Brevibacterium aurantiacum is an actinobacterium that confers key organoleptic properties to washed-rind cheeses during the ripening process. Although this industrially relevant species has been gaining an increasing attention in the past years, its genome plasticity is still understudied due to the unavailability of complete genomic sequences. To add insights on the mobilome of this group, we sequenced the complete genomes of five dairy Brevibacterium strains and one non-dairy strain using PacBio RSII. We performed phylogenetic and pan-genome analyses, including comparisons with other publicly available Brevibacterium genomic sequences. Our phylogenetic analysis revealed that these five dairy strains, previously identified as Brevibacterium linens, belong instead to the B. aurantiacum species. A high number of transposases and integrases were observed in the Brevibacterium spp. strains. In addition, we identified 14 and 12 new insertion sequences (IS) in B. aurantiacum and B. linens genomes, respectively. Several stretches of homologous DNA sequences were also found between B. aurantiacum and other cheese rind actinobacteria, suggesting horizontal gene transfer (HGT). A HGT region from an iRon Uptake/Siderophore Transport Island (RUSTI) and an iron uptake composite transposon were found in five B. aurantiacum genomes. These findings suggest that low iron availability in milk is a driving force in the adaptation of this bacterial species to this niche. Moreover, the exchange of iron uptake systems suggests cooperative evolution between cheese rind actinobacteria. We also demonstrated that the integrative and conjugative element BreLI (Brevibacterium Lanthipeptide Island) can excise from B. aurantiacum SMQ-1417 chromosome. Our comparative genomic analysis suggests that mobile genetic elements played an important role into the adaptation of B. aurantiacum to cheese ecosystems. Micrococcaceae, Corynebacteriaceae, and Brevibacteriaceae families (The coexistence and succession of diverse microorganisms over time lead to the development of distinct organoleptic properties in a wide variety of aged cheeses . During families . The comfamilies .Brevibacterium linens have been acknowledged as widely used for washed rind cheese ripening as they produce volatile sulfur compounds (VSCs)  , carotens (VSCs) , lipolyts (VSCs) , which dotenoids  but theyeristics . A recen cheeses , suggestBrevibacterium spp. genomes, including 12 isolates from cheeses, identified genetic determinants involved in adaptation to the cheese habitat  and sequenced using a MiSeq reagent kit v2  on a MiSeq system. De novo assembly was performed with Ray assembler version 2.2.0  were identified with GeneMark.hmm (cas) genes was assessed using CRISPRCasFinder . We then aligned the sequences with Multiple Sequence Comparison by Log-Expectation (MUSCLE) (We used a broad phylogenetic distribution of 82 software . Glutami(MUSCLE) . The phy(MUSCLE)  was cons(MUSCLE) . We perf(MUSCLE) . The treB. aurantiacum were extracted from GenBank and added to the pan-genome analyses. All genomes used in our analyses were annotated with the NCBI pipeline. The clustering of the protein sequences in orthologous groups was conducted with USEARCH  were not validated and removed from the dataset. The other IS elements were validated by the ISFinder database. Complete ISs, transposases, and repeat sequences are available in the ISFinder database. To identify putative HGT regions, whole Brevibacterium spp. genomes were aligned against the NCBI database using BLASTn. Alignment hits were considered as probable HGT regions when nucleotide sequences larger than 2,000 bp shared more than 90% identity with other bacterial species.ISFinder database was used to identify ISBli1-ISBli5 in the genomes while ISBli6-ISBli35 were identified manually, as follow. We searched for transposase and mobile element annotations with identical lengths, and the corresponding CDS were extracted with 1000 bp upstream and downstream, as previously described . These sB. aurantiacum strains as described elsewhere (B. aurantiacum SMQ-1335 and other five B. aurantiacum strains (SMQ-1417 – SMQ-1421) were grown in Elliker media with different concentrations of mitomycin C  and incubated at 20°C with 200 rpm agitation. The lysogenic strain Lactobacillus paracasei A was used as a positive control, while each culture without mitomycin C was used as negative controls.Prophage induction assays with mitomycin C was performed with six lsewhere . B. auraBrevibacterium linens ATCC 19391 and B. aurantiacum strains  were grown in mineral salts media (MSM) supplemented with trace elements solution  (600) for 21 h.l2.6H2O)  for 24 hB. aurantiacum was cultivated in BHI containing 1% (w/v) agar for 48 h at 30°C and one colony was used for each PCR reaction. PCR was performed using Taq polymerase Mastermix (Feldan) according to the manufacturer’s instructions. All primers used are listed in To verify the excision of iRon Uptake/Siderophore Transport Island (RUSTI) , we desiIn vitro production and activity of antimicrobial peptides was tested for actinobacteria and lactic acid bacteria. B. linens ATCC 19391, Corynebacterium glutamicum ATCC 21086, B. aurantiacum SMQ-1417 to SMQ-1421, SMQ-1335, and JB5 were cultivated in Elliker broth at 30°C, 200 rpm. The antimicrobial activity of the filtered supernatants of these strains was tested by spotting 5 μl on a lawn of each of the strains. Additionally, activity was tested against Micrococcus luteus HER1157, C. glutamicum HER1229, Arthrobacter arilaitensis LMA-1184, Lactococcus lactis MG1363 and SMQ-1200, Streptococcus thermophilus HER1458 and HER1368. The nisin-producing strain L. lactis SMQ-1200 was grown in Elliker and incubated overnight at 30°C. This strain was used as a positive control for the experiments against all the bacteria mentioned above.Brevibacterium aurantiacum strains with a SMQ number are available upon request. All IS elements were validated by the ISFinder database under the name ISBli6-ISBli35. Genomes have been deposited in the NCBI genome database , including type strains for the different species of Brevibacterium  have been previously classified as B. linens, their 16S rRNA phylogenetic analysis grouped them with the type strain Brevibacterium epidermidis (P159), suggesting that these strains should have their taxonomic classification reassessed. Surprisingly, only three strains grouped with B. linens type strain ATCC 9172, which are B. linens ATCC 19391, VCM10, and Mu101. Only B. linens Mu101 and ATCC 9172 were isolated from cheeses. These results suggest that most of the strains used for commercial cheese ripening belong to the B. aurantiacum species. For years, B. linens has been recognized as a key player on the production of orange-pigmented washed-rind cheeses  were genetically distinct, we first performed PFGE. The seven strains presented a unique PFGE profile using restrictions enzymes AseI and PsiI (data not shown). Then to shed light on their genomic diversity, we sequenced the genomes of six strains  using PacBio RSII. Before performing bioinformatic analyses on these six complete genomes, we added to our analyses three other complete genomes (B. aurantiacum SMQ-1335 and JB5 as well as B. epidermidis BS258), previously sequenced with the same technology  and higher G+C contents (64.16 and 64.79%). The number of predicted coding sequences (CDS) in these two genomes was also lower than in B. aurantiacum, correlating with the smaller genome sizes. Four rRNA operons were observed in all Brevibacterium strains analyzed. The average number of CDS per B. aurantiacum genome was 3,805, of which 79.3% could be assigned a predicted function. We also investigated the presence of CRISPR-Cas system in B. aurantiacum or B. linens ATCC 19391, but no CRISPR array with the associated Cas genes was predicted in any of the genomes.To confirm that our seven chnology . The geno 62.79% . B. lineBrevibacterium is rare with only a few described in the literature , suggesting genetic exchange between these two closely related species  and genes encoding an additional non-essential replication protein and a hypothetical protein (ORF16). Of note, we found orthologs of JetA, JetB, JetC, and JetD of the recently described anti-plasmid system Wadjet  and the orphan genes (k = 1 genome), with 2,612, and 1,790 genes, respectively , DNA and RNA metabolism (99 genes), and transcriptional regulators (83 genes) also accounted for several ORFans. A total of 20 Type I restriction-modification (R-M) systems and 6 Type III R-M methylation subunits were identified in the DNA and RNA metabolism category, perhaps offering broad protection against phage infection. Another intriguing category among B. aurantiacum ORFans was ATP-Binding Cassette (ABC) transporter proteins, such as components of iron siderophore transport system. In the cheese environment where iron is poorly available, these genes could improve growth on cheese rinds. Furthermore, we analyzed the genomic context of the ORFans and found that these genes were dispersed into B. aurantiacum genomes (ori region (upstream dnaA) in all eleven B. aurantiacum genomes suggesting that this chromosomal region is less conserved in this species.To determine the genetic diversity of chnology . Our ana59 genes . We alsoectively . Hence, e strain . The orp by RAST . Of 1,79 by RAST . In RAST genomes . A slighB. aurantiacum pan-genome, we analyzed the B. aurantiacum mobilome. In the largest B. aurantiacum genome (strain SMQ-1417), we identified 116 transposases and 68 integrases indicating that this species contains several mobile genetic elements, such as prophages and insertion sequences (IS). A putative prophage was previously identified in the genome of SMQ-1335  was detected in the genome of B. aurantiacum SMQ-1419. Although no structural gene was predicted, we performed a manual inspection of the regions flanking the putative prophage and found a gene encoding a putative tail tape measure protein (TMP) located a few ORFs upstream of PHASTER predicted attachment site attL. The BLASTp analysis of other ORFs surrounding the TMP also showed the presence of other genes involved in phage morphogenesis, such as putative genes coding for minor tail proteins, tail assembly chaperone and major capsid protein. We also identified genes involved in phage DNA packaging . This extended 45-kbp putative prophage in SMQ-1419 was not found in other B. aurantiacum strains.Once the genome was annotated, we observed genes encoding putative phage proteins  spread in the genomes of all six B. aurantiacum mobilome, IS also play an important role in bacterial evolution by spreading into a genome and creating genetic variations . A total of 26 ISs were validated by the ISFinder database, whereas four  were not validated for not being discriminating enough to the others and were then removed from our analysis. We observed a dichotomy in the abundance and the type of ISs detected in B. aurantiacum and B. linens genomes (B. aurantiacum genomes (5 to 13 copies/genome), only one copy was identified in B. linens ATCC 19391.As part of riations . They cariations . Moreoveriations . Using t genomes , suggestB. aurantiacum SMQ-1417, SMQ-1419, SMQ-1421, and SMQ-1335  infecting dairy cultures are well documented , but without the IS (We found ISs from two other actinobacteria species used for cheese production in bacteria . In-dept ISAar39  in both ansposon . Interest the IS . We hypoB. aurantiacum genomes contained homologous nucleotide sequences (92–99% identity) with other species of cheese rind actinobacteria (B. aurantiacum genomes (C. casei LMG S-19264. This region has been previously described as the Brevibacterium Lanthipeptide Island (BreLI)  . It is nB. aurantiacum