Protein tyrosine phosphorylation is crucial in many cellular regulatory mechanisms. However, regulatory mechanisms of plant protein tyrosine phosphatases (PTPs) in controlling responses to abiotic stress remain limited. We report here the identification of a Tyrosine (Tyr)-specific phosphatase, PdPTP1, from NE19 (Populus nigra × (P. deltoides × P. nigra). Transcript levels of PdPTP1 were upregulated significantly by NaCl treatment and oxidative stress. PdPTP1 was found both in the nucleus and cytoplasm. Under NaCl treatment, transgenic plants overexpressing PdPTP1 (OxPdPTP1) accumulated more Na+ and less K+. In addition, OxPdPTP1 poplars accumulated more H2O2 and O2·−, which is consistent with the downregulation of enzymatic ROS-scavengers activity. Furthermore, PdPTP1 interacted with PdMAPK3/6 in vivo and in vitro. In conclusion, our findings demonstrate that PdPTP1 functions as a negative regulator of salt tolerance via a mechanism of affecting Na+/K+ and ROS homeostasis.High concentrations of Na Soil salinity is one of the major abiotic stresses that imposes severe threats to the growth and yield of diverse plants. More than 6% of the world’s total land area suffers from overabundance of salt that impedes plant absorption of water and nutrients from the soil . Salt st2+ concentrations, which is perceived by a calcium-binding protein, SOS3. SOS3 physically translates to Ser/Thr protein kinase SOS2. The SOS2–SOS3 complex phosphorylate and activate Na+/H+ exchanger SOS1 located in the plasma membrane, finally transporting Na+ out of the cytosol [+ often leads to K+ deficiency under salt stress [+ concentrations is crucial for plant salinity tolerance [+/Na+ is an important mechanism used by plants to adapt to salt stress. Genetic analyses have indicated that the salt overly sensitive (SOS) pathway is crucial in regulating ionic homeostasis and helps plants adapt to salt stress. In this pathway, exposure to NaCl would elicit a transit increase in cytosolic Ca cytosol . In glyct stress . As a maolerance . Therefo2·−), hydroxyl radicals (·OH), and hydrogen peroxide (H2O2). Low ROS concentrations have been considered to be a signal that activates specific downstream responses, while high ROS concentrations can have a detrimental effect on DNA, proteins, lipids, and carbohydrates [Salt-induced secondary oxidative stresses generate ROS, including superoxide anion radical  [4A (HSFA4A) and thereby displayed increased salt resistance [Mitogen activated protein kinase (MAPK) cascades participate in ionic, osmotic, and oxidative stress signaling in plants . AtMPK6 mulation . MKK4-MPype (WT) . MPK3/6 sistance . Arabidopsis and other species [Arabidopsis, Chlamydomonas reinhardtii, and Ostreococcus tauri [Tyr phosphorylation plays a crucial role in animal cells, as it regulates many cellular processes, including division, growth, and differentiation. Tyr phosphorylation in plants, however, was ignored originally due to the lack of a typical tyrosine kinase gene . Even th species ,15. The  species . Notablyus tauri ,18, suggArabidopsis, the expression of AtPTP1, a Tyr specific PTP, was found to be upregulated by salt, and its involvement in oxidative stress signaling was suggested [Arabidopsis MAPK phosphatase 1 (MKP1), a DsPTP, participates in salt-stress response, which can be indicated by increased resistance of mkp1 mutant plants to salinity [mkp1 mutant is also hypersensitive to UV radiation [Commelina communis by reopening them [Studies have shown that PTPs are involved in abiotic stress responses. In uggested ,19. Arabsalinity . Furtheradiation . Moreoveing them . These sArabidopsis identified MKP1 essential for UV resistance can interact with stress-activated MAPK3, MAPK4, and MAPK6 [mkp1 is strongly enhanced by the ptp1 null mutation, which could be genetically attributed to increased levels of active MPK3 and MPK6 [PTPs are known to be dedicated MAPK phosphatases (MKPs) . Both PTnd MAPK6 . The conand MPK6 . Taken tPdPTP1 reduced salinity tolerance in poplar, as a consequence of impairing re-establishment of cellular ionic and ROS homeostasis. Furthermore, we also provided evidence that PdPTP1 interacts with the stress-responsive kinase PdMAPK3/6. This study provides important new information for engineering salt-tolerant poplar.In the poplar genome, 38 PTPs superfamily members had been identified by using the complete human genome as reference. Like most of the other plant species, there is also only one Tyr specific phosphatase in poplar . HoweverP. trichocarpa PtPTP1 gene, the PdPTP1 gene in NE19 was isolated by using the polymerase chain reaction (PCR) approach, using the specific primers  AtPTP1, soybean  GmPTP1, and pea  PsPTP1, suggesting that PdPTP1 may be a member of Tyr-specific PTPs. The phylogenetic tree of PdPTP1 protein and other PTPs from eukaryotes was constructed (Based on the CDS sequences of the  primers . The fulstructed . Furtherstructed . Moreovestructed B. These GFP)-PdPTP1 fusion protein, and then the transformed protoplasts, tobacco, and transgenic poplar leaves were observed by laser confocal fluorescence microscopy. The green fluorescence protein was observed in both the cytosol and nucleus  was produced by introducing the construct pSuper:PdPTP1:GFP into poplar 84K. Putative transgenic plants were selected for confirming the integration of transgene by PCR analysis, using the primers of the hygromycin phosphotransferase gene (Hyg)  were selected for further functional analysis.In order to investigate the function of PdPTP1 proteins, transgenic poplar overexpressing ne (Hyg) . Furtheretection . Three tPdPTP1 was a salt induced gene, it is probable that PdPTP1 functions in a plant’s response to salt. To confirm this assumption, we next treated WT and OxPdPTP1 poplars with 350 mM of NaCl. After five days, OxPdPTP1 poplars showed more severe shrinking of the leaves than WT poplars  (dark brown)  A,C and nk brown) B,D. The T plants C,D after poplars E. Furthe poplars F. SOD ac of NaCl G. Taken PdPTP1 overexpression on ion homeostasis, the Na+ and K+ contents of OxPdPTP1 and WT plants were measured after exposure to 350 mM of NaCl for five days. Under nonstress growing condition, there was no significant variation in the Na+ and K+ contents between OxPdPTP1 and WT plants HCXAGXXR(S/T)G defined the large PTP superfamily, which now, in addition to DsPTPs, includes Tyr-specific PTPs that only dephosphorylate tyrosine . In the PdPTP1 was induced by NaCl and oxidative treatment  in OxPdPTP1 poplars compared with WT poplars after salt treatment  seedlings were cultured in half-concentrated Murashige and Skoog (MS) medium supplemented with 0.05 mg/L of IBA and 0.05 mg/L of NAA  [PdPTP1 in different tissues, salinity stress, and oxidative stress. For oxidative stress, 100 µM of Methylviologen (MV)  was sprayed once on the leaves of NE19. The leaves of treated poplars were harvested at 0, 1, 4, 6, 12, and 24 h, after MV treatment. For salinity stress, NE19 plants were subjected to 350 mM of NaCl. The leaves of treated poplars were harvested at 0, 1, 4, 6, 12, and 24 h, after salinity treatment. The harvested samples were immediately frozen in liquid nitrogen and stored at −80 degrees.MO, USA) , and NE1® Premix Ex Taq™ GC  with gene-specific primers and the internal control gene (ACTIN). The relative value for the expression level of each gene was calculated by the 2−ΔΔCt method [The leaves of control and treated poplars, as described above, were collected to extract total RNA by using the CTAB reagent, a method described previously . Then, ct method . PrimersOxPdPTP1 lines were treated with short-term salt treatment and long-term salt treatment. To short-term salt treatment, WT lines and OxPdPTP1 lines with similar size and growth status were chosen and subjected to 0 or 350 mM of NaCl for 5 days. Net photosynthetic rate, transpiration, stomatal conductance and intercellular CO2 concentration in the 9th to 11th leaves of OxPdPTP1 and WT poplars were measured by the Li-6400 Photosynthesis System  at day 0, day 3, and day 5. Five days after exposure to 350 mM of NaCl, photosynthetic activity in the 10th to 13th leaves of WT lines and OxPdPTP1 lines was monitored by Maximal PSII quantum yield (Fv/Fm) values, using a PAM chlorophyll fluorometer (PAM100) after 20 min of dark adaptation. Relative electrolyte leakage (REL), leaf relative water content (RWC), Malondialdehyde (MDA) content, and root reduction activity of WT lines and OxPdPTP1 lines were determined as described through previous protocol [OxPdPTP1 lines with similar size and growth status were chosen and subjected to 1/8 MS solution supplemented with or without 200 mM of NaCl, every 4 days for 4 weeks. The height of WT lines and OxPdPTP1 lines was measured weekly. Net photosynthetic rate, transpiration, stomatal conductance and intercellular CO2 concentration in the 9th to 11th leaves of WT plants and transgenic lines were measured by the Li-6400 Photosynthesis System  weekly. After 4 weeks, leaf RWC, REL, shoot biomass, root biomass, and chlorophyll content of each poplar were measured. All of the plants (height 20–30 cm) were grown in suitably sized pots  of the same soil , and each pot had a tray. In order to investigate the functional role of PdPTP1 in response to salt stress, potted WT lines and protocol . Root reprotocol . For the+ and K+ assay, as described previously [+ and K+ contents were determined by using an atomic absorption spectrometer .At the end of 350 mM of NaCl treatment for 5 days, the leaves, stems, and roots were harvested for Naeviously ,58. BriepSuper:PdPTP1:GFP, and pSuper:GFP fusion constructs were transformed into tobacco epidermal cells by the previously published