A Novel Plasmid Entry Exclusion System in pKPC_UVA01, a Promiscuous Conjugative Plasmid Carrying the blaKPC Carbapenemase Gene.

Conjugative plasmids are the principal mediator in the emergence and spread of antibiotic resistance genes in Enterobacterales. Plasmid entry exclusion (EEX) systems can restrict their transfer into the recipient bacteria carrying closely related plasmids. In this study, we identified and characterized a novel plasmid entry exclusion system in a carbapenem resistance plasmid pKPC_UVA01, which is responsible for widespread dissemination of the blaKPC carbapenemase gene among Enterobacterales in the United States. The identified eex gene in the recipient strain of different Enterobacterales species inhibited the conjugation transfer of pKPC_UVA01 plasmids at a range of 200- to 400-fold, and this inhibition was found to be a dose-dependent function of the EEX protein in recipient cells. The C terminus truncated version of eex or eex with an early termination codon at the C terminus region alleviated the inhibition of conjugative transfer. Unlike the strict specificity of plasmid exclusion by the known EEX protein, the newly identified EEX in the recipient strain could inhibit the transfer of IncP and IncN plasmids. The eex gene from the plasmid pKPC_UVA01 was not required for conjugative transfer but was essential in the donor bacteria for entry exclusion of this plasmid. This was a novel function of a single protein that is essential in both donor and recipient bacteria for the entry exclusion of a plasmid. This eex gene is found to be distributed in multidrug resistance plasmids similar to pKPC_UVA01 in different Enterobacterales species and may contribute to the stability of this plasmid type by controlling its transfer.

Relative Strengths of Promoters Provided by Common Mobile Genetic Elements Associated with Resistance Gene Expression in Gram-Negative Bacteria.

Comparison of green fluorescent protein expression from outward-facing promoters (POUT) of ISAba1, ISEcp1, and ISAba125 revealed approximate equivalence in strength, intermediate between PCS (strong) and PCWTGN-10 (weak) class 1 integron promoter variants, >30-fold stronger than POUT of ISCR1, and >5 times stronger than Ptac. Consistent with its usual role, PCWTGN-10 produces more mRNA from a "downstream" gfp gene transcriptionally linked to a "usual" PCWTGN-10-associated gene cassette than does POUT of ISAba1.

Editorial for Special Issue "Bacterial Toxin-Antitoxin Systems".

Toxin antitoxin systems (TAS) are widely distributed in bacterial chromosomes as well as on mobile genetic elements [...].

A Streamlined Approach for Fluorescence Labelling of Low-Copy-Number Plasmids for Determination of Conjugation Frequency by Flow Cytometry.

Bacterial conjugation plays a major role in the dissemination of antibiotic resistance and virulence traits through horizontal transfer of plasmids. Robust measurement of conjugation frequency of plasmids between bacterial strains and species is therefore important for understanding the transfer dynamics and epidemiology of conjugative plasmids. In this study, we present a streamlined experimental approach for fluorescence labelling of low-copy-number conjugative plasmids that allows plasmid transfer frequency during filter mating to be measured by flow cytometry. A blue fluorescent protein gene is inserted into a conjugative plasmid of interest using a simple homologous recombineering procedure. A small non-conjugative plasmid, which carries a red fluorescent protein gene with a toxin-antitoxin system that functions as a plasmid stability module, is used to label the recipient bacterial strain. This offers the dual advantage of circumventing chromosomal modifications of recipient strains and ensuring that the red fluorescent protein gene-bearing plasmid can be stably maintained in recipient cells in an antibiotic-free environment during conjugation. A strong constitutive promoter allows the two fluorescent protein genes to be strongly and constitutively expressed from the plasmids, thus allowing flow cytometers to clearly distinguish between donor, recipient, and transconjugant populations in a conjugation mix for monitoring conjugation frequencies more precisely over time.

Biological Functions of Type II Toxin-Antitoxin Systems in Bacteria.

