A novel major facilitator superfamily-type tripartite efflux system CprABC mediates resistance to polymyxins in Chryseobacterium sp. PL22-22A.

Background: Polymyxin B (PMB) and polymyxin E (colistin, CST) are polymyxin antibiotics, which are considered last-line therapeutic options against multidrug-resistant Gram-negative bacteria in serious infections. However, there is increasing risk of resistance to antimicrobial drugs. Effective efflux pump inhibitors (EPIs) should be developed to help combat efflux pump-mediated antibiotic resistance. Methods: Chryseobacterium sp. PL22-22A was isolated from aquaculture sewage under selection with 8 mg/L PMB, and then its genome was sequenced using Oxford Nanopore and BGISEQ-500 platforms. Cpr (Chryseobacterium Polymyxins Resistance) genes encoding a major facilitator superfamily-type tripartite efflux system, were found in the genome. These genes, and the gene encoding a truncation mutant of CprB from which sequence called CprBc was deleted, were amplified and expressed/co-expressed in Escherichia coli DH5α. Minimum inhibitory concentrations (MICs) of polymyxins toward the various E. coli heterologous expression strains were tested in the presence of 2-128 mg/L PMB or CST. The pumping activity of CprABC was assessed via structural modeling using Discovery Studio 2.0 software. Moreover, the influence on MICs of baicalin, a novel MFS EPI, was determined, and the effect was analyzed based on homology modeling. Results: Multidrug-resistant bacterial strain Chryseobacterium sp. PL22-22A was isolated in this work; it has notable resistance to polymyxin, with MICs for PMB and CST of 96 and 128 mg/L, respectively. A novel MFS-type tripartite efflux system, named CprABC, was identified in the genome of Chryseobacterium sp. PL22-22A. Heterologous expression and EPI assays indicated that the CprABC system is responsible for the polymyxin resistance of Chryseobacterium sp. PL22-22A. Structural modeling suggested that this efflux system provides a continuous conduit that runs from the CprB funnel through the CprC porin domain to pump polymyxins out of the cell. A specific C-terminal α-helix, CprBc, has an activation function on polymyxin excretion by CprB. The flavonoid compound baicalin was found to affect the allostery of CprB and/or obstruct the substrate conduit, and thus to inhibit extracellular polymyxin transport by CprABC. Conclusion: Novel MFS-type tripartite efflux system CprABC in Chryseobacterium sp. PL22-22A mediates resistance to polymyxins, and baicalin is a promising EPI.

An efflux pump in genomic island GI-M202a mediates the transfer of polymyxin B resistance in Pandoraea pnomenusa M202 / Una bomba de eflujo en la isla genómica GI-M202a media la transferencia de resistencia a la polimixina B en Pandoraea pnomenusa M202

Background: Polymyxin B is considered a last-line therapeutic option against multidrug-resistant gram-negative bacteria, especially in COVID-19 coinfections or other serious infections. However, the risk of antimicrobial resistance and its spread to the environment should be brought to the forefront. Methods: Pandoraea pnomenusa M202 was isolated under selection with 8 mg/L polymyxin B from hospital sewage and then was sequenced by the PacBio RS II and Illumina HiSeq 4000 platforms. Mating experiments were performed to evaluate the transfer of the major facilitator superfamily (MFS) transporter in genomic islands (GIs) to Escherichia coli 25DN. The recombinant E. coli strain Mrc-3 harboring MFS transporter encoding gene FKQ53_RS21695 was also constructed. The influence of efflux pump inhibitors (EPIs) on MICs was determined. The mechanism of polymyxin B excretion mediated by FKQ53_RS21695 was investigated by Discovery Studio 2.0 based on homology modeling. Results: The MIC of polymyxin B for the multidrug-resistant bacterial strain P. pnomenusa M202, isolated from hospital sewage, was 96 mg/L. GI-M202a, harboring an MFS transporter-encoding gene and conjugative transfer protein-encoding genes of the type IV secretion system, was identified in P. pnomenusa M202. The mating experiment between M202 and E. coli 25DN reflected the transferability of polymyxin B resistance via GI-M202a. EPI and heterogeneous expression assays also suggested that the MFS transporter gene FKQ53_RS21695 in GI-M202a was responsible for polymyxin B resistance. Molecular docking revealed that the polymyxin B fatty acyl group inserts into the hydrophobic region of the transmembrane core with Pi-alkyl and unfavorable bump interactions, and then polymyxin B rotates around Tyr43 to externally display the peptide group during the efflux process, accompanied by an inward-to-outward conformational change in the MFS transporter...(AU)

An efflux pump in genomic island GI-M202a mediates the transfer of polymyxin B resistance in Pandoraea pnomenusa M202.

