Curing of a field strain of Salmonella enterica serovar Infantis isolated from poultry from its highly stable pESI like plasmid.

Salmonella enterica serovar Infantis (S. infantis) is an important emerging pathogen, associated with poultry and poultry products and related to an increasing number of human infections in many countries. A concerning trend among S. infantis isolates is the presence of plasmid-mediated multidrug resistance. In many instances, the genes responsible for this resistance are carried on a megaplasmid known as the plasmid of emerging S. infantis (pESI) or pESI like plasmids. Plasmids can be remarkably stable due to the presence of multiple replicons and post-segregational killing systems (PSKs), which contribute to their maintenance within bacterial populations. To enhance our understanding of S. infantis and its multidrug resistance determinants toward the development of new vaccination strategies, we have devised a new method for targeted plasmid curing. This approach effectively overcomes plasmid addiction by leveraging the temporal overproduction of specific antitoxins coupled with the deletion of the partition region. By employing this strategy, we successfully generated a plasmid-free strain from a field isolate derived from S. infantis 119,944. This method provides valuable tools for studying S. infantis and its plasmid-borne multidrug resistance mechanisms and can be easily adopted for plasmid curing from other related bacteria.

Characteristics and phylogenetic distribution of megaplasmids and prediction of a putative chromid in Pseudomonas aeruginosa.

Research on megaplasmids that contribute to the spread of antimicrobial resistance (AMR) in Pseudomonas aeruginosa strains has grown in recent years due to the now widely used technologies allowing long-read sequencing. Here, we systematically analyzed distinct and consistent genetic characteristics of megaplasmids found in P. aeruginosa. Our data provide information on their phylogenetic distribution and hypotheses tracing the potential evolutionary paths of megaplasmids. Most of the megaplasmids we found belong to the IncP-2-type, with conserved and syntenic genetic backbones carrying modules of genes associated with chemotaxis apparatus, tellurite resistance and plasmid replication, segregation, and transmission. Extensively variable regions harbor abundant AMR genes, especially those encoding ß-lactamases such as VIM-2, IMP-45, and KPC variants, which are high-risk elements in nosocomial infection. IncP-2 megaplasmids act as effective vehicles transmitting AMR genes to diverse regions. One evolutionary model of the origin of megaplasmids claims that chromids can develop from megaplasmids. These chromids have been characterized as an intermediate between a megaplasmid and a chromosome, also containing core genes that can be found on the chromosome but not on the megaplasmid. Using in silico prediction, we identified the "PABCH45 unnamed replicon" as a putative chromid in P. aeruginosa, which shows a much higher similarity and closer phylogenetic relationship to chromosomes than to megaplasmids while also encoding plasmid-like partition genes. We propose that such a chromid could facilitate genome expansion, allowing for more rapid adaptations to novel ecological niches or selective conditions, in comparison to megaplasmids.

Development of a genetic system for Haloferax gibbonsii LR2-5, model host for haloarchaeal viruses.

Archaeal viruses are among the most enigmatic members of the virosphere, and their diverse morphologies raise many questions about their infection mechanisms. The study of molecular mechanisms underlying virus-host interactions hinges upon robust model organisms with a system for gene expression and deletion. Currently, there are only a limited number of archaea that have associated viruses and have a well-developed genetic system. Here, we report the development of a genetic system for the euryarchaeon Haloferax gibbonsii LR2-5. This strain can be infected by multiple viruses and is a model for the study of virus-host interactions. We created a Hfx. gibbonsii LR2-5 ∆pyrE strain, resulting in uracil auxotrophy, which could be used as a selection marker. An expression plasmid carrying a pyrE gene from the well-established Haloferax volcanii system was tested for functionality. Expression of a GFP-MinD fusion under a tryptophan inducible promoter was fully functional and showed similar cellular localization as in Hfx. volcanii. Thus, the plasmids of the Hfx. volcanii system can be used directly for the Hfx. gibbonsii LR2-5 genetic system, facilitating the transfer of tools between the two. Finally, we tested for the functionality of gene deletions by knocking out two genes of the archaeal motility structure, the archaellum. These deletion mutants were as expected non-motile and the phenotype of one deletion could be rescued by the expression of the deleted archaellum gene from a plasmid. Thus, we developed a functional genetic toolbox for the euryarchaeal virus host Hfx. gibbonsii LR2-5, which will propel future studies on archaeal viruses. IMPORTANCE: Species from all domains of life are infected by viruses. In some environments, viruses outnumber their microbial hosts by a factor of 10, and viruses are the most important predators of microorganisms. While much has been discovered about the infection mechanisms of bacterial and eukaryotic viruses, archaeal viruses remain understudied. Good model systems are needed to study their virus-host interactions in detail. The salt-loving archaeon Haloferax gibbonsii LR2-5 has been shown to be infected by a variety of different viruses and, thus, is an excellent model to study archaeal viruses. By establishing a genetic system, we have significantly expanded the toolbox for this model organism, which will fuel our understanding of infection strategies of the underexplored archaeal viruses.

