Pathogen invasion-dependent tissue reservoirs and plasmid-encoded antibiotic degradation boost plasmid spread in the gut.

Many plasmids encode antibiotic resistance genes. Through conjugation, plasmids can be rapidly disseminated. Previous work identified gut luminal donor/recipient blooms and tissue-lodged plasmid-bearing persister cells of the enteric pathogen Salmonella enterica serovar Typhimurium (S.Tm) that survive antibiotic therapy in host tissues, as factors promoting plasmid dissemination among Enterobacteriaceae. However, the buildup of tissue reservoirs and their contribution to plasmid spread await experimental demonstration. Here, we asked if re-seeding-plasmid acquisition-invasion cycles by S.Tm could serve to diversify tissue-lodged plasmid reservoirs, and thereby promote plasmid spread. Starting with intraperitoneal mouse infections, we demonstrate that S.Tm cells re-seeding the gut lumen initiate clonal expansion. Extended spectrum beta-lactamase (ESBL) plasmid-encoded gut luminal antibiotic degradation by donors can foster recipient survival under beta-lactam antibiotic treatment, enhancing transconjugant formation upon re-seeding. S.Tm transconjugants can subsequently re-enter host tissues introducing the new plasmid into the tissue-lodged reservoir. Population dynamics analyses pinpoint recipient migration into the gut lumen as rate-limiting for plasmid transfer dynamics in our model. Priority effects may be a limiting factor for reservoir formation in host tissues. Overall, our proof-of-principle data indicates that luminal antibiotic degradation and shuttling between the gut lumen and tissue-resident reservoirs can promote the accumulation and spread of plasmids within a host over time.

Episome partitioning and symmetric cell divisions: Quantifying the role of random events in the persistence of HPV infections.

Human Papillomaviruses (HPV) are one of the most prevalent sexually transmitted infections (STI) and the most oncogenic viruses known to humans. The vast majority of HPV infections clear in less than 3 years, but the underlying mechanisms, especially the involvement of the immune response, are still poorly known. Building on earlier work stressing the importance of randomness in the type of cell divisions in the clearance of HPV infection, we develop a stochastic mathematical model of HPV dynamics that combines the previous aspect with an explicit description of the intracellular level. We show that the random partitioning of virus episomes upon stem cell division and the occurrence of symmetric divisions dramatically affect viral persistence. These results call for more detailed within-host studies to better understand the relative importance of stochasticity and immunity in HPV infection clearance.

Colistin resistance and plasmid-mediated mcr genes in Escherichia coli and Salmonella isolated from pigs, pig carcass and pork in Thailand, Lao PDR and Cambodia border provinces.

BACKGROUND: Colistin and carbapenem-resistant bacteria have emerged and become a serious public health concern, but their epidemiological data is still limited. OBJECTIVES: This study examined colistin and carbapenem resistance in Escherichia coli and Salmonella from pigs, pig carcasses, and pork in Thailand, Lao PDR, and Cambodia border provinces. METHODS: The phenotypic and genotypic resistance to colistin and meropenem was determined in E. coli and Salmonella obtained from pigs, pig carcasses, and pork (n = 1,619). A conjugative experiment was performed in all isolates carrying the mcr gene (s) (n = 68). The plasmid replicon type was determined in the isolates carrying a conjugative plasmid with mcr by PCR-based replicon typing (n = 7). The genetic relatedness of mcr-positive Salmonella (n = 11) was investigated by multi-locus sequence typing. RESULTS: Colistin resistance was more common in E. coli (8%) than Salmonella (1%). The highest resistance rate was found in E. coli (17.8%) and Salmonella (1.7%) from Cambodia. Colistin-resistance genes, mcr-1, mcr-3, and mcr-5, were identified, of which mcr-1 and mcr-3 were predominant in E. coli (5.8%) and Salmonella (1.7%), respectively. The mcr-5 gene was observed in E. coli from pork in Cambodia. Two colistin-susceptible pig isolates from Thailand carried both mcr-1 and mcr-3. Seven E. coli and Salmonella isolates contained mcr-1 or mcr-3 associated with the IncF and IncI plasmids. The mcr-positive Salmonella from Thailand and Cambodia were categorized into two clusters with 94%-97% similarity. None of these clusters was meropenem resistant. CONCLUSIONS: Colistin-resistant E. coli and Salmonella were distributed in pigs, pig carcasses, and pork in the border areas. Undivided-One Health collaboration is needed to address the issue.