After the first discovery in the 1980s in F-plasmids as a plasmid maintenance system, a myriad of toxin-antitoxin (TA) systems has been identified in bacterial chromosomes and mobile genetic elements (MGEs), including plasmids and bacteriophages. TA systems are small genetic modules that encode a toxin and its antidote and can be divided into seven types based on the nature of the antitoxin molecules and their mechanism of action to neutralise toxins. Among them, type II TA systems are widely distributed in chromosomes and plasmids and the best studied so far. Maintaining genetic material may be the major function of type II TA systems associated with MGEs, but the chromosomal TA systems contribute largely to functions associated with bacterial physiology, including the management of different stresses, virulence and pathogenesis. Due to growing interest in TA research, extensive work has been conducted in recent decades to better understand the physiological roles of these chromosomally encoded modules. However, there are still controversies about some of the functions associated with different TA systems. This review will discuss the most current findings and the bona fide functions of bacterial type II TA systems.

The higBA-Type Toxin-Antitoxin System in IncC Plasmids Is a Mobilizable Ciprofloxacin-Inducible System.

A putative type II toxin-antitoxin (TA) module almost exclusively associated with conjugative IncC plasmids is homologous to the higBA family of TA systems found in chromosomes and plasmids of several species of bacteria. Despite the clinical significance and strong association with high-profile antimicrobial resistance (AMR) genes, the TA system of IncC plasmids remains largely uncharacterized. In this study, we present evidence that IncC plasmids encode a bona fide HigB-like toxin that strongly inhibits bacterial growth and results in cell elongation in Escherichia coli. IncC HigB toxin acts as a ribosome-dependent endoribonuclease that significantly reduces the transcript abundance of a subset of adenine-rich mRNA transcripts. A glycine residue at amino acid position 64 is highly conserved in HigB toxins from different bacterial species, and its replacement with valine (G64V) abolishes the toxicity and the mRNA cleavage activity of the IncC HigB toxin. The IncC plasmid higBA TA system functions as an effective addiction module that maintains plasmid stability in an antibiotic-free environment. This higBA addiction module is the only TA system that we identified in the IncC backbone and appears essential for the stable maintenance of IncC plasmids. We also observed that exposure to subinhibitory concentrations of ciprofloxacin, a DNA-damaging fluoroquinolone antibiotic, results in elevated higBA expression, which raises interesting questions about its regulatory mechanisms. A better understanding of this higBA-type TA module potentially allows for its subversion as part of an AMR eradication strategy. IMPORTANCE Toxin-antitoxin (TA) systems play vital roles in maintaining plasmids in bacteria. Plasmids with incompatibility group C are large plasmids that disseminate via conjugation and carry high-profile antibiotic resistance genes. We present experimental evidence that IncC plasmids carry a TA system that functions as an effective addiction module and maintains plasmid stability in an antibiotic-free environment. The toxin of IncC plasmids acts as an endoribonuclease that targets a subset of mRNA transcripts. Overexpressing the IncC toxin gene strongly inhibits bacterial growth and results in cell elongation in Escherichia coli hosts. We also identify a conserved amino acid residue in the toxin protein that is essential for its toxicity and show that the expression of this TA system is activated by a DNA-damaging antibiotic, ciprofloxacin. This mobile TA system may contribute to managing bacterial stress associated with DNA-damaging antibiotics.

Specialised functions of two common plasmid mediated toxin-antitoxin systems, ccdAB and pemIK, in Enterobacteriaceae.