BACKGROUND: Polymyxin B is considered a last-line therapeutic option against multidrug-resistant gram-negative bacteria, especially in COVID-19 coinfections or other serious infections. However, the risk of antimicrobial resistance and its spread to the environment should be brought to the forefront. METHODS: Pandoraea pnomenusa M202 was isolated under selection with 8 mg/L polymyxin B from hospital sewage and then was sequenced by the PacBio RS II and Illumina HiSeq 4000 platforms. Mating experiments were performed to evaluate the transfer of the major facilitator superfamily (MFS) transporter in genomic islands (GIs) to Escherichia coli 25DN. The recombinant E. coli strain Mrc-3 harboring MFS transporter encoding gene FKQ53_RS21695 was also constructed. The influence of efflux pump inhibitors (EPIs) on MICs was determined. The mechanism of polymyxin B excretion mediated by FKQ53_RS21695 was investigated by Discovery Studio 2.0 based on homology modeling. RESULTS: The MIC of polymyxin B for the multidrug-resistant bacterial strain P. pnomenusa M202, isolated from hospital sewage, was 96 mg/L. GI-M202a, harboring an MFS transporter-encoding gene and conjugative transfer protein-encoding genes of the type IV secretion system, was identified in P. pnomenusa M202. The mating experiment between M202 and E. coli 25DN reflected the transferability of polymyxin B resistance via GI-M202a. EPI and heterogeneous expression assays also suggested that the MFS transporter gene FKQ53_RS21695 in GI-M202a was responsible for polymyxin B resistance. Molecular docking revealed that the polymyxin B fatty acyl group inserts into the hydrophobic region of the transmembrane core with Pi-alkyl and unfavorable bump interactions, and then polymyxin B rotates around Tyr43 to externally display the peptide group during the efflux process, accompanied by an inward-to-outward conformational change in the MFS transporter. Additionally, verapamil and CCCP exhibited significant inhibition via competition for binding sites. CONCLUSIONS: These findings demonstrated that GI-M202a along with the MFS transporter FKQ53_RS21695 in P. pnomenusa M202 could mediate the transmission of polymyxin B resistance.

Novel mechanism of hydrogen peroxide for promoting efficient natamycin synthesis in Streptomyces.

The mechanism of regulation of natamycin biosynthesis by Streptomyces in response to oxidative stress is unclear. Here, we first show cholesterol oxidase SgnE, which catalyzes the formation of H2O2 from sterols, triggered a series of redox-dependent interactions to stimulate natamycin production in S. gilvosporeus. In response to reactive oxygen species, residues Cys212 and Cys221 of the H2O2-sensing consensus sequence of OxyR were oxidized, resulting in conformational changes in the protein: OxyR extended its DNA-binding domain to interact with four motifs of promoter p sgnM . This acted as a redox-dependent switch to turn on/off gene transcription of sgnM, which encodes a cluster-situated regulator, by controlling the affinity between OxyR and p sgnM , thus regulating the expression of 12 genes in the natamycin biosynthesis gene cluster. OxyR cooperates with SgnR, another cluster-situated regulator and an upstream regulatory factor of SgnM, synergistically modulated natamycin biosynthesis by masking/unmasking the -35 region of p sgnM depending on the redox state of OxyR in response to the intracellular H2O2 concentration. IMPORTANCE Cholesterol oxidase SgnE is an indispensable factor, with an unclear mechanism, for natamycin biosynthesis in Streptomyces. Oxidative stress has been attributed to the natamycin biosynthesis. Here, we show that SgnE catalyzes the formation of H2O2 from sterols and triggers a series of redox-dependent interactions to stimulate natamycin production in S. gilvosporeus. OxyR, which cooperates with SgnR, acted as a redox-dependent switch to turn on/off gene transcription of sgnM, which encodes a cluster-situated regulator, by masking/unmasking its -35 region, to control the natamycin biosynthesis gene cluster. This work provides a novel perspective on the crosstalk between intracellular ROS homeostasis and natamycin biosynthesis. Application of these findings will improve antibiotic yields via control of the intracellular redox pressure in Streptomyces.