protocol [For subcellular localization of PdPTP1, protocol  and tobaprotocol . The traPdMAPK1, PdMAPK3, PdMAPK4, PdMAPK6, PdMAPK7, and PdMAPK9 cDNA fragments were introduced to pGADT7 vectors  and used as the prey, and the full length of PdPTP1 coding sequences cDNA fragment was introduced to pGBKT7 vectors (Clontech) and used as the bait, to identify the interacting proteins. Yeast strain AH109 was co-transformed with bait and prey constructs. Yeast cells with the two constructs were selected on minimal medium without leucine (Leu) and tryptophan (Trp) medium. Positive clones were confirmed by growing on minimal medium containing 6 mM of 3-amino-1,2,4-triazole without Leu, Trp, and histidine (His), and minimal medium without Leu, Trp, His, and adenine (Ade). Yeast two-hybrid analysis was performed, using the protocol described by the previously published protocol . The fulPdPTP1 coding sequences were recombined into the pSPYNE vector to produce YN-PdPTP1, PdMAPK1, PdMAPK3, PdMAPK4, PdMAPK6, PdMAPK7, and PdMAPK9 coding sequences, which were then recombined into pSPYCE vectors, to produce YC-PdMPK1, YC-PdMPK3, YC-PdMAPK4, YC-PdMPK6, YC-PdMPK7, and YC-PdMPK9 [YN-PTP1 or YN-Empty, in combination with YC-Empty, YC-PdMPK1, YC-PdMPK3, YC-PdMAPK4, YC-PdMPK6, YC-PdMPK7, and YC-PdMPK9, was also performed in tobacco protoplasts by means of PEG treatment. The fluorescence was detected by using confocal microscopy . The LAS-AF software was used to record the images. For BiFC assay, C-PdMPK9 . Co-tranhttps://www.ncbi.nlm.nih.gov/), and sequences data of the other proteins shown in the diagram can be found in the GenBank database, under the accession numbers  [The MtPTP1, NtPTP1, and LePTP1 protein sequences were obtained from NCBI  .p < 0.05 (*) and p < 0.01 (**).The experiment was repeated at least three times, independently. The results are presented as the means ± SDs. Data were analyzed through LSD multiple range tests in the one or two way ANOVA program of SPSS (IBM SPSS17.0). Significance was defined as


Acinetobacter baumannii, including antibiotic resistance (AR) genes and virulence factors (VF), whole-genome shotgun sequencing was performed on 19 Tgc-resistant (TgcR) A. baumannii strains collected in a tertiary hospital during the early phase of the clinical introduction of Tgc in China from late 2012 to mid-2014. The major sample types containing TgcR strains were sputum and drain fluid. Data from an average of 624 Mbp of sequence was generated on each bacterial genome, with Q30 quality of 90%, and an average coverage of 96.6%. TCDC-AB0715 was used as a reference genome. The genome sequences were annotated for functional elements including AR genes, VFs, genome islands, and inserted sequences before they were comparatively analyzed. The antibiotic susceptibility phenotypes of the strains were examined by a broth microdilution method to determine the minimal inhibitory concentration (MIC) of strains against major clinical antibiotics. The AR genes (ARGs) were annotated using the Comprehensive Antibiotic Resistance Database (CARD). Thirty-three ARGs were shared by all 19 TgcR strains, and 24 ARGs were distributed differently among strains. A total of 391 VFs were found to be diversely distributed in all TgcR strains. Based on ARG number distribution, the 19 TgcR strains were divided into several groups. Highly differentiated genes included gpi, mphG, armA, msrE, adec, catB8, aadA, sul1, blaOXA–435, aph3i, and blaTEM–1, which may represent gene markers for TgcR A. baumannii sub-types. In addition, when compared with Tgc-sensitive (TgcS) strains collected during the same period, TgcR strains featured enrichment of ARGs including aph6id, aph3ib, and teta. Compared with 26 other whole-genome sequences of A. baumannii deposited in GeneBank, TgcR strains in this study commonly lacked the EF-Tu mutation for elfamycin resistance. Previous investigation of three A. baumannii strains isolated from one patient indicated genomic exchange and a homologous recombination event associated with generation of tigecycline resistance. This study further analyzed additional TgcR strains. Phylogenetic analysis revealed a close evolutionary relationship between 19 TgcR strains and to isolates in East and Northeast China. In short, the comprehensive functional and comparative genomic analysis of 19 clinical TgcR A. baumannii strains isolated in the early stage of Tgc usage in China revealed their close phylogenetic relationship yet variable genetic background involving multiple resistance mechanisms. Using a simple ARG or VF gene number diversity method and marker genes, TgcR strain sub-types can be identified. The distinct characteristics of TgcR A. baumannii strains with versatile genomic resistance and regulation patterns raise concern regarding prediction and control of Tgc resistance in the clinic.To assess the genomic profiles of tigecycline (Tgc)-resistant  Acinetobacter baumannii is a nosocomial pathogen that is often detected in intensive care settings .settings . Infectisettings . The rapl agents . In addievalence  and tranevalence . The gloA. baumannii strains has been reported (A. baumannii is associated with the overexpression of resistance-nodulation-division (RND) efflux pumps, such as adeABC  is a novel 9-t-butyl-substituted minocycline derivative and is effective against a broad spectrum of Gram-positive and Gram-negative pathogens, a significant proportion of which are resistant to many currently available antibiotics . In recereported . The abireported . Previous adeABC , adeIJK   were retrieved and analyzed.Nineteen TM 2000  in the paired-end mode with a read length of 100 bp. The Illumina sequence data was assembled using the SOAPdenovo (version 2.04) package (DNA was isolated using a QiaAmp DNA Mini kit. Paired-end libraries were constructed using a NEBNext DNA library preparation kit. Each library was sequenced by Wuxi AppTec using a HiSeq package . The tot1 (The assembled genome sequence was annotated using the NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP). Glimmer software (version 3.0) was used for the identification of protein-coding genes , tRNAsca1 . The dat2, including complete sequences and annotation of A. baumannii isolates. Genome level differences between different isolates were analyzed. SNPs were identified with SNPFinder and Mauve  and unique genes among the isolated strains using the PanOCT program . Data usnd Mauve . IS seqund Mauve . The phyThe data from this whole-genome shotgun project have been deposited at GenBank.No animal studies are presented in this manuscript. No human studies are presented in this manuscript. No potentially identifiable human images or data is presented in this study.A. baumannii.From late 2012 to mid-2014, 19 TgcR (MIC ≥ 4) and 37 Tgc-intermediate (2 ≤ MIC < 4) strains were isolated using a broth microdilution method in a tertiary hospital in Eastern China. Three TgcR strains were previously reported with TgcR-related genomic mutation and transcriptomic regulation . In thisMolecular epidemiological analysis showed that ST195  was the most prevalent type, followed by ST208 , ST191 , and ST784  . Other sThe minimal inhibitory concentrations (MICs) against major clinical antibiotics were measured for 19 TgcR strains . All strA. baumannii strains was 38.86%.Sequencing data and genome assembly results are shown in A. baumannii genomes were functionally annotated for ARGs, VFs, mobile elements including ISs and Genomic Islands  and common traits. Unique characteristics of strains were compared and analyzed.The 19 TgcR A. baumannii strains . The first and earliest-isolated strain was considered the intra-group reference for detection of SNPs, InDels, and large structural variations by colinearity analysis within each group (patient).SnatA, glycosyl hydrolase, two Vi polysaccharide biosynthesis proteins, VipA, TviB, heme-binding protein A, and a probable tape measure protein according to SwissProt annotation. Multiple genes discovered to be mutated were related to cell surface structure  and virulence.In Group 1, 1,034 synonymous and 344 non-synonymous mutations were identified in strains 2015ZJAB12 and 2015ZJAB13 . These Strans-acting regulatory protein HvrA, UPF0056 membrane protein BU449, outer membrane protein OprM, and probable tape measure protein OS.Of the Group-2 strains, 2015ZJAB23 may derive from a different strain than 2015ZJAB22, indicating a possible re-infection. Strain 2015ZJAB23 contained four non-synonymous start mutations and seven non-synonymous stop mutations. Proteins with premature stop codons included stearoyl-CoA 9-desaturase electron transfer partner, UTP-glucose-1-phosphate uridylyltransferase, tyrosine-protein kinase PTK, Na(+)/H(+) ion antiporter NhaA, Colinearity analysis revealed large structural changes of genomes as shown in A. baumannii genomes. GO terms, COG categories, and KEGG pathways were analyzed to reveal metabolic characteristics.Functional CDS, ARGs, VFs, GIs, ISs, and integron elements were annotated and compared between TgcS and other MDR aph33ib, aph6id, aph3ia, bl2b_tem1, and sul1 genes, which are related to antimicrobial resistance to aminoglycoside, beta-lactam, and sulfonamide.The distribution of GIs in each TgcR strain is shown in Using a query coverage of 70% as a cutoff, plasmids homologous to p2BAB07104, pMDR-ZJ06, p1ABIBUN, and pAB120 were revealed.Vsa3 (19/19) and ISAba1 (18/19) were the most prevalent IS elements. ISAba26, ISAba33, IS26, IS15DI, IS15DII, and IS15DIV were found in 17 out of 19 strains. The least common IS types were ISAha1, ISAha2, ISAha3, ISAba5, ISAba7, ISAba12, ISAba13, IS17, ISAjo1, and ISAlw1, which were only detected in strain 2015ZJAB13, indicating