In vitro antibacterial activity of danofloxacin against Escherichia coli isolated from pigeons and its pharmacokinetic in pigeons.

This experiment aimed to investigate the in vitro antimicrobial activity of danofloxacin against Escherichia coli (E. coli) isolated from pigeons, as well as the pharmacokinetics of danofloxacin in pigeons following oral (PO), intramuscular (IM), and intravenous (IV) administration. The minimum inhibitory concentration (MIC) of danofloxacin was first determined for 38 clinical E. coli strains using the micro broth dilution method. Subsequently, 30 healthy pigeons were weighed and randomly divided into 3 groups: IM, IV, and PO, with 10 pigeons in each group. Danofloxacin was given at 5 mg/kg body weight (BW) through 3 different routes. Blood was collected, and plasma was separated at various time points from 0 to 48 h. Plasma samples were analyzed for danofloxacin concentrations using a validated HPLC method. Pharmacokinetic analysis was performed using Phoenix software and a noncompartmental analytical (NCA) method. The results indicated that danofloxacin had a strong antibacterial effect on E. coli, with a MIC50 of 0.5 µg/mL. The noncompartmental analysis showed that after PO and IM administration at 5 mg/kg in pigeons, peak plasma concentrations (Cmax) of 0.61 and 1.62 µg/mL were reached at 4.5 and 0.53 h, respectively. The oral and intramuscular bioavailability (F) were 68.08% ± 24.82% and 87.82% ± 25.36%, respectively. Following IV administration, danofloxacin was widely distributed in pigeons, with volume of distribution (VZ) and volume of distribution at steady state (VSS) values of 6.11 ± 2.01 and 4.65 ± 1.62 L/kg, respectively, and was eliminated slowly, with an elimination half-life (t1/2λz) of 6.41 ± 2.15 h. Based on the calculated ratio values of AUC/MIC, the current IV, IM, and PO doses of 5 mg/kg of danofloxacin would be expected to effectively treat pigeons infected with E. coli strains with MIC values equal to or less than 0.5 µg/mL.

Research Note: Pharmacokinetics of bromhexine hydrochloride in broilers after single oral and intravenous administration.

The current study aimed to investigate the pharmacokinetics of bromhexine hydrochloride in broilers after single intravenous (IV) and oral (PO) administration at 2.5 mg/kg body weight (BW). The trial adopted a randomized, parallel-controlled design, where 20 twelve-wk-old broilers were randomly assigned to either the PO or IV group. Blood samples were collected at predetermined time points, and plasma was further separated for analysis. The bromhexine hydrochloride concentrations in plasma samples were determined using an ultra-performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC-MS/MS) method. Noncompartmental analysis (NCA) using Phoenix software was conducted to analyze the concentration versus time data of bromhexine hydrochloride in every chicken. Subsequently, the main pharmacokinetic parameters between the 2 groups were statistically analyzed using SPSS software. Results from NCA revealed that after oral administration at 2.5 mg/kg BW, bromhexine hydrochloride exhibited slow absorption, reaching an average peak concentration of 32.72 ng/mL at 1.78 h. However, incomplete absorption was observed, with an absolute bioavailability of only 20.06% ± 10.84%. Additionally, bromhexine hydrochloride displayed wide distribution, with a steady-state distribution volume (VSS) of 22.55 ± 13.45 L/kg, and slow elimination, with a clearance (Cl) of 1.52 ± 0.38 L/h/kg. Furthermore, gender effects were assessed on the pharmacokinetics of bromhexine hydrochloride in broilers, revealing better absorption in male broilers compared to females. This disparity may be attributed to the faster blood flow and richer blood volume typically found in male broilers.

Comparison of gamithromycin residue depletion in yellow-feather and white-feather broilers after one single subcutaneous injection.