Glabridin inhibited the spread of polymyxin-resistant Enterobacterium carrying ICEMmoMP63.

Introduction: The role of integrative and conjugative elements (ICEs) in antibiotic resistance in Morganella morganii is unknown. This study aimed to determine whether an ICE identified in the M. morganii genome contributed to the polymyxin resistance. Methods: Whole-genome sequencing was performed followed by bioinformatics analyses to identify ICEs and antibiotic resistance genes. Conjugation assays were performed to analyze the transferability of a discovered ICE. A drug transporter encoded on the ICE was heterogeneously expressed in Escherichia coli, minimum inhibitory concentrations of antibiotics were determined, and a traditional Chinese medicine library was screened for potential efflux pump inhibitors. Results: An antibiotic resistance-conferring ICE, named ICEMmoMP63, was identified. ICEMmoMP63 was verified to be horizontally transferred among Enterobacteriaceae bacteria. G3577_03020 in ICEMmoMP63 was found to mediate multiple antibiotic resistances, especially polymyxin resistance. However, natural compound glabridin was demonstrated to inhibit polymyxin resistance. Discussion: Our findings support the need for monitoring dissemination of ICEMmoMP63 in Enterobacteriaceae bacteria. Combined glabridin and polymyxin may have therapeutic potential for treating infections from multi-drug resistant bacteria carrying ICEMmoMP63.

Bifunctional protein ArsRM contributes to arsenite methylation and resistance in Brevundimonas sp. M20.

BACKGROUND: Arsenic (As) with various chemical forms, including inorganic arsenic and organic arsenic, is the most prevalent water and environmental toxin. This metalloid occurs worldwide and many of its forms, especially arsenite [As(III)], cause various diseases including cancer. Organification of arsenite is an effective way for organisms to cope with arsenic toxicity. Microbial communities are vital contributors to the global arsenic biocycle and represent a promising way to reduce arsenite toxicity. METHODS: Brevundimonas sp. M20 with arsenite and roxarsone resistance was isolated from aquaculture sewage. The arsHRNBC cluster and the metRFHH operon of M20 were identified by sequencing. The gene encoding ArsR/methyltransferase fusion protein, arsRM, was amplified and expressed in Escherichia coli BL21 (DE3), and this strain showed resistance to arsenic in the present of 0.25-6 mM As(III), aresenate, or pentavalent roxarsone. The methylation activity and regulatory action of ArsRM were analyzed using Discovery Studio 2.0, and its functions were confirmed by methyltransferase activity analysis and electrophoretic mobility shift assays. RESULTS: The minimum inhibitory concentration of the roxarsone resistant strain Brevundimonas sp. M20 to arsenite was 4.5 mM. A 3,011-bp arsenite resistance ars cluster arsHRNBC and a 5649-bp methionine biosynthesis met operon were found on the 3.315-Mb chromosome. Functional prediction analyses suggested that ArsRM is a difunctional protein with transcriptional regulation and methyltransferase activities. Expression of ArsRM in E. coli increased its arsenite resistance to 1.5 mM. The arsenite methylation activity of ArsRM and its ability to bind to its own gene promoter were confirmed. The As(III)-binding site (ABS) and S-adenosylmethionine-binding motif are responsible for the difunctional characteristic of ArsRM. CONCLUSIONS: We conclude that ArsRM promotes arsenite methylation and is able to bind to its own promoter region to regulate transcription. This difunctional characteristic directly connects methionine and arsenic metabolism. Our findings contribute important new knowledge about microbial arsenic resistance and detoxification. Future work should further explore how ArsRM regulates the met operon and the ars cluster.

Rich Organic Nitrogen Impacts Clavulanic Acid Biosynthesis through the Arginine Metabolic Pathway in Streptomyces clavuligerus F613-1.