Genomic characterisation of the population structure and antibiotic resistance of Salmonella enterica serovar Infantis in Chile, 2009-2022.

Background: Multidrug-resistant (MDR) Salmonella Infantis has disseminated worldwide, mainly linked to the consumption of poultry products. Evidence shows dissemination of this pathogen in Chile; however, studies are primarily limited to phenotypic data or involve few isolates. As human cases of Salmonella Infantis infections have substantially increased in recent years, this study aimed to characterise the genomic epidemiology and antimicrobial-resistance profiles of isolates obtained from different sources, aiming to inform effective surveillance and control measures. Methods: We sequenced 396 Salmonella Infantis genomes and analysed them with all publicly available genomes of this pathogen from Chile (440 genomes in total), representing isolates from environmental, food, animal, and human sources obtained from 2009 to 2022. Based on bioinformatic and phenotypic methods, we assessed the population structure, dissemination among different niches, and antimicrobial resistance (AMR) profiles of Salmonella Infantis in the country. Findings: The genomic and phylogenetic analyses showed that Salmonella Infantis from Chile comprised several clusters of highly related isolates dominated by sequence type 32. The HC20_343 cluster grouped an important proportion of all isolates. This was the only cluster associated with pESI-like megaplasmids, and up to 12 acquired AMR genes/mutations predicted to result in an MDR phenotype. Accordingly, antimicrobial-susceptibility testing revealed a strong concordance between the AMR genetic determinants and their matching phenotypic expression, indicating that a significant proportion of HC20_343 isolates produce extended-spectrum ß-lactamases and have intermediate fluoroquinolone resistance. HC20_343 Salmonella Infantis were spread among environmental, animal, food, and human niches, showing a close relationship between isolates from different years and sources, and a low intra-source genomic diversity. Interpretation: Our findings show a widespread dissemination of MDR Salmonella Infantis from the HC20_343 cluster in Chile. The high proportion of isolates with resistance to first-line antibiotics and the evidence of active transmission between the environment, animals, food, and humans highlight the urgency of improved surveillance and control measures in the country. As HC20_343 isolates predominate in the Americas, our results suggest a high prevalence of ESBL-producing Salmonella Infantis with intermediate fluoroquinolone resistance in the continent. Funding: Partially supported by the Food and Drug Administration (FDA) of the U.S. Department of Health and Human Services as part of an award, FDU001818, with 30% percent funded by FDA/HHS; and by Agencia de Investigación y Desarrollo de Chile (ANID) through FONDECYT de Postdoctorado Folio 3230796 and Folio 3210317, FONDECYT Regular Folio 1231082, and ANID-Millennium Science Initiative Program-ICN2021_044.

Phenotypic and genomic characterization of Pseudomonas aeruginosa isolates recovered from catheter-associated urinary tract infections in an Egyptian hospital.