This study aimed to compare the residue depletion of gamithromycin in yellow-feather and white-feather broilers, using Sanhuang and Arbor Acres chickens as typical examples, respectively. Each breed (54 chickens) received a single subcutaneous dose of gamithromycin at 7.5 mg/kg bodyweight (BW). Tissues, including muscle, skin + fat, liver, kidney, and injection site, were collected at 6 h, 3, 5, 7, 10, 14, 21, 28, and 35 d postdrug administration. Gamithromycin concentrations in these tissues were determined using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The kinetics of gamithromycin were analyzed in different tissues using a noncompartmental method in the Phoenix software. Differences were observed in gamithromycin concentrations and kinetic characteristics in both breeds of chickens, with higher residue concentrations and longer residue times found in yellow-feathered broilers. In Sanhuang broilers, the elimination rates of gamithromycin followed this order: injection site > muscle > liver > kidney > skin + fat. The corresponding elimination half-lives (t1/2λzs) in these samples were 1.22, 1.30, 1.71, 2.04, and 2.52 d, respectively. In contrast, in Arbor Acres broilers, a different order was noted: muscle > injection site > kidney > liver > skin + fat, with corresponding t1/2λzs of 1, 1.23, 1.88, 1.93, and 2.21 d, respectively. These differences may be related to variations in pigments in various tissues of chickens of the 2 breeds. However, further investigations are warranted to discern the underlying reasons.

Molecular characteristic of mcr-1 gene in Escherichia coli from aquatic products in Guangdong, China.

OBJECTIVES: Aquatic ecosystems serve as a dissemination pathway and a reservoir of both antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study aimed to determine the prevalence of colistin-resistant mcr-like genes in Enterobacteriales in aquatic products, which may be contribute to the transfer of ARGs in water environments. METHODS: The mcr-1-positive Escherichia coli were recovered from 123 freshwater fish and 34 cultured crocodile cecum samples from 10 farmers' markets in Guangdong, China. Minimum inhibitory concentration (MIC) was determined using the agar dilution method. Genotyping was performed using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Conjugation assay was carried out to investigate the transferability of mcr-1. Genomic information was obtained by whole genome sequencing (WGS) and bioinformatic analysis. RESULTS: Forty-four mcr-1 positive isolates showed co-resistance to tetracycline, trimethoprim/sulfamethoxazole, and gentamicin, while they were all sensitive to tigecycline, meropenem, and amikacin. They were typed into sixteen PFGE clusters. ST10 and ST117 were the most popular sequence types, followed by ST1114. S1-PFGE verified the presence of the mcr-1 gene on plasmids in sizes of ∼60 kb (n = 1) and ∼240 kb (n = 3). Whole genome sequencing-based analysis identified mcr-1 integrated in IncHI2 plasmid (n = 3), IncI2 plasmid (n = 2), and bacterial chromosome in two copies (n = 1). In addition to mcr-1, they carried several other antibiotic resistance genes, such as blaCTX-M-14, fosA3, and aac(6')-Ib-cr. CONCLUSION: These data suggest that aquatic products are an important antibiotic resistance reservoir and highlight possible risks regarding food safety.

Effects of chitooligosaccharide on the in vitro antibacterial activity against avian Escherichia coli and the pharmacokinetics of florfenicol in healthy chickens.

This study investigates the combined effects of chitooligosaccharide (COS) and florfenicol (FLO) on the inhibition of Escherichia coli in vitro, as well as the pharmacokinetic interactions between these compounds in healthy chickens. The minimum inhibitory concentration (MIC) of COS and FLO alone and the fractional inhibitory concentration index (FICI) after combined treatment were determined using the broth microdilution method and checkerboard method, respectively. Additionally, we evaluated the pharmacokinetic interactions between the 2 types of COS and FLO in healthy chickens. Thirty chickens were randomly divided into 3 groups: Florfenicol group (30 mg/kg), COS J85 group (COS J85 20 mg/kg + florfenicol 30 mg/kg), COS H85 group (COS H85 20 mg/kg + florfenicol 30 mg/kg). Either FLO or COS was orally administered by gavage. The concentrations of FLO in chicken plasma were measured at different time points after the drug withdrawal using high-performance liquid chromatography (HPLC), and pharmacokinetic parameters were calculated by a compartmental method. The results showed that COS J85 and COS H85, when combined with FLO, had FICI values of 0.1875 to 0.75 and 0.3125 to 1, respectively, indicating good synergistic or additive effects against Escherichia coli. The pharmacokinetics of FLO alone and in combination with COS followed a 1-compartment model with first-order absorption and elimination. Furthermore, the pharmacokinetic analysis revealed that the elimination half-life (t1/2ke) of florfenicol was significantly increased in the COS H85 group compared to oral administration of florfenicol alone (P < 0.05). Other pharmacokinetic parameters did not show significant changes (P > 0.05), except between the 2 combined treatment groups, where statistical differences were observed for various parameters, excluding the area under the concentration-time curve from the time of dosing to infinity (AUC) and peak concentration (Cmax).