Activity of Plasmodium vivax promoter elements in Plasmodium knowlesi, and a centromere-containing plasmid that expresses NanoLuc throughout the parasite life cycle.

BACKGROUND: Plasmodium knowlesi is now the major cause of human malaria in Malaysia, complicating malaria control efforts that must attend to the elimination of multiple Plasmodium species. Recent advances in the cultivation of P. knowlesi erythrocytic-stage parasites in vitro, transformation with exogenous DNA, and infection of mosquitoes with gametocytes from culture have opened up studies of this pathogen without the need for resource-intensive and costly non-human primate (NHP) models. For further understanding and development of methods for parasite transformation in malaria research, this study examined the activity of various trans-species transcriptional control sequences and the influence of Plasmodium vivax centromeric (pvcen) repeats in plasmid-transfected P. knowlesi parasites. METHODS: In vitro cultivated P. knowlesi parasites were transfected with plasmid constructs that incorporated Plasmodium vivax or Plasmodium falciparum 5' UTRs driving the expression of bioluminescence markers (firefly luciferase or Nanoluc). Promoter activities were assessed by bioluminescence, and parasites transformed with human resistant allele dihydrofolate reductase-expressing plasmids were selected using antifolates. The stability of transformants carrying pvcen-stabilized episomes was assessed by bioluminescence over a complete parasite life cycle through a rhesus macaque monkey, mosquitoes, and a second rhesus monkey. RESULTS: Luciferase expression assessments show that certain P. vivax promoter regions, not functional in the more evolutionarily-distant P. falciparum, can drive transgene expression in P. knowlesi. Further, pvcen repeats may improve the stability of episomal plasmids in P. knowlesi and support detection of NanoLuc-expressing elements over the full parasite life cycle from rhesus macaque monkeys to Anopheles dirus mosquitoes and back again to monkeys. In assays of drug responses to chloroquine, G418 and WR9910, anti-malarial half-inhibitory concentration (IC50) values of blood stages measured by NanoLuc activity proved comparable to IC50 values measured by the standard SYBR Green method. CONCLUSION: All three P. vivax promoters tested in this study functioned in P. knowlesi, whereas two of the three were inactive in P. falciparum. NanoLuc-expressing, centromere-stabilized plasmids may support high-throughput screenings of P. knowlesi for new anti-malarial agents, including compounds that can block the development of mosquito- and/or liver-stage parasites.

Population genomics and antimicrobial resistance dynamics of Escherichia coli in wastewater and river environments.

Aquatic environments are key niches for the emergence, evolution and dissemination of antimicrobial resistance. However, the population diversity and the genetic elements that drive the dynamics of resistant bacteria in different aquatic environments are still largely unknown. The aim of this study was to understand the population genomics and evolutionary events of Escherichia coli resistant to clinically important antibiotics including aminoglycosides, in anthropogenic and natural water ecosystems. Here we show that less different E. coli sequence types (STs) are identified in wastewater than in rivers, albeit more resistant to antibiotics, and with significantly more plasmids/cell (6.36 vs 3.72). However, the genomic diversity within E. coli STs in both aquatic environments is similar. Wastewater environments favor the selection of conserved chromosomal structures associated with diverse flexible plasmids, unraveling promiscuous interplasmidic resistance genes flux. On the contrary, the key driver for river E. coli adaptation is a mutable chromosome along with few plasmid types shared between diverse STs harboring a limited resistance gene content.

The Rcs stress response inversely controls surface and CRISPR-Cas adaptive immunity to discriminate plasmids and phages.