Catheter-associated urinary tract infections (CAUTIs) represent one of the major healthcare-associated infections, and Pseudomonas aeruginosa is a common Gram-negative bacterium associated with catheter infections in Egyptian clinical settings. The present study describes the phenotypic and genotypic characteristics of 31 P. aeruginosa isolates recovered from CAUTIs in an Egyptian hospital over a 3 month period. Genomes of isolates were of good quality and were confirmed to be P. aeruginosa by comparison to the type strain (average nucleotide identity, phylogenetic analysis). Clonal diversity among the isolates was determined; eight different sequence types were found (STs 244, 357, 381, 621, 773, 1430, 1667 and 3765), of which ST357 and ST773 are considered to be high-risk clones. Antimicrobial resistance (AMR) testing according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines showed that the isolates were highly resistant to quinolones [ciprofloxacin (12/31, 38.7 %) and levofloxacin (9/31, 29 %) followed by tobramycin (10/31, 32.5 %)] and cephalosporins (7/31, 22.5 %). Genotypic analysis of resistance determinants predicted all isolates to encode a range of AMR genes, including those conferring resistance to aminoglycosides, ß-lactamases, fluoroquinolones, fosfomycin, sulfonamides, tetracyclines and chloramphenicol. One isolate was found to carry a 422 938 bp pBT2436-like megaplasmid encoding OXA-520, the first report from Egypt of this emerging family of clinically important mobile genetic elements. All isolates were able to form biofilms and were predicted to encode virulence genes associated with adherence, antimicrobial activity, anti-phagocytosis, phospholipase enzymes, iron uptake, proteases, secretion systems and toxins. The present study shows how phenotypic analysis alongside genomic analysis may help us understand the AMR and virulence profiles of P. aeruginosa contributing to CAUTIs in Egypt.

Characterization of two novel VIM-type metallo-ß-lactamases, VIM-84 and VIM-85, associated with the spread of IncP-2 megaplasmids in Pseudomonas aeruginosa.

This study aimed to characterize two novel VIM-type metallo-ß-lactamases, VIM-84 and VIM-85, and reveal the important role of the IncP-2 type megaplasmids in the spread of antimicrobial resistance (AMR) genes. VIM-84 and VIM-85 were encoded by two novel genes bla VIM-84 and bla VIM-85 which showed similarity to bla VIM-24. Both bla VIM-84 and bla VIM-85 are harbored into class 1 integrons embedded into the Tn1403 transposon. The bla VIM-85 gene was identified in a megaplasmid, which was related to 17 megaplasmid sequences with sizes larger than 430 kb, deposited previously in Genbank. A comparative analysis of complete plasmid sequences showed highly similar backbone regions and various AMR genes. A phylogenetic tree revealed that these megaplasmids, which were widely distributed globally, were vehicles for the spread of AMR genes. The bla VIM-24, bla VIM-84, and bla VIM-85 genes were cloned into pGK1900, and the recombinant vectors were further transformed into Escherichia coli DH5α and Pseudomonas aeruginosa PAO1. The antimicrobial susceptibility test of the cloning strains showed high levels of resistance to ß-lactams while they remained susceptible to aztreonam. Enzymatic tests revealed that both, VIM-84 and VIM-85, exhibited higher activity in hydrolyzing ß-lactams compared to VIM-24. A D117N mutation found in VIM-24 affected binding to the antibiotics. IMPORTANCE The metallo-ß-lactamases-producing Pseudomonas aeruginosa strains play an important role in hospital outbreaks and the VIM-type enzyme is the most prevalent in European countries. Two novel VIM-type enzymes in our study, VIM-84 and VIM-85, have higher levels of resistance to ß-lactams and greater hydrolytic activities for most ß-lactams compared with VIM-24. Both bla VIM-84 and bla VIM-85 are harbored into class 1 integrons embedded into the Tn1403 transposon. Notably, the genes bla VIM-85 are carried by three different IncP-2-type megaplasmids which are distributed locally and appear responsible for the spread of antimicrobial resistance genes in hospital settings.

Inter-species diversity and functional genomic analyses of closed genome assemblies of clinically isolated, megaplasmid-containing Enterococcus raffinosus Er676 and ATCC49464.