Egg residue and depletion of meloxicam in Jing Hong laying hens following multiple oral doses.

Meloxicam is a nonsteroidal anti-inflammatory drug (NSAID) commonly used in an extra-label manner in commercial laying hens for the treatment of foot lesions, which are a common issue in this species. The present study aimed to determine the depletion profiles of meloxicam in eggs with multiple oral administration under 2 different dosing regimens and to further recommend reasonable withdrawal intervals (WDIs). Meloxicam (1 mg/kg) was administered orally to laying hens under 2 dosing schedules: 10 doses at 24-h intervals and 15 doses at 12-h intervals. Eggs were collected daily after the first dosing, and meloxicam concentrations in both yolk and white were determined by a high-performance liquid chromatography (HPLC) method. The weight ratio of white to yolk in the whole egg was 1.54 (the mean of 20 eggs with repeated tests), and this value combined with the meloxicam concentrations in white and yolk were used to calculate the drug concentrations in whole eggs. Meloxicam was quickly eliminated from egg white, and its concentrations could only be quantified at 2 time points during the elimination phase. The elimination half-lives in yolk and whole egg were 3.07 ± 1.00 and 2.98 ± 0.88 d, respectively, after 10 repeated doses. And the corresponding elimination half-lives were 2.30 ± 0.83 and 2.18 ± 0.67 d, respectively, after repeated 15 doses. Considering the time when meloxicam was not detectable in eggs with the time of ovum development and maturation, a withdrawal interval (WDI) was suggested as 17 d for both dosing schedules. The current results enriched the study on the residue of meloxicam in domestic Jing Hong laying hens and provided WDIs to help ensure animal-derived food safety.

Development and Application of a Physiologically Based Pharmacokinetic Model for Diclazuril in Broiler Chickens.

Withdrawal periods for diclazuril in broilers have traditionally been determined through regression analysis. However, over the last two decades, the physiologically based pharmacokinetic (PBPK) model has gained prominence as a predictive tool for veterinary drug residues, which offers an alternative method for establishing appropriate withdrawal periods for veterinary drugs. In this current study, a flow-limited PBPK model was developed to predict diclazuril concentrations in broilers following long-duration administration via medicated feed and water. This model consists of nine compartments, including arterial and venous plasma, lung, muscle, skin + fat, kidney, liver, intestine contents, and the rest of the body compartment. Physiological parameters such as tissue weights (Vcxx) and blood flow (Qcxx) were gathered from published studies, and tissue/plasma partition coefficients (Pxx) were calculated through the area method or parameter optimization. Published diclazuril concentrations were compared to the predicted values, indicating the accuracy and validity of the model. The sensitivity analysis showed that parameters associated with cardiac output, drug absorption, and elimination significantly affected diclazuril concentrations in the muscle. Finally, a Monte Carlo analysis, consisting of 1000 iterations, was conducted to calculate the withdrawal period. Based on the Chinese MRL values, we calculated a withdrawal period of 0 days for both recommended dosing regimens (through mediated water and feed at concentrations of 0.5-1 mg/L and 1 mg/kg, respectively). However, based on the European MRLs, longer periods were determined for the mediated feed dosing route. Our model provides a foundation for scaling other coccidiostats and poultry species.

Longitudinal surveillance and comparative characterization of Escherichia albertii in wild raccoons in the United States.