genomes and clustered them into functional groups based on the RAST subsystem categories , we performed growth curves in a MSM with limited iron availability and the presence of increasing concentrations of EDDHA as a chelating agent  and cultures with the addition of chelating agent. Since SMQ-1421 is the strain with the lower number of iron-uptake components, this result supports the premise that the presence of genes/proteins cluster involved in iron acquisition is likely to confer adaptive growth on the cheese surface. However, they are not directly proportional as can be seen with JB5, which is the strain with more iron acquisition components. JB5 presented a similar growth in all three EDDHA concentrations, but not as much as the control culture that grew faster and reached a higher optical density. These results suggest that most of Brevibacterium strains are adapted to the growth in iron-limited medium.Considering the diverse amount of iron uptake gene clusters observed in ng agent . We hypoly shown  that 100Brevibacterium strains  (Brevibacterium Lanthipeptide Island (BreLI), was also observed in C. casei LMG S-19264 (B. aurantiacum SMQ-1417. ICE elements can be identified by the presence of signature genes/proteins associated with the core conjugation functions of (i) integration and excision from the host genome, (ii) replication as an extrachromosomal element, and (iii) conjugation between host and recipient cells (B. aurantiacum SMQ-1417 BreLI region. An integrase (CXR23_14705) and a site-specific integrase (CXR23_15110) were observed at the two borders of BreLI, directly beside the repeats. Two other integrases , and one excisionase (CXR23_14930) were also identified and these five genes are likely involved in the integration and excision of BreLI.Another factor that can influence the microbial community on cheese surface is the production of antimicrobials, such as bacteriocins. For example, lanthionine-containing peptides (lanthipeptides) can inhibit the growth of undesirable microorganisms  and confd Mu101) . This 96 S-19264 . This IC S-19264 . We idennt cells . Genes iB. aurantiacum SMQ-1417 ICE element , a DNA polymerase III subunit alpha (CXR23_14790) and a DNA primase (CXR23_14925). The majority of actinobacterial ICEs utilizes a FtsK homolog-based conjugative DNA-translocation system to exchange double-stranded DNA  and linaridin modification and transport, whereas SMQ-1420 and SMQ-1421 only have the genes involved in biosynthesis. SMQ-1335, SMQ-1418, and SMQ-1419 contain a putative gene cluster involved in sactipeptide production, although they do not encode putative antimicrobial peptide precursors.We used Bagel4 to find genes involved in the biosynthesis of bacteriocins or bactericidal post-translational modified peptides in the genome of in vitro antimicrobial activity of Brevibacterium spp. filtered supernatant against different bacterial species. We used the class I lantibiotic nisin-producing strain L. lactis SMQ-1220 as a positive control in our experiments. Although nisin-containing supernatants of SMQ-1220 were active against the bacterial strains tested , we did not observe antimicrobial activity for the filtered supernatants of Brevibacterium strains in the conditions tested. Perhaps, the lack of activity is related to experimental parameters, but it can also be due to genetic features of the host cells that prevent the expression of the laterally shared genes, such as promoter incompatibilities (We tested the bilities  or absenbilities .S. thermophilus (L. lactis (C. variabile (G. arilaitensis (P. freudenreichii (Brevibacterium strains also provided insights into their adaptation to the cheese habitat (Comparative genomic studies have led to a better understanding of the genetic adaptation of dairy bacteria, such as mophilus , L. lact. lactis , C. variariabile , G. arilaitensis , and P. nreichii  to chees habitat . OverallBrevibacterium spp. and other cheese actinobacteria (Brevibacterium genome sequences and added information on the mobilome of these bacteria. Our phylogenetic analysis support previous findings (Brevibacterium strains used for cheese production belong to the B. aurantiacum species, making this species a key player in the dairy industry. Therefore, the taxonomic position of commercial Brevibacterium strains should be revisited.Given the prevalence of mobile genetic elements in bacteria , we usedfindings  and showB. linens and B. epidermidis to compare with B. aurantiacum dairy strains and understand the cheese domestication of B. aurantiacum. Despite being the most prevalent (B. aurantiacum is not the only Brevibacterium species able to grow on cheese. For example, B. antiquum, B. casei, and B. linens have been isolated from cheeses (B. aurantiacum in the dairy ecosystem. Multiple HGTs have been documented between cheese rind bacteria (B. aurantiacum dairy strains and environmental strains of B. linens and B. epidermidis. B. aurantiacum strains seem to have acquired heterologous genes from other dairy actinobacteria, such as G. arilaitensis, C. variabile, and C. casei. The high percentage of identity between these DNA segments suggests that these genes were recently transferred. Moreover, mobile genetic elements are expanding B. aurantiacum pan-genome and they contribute to its genetic diversity. It is noteworthy that B. aurantiacum pan-genome is still open and more genomes should be analyzed to appreciate the diversity of its gene repertoire.We used environmental strains of revalent , B. aura cheeses . Howeverbacteria  and betwbacteria , suggestBrevibacterium spp. genomes. PCR amplifications of BreLI confirmed that this ICE was still active in B. aurantiacum SMQ-1417. Although in vitro tests did not confirm antimicrobial activity, it is still likely that an efficient production of active lanthipeptides could confer a selective advantage for the producing strain. Also observed in C. casei LMG S-19264, BreLI is likely to have been mobilized from a Brevibacterium spp., perhaps during cheese ripening (B. aurantiacum is likely a player in the widespread distribution of mobile genetic elements.The use of commercial ripening cultures with active mobile genetic elements could explain the prevalence of nearly identical DNA sequences between cheese rind actinobacteria , such asripening . With thB. aurantiacum genomes. As previously showed (Brevibacterium strains in media with limited iron availability. Except with SMQ-1419, that was not able to grow in the media, all the other strains grew in increasing amount of iron chelating agent, with variability in the growth pattern. The strain SMQ-1421 was the less fit in iron-limited media, which corroborates with the lower abundance of iron acquisition and siderophore biosynthesis genes. On the other hand, the environmental strain B. linens ATCC 19391 was the better adapted when growing in the presence or absence of chelating agent.Here, we found horizontally transferred regions involved in iron uptake in five out of seven y showed , genes aBrevibacterium strains to grow, alone or in combination, in iron-depleted environments. Some B. aurantiacum strains with high aromatic potential could be auxotrophic for iron siderophores and dependent on other strains or species to grow well on cheese. Cheesemakers are sometimes faced with unstable ripening activity or the growth of undesirable microorganisms (Growth on the surface of smear-ripened cheeses is dependent of the capability to use substrates available in the cheese habitat. Additionally, it depends on its interactions with members of the microbial community present during ripening. Further studies could be done to explore the capacity of different rganisms . The optBrevibacterium spp. allowed an in-depth analysis of the mobilome of commercial B. aurantiacum strains and complement previous comparative analysis targeting cheese-related actinobacteria. Our phylogenetic analysis demonstrated that the industrial strains used for cheese production belong to the B. aurantiacum species. Our study also revealed that mobile genetic elements are widespread among the strains analyzed here and contribute to B. aurantiacum genetic diversity. Moreover, iron uptake HGT demonstrate the cooperative evolution of cheese actinobacteria, allowing their niche adaptation to the iron-depleted cheese surface. Regardless of the strong selective pressure exerted on the surface of smear-ripened cheeses, B. aurantiacum strains seem to be well adapted to thrive in this ecological niche. This comprehensive genomic information will serve as a tool to continue improving our understanding of the complex interactions taking place in smear-ripened cheese microbial communities.In conclusion, the addition of six complete genomic sequences of B. aurantiacum genomes were available in public databases prior to this study. B. aurantiacum genomes contain a high number of transposable elements and their genome sequences remain at the draft level when sequenced with short-read sequencing technologies, which precludes in-depth analysis of mobile genetic elements and genome plasticity. Here, we used long-read sequencing technology to provide six new complete Brevibacterium genomes. We identified a high number of mobile genetic elements that could be involved in the growth fitness of B. aurantiacum on the surface of smear-ripened cheeses. These genetic traits are shared between cheese rind actinobacteria and they seem involved in iron acquisition. Our study shows that mobile genetic elements are widespread in B. aurantiacum and contribute to its genomic diversity.Only two complete The datasets generated for this study can be found in GenBank, CP025330, CP025331, CP025332, CP025333, CP025334, and CP026734.SL and SM designed the research protocol. SL, AM, and SJL performed the research. SJL wrote the in-house python and R scripts and performed the pan/core-genome analysis. SL and AM analyzed the data. SL, AM, and SM wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Antibiotic-resistant bacterial pathogens are increasingly implicated in hospital- and community-acquired infections. Recent advances in monoclonal antibody (mAb) production and engineering have led to renewed interest in the development of antibody-based therapies for treatment of drug-resistant bacterial infections. Currently, there are three antibacterial mAb products approved by the Food and Drug Administration (FDA) and at least nine mAbs are in clinical trials. Antibacterial mAbs are typically developed to kill bacteria or to attenuate bacterial pathological activity through neutralization of bacterial toxins and virulence factors. Antibodies exhibit distinct pharmacological mechanisms from traditional antimicrobials and, hence, cross-resistance between small molecule antimicrobials and antibacterial mAbs is unlikely. Additionally, the long biological half-lives typically found for mAbs may allow convenient dosing and vaccine-like prophylaxis from infection. However, the high affinity of mAbs and the involvement of the host immune system in their pharmacological actions may lead to complex and nonlinear pharmacokinetics and pharmacodynamics. In this review, we summarize the pharmacokinetics and pharmacodynamics of the FDA-approved antibacterial mAbs and those are currently in clinical trials. Challenges in the development of antibacterial mAbs are also discussed. Corynebacterium diphtheria, Streptococcus pneumoniae, Neisseria meningitides, Haemophilus influenzae, Group A Streptococcus, and Clostridium tetani [Acinetobacter and Pseudomonas were identified [The clinical application of antibodies for the treatment of infectious diseases was first introduced in the form of serum therapy in early 1890s by Emil von Behring and Shibasaburo Kitasato [m tetani . Althougm