Enterococcus raffinosus is an understudied member of its genus possessing a characteristic megaplasmid contributing to a large genome size. Although less commonly associated with human infection compared to other enterococci, this species can cause disease and persist in diverse niches such as the gut, urinary tract, blood and environment. Few complete genome assemblies have been published to date for E. raffinosus . In this study, we report the complete assembly of the first clinical urinary E. raffinosus strain, Er676, isolated from a postmenopausal woman with history of recurrent urinary tract infection. We additionally completed the assembly of clinical type strain ATCC49464. Comparative genomic analyses reveal inter-species diversity driven by large accessory genomes. The presence of a conserved megaplasmid indicates it is a ubiquitous and vital genetic feature of E. raffinosus . We find that the E. raffinosus chromosome is enriched for DNA replication and protein biosynthesis genes while the megaplasmid is enriched for transcription and carbohydrate metabolism genes. Prophage analysis suggests that diversity in the chromosome and megaplasmid sequences arises, in part, from horizontal gene transfer. Er676 demonstrated the largest genome size reported to date for E. raffinosus and the highest probability of human pathogenicity. Er676 also possesses multiple antimicrobial resistance genes, of which all but one are encoded on the chromosome, and has the most complete prophage sequences. Complete assembly and comparative analyses of the Er676 and ATCC49464 genomes provide important insight into the inter-species diversity of E. raffinosus that gives it its ability to colonize and persist in the human body. Investigating genetic factors that contribute to the pathogenicity of this species will provide valuable tools to combat diseases caused by this opportunistic pathogen.

Plasmid - Chromosome interplay in natural and non-natural hosts: global transcription study of three Bacillus cereus group strains carrying pCER270 plasmid.

The Bacillus cereus group comprises genetically related Gram-positive spore-forming bacteria that colonize a wide range of ecological niches and hosts. Despite their high degree of genome conservation, extrachromosomal genetic material diverges between these species. The discriminating properties of the B. cereus group strains are mainly due to plasmid-borne toxins, reflecting the importance of horizontal gene transfers in bacterial evolution and species definition. To investigate how a newly acquired megaplasmid can impact the transcriptome of its host, we transferred the pCER270 from the emetic B. cereus strains to phylogenetically distant B. cereus group strains. RNA-sequencing experiments allowed us to determine the transcriptional influence of the plasmid on host gene expression and the impact of the host genomic background on the pCER270 gene expression. Our results show a transcriptional cross-regulation between the megaplasmid and the host genome. pCER270 impacted carbohydrate metabolism and sporulation genes expression, with a higher effect in the natural host of the plasmid, suggesting a role of the plasmid in the adaptation of the carrying strain to its environment. In addition, the host genomes also modulated the expression of pCER270 genes. Altogether, these results provide an example of the involvement of megaplasmids in the emergence of new pathogenic strains.

Emergence and clonal dissemination of KPC-3-producing Pseudomonas aeruginosa in China with an IncP-2 megaplasmid.

BACKGROUND: Despite the global prevalence of Klebsiella pneumoniae Carbapenemase (KPC)-type class A ß-lactamases, occurrences of KPC-3-producing isolates in China remain infrequent. This study aims to explore the emergence, antibiotic resistance profiles, and plasmid characteristics of blaKPC-3-carrying Pseudomonas aeruginosa. METHODS: Species identification was performed by MALDI-TOF-MS, and antimicrobial resistance genes (ARGs) were identified by polymerase chain reaction (PCR). The characteristics of the target strain were detected by whole-genome sequencing (WGS) and antimicrobial susceptibility testing (AST). Plasmids were analyzed by S1-nuclease pulsed-field gel electrophoresis(S1-PFGE), Southern blotting and transconjugation experiment. RESULTS: Five P. aeruginosa strains carrying blaKPC-3 were isolated from two Chinese patients without a history of travelling to endemic areas. All strains belonged to the novel sequence type ST1076. The blaKPC-3 was carried on a 395-kb IncP-2 megaplasmid with a conserved structure (IS6100-ISKpn27-blaKPC-3-ISKpn6-korC-klcA), and this genetic sequence was identical to many plasmid-encoded KPC of Pseudomonas species. By further analyzing the genetic context, it was supposed that the original of blaKPC-3 in our work was a series of mutation of blaKPC-2. CONCLUSIONS: The emergence of a multidrug resistance IncP-2 megaplasmid and clonal transmission of blaKPC-3-producing P. aeruginosa in China underlined the crucial need for continuous monitoring of blaKPC-3 for prevention and control of its further dissemination in China.

A broadly distributed predicted helicase/nuclease confers phage resistance via abortive infection.