Escherichia albertii is an emerging enteric bacterial pathogen causing watery diarrhea, abdominal distension, vomiting and fever in humans. E. albertii has caused many foodborne outbreaks in Japan and was also reported in other countries worldwide. However, the important animal reservoirs of this pathogen are still largely unknown, impeding us to combat this emerging pathogen. Recently, we reported that wild raccoons (Procyon lotor) and broiler chickens are significant reservoirs of E. albertii in Japan and the U.S., respectively. Here, we performed a longitudinal surveillance to monitor prevalence of E. albertii in wild raccoons in the U.S. and conducted comprehensive comparative analyses of the E. albertii of different origins. A total of 289 fecal swab samples were collected from wild raccoons in Tennessee and Kentucky in the U.S. (2018-2020). Approximately 26% (74/289) of the raccoons examined were PCR-positive for E. albertii and eventually 22 E. albertii isolates were obtained. PFGE analysis showed the U.S. raccoon E. albertii were phylogenetically distant even though the corresponding raccoons were captured from a small area. Unlike the high prevalence of multidrug resistance (83%) observed in previous chicken E. albertii survey, antibiotic resistance was rarely observed in all the U.S. raccoon and 22 Japan raccoon strains with only one Japan strain displaying multidrug resistance (2%). Whole genome sequencing of 54 diverse E. albertii strains and subsequent comparative genomics analysis revealed unique clusters that displayed close evolutionary relationships and similar virulence gene profiles among the strains of different origins in terms of geographical locations (e.g., U.S. and Japan) and hosts (raccoon, chicken, swine, and human). Challenge experiment demonstrated raccoon E. albertii strains could successfully colonize in the chicken intestine at 3 and 8 days postinfection. A pilot environmental survey further showed all the four tested water samples from Tennessee river were E. albertii-positive; two different E. albertii strains, isolated from a single water sample, showed close relationships to those of human origin. Together, the findings from this study provide new insights into the ecology, evolution, and pathobiology of E. albertii, and underscore the need to control the emerging E. albertii in a complex ecosystem using One Health approach.

Identification of the Plasmid-Mediated Colistin Resistance Gene mcr-1 in Escherichia coli Isolates From Migratory Birds in Guangdong, China.

We determined the prevalence and transmission characteristics of mcr-1-positive Escherichia coli (MCRPEC) isolates from migratory birds Anser indicus in Guangdong, China. We identified 22 MCRPEC from 303 A. indicus fecal samples (7.3%) in Guangzhou, Zhaoqing, and Futian. The mcr-1 gene coexisted with 24 other types of antibiotic resistance genes (ARG), and 11 ARGs were highly prevalent at levels >50%. The MCRPEC displayed a diversity of sequence types (ST), and 19 distinct STs were identified with ST10, ST1146, and ST1147 as the most prevalent. In addition, these MCRPEC from birds were closely related phylogenetically to those from other sources in China. Whole-genome sequencing analysis demonstrated that mcr-1 was located on IncX4 (n=9, 40.9%), IncI2 (n=5, 22.7%) and IncP (n=1, 4.5%) plasmids and the latter shared an identical plasmid backbone with other sources. These results highlight the significance of migratory birds in the transmission of antibiotic resistance and provide powerful evidence that migratory birds are potential transmitters of antibiotic resistance.

Critical Role of 3'-Downstream Region of pmrB in Polymyxin Resistance in Escherichia coli BL21(DE3).