frequent genomic information exchange in its evolutionary history. In the phylogenetic tree, it is shown that 2015ZJAB13 was the most divergent strain from the other TgcR isolates, which was possibly related to frequent insertion of exogenous sequences.In total, 82 ISs belonging to seven families were identified in TgcR strains . ISVsa3 blaOXA–66, gyrA, adeR, adeS, mphG, armA, msrE, adeN, blaOXA–23, lrfA, blaTEM–1, aph3ia, blaOXA–435, catB8, aadA, sul1, blaTEM–122, blaCTX–M–55, gyrB, EF-Tu mutant, blaOXA–316, mel, sul2, and adeC). Among these varied ARGs, lrfA and adeC were present in all strains, including 2015ZJAB9, but with different numbers. OXA-66 was only absent in 2015ZJAB9. Propionibacterium acnes gyrA (17/19), adeR (18/19), adeS (16/19), mphG (15/19), armA (15/19), and adeN (15/19) were also widely distributed among strains. APH3’1a and OXA-435 genes were present in approximately half of the TgcR strains. The least common ARGs were sul1 (5/19), catb8 (4/19), and aadA (3/19). EF-Tu mutants (2/19) were only present in strains 2015ZJAB9 and 2015ZJAB13. Mutants of mel (2/19) were only present in strains 2015ZJAB21 and 2015ZJAB22. Those of ctx-M-55 were only found in one strain, 2015ZJAB17. Thus, this simple gene number method can efficiently divide the strains into at least three sub-groups  in the two strains of Patient 1, with strain 2015ZJAB13 having the highest number of ARGs. Between TgcR strains 2015ZJAB19 and 2015ZJAB20 isolated from Patient 3, only sul2 number distribution was different, which was in accordance with a prior intra-group comparison showing high similarity between the two genomes. In strains isolated from Patient 2, only sul2 gene number was different between 2015ZJAB21 and 2015ZJAB22. ARGs were distributed differently in 2015ZJAB23. Among all isolates, 2015ZJAB9 had the lowest number of ARGs. With CARD annotation, the numbers of different ARGs in the three patients were 11, 9, and 2, respectively.There were eight different resistance types (A. baumannii genome sequences (an unpublished lab resource), TgcR contained more ARG elements, especially aph6id, aph3ib, and teta, which encode aminoglycoside O-phosphotransferase, and major facilitator superfamily transporter tetracycline efflux pump, respectively .Compared to TgcS ectively . Compareanalysis , the 19 Y and cylG were the most annotated VF among all strains. Isolates from Patient 1 had 84 different VFs, whereas those from patients 2 and 3 had 59 and 9 different VFs, respectively. Based on the VF number distribution, 74 VFs were present with multiple VFs in all strains except 2015ZJAB9, whose VF profile was unique. The 19 TgcR strains can be divided into four sub-groups based on VF number distribution  and 2015ZJAB23 having the least (263). In total, 391 VFs were found to be diversely distributed in all TgcR strains. The transcriptional regulator ribution . VF pattA. baumannii involved seven categories of elements . Although the VFs were diversely distributed among the 19 TgcR strains, the first two sub-groups and the latter two sub-groups could be differentiated using VFs R033541, R012040, R010421, R004310, R009821, R010419, R006935, R010422, R007617, R002520, and R007621, representing cdiB, pvdN, wbpD, pilA, 1sgF, wbpB, mip, wbpE, wbmB, cap8J, and BAV0081 genes, coding for hemolysin activator Hly, pvdN in Pseudomonas_aeruginosa, probable acetyltransferase WbpD, fimbrial protein, probable aminotransferase, putative UDP-galactose–lipooligosaccharide galactosyltransferase, probable oxidoreductase WpbB, macrophage infectivity potentiator (Mip), probable aminotransferase WbpE, wbmB hypothetical protein, capsular polysaccharide synthesis enzyme Cap8J, and putative O-antigen translocase, respectively. Compared to the 19 TgcR strains and geographically proximal isolates from Eastern and Northeast China, the remaining reference genomes lacked 36 VFs from R021300 to R030574 . Notably, BJAB0715 and 2015ZJAB9 were clustered with strains isolated from western countries, indicating a western origin and epidemical spread to China. ACICU, AC29, and AC30 from Rome and Malaysia were clustered with our strains and isolates from East and Northeast China and Taiwan, indicating their close evolutionary relationship.Major virulence factors in  R030574 , but posGO terms of the 20 strains were annotated. In all, 39 GO terms were annotated: 26 in Group 1 (Patient 1), 23 in Group 2, and 21 in Group 3. COG categories were analyzed and compared.KEGG pathway metabolic characteristics of the strains were analyzed. Comparatively, KEGG pathways varied the most in Group 1, and the most variable pathways were Replication and repair, Drug resistance, Metabolism of cofactors and vitamins, and Nucleotide metabolism.A. baumannii strains (tested by disk diffusion) collected during the first 18 months of Tgc usage in the hospital revealed that the most prevalent ST was ST195 (57.8%), followed by ST208, ST191, and ST784. In comparison to traditional strain typing approaches, such as PCR and sequencing-based methods targeting a few conserved genes, genome-wide comparative analysis provides a comprehensive and SNP-level resolution. Currently, the gene gpi is the most efficient and widely used locus for typing A. baumannii clinical strains.Sequence types analysis of the 90 TgcR gpi gene. The two strains derived from Patient 2 had highly similar genomes. In comparison, the strains from Patient 3 were vastly different, suggesting that they might stem from separate infections.When strains isolated from the same patient during antibiotic treatment were grouped together, a comparison within each group revealed their intricate genomic relationship and elucidated possible gene transfer events (21). For example, the two strains obtained from Patient 1 (2015ZJAB12 and 2015ZJAB13) had a limited number of SNPs  associated with different STs, indicating exchange of genomic sequences that possibly contained the conserved ST A. baumannii strains were isolated from sputum, drainage fluid and blood, which were obtained from ICU, sICU, and Department of Internal Medicine settings , annotated by CARD, were present with different ARG number distribution patterns in the 19 TgcR isolates. This provided a genotyping method without consideration of SNPs. The 19 TgcR strains can, thus, be divided into three sub-groups (lrfA and adeC (19/19) , blaOXA–66 (1/19) (absent only in 2015ZJAB9), adeR (18/19), gyrA (17/19), adeS (16/19), mphG (15/19), armA (15/19), adeN (15/19), APH3’1a (10/19), blaOXA–435 (10/19), sul1 (5/19), catb8 (4/19), aadA (3/19), and EF-Tu mutant (2/19) (present only in strains 2015ZJAB9 and 2015ZJAB13), mel (2/19) (present only in strains 2015ZJAB21 and 2015ZJAB22) and blaTEM–122, and blaCTX–M–55 found only in 2015ZJAB17. In the grouping analysis, sub-group 1 was characterized by the presence of blaOXA–23 and absence of blaOXA–316. ARGs mel, blaTEM–122, blaCTX–M–55, and blaOXA–23 were only present in the first sub-group strains. Sul2 was a differentiating gene present in about half of sub-group 1 strains that appeared only once in sub-group 2. The unique and most abundant ARGs carried by the third sub-group were catB8, aadA, gryB, and EF-Tu. A lack of mphG and armA was a marker of the second sub-group.In total, 24 ARGs  can differentiate between the first two sub-groups and the last two sub-groups. Compared to the TgcR strains and geographically proximal isolates from East and Northeast China, the remaining reference genomes lacked 36 VFs from R021300 to R030574  and 2015ZJAB23 having the least (188 VFs). The transcriptional regulator  R030574 , but posTaken together, ARGs and VFs may have coordinated interactions and interplay in survival and environmental adaptation. Metabolic activities and pathways in three strains under different concentrations of tigecycline were analyzed, and the differential transcript expression profiles suggested relationship with Tgc resistance. Further transcriptome analysis in all TgcR strains may provide more comprehensive information about gene regulation under antibiotic stress.A. baumannii isolates in a tertiary hospital in Eastern China during the first 2 years of adoption of Tgc in clinical use. We analyzed important functional ARG and VF elements, highlighting a dynamic genomic background in 19 TgcR A. baumannii strains. Herein, we demonstrate the possibility of using a simple approach of ARG number distribution analysis to sub-group TgcR A. baumannii. This facilitates the resistome profile characterization and comparison. The distinct yet traceable occurrence of TgcR A. baumannii has raised concern for medical workers. Genome plasticity revealed by our study provided a basis for the development of Tgc resistance, especially under environment pressure such as antibiotic treatment. Possible mechanisms include frequent genetic material exchange, homologous recombination of large genomic fragments, re-infection of resistant bacteria, changes in transcriptional regulation of efflux pumps, or induction by other antibiotics. Elucidation of these mechanisms requires further investigation.In summary, this study provides a survey of clinical TgcR PRJNA593931 and PRNJNA307212.The datasets generated for this study can be found in the NCBI with accession numbers LL and YX designed the study. LL managed the project, analyzed the data, and wrote the manuscript. LL, PS, BZ, WY, and JJ collected the samples and performed the bacterial antibiotic susceptibility tests. LL and PS preformed the DNA extraction, library construction, and sequencing experiments. YX revised the manuscript. All authors contributed to the article and approved the submitted version.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.