There is strong selection for the evolution of systems that protect bacterial populations from viral attack. We report a single phage defense protein, Hna, that provides protection against diverse phages in Sinorhizobium meliloti, a nitrogen-fixing alpha-proteobacterium. Homologs of Hna are distributed widely across bacterial lineages, and a homologous protein from Escherichia coli also confers phage defense. Hna contains superfamily II helicase motifs at its N terminus and a nuclease motif at its C terminus, with mutagenesis of these motifs inactivating viral defense. Hna variably impacts phage DNA replication but consistently triggers an abortive infection response in which infected cells carrying the system die but do not release phage progeny. A similar host cell response is triggered in cells containing Hna upon expression of a phage-encoded single-stranded DNA binding protein (SSB), independent of phage infection. Thus, we conclude that Hna limits phage spread by initiating abortive infection in response to a phage protein.

Erratum: Comparative analysis reveals the modular functional structure of conjugative megaplasmid pTTS12 of Pseudomonas putida S12: A paradigm for transferable traits, plasmid stability, and inheritance?

[This corrects the article DOI: 10.3389/fmicb.2022.1001472.].

The parABSm system is involved in megaplasmid partitioning and genome integrity maintenance in Thermus thermophilus.

The characteristics of the parABS system in polyploid bacteria are barely understood. We initially analyzed the physiological functions and mechanisms of the megaplasmid parABSm system in the thermophilic polyploid bacterium Thermus thermophilus. Deletion of parABm was possible only when a plasmid-born copy of parABm was provided, indicating that these genes are conditionally essential. The cell morphology of the parABm deletion mutant (ΔparABm) was changed to some extent, and in certain extra-large or twisted cells, the nucleoids were dispersed and damaged. Compared with that of the wild type, the frequency of anucleate cells was significantly increased. Genome content analyses showed that ΔparABm had lost ∼160 kb of megaplasmid and ∼23 kb of chromosomal sequences, respectively. Genome fluorescent tagging and PFGE experiments demonstrated that the truncated megaplasmid was frequently interlinked and could not be segregated correctly; thus, certain daughter cells eventually lost the entire megaplasmid and became twisted or enlarged with damaged nucleoids. Further, we found that when the megaplasmid was lost in these cells, the toxins encoded by the megaplasmid toxin-antitoxin (TA) systems (VapBC64_65 and VapBC142_143) would exert detrimental effects, such as to fragment DNA. Thus, parABSm might ensure the existence of these TA systems, thereby preventing genomic degradation. Together, our results suggested that in T. thermophilus, the megaplasmid-encoded parABS system plays an essential role in the megaplasmid partitioning process; also it is an important determination factor for the genome integrity maintenance.

Evidence of structural rearrangements in ESBL-positive pESI(like) megaplasmids of S.Infantis.

The increasing prevalence of pESI(like)-positive, multidrug-resistant (MDR) S. Infantis in Europe is a cause of major concern. As previously demonstrated, the pESI(like) megaplasmid is not only a carrier of antimicrobial resistant (AMR) genes (at least tet, dfr, and sul genes), but also harbours several virulence and fitness genes, and toxin/antitoxin systems that enhance its persistence in the S. Infantis host. In this study, five prototype pESI(like) plasmids, of either CTX-M-1 or CTX-M-65 ESBL-producing strains, were long-read sequenced using Oxford Nanopore Technology (ONT), and their complete sequences were resolved. Comparison of the structure and gene content of the five sequenced plasmids, and further comparison with previously published pESI(like) sequences, indicated that although the sequence of such pESI(like) 'mosaic' plasmids remains almost identical, their structures appear different and composed of regions inserted or transposed after different events. The results obtained in this study are essential to better understand the plasticity and the evolution of the pESI(like) megaplasmid, and therefore to better address risk management options and policy decisions to fight against AMR and MDR in Salmonella and other food-borne pathogens. Graphical representation of the pESI-like plasmid complete sequence (ID 12037823/11). Block colours indicate the function of the genes: red: repB gene; pink: class I integrons (IntI); yellow; mobile elements; blue: resistance genes; green: toxin/anti-toxin systems; grey: mer operon; light green: genes involve in conjugation.