tetani ,3,4,5. Im tetani , and them tetani . In 2004entified . Unfortuentified . The discovery of hybridoma technology in 1975 and recent advances in monoclonal antibody (mAb) engineering, which make production of unlimited amount of human mAbs possible ,9, have In this review, we discuss important considerations of mAb-based therapies for the treatment of bacterial infections, including unique challenges, pharmacokinetic (PK) properties, and pharmacodynamic (PD) mechanisms of action. The PK/PD characteristics of FDA-approved antibacterial mAbs and those in clinical trial are also summarized.All FDA-approved antibacterial mAbs and the majority of those in clinical trials are of the immune gamma globulin (IgG) isotype. IgG is the predominant immunoglobulin isotype, comprising approximately 80% of immunoglobulin in human serum. An intact IgG has a molecular weight of ~150 kDa, with two antigen binding domains and a highly conserved crystallizable region (Fc) that is responsible for binding to Fc gamma receptors (FcγRs) on immune cells and activating Fc-mediated effector functions. IgG typically exhibits linear pharmacokinetics  is directly proportional to the dose) in healthy human subjects, with small volumes of distribution (3–9 L), relatively slow clearance (8–12 mL/h), and long half-lives (20–25 days) . The lonTarget mediated drug disposition describes the phenomenon where binding of a high affinity drug to its pharmacological target affects the PK characteristics of the drug  . At relaBacillus anthracis, showed ~2-fold faster clearances in rabbits and monkeys challenged with B. anthracis spores compared to those in non-infected animals [Staphylococcus aureus also demonstrated significantly increased clearance (12.1–15.8 mL/day/kg) and decreased half-life (3.74–5.28 days) in S. aureus infected mice compared to those in non-infected mice  [Acinetobacter baumannii did not impact the PK of an anti-K2 capsule mAb in mouse blood, although a substantial accumulation of mAb (5.64–36.1 fold higher amount) was observed in tissues  with high bacterial load compared to values found in non-infected mice [All marketed antibacterial mAbs and those in clinical trials were developed to kill bacteria or attenuate bacterial pathological activity via antibody-mediated effector functions or toxin neutralization . Antibod animals . Similarctively) . In contStaphylococcus epidermis, that act as decoys to reduce mAb molecules reaching the bacteria [The tissue disposition of anti-bacterial mAbs may be complex, involving extravasation of mAb molecules from blood to tissue interstitial fluids, diffusion of the molecule in the interstitial fluids to bacterial targets, binding to bacterial targets, and elimination of mAbs from tissue via convective drainage through the lymphatics and via catabolism. Extravasation of IgG antibodies is typically thought to be governed by both diffusion and convection , but convection has been estimated to contribute more than 98% of the total transport ,17,18. Bbacteria . TherefoAntibacterial mAbs have been developed against a variety of bacterial cell surface targets  and soluble exotoxins . The potPseudomonas aeruginosa type III toxins to host cells [P. aeruginosa in a mouse pneumonia infection model [Antibacterial mAbs that act through the mechanism of neutralization are typically directed against exotoxins. mAb binding to soluble exotoxins leads to the formation of antibody-toxin complexes, which are primarily cleared by the reticuloendothelial system. All three marketed antibacterial mAbs achieve effects via toxin neutralization . The effst cells . A singlon model ,25. 2  is required to trigger antibody-dependent phagocytosis [Acinetobacter baumannii, which showed potent inhibitory effects on bacterial growth in a rat soft tissue infection model. Antibody-dependent phagocytic killing has been found to be the primary bactericidal mechanism in this study [Opsonophagocytosis has been considered as one of the key bactericidal mechanisms of the innate immune system. Antibody-mediated opsonophagocytosis involves antibody binding to bacterial surface antigens, followed by the engagement of FcγRs on the surface of professional phagocytes , which in turn trigger actin-myosin driven endocytosis of antibody-bacteria complexes . Phagosoocytosis . In one is study . Staphylococcus aureus (MRSA) was generated by An et al. with strong binding to MRSA and mannose-binding lectin (MBL), which in turn activated the classical and MBL complement pathways and led to potent bactericidal activity [Antibody-dependent  complement activation is another important bactericidal mechanism of the innate immune system. Binding of antibodies on the bacterial surface enhances the recruitment and binding of soluble complement factors, including C1q, to the Fc domain of the mAb, which leads to the activation of the complement cascade , formation of the membrane attack complex, thus leading to bacterial killing. Activation of the antibody-dependent pathway requires interaction of C1q with at least two IgG molecules . Based oactivity . Passiveactivity .Borrelia burgdorferi for the treatment of Lyme disease [B. burgdorferi, which resulted in osmotic lysis of bacterial cells. However, this bactericidal effect was not transferable to Escherichia coli expressing recombinant OspB, suggesting a unique interaction between CB2 and B. burgdorferi [B. burgdorferi outer membrane that exist as temperature-sensitive lipid raft-like microdomains [In addition to the mechanisms of action discussed above, antibacterial mAbs showing direct bactericidal activity have been occasionally identified. Binding of these mAbs may trigger lysis of bacterial cells directly . For instance, LaRocca et al. developed an IgG1 mAb (CB2) that targets outer surface protein B (OspB) of  disease . CB2 exhgdorferi . The undodomains .Staphylococcus aureus [S. aureus and mediates uptake into phagolysosomes, where a potent antibiotic payload is released, allowing efficient killing of intracellular bacteria [S. aureus, and it was especially efficacious for bacteria with an intracellular life cycle [The use of mAbs to deliver highly potent payloads has been successfully applied in cancer treatment . This sts aureus . The antbacteria . This AAfe cycle . Antibod213Bi-D11 showed dose-dependent bacterial killing in vitro and in a mouse bacteremia model, without detectable hematological toxicity [Radioimmunoconjuates, which link radionuclides to mAbs, may allow targeted delivery of bactericidal radiation to bacteria. As a proof-of-principle, Dadachova et al. developed a radioimmunoconjugate with Bismuth-213 linked to a mAb (D11) targeting the pneumococcal capsular polysaccharide . Administoxicity . In additoxicity . These d+ and CD8+ T cells in TB patients after standard-of-care therapy [Immunomodulatory mAbs, such as T-cell engaging antibodies and antibodies targeting programmed cell death protein 1 (PD-1) or cytotoxic T lymphocyte-associated protein 4 (CRLA-4), have gained great success in the treatment of cancer. However, their application in bacterial infections has not been well explored. Akin to cancer, chronic exposure of antigens to T-cells during persistent infections leads to cellular exhaustion of effector functions . Thus, i therapy . Treatme therapy .Development of antibacterial mAbs has been progressing relatively slowly. Only three antibacterial mAbs have been marketed in the United States , and ninEscherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii [Staphylococcus aureus and Streptococcus pneumoniae, respectively [P. aeruginosa  of an unencapsulated strains . Incubation with these antimicrobial peptides also stimulated the release of capsular polysaccharide [A. baumannii appear to neutralize free anti-capsule mAb molecules and may contribute to the lack of efficacy of mAb treatment in an A. baumannii mouse pneumonia infection model [Antibacterial mAbs have been primarily developed to target either bacterial cell surface targets or secreted exotoxins. Although anti-exotoxin antibodies have been successful, these mAbs only attenuate bacterial pathological activity though neutralization of toxins, and are not expected to provide bactericidal activities. Anti-exotoxin mAbs, therefore, are typically indicated for prophylaxis or as adjunctive therapies to antibiotics. Antibacterial mAbs directed against bacterial surface epitopes have primarily been developed for binding to outer membrane proteins (OMPs) or exopolysaccharides  as shown in aumannii ,43. Howeaumannii ,46,47,48ectively ,50. A siruginosa  and, theccharide . Shed caon model . Streptococcus pneumoniae cleave the anti-capsule IgA1 mAb that is bound on the bacterial exopolysaccharide. Positive charges on the IgA1 Fab fragments neutralize the negative charges on the polysaccharide, which unmasks phosphorylcholine underneath the capsular polysaccharide, thus enhancing S. pneumoniae binding to epithelial cells [S. pneumoniae adherence was found to be directly correlated with the isoelectric points of the IgA1 Fab fragments [Mycobacterium tuberculosis infection of human lung epithelial cells and promote bacterial replication in macrophages [M. tuberculosis mAbs (protective vs. non-protective) was shown to be correlated with both antibody isotype and glycosylation patterns [Acinetobacter baumannii, one of the three top priority pathogens listed by World Health Organization, also demonstrated ADE of infection. Our laboratory had shown that an anti-capsule mAb IgG3 enhances A. baumannii adherence/invasion to macrophages and human lung epithelial cells through IgG engagement of FcγRs, and this ADE of infection leads to a significant increase of animal mortality in a mouse pneumonia infection model [Although the role of immunoglobulin in host defense against invading microorganisms via activation of effector cells and complement is undisputed, accumulating evidence supports that, in some instances, antibody augments microorganism infection via assisting in their colonization and invasion to host cells. Antibody-dependent enhancement (ADE) of infection was first discovered in Murray Valley encephalitis virus by Hawkes , and it al cells . The incragments . Recentlrophages . Additiopatterns ,63. Monoon model .S. aureus and protein G of Streptococcus, are membrane proteins that bind antibody Fc domain and thus impede opsonophagocytosis and complement activation [Streptococcus pyogenes, for example, secrete IdeS (Immunoglobulin G-degrading enzyme of S. pyogenes) that specifically cleaves the γ-chain of human IgG in the hinge region, and SpeB  that has a broad immunoglobulin-degrading activity toward IgG, IgA, IgM, IgD, and IgE [2) may compete for binding with intact antibodies and further impede antibody-mediated bactericidal activities [S. aureus, P. aeruginosa, Streptococcus pneumoniae, and Haemophilus influenzae [M. tuberculosis, for instance, inhibits fusion of lysosomes with phagosomes in macrophages, which protects bacteria from killing mediated by lysosomal constituents [Rickettsia prowazekii escapes from phagosome vacuoles before the phagosome-lysosome fusion, likely via phospholipase-mediated dissolution of phagosome membrane [S. aureus is resistant to killing by phagolysosomal catabolism through neutralization of toxic oxygen radicals by released catalase, superoxide dismutase, and carotenoids [Although antibacterial mAbs exploit pharmacological mechanisms that are distinct from those of antimicrobials and hence cross-resistance with small-molecule antibiotics is unlikely, host immunoglobulin has applied “selection pressure” to bacteria for millennia, which has led to the evolution of a variety of bacterial defense mechanisms. Countermeasures against host immunoglobulin may, in many cases, provide defense against