Polymyxins, such as colistin and polymyxin B, are the drugs used as a last resort to treat multidrug-resistant Gram-negative bacterial infections in humans. Increasing colistin resistance has posed a serious threat to human health, warranting in-depth mechanistic research. In this study, using a functional cloning approach, we examined the molecular basis of colistin resistance in Escherichia coli BL21(DE3). Five transformants with inserts ranging from 3.8 to 10.7 kb displayed significantly increased colistin resistance, three of which containing pmrB locus and two containing pmrD locus. Stepwise subcloning indicated that both the pmrB with a single G361A mutation and at least a 103 bp downstream region of pmrB are essential for conferring colistin resistance. Analysis of the mRNA level and stability showed that the length of the downstream region drastically affected the pmrB mRNA level but not its half-life. Lipid A analysis, by mass spectrometry, revealed that the constructs containing pmrB with a longer downstream region (103 or 126 bp) have charge-altering l-4-aminoarabinose (Ara4N) and phosphoethanolamine (pEtN) modifications in lipid A, which were not observed in both vector control and the construct containing pmrB with an 86 bp downstream region. Together, the findings from this study indicate that the 3'-downstream region of pmrB is critical for the PmrB-mediated lipid A modifications and colistin resistance in E. coli BL21(DE3), suggesting a novel regulatory mechanism of PmrB-mediated colistin resistance in E. coli.

Isolation and characterization of Escherichia albertii in poultry at the pre-harvest level.

Escherichia albertii, often misidentified as Escherichia coli, has become an emerging foodborne human enteric pathogen. However, the prevalence and major animal reservoirs of this significant pathogen are still not clear. Here, we performed comprehensive microbiological, molecular, comparative genomics and animal studies to understand the status and features of E. albertii in the US domestic and food animals. Although no E. albertii was identified in a total of 1,022 diverse E. coli strains isolated from pets and food animals in a retrospective screening, in a pilot study, E. albertii was successfully isolated from a broiler farm (6 out of 20 chickens). The chicken E. albertii isolates showed clonal relationship as indicated by both pulsed-field gel electrophoresis (PFGE) and whole-genome sequence analysis. The isolated chicken E. albertii displayed multidrug resistance; all the resistance determinants including the extended-spectrum beta-lactamase gene, carried by plasmids, could be conjugatively transferred to E. coli, which was further confirmed by S1-PFGE and Southern hybridization. Whole-genome sequence-based phylogenetic analysis showed the chicken E. albertii strains were phylogenetically close to those of human origins. Challenge experiment demonstrated that the E. albertii strains isolated from human and wild bird could successfully colonize in the chicken intestine. Together, this study, for the first time, reported the isolation of E. albertii in poultry at the pre-hrvest level. The findings from multi-tier characterization of the chicken E. albertii strains indicated the importance of chickens as a reservoir for E. albertii. A large scale of E. albertii survey in poultry production at the pre-harvest level is highly warranted in the future.

ISKpn40-Mediated Mobilization of the Colistin Resistance Gene mcr-3.11 in Escherichia coli.

The mobile colistin resistance gene mcr-3 has globally disseminated since it was first reported in 2017 in Escherichia coliIn vitro mobilization assays in this study demonstrate the functionality of the composite transposon structure ISKpn40-mcr-3.11-dgkA-ISKpn40 in wild-type and recA-E. coli strains. These transpositions generated 4-bp duplications at the target sites. This is the first report demonstrating the mobility of the mcr-3.11 gene by transposition.

Emerging High-Level Tigecycline Resistance: Novel Tetracycline Destructases Spread via the Mobile Tet(X).

Antibiotic resistance in bacteria has become a great threat to global public health. Tigecycline is a next-generation tetracycline that is the final line of defense against severe infections by pan-drug-resistant bacterial pathogens. Unfortunately, this last-resort antibiotic has been challenged by the recent emergence of the mobile Tet(X) orthologs that can confer high-level tigecycline resistance. As it is reviewed here, these novel tetracycline destructases represent a growing threat to the next-generation tetracyclines, and a basic framework for understanding the molecular epidemiology and resistance mechanisms of them is presented. However, further large-scale epidemiological and functional studies are urgently needed to better understand the prevalence and dissemination of these newly discovered Tet(X) orthologs among Gram-negative bacteria in both human and veterinary medicine.

Global clonal spread of mcr-3-carrying MDR ST34 Salmonella enterica serotype Typhimurium and monophasic 1,4,[5],12:i:- variants from clinical isolates.