The Contribution of Actinobacteria to the Degradation of Chlorinated Compounds: Variations in the Activity of Key Degradation Enzymes.

Bacteria make a huge contribution to the purification of the environment from toxic stable pollutants of anthropogenic and natural origin due to the diversity of their enzyme systems. For example, the ability to decompose 3-chlorobenzoate (3CBA) by the four representative genera of Actinobacteria, such as Rhodococcus, Gordonia, Microbacterium, and Arthrobacter, was studied. In most cases, the formation of 4-chlorocatechol as the only key intermediate during the decomposition of 3CBA was observed. However, Rhodococcus opacus strain 1CP was an exception, whose cells decomposed 3CBA via both 3-chloro- and 4-chlorocatechol. The enzyme 3-Chlorobenzoate 1,2-dioxygenase (3CBDO) induced during the growth of these bacteria in the presence of 3CBA differed significantly in substrate specificity from the benzoate dioxygenases induced upon growth in the presence of benzoate. The R. opacus 6a strain was found to contain genes encoding chlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and dienelactone hydrolase, whose nucleotide sequence was 100% consistent with the sequences of the corresponding genes encoding the enzymes of the modified 4-chlorocatechol ortho-cleavage pathway of the strain R. opacus 1CP. However, the gene encoding chloromuconolactone dehalogenase (clcF) was not found in the representatives of the actinomycete genera, including Gordonia and Arthrobacter. A linear mega-plasmid carrying 3-chlorocatechol degradation genes remained stable after maintaining the R. opacus 1CP strain on an agar-rich medium for 25 years. In general, a similar plasmid was absent in actinobacteria of other genera, as well as in closely related species of R. opacus 6a.

Evaluation of Safety and Probiotic Traits from a Comprehensive Genome-Based In Silico Analysis of Ligilactobacillus salivarius P1CEA3, Isolated from Pigs and Producer of Nisin S.

Ligilactobacillus salivarius is an important member of the porcine gastrointestinal tract (GIT). Some L. salivarius strains are considered to have a beneficial effect on the host by exerting different probiotic properties, including the production of antimicrobial peptides which help maintain a healthy gut microbiota. L. salivarius P1CEA3, a porcine isolated strain, was first selected and identified by its antimicrobial activity against a broad range of pathogenic bacteria due to the production of the novel bacteriocin nisin S. The assembled L. salivarius P1CEA3 genome includes a circular chromosome, a megaplasmid (pMP1CEA3) encoding the nisin S gene cluster, and two small plasmids. A comprehensive genome-based in silico analysis of the L. salivarius P1CEA3 genome reveals the presence of genes related to probiotic features such as bacteriocin synthesis, regulation and production, adhesion and aggregation, the production of lactic acid, amino acids metabolism, vitamin biosynthesis, and tolerance to temperature, acid, bile salts and osmotic and oxidative stress. Furthermore, the strain is absent of risk-related genes for acquired antibiotic resistance traits, virulence factors, toxic metabolites and detrimental metabolic or enzymatic activities. Resistance to common antibiotics and gelatinase and hemolytic activities have been discarded by in vitro experiments. This study identifies several probiotic and safety traits of L. salivarius P1CEA3 and suggests its potential as a promising probiotic in swine production.

Geographical and temporal distribution of multidrug-resistant Salmonella Infantis in Europe and the Americas.