Clavulanic acid (CA) is the preferred clinical drug for the treatment of infections by ß-lactam antibiotic-resistant bacteria. CA is produced by Streptomyces clavuligerus, and although there have been many reports on the effects of carbon and nitrogen sources on CA production, the mechanisms involved remain unclear. In this study, we found that CA accumulation in S. clavuligerus F613-1 was increased significantly in MH medium, which is rich in organic nitrogen, compared with that in ML medium, which contains half the amount of organic nitrogen present in MH medium. Transcriptome analysis revealed that genes involved in CA biosynthesis, such as ceas1, ceas2, bls1, bls2, cas2, pah2, gcaS, and cad, and arginine biosynthesis, such as argB, argC, argD, argG, argH, argJ, and argR, were upregulated under rich organic nitrogen. Metabolome data revealed notable differences between cultures of F613-1 grown in MH and ML media with regard to levels of key intracellular metabolites, most of which are involved in arginine metabolic pathways, including arginine, glutamine, and glutamic acid. Additionally, supplementation of ML medium with arginine, glutamine, or glutamic acid resulted in increased CA production by S. clavuligerus F613-1. Our results indicate that rich organic nitrogen mainly affects CA biosynthesis by increasing the levels of amino acids associated with the arginine metabolic pathway and activating the expression of the CA biosynthetic gene cluster. These findings provide important insights for improving medium optimization and engineering of S. clavuligerus F613-1 for high-yield production of CA. IMPORTANCE The bacterium Streptomyces clavuligerus is used for the industrial production of the broad-spectrum ß-lactamase inhibitor clavulanic acid (CA). However, much remains unknown about the factors which affect CA yields. We investigated the effects of different levels of organic nitrogen on CA production. Our analyses indicate that higher organic nitrogen levels were associated with increased CA yields and increased levels of arginine biosynthesis. Further analyses supported the relationship between arginine metabolism and CA production and demonstrated that increasing the levels of arginine or associated amino acids could boost CA yields. These findings suggest approaches for improving the production of this clinically important antibiotic.

[ABC transporter SgnA/B promotes extracellular transport and efficient production of natamycin].

Natamycin is a natural, broad spectrum and highly efficient antifungal compound that belongs to polyene macrolide antibiotics. It has been used in prevention of food fungal contamination and treatment of clinical fungal infection. The extracellular transport efficiency of natamycin may be an important factor hampering the yield of natamycin produced by Streptomyces gilvosporeus. The extracellular transporter SgnA/B of natamycin was analyzed by bioinformatics tools and molecular docking techniques. This ATP-binding cassette transporter, consisted of SgnA and SgnB, is a heterodimers with inward-facing conformation. The difference between the natamycin combining efficiency of the two drug-binding cavities in SgnA/B is favorable for natamycin extracellular transport. sgnA/B gene was overexpressed in S. gilvosporeus F607 and the effects of sgnA/B gene overexpression on natamycin synthesis and extracellular transport were analyzed. In F-EX strain, the extracellular/intracellular ratio of natamycin in logarithmic synthesis stage was increased, and the total fermentation yield at 120 h was increased by 12.5% and reached to 7.38 g/L. Moreover, transcriptome sequencing analysis showed that sgnA/B gene overexpression affected the expression of genes involved in the metabolism of various amino acids, propionate, glucose, C5-branched dibasic acid and TCA cycle. This research demonstrated that the enhanced extracellular transport increased the synthesis of natamycin by S. gilvosporeus, and S. gilvosporeus F-EX showed good potential for the industrial production of natamycin.

MacRS controls morphological differentiation and natamycin biosynthesis in Streptomyces gilvosporeus F607.

Streptomyces gilvosporeus F607 produces large amounts of natamycin in a process regulated by multiple networks, including two-component systems (TCSs). The macR and macS genes, which are annotated as rs12540 and rs12545, respectively, in S. gilvosporeus F607, affect natamycin biosynthesis and sporulation. The findings of this study indicate that deletion of macRS from S. gilvosporeus F607 prevents the production of natamycin, delays spore formation (according to scanning electron microscopy), and results in aerial hyphae lacking compartments separated by septa (according to transmission electron microscopy). Real-time quantitative polymerase chain reaction (RT-qPCR) analyses revealed that the expression levels of natamycin biosynthesis-related genes and genes essential for septum formation during sporulation were affected in the ΔmacRS mutant strain. Molecular simulations and electrophoretic mobility shift assays (EMSAs) suggested MacR not only interacted with the intergenic region of sgnM and sgnR, but also with the promoter of penicillin-binding protein gene ftsL required for cell division. sgnR promoter was presumed to be the binding target of MacR based on the RT-qPCR results. MacR had different affinity with two binding sites: one was located at ftsL promoter region with a perfect inverted repeats 'TGAGTACGCGTACTCA', the other was located at the presumed sgnR promoter with an imperfect inverted repeats 'TGAAGGTGCTGGACTCA'. We propose a hypothesis of a three-level regulatory pathway based on pleiotropic transcriptional regulator MacR and its target genes sgnR and ftsL; the pathway activates natamycin biosynthesis and influences septum development via direct and indirect effects in S. gilvosporeus F607.