therapeutic monoclonal antibacterial antibodies. Antibody neutralizing proteins, for instance, protein A of tivation ,66,67. A and IgE ,69. Folltivities . Similarfluenzae ,72,73,74tituents . Rickettmembrane . S. aureotenoids ,77,78. Totenoids .Bacillus anthracis. Raxibacumab is approved for use in combination with appropriate antibacterial drugs, and for prophylaxis of inhalational anthrax when alternative therapies are not available or are not appropriate. Raxibacumab binds free PA with a high affinity (equilibrium dissociation constant Kd = 2.78 nM), which inhibits engagement of PA to its cellular receptors on macrophages. The antibody impedes intracellular entry of anthrax lethal factor and edema factor, which contribute substantially to the pathogenic effects of anthrax toxin [B. anthracis bacteremia, did not affect the PK of raxibacumab [Raxibacumab (Abthrax) is an anti-protective antigen (PA) mAb that has been approved for the treatment of adult and pediatric patients with inhalational anthrax due to ax toxin ,80. Raxiax toxin ,82. Co-aB. anthracis spores. Significantly improved survival was demonstrated in infected New Zealand White (NZW) rabbits and cynomolgus macaques , when treated with 40 mg/kg raxibacumab compared to placebo groups  [B. anthracis challenged NZW rabbits [Raxibacumab is the first biologic product that was developed and approved under the FDA Animal Rule that may be applied when it is not ethical or feasible to conduct controlled clinical trials in humans. The effectiveness of raxibacumab for treatment of inhalational anthrax thus is based on efficacy studies in rabbits and monkeys. Treatment with raxibacumab was initiated when PA was detected in serum (28–42 h) or when body temperature was sustained above baseline for 2 h in animals after challenge with aerosolized urvival) . In addi rabbits . Based od = 0.33 nM) protects against anthrax toxin through inhibition of PA binding to cellular receptors on host cells [B. anthracis led to significantly faster clearance (17.0 mL/day/kg and 8.6 mL/day/kg) compared to values observed in non-infected animals  [Obiltoxaximab (Anthim) is also an anti-PA mAb that was approved for the same indication and usage as raxibacumab. However, premedication with diphenhydramine is recommended and close monitoring of individuals who receive obiltoxaximab is also required due to common adverse reactions including hypersensitivity  and anaphylaxis (0.9% cases) observed in Phase 1 clinical trials . Obiltoxst cells . Obiltoxst cells , infectictively) . These dctively) . Low titctively) . Furtherctively) . B. anthracis spores. The mAb was administered after animals exhibited clinical signs of systemic anthrax , and intravenous obiltoxaximab at 16 mg/kg was able to significantly improve survival in NZW rabbits (62–93%) and cynomolgus macaques (31–47%) compared to 0–6% survival in placebo groups [B. anthracis infection provided full protection  in cynomolgus macaques versus 10% survival in control animals [B. anthracis infected animals [Obiltoxaximab was also approved under the US FDA Animal Rule. Therapeutic efficacy of obiltoxaximab was assessed in animals challenged with aerosolized o groups . In prop animals . Further animals . Clostridium difficile infection (CDI) in patients ≥18 years of age who are receiving antibacterial drugs for CDI and are at high risk for CDI recurrence. Bezlotoxumab binds with high affinity (Kd < 1 nM) to toxin B, a pivotal virulence factor of C. difficile. The mAb inhibits toxin B binding to host cells and hence prevents toxin B-mediated inactivation of Rho GTPases and downstream signaling pathways in cells [C. difficile-infected patients in Phase 3 clinical trials, with estimated mean clearance, volume of distribution, and half-life of 0.317 L/day, 7.33 L, and 19 days, respectively [C. difficile-associated diarrhea following clinical cure of the presenting CDI episode) was significantly lower in patients receiving 10 mg/kg bezlotoxumab with SoC (17.4% and 15.7%) than the subjects receiving placebo with SoC therapy (27.6% and 25.7%) in two Phase 3 studies [C. difficile-infected patients compared to the SoC group [Bezlotoxumab (Zinplava) is a human IgG1 that has been approved for use to reduce recurrence of in cells . The PK ectively . Recurre studies . HoweveroC group . Thus beStaphylococcus aureus, three are targeting Pseudomonas aeruginosa, and one is for Escherichia coli. Released data from preclinical and clinical studies are summarized here to give a broad overview of the products that may be clinically available in the next few years. In addition to the three marketed mAb products, there are nine mAbs that are currently being investigated in clinical trials. Among the nine products listed in Staphylococcus aureus and hence inhibits AT-mediated cytotoxic activity toward host cells [S. aureus infections including sepsis, skin and soft tissue infection, and pneumonia [MEDI4893 is a human IgG1 mAb that specifically binds to and neutralizes alpha-toxin (AT) of st cells . AT is ast cells . Animal neumonia ,92,93. Aneumonia . In an aneumonia ,95. In aneumonia . S. aureus pneumonia (VASP). ASN-1 targets alpha-toxin and four leukocidins including gamma hemolysins (HIgAB and HIgCB), Panton-Valentine leukocidin (LukSF or PVL), and LukED. ASN-2 binds another leukocidin LukGH (LukAB) [S. aureus clinical isolates. LukED is expressed in 50–75% isolates, while LukSF is only present in 5–10% isolates but is correlated with more severe infections [ASN100 is a mAb combination of two human IgG1, ASN-1 and ASN-2, which is in development for the prevention of ventilator-associated  (LukAB) ,98. Leuk (LukAB) ,102. Thefections . ASN-1 afections . Estimatfections . S. aureus THIOMAB™  antibody and a potent antibiotic, 4-dimethylamino piperidino-hydroxybenzoxazino rifamycin (dmDNA31), linked through a protease cleavable valine-citrulline linker [S. aureus can survive inside neutrophils and turn them into “Trojan horses”, which assist in systemic bacterial dissemination and contribute to recurrence of infection following antibacterial therapy [S. aureus both in vitro and in a mouse bacteremia model [O-linked N-acetylglucosamine sugar modifications on cell wall teichoic acid residues of S. aureus and is responsible for opsonization of bacteria. Once the opsonized bacteria are taken up into phagolysosomes, proteases such as cathepsins cleave the linker and release the potent dmDNA31 antibiotic, which eradicates intracellular S. aureus [S. aureus bacteremia mice [S. aureus had negligible impact on plasma PK of TAb and ac-dmDNA31 over the efficacious dose range of 25–50 mg/kg, with mean clearance of 4.95 vs. 6.08 mL/day/kg, volume of distribution of 94.9 vs. 119 mL/day/kg, and half-life of 14.3 vs. 13.9 days for TAb in non-infected and infected mice, respectively [DSTA4637S is a THIOMAB™  antibody antibiotic conjugate (AAC) that is comprised of an anti-e linker . It has  therapy . DSTA463ia model . The ant. aureus . Total cmia mice . InfectiS. aureus pneumonia [Salvecin (AR-301) is a mAb developed as an adjunctive therapy to SoC antibiotics for ventilator-associated neumonia . It bindneumonia . In addineumonia . Staphylococcus Protein A (SpA), a key virulence determinant of S. aureus that is expressed in all clinical isolates [S. aureus cell wall envelope and is released during bacterial growth [S. aureus from antibody-dependent phagocytic killing [H3 domain [S. aureus surface and enhances opsonophagocytosis or other mechanisms of immune clearance of bacteria [S. aureus bacteremia, treatment with 40 mg/kg 514G3 led to 49% reduction in relative risk of overall incidence of serious adverse events (SAEs) and 56% relative risk reduction in S. aureus related SAEs compared to the placebo group [514G3 is a human mAb targeting isolates . SpA is l growth . SpA bin killing ,110. Add3 domain ,112. 514bacteria . In a pibo group . More imbo group . Pseudomonas aeruginosa [P. aeruginosa colonization/attachment to mammalian cells and formation of biofilms [P. aeruginosa clinical isolates express Psl (89.8–91.2%) and PcrV (87.7–90.2%), and at least one of the targets were identified in 97.3–100% of isolates [P. aeruginosa attachment to host cells and enhances opsonophagocytic killing of bacteria. Intravenous administration of MEDI3902 (5 or 15 mg/kg) at 24 h before or 1 h after lethal P. aeruginosa challenge conferred 100% survival and significant reductions on tissue bacterial burdens in acute pneumonia and bacteremia animal models including mice, New Zealand rabbits, and pigs [MEDI3902 is a bispecific mAb that targets both type III secretion injectisome PcrV anchored on bacterial cell wall and serotype-independent Psl exopolysaccharide of ruginosa . PcrV isbiofilms ,116. Theisolates . While band pigs ,117,118.P. aeruginosa lipopolysaccharide serotype O11, which accounts for ~20% of clinical isolates. Aerumab is being developed as an adjunctive immunotherapy to SoC antibiotics for ventilator associated pneumonia caused by P. aeruginosa [P. aeruginosa leads to enhanced bacterial clearance through either phagocytosis or complement-mediated bacterial killing [P. aeruginosa infection in patients did not affect the PK of Aerumab following IV doses of 1.2 mg/kg, where estimated clearance, volume of distribution, and half-life are 0.0579 L/h, 7.5 L, and 102 h, respectively [Aerumab (AR-101), previously known as panobacumab, is a human IgM mAb directed against the O-antigen of ruginosa ,120. Bin killing . P. aeruectively . Furtherectively ,123.P. aeruginosa clinical isolates. Aerucin is a second generation anti-P. aeruginosa mAb that binds to alginate  in greater than 90% of clinical isolates [P. aeruginosa, and binding of Aerucin is also expected to augment the opsonophagocytic killing and complement-dependent bactericidal activity against P. aeruginosa [As indicated above, Aerumab can only recognize ~20% of isolates . Aerucinruginosa . HoweverEscherichia coli (STEC) is the major cause of hemorrhagic colitis by infectious agents in the United States. A serious consequence of STEC infection is hemolytic uremic syndrome (HUS) that can lead to renal failure and death in 5–15% of infected children [Shiga toxin (Stx)-producing children . Treatmechildren . Shigamachildren . Treatmechildren . In addichildren . Shigamachildren . Pathogen-specific antibacterial mAbs have become an appealing therapeutic option due to recent advances in mAb production and engineering technologies. Antibodies kill bacteria or attenuate bacterial pathological activity via various mechanisms, including opsonophagocytosis, complement-mediated bactericidal activity, antibody-dependent cellular cytotoxicity, and neutralization of bacterial toxins. These pharmacodynamic mechanisms are distinct from those of small-molecule antimicrobials and therefore, such mAbs provide an attractive therapeutic option for antimicrobial resistant strains. The high specificity of mAbs may be expected to allow less disturbance to normal flora and less selective pressure for cross-resistance. Extended half-lives of mAbs may allow less frequent dosing and long-term prophylaxis. Antibacterial mAbs may also exhibit pharmacokinetic properties such as target-mediated drug disposition due to opsonophagocytosis or formation of antibody-toxin complexes, and there is some potential for complicated tissue distribution during the course of bacterial infection. These possible complexities require further study. Though there are only three mAbs marketed for prophylaxis or treatment of bacterial infection as of today, there is promise for a more prominent future role for antibacterial mAbs in view of their many advantages over traditional antimicrobials, and in view of the positive findings from clinical investigations of several mAbs in development.