Bacteria harbour multiple innate defences and adaptive CRISPR-Cas systems that provide immunity against bacteriophages and mobile genetic elements. Although some bacteria modulate defences in response to population density, stress and metabolic state, a lack of high-throughput methods to systematically reveal regulators has hampered efforts to understand when and how immune strategies are deployed. We developed a robust approach called SorTn-seq, which combines saturation transposon mutagenesis, fluorescence-activated cell sorting and deep sequencing to characterize regulatory networks controlling CRISPR-Cas immunity in Serratia sp. ATCC 39006. We applied our technology to assess csm gene expression for ~300,000 mutants and uncovered multiple pathways regulating type III-A CRISPR-Cas expression. Mutation of igaA or mdoG activated the Rcs outer-membrane stress response, eliciting cell-surface-based innate immunity against diverse phages via the transcriptional regulators RcsB and RcsA. Activation of this Rcs phosphorelay concomitantly attenuated adaptive immunity by three distinct type I and III CRISPR-Cas systems. Rcs-mediated repression of CRISPR-Cas defence enabled increased acquisition and retention of plasmids. Dual downregulation of cell-surface receptors and adaptive immunity in response to stress by the Rcs pathway enables protection from phage infection without preventing the uptake of plasmids that may harbour beneficial traits.

Beyond horizontal gene transfer: the role of plasmids in bacterial evolution.

Plasmids have a key role in bacterial ecology and evolution because they mobilize accessory genes by horizontal gene transfer. However, recent studies have revealed that the evolutionary impact of plasmids goes above and beyond their being mere gene delivery platforms. Plasmids are usually kept at multiple copies per cell, producing islands of polyploidy in the bacterial genome. As a consequence, the evolution of plasmid-encoded genes is governed by a set of rules different from those affecting chromosomal genes, and these rules are shaped by unusual concepts in bacterial genetics, such as genetic dominance, heteroplasmy or segregational drift. In this Review, we discuss recent advances that underscore the importance of plasmids in bacterial ecology and evolution beyond horizontal gene transfer. We focus on new evidence that suggests that plasmids might accelerate bacterial evolution, mainly by promoting the evolution of plasmid-encoded genes, but also by enhancing the adaptation of their host chromosome. Finally, we integrate the most relevant theoretical and empirical studies providing a global understanding of the forces that govern plasmid-mediated evolution in bacteria.

Collateral sensitivity associated with antibiotic resistance plasmids.

Collateral sensitivity (CS) is a promising alternative approach to counteract the rising problem of antibiotic resistance (ABR). CS occurs when the acquisition of resistance to one antibiotic produces increased susceptibility to a second antibiotic. Recent studies have focused on CS strategies designed against ABR mediated by chromosomal mutations. However, one of the main drivers of ABR in clinically relevant bacteria is the horizontal transfer of ABR genes mediated by plasmids. Here, we report the first analysis of CS associated with the acquisition of complete ABR plasmids, including the clinically important carbapenem-resistance conjugative plasmid pOXA-48. In addition, we describe the conservation of CS in clinical E. coli isolates and its application to selectively kill plasmid-carrying bacteria. Our results provide new insights that establish the basis for developing CS-informed treatment strategies to combat plasmid-mediated ABR.

Borrelia miyamotoi strain LB-2001 retains plasmids and infectious phenotype throughout continuous culture passages as evaluated by multiplex PCR.

Borrelia miyamotoi is a tick-borne spirochete of the relapsing fever borrelia group and an emerging pathogen of public health significance. The genomes of relapsing fever borreliae and Lyme disease borreliae consist of multiple linear and circular plasmids in addition to the chromosome. Previous work with B. burgdorferi sensu lato found diminished infectivity upon continuous in vitro culture passage that was attributable to plasmid loss. The effect of long-term culture passage on B. miyamotoi is not known. We generated a series of plasmid-specific primer sets and developed a multiplex PCR assay to detect the 14 known plasmids of B. miyamotoi North American strains LB-2001 and CT13-2396. We assessed the plasmid content of B. miyamotoi LB-2001 over 64 culture passages spanning 15 months and determined that strain LB-2001 retained all plasmids upon prolonged in vitro cultivation and remained infectious in mice. We also found that strain LB-2001 lacks plasmid lp20-1 which is present in strain CT13-2396. These results suggest that B. miyamotoi remains genetically stable when cultured and passaged in vitro.