Deletion of the Response Regulator PhoP Accelerates the Formation of Aerial Mycelium and Spores in Actinosynnema pretiosum.

PhoPR is an important two-component signal transduction system (TCS) for microorganisms to sense and respond to phosphate limitation. Although the response regulator PhoP controls morphological development and secondary metabolism in various Streptomyces species, the function of PhoP in Actinosynnema pretiosum remains unclear. In this study, we showed that PhoP significantly represses the morphological development of the A. pretiosum X47 strain. Production of aerial mycelium and spore formation occurred much earlier in the ΔphoP strain than in X47 during growth on ISP2 medium. Transcription analysis indicated that 222 genes were differentially expressed in ∆phoP compared to strain X47. Chemotaxis genes (cheA, cheW, cheX, and cheY); flagellum biosynthesis and motility genes (flgBCDGKLN, flaD, fliD-R, motA, and swrD); and differentiation genes (whiB and ssgB) were significantly upregulated in ∆phoP. Gel-shift analysis indicated that PhoP binds to the promoters of flgB, flaD, and ssgB genes, and PHO box-like motif with the 8-bp conserved sequence GTTCACGC was identified. The transcription of phoP/phoR of X47 strain was induced at low phosphate concentration. Our results demonstrate that PhoP is a negative regulator that controls the morphological development of A. pretiosum X47 by repressing the transcription of differentiation genes.

Cadmium stress efficiently enhanced meropenem degradation by the meropenem- and cadmium-resistant strain Pseudomonas putida R51.

The ß-lactam antibiotic meropenem (MEM) is widely used in infectious disease treatment and consequently can be released into the environment, causing environmental pollution. In this study, Pseudomonas putida strain R51 was isolated from the wastewater of a poultry farm and found to efficiently degrade MEM. The genome of strain R51 contains a variety of heavy metal and antibiotic resistance genes, including the metallo-ß-lactamase gene (JQN61_03315) and cadmium resistance gene cadA (JQN61_19995). Under cadmium stress, the degradation rate of MEM increased significantly in strain R51. Transcriptional analysis revealed that the expression of JQN61_03315 and cadA significantly increased under cadmium stress and that the expression of many genes associated with heavy metal and antibiotic resistance also changed significantly. Molecular docking analysis suggested that metallo-ß-lactamase JQN61_03315 binds to MEM. In addition, no plasmid was found in strain R51, and no mobile genetic elements were found nearby JQN61_03315. In conclusion. we proposed that JQN61_03315 was responsible for the degradation of MEM, that the expression of this gene was induced under cadmium stress, and that strain R51 can be used for bioremediation of MEM without the risk for the transmission of the MEM resistance gene. These findings will have importance for studying the microbial degradation of MEM in the presence of heavy metal pollutants.

Use of elicitors to enhance or activate the antibiotic production in streptomyces.

Streptomyces is the largest and most significant genus of Actinobacteria, comprising 961 species. These Gram-positive bacteria produce many versatile and important bioactive compounds; of these, antibiotics, specifically the enhancement or activation of their production, have received extensive research attention. Recently, various biotic and abiotic elicitors have been reported to modify the antibiotic metabolism of Streptomyces, which promotes the production of new antibiotics and bioactive metabolites for improvement in the yields of endogenous products. However, some elicitors that obviously contribute to secondary metabolite production have not yet received sufficient attention. In this study, we have reviewed the functions and mechanisms of chemicals, novel microbial metabolic elicitors, microbial interactions, enzymes, enzyme inhibitors, environmental factors, and novel combination methods regarding antibiotic production in Streptomyces. This review has aimed to identify potentially valuable elicitors for stimulating the production of latent antibiotics or enhancing the synthesis of subsistent antibiotics in Streptomyces. Future applications and challenges in the discovery of new antibiotics and enhancement of existing antibiotic production using elicitors are discussed.