Eclipta prostrata (L.) is an important and well-known medicinal plant due to its valuable bioactive compounds. Microorganisms, including arbuscular mycorrhizal fungi (AMF), and salinity could directly impact plant metabolome, thus influencing their secondary metabolites and the efficacy of herbal medicine. In this study, the role of different single AMF species  and a mixture of six AMF species in plant growth and physio-biochemical characteristics of E. prostrata under non-saline conditions was investigated. Next, the most suitable AM treatment was chosen to examine the impact of AMF on physio-biochemical features and polyphenol profiles of E. prostrata under saline conditions (100 and 200 mM NaCl). The findings indicated that AMF mixture application resulted in more effective promotion on the aboveground part of non-saline plants than single AMF species. AM mixture application improved growth and salt tolerance of E. prostrata through increasing the activity of catalase, peroxidase (at 4 weeks), proline, and total phenolic content (at 8 weeks). Such benefits were not observed under high salinity, except for a higher total phenolic concentration in mycorrhizal plants at 8 weeks. Through high-performance liquid chromatography, 14 individual phenolic compounds were analyzed, with wedelolactone and/or 4,5-dicaffeoylquinic acid abundant in all treatments. Salinity and mycorrhizal inoculation sharply altered the polyphenol profiles of E. prostrata. Moderate salinity boosted phenolic compound production in non-AM plants at 4 weeks, while at 8 weeks, the decline in the content of phenolic compounds occurred in uncolonized plants subjected to both saline conditions. Mycorrhization augmented polyphenol concentration and yield under non-saline and saline conditions, depending on the growth stages and salt stress severity. Plant age influenced polyphenol profiles with usually a higher content of phenolic compounds in older plants and changed the production of individual polyphenols of both non-AM and AM plants under non-stress and salt stress conditions. A better understanding of factors  affecting the phenolic compounds of E. prostrata facilitates the optimization of individual polyphenol production in this medicinal plant. Eclipta prostrata (L.), belonging to a family of medicinal plants (Asteraceae), is a native plant of Asia but is widely distributed in subtropical, tropical, and warm temperate regions on the globe , nutritional imbalance, pigment degradation and inhibition of photosynthesis, oxidative and osmotic stress, limited leaf diffusion (flux) of CO2, modification of metabolic pathways, cell deformation, premature senescence, and ultimately cell death in the plant , and the ratios of total chlorophyll:carotenoids, Ca2+:Na+, Mg2+:Na+, and K+:Na+ in the shoots of the medicinal plant Valeriana officinalis under salinity stress , one of the prevalent soil microbes, can colonize most terrestrial plant species’ roots. These symbiotic fungi have been reported to considerably offer various benefits to their host plants, such as enhanced uptake of mineral nutrients and water and increased tolerance to stressful environments . Remarkay stress . The autThymus vulgaris (L.) and T. daenensis Celak was inclined by approximately 20% after treatment with 60 mM NaCl, while leaf flavonoid content was enhanced by 38.6 and 36.6% in plants in response to 60 and 90 mM NaCl, respectively. Cinnamic acid, the main constituent in both plant species, was elevated by 31.4% in T. vulgaris in the presence of 60 mM NaCl  that mycorrhizal colonization could improve plant growth and tolerance to salt stresses, particularly moderate salinity, and (2) that AMF and salinity had interactive effects on the polyphenol profiles of this medicinal plant, which depended on plant age.It is noteworthy that the synergistic effects of salt stress with other environmental factors on bioactive compounds in medicinal plants have been poorly understood . To the stresses . Thus, tE. prostrata (L.) from Hong Dai Viet Ltd (Vietnam) were used in our experiments. The mycorrhizal commercial inoculant Symbivit containing six AMF species, Claroideoglomus etunicatum, Rhizoglomus microaggregatum, Rhizophagus intraradices, Claroideoglomus claroideum, Funneliformis mosseae, and Funneliformis geosporum, was provided by Symbiom Ltd., Czechia. Septoglomus deserticola BEG 73 and Acaulospora lacunose BEG 78 were obtained from the International Bank for the Glomeromycota. F. mosseae SZIE originated from the collection of Szent István University. Three single AMF species were separately propagated by using Medicago truncatula and Zea mays as host plants cultured in autoclaved sand for 6 months. A mixture of spores, mycelia, infected root fragments, and sand from cultures was harvested for mycorrhizal inoculation. The inoculation dosage was 15 g of inocula per pot with about 2,400 infective propagules evaluated by the most probable number test  and 3 ml of a filtrate (<20 μm) of this combined inoculum to provide a microbial population with non-AM propagules. In the salt stress experiment, 15 g of sterilized Symbivit and 3 ml of a filtrated solution of Symbivit were used for pots without mycorrhizal inoculation.Seeds of ber test . Mycorrh–1, K 2,819 mg kg–1, carbonate (%) 17.18%, and dry matter content (m/m%) 54%  plants inoculated with A. lacunose, (3) plants inoculated with F. mosseae, (4) plants inoculated with a mixture of six AMF species (Symbivit), and (5) plants without AM inoculation (control). Each treatment had 12 biological replicates, equivalent to 12 pots (one plant per pot). Therefore, five treatments (four different kinds of AM treatment and control) with 12 replicates resulted in a total of 60 pots. Pots were put in a climatic chamber EKOCHL 1500  and watered once a week. At 4 and 8 weeks of growth, plants were harvested for measurements. Root colonization, plant height, fresh root and shoot weight, leaf number, and leaf area were examined. Fully expanded leaves (excluding petioles) were immediately frozen in liquid nitrogen and stored at −80°C until total phenolics and proline content determination.The seeds were sterilized with NaOCl 1%, then washed with distilled water several times and put on a filter paper in Petri dishes at 26°C for germination for 3 days. Germinated seeds were placed in 0.5-L plastic pots filled with an autoclaved mixture of sand and peat (60:40%) (v/v). The chemical properties of the sand:peat substrate have pH 6.9, N (%) 0.6%, P 681.29 mg kg/m%) 54% . The AM –2 s–1). During plant growth (8 weeks), non-stress plants were watered with 100 ml of tapping water per pot once a week, while salt stress treatments were applied by watering with 100 ml of 100 or 200 mM NaCl for each pot once a week. Fresh shoot and root weight, plant height, leaf number, leaf area, stem diameter, chlorophyll fluorescence, and mycorrhizal colonization rate were examined at 4 and 8 weeks of growth. The fully expanded leaves (excluding petioles) were immediately frozen in liquid nitrogen and stored at -80°C until analysis of proline, superoxide dismutase, peroxidase, catalase, and polyphenol components.Based on the preliminary experiment results, the mixture of six AMF species (Symbivit) was chosen for AM treatment in this experiment. A factorial experiment was performed using a randomized complete block design with two factors: (1) salinity levels   and (2) Five plants per treatment were randomly selected, then their roots were washed to remove the substrate and cleared with 10% KOH for 10 min, acidified using 2% HCl and 0.05% Trypan blue in 1:1:1 water:glycerin:lactic acid overnight. Thirty root fragments (1 cm long) were mounted on a glass slide, and four glass slides per plant were examined according to Fv/Fm), a chlorophyll fluorescence parameter, was measured after 30 min of dark adaptation using a Walz-PAM 2500 (Germany) fluorometer according to the method of Leaf area was determined according to the method of Proline content was quantified by the acid ninhydrin procedure of 2CO3 was added after 3 min at room temperature. The mixture was completed to 10 ml with distilled water and incubated for 30 min at room temperature. The absorbance at 760 nm was recorded, and total phenolics content was presented as mg gallic acid per g fresh weight.Total phenolic concentration was measured by the Folin–Ciocalteu assay . Briefly2EDTA and 7.5% (w/v) polyvinylpyrrolidone K25 and centrifuged at 10,000 × g at 4°C for 20 min. The supernatants were used for peroxidase, superoxide dismutase, and catalase assays. The protein content of all leaf extracts was estimated by the method of Frozen leaves (0.5 g) were homogenized in 3 ml of 50 mM Tris–HCl buffer (pH 7.8) containing 1 mM NaPeroxidase  activity was measured by the method of 2O2, and 100 μl of 50 mM guaiacol with 10 μl of plant extract was used to measure the POD activity at 436 nm in 5 min. The enzyme activity was expressed as the changes in absorbance mg–1 protein min–1.Superoxide dismutase  activity was determined spectrophotometrically at 560 nm following by the method of L-methionine, 20 μl of crude extract, and 20 μl of 1 mM riboflavin. The absorbance was read at 560 nm. One unit of SOD activity (U) was defined as the required enzyme volume to lead to 50% inhibition of the NBT decline under the assay conditions.Catalase  activity was measured by the method of 2O2. The enzyme activity was presented as the changes in absorbance mg–1 protein min–1.E. prostrata, a 0.5-g sample was taken and crushed in a crucible mortar with quartz sand. Twenty milliliters of a mixture of 44% EtOH, 4% MeOH, 10% water, and 2% acetic acid was gradually added with crushing and then transferred to a 100-ml Erlenmeyer flask. The macerate was subjected to an ultrasonication force using an ultrasonic water bath device  for 4 min, followed by mechanical shaking (GLF3005) for 15 min. The mixture was kept overnight at 4°C and filtered through Albet-DF400125 type filter paper. Before injection onto the HPLC column, it was further cleaned up by passing through a 0.22-mm PTFE HPLC syringe filter. Nucleosil C18-100, 3 μm, 240 × 4.6 mm Protect-1 HPLC column  was used to separate phenolic compounds using a gradient elution of 1% formic acid in water (A) and acetonitrile (B) with a flow rate of 0.6 ml min–1. Gradient elution began with 2% B, changed to 13, 25, and 40% B in 10, 5, and 15 min, respectively, and finally turned to 2% B in 5 min. The HPLC determination was performed using a Hitachi Chromaster HPLC with a Model 5160 pump, a Model 5260 autosampler, a Model 5310 column oven, and a Model 5430 diode-array detector. The separation and data processing were operated by OpenLab CDS software. The peaks were identified by comparing their retention times and spectral characteristics with available standards such as