Controlled expression of lysis gene E by a mutant of the promoter pL of the thermo-inducible λcI857-pL system.

AIMS: To identify a lambda promoter pL mutant that could extend the thermal stability of the thermo-inducible λcI857-pR/pL system and to evaluate the effects of the modified system for the controlled expression of lysis gene E during the production of bacterial ghosts (BGs). METHODS AND RESULTS: The promoter pL mutant was identified by random mutagenesis and site-directed mutagenesis. The results showed that a T â†’ 35C mutation in the pL promoter was responsible for the phenotype alteration. Under the same induction conditions, the lysis rates of the modified lytic system on Escherichia coli and Salmonella enteritidis were significantly lower than that of the control, while the lysis rates of Escherichia coli with the thermo-inducible lytic system were significantly higher than that of S. enteritidis with the corresponding plasmid (P < 0·05). CONCLUSIONS: Increasing the heat stability of the thermo-inducible lytic systems decreased lysis efficiency during the production of BGs. There exist differences in the lysis efficiency of thermo-inducible lytic systems between different bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings enrich current knowledge about modifications to thermo-inducible systems and provide a reference for the application of these modified systems for the production of BGs and controlled gene expression in bacteria.

Occurrence and characteristics of Escherichia coli mcr-1-like in rabbits in Shandong, China.

Polymyxin is regarded as the last retort to fight against multidrug-resistant (MDR) Enterobacteriaceae. The emergency and spread of polymyxin-associated resistance gene mcr-1 evoked great panic of no medicine to cure the bacterial infection in society. mcr-1 is widespread in domestic and wild animals. Therefore, continuous monitoring of its prevalence and characteristics is required. In this study, we used a polymerase chain reaction (PCR)-based method to detect the mcr-1 of Escherichia coli isolated from rabbits of Tai'an, China, and determined the characteristics of mcr-1-bearing plasmids. A total of 55 non-duplicated E. coli was recovered from the swabs of rabbit faeces. Plasmid profiling, plasmid and chromosome PCR, complete genome sequencing, a conjugation experiment, lactose fermentation experiment, multilocus sequence typing and polymyxin resistance tests were performed to determine the characteristics of mcr-1-bearing plasmids. 14.6% (8/55) of the specimens were mcr-1 positive. The mcr-1-positive E. coli harboured more drug-resistant genes compared with the mcr-1-negative specimens, and results showed four sequence types. Overall, these findings suggested the possible threat of the transmission of mcr-1 from rabbits to humans, especially since the gene is located on transferable plasmids making horizontal transfer relatively easy. Since food-producing animals are necessary for our daily diet, worldwide cooperation is needed in fighting the spread of this drug resistance gene to avoid human infections with MDR pathogenic bacteria.

LncRNA PART1 promotes cell proliferation and progression in non-small-cell lung cancer cells via sponging miR-17-5p.

It has been demonstrated in previous studies that lncPART1 is dysregulated in non-small cell lung cancer (NSCLC). However, the function of lncPART1 in NSCLC is unclear. Therefore, this experimental design was based on LncPART1 to explore the pathogenesis of NSCLC. Real-time polymerase chain reaction was used to detect the expression of lncPART1 and miR-17-5p in NSCLC. Cell Counting Kit -8, colony formation, and transwell assays were used to examine the effects of lncPART1 and miR-17-5p on NSCLC cell proliferation and migration invasiveness. Target gene prediction, luciferase reporter assays were used to validate downstream target genes for lncPART1 and miR-17-5p. Western blot analysis was used to detect the expression of TGFBETAR2. LncPART1 was highly expressed in NSCLC. LncPART1 significantly promoted cell proliferation of NSCLC cells. miR-17-5p was down-expressed in NSCLC. miR-17-5p overexpression inhibited cell proliferation and migration invasion in NSCLC cells. LncPART1 was able to inhibit miR-17-5p expression and upregulate the expression level of TGFBETAR2. The results of in vivo animal models confirmed that lncPART1 promoted NSCLC progression by miR-17-5p/TGFBETAR2 axis. LncPART1 promoted the progression of NSCLC by miR-17-5p/TGFBETAR2 axis.