The Integrative and Conjugative Element ICECspPOL2 Contributes to the Outbreak of Multi-Antibiotic-Resistant Bacteria for Chryseobacterium Spp. and Elizabethkingia Spp.

Antibiotic resistance genes (ARGs) and horizontal transfer of ARGs among bacterial species in the environment can have serious clinical implications as such transfers can lead to disease outbreaks from multidrug-resistant (MDR) bacteria. Infections due to antibiotic-resistant Chryseobacterium and Elizabethkingia in intensive care units have been increasing in recent years. In this study, the multi-antibiotic-resistant strain Chryseobacterium sp. POL2 was isolated from the wastewater of a livestock farm. Whole-genome sequencing and annotation revealed that the POL2 genome encodes dozens of ARGs. The integrative and conjugative element (ICE) ICECspPOL2, which encodes ARGs associated with four types of antibiotics, including carbapenem, was identified in the POL2 genome, and phylogenetic affiliation analysis suggested that ICECspPOL2 evolved from related ICEEas of Elizabethkingia spp. Conjugation assays verified that ICECspPOL2 can horizontally transfer to Elizabethkingia species, suggesting that ICECspPOL2 contributes to the dissemination of multiple ARGs among Chryseobacterium spp. and Elizabethkingia spp. Because Elizabethkingia spp. is associated with clinically significant infections and high mortality, there would be challenges to clinical treatment if these bacteria acquire ICECspPOL2 with its multiple ARGs, especially the carbapenem resistance gene. Therefore, the results of this study support the need for monitoring the dissemination of this type of ICE in Chryseobacterium and Elizabethkingia strains to prevent further outbreaks of MDR bacteria. IMPORTANCE Infections with multiple antibiotic-resistant Chryseobacterium and Elizabethkingia in intensive care units have been increasing in recent years. In this study, the mobile integrative and conjugative element ICECspPOL2, which was associated with the transmission of a carbapenem resistance gene, was identified in the genome of the multi-antibiotic-resistant strain Chryseobacterium sp. POL2. ICECspPOL2 is closely related to the ICEEas from Elizabethkingia species, and ICECspPOL2 can horizontally transfer to Elizabethkingia species with the tRNA-Glu-TTC gene as the insertion site. Because Elizabethkingia species are associated with clinically significant infections and high mortality, the ability of ICECspPOL2 to transfer carbapenem resistance from environmental strains of Chryseobacterium to Elizabethkingia is of clinical concern.

The carbapenem resistance gene blaOXA-23 is disseminated by a conjugative plasmid containing the novel transposon Tn6681 in Acinetobacter johnsonii M19.

BACKGROUND: Carbapenem resistant Acinetobacter species have caused great difficulties in clinical therapy in the worldwide. Here we describe an Acinetobacter johnsonii M19 with a novel blaOXA-23 containing transposon Tn6681 on the conjugative plasmid pFM-M19 and the ability to transferand carbapenem resistance. METHODS: A. johnsonii M19 was isolated under selection with 8 mg/L meropenem from hospital sewage, and the minimum inhibitory concentrations (MICs) for the representative carbapenems imipenem, meropenem and ertapenem were determined. The genome of A. johnsonii M19 was sequenced by PacBio RS II and Illumina HiSeq 4000 platforms. A homologous model of OXA-23 was generated, and molecular docking models with imipenem, meropenem and ertapenem were constructed by Discovery Studio 2.0. Type IV secretion system and conjugation elements were identified by the Pathosystems Resource Integration Center (PATRIC) server and the oriTfinder. Mating experiments were performed to evaluate transfer of OXA-23 to Escherichia coli 25DN. RESULTS: MICs of A. johnsonii M19 for imipenem, meropenem and ertapenem were 128 mg/L, 48 mg/L and 24 mg/L, respectively. Genome sequencing identified plasmid pFM-M19, which harbours the carbapenem resistance gene blaOXA-23 within the novel transposon Tn6681. Molecular docking analysis indicated that the elongated hydrophobic tunnel of OXA-23 provides a hydrophobic environment and that Lys-216, Thr-217, Met-221 and Arg-259 were the conserved amino acids bound to imipenem, meropenem and ertapenem. Furthermore, pFM-M19 could transfer blaOXA-23 to E. coli 25DN by conjugation, resulting in carbapenem-resistant transconjugants. CONCLUSIONS: Our investigation showed that A. johnsonii M19 is a source and disseminator of blaOXA-23 and carbapenem resistance. The ability to transfer blaOXA-23 to other species by the conjugative plasmid pFM-M19 raises the risk of spread of carbapenem resistance. The carbapenem resistance gene blaOXA-23 is disseminated by a conjugative plasmid containing the novel transposon Tn6681 in Acinetobacter johnsonii M19.