Recently emerged S. Infantis strains carrying resistance to several commonly used antimicrobials have been reported from different parts of the globe, causing human cases of salmonellosis and with occurrence reported predominantly in broiler chickens. Here, we performed phylogenetic and genetic clustering analyses to describe the population structure of 417 S. Infantis originating from multiple European countries and the Americas collected between 1985 and 2019. Of these, 171 were collected from 56 distinct premises located in England and Wales (E/W) between 2009 and 2019, including isolates linked to incursions of multidrug-resistant (MDR) strains from Europe associated with imported poultry meat. The analysis facilitated the comparison of isolates from different E/W sources with isolates originating from other countries. There was a high degree of congruency between the outputs of different types of population structure analyses revealing that the E/W and central European (Germany, Hungary, and Poland) isolates formed several disparate groups, which were distinct from the cluster relating to the United States (USA) and Ecuador/Peru, but that isolates from Brazil were closely related to the E/W and the central European isolates. Nearly half of the analysed strains/genomes (194/417) harboured the IncFIB(pN55391) replicon typical of the "parasitic" pESI-like megaplasmid found in diverse strains of S. Infantis. The isolates that contained the IncFIB(pN55391) replicon clustered together, despite originating from different parts of the globe. This outcome was corroborated by the time-measured phylogeny, which indicated that the initial acquisition of IncFIB(pN55391) likely occurred in Europe in the late 1980s, with a single introduction of IncFIB(pN55391)-carrying S. Infantis to the Americas several years later. Most of the antimicrobial resistance (AMR) genes were identified in isolates that harboured one or more different plasmids, but based on the short-read assemblies, only a minority of the resistance genes found in these isolates were identified as being associated with the detected plasmids, whereas the hybrid assemblies comprising the short and long reads demonstrated that the majority of the identified AMR genes were associated with IncFIB(pN55391) and other detected plasmid replicon types. This finding underlies the importance of applying appropriate methodologies to investigate associations of AMR genes with bacterial plasmids.

Replication Origin Deletion Enhances Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloarchaea.

Although the use of multiple replication origins for chromosome replication has been widely characterized in haloarchaea, whether it is possible to manipulate the chromosome copy number by their genetic engineering is not known, and how it would affect the cell functioning is poorly understood. Here, we demonstrate that deletion of the three active chromosomal origins in Haloferax mediterranei remarkably reduces its DNA amounts and ploidy numbers. Consequently, the mutant strain H. mediterranei Δ123 is more sensitive to UV and mitomycin C. Surprisingly, the cell size increases by 21.2%, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production in shake flask culture enhances from 7.23 to 8.11 g/L in ΔEPSΔ123, although there is also a decrease in cell growth. In this mutant, the chromosomal copy number decreases, whereas the pha-encoding pHM300 megaplasmid copy number increases. Moreover, our transcriptome analysis reveals that the genes involved in primary metabolisms are significantly downregulated in ΔEPSΔ123, whereas those responsible for starch utilization and precursor supplying for PHBV monomers are upregulated. This indicates that more energy and carbon flux is redirected from primary metabolism to PHBV synthesis, thereby enhancing its PHBV accumulation. These findings may therefore provide a rational design to enhance PHBV synthesis by simply tuning the replication origins to modulate the chromosome/megaplasmid copy number ratio and subsequently influence cellular metabolism and physiological functions. IMPORTANCE The haloarchaeon Haloferax mediterranei is a potential producer of PHBV (100% biodegradable plastic) from inexpensive carbon sources. We previously reported that H. mediterranei possessed three active chromosomal origins and, when these origins were deleted, a dormant origin was activated to initiate the replication of chromosome. In this context, in the present study, we first found a close connection between replication initiation and PHBV accumulation. We describe the potential industrial advantages of the strain H. mediterranei ΔEPSΔ123, which includes the enlargement of cell volume by 21.2% and enhancement of PHBV production by 11.2%. We further reveal the possible mechanism that contributes to the greater PHBV production in the ΔEPSΔ123 strain. Overall, we provide here a conceptual advance in the field of synthetic biology by modulating chromosome replication to improve the production of bio-based chemicals.

Comparative analysis reveals the modular functional structure of conjugative megaplasmid pTTS12 of Pseudomonas putida S12: A paradigm for transferable traits, plasmid stability, and inheritance?