OBJECTIVES: To investigate the prevalence and transmission of mcr-3 among Salmonella enterica serotype Typhimurium and 1,4,[5],12:i:-. METHODS: A total of 4724 clinical Salmonella isolates were screened for the presence of mcr-3 in China during 2014-19. The clonal relationship of the mcr-3-positive isolates and their plasmid contents and complete sequence were also characterized based on WGS data from the Illumina and MinION platforms. RESULTS: We identified 10 mcr-3-positive isolates, and all were MDR, mostly resistant to colistin, cefotaxime, ciprofloxacin, doxycycline and florfenicol. mcr-3 was co-present with blaCTX-M-55-qnrS1 on hybrid ST3-IncC-FII conjugatable plasmids (n = 6) and an ST3-IncC non-conjugatable plasmid (n = 1) and embedded into a pCHL5009T-like IncFII plasmid on the Salmonella chromosome (n = 3). Four distinctive genetic contexts surrounded mcr-3 and all but one were closely related to each other and to the corresponding region of IncFII plasmid pCHL5009T. IS15DI was most likely the vehicle for integration of mcr-3-carrying IncFII plasmids into ST3-IncC plasmids and the chromosome and for shaping the MDR regions. In addition, a phylogenetic tree based on the core genome revealed a unique Salmonella lineage (≤665 SNPs) that contained these 10 mcr-3-positive isolates and another 38 (33 from patients) mcr-3-positive Salmonella from five countries. In particular, most of the 51 mcr-3-positive isolates belonged to ST34 and harboured diverse antibiotic resistance genes (ARGs), including mcr-3-blaCTX-M-55-qnrS1, and possessed similar ARG profiles. CONCLUSIONS: Our findings revealed global clonal spread of MDR ST34 Salmonella from clinical isolates co-harbouring mcr-3 with blaCTX-M-55 and qnrS1 and a flexibility of mcr-3 co-transmittance with other ARGs mediated by mobile genetic elements.

Within-host heterogeneity and flexibility of mcr-1 transmission in chicken gut.

OBJECTIVES: To characterize the colistin-resistant bacterial population in the gut and assess diversity of mcr-1 transmission within a single individual. METHODS: Large numbers of isolates (>100 colonies/chicken cecum sample) were collected from nine randomly selected mcr-1-positive chickens in China and used for comprehensive microbiological, molecular and comparative genomics analyses. RESULTS: Of 1273 colonies, 968 were mcr-1 positive (962 Escherichia coli, two Escherichia fergusonii, two Klebsiella pneumoniae and two Klebsiella quasipneumoniae). One to six colistin-resistant species and three to 10 E. coli pulsed-field gel electrophoresis (PFGE) clusters could be identified from each sample. Whole-genome sequencing (WGS) analysis of the representative E. coli strains revealed three to nine sequence types observed in a single chicken host. The mcr-1 genes are located in either chromosomes or plasmids of different types, including IncI2 (n=30), IncHI2 (n=14), IncX4 (n=4), p0111(n=2) and IncHI1(n=1). Strikingly, in single cecum samples, one to five Inc type plasmids harbouring mcr-1 could be identified. Great diversity was also observed for the same IncI2 plasmid within a single chicken host. In addition, up to eight genetic contexts of the mcr-1 gene occurred within a single chicken. CONCLUSIONS: There is extensive heterogeneity and flexibility of mcr-1 transmission in chicken gut due to bacterial species differences, distant clonal relatedness of isolates, many types and variations of mcr-positive plasmids, and the flexible genetic context of the mcr-1 gene. These compelling findings indicate that the gut is a 'melting pot' for active horizontal transfer of the mcr-1 gene.

ISEc69-Mediated Mobilization of the Colistin Resistance Gene mcr-2 in Escherichia coli.

OBJECTIVES: The emergence of mobile colistin resistance genes has compromised the efficacy of the last resort antibiotic, colistin, in clinical treatment. The mcr-2 gene was first identified in Belgium in association with the insertion sequence ISEc69. However, the molecular mechanisms of mcr-2 mobilization are not well understood. METHODS: To further explore the mobilization of mcr-2 gene via ISEc69, we constructed a conjugative plasmid that carries an intact composite transposon Tn7052. Transposition assays were performed by conjugation, the transposition sites were characterized by arbitrary primed PCR and DNA sequencing. RESULTS: In this study, we experimentally demonstrated that mcr-2 could be mobilized as a composite transposon Tn7052 and its transposition generated 8-bp AT-rich duplications in the host genome. CONCLUSION: These results indicate that mcr-2 gene could be mobilized by ISEc69, the current investigations provide mechanistic insights in the transposition of mcr-2.

