Assessment of Bacillus subtilis Plasmid pLS20 Conjugation in the Absence of Quorum Sensing Repression

Bacillus subtilis conjugative plasmid pLS20 uses a quorum-sensing mechanism to control expression levels of its conjugation genes, involving the repressor RcopLS20, the anti-repressor RappLS20, and the signaling peptide Phr*pLS20. In previous studies, artificial overexpression of rappLS20 in the donor cells was shown to enhance conjugation efficiency. However, we found that the overexpression of rappLS20 led to various phenotypic traits, including cell aggregation and death, which might have affected the correct determination of the conjugation efficiency when determined by colony formation assay. In the current study, conjugation efficiencies were determined under different conditions using a two-color fluorescence-activated flow cytometry method and measuring a single-round of pLS20-mediated transfer of a mobilizable plasmid. Under standard conditions, the conjugation efficiency obtained by fluorescence-activated flow cytometry was 23-fold higher than that obtained by colony formation. Furthermore, the efficiency difference increased to 45-fold when rappLS20 was overexpressed.

A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection

The use of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) for sequence-specific elimination of bacteria or resistance genes is a powerful tool for combating antibiotic resistance. However, this approach requires efficient delivery of CRISPR/Cas DNA cassette(s) into the targeted bacterial population. Compared to phage transduction, plasmid conjugation can deliver DNA to a broader host range but often suffers from low delivery efficiency. Here, we developed multi-plasmid conjugation systems for efficient CRISPR/Cas delivery, target DNA elimination and plasmid replacement. The CRISPR/Cas system, delivered via a broad-host-range R1162 mobilizable plasmid, specifically eliminated the targeted plasmid in recipient cells. A self-transmissible RK2 helper plasmid facilitated the spread of mobilizable CRISPR/Cas. The replacement of the target plasmid with another plasmid from the same compatibility group helped speed up target plasmid elimination especially when the target plasmid was also mobilizable. Together, we showed that up to 100% of target plasmid from the entire recipient population could be replaced even at a low (1:180) donor-to-recipient ratio and in the absence of transconjugant selection. Such an ability to modify genetic content of microbiota efficiently in the absence of selection will be critical for future development of CRISPR antimicrobials as well as genetic tools for in situ microbiome engineering.

Antimicrobial Resistance Genes in Bacteria Isolated From Japanese Honey, and Their Potential for Conferring Macrolide and Lincosamide Resistance in the American Foulbrood Pathogen Paenibacillus larvae

American foulbrood (AFB) is the most serious bacterial disease of honey bee brood. Spores of the causative agent Paenibacillus larvae are ingested by bee larvae via brood foods and germinated cells proliferate in the larval midgut. In Japan, a macrolide antibiotic, tylosin, is used as the approved prophylactic for AFB. Although tylosin-resistant P. larvae has yet to be found in Japan, it may emerge in the future through the acquisition of macrolide resistance genes from other bacteria, and bacteria latent in brood foods, such as honey, may serve as a source of resistance genes. In this study, to investigate macrolide resistance genes in honey, we attempted to isolate tylosin-resistant bacteria from 53 Japanese honey samples and obtained 209 isolates from 48 samples in the presence of 1 μg/ml of tylosin. All isolates were Gram-positive spore-forming bacteria mainly belonging to genera Bacillus and Paenibacillus, and 94.3% exhibited lower susceptibility to tylosin than Japanese P. larvae isolates. Genome analysis of 50 representative isolates revealed the presence of putative macrolide resistance genes in the isolates, and some of them were located on mobile genetic elements (MGEs). Among the genes on MGEs, ermC on the putative mobilizable plasmid pJ18TS1mac of Oceanobacillus strain J18TS1 conferred tylosin and lincomycin resistance to P. larvae after introducing the cloned gene using the expression vector. Moreover, pJ18TS1mac was retained in the P. larvae population for a long period even under non-selective conditions. This suggests that bacteria in honey is a source of genes for conferring tylosin resistance to P. larvae; therefore, monitoring of bacteria in honey may be helpful to predict the emergence of tylosin-resistant P. larvae and prevent the selection of resistant strains.