Escherichia coli resistance using a nationwide database. Nationwide data on antimicrobial consumption for 12 years (2002 to 2013) were acquired from a database of subjects  included in the National Health Insurance Service-National Sample Cohort. National antimicrobial resistance rates of E. coli were obtained from the Korean Antimicrobial Resistance Monitoring System, which has been administered by the Korean Centers for Disease Control and Prevention since 2002. Fluoroquinolone-resistance rates of E. coli isolated from general hospitals have continuously increased since 2002 and were correlated with nationwide fluoroquinolone use  or ciprofloxacin use . Cefotaxime-resistance rates of E. coli isolated from general hospitals markedly increased since 2008 and were correlated with nationwide cefotaxime use  or third-generation cephalosporin use . Cefoxitin-resistance rates of E. coli isolated from general hospitals peaked in 2010 and significantly correlated with cephamycin use at a two-year interval . In conclusion, consumption of antimicrobials such as fluoroquinolone, cefoxitin, and cefotaxime is well correlated with the resistance rates of E. coli to these agents. This study provides background data for national antimicrobial management policies to reduce antimicrobial resistance.The purpose of this study is to determine the correlation between use of antimicrobials, such as fluoroquinolone, cefoxitin, and cefotaxime, and  Appropriate antimicrobial use is essential to control the development of microbial resistance. Higher rates of resistance have been reported in high antimicrobial consuming countries . The volBecause antimicrobial selective pressures differ among countries not only due to different resistance rates, but also because of discrepancies in medical systems, economic status, and sociological backgrounds, there is a need to investigate the correlation between antimicrobial use and resistance in each unique situation. Although associations between antimicrobial resistance and antimicrobial use according to geographic difference have been reported ,4, studiAntimicrobial resistance in South Korea is higher than in advanced regions such as the United States and Europe –11. In SEscherichia coli resistance rates. We focused on E. coli because the wide spread multidrug-resistant E. coli in community and healthcare facilities has become a serious situation in South Korea [In our 12-year ecological study using a nationwide surveillance and antimicrobial prescription database, we investigated the correlation between changes in use of third-generation cephalosporin, cephamycin, and fluoroquinolone and th Korea . We hopeThe study was approved by National Health Insurance Service Ilsan Hospital IRB (NHIMC 2017-03-027). All methods were performed in accordance with the relevant guidelines and regulations. This study used National Health Insurance Service-National Sample Cohort with permission (REQ0000007624).Antimicrobial use in South Korea for 12 years (2002 to 2013) was acquired from a database of subjects from the National Health Insurance Service-National Sample Cohort (NHIS-NSC), a population-based cohort established to provide public health researchers and policy makers with representative, useful information regarding citizens’ utilization of health insurance and health examinations . InformeAfter separation of dispensing and prescribing function policy has been compelled by law in 2000 and all antimicrobials should be used by prescriptions which is electronically transferred to national health insurance service and accumulated in systemic database in South Korea . TherefoThe data of antimicrobial resistance rates in South Korea were obtained from the Korean Antimicrobial Resistance Monitoring System , which hSAS software, version 9.2 was used for the statistical analysis . The correlations between antimicrobial use and resistance were evaluated by correlation analysis, and a p value less than 0.05 was regarded as significant.E. coli isolated from general hospitals has continuously increased from 30% in 2002 to 42.1% in 2013 and nationwide fluoroquinolone use increased from 1.445 DID in 2002 to 2.565 DID in 2013. Fluoroquinolone-resistance rates of E. coli correlated with nationwide total fluoroquinolone use  and ciprofloxacin use .Total fluoroquinolone prescriptions in South Korea increased from 1.445 DID in 2002 to 2.565 DID in 2013. Ciprofloxacin prescriptions in South Korea increased from 0.191 DID in 2002 to 0.675 DID in 2013. Fluoroquinolone-resistance rates of E. coli isolated from general hospitals has continuously increased from 10% in 2004 to 28.7% in 2013. Cefotaxime-resistance rate correlated with nationwide third-generation cephalosporin use  and cefotaxime use . Cefotaxime-resistance rate of E. coli also correlated with summation of nationwide third- and fourth-generation cephalosporin use .Total third-generation cephalosporin prescriptions in South Korea increased from 0.338 DID in 2002 to 1.457 DID in 2013. Cefotaxime prescriptions in South Korea increased from 0.020 DID in 2002 to 0.053 DID in 2013. Total fourth-generation cephalosporin prescriptions in South Korea increased from 0.002 DID in 2002 to 0.046 DID in 2013. Cefotaxime-resistance rate of E. coli isolated from general hospitals peaked in 2010. This did not correlate with nationwide cephamycin use , but significantly correlated with cephamycin use at a two-year interval . Cefoxitin-resistance rate of E. coli correlated with nationwide cefoxitin use .Total cephamycin use was 0.027 DID in 2002 and peaked at 0.071 DID in 2007. After 2007, cephamycin use decreased to 0.046 DID in 2013. Cefoxitin use was 0.003 DID in 2002 and increased to 0.010 DID in 2008. After 2008, cefoxitin use decreased to 0.008 DID in 2013. Cefoxitin-resistance rates of E. coli isolated from general hospitals have peaked in 2010 and decreased after that, which did not correlate with nationwide cephamycin use , but significantly correlated with cephamycin use at 2 year interval . Cefoxitin-resistance rates of E. coli correlated with nationwide cefoxitin use .Total cephamycin use was 0.027 DID in 2002, and peaked to 0.071 DID in 2007. After 2007, cephamycin use decreased to 0.046 DID in 2013. Cefoxitin use was 0.003 DID in 2002, and increased to 0.010 DID in 2008. After 2008, cefoxitin use decreased to 0.008 DID in 2013. Cefoxitin-resistance rates of E. coli resistance in a 12-year ecological study using nationwide surveillance and an antimicrobial prescription database.We identified significant correlations between third-generation cephalosporin, cephamycin, and fluoroquinolone use and E. coli regardless of healthcare-associated or community-associated infections [Previous antimicrobial exposure is a well-known risk factor for infection with ESBL- or AmpC-producing fections –21. Singfections ,23. Thisfections . The sprfections –30, and E. coli has been widespread in hospitals around the world since the late 1980s [H30Rx subsets within the ST131-O25-H30 subclone were associated specifically with fluoroquinolone resistance, and CTX-M-15 was widely detected in South Korea [E. coli resistance. E. coli is a major pathogen involved in urinary tract infection and shows good clinical outcomes [E. coli, have increased in South Korea, becoming a concerning problem [.Resistance to third-generation cephalosporins such as cefotaxime or ceftazidime via extended-spectrum β-lactamase (ESBL)-producing te 1980s , but a ste 1980s ,33. A reth Korea , the sproutcomes ,35. Howe problem ,36,37.E. coli clinical isolates, which shares ESBL and mutations within the fluoroquinolone resistance-determining regions of gyrA and parC [Klebsiella pneumoniae with transferrable pAmpC genes was first detected in South Korea in 1988 [E. coli with cefoxitin resistance has increased in South Korea [It is well-known that a single ST131 strain rapidly expanded and disseminated in most current fluoroquinolone-resistant and parC . Therefoand parC . Since K in 1988 , pAmpC βth Korea .E. coli isolated from general hospitals have markedly decreased since 2011 in South Korea, regardless of clinical breakpoint changes in CLSI guidelines 2010/2011 [E. coli to cefoxitin did not decrease until 2011. Cefoxitin-resistance rates of E. coli did not significantly correlate with nationwide cephamycin use , but showed significant correlation with nationwide cephamycin use  at a two-year interval.Cefoxitin-resistance rates of 010/2011 . In thisAlthough the time lag between antibiotic consumption and changes in resistance patterns on a national level is unknown, a period of one to two years has been suggested by other ecologic studies ,4,43. WeThe strong point of this study is analysis based on nationwide surveillance and an antimicrobial prescription database over a long period. Although associations between antimicrobial resistance and antimicrobial use according to geographic difference have been reported several times ,4, studiE. coli resistance to these agents were observed in this study. This provides background data for national antimicrobial management policies to reduce antimicrobial resistance.In conclusion, correlations between use of antimicrobials such as fluoroquinolone, cefoxitin, and cefotaxime and S1 Table(XLSX)Click here for additional data file.


