AVI treatment. The CAZ/AVI-resistant Carbapenem-resistant Klebsiella pneumoniae (CR-KP) (KP137060) and earlier CAZ/AVI-susceptible isolate (KP135194) from the same hospitalized patient were collected at Fujian Medical University Union Hospital between October and November 2019. In this study, CAZ/AVI MICs of CAZ/AVI-susceptible and -resistant isolates (KP135194 and KP137060) were 4 mg/L and 128 mg/L, respectively; and the two isolates had the same antibiotic resistance pattern to other carbapenems. Two strains were then submitted for whole-genome sequencing and bioinformatic analysis. ompK36 was not detected in two isolates. No mutation was observed in bla KPC-2, ompK35 and ompK37 in this study and there was no significant difference of the expression in bla KPC-2, ompK35 and ompK37 between the two isolates (p>0.05). Two isolates were sequence type 11 and harbored bla KPC-2, bla SHV-182 and bla TEM-1B. Compared with KP135194, KP137060 harbored an additional bla NDM-5 positive plasmid. bla NDM-5 gene could be successfully transferred into E. coli J53 at a conjugation frequency of 1.14×10-4. Plasmid stability testing showed that bla KPC-2- and bla NDM-5-harboring plasmids were still stably maintained in the hosts. Growth assay and growth competition experiments showed there was no significant difference in fitness cost between two CR-KP isolates. Our study described the acquisition of a bla NDM-5-harboring plasmid leading to resistance to ceftazidime/avibactam in KPC-2-producing Klebsiella pneumoniae during treatment. This phenomenon deserves further exploration.