Studies on the role of IS1216E in the formation and dissemination of poxtA-carrying plasmids in an Enterococcus faecium clade A1 isolate

Objectives To analyse the role of IS1216E in the dissemination of the phenicol-oxazolidinone-tetracycline resistance gene poxtA in an Enterococcus faecium clade A1 isolate. Methods MICs were determined by broth microdilution. The poxtA-positive isolate was typed by MLST. The two plasmids were characterized by PCR, conjugation, S1-PFGE, Southern blot hybridization and WGS analysis. The presence of translocatable units (TUs) was examined by PCR and sequencing. Results Isolate E1077 contains the 217661 bp conjugative plasmid pE1077-217 and the 23710 bp mobilizable plasmid pE1077-23. pE1077-217 harbours erm(B), aac(A)-aph(D), aadE, spw, lsa(E), lnu(B), aphA3 and dfrG, whereas pE1077-23 carries a Tn6657-like transposon containing poxtA and fexB. pE1077-23 was apparently formed by an IS1216E-mediated composite transposon–plasmid fusion event, involving a replicative transposition process. Conjugation experiments showed that pE1077-23 is mobilizable by pE1077-217. Moreover, a novel 31742 bp plasmid, pT-E1077-31, was found in a transconjugant. WGS analysis indicated that pT-E1077-31 was formed by the integration of a Tn6657-derived, IS1216E-based translocatable unit, which carried fexB and poxtA, into a copy of pE1077-23. Conclusions This study showed the presence of two cointegrate formation events in the formation and spread of a poxtA/fexB-carrying plasmid in E. faecium. One was the integration of a transposon into a plasmid while the other was the integration of a TU into a different site of the same type of plasmid-borne transposon from which it originated. In both events, IS1216E played a major role, suggesting that IS1216E-mediated transposition and translocation processes aid the dissemination and persistence of important antimicrobial resistance genes, such as poxtA, among enterococci.

A Novel Mobilizing Tool Based on the Conjugative Transfer System of the IncM Plasmid pCTX-M3

ABSTRACT Conjugative plasmids are the main players in horizontal gene transfer in Gram-negative bacteria. DNA transfer tools constructed on the basis of such plasmids enable gene manipulation even in strains of clinical or environmental origin, which are often difficult to work with. The conjugation system of the IncM plasmid pCTX-M3 isolated from a clinical strain of Citrobacter freundii has been shown to enable efficient mobilization of oriTpCTX-M3-bearing plasmids into a broad range of hosts comprising Alpha-, Beta-, and Gammaproteobacteria. We constructed a helper plasmid, pMOBS, mediating such mobilization with an efficiency up to 1,000-fold higher than that achieved with native pCTX-M3. We also constructed Escherichia coli donor strains with chromosome-integrated conjugative transfer genes: S14 and S15, devoid of one putative regulator (orf35) of the pCTX-M3 tra genes, and S25 and S26, devoid of two putative regulators (orf35 and orf36) of the pCTX-M3 tra genes. Strains S14 and S15 and strains S25 and S26 are, respectively, up to 100 and 1,000 times more efficient in mobilization than pCTX-M3. Moreover, they also enable plasmid mobilization into the Gram-positive bacteria Bacillus subtilis and Lactococcus lactis. Additionally, the constructed E. coli strains carried no antibiotic resistance genes that are present in pCTX-M3 to facilitate manipulations with antibiotic-resistant recipient strains, such as those of clinical origin. To demonstrate possible application of the constructed tool, an antibacterial conjugation-based system was designed. Strain S26 was used for introduction of a mobilizable plasmid coding for a toxin, resulting in the elimination of over 90% of recipient E. coli cells. IMPORTANCE The conjugation of donor and recipient bacterial cells resulting in conjugative transfer of mobilizable plasmids is the preferred method enabling the introduction of DNA into strains for which other transfer methods are difficult to establish (e.g., clinical strains). We have constructed E. coli strains carrying the conjugation system of the IncM plasmid pCTX-M3 integrated into the chromosome. To increase the mobilization efficiency up to 1,000-fold, two putative regulators of this system, orf35 and orf36, were disabled. The constructed strains broaden the repertoire of tools for the introduction of DNA into the Gram-negative Alpha-, Beta-, and Gammaproteobacteria, as well as into Gram-positive bacteria such as Bacillus subtilis and Lactococcus lactis. The antibacterial procedure based on conjugation with the use of the orf35- and orf36-deficient strain lowered the recipient cell number by over 90% owing to the mobilizable plasmid-encoded toxin.