quercetin-3-arabinoside, luteolin-glucoside, luteolin-7-O-glucoside, luteolin, wedelolactone, demethyl wedelolactone, caffeic acid, 3,4-O-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4-O-caffeoylquinic acid, 4,5-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, ferulic acid, and feruloylquinic acid . For the quantification of phenolic compounds, each peak area was integrated at the maximum absorption wavelength, and the concentrations were calculated by relating the areas of the peaks to those of the available external standards  was applied with explanatory variables (factors) of AM inoculation and salt stress levels as well as their interaction. Moreover, Tukey post hoc tests were applied. In addition, two-sample, two-tailed t-test was applied using MS Excel to compare each particular treatment result at 4 and 8 weeks. PCA was carried out by the XLSTAT program to identify patterns, i.e., interactions among the studied variables and treatments, in polyphenolic data of E. prostrata with and without AMF under different salinity stress levels including no salinity stress.Statistical analysis was implemented using the SAS 9.1  package for Windows. In the preliminary experiment, differences in plant growth traits, mycorrhizal colonization, proline, and total phenolics among AMF treatments were analyzed by one-way analysis of variance and Tukey A. lacunose was lowest (22.9%) and significantly lower than plants treated with other single AMF species and the mixture of AMF (from 47.8 to 54.1%) 4 weeks after inoculation. Interestingly, no significant differences in this rate among 8-week plants treated with different mycorrhizal inoculants (ranged from 49.5 to 59.5%) were found (Microscopic observation of the roots showed that no AM colonization in non-AM plants (control plants) was detected. In contrast, plants in mycorrhizal treatments were successfully colonized by three single AMF species and the mixed AM inoculant. The colonization rate of plants infected by re found .S. deserticola displayed a substantial decline in fresh shoot weight by 52.9 and 47.5% after 4 and 8 weeks of plant growth, respectively, in comparison with that of the corresponding non-mycorrhizal plants. Notably, inoculation of F. mosseae, S. deserticola, and A. lacunose considerably reduced plant height compared with uncolonized plants at 4 weeks, while the decline did not occur in the mixed AM treatment at both stages of plant growth. In terms of leaf area, no beneficial effects of different AM inoculations were observed in host plants.Regarding plant growth parameters, there were no significant differences in fresh root weight and leaf number among all treatments at 4 and 8 weeks . TreatmeS. deserticola (by 64.6%) and A. lacunose (49.3%) at the later stage compared with plants at the early stage of plant growth (P < 0.05). Similarly, there were no significant differences in total phenolic content between control plants and mycorrhizal plants after 4 weeks of growth . No significant differences in total phenolic content among control plants and plants treated with A. lacunose or Symbivit were detected 8 weeks after inoculation. Conversely, the total phenolic level in S. deserticola colonized plants at 8 weeks considerably declined by 31.2% (P < 0.05) in relation to that at 4 weeks. Moreover, S. deserticola caused a sharp reduction in total phenolic concentration in plants, as compared with non-AM plants (by 37.8%) and plants colonized by other fungal symbionts  after 8 weeks of growth.Under non-stress conditions, inoculation with different species of AMF did not induce substantially higher proline concentrations in plants compared with non-AM plants during plant growth . Remarkaf growth . NonetheP < 0.05) relative to those at the early stage.Non-AM plants had no mycorrhizal colonization during plant growth. After 4 weeks of growth, the mycorrhizal colonization rate of AM plants obtained 54% under non-stress conditions, while the rate was 58.4% in those treated with 100 mM NaCl . No signE. prostrata plants to salt stresses, particularly at 200 mM NaCl, led to a considerable decrement in most growth parameters tested at both plant growth stages (Exposure of h stages . Under nFv/Fm) in plants, no significant differences between mycorrhizal and non-mycorrhizal plants were found under the same conditions at 4 and 8 weeks of growth (Fv/Fm at 4 and 8 weeks (P < 0.01), whereas the effect of salt stress was statistically significant on this parameter at 4 weeks (P < 0.05).Although salt stresses slightly increased the maximal photochemical efficiency of photosystem II (f growth . MycorrhP < 0.05) with the existence of interaction between the two factors at 8 weeks (P < 0.05).Salinity heightened proline concentrations in mycorrhizal and non-mycorrhizal plants at 4 weeks . In detaP < 0.05) in non-AM plants under non-stress conditions and  in moderate-salted mycorrhizal plants, but it substantially leaped  in uncolonized plants exposed to 100 mM NaCl. Salt treatments remarkably induced almost three- and seven-folds higher POD activity in non-stress uncolonized plants subjected to 100 and 200 mM NaCl, respectively, at 8 weeks. In contrast, both saline levels did not elevate POD activity in colonized plants. However, AM inoculation triggered an increase in POD activity by nearly sixfold in non-stress plants. Under moderate and high salt stress, no significant differences in POD activity were found between non-AM and AM plants. Mycorrhizal treatment markedly impacted POD at 4 weeks (P < 0.01), while salinity remarkably affected POD at 8 weeks (P < 0.05).At the early stage of plant growth, mycorrhizal plants gained the highest POD activity under moderate salt stress, while the activity of this enzyme was lowest in non-AM plants subjected to 100 mM NaCl . No signP < 0.05), non-stress AM plants , and mycorrhizal plants exposed to moderate saline conditions  at the later stage versus the early stage of plant growth. Salinity considerably affected SOD at 4 weeks (P < 0.05).In terms of SOD activity, the observed differences between mycorrhizal and non-mycorrhizal plants under non-stress and salt levels were not statistically significant at 4 and 8 weeks . There wP < 0.001) and AM plants exposed to moderate salt stress  8 versus 4 weeks after inoculation were observed. Salinity remarkably affected CAT at 4 (P < 0.001) and 8 weeks (P < 0.05).Under non-stress conditions, mycorrhizal application significantly dropped CAT activity in plants at 4 weeks . ModeratE. prostrata were implemented by HPLC-DAD analysis. The gradient elution applied was able to efficiently separate 14 phenolic constituents in plants 4 weeks after growth, namely eight hydroxycinnamates , four flavonoids , and two coumarins (wedelolactone and demethyl wedelolactone) (P < 0.05), five hydroxycinnamic acids (at least P < 0.01), and demethyl wedelolactone (P < 0.001). Salinity had a substantial impact on the level of all polyphenol compounds tested (at least P < 0.05), except demethyl wedelolactone and 5-O-caffeoylquinic acid. Interactions between two main effects on 3,5-dicaffeoylquinic acid (P < 0.001), ferulic acid (P < 0.01), feruloylquinic acid (P < 0.001), 4,5-dicaffeoylquinic acid (P < 0.05), and luteolin (P < 0.001) were found. When plants reached 8 weeks of age, mycorrhizal colonization significantly influenced all polyphenol compounds (at least P < 0.05), except demethyl wedelolactone. Likewise, salinity elicited sharp changes in all polyphenols (with at least P < 0.01). Interactions between two main effects on most polyphenols were recorded .The quantitative and qualitative measurements of polyphenols in the leaves of lactone) , whereaslactone) . Among plactone) , 8. In dO-caffeoylquinic acid (336%), caffeic acid (171%), and luteolin (287%) in uncolonized plants at 4 weeks, while these increments were not found under high salt stress, except ferulic acid, 5-O-caffeoylquinic acid, and luteolin. By contrast, under both salt stresses, the decrement trend was seen in the content of total polyphenols and wedelolactone, luteolin-7-glucoside, and feruloylquinic acid in mycorrhizal plants, being more severe under high salt stress, whereas there were no significant changes in the concentrations of ferulic acid, 4,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, quercentin-3-arabinoside, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, and 3,4-O-dicaffeoylquinic acid in colonized plants as compared with the counterparts of non-stress mycorrhizal ones. Noticeably, under moderate salinity, the concentrations of wedelolactone, ferulic acid, and 4-O-caffeoylquinic acid were substantially higher in non-AM plants than in AM plants. Nevertheless, the fungal symbiont markedly enhanced the content of luteolin (62.3%) in the host plants in relation to those of non-AM plants. Besides, demethyl wedelolactone was under the detection limit in colonized plants under such stress, but their feruloylquinic acid was detectable. When exposed to high salinity (200 mM NaCl), fungal colonization positively influenced the level of 3,5-dicaffeoylquinic acid (37-folds more than that of the corresponding uncolonized plants), 4,5-dicaffeoylquinic acid (17-folds), feruloylquinic acid (detectable versus undetectable), and luteolin-glucoside (detectable), but negatively affected the content of ferulic acid (decreased by 268% over the corresponding uncolonized plants) in colonized plants at 4 weeks.After 4 weeks of growth, mycorrhizal colonization resulted in a significant increase in the total polyphenols (by 139%) in non-stress plants. Such a tendency was observed in the content of wedelolactone (105%), 3,5-dicaffeoylquinic acid (404%), 4,5-dicaffeoylquinic acid , and feruloylquinic acid . Moderate salinity significantly induced higher total phenolics (166%) and seven individual polyphenols such as wedelolactone (134%), ferulic acid (239%), 3,5-dicaffeoylquinic acid (842%), 4,5-dicaffeoylquinic acid , 4-O-caffeoylquinic acid, and luteolin-glucoside was even profoundly enhanced by 93.7, 204, and 74%, respectively, in AM plants exposed to high salinity relative to non-stress AM plants. Noticeably, after 8 weeks of growth in the presence of 200 mM NaCl, the concentrations of all phenolic compounds were sharply inclined in mycorrhizal plants in relation to the counterparts in non-AM plants, except demethyl wedelolactone. The highest and lowest increases induced by AMF were 4-O-caffeoylquinic acid (more than 10-folds) and luteolin-glucoside (160%), respectively.After 8 weeks of growth, salinity led to a significant reduction in the content of total polyphenols and 11 phenolic compounds in non-AM plants, being more severe under high saline conditions. In 8-week mycorrhizal plants, moderate salinity also depressed the content of total polyphenols and eight phenolic substances, but the descending trend was alleviated in most bioactive compounds under high