The ISApl1 2 Dimer Circular Intermediate Participates in mcr-1 Transposition.

Objectives: The mobile colistin resistance gene mcr-1 is a serious threat to global human and animal health. The composite transposon Tn6330 and its circular intermediate were proposed to be involved in the spread of mcr-1 but their roles remain poorly understood. Methods: To further explore the intermediates during the transposition of Tn6330, we engineered Escherichia coli strains that carry an intact Tn6330 transposon or its deletion derivatives. PCR assays were performed to detect IR-IR junctions and possible circular intermediates. We carried out transposition experiments to calculate transposition frequency. The transposition sites were characterized by whole genome sequence and ISMapper-based analyses. Results: The presence of an intact Tn6330 was demonstrated to be essential for the successful transposition of mcr-1, although both Tn6330 and Tn6330-ΔIR could form circular intermediates. The insertion sequence junction structure was observed in all constructed plasmids but the ISApl1 dimer was only formed in one construct containing an intact Tn6330. The average frequency of mcr-1 transposition in an E. coli strain possessing an intact Tn6330 was ∼10-6 per transformed cell. We identified 27 integration sites for the Tn6330 transposition event. All the transposition sites were flanked by 2 bp target duplications and preferentially occurred in AT-rich regions. Conclusion: These results indicate that mcr-1 transposition relies on the presence of an intact Tn6330. In addition, formation of the tandem repeat ISApl1 2 could represent a crucial intermediate. Taken together, the current investigations provide mechanistic insights in the transposition of mcr-1.

ISEcp1-mediated transposition of chromosome-borne blaCMY-2 into an endogenous ColE1-like plasmid in Escherichia coli.

BACKGROUND: CMY-2 is the most prevalent pAmpC ß-lactamase, but the chromosomal blaCMY-2 gene transfer via horizontal transmission has been seldom reported. This study aimed to describe an ISEcp1-mediated transposition of a chromosomal blaCMY-2 gene from Escherichia coli into a small endogenous ColE1-like plasmid, resulting in elevated resistance to extended-spectrum cephalosporins. METHODS: Three ESCs-resistant ST641 E. coli strains EC6413, EC4103 and EC5106 harbored the blaCMY-2 gene. S1-PFGE, I-ceu I-PFGE, Southern blotting and electroporation experiments were performed to investigate the location and transferability of blaCMY-2. The genetic context and gene expression of blaCMY-2 in the original isolates and the corresponding electroporants were explored by PCR mapping, primer walking strategy and RT-qPCR. RESULTS: The blaCMY-2-containing region (ISEcp1-blaCMY-2-∆blc-∆yggR-∆tnp1-orf7-orf8-orf9-∆tnp2-∆hsdR) was transposed into endogenous ColE1-like plasmid pSC137 in the process of electroporation at very low frequencies (10-8-10-9). The transpositions resulted in novel larger blaCMY-2-harboring ColE1-like plasmids with size of 14,845 bp, enabling increase in MICs of 2 to 8-fold for cefotaxime, ceftiofur, and ceftazidime in recipient strains over their respective original counterparts. Transcriptional level analysis revealed that the increased blaCMY-2 expression was correlated with elevated MIC values of cephalosporins. The blaCMY-2 transposition unit was identical to that in a clinical isolate E. coli TN44889 from France isolated in 2004. CONCLUSIONS: Our results firstly demonstrated that ISEcp1 mediated a transposition of chromosome-borne blaCMY-2 into an endogenous ColE1-like plasmid by electroporation. Amplification of the blaCMY-2 gene facilitates the strain adaptation to a changed environment with an elevated antibiotic pressure.