Toxin-antitoxin systems (TAS) are commonly found on bacterial plasmids and are generally involved in plasmid maintenance. In addition to plasmid maintenance, several plasmid-mediated TAS are also involved in bacterial stress response and virulence. Even though the same TAS are present in a variety of plasmid types and bacterial species, differences in their sequences, expression and functions are not well defined. Here, we aimed to identify commonly occurring plasmid TAS in Escherichia coli and Klebsiella pneumoniae and compare the sequence, expression and plasmid stability function of their variants. 27 putative type II TAS were identified from 1063 plasmids of Klebsiella pneumoniae in GenBank. Among these, ccdAB and pemIK were found to be most common, also occurring in plasmids of E. coli. Comparisons of ccdAB variants, taken from E. coli and K. pneumoniae, revealed sequence differences, while pemIK variants from IncF and IncL/M plasmids were almost identical. Similarly, the expression and plasmid stability functions of ccdAB variants varied according to the host strain and species, whereas the expression and functions of pemIK variants were consistent among host strains. The specialised functions of some TAS may determine the host specificity and epidemiology of major antibiotic resistance plasmids.

Plasmid DNA Isolation and Visualization: Isolation and Characterization of Plasmids from Clinical Samples.

Plasmids are important in carrying antibiotic resistance and other genes between bacterial cells, and a number of methods can be employed to characterize plasmids from clinical isolates. Single colonies typically obtained as part of hospital workflow can undergo S1 nuclease treatment to linearize plasmids followed by pulsed-field gel electrophoresis to enable determination of the number and sizes of plasmids present. Hybridization of S1/PFGE gels can be used to associate replicon types and passenger genes, such as those conferring antibiotic resistance, with a particular plasmid band. Individual plasmids, obtained by conjugation or transformation, can be compared by gel electrophoresis following restriction digestion of plasmid DNA prepared by alkaline lysis methods, including using specialized kits.

Potentiation of curing by a broad-host-range self-transmissible vector for displacing resistance plasmids to tackle AMR.

Plasmids are potent vehicles for spread of antibiotic resistance genes in bacterial populations and often persist in the absence of selection due to efficient maintenance mechanisms. We previously constructed non-conjugative high copy number plasmid vectors that efficiently displace stable plasmids from enteric bacteria in a laboratory context by blocking their replication and neutralising their addiction systems. Here we assess a low copy number broad-host-range self-transmissible IncP-1 plasmid as a vector for such curing cassettes to displace IncF and IncK plasmids. The wild type plasmid carrying the curing cassette displaces target plasmids poorly but derivatives with deletions near the IncP-1 replication origin that elevate copy number about two-fold are efficient. Verification of this in mini IncP-1 plasmids showed that elevated copy number was not sufficient and that the parB gene, korB, that is central to its partitioning and gene control system, also needs to be included. The resulting vector can displace target plasmids from a laboratory population without selection and demonstrated activity in a mouse model although spread is less efficient and requires additional selection pressure.

A ParDE-family toxin antitoxin system in major resistance plasmids of Enterobacteriaceae confers antibiotic and heat tolerance.

Toxin-antitoxin (TA) systems were initially discovered as plasmid addiction systems on low-copy-number plasmids. Thousands of TA loci have since been identified on chromosomes, plasmids and mobile elements in bacteria and archaea with diverse roles in bacterial physiology and in maintenance of genetic elements. Here, we identified and characterised a plasmid mediated type II TA system in Enterobacteriaceae as a member of the ParDE super family. This system (hereafter, ParDEI) is distributed among IncI and IncF-type antibiotic resistance and virulence plasmids found in avian and human-source Escherichia coli and Salmonella. It is found that ParDEI is a plasmid stability and stress response module that increases tolerance of aminoglycoside, quinolone and ß-lactam antibiotics in E. coli by ~100-1,000-fold, and thus to levels beyond those achievable in the course of antibiotic therapy for human infections. ParDEI also confers a clear survival advantage at 42 °C and expression of the ParEI toxin in trans induces the SOS response, inhibits cell division and promotes biofilm formation. This transmissible high-level antibiotic tolerance is likely to be an important factor in the success of the IncI and IncF plasmids which carry it and the important pathogens in which these are resident.

CRISPR-Cas System in Antibiotic Resistance Plasmids in Klebsiella pneumoniae.

CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated protein) is a microbial adaptive immune system involved in defense against different types of mobile genetic elements. CRISPR-Cas systems are usually found in bacterial and archaeal chromosomes but have also been reported in bacteriophage genomes and in a few mega-plasmids. Klebsiella pneumoniae is an important member of the Enterobacteriaceae with which they share a huge pool of antibiotic resistance genes, mostly via plasmids. CRISPR-Cas systems have been identified in K. pneumoniae chromosomes, but relatively little is known of CRISPR-Cas in the plasmids resident in this species. In this study, we searched for CRISPR-Cas system in 699 complete plasmid sequences (>50-kb) and 217 complete chromosomal sequences of K. pneumoniae from GenBank and analyzed the CRISPR-Cas systems and CRISPR spacers found in plasmids and chromosomes. We found a putative CRISPR-Cas system in the 44 plasmids from Klebsiella species and GenBank search also identified the identical system in three plasmids from other Enterobacteriaceae, with CRISPR spacers targeting different plasmid and chromosome sequences. 45 of 47 plasmids with putative type IV CRISPR had IncFIB replicon and 36 of them had an additional IncHI1B replicon. All plasmids except two are very large (>200 kb) and half of them carried multiple antibiotic resistance genes including bla CTX-M , bla NDM , bla OXA . To our knowledge, this is the first report of multi drug resistance plasmids from Enterobacteriaceae with their own CRISPR-Cas system and it is possible that the plasmid type IV CRISPR may depend on the chromosomal type I-E CRISPRs for their competence. Both chromosomal and plasmid CRISPRs target a large variety of plasmids from this species, further suggesting key roles in the epidemiology of large plasmids.

Detection and characterization of a functional insertion sequence, ISVpa2, in Vibrio parahaemolyticus.

PCR analysis of the pandemic strain of Vibrio parahaemolyticus, KX-V237 (total genome sequenced) showed a subculture where the size of the amplicons had increased. The purpose of this study was to analyze the mechanism of this change. We found a 1,243-bp DNA sequence inserted in one of the pandemic marker genes in this strain. The inserted DNA sequence possessed the genetic structures shared by insertion sequences (ISs) of the IS3 family. This IS had 26-bp imperfect terminal inverted repeats (IRs) and two partially overlapping reading frames, orfA and orfB. OrfA codes for a helix-turn-helix, OrfA and OrfAB produced by translational frameshifting code for leucine zipper motifs, and OrfB codes for a DDE motif. orfA and orfB were homologous to those in the IS3 family. This IS was named ISVpa2. Southern blot analysis showed the copy number of ISVpa2 in our stock culture and its subculture of KX-V237 was three and four, respectively; whereas it was only one in the reported genome sequence. Analysis of the flanking sequences for seven ISVpa2 copies showed ISVpa2 is capable of inserting at multiple sites and ISVpa2 causes genetic rearrangements including insertional inactivation of the target gene and adjacent deletion. ISVpa2 created 3-base duplications upon insertion. PCR, hybridization, and nucleotide sequence analyses showed ISVpa2 homologs were detected in all of the 62 other strains of V. parahaemolyticus examined; and in some strains of Vibrio vulnificus (98% identity), Vibrio penaeicida (86% identity), and Vibrio splendidus (87% identity); but was not in 25 other species in the genus Vibrio. The data demonstrate that ISVpa2 is a transpositionally active IS discovered for the first time in V. parahaemolyticus and suggest that ISVpa2 may be transferred among the species of the genus Vibrio.

Detection of a functional insertion sequence responsible for deletion of the thermostable direct hemolysin gene (tdh) in Vibrio parahaemolyticus.