The Conjugation Window in an Escherichia coli K-12 Strain with an IncFII Plasmid.

Many studies have examined the role that conjugation plays in disseminating antibiotic resistance genes in bacteria. However, relatively little research has quantitively examined and modeled the dynamics of conjugation under growing and nongrowing conditions beyond a couple of hours. We therefore examined growing and nongrowing cultures of Escherichia coli over a 24-h period to understand the dynamics of bacterial conjugation in the presence and absence of antibiotics with pUUH239.2, an IncFII plasmid containing multiantibiotic- and metal-resistant genes. Our data indicate that conjugation occurs after E. coli cells divide and before they have transitioned to a nongrowing phase. The result is that there is only a small window of opportunity for E. coli to conjugate with pUUH239.2 under both growing and nongrowing conditions. Only a very small percentage of the donor cells likely are capable of even undergoing conjugation, and not all transconjugants can become donor cells due to molecular regulatory controls and not being in the correct growth phase. Once a growing culture enters stationary phase, the number of capable donor cells decreases rapidly and conjugation slows to produce a plateau. Published models did not provide accurate descriptions of conjugation under nongrowing conditions. We present here a modified modeling approach that accurately describes observed conjugation behavior under growing and nongrowing conditions.IMPORTANCE There has been growing interest in horizontal gene transfer of antibiotic resistance plasmids as the antibiotic resistance crisis has worsened over the years. Most studies examining conjugation of bacterial plasmids focus on growing cultures of bacteria for short periods, but in the environment, most bacteria grow episodically and at much lower rates than in the laboratory. We examined conjugation of an IncFII antibiotic resistance plasmid in E. coli under growing and nongrowing conditions to understand the dynamics of conjugation under which the plasmid is transferred. We found that conjugation occurs in a narrow time frame when E. coli is transitioning from a growing to nongrowing phase and that the conjugation plateau develops because of a lack of capable donor cells in growing cultures. From an environmental aspect, our results suggest that episodic growth in nutrient-depleted environments could result in more conjugation than sustained growth in a nutrient rich environment.

Chlamydia trachomatis plasmid-encoded protein pORF5 activates unfolded protein response to induce autophagy via MAPK/ERK signaling pathway.

Chlamydia trachomatis (C. trachomatis) is an obligate intracellular organism that depends on nutrients from the host cell for their replication and proliferation. Therefore, the interaction between this pathogen and host induces sustained endoplasmic reticulum (ER) stress in the infected cells. Unfolded protein response (UPR) has been demonstrated to be activated by chlamydial secreted effectors, allowing host cells to recover from the stressful state. In this study, we attempted to explore the role of the only secreted plasmid-encoded protein pORF5 of C. trachomatis between UPR and autophagy induction. The results showed that three branches of UPR (PERK, IRE1, and ATF6) were activated by pORF5. pORF5-induced autophagy was repressed by UPR inhibitors GSK2606414 and 4µ8C, while the autophagy inhibition was failed to influence pORF5-induced UPR significantly. MAPK/ERK inhibitor PD98059 partially suppressed the pORF5-induced autophagy, but had little effect on UPR, indicating that pORF5 actives UPR to induce autophagy via the MAPK/ERK signaling pathway. These observations provide clues on how the host maintains the cellular homeostasis during C. trachomatis infection.

Investigating Neurogenesis in Birds.