Bacillus thuringiensis toxin gene is a potential environmental stressor. We compared the size of antennae, forewings, and tibia, as well as their FA values, in two parasitoids developed on two East African host species feeding on non-transgenic vs. transgenic maize. The two lepidopteran stem-borer hosts were the native Sesamia calamistis Hampson (Lepidoptera: Noctuidae) and a recent invader, Chilo partellus Swinhoe (Lepidoptera: Crambidae). The two braconid parasitoids were the native, gregarious larval endoparasitoid Cotesia sesamiae and the recently introduced Cotesia flavipes. Both parasitoids attacked both hosts, creating evolutionarily old vs. novel interactions. Transient feeding of hosts on transgenic maize had various effects on FA, depending on trait as well as the host and parasitoid species. These effects were usually stronger in evolutionarily novel host–parasitoid associations than in the older, native ones. These parameters have capacity to more sensitively indicate the effects of potential stressors and merit further consideration.Environmental stress can affect trait size and cause an increase in the fluctuating asymmetry (FA) of bilateral morphological traits in many animals. For insect parasitoids, feeding of hosts on transgenic maize, expressing a  Bacillus thuringiensis Berliner. One of the first such plants is maize, Zea mays L., expressing the (activated) Bt toxin [Ostrinia nubilalis Hubner , is difficult to control by conventional pesticides, agronomic measures, or biological control agents [The use of genetic transformation technology in crops allowed several crop plants to express genes that encode insecticidal proteins (delta endotoxins) from the bacterium Bt toxin , becausel agents . Bt maizl agents .Maize is an important staple crop in sub-Saharan Africa (SSA), and is much affected by insect pests there . In AfriThe procedure before a field release of transgenic crop requires an environmental risk assessment . Kenya iNatural pest control is one such ecosystem service  and therInsect-resistant transgenic plants may kill immature parasitoids indirectly by killing their host , or by rTests of the impact of transgenic plants on natural enemies usually measure life history variables such as growth, mortality, or development time. In a few experiments, behavioral  or physiMalacosoma disstria Hubner (Lepidoptera: Lasiocampidae) was shortened as the degree of FA of the first segment of foreleg tarsi increased [Generally, there is a negative correlation between the degree of FA and population fitness. For instance, the lifespan of ncreased . Furtherncreased ,37. In HEoreuma loftini Dyar (Lepidoptera: Crambidae) had significant negative effects on certain fitness parameters of the parasitoid Parallorhogas pyralophagus Marsh (Hymenoptera: Braconidae) [Spodoptera frugiperda (Lepidoptera: Noctuidae) on its braconid parasitoid Cotesia marginiventris Cresson (Hymenoptera: Braconidae) [Parasitoid size and fitness may be positively or negatively correlated to host condition, which, in turn, can be strongly dependent on the quantity and quality of food . Bt-intoconidae) , as did conidae) .In our laboratory experiments, we hypothesized that feeding on transgenic Bt maize would constitute a feeding stress for lepidopteran larvae, and their quality as hosts for the parasitoid would decrease. We expected that this would influence morphological parameters such as body size, as well as cause an increase in FA.We found that transient feeding on transgenic Bt maize had a negative effect on some body size parameters and, to a lesser degree, on FA in two hymenopteran parasitoids kept on two species of African stem borers, and these effects were stronger in the evolutionarily novel host–parasitoid associations than in the evolutionarily older, native ones.Plant material was obtained from Event 216 (Bt maize) (described in ). The isChilo partellus Swinhoe (Lepidoptera: Crambidae) and Sesamia calamistis Hampson (Lepidoptera: Noctuidae). Both are among the most damaging stem borer species in Kenya [C. partellus is an Asian species introduced to East Africa in the early 1930s [S. calamistis is a native stem borer species that occurs in all areas of East Africa up to 2400 m above sea level [Two species of African stem borers were used as hosts: in Kenya . C. partly 1930s , while SC. partellus and S. calamistis larvae used in this study were obtained as eggs from the insectaries at the International Centre of Insect Physiology and Ecology (ICIPE), Nairobi, Kenya and KARI, Katumani Centre, Machakos, Kenya. Both originated from colonies maintained on artificial diet of Ochieng et al. [The g et al. . The eggCotesia sesamiae Cameron (Hymenoptera: Braconidae) is an indigenous species that attacks mid- to late-instar stem borer larvae [Cotesia flavipes Cameron (Hymenoptera: Braconidae) is an introduced species, attacking both C. partellus and S. calamistis [C. sesamiae [Two larval parasitoids were selected as natural enemies. The gregarious larval endoparasitoid r larvae , and is r larvae . Cotesialamistis ,47. Thissesamiae . Parasitsesamiae .S. calamisits and Chilo partellus to mimic what would prevail in nature, where they would be exposed to different parasitoids as the stem borers occur in complexes with overlapping spatial and temporal distributions [3 Perspex cages and provided with a 20% honey solution; they were left to mate for 24 h prior to host exposure.The parasitoids used in this study originated from a laboratory-reared population maintained at the International Centre for Insect Physiology and Ecology, Nairobi, Kenya. These colonies were periodically “refreshed” by introducing field collected adults to maintain genetic diversity in the rearing. The parasitoids were reared on a combination of ibutions . Parasitibutions . Newly eC. partellus and S. calamistis, respectively [Fourth-instar larvae of each of the two stem borer species were subjected to transient feeding on Bt maize for 24 h.To enhance feeding on Bt maize (toxin ingestion), the larvae were starved for 24 h prior to exposure to the treatments. The larvae were transferred individually onto pieces of plant material in moist filter paper. After 24 h, the larvae were held individually using forceps and exposed to 24 h-old mated female parasitoids in Perspex cages. The parasitoids readily attacked the host and usually oviposited in a few seconds, after which the host larvae were individually placed on diet containing non-transgenic plant material . This trectively . Each paParasitized larvae were reared in Petri dishes on pieces of fresh, non-transgenic control maize stems (which were changed every 3–4 days) and inspected daily until parasitoid cocoon production. Emerging cocoons were collected separately from each larva, and incubated in the laboratory in transparent glass vials stopped with cotton wool.Twenty female wasps were randomly selected from each treatment and measured using Leica Application Suite Educational Zoom  software. Three traits were measured (±0.01 µm) three times on both sides: hind tibia length (measured from the femur/tibia joint to the tibia/tarsus joint), wing length, and antennal length. These traits are good indicators of the field quality of hymenopteran wasps .The FA value was calculated using the formula : FA=meanC. flavipes and C. sesamiae among the larvae subjected to transient feeding on Bt maize and those reared exclusively on non-Bt maize. When ANOVA indicated significant (p < 0.05) differences, Student–Newman–Keuls (SNK) tests were used to identify treatments that were different.Analysis of variance was used to compare FA of Additionally, in an approach akin to that adopted by Lövei et al. , we asseF = 49.3, d.f. = 1.38, p = 0.0005 for C. flavipes on C. partellus; F = 5.7, d.f. = 1.38, p = 0.022 for C. sesamiae on C. partellus; F = 13.6, d.f. = 1.38, p = 0.001 for C. flavipes on S. calamistis). The reduction in wing length in C. sesamiae developed in S. calamistis was non-significant . Conversely, antennal length was significantly increased in C. sesamiae developing on Bt-fed S. calamistis hosts . For the two “evolutionarily novel host-parasitoid combinations” , there was a non-significant decrease.Only two of the host–parasitoid–host plant combinations caused significant changes in antennal length: C. flavipes developing on Bt-fed S. calamistis  and in C. sesamiae developing on Bt-fed C. partellus  FA values compared to those developing on non-Bt maize-fed hosts  compared to those developing on the non-transgenic control. Conversely, when the same parasitoid was developing on its Bt-fed original host, S. calamistis, the adult parasitoids had significantly lower FA values  compared to the non-transgenic control  has more success on its host Helicoverpa armigera Hubner (Lepidoptera: Noctuidae), fed on less toxic Bt-amended diets [Tranosema rostrale Brishke (Hymenoptera: Ichneumonidae) developing on the Bt-fed spruce budworm Choristoneura fumiferana Clemens (Lepidoptera: Tortricidae) [Wing length in all host–parasitoid combinations was reduced following transient host feeding on Bt maize. The reduction was not significant for s occurs ,63 but ss occurs , which cs occurs ,66,67. Ts is 55% . The obssesamiae  were atted diets , as doesricidae) .Venturia canescens Gravenhorst (Hymenoptera: Ichneumonidae), when developing in Bt fed Plodia interpunctella Hubner , also develops shorter wings [C. flavipes developing on S. calamistis was most affected: both wing length and its FA were significantly and negatively affected when the parasitoid was on Bt-fed hosts. S. calamistis is not a very good host for C. flavipes [The shorter parasitoid wings in the other host–parasitoid combinations were possibly due to the novelty of these associations. The ichneumonid er wings . C. flavflavipes ,74 and, C. flavipes developing on S. calamistis and C. sesamiae developing on C. partellus. The lack of significant effects on tibial length, as well as FA in C. sesamiae developing on S. calamistis, could have resulted from the weakening of the host’s defenses by the Bt toxin. As S. calamistis is not a very suitable host for C. flavipes, the additional stressor, whether in the form of the Bt toxin or biochemical changes in the host, could contribute to the negative impact. The changes in FA for tibia were not significant in any host–parasitoid combinations, indicating that hind tibia symmetry is either unaffected or not sensitive to stress factors emerging from hosts feeding on Bt maize.Hind tibia lengths in parasitoids were reduced following host exposure to Bt maize, with the reduction being significant for the evolutionarily novel combinations: C. sesamiae developing on S. calamistis larvae (possibly due to the reasons mentioned earlier). In the C. flavipes/C. partellus combination, it was significantly reduced, possibly due to the evolutionarily novel host–parasitoid association.Antennal length was significantly increased in The lack of consistency in FA responses across traits may reflect the variation between trait types in their susceptibility to environmental stress . In mostBy studying evolutionarily “old” vs. “novel” host–parasitoid associations, we found that parasitoids in novel associations were more sensitive to host quality, lowered by the Bt treatment, and can be negatively affected. These results underline the importance of considering multiple factors when assessing the impact of genetically modified plants on ecosystem service providers . The parFactors that negatively impact on fitness of such biocontrol agents could result in a lower level of ecosystem services. The effect on these ecological processes, which operate on vast scales and from which we derive substantial benefits, should be evaluated when harmonization of the GM technology and biological control is sought .