The thermostable direct hemolysin coded by the tdh gene is a marker of virulent strains of Vibrio parahaemolyticus. The tdh genes are flanked by insertion sequences collectively named as ISVs or their remnants; but the ISVs so far examined have accumulated mutations in the transposase genes and underwent structural arrangements and their transposition activity could not be expected; the tdh gene was thus considered to have been acquired by V. parahaemolyticus through horizontal transfer in the past during evolution. We recently isolated from the same patient tdh+ strains and a tdh(-) strain (PCR examination) that were otherwise indistinguishable. The purpose of this study was to examine the hypothesis that the tdh(-) strain was derived from the tdh+ strain by a deletion of the tdh gene mediated by a functional ISV. Southern blot hybridization showed tdh+ sequences in the tdh(-) strain (PSU-1466). Nucleotide sequence analysis of the tdh and its flanking sequences revealed the tdh gene was split into two parts and they were located 3182-bp apart in PSU-1466. The two tdh sequences were flanked by one of the ISVs, named as ISVpa3, in PSU-1466. This genetic structure could be explained by an ISVpa3-mediated partial tdh deletion from a tdh+ strain followed by transposition of the duplicated ISVpa3 and the deleted tdh sequence into a neighboring location. The ISVpa3 of PSU-1466 coded for a full-length transposase and a DDE motif. We were able to demonstrate transposition activity of the ISVpa3 cloned from PSU-1466 using the replicon fusion assay with the conjugal transfer of a cointegrate from Escherichia coli to V. parahaemolyticus. Our data support ISVpa3-mediated partial tdh deletion resulted in the emergence of the tdh(-) strain.

On the origin and function of an insertion element VPaI-1 specific to post-1995 pandemic Vibrio parahaemolyticus strains.

Since 1995, new virulent strains of Vibrio parahaemolyticus have emerged and spread throughout the world. These "pandemic" strains have four strain specific genomic islands (GIs), which are considered to be potential factors of the pandemicity. We investigated the origin and function of 24 genes in the so-called VPaI-1, one of the four GIs, by searching homologs in various species in Bacteria and Archaea. Of these 24 genes, two are found only in Vibrio vulnificus CMCP6 and Shewanella sp. MR-7. The genomic segment (- 8 kb) encompassing the two genes shows the synteny among the three species. Since many of the Shewanella species can grow at 4 degrees C, these two genes may be candidates of adaptation to temperature stress. Further, we found a candidate for a swarming gene, which is reported as the V. cholerae virulence gene. Based on these findings, we hypothesized the emergence of pandemicity and discuss the mechanism for how these strains spread throughout the world.

Isolation and characterization of the Shiga toxin gene (stx)-bearing Escherichia coli O157 and non-O157 from retail meats in Shandong Province, China, and characterization of the O157-derived stx2 phages.

Infection by Shiga toxin (Stx)-producing Escherichia coli of non-O157 and O157 serotypes are rare in China, but infection by O157 serotype was found in Shandong Province and three other provinces in China. To understand the reason for these rare infections and to determine the safety of retail meats in Shandong Province, we examined the distribution of Shiga toxin gene (stx)-bearing E. coli in retail meats and characterized the isolated stx-bearing strains. We used hybridization with DNA probes and isolated stx1- and/or stx2-positive E. coli from 31 (58%) of 53 retail meat samples, with beef showing the highest frequency (68%). Of 42 stx-positive isolates, none belonged to O157. Using the O157-specific immunomagnetic bead technique, we isolated E. coli O157 carrying the eae and stx2 genes from eight beef samples (26%). These strains produced little or no Stx2 and carried a unique q gene. Replication of the stx2 phages was detected in these strains, whereas stx2 phage replication was not detected in our previous study in which we examined similar stx2-bearing E. coli O157 strains from other Asian countries. Analysis of E. coli C600 lysogenized with the stx2 phages found in this study suggests that the lack of Stx2 production is due to changes in non-q gene region(s) of the phage genome or chromosomal mutation(s) in the host. Our data and reports by other workers suggest it is necessary to determine if various stx2-bearing E. coli O157 strains producing Stx2 to varying degrees are distributed in meats in various locations in China.

Plasmid interference for curing antibiotic resistance plasmids in vivo.

Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing ('addiction') systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative 'interference plasmids' were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored.