The macula and fovea make human vision unique among mammals. An understanding of the genetic network underlying the development and maintenance of this highly specialized region is instrumental to address issues about human macula-related retinopathies. The pigeon retina, unlike currently available animal models, shares numerous key characteristics of the primate macula and represents a promising new model for the study of retinal development. We provide key elements to take advantage of this new model for the study of retina and brain development. This includes precise embryo staging, transfection of genetic material (reporter plasmid, expression vectors, siRNAs) using in ovo and ex vivo electroporation, live imaging, high-resolution confocal imaging, and data layout and instructions for data analysis.

Multiple Megaplasmids Confer Extremely High Levels of Metal Tolerance in Alteromonas Strains.

Alteromonas is a widely distributed genus of marine Gammaproteobacteria, with representatives shown to be key players in diverse processes, including biogeochemical cycling and biofouling of marine substrata. While Alteromonas spp. are early colonizers of copper-based antifouling paints on marine vessels, their mechanism of tolerance is poorly understood. PacBio whole-genome sequencing of Alteromonas macleodii strains CUKW and KCC02, isolated from Cu/Ni alloy test coupons submerged in oligotrophic coastal waters, indicated the presence of multiple megaplasmids (ca. 200 kb) in both. A pulsed-field gel electrophoresis method was developed and used to confirm the presence of multiple megaplasmids in these two strains; it was then used to screen additional Alteromonas strains for which little to no sequencing data exist. Plasmids were not detected in any of the other strains. Bioinformatic analysis of the CUKW and KCC02 plasmids identified numerous genes associated with metal resistance. Copper resistance orthologs from both the Escherichia coli Cue and Cus and Pseudomonas syringae Cop systems were present, at times as multiple copies. Metal growth assays in the presence of copper, cobalt, manganese, and zinc performed with 10 Alteromonas strains demonstrated the ability of CUKW and KCC02 to grow at metal concentrations inhibitory to all the other strains tested. This study reports multiple megaplasmids in Alteromonas strains. Bioinformatic analysis of the CUKW and KCC02 plasmids indicate that they harbor elements of the Tra system conjugation apparatus, although their type of mobility remains to be experimentally verified.IMPORTANCE Copper is commonly used as an antifouling agent on ship hulls. Alteromonas spp. are early colonizers of copper-based antifouling paint, but their mechanism of tolerance is poorly understood. Sequencing of A. macleodii strains isolated from copper test materials for marine ships indicated the presence of multiple megaplasmids. Plasmids serve as key vectors in horizontal gene transfer and confer traits such as metal resistance, detoxification, ecological interaction, and antibiotic resistance. Bioinformatic analysis identified many metal resistance genes and genes associated with mobility. Understanding the molecular mechanisms and capacity for gene transfer within marine biofilms provides a platform for the development of novel antifouling solutions targeting genes involved in copper tolerance and biofilm formation.

Extremely fast amelioration of plasmid fitness costs by multiple functionally diverse pathways.

The acquisition of plasmids is often accompanied by fitness costs such that compensatory evolution is required to allow plasmid survival, but it is unclear whether compensatory evolution can be extensive or rapid enough to maintain plasmids when they are very costly. The mercury-resistance plasmid pQBR55 drastically reduced the growth of its host, Pseudomonas fluorescens SBW25, immediately after acquisition, causing a small colony phenotype. However, within 48 h of growth on agar plates we observed restoration of the ancestral large colony morphology, suggesting that compensatory mutations had occurred. Relative fitness of these evolved strains, in lab media and in soil microcosms, varied between replicates, indicating different mutational mechanisms. Using genome sequencing we identified that restoration was associated with chromosomal mutations in either a hypothetical DNA-binding protein PFLU4242, RNA polymerase or the GacA/S two-component system. Targeted deletions in PFLU4242, gacA or gacS recapitulated the ameliorated phenotype upon plasmid acquisition, indicating three distinct mutational pathways to compensation. Our data shows that plasmid compensatory evolution is fast enough to allow survival of a plasmid despite it imposing very high fitness costs upon its host, and indeed may regularly occur during the process of isolating and selecting individual plasmid-containing clones.