Assembly of an atrazine catabolic operon and its introduction to Gram-negative hosts for robust and stable degradation of triazine herbicides

ABSTRACT In 1995, Pseudomonas sp. ADP, capable of metabolizing atrazine, was isolated from contaminated soil. Genes responsible for atrazine mineralization were found scattered in the 108.8 kb pADP-1 plasmid carried by this strain, some of them flanked by insertion sequences rendering them unstable. The goal of this work was to construct a transcriptional unit containing the atz operon in an easy to transfer manner, to be introduced and inherited stably by Gram-negative bacteria. atz genes were PCR amplified, joined into an operon and inserted onto the mobilizable plasmid pBAMD1–2. Primers were designed to add efficient transcription and translation signals. Plasmid bearing the atz operon was transferred to different Gram-negative strains by conjugation, which resulted in Tn5 transposase-mediated chromosomal insertion of the atz operon. To test the operon activity, atrazine degradation by transposants was assessed both colorimetrically and by high-performance liquid chromatography (HPLC). Transposants mineralized atrazine more efficiently than wild-type Pseudomonas sp. ADP and did not accumulate cyanuric acid. Atrazine degradation was not repressed by simple nitrogen sources. Genes conferring atrazine-mineralizing capacities were stable and had little or null effect on the fitness of different transposants. Introduction of catabolic operons in a stable fashion could be used to develop bacteria with better degrading capabilities useful in bioremediation.

Interspecies Dissemination of a Mobilizable Plasmid HarboringblaIMP-19and the Possibility of Horizontal Gene Transfer in a Single Patient

ABSTRACTCarbapenemase-producing Gram-negative bacilli have been a global concern over the past 2 decades because these organisms can cause severe infections with high mortality rates. Carbapenemase genes are often carried by mobile genetic elements, and resistance plasmids can be transferred through conjugation. We conducted whole-genome sequencing (WGS) to demonstrate that the same plasmid harboring a metallo-β-lactamase gene was detected in two different species isolated from a single patient. Metallo-β-lactamase-producingAchromobacter xylosoxidans(KUN4507), non-metallo-β-lactamase-producingKlebsiella pneumoniae(KUN4843), and metallo-β-lactamase-producingK. pneumoniae(KUN5033) were sequentially isolated from a single patient and then analyzed in this study. Antimicrobial susceptibility testing, molecular typing (pulsed-field gel electrophoresis and multilocus sequence typing), and conjugation analyses were performed by conventional methods. Phylogenetic and molecular clock analysis ofK. pneumoniaeisolates were performed with WGS, and the nucleotide sequences of plasmids detected from these isolates were determined using WGS. Conventional molecular typing revealed that KUN4843 and KUN5033 were identical, whereas the phylogenetic tree analysis revealed a slight difference. These two isolates were separated from the most recent common ancestor 0.74 years before they were isolated. The same resistance plasmid harboringblaIMP-19was detected in metallo-β-lactamase-producingA. xylosoxidansandK. pneumoniae. Although this plasmid was not self-transferable, the conjugation of this plasmid fromA. xylosoxidansto non-metallo-β-lactamase-producingK. pneumoniaewas successfully performed. The susceptibility patterns for metallo-β-lactamase-producingK. pneumoniaeand the transconjugant were similar. These findings supported the possibility of the horizontal transfer of plasmid-borneblaIMP-19fromA. xylosoxidanstoK. pneumoniaein a single patient.

Complete Genome Sequence of Helicobacter pylori Strain 7C Isolated from a Mexican Patient with Chronic Gastritis

Helicobacter pylori -induced gastritis is a risk factor for developing gastric pathologies. Here, we report the complete genome sequence of a multidrug-resistant H. pylori strain isolated from a chronic gastritis patient in Mexico City, Mexico. Nonvirulent VacA and cag -pathogenicity island (PAI) genotypes were found, but the presence of a potential mobilizable plasmid carrying an IS 605 element is of outstanding interest.

Molecular Characterization of Streptococcus agalactiae Isolates Harboring Smallerm(T)-Carrying Plasmids

ABSTRACTAmong 1,827 group BStreptococcus(GBS) strains collected between 2006 and 2013 by the French National Reference Center for Streptococci, 490 (26.8%) strains were erythromycin resistant. Theerm(T) resistance gene was found in six strains belonging to capsular polysaccharides Ia, III, and V and was carried by the same mobilizable plasmid, which could be efficiently transferred by mobilization to GBS andEnterococcus faecalisrecipients, thus promoting a broad dissemination oferm(T).