Originating from various environmental niches, large numbers of bacterial plasmids have been found carrying heavy metal and antibiotic resistance genes, degradation pathways and specific transporter genes for organic solvents or aromatic compounds. Such genes may constitute promising candidates for novel synthetic biology applications. Our systematic analysis of gene clusters encoded on megaplasmid pTTS12 from Pseudomonas putida S12 underscores that a large portion of its genes is involved in stress response to increase survival under harsh conditions like the presence of heavy metal and organic solvent. We investigated putative roles of genes encoded on pTTS12 and further elaborated on their roles in the establishment and maintenance under several stress conditions, specifically focusing on solvent tolerance in P. putida strains. The backbone of pTTS12 was found to be closely related to that of the carbapenem-resistance plasmid pOZ176, member of the IncP-2 incompatibility group, although the carbapenem resistance cassette is absent from pTTS12. Megaplasmid pTTS12 contains multiple transposon-flanked cassettes mediating resistance to various heavy metals such as tellurite, chromate (Tn7), and mercury (Tn5053 and Tn5563). Additionally, pTTS12 also contains a P-type, Type IV secretion system (T4SS) supporting self-transfer to other P. putida strains. This study increases our understanding in the modular structure of pTTS12 as a member of IncP-2 plasmid family and several promising exchangeable gene clusters to construct robust microbial hosts for biotechnology applications.

First report of a multidrug-resistant Salmonella enterica Serovar Infantis carrying pESI megaplasmid isolated from marine shrimp in India.

BACKGROUND: Non-typhoidal Salmonella enterica (NTS) in seafood is an important human health concern. An emerging strain of NTS serovar Infantis carrying a megaplasmid pESI and resistant to multipe drugs has been responsible for frequent food-borne human infections worldwide. METHODS: S. enterica strain JS5 was isolated from a sample of shrimp from the retail market on XLD agar after enrichment in the RV medium. The genomic DNA was isolated and sequenced using the Illumina platform. The draft whole genome sequence of Salmonella Infantis JS5 revealed the presence of a plasmid. RESULTS: The genome size was 4,977,731 bp with 4663 open reading frames. The bacterium harboured a megaplasmid similar to the pESI plasmid reported in the emerging S. Infantis. The 285 kb plasmid contained the characteristic genes of the pESI plasmids, such as the mercury operon, yersiniabactin siderophore operon, fimbriae, toxin-antitoxin systems and the hypothetical protein backbone. The antibiotic resistance genes tet(A), dfrA14, aadA, qacEdelta1, and sul1 were detected. To the best of our knowledge, this is the first report on pESI plasmid carrying S. Infantis from India.

Genomic Analysis of Two MDR Isolates of Salmonella enterica Serovar Infantis from a Spanish Hospital Bearing the blaCTX-M-65 Gene with or without fosA3 in pESI-like Plasmids.

Salmonella enterica serovar Infantis (S. Infantis) is a broiler-associated pathogen which ranks in the fourth position as a cause of human salmonellosis in the European Union. Here, we report a comparative genomic analysis of two clinical S. Infantis isolates recovered in Spain from children who just returned from Peru. The isolates were selected on the basis of resistance to cefotaxime, one of the antibiotics of choice for treatment of S. enterica infections. Antimicrobial susceptibility testing demonstrated that they were resistant to eight classes of antimicrobial agents: penicillins, cephalosporins, phenicols, aminoglycosides, tetracyclines, inhibitors of folate synthesis, (fluoro)quinolones and nitrofurans, and one of them was also resistant to fosfomycin. As shown by whole-genome sequence analysis, each isolate carried a pESI-like megaplasmid of ca. 300 kb harboring multiple resistance genes [blaCTX-M-65, aph(4)-Ia, aac(3)-IVa, aph(3')-Ia, floR, dfrA14, sul1, tet(A), aadA1 ± fosA3], as well as genes for resistance to heavy metals and disinfectants (mer, ars and qacEΔ1). These genes were distributed in two complex regions, separated by DNA belonging to the plasmid backbone, and associated with a wealth of transposable elements. The two isolates had a D87Y amino acid substitution in the GyrA protein, and truncated variants of the nitroreductase genes nfsA and nsfB, accounting for chromosomally encoded resistances to nalidixic acid and nitrofurantoin, respectively. The two S. Infantis isolates were assigned to sequence type ST32 by in silico multilocus sequence typing (MLST). Phylogenetic analysis revealed that they were closely related, differing only by 12 SNPs, although they were recovered from different children two years apart. They were also genetically similar to blaCTX-M-65-positive ± fosA3 isolates obtained from humans and along the poultry production chain in the USA, South America, as well as from humans in several European countries, usually associated with a travel history to America. However, this is the first time that the S. Infantis blaCTX-M-65 ± fosA3 MDR clone has been reported in Spain.