Ostrya (Betulaceae) contains eight species and four of them are distributed in China. However, studies based on limited informative sites of several chloroplast markers failed to resolve interspecific delimitation and relationships among the four Chinese species. In this study, we aimed to use the whole chloroplast genomes to address these two issues.The genus We assembled and annotated 33 complete chloroplast genomes (plastomes) of the four Chinese species, representing 17 populations across most of their geographical distributions. Each species contained samples of several individuals that cover most of geographic distributions of the species. All plastomes are highly conserved in genome structure and gene order, with a total length of 158–159 kb and 122 genes. Phylogenetic analyses of whole plastomes, non-coding regions and protein-coding genes produced almost the same topological relationships. In contrast to the well-delimitated species boundary inferred from the nuclear ITS sequence variations, three of the four species are non-monophyletic in the plastome trees, which is consistent with previous studies based on a few chloroplast markers.The high incongruence between the ITS and plastome trees may suggest the widespread occurrences of hybrid introgression and incomplete lineage sorting during the divergence of these species. In addition, the plastomes with more informative sites compared with a few chloroplast markers still failed to resolve the phylogenetic relationships of the four species, and further studies involving population genomic data may be needed to better understand their evolutionary histories.The online version of this article (10.1186/s12863-019-0733-0) contains supplementary material, which is available to authorized users. Ostrya is a small genus belongs to the birch family Betulaceae and merely consists of eight species native in southern Europe, southwest and eastern Asia, and North and Central America [Ostrya woods have been used for various purposes such as furniture, axles, fuel wood and charcoal [http://www.efloras.org) until Lu et al. (2016) [Ostrya yunnanensis was nested within O. multinervis in both phylogenetic trees and morphological clustering. The two species were therefore recognized as the same species and renamed as O. chinensis [O. japonica is mainly distributed in China, Japan and Korea, while the remaining three species, O. rehderiana, O. chinensis and O. trichocarpa, are endemic to China. The amount of these wild plants has been decreased rapidly due to overexploitation, habitat destruction and ecosystem deterioration. The famous endangered species, O. rehderiana, has been reported that only five mature trees survive and are conserved in the Tianmu Mountain, Zhejiang [Ostrya species own excellent wood and are regarded as ecologically and economically valuable foundation for future forests, the interspecific delimitation and relationships remain unresolved. Previous study failed to discern the four Ostrya species based on four chloroplast (cp) fragments  intron, trnH–psbA, and trnL–trnF) while the ITS sequence variations did [Ostrya species based on plastomes. We selected multiple individuals per species to cover the most of geographic distributions of the species to reduce the sampling bias. We also extracted three datasets, i.e., the whole plastomes, non-coding regions and protein-coding genes, for phylogenetic analyses and compared these plastome trees with the ITS tree. Specifically, the aims of our study are to: 1) investigate whether the three plastome datasets result in consistent phylogenies and whether they can discern the four species and 2) test if phylogeny based on plastome datasets congruent with the ITS tree. America –3. Thesecharcoal , 4. It h. 2016)  found th  found tZhejiang . Althougions did . It has ions did –10. By cions did –16. And ions did , 17, 18.ions did , 19. TheOstrya species that occur in China. For each species, we selected samples that cover most of its geographic distribution  to generate the fastq format files from the bam file. Then, we assembled the plastomes using Velvet v1.2.10 [https://sourceforge.net/projects/soapdenovo2/files/GapCloser/). Moreover, we used Plann v.1.1.2 [O. rehderiana as a reference. Then we corrected the start codons, stop codons and intron/exon boundaries manually using Sequin v.15.10 (http://www.ncbi.nlm.nih.gov/Sequin/) as well as a visual software Geneious v.R.8.1.4 [http://ogdraw.mpimp-golm.mpg.de/) [Raw reads of all samples were trimmed with a filter standard (Q < = 5 or N base content > 10%). Afterwards, we used Trimmomatic  to furth v1.2.10  and fill v.1.1.2  to annot.R.8.1.4 . Ultimatmpg.de/) .Ostrya species: dispersed, palindromic and tandem repeat. The former two types were visualized and located by an online program REPuter [In order to show interspecific variations, the alignments of all plastomes were plotted using the mVISTA program with LAGAN mode . All val REPuter  with a m REPuter . Tandem  REPuter  was usedCarpinus could not be separated well from Ostrya and there were no published Ostryopsis plastome sequences available in GenBank. We therefore downloaded a plastome of Corylus chinensis (GenBank accession number: NC_032351.1) from GenBank as outgroup to reconstruct the phylogenetic tree of the four Ostrya species. In order to test whether different plastome regions resulted in consistent phylogenies during the phylogenetic reconstructions, we used three plastome datasets: (1) the whole plastomes, (2) the non-coding regions and (3) the protein-coding genes (PCGs). In addition, we downloaded from GenBank the same ITS dataset as in Lu et al. (2016) [Ostrya plastomes and Corylus chinensis and translated their nucleotide sequences into amino acid (aa) sequences for phylogenetic analysis in order to reduce the tree artifacts due to high DNA divergence. Multiple alignment of aa sequence of each gene was achieved by T-coffee [According to Grimm & Renner (2013) , the mos. (2016)  and trim. (2016) . The latT-coffee  to do auT-coffee  was usedPhylogenetic analyses were performed by both maximum likelihood (ML) and Bayesian inference (BI) methods. For whole plastomes, non-coding regions and ITS datasets, GTR + I + G as the best-fit model selected by JModeltest v.2.1.1  was usedOstrya species using the web tool GGDC [http://ggdc.dsmz.de/distcalc2.php) to further compare the inter- and intra-specific variation. As individuals from the same population were grouped together in the phylogenetic trees (see Results), only one individual from each population of the four species was selected for this analysis.The node supports were determined with 1000 bootstrap replicates in ML analyses. For BI analysis, two independent parallel runs and four chains (one cold and three hot) were running for 10,000,000 generations with trees sampled every 500 generations. Then we determined convergence by examining trace plots of the log likelihood values for each parameter in Tracer . In addiool GGDC   divided by a large single-copy region  and a small single-copy region  , and 6 PCG species  contained a single intron, and three genes  had two introns. Intergenic spacers showed moderate genetic divergences. The intergenic spacers of trnQ-psbK, trnS-trnR and psbZ-trnG were identified as the most divergence hotspots  and one nuclear ITS sequences were used in this study for phylogenetic analyses. The whole plastome dataset comprised 157,116 bp, 2209 of which were variable and 456 were parsimony-informative. The non-coding dataset comprised 64,479 bp, 1332 of which were variable and 244 were parsimony-informative. 76 PCGs shared by all O. rehderiana that collected from the same population, the other three species were all non-monophyletic in the plastome trees  formed another two subclades, respectively. Moreover, some samples that were collected from adjacent regions regardless of species may be clustered together in the plastome trees. For example, population Otr1 of O. trichocarpa was geographically close to populations Och2 and Och3 of O. chinensis Additional file 2:Table S2. 33 complete chloroplast genomes of four Ostrya species from GenBank. (DOCX 17 kb)Additional file 3:Table S3. Best-fit models for each of the amino acid sequence of PCGs. (DOCX 27 kb)Additional file 4:Table S4. Gene list of plastomes of four Ostrya species. (DOCX 21 kb)Additional file 5:Table S5. Repeats distribution in Ostrya chloroplast genome. (XLSX 17 kb): Additional file 6:Figure S1. Sample information of Ostrya populations. Different dot color indicates the different taxa. Green: O. rehderiana. Orange: O. japonica. Red: O. chinensis. Blue: O. trichocarpa. The permission of the graph is not required. (PDF 9547 kb)Additional file 7:Figure S2. Heatmap of plastome distances among 17 Ostrya populations. For populations that have multiple samples, we only select one sample in this analysis. (PDF 375 kb)


Natural halogenated compounds  are produced mainly by marine organisms, including marine Actinobacteria. Many commercially important compounds for pharmaceuticals contain halogen, and the halogen is responsible for the physical and chemical properties as well as bioactivities and toxicities. In the exploration of marine environment that is supported by advanced structure elucidation, varied panel bioassays and high-throughput screening have accelerated number of halometabolites isolated from marine Actinobacteria to date. The metabolites exhibited unique structures and promising bioactivities. This review focuses on the chemodiversity and bioactivities of marine halometabolites from marine Actinobacteria reported in the last 15 years (2003–2018). Halometabolites are a group of compounds contain halogen substituents . To date, there are more than 5000 halogenated compounds with high degree of structural variability containing a single or several halogen atoms . HalogenHalometabolites in nature have several functions in physiological, biochemical, or defensive role for their host including communication (quorum sensing) and production of growth hormones, sex pheromone, toxin, or antibiotics. The role of substituent halogen in organic compounds is related to the bioactivity, bioavailability, and stability of the compounds. Streptomyces griseus. A number of drugs derived from Actinobacteria such as antibiotic and anticancer are on the market today. Chlorinated antibiotics such as chloramphenicol and vancomycin played important roles for the eradication of infectious diseases in human. Chloramphenicol is a broad-spectrum antibiotic used to treat bacterial infections. Chloramphenicol antibiotic is on the WHO  list of essential medicine. Chlortetracycline is a member of the tetracycline family and produced by Streptomyces aureofaciens. Chlortetracycline was used clinically in 1948 and is used to prevent, control, and treat animal health problems and increase growth rate in chickens, turkeys, ducks, swine, calves, beef cattle, and others. Calicheamicin is a group of enediyne metabolite with iodine and has remarkable activity as anticancer produced by Micromonospora echinospora. Linking calicheamicin to monoclonal antibody is used for therapy of acute myeloid leukemia. Rebeccamycin is produced by Streptomyces sp. and is a derivate of staurosporine with chlor attached to it. Rebeccamycin showed in vitro antitumor activity at Inhibitory Concentration (IC50) 480 nM against P388 leukemia cells. Complestatin is a cyclic halogenated peptide produced by Streptomyces lavendulae anti and exhibited activity as HIV-1 integrase inhibitor. Vancomycin is a halogenated glycopeptide active against Staphylococcus aureus (including methicillin-resistant strains), S. epidermidis (including multiple-resistant strains), Streptococcus pneumoniae (including multiple-resistant strains), S. pyogenes, S. agalactiae, S. bovis, S. mutans, Clostridium spp., Listeria monocytogenes, Actinomyces spp., and Lactobacillus spp. spiro-ring showed moderate activity against HepG2, B16, and H460  indene ring. In the bioassay against resistant strain, cyanosporoside A was inactive as antibacterial agent against MRSA, VREF , and amphotericin-resistant Candida albicans (ARCA). The activity against HCT-116 was weak at IC50 30 μg/mL [Salinispora pacifica CNS-143 and Streptomyces sp. CNT-179 [Two novel compounds cyanosporaside A and B  were iso CNT-179 .Streptomyces CNH-990. The compounds have no significant activity as antibiotic against MRSA and VREF and antifungal against ARCA. Marmycin A  was less potent at IC50 1.09 μM [Marmycins, an angucycline class of compounds, were isolated from marine Actinomycetes belonging to rmycin A  showed a 1.09 μM . This faNocardiopsis isolated from sediment near Beqa Island in Beqa Lagoon, Fiji, produce Fijiolide A and B . Profiling chemistry of fermentation broth yielded identification of fijiolide A and B along with articoside, C-1027-chromophore V, and C-1027-chromophore-III. The compounds were tested for bioactivity against Candida albicans and antiproliferative activities against human carcinoma cell lines. C-1027-chromophore V and C-1027-chromophore-III showed bioactivity against C. albicans at IC50 37.9 μM and 25.6 μM, respectively, but fijiolides showed no activity. The difference in the benzoxazine that counts for the activity. Antiproliferative activities of compounds were ranged from moderate to strong against cancer cell lines HCT-116, A549, SNU638, SK-HEP1, K562, and MDA-MB231 at IC50 0.6–44 μM [Marine-derived Actinobacteria genus  A and B . Fijioliactivity . Arctic Streptomyces sp. OUCMDZ-1703 associated with unidentified soft coral. Both compounds have modest antimicrobial activity against P. aeruginosa, E. coli, and S. aureus but no activity against MRSA. Streptochloride A and B demonstrated cytotoxicity against MCF-7 cell line at IC50 9.9 and 20.2 μmol/L, respectively [Chlorinated polyketide compound streptochlorides  were isoectively .Marine Actinobacteria have shown as producer of varied diversity of halometabolites compounds. The compounds range from simple to complicated structures with group under polyketides, peptides, alkaloid, and terpenoid. Some compounds demonstrate intriguing structure special for marine compounds. The compounds exhibited enormous potential for the discovery of new therapeutic leads in the development of drugs to fight the current antibiotic resistance threats, anticancer, and other bioactivities. Marine Actinobacteria produce more chlorometabolites than bromometabolites in contrast with sponges and red algae which are rich in bromometabolites.2-dependent halogenase is the biggest group of halogenating enzymes, thus can be used as target in the bioprospecting of halometabolites from marine Actinobacteria. Genome mining by employing gene that encodes FADH2-dependent halogenase as an indicator has enabled to screen 555 genetic potentials of actinomycetes for halogenated natural products [2-dependent halogenase resulted in 26 halogenase-positive strain among 163 isolates [2-dependent halogenase gene of Arctic marine Actinobacteria. The study concluded that Arctic marine Actinobacteria are potential in halometabolites production [2-dependent halogenase gene in filamentous actinomycetes and the potential for producing halometabolites.To date, there are several bioprospecting programs with target marine biodiversity for novel bioactive metabolites including halometabolites. FADHproducts . Gao andproducts . Screeniisolates . PCR-basoduction . Three noduction . There iComparative genome studies showed that Actinobacteria are rich in secondary metabolites genes that never been explored, so the chance to discover new bioactive halometabolites is still wide open. Further research into mechanisms of biological halogenation will provide insight and a greater understanding of biosynthesis of halometabolites. Furthermore, understanding the genes encoding halogenase enzymes may be used to generate recombinant organisms to produce derivative new natural product. Advance technology in exploration and collection, compound isolation, purification, structure elucidation, bioassay, and high-throughput screening will ensure and enable to identify potential halometabolites from marine Actinobacteria for benefit to humankind.