Control of Viral Latency by Episome Maintenance Proteins.

The human DNA tumor viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV) share the common property of persisting as multicopy episomes in the nuclei of rapidly dividing host cells. These episomes form the molecular basis for viral latency and are etiologically linked to virus-associated cancers. Episome maintenance requires epigenetic programming to ensure the proper control of viral gene expression, DNA replication, and genome copy number. For these viruses, episome maintenance requires a dedicated virus-encoded episome maintenance protein (EMP), namely LANA (KSHV), EBNA1 (EBV), and E2 (HPV). Here, we review common features of these viral EMPs and discuss recent advances in understanding how they contribute to the epigenetic control of viral episome maintenance during latency.

The Cryptic Plasmid Improves Chlamydia Fitness in Different Regions of the Gastrointestinal Tract.

The cryptic plasmid is important for chlamydial colonization in the gastrointestinal tract. We used a combination of intragastric, intrajejunal, and intracolon inoculations to reveal the impact of the plasmid on chlamydial colonization in distinct regions of gastrointestinal tract. Following an intragastric inoculation, the plasmid significantly improved chlamydial colonization. At the tissue level, plasmid-positive Chlamydia produced infectious progenies throughout gastrointestinal tract. However, to our surprise, plasmid-deficient Chlamydia failed to produce infectious progenies in small intestine, although infectious progenies were eventually detected in large intestine, indicating a critical role of the plasmid in chlamydial differentiation into infectious particles in small intestine. The noninfectious status may represent persistent infection, since Chlamydia genomes proliferated in the same tissues. Following an intrajejunal inoculation that bypasses the gastric barrier, plasmid-deficient Chlamydia produced infectious progenies in small intestine but was 530-fold less infectious than plasmid-positive Chlamydia, suggesting that (i) the noninfectious status developed after intragastric inoculation might be induced by a combination of gastric and intestinal effectors and (ii) chlamydial colonization in small intestine was highly dependent on plasmid. Finally, following an intracolon inoculation, the dependence of chlamydial colonization on plasmid increased over time. Thus, we have demonstrated that the plasmid may be able to improve chlamydial fitness in different gut regions via different mechanisms, which has laid a foundation to further reveal the specific mechanisms.

Plasmid-mediated quinolone resistance in Escherichia coli isolates from commercial broiler chickens and selection of fluoroquinolone-resistant mutants.

Plasmid-mediated quinolone resistance (PMQR) is a potential concern for animal husbandry and public health. Escherichia coli isolates from a total of 109 fecal samples collected from 6 commercial broiler farms between 2007 and 2011 were examined for PMQR genes, and transfer of these genes was tested by conjugation analysis to elucidate the prevalence and spread of PMQR in broiler chickens. Two isolates from 2 farms harbored the aac(6')-Ib-cr gene that was not detected in plasmids using Southern blot analysis of S1 nuclease-digested genomic DNA separated by pulsed-field gel electrophoresis. In these 2 isolates, nucleotide mutations in the gyrA and parC genes that result in amino acid substitutions were detected. Additionally, a total of 6 isolates originating from 6 chickens from the 2 farms were positive for the qnrS1 gene. In 2 of the 6 isolates, the qnrS1 gene was transferred to a recipient strain. Two transconjugants harboring the qnrS1 gene were cultured on media supplemented with successively higher concentrations of enrofloxacin (ERFX). After a 5-time subcultivation, the ERFX MICs reached 8 and 16 µg/mL, and no nucleotide mutations were detected in the gyrA, gyrB, parC, and parE genes. Our results suggest that the prevalence of PMQR was relatively low in broiler chickens and that exposure of bacteria carrying PMQR genes to the selective pressure of fluoroquinolones can result in resistance to fluoroquinolone, which is not caused by mutations in genes encoding topoisomerases.