Novel Mobilizable Genomic Island GEI-D18A Mediates Conjugational Transfer of Antibiotic Resistance Genes in the Multidrug-Resistant Strain Rheinheimera sp. D18.

Aquatic environments act as reservoirs of antimicrobial-resistant bacteria and antimicrobial resistance (AMR) genes, and the dissemination of antibiotic resistance from these environments is of increasing concern. In this study, a multidrug-resistant bacterial strain, identified as Rheinheimera sp. D18, was isolated from the sea water of an industrial maricultural system in the Yellow Sea, China. Whole-genome sequencing of D18 revealed the presence of a novel 25.8 kb antibiotic resistance island, designated GEI-D18A, which carries several antibiotic resistance genes (ARGs), including aadA1, aacA3, tetR, tet(B), catA, dfrA37, and three sul1 genes. Besides, integrase, transposase, resolvase, and recombinase encoding genes were also identified in GEI-D18A. The transferability of GEI-D18A was confirmed by mating experiments between Rheinheimera sp. D18 and Escherichia coli 25DN, and efflux pump inhibitor assays also suggested that tet(B) in GEI-D18A was responsible for tetracycline resistance in both D18 and the transconjugant. This study represents the first characterization of a mobilizable antibiotic resistance island in a species of Rheinheimera and provides evidence that Rheinheimera spp. could be important reservoirs and vehicles for ARGs in the Yellow Sea area.

Identification of a mobilizable, multidrug-resistant genomic island in Myroides odoratimimus isolated from Tibetan pasture.

Strains of the environmental bacterium Myroides odoratimimus can cause human infections. However, treating M. odoratimimus infections can be difficult because of multidrug resistance in this organism. In this study, we isolated strain M. odoratimimus G13 from pastureland in Tibet, China. The minimum inhibitory concentration analysis suggested that strain G13 has resistance to multiple antibiotics, with an MIC for tetracycline of 168 mg/L. Whole-genome sequencing and bioinformatic analysis revealed that the genome of G13 was rich in virulence factor-encoding genes and antibiotic resistance genes (ARGs). The mobilizable genomic island MGI1313 was also identified and characterized, and six resistance genes related to four types of antibiotics were annotated in MGI1313. Conjugation assays indicated that MGI1313 could be transferred from G13 to Escherichia coli 25DN by horizontal gene transfer, resulting in multidrug-resistant E. coli conjugants. In conclusion, multidrug-resistant M. odoratimimus G13 and the mobility of MGI1313 raise the risk of difficult-to-treat bacterial infections and should be under close surveillance.

A Novel Mobile Element ICERspD18B in Rheinheimera sp. D18 Contributes to Antibiotic and Arsenic Resistance.

Antibiotics and organoarsenical compounds are frequently used as feed additives in many countries. However, these compounds can cause serious antibiotic and arsenic (As) pollution in the environment, and the spread of antibiotic and As resistance genes from the environment. In this report, we characterized the 28.5 kb genomic island (GI), named as ICERspD18B, as a novel chromosomal integrative and conjugative element (ICE) in multidrug-resistant Rheinheimera sp. D18. Notably, ICERspD18B contains six antibiotic resistance genes (ARGs) and an arsenic tolerance operon, as well as genes encoding conjugative transfer proteins of a type IV secretion system, relaxase, site-specific integrase, and DNA replication or partitioning proteins. The transconjugant strain 25D18-B4 was generated using Escherichia coli 25DN as the recipient strain. ICERspD18B was inserted into 3'-end of the guaA gene in 25D18-B4. In addition, 25D18-B4 had markedly higher minimum inhibitory concentrations for arsenic compounds and antibiotics when compared to the parental E. coli strain. These findings demonstrated that the integrative and conjugative element ICERspD18B could mediate both antibiotic and arsenic resistance in Rheinheimera sp. D18 and the transconjugant 25D18-B4.

The two-component system CepRS regulates the cephamycin C biosynthesis in Streptomyces clavuligerus F613-1.