Aspergillus oryzae is an industrially useful species, of which various strains have been identified; however, their genetic relationships remain unclear. A. oryzae was previously thought to be asexual and unable to undergo crossbreeding. However, recent studies revealed the sexual reproduction of Aspergillus flavus, a species closely related to A. oryzae. To investigate potential sexual reproduction in A. oryzae and evolutionary history among A. oryzae and A. flavus strains, we assembled 82 draft genomes of A. oryzae strains used practically. The phylogenetic tree of concatenated genes confirmed that A. oryzae was monophyletic and nested in one of the clades of A. flavus but formed several clades with different genomic structures. Our results suggest that A. oryzae strains have undergone multiple inter-genomic recombination events between A. oryzae ancestors, although sexual recombination among domesticated species did not appear to have occurred during the domestication process, at least in the past few decades. Through inter- and intra-cladal comparative analysis, we found that evolutionary pressure induced by the domestication of A. oryzae appears to selectively cause non-synonymous and gap mutations in genes involved in fermentation characteristics, as well as intra-genomic rearrangements, with the conservation of industrially useful catalytic enzyme-encoding genes. These strains show diversity in colour and fermentation function and are managed for different applications such as in sake, miso (soybean paste), and shoyu (soy sauce). However, the relationship between the diversity of A. oryzae species and genetic factors remains unclear.A. oryzae RIB40, a wild-type strain, was sequenced.Aspergillus nidulans and Aspergillus fumigatus revealed that the A. oryzae genome was 7–9 Mb larger.,In 2005, the whole genome of Aspergillus flavus and A. oryzae are genetically very closely related, with their genomes showing 99.5% similarity in coding regions,Aspergillus flavus is an important species linked to food safety, as some strains produce fungal toxins, particularly aflatoxin,,A. oryzae based on morphological differences and toxicity.,A. oryzae strains contain all or parts of the aflatoxin biosynthetic gene cluster, although they are non-aflatoxigenic.A. oryzae can be detoxified and differentiated from A. flavus by domestication.,,,A. oryzae was shown to form a monophyletic clade derived from one clade of A. flavus.Some researchers suggested that Aspergillus oryzae and A. flavus have long been considered asexual species with no sexual reproduction cycle.A. flavus revealed that sexual reproduction occurs in laboratory and field environments.,,A. oryzae and A. flavus possess one mating type (MAT type) locus in the genome, at which either MAT1-1 or MAT1-2 is encoded.,A. oryzae. Breeding currently carried out by tane-koji manufacturers utilizes a single strain with mutations or recombination, but crossbreeding has not been successful. Genome analysis suggested that recombination occurred between the ancestors of A. oryzae based on the linkage disequilibrium between MAT types and the phylogeny of a single gene.A. oryzae isolates, we acquired 82 industrial strains from five independent Japanese tane-koji manufacturers in different locations and conducted whole-genome sequencing to determine their draft genomes. For the classification of these strains, we performed orthologue clustering of predicted genes from each genome, phylogenetic tree inference of the chromosomal genome, and chromosome recombination analysis. Through these analyses, we hypothesized that A. oryzae strains have undergone multiple inter-genomic recombination events between A. oryzae ancestors, and that evolutionary pressure by A. oryzae domestication is extremely limited to intra-genomic mutations and rearrangements. Moreover, we identified genes that are mutated/duplicated/deleted within clades, which might reflect the fact that Japanese tane-koji manufacturers have passaged their strains to prevent changes in industrially useful traits in parallel with breeding.In this study, to uncover genomic diversity and evolutionary relationships among 2.A full description of the methods, including software versions and parameters, is available in A. oryzae and three Aspergillus sojae (as an out group) industrially used strains were collected from five independent tane-koji manufacturers in Japan  and 550 bp (for run no. 2–5) on average and sequenced on an Illumina HiSeq2500 system using 150 bp (for run no. 1) and 250 bp (for run no. 2–5) paired-end runs. Quality filtering and assembly of the paired-end reads were performed with Platanus.Aspergillus strains were used  of the newly sequenced or NCBI GenBank ere used . Gene coAlignments of degapped gene sequences (DGSs) of single-copy OGs (SCGs), which were common to 152 protein sets, were generated with MAFFT3.A. oryzae and 45–71 and 40.0–40.1 Mb for A. sojae, respectively  used to produce yatalase was contaminated.The number of scaffolds  and total lengths were 36–256 and 35.9–38.66 Mb for ectively . The twoA. oryzae and A. sojae were 11, 196–11, 716 and 13, 309–13, 317, respectively (Gene prediction for our sample showed that the number of predicted genes of A. oryzae RIB40 reference proteins (s01-m08-r29 or s01-m09-r06).By orthologue clustering, 15,614 OGs were created from 1,882,788 proteins, while 1,395 proteins became unclustered. Of these, 3,951 were common SCGs. We succeeded in the annotation of the KEGG KO and protein domains/motifs at 28.6%  and 88.0%  , and assA. oryzae was monophyletic and nested in a clade of A. flavus  were nested in the A. oryzae clade. However, WRRL1519,A. oryzae-type aflatoxin gene clusters . Thus, considering the location in the phylogenetic tree and toxigenicity, these strains were reclassified as A. oryzae.The length of the sequence of the concatenated DGSs of 4,361 SCGs was 5,677,852 columns (bp), corresponding to approximately 15% of the total genome length. We confirmed that . flavus . This isA. oryzae formed several clades, showing an intra-cladal DGS dissimilarity within 0.01%. For further analysis, we defined clades A–H to which Japanese industrial strains of A. oryzae belong. Although koji manufacturers do not share their strains, interestingly, many strains clustered in the same clade. Particularly, clade E included several strains provided by four different tane-koji manufacturers, one strain collected in Thailand (BCC7051),Industrial strains of A. oryzae strains were not clustered based on their industrial uses. However, all strains in clades D, E, and H (and a single strain TK-29) were those used in shoyu production. Strains used for other purposes such as in miso, sake, sake/miso, mirin, or other products were mixed in the same clade, which is consistent with classification by appearance and enzymatic activity.Industrial Of the 152 genomic sequences, MAT regions were uniquely detected as either MAT1-1 or MAT1-2 from 146 sequences but not from six downloaded sequences . We examytk6/OG0002894/AO090023000584 to that of mdlB/OG0004275/AO090701000644, the combinations of clades with the same sequence were different  shared 46% of the exact same gene sequences with clade B, but the homologous genes were distributed in a mosaic manner  began in China over 3,000–2,000 years ago, and stocking and selling of these products began in Japan 700–500 years ago.A. oryzae in the last 3,000 or 700 years after clade divergence  divergence in vergence .A. flavus and A. oryzaeIn a previous study, mutational pressure was estimated by comparing the SNP frequencies of A. oryzae and much higher than those of intra-species mutations in A. flavus, with a similar tendency for gap/synonymous mutations , suggesting that selection pressure for gene duplication is not explained by gene function. The estimated copy numbers of OG0000041/PF03221/Tc5 transposase and OG0000321/PF14529/endonuclease-reverse transcriptase, which are transposon-derived genes, had changed more than twice as compared with that in RIB40; in particular, the clade B and G strains showed 15–20 times the estimated copy number. Furthermore, rRNA genes exhibited a wide range in estimated copy number . However, the selective pressure on rRNA gene duplication is hard to estimate, because a previous report showed strain-dependent copy number variation in rRNA genes in A. fumigatus by quantitative PCR,We estimated gene duplication/deletion by direct read mapping and calculation of the normalized depth, equal to the copy number of genes . As a reAs a general trend, changes in the sequence depth were more frequent in the sub-telomeric region . Change amyA/amyB/amyC/OG0011956) have been detected in RIB40.A. sojae strains TK-83, TK-84, and TK-85 had one copy, which is consistent with previous studies.,Three copies of α-amylase  and strains estimated to be A. oryzae in this study, were classified into three groups as defined in a previous study.A. oryzae/A. flavus had occurred in parallel. This is consistent with a previous report inferring that the selective pressure against toxins was lost in nature.A. oryzae is phylogenetically guaranteed, tane-koji manufacturers might have distinguished A. oryzae from A. flavus based on their growth ability on rice and incidentally selected aflatoxicity.The types of aflatoxin biosynthetic gene clusters in tane-koji manufacturers and conducted whole-genome sequencing to determine their draft genomes. Through phylogenetic tree-based inferences of the chromosomal genome and chromosome recombination analysis, we showed that A. oryzae strains have undergone multiple inter-genomic recombination events between A. oryzae ancestors. However, sexual recombination among domesticated species did not appear to have occurred during the domestication process, at least in the past few decades; therefore, we hypothesized that evolutionary pressure introduced by the domestication of A. oryzae is extremely limited to intra-genomic mutation and rearrangements. Through intra- and inter-cladal comparative analysis, we showed that the evolutionary pressure of domestication selectively caused non-synonymous and gap mutations and intra-genomic recombination. Our results suggest that the domestication process might have contributed to altering strain traits while maintaining the activity of industrially useful catalytic enzyme genes by applying selective pressure to peripheral genes involved in fermentation rather than the enzyme-encoding genes themselves.We acquired 82 industrial strains from five Japanese A. oryzae, and importantly, the whole genomic data and phylogenetic tree will help to develop breeding methods based on sexual reproduction using industrial strains.Our study provides suggestions on the relationship between the evolution and domestication of dsz024_Supplementary_DataClick here for additional data file.


Many older Veterans are physically debilitated, with a reduced quality of life (QOL.) Use of assistive devices during ambulation is indicative of fall risk. We describe two case studies following six-months of participation in Gerofit, a structured exercise program. Ms. M., a 75 y/o Veteran with chronic PTSD and joint pain could walk 237 yards in six-minutes using a cane. Mr. D., an 86 y/o Veteran, came to Gerofit using a Front Wheeled Walker and could walk 307 yards in 6-minutes. After six-months of Gerofit, neither Veteran used assistive devices during testing, and both reported higher QOL. Ms. M. now walks 514 yards in six minutes, while Mr. D. can walk 375 yards in six minutes. Both Veterans exhibit improved balance and endurance. They tell us: “Gerofit gave me my life back!” and “Gerofit gets me out of the house and off the couch.”