saline conditions. The level of few metabolites such as ferulic acid, 4-Interestingly, significant changes in the content of phenolic compounds in non-AM and AM plants were observed over time. Under non-stress conditions, there were substantial increases in the content of most polyphenols in AM (eight phenolics) and non-AM plants (10 phenolics) at 8 weeks versus their levels in the corresponding plants at 4 weeks. Considerable decreases in the content of seven individual phenolics were found in uncolonized plants treated with 100 mM NaCl 8 weeks after growth versus those 4 weeks after growth. By contrast, substantial inclines in the concentration of three polyphenols and a dramatic decrement in luteolin level (73%) were detected in colonized plants exposed to moderate salinity at 8 weeks relative to their counterparts at 4 weeks. A significant augmentation in the level of two polyphenols and remarkable declines in four phenolic concentrations were found in uncolonized plants subjected to 200 mM NaCl at 8 versus 4 weeks. Contrariwise, pronounced increases in the concentration of all phenolic compounds were recorded in colonized plants exposed to high salinity at the early stage of plant growth relative to those at the later stage.O-caffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and luteolin-glucoside. PC2, covering 21.9%, was contributed primarily by caffeic acid (positive association) and feruloylquinic acid (negative association). In the next stage of plant growth (8 weeks), as much as 67.5% of the total variation was covered by the PC1, which was positively influenced by all phenolic compounds . PC2 explaining 13.3% of the total variance is positively influenced mainly by luteolin-glucoside and luteolin but negatively impacted principally by demethyl wedelolactone. At 4 weeks, high positive correlations between ferulic acid and 5-O-caffeoylquinic acid, demethyl wedelolactone and caffeic acid, 4-O-caffeoylquinic acid and 3,5-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid and luteolin-glucoside, luteolin-glucoside and 4,5-O-dicaffeoylquinic acid, quercetin-3-arabinoside and 3,4-O-dicaffeoylquinic acid, and luteolin-7-O-glucoside and feruloylquinic acid could be seen, whereas there were negative associations between feruloylquinic acid/luteolin-7-O-glucoside and ferulic acid/5-O-caffeoylquinic acid  and mycorrhizal treatment (A0) under non-saline conditions, the control treatment under 200 mM NaCl (C200), and the other group (A100 + A200 + C100) at 4 weeks. Differences among C200, C0, and A0 groups were distinguished by PC1, while PC2 discriminated between the salinity stresses and non-saline groups at 4 weeks. Four different clusters—C0, A100 (AM inoculation under 100 mM NaCl), A0 + A200 , and C100 + C200 (control treatment in the presence of 100 and 200 mM NaCl)—were recognized at 8 weeks. Obviously, AM inoculation under non-saline and high saline conditions influencing individual polyphenols was different from the other groups at the later stage of plant growth.Principal component analysis of individual polyphenols were performed, independently for each harvest time, to correlate variables determined under different conditions at 4 and 8 weeks. Four (at 4 weeks) and three (at 8 weeks) components showed eigenvalues higher than 1 , S2. Thenic acid . At 8 welucoside . The bipA. lacunose treatment had considerably lower mycorrhizal colonization than others at 4 weeks, no significant differences in AM colonization among colonized plants in different AM treatments were recognized after 8 weeks of growth. In contrast to observations in artichoke, six AMF isolates exhibited different colonization dynamics at 7, 12, and 23 weeks after inoculation in two cultivars  was chosen for mycorrhizal inoculation in our salt stress experiment.In our preliminary experiment, although ultivars . Indeed,ultivars . Howeverultivars . Our finultivars . S. deserasitism  due to trasitism . InteresE. prostrata plants to salinity, especially in terms of bioactive compounds, have not been addressed. In the present work, mycorrhizal colonization rate was markedly reduced after 4 weeks of plant growth due to the high saline level (200 mM NaCl), but not at the later stage of plant growth (Phoenix dactylifera L.)   and/or slightly and moderately saline soils (EC of the saturation extract from 4 to 8 dS m–1).Although AMF have been shown to improve plant performance under salinity in many plant species , responst growth . The advt growth , but subt growth . Plants t growth . Plant bt growth , date pafera L.) , and thelis (L.) . These flis (L.) . Furtherthe host . A substsalinity , which mV. officinalis inoculated by either R. irregularis or F. mosseae, which may indicate mitigation of the stress  upstream of proline synthesis  was not affected by salinity, indicating that salinity did not impair the photosynthetic system under our experimental conditions. Contrast observations were obtained in early investigations  or singlet oxygen (1O2) to H2O2 and O2 . According to Salt stress stimulates phenolic compound accumulation in plants as a defense mechanism to stress . TherefoE. prostrata. The findings illustrated that the positive effect of AM inoculation on polyphenol profiles was dependent on stress severity and plant growth stage. Mycorrhizal inoculation under moderate salinity showed a higher plant tolerance during plant growth, but under high saline conditions, the higher accumulated content of phenolic compounds was achieved at the later plant growth stage. Hence, mycorrhizal application individually or in combination with salinity and harvest time would be a practical approach for optimizing individual polyphenol production in this medicinal plant.In this work, mycorrhization and co-treatment of AMF and salinity elicited significant changes in the accumulation of phenolic compounds in the medicinal plant This study shows how important the selection of the right date of harvest is for obtaining the optimal composition of phenolic compounds for particular purposes ; moreover, AMF and moderate salt stress can be used to manipulate the pattern of individual polyphenol production. Further studies should investigate other bioactive compounds in this medicinal plant colonized by AMF and/or exposure to different abiotic stresses to optimize their production.The original contributions presented in the study are included in the article/ND: propagation of single AMF species for inoculants, data analysis, data presentation, writing of the original draft, revision, and editing. AV: conceptualization, experimental design, mycorrhizal inoculation, plant care, stress treatments, collecting samples, measurement of AM colonization, growth parameters, chlorophyll fluorescence, proline and total phenolics, extraction of leaf samples for enzyme measurements, sample preparation for HPLC, data curation, and data analysis. IH: measurement of AM colonization, enzymes, proline, and phenolic compounds (HPLC), extraction of leaf samples for enzyme measurements, sample preparation for HPLC, and preparation of 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.


Chemotherapy-induced peripheral neuropathy (CIPN), one of major dose-limiting side effects of first-line chemotherapeutic agents such as paclitaxel, oxaliplatin, vincristine, and bortezomib is resistant to most of existing medicines. The molecular mechanisms of CIPN have not been fully understood. High mobility group box 1 (HMGB1), a nuclear protein, is a damage-associated molecular pattern protein now considered to function as a pro-nociceptive mediator once released to the extracellular space. Most interestingly, HMGB1 plays a key role in the development of CIPN. Soluble thrombomodulin (TMα), known to degrade HMGB1 in a thrombin-dependent manner, prevents CIPN in rodents treated with paclitaxel, oxaliplatin, or vincristine and in patients with colorectal cancer undergoing oxaliplatin-based chemotherapy. In this review, we describe the role of HMGB1 and its upstream/downstream mechanisms in the development of CIPN and show drug candidates that inhibit the HMGB1 pathway, possibly useful for prevention of CIPN. De. De9]. DAntagonists of RAGE or CXCR4, membrane receptors targeted by at-HMGB1, prevent the development of CIPN caused by paclitaxel  ,22, stroO-glycosylated serine-threonine-rich domain), D4 (transmembrane domain), and D5 (cytoplasmic domain). Thrombin binding to the D2 proteolytically converts protein C and TAFI into activated protein C (APC) and the activated form of TAFI (TAFIa), respectively , D2 (EGF-like domain), D3  has been demonstrated by nuclear magnetic resonance (NMR) and fluorescence studies (Table 1), and the IC50 of glycyrrhizin is 50 µM in inhibiting cell migration of 3T3 fibroblasts stimulated with HMGB1 at 1 nM [Glycyrrhizin (glycyrrhizic acid), a component of licorice root, possesses anti-inflammatory and antiviral activities . Direct  at 1 nM . It has  at 1 nM ,105, der at 1 nM , chemoth at 1 nM ,108, bra at 1 nM ,110, etcMethotrexate, a folic acid antagonist, is used in chemotherapy of tumors and autoimmune diseases including rheumatoid arthritis. Direct binding of methotrexate to two independent sites of HMGB1 has been demonstrated by surface plasmon resonance (SPR) analysis and electrophoretic mobility shift assay (EMSA) (Table 1) . MethotrMetformin, a biguanide derivative, is the first-line drug in the treatment of type 2 diabetes, and has also an anti-inflammatory activity. There is evidence that metformin directly binds to the C-terminal acidic tail of HMGB1, as demonstrated by a pull-down assay using full-length and C-terminal acidic tail-lacking HMGB1 (Table 1), an effect contributing to its anti-inflammatory effects . Metform(−)-Epigalocatechin-3-gllate (EGCG), a major effective component of green tea, is associated with many health benefits against multiple inflammatory diseases including rheumatoid arthritis . EGCG isKi = 25 nM), S100B (Ki = 230 nM), and HMGB1 (Ki = 148 nM) [SW/0APP mice [FPS-ZM1 was developed as a high-affinity RAGE-specific blocker through screening of 5000 compounds (Table 1) ,121. In W/0 mice  and bladW/0 mice  pain. FPW/0 mice  or oxaliKd value of 17 nM, as determined by SPR assay [Low molecular weight heparin , an anticoagulant that preferentially inhibits factor Xa rather than factor IIa (thrombin), binds to RAGE at PR assay , althougPR assay . LMWH stPR assay . LMWH alPR assay  and neurPR assay . As doesPR assay  or oxaliPR assay  , an orally bioavailable small molecule antagonist of RAGE that can penetrate BBB, is now being evaluated for efficacy and safety in patients with Alzheimer’s disease, because stimulation of RAGE by amyloid β is involved in neurodegeneration (Table 1) ,125,126.Rhodobacter sphaeroides (LPS-RS), one of the best-known TLR4 antagonists [Lipopolysaccharide of agonists  , a small molecule, selectively blocks CXCR4 (Table 1) . AMD3100As described above, ROS-dependent release of HMGB1 from macrophages plays a critical role in the development of CIPN in mice treated with paclitaxel , but notHMGB1 is now considered a key molecule responsible for the development of pathological pain including CIPN. HMGB1 and its upstream/downstream molecules are thus considered promising targets to develop agents to prevent CIPN. In particular, the clinical effectiveness of TMα, capable of inactivating HMGB1, in preventing the CIPN due to oxaliplatin in humans  is encou