During industrial fermentation, Streptomyces clavuligerus F613-1 simultaneously produces primary product clavulanic acid (CA) and cephamycin C. The cephamycin C biosynthetic gene cluster and pathway have been basically elucidated and the CcaR positive regulator was found to control the cephamycin genes expression. However, additional mechanisms of regulation cannot be excluded. The BB341_RS13780/13785 gene pair in S. clavuligerus F613-1 (annotated as SCLAV_2960/2959 in S. clavuligerus ATCC27064) encodes a bacterial two-component system (TCS) and were designated as CepRS (for cephamycin regulator/sensor). CepRS significantly affects cephamycin C production but only slightly affects CA production. To further understand the regulation of cephamycin C biosynthesis, the cepRS genes were deleted from S. clavuligerus F613-1. The deletion mutant resulted in decreased cephamycin C production but had no phenotypic effects. Real-time quantitative polymerase chain reaction analysis revealed that CepRS regulates the expression of most genes involved in cephamycin C biosynthesis, with electrophoretic mobility shift assays showing that CepR interacts with the cefD-cmcI intergenic region. These results demonstrate that the CepR response regulator serves as a transcriptional activator of cephamycin C biosynthesis, which may provide an approach for metabolic engineering methods for CA production by S. clavuligerus F613-1 in future.

The CagRS Two-Component System Regulates Clavulanic Acid Metabolism via Multiple Pathways in Streptomyces clavuligerus F613-1.

Streptomyces clavuligerus F613-1 produces a clinically important ß-lactamase inhibitor, clavulanic acid (CA). Although the biosynthesis pathway of CA has essentially been elucidated, the global regulatory mechanisms of CA biosynthesis remain unclear. The paired genes cagS and cagR, which are annotated, respectively, as orf22 and orf23 in S. clavuligerus ATCC 27064, encode a bacterial two-component regulatory system (TCS) and were found next to the CA biosynthetic gene cluster of S. clavuligerus F613-1. To further elucidate the regulatory mechanism of CA biosynthesis, the CagRS TCS was deleted from S. clavuligerus F613-1. Deletion of cagRS resulted in decreased production of CA, but the strain phenotype was not otherwise affected. Both transcriptome and ChIP-seq data revealed that, in addition to CA biosynthesis, the CagRS TCS mainly regulates genes involved in primary metabolism, such as glyceraldehyde 3-phosphate (G3P) metabolism and arginine biosynthesis. Notably, both G3P and arginine are precursors of CA. Electrophoretic mobility shift assays demonstrated that the response regulator CagR could bind to the intergenic regions of argG, argC, oat1, oat2, ceaS1, and claR in vitro, suggesting that CagR can directly regulate genes involved in arginine and CA biosynthesis. This study indicated that CagRS is a pleiotropic regulator that can directly affect the biosynthesis of CA and indirectly affect CA production by regulating the metabolism of arginine and G3P. Our findings provide new insights into the regulation of CA biosynthetic pathways and provide an innovative approach for future metabolic engineering efforts for CA production in S. clavuligerus.

Complete Genome Sequence of Actinosynnema pretiosum X47, An Industrial Strain that Produces the Antibiotic Ansamitocin AP-3.

Ansamitocins are extraordinarily potent antitumor agents. Ansamitocin P-3 (AP-3), which is produced by Actinosynnema pretiosum, has been developed as a cytotoxic drug for breast cancer. Despite its importance, AP-3 is of limited applicability because of the low production yield. A. pretiosum strain X47 was developed from A. pretiosum ATCC 31565 by mutation breeding and shows a relatively high AP-3 yield. Here, we analyzed the A. pretiosum X47 genome, which is ~8.13 Mb in length with 6693 coding sequences, 58 tRNA genes, and 15 rRNA genes. The DNA sequence of the ansamitocin biosynthetic gene cluster is highly similar to that of the corresponding cluster in A. pretiosum ATCC 31565, with 99.9% identity. However, RT-qPCR analysis showed that the expression levels of ansamitocin biosynthetic genes were significantly increased in X47 compared with the levels in the wild-type strain, consistent with the higher yield of AP-3 in X47. The annotated complete genome sequence of this strain will facilitate understanding the molecular mechanisms of ansamitocin biosynthesis and regulation in A. pretiosum and help further genetic engineering studies to enhance the production of AP-3.