Characterization of multidrug-resistant Klebsiella pneumoniae from Australia carrying blaNDM-1.

blaNDM genes, encoding metallo-ß-lactamases providing resistance to carbapenems, have been reported in many locations since the initial report in 2008, including in several Enterobacteriaceae isolates in Australia/New Zealand. Here, we compare 4 additional carbapenem-resistant Klebsiella pneumoniae carrying blaNDM-1 isolated in Australia. Two are sequence type ST147, previously associated with blaNDM in Australia and elsewhere. They carry blaNDM-1 and different 16S rRNA methylase genes (armA or rmtC) on different conjugative plasmids, in 1 case with an IncFIIY replicon. One isolate belongs to the globally important ST11 but did not transfer a plasmid to Escherichia coli. The fourth isolate belongs to the novel ST1068 and transferred blaNDM-1, armA, and an IncA/C plasmid. Amplification and sequencing of ompK porin genes suggest that, unlike the case for other carbapenemase genes, ompK36 defects may not be required for NDM to cause clinically relevant levels of carbapenem resistance.

In vitro and in vivo bactericidal activity of Vitex negundo leaf extract against diverse multidrug resistant enteric bacterial pathogens.

OBJECTIVE: To investigate in vitro and in vivo antibacterial potentials of Vitex negundo (V. negundo) leaf extracts against diverse enteric pathogens. METHODS: Water and methanol extracts of V. negundo leaves were evaluated against enteric bacterial pathogens by using standard disc diffusion, viable bacterial cell count methods, determination of minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). RESULTS: Methanol extract of V. negundo leaves showed potent antibacterial activity (inhibition zone: 9.9-22.6 mm, MIC: 200-3200 µg/mL, MBC: 200-6400 µg/mL) against all the pathogenic enteric bacteria (Vibrio cholerae, Vibrio parahaemolyticus, Vibrio mimicus, Echerichia coli, Shigella spps., and Aeromonas spps) tested. Methanol extract of V. negundo leaves showed potent bactericidal activity both in vitro laboratory conditions (MBC, 200-400 µg/mL) and in the intestinal environment (Dose, 1-2 mg/mL) of infant mice against pathogenic Vibrio cholerae, the major causative agent of cholera. Furthermore, assays using the mice cholera model showed that V. negundo methanol extract can protect mice from Vibrio cholerae infection and significantly decrease the mortality rate (P<0.0001). CONCLUSIONS: For the first time we showed that methanol extract of V. negundo leaves exhibited strong vibriocidal activity both in vitro and in vivo conditions. Therefore, it will be useful to identify and isolate the active compounds of this extract that could be a good alternative of antibiotics to treat cholera.

Variability of properties of Vibrio parahaemolyticus strains isolated from individual patients.

Infections by strains belonging to the O3:K6 pandemic clone of Vibrio parahaemolyticus are prevalent in southern Thailand, and serovariants of these strains have also been detected. V. parahaemolyticus strains lacking important virulence genes (tdh and trh) were isolated from 6.5 to 10.9% of clinical specimens during the period from 2000 to 2003. In order to understand whether changes to the characteristics of V. parahaemolyticus occur during infection, 10 isolates collected from each of 63 patients who presented with diarrhea at the Hat Yai hospital from 2003 to 2004 were examined for the presence of the tdh and trh genes, the O:K serotype, and genetic markers for the pandemic clone. A total of 42 patients (66.7%) yielded identical isolates (homogeneous populations), and 21 of the patients (33.3%) yielded isolates that differed in at least one character from the other isolates (heterogeneous populations). The DNA fingerprints (examined by arbitrarily primed PCR and pulsed-field gel electrophoresis) of some, but not all, of the heterogeneous populations from single patients were indistinguishable. The results indicated that some patients were infected with a unique strain and that in vivo changes (tdh deletion or serotype conversion) might have occurred in certain individuals. It is therefore important to bear in mind that epidemiological studies based on the analysis of a single colony from a single patient might lead to misleading conclusions. Finally, the present study did not rule out the possibility that isolates lacking tdh and trh have unknown virulence mechanisms other than the tdh and trh genes.