Campylobacter spp., Escherichia coli, Salmonella, and Shigella spp.) from the USA, Peru, Egypt, Cambodia, and Kenya for the presence/absence of over 200 AMR determinants. We detected a total of 55 different determinants conferring resistance to ten different categories of antimicrobials: genes detected in ≥ 25 samples included blaTEM, tet(A), tet(B), mac(A), mac(B), aadA1/A2, strA, strB, sul1, sul2, qacEΔ1, cmr, and dfrA1. The number of determinants per strain ranged from none  to sixteen, with isolates from Egypt harboring a wider variety and greater number of genes per isolate than other sites. Two samples harbored carbapenemase genes, blaOXA-48 or blaNDM. Genes conferring resistance to azithromycin (ere(A), mph(A)/mph(K), erm(B)), a first-line therapeutic for severe diarrhea, were detected in over 10% of all Enterobacteriaceae tested: these included >25% of the Enterobacteriaceae from Egypt and Kenya. Forty-six percent of the Egyptian Enterobacteriaceae harbored genes encoding CTX-M-1 or CTX-M-9 families of extended-spectrum β-lactamases. Overall, the data provide cross-comparable resistome information to establish regional trends in support of international surveillance activities and potentially guide geospatially informed medical care.Infectious diarrhea affects over four billion individuals annually and causes over a million deaths each year. Though not typically prescribed for treatment of uncomplicated diarrheal disease, antimicrobials serve as a critical part of the armamentarium used to treat severe or persistent cases. Due to widespread over- and misuse of antimicrobials, there has been an alarming increase in global resistance, for which a standardized methodology for geographic surveillance would be highly beneficial. To demonstrate that a standardized methodology could be used to provide molecular surveillance of antimicrobial resistance (AMR) genes, we initiated a pilot study to test 130 diarrheal pathogens ( Diarrheal disease ranks among the top 10 leading causes of death for all ages and fifth for children under five years of age, affecting over four billion individuals globally each year in 2016 and 2017 . Low-incMany cases of intestinal disease are self-limiting and can be effectively treated with oral rehydration solutions and antisecretory/antimotility drugs ,8. WhileVeterinary and aquaculture use is also responsible for a large proportion of global antimicrobial consumption ,16,17,18Pertinent to the current study, there has been an alarming increase in resistance to drugs commonly used to treat diarrheal illness throughout the world ,10,24. TCompounding the problem of global AMR are geographical differences in pathogen and resistance profiles. Therefore, selecting an appropriate and effective therapeutic strategy for infectious diarrhea—when needed—requires situational awareness of the prevalence and types of AMR pathogens and mechanisms for resistance within each geographic region. To demonstrate that a standardized methodology could be used to provide cross-comparable molecular surveillance of AMR genes, we initiated a pilot study to test four genera of diarrheal pathogens for > 200 ARDs. Human clinical isolates were obtained from five geographic locations spanning four continents . Not surprisingly, testing in this manner elucidated some generalized trends in AMR in the three Enterobacteriaceae in various geographic regions.The United States (US) Department of Defense has established its Global Emerging Infections Surveillance and Response System (GEIS) to provide geographically relevant disease surveillance outside the continental US. While the GEIS centralized laboratories have developed standardized protocols for molecular characterization of infectious diseases, many of the surveillance laboratories implement their own protocols for disease detection, identification, and analysis, making comparisons between partner laboratories challenging. For this reason, we sought to demonstrate a harmonized strategy for molecular AMR surveillance in isolates collected at the US Centers for Disease Control and Prevention (CDC) and four GEIS laboratories with their own laboratory-specific and/or narrow spectrum protocols.Campylobacter spp., Escherichia coli, Salmonella, and Shigella spp. These genera were chosen to reflect the top four global bacterial diarrheal pathogens and were responsible for over 430,000 deaths in 2016 [In this study, we tested a set of diarrheal isolates collected at clinical sites in the USA, Cambodia, Egypt, Peru, and Kenya. Isolates included  in 2016 ,18,27,28 in 2016 . As a reblaGES (including extended-spectrum β-lactamase variants), blaIMI, blaIMP, blaKPC, blaOXA-23, blaOXA-24, blaOXA-51, blaOXA-58, blaSIM, blaSME, blaVIM. However, genes for OXA-48- and NDM-type carbapenemases were detected in one isolate each (described below). Given the TET-resistant selection criteria for inclusion in the study, we were somewhat surprised that fifteen isolates  were negative for all 38 TET-specific ARDs represented on the microarray. However, phenotypic TET resistance can arise from multiple intrinsic mechanisms not interrogated or detected by the microarray used here  v.2 used in this study [We detected a total of 55 different ARDs conferring resistance to eleven different categories of antimicrobials: β-lactams, aminoglycosides, macrolides, TETs, phenicols, streptothricins, sulfonamides, diaminopyrimidine (trimethoprim), ansamycins, and quaternary amines A. None oiewed by ,32): eliis study ,34,35,36Campylobacter spp. strains) to sixteen, with median and mean values of seven and 6.58 genes per isolate, respectively   B. Resist content .tet(O)/tet(32) (83%), the most common ARD for TET resistance in Campylobacter spp. Two samples were negative for all 38 TET resistance determinants on the microarray, and the third was positive for tet(M). It is likely that the two negative samples harbored tet(44) [cmeABC, or over-expression of one or more efflux pumps [cmeABC also contributes to fluoroquinolone resistance, which was also observed in 65% of the Camplyobacter spp. isolates where phenotypic data were reported. The tet(M)-positive sample was potentially a false-positive, as this gene has never been detected in Campylobacter spp.; however, this gene has the broadest host range of all TET ARDs [tet(M) reference sequence is 80% identical to tet(O)/tet(32) over the entire gene and some regions have significantly higher identity. Understanding that the microarray used here requires approx. 85–90% sequence identity for detection of similar genes (or families of genes) [All but three isolates harbored  tet44)   ,44, it i   was also observed in one of these isolates; this gene confers high-level resistance to macrolides such as azithromycin (AZM), which is a first-line therapeutic for campylobacteriosis in locations where fluoroquinolone (FQ) resistance is prevalent [erm(B)-aadE-aphA3-tet(O) clusters have been documented in a number of plasmids [erm(B) amongst Campylobacter spp., while not historically high, has recently increased in East Asia and Southern Africa [Four additional ARDs were detected amongst the  strains ,46. Erm, aph(2)-Ic) or not included in the microarray content (tet(44), ant(6)-Ib, blaOXA-61; aacA/aphD, aac, aad6, aad9, aph(2)-3a).Other ARDs occasionally observed in E. coli are responsible for over 100 million illnesses and 60,000 deaths each year [E. coli samples provided, 12 were categorized into pathotypes based on their mechanism of virulence: enterotoxigenic—three from Kenya, two from Cambodia, two from Peru; enteroaggregative—one each from Kenya and Cambodia; enterohemorrhagic—one from Kenya; enteropathogenic—one each from Cambodia and Peru. Pathotypes for the remaining samples were not provided.Diarrheagenic ach year . Of the E. coli strains (E. coli strains: tet(A) , tet(B) , tet(C) , and tet(D) . Two Egyptian isolates harbored both tet(A) and tet(B) genes, and four isolates were negative for all 38 TET genes represented on the microarray. Other genes with high prevalence included blaTEM (64%), strA and strB (70% each), mac(A) and mac(B) , cmr (83%), and sul2 (67%) (mph(A)/mph(K) in eight isolates (27%); genes in this family confer resistance to AZM, an alternative therapeutic for severe travelers’ diarrhea and shigellosis in both children and adults [E. coli infections, interspecies transfer of AZM resistance plasmids has been documented between E. coli and Shigella spp. [E. coli as a potential reservoir for ARDs causing clinically relevant drug resistance in Shigella spp.Thirty-seven unique ARDs were detected among the tested  strains . As expel2 (67%) C. Of potd adults ,50,51,52lla spp. ,55, furtE. coli strains (20%) harbored a gene encoding a CTX-M type of extended-spectrum β-lactamase (ESBL). Furthermore, some blaTEM and blaSHV alleles—detected in 63% of all E. coli strains—may also encode ESBLs. ESBL phenotype is conferred to TEM and SHV β-lactamases by a wide variety of single nucleotide polymorphisms . The microarray used here is unable to detect these SNPs and therefore cannot identify the blaTEM and blaSHV alleles. We anticipate that our estimate of approx. 20% ESBL carriage in E. coli  may be an underestimate; analogous situations hold for Salmonella and Shigella spp. strains. These results illustrate that techniques offering greater sequence resolution, such as amplicon or genome sequencing, may provide additional information that is important for molecular surveillance.Of additional concern, six of the blaNDM carbapenemase gene was detected in a single Egyptian E. coli isolate. Coincidentally, many of the ARDs often found on plasmids with blaNDM -Ib) were also observed in this isolate, suggesting that this gene might be plasmid-borne. However, we did not attempt to isolate or sequence plasmids from any of the isolates tested.The E. coli strains harbored both qacEΔ and sul1, which are components of the 3′-conserved region of many, but not all, class 1 integrons. Similarly, three E. coli isolates co-carried three genes commonly associated with class 2 integrons—dfrA1, aadA1/A2, and sat2—suggesting the presence of class 2 integrons within these isolates. Integrase-specific PCRs confirmed the presence of class 1 (intI1) and class 2 (intI2) integrons within all samples that co-carried these ARDs . These numbers likely underestimate the carriage of class 1 and class 2 integrons within this population, as they did not account for integrons with alternative structures lacking one or more “marker” genes [Four r” genes ,56,57,58strA, strB, and sul2 were highly correlated (p < 0.001) and co-carriage was observed in 64% of the E. coli strains. These genes are commonly observed as a cluster in plasmids of clinical Enterobacteriaceae isolates, as well as in integrative and conjugative elements in Vibrio spp. [mac(A) was highly correlated with mac(B) (p < 0.001)—not surprising, since these genes are typically found in a single operon [cmr (p < 0.001); while also part of the E. coli chromosome, cmr is found up to 40 kb away from the macAB operon.Other possible gene assemblages were detected. Positive results for rio spp. ,60. Simie operon . CarriagShigella spp. have a low infectious dose (only 10–100 organisms) compared to other diarrheagenic organisms and were the second leading cause of global diarrheal mortality in 2016 [Shigella spp. resistant to first-line therapeutics are therefore of tremendous concern, particularly in Asia and Africa [ in 2016 ,62. In cS. flexneri (13 isolates), S. sonnei (13 isolates), and Shigella sp. (12 isolates), with the remaining strains split between S. boydii (four isolates) and S. dysenteriae (three isolates). Thirty unique ARDs were detected amongst the Shigella spp. isolates .The 45 shigellae tested here were roughly divided between isolates —somewhatlmonella . The perE. coli are very closely related [Shigella spp. with similar frequencies as in E. coli: strA (56%), strB (58%), mac(A) (64%), mac(B) (58%), cmr (82%), and sul2 (64%) (tet(A) (39%) and tet(B) (59%); three strains were negative for all tested TET ARDs. Plasmid-borne FQ ARD, qnrS, was observed in two isolates, and the ansamycin resistance gene, arr, was detected in a Cambodian S. dysenteriae isolate. Notably, blaCTX-M-1 and blaCTX-M-9 family genes were detected at a much lower rate in the Shigella spp. strains than in E. coli—2% and 5%, respectively. Other notable differences between Shigella spp. and the closely related E. coli were the lower carriage of blaTEM (27% versus 63%) and mph(A)/mph(K) (9% versus 27%) and higher carriage of the blaOXA-1 family (34% versus 3%) in Shigella spp. isolates.As shigellae and  related , we werel2 (64%) C. TET ARE. coli, the presence of some sets of genes was highly correlated: strA, strB, and sul2 (p < 0.001); mac(A), mac(B), and cmr (p < 0.001); qacEΔ and sul2 (p < 0.001), and aadA1/A2, sat2, and dfrA1 (p < 0.005). IntI1-specific PCR confirmed the presence of class 1 integrons in 7% of the Shigella spp. strains, similar to observations in E. coli. However, intI2-specific PCR detected class 2 integrons in a much larger proportion of shigellae (29%). As only those isolates with integron-associated markers were tested, these numbers are likely underestimates. As with Shigella spp. strains (27%) carried the same four genes found in the Shigella resistance island , and catA1), a chromosomal pathogenicity island found in S. flexneri, S. sonnei, and S. dysenteriae with wide global distribution [Twelve ribution . No sampSalmonella gastroenteritis affects nearly 200 million persons, causing over 80,000 deaths in 2016 [S. enterica subsp. enterica serotype Typhi isolates were included amongst the 32 samples tested here. Serotypes for 21 additional non-typhoidal Salmonella (NTS) isolates were also provided: five isolates of serotype B  and one strain each of serotypes Newport, Anatum, Heidelberg, Dublin, and Agona ; one isolate each of serotype A and O (Kenya); one isolate of serogroup 3 (Egypt), and three isolates each from serogroups B and D, and two from serogroup C1 (Peru). in 2016 . As typh in 2016 ,66, fourSalmonella strains  (47%), tet(B) (22%), tet(C) (3%), tet(D) (6%), and tet(G) (6%). The following genes were also detected in a significant proportion of the Salmonella isolates: strB (63%), blaTEM (56%), strA (56%), qacEΔ, (53%), sul1 (53%), sul2 (50%), and aadA1/A2 (50%)  C.Salmonella strains (< 10%): blaCMY/LAT, PSE/CARBbla, blaOXA-48-like, aac(3)-Id, aac(3)-III, aadA7, rmtD, tet(G), floR, and sul3.A variety of genes not detected amongst the other Enterobacteriaceae isolates was observed in a few Salmonella isolates, conferring resistance to first-line therapeutics for NTS diarrhea in areas with multidrug resistance (MDR) [rmtD—detected in two isolates—is not clinically relevant for Salmonella-derived infections but confers resistance to all aminoglycosides and can be transferred horizontally to other species for which aminoglycosides serve as common therapeutics. Also alarming was detection of the carbapenemase gene, blaOXA-48, in a Kenyan Salmonella isolate. While its presence in Enterobacteriaceae in East Africa has recently been reported [Of clinical concern, genes encoding CTX-M-type ESBLs were detected in 12% of the ce (MDR) . On the reported ,68,69,70qacEΔ and sul1 genes were co-harbored in over half of the Salmonella strains, and the presence of intI1 in these strains was confirmed by PCR. The prevalence of class 1 integrons in the Salmonella strains was therefore significantly higher (51%) than observed in E. coli (13%) or Shigella spp. strains (7%). However, no Salmonella isolates co-harbored the combination of aadA1/A2 + sat2 + dfrA1, supporting previous observations that class 2 integrons are infrequently harbored by Salmonella [The lmonella ,72,73.Salmonella genomic island 1 , blaPSE/CARB) [aac(3)-id, aadA7, qacEΔ, sul1, tet(A), blaTEM) [Several isolates harbored ARDs commonly associated with SE/CARB)  or its S blaTEM) .E. coli (30), in Salmonella (23), and overall (39)  of the Egyptian Enterobacteriaceae harbored genes conferring resistance to AZM (two isolates with ere(A), five with mph(A)/mph(K)); Kenya was the only other site where AZM ARDs were carried at a similar rate (30% in Enterobacteriaceae).As a pilot-scale study, the numbers of isolates obtained per genus and per site hindered attempts to draw strong statistical conclusions from the data. However, a number of general trends were observed. Of the five collection sites surveyed, Egypt had the largest total number of ARDs detected in all (39) . Not surall (39) . The higall (39) . Over hacarriage ,77 and mcarriage ,79. FurtShigella spp., Salmonella spp., and all genera combined were also observed in the Cambodian samples -id, aadA7, qacEΔ, sul1, tet(A), blaTEM; [E. coli: aadA1, strA, strB, blaTEM, catA1, dfrA1, sul1, sul2, qacEΔ; [Other interesting observations included the detection of ARDs associated with SGI1-K  or plas, qacEΔ; ) in Afri, qacEΔ; ,84. WhilShigella spp. isolates also harbored ARDs associated with SRL, but carriage rates were not consistent between collection sites: the rates of co-carriage were much higher amongst Egyptian and Peruvian strains . These different carriage rates may be related to the prevalence of the various global Shigella spp. lineages (and related absence/presence of SRL) within each site [Twenty-seven percent (27%) of the ach site .sul and dfrA genes in Kenyan Enterobacteriaceae , species , serotypes , or sampled populations  vary between sites or were not fully characterized. Furthermore, Cambodian and Kenyan subject populations  tended to be younger than those from the USA or Peru , mac(B), cmr) were detected at lower-than-expected rates in Kenyan samples. More importantly, with regard to study design, it is also unclear whether the sample set used here—selected for TET resistance—accurately reflects the overall prevalence of various ARDs within the larger populations, including both resistant and susceptible pathogens from these collection sites; TET ARDs are commonly found in assemblages with other ARDs. Future study designs will emphasize a single species or serotype, with a larger number of samples collected from each site. Integrating a whole genome sequencing approach into a follow-on study would improve both the analytical depth and breadth. Whole genome sequencing—while more computationally intense—would enable rapid discrimination of closely related alleles or variants conferring different phenotypes -ib versus aac(6’)-ib-cr) and detect chromosomal mutations conferring resistance ; the microarray used here does not provide this capability. Next generation sequencing can further provide high resolution epidemiological tracking and “One Health” linkages [A key limitation of the current study is the low numbers and variability of isolates of each species obtained from the five sampling sites, constraining our ability to perform robust statistical comparisons. This limitation is further compounded when pathotypes . While the prevalence of ESBLs was alarming—especially in Egypt—we observed only two samples that harbored carbapenemase genes; other studies have reported much higher levels of carbapenemase-producing Enterobacteriaceae within fecal samples [The current study does address the presence of a large number of resistance markers covering multiple classes of antimicrobials across a diverse collection of organisms comprising four genera using a standardized and broad-spectrum detection platform. This approach allowed us to detect possible assemblages and make some surprising observations. With the exception of  samples ,104.qnrS, observed from all collection sites mirrored results from other studies [gyrA and parC in Enterobacteriaceae, which are not detectable by the microarray technology used here; the ability to interrogate samples for such point mutations would be an advantage of any sequencing-based approach.Genes conferring resistance to AZM were observed in > 10% of the entire population, with Enterobacteriaceae strains from Egypt and Kenya having the highest rates of carriage . The high prevalence of these ARDs is particularly concerning, as AZM has replaced FQs as a first-line therapeutic for moderate travelers’ diarrhea in Southeast Asia and for severe travelers’ diarrhea globally . The low studies ,107,108. studies ,109. HigWhile the selection criteria used here (TET resistance) may have artificially raised the overall prevalence of ARDs directed against other antimicrobials, we were somewhat surprised to see such a broad variety and high carriage rate of many ARDs in isolates collected from all sites. In particular, the large numbers of strains among the Egyptian samples positive for ARDs directed against WHO’s critically important antimicrobials  suggest an urgent need for improving awareness of AMR, more in-depth surveillance for resistant pathogens, and improved antimicrobial stewardship within this region.Campylobacter spp., 30 Escherichia coli, 32 Salmonella spp., and 45 Shigella spp. diarrheal isolates were selected from pre-existing collections at the US Centers for Disease Control and Prevention , US Army Medical Research Directorate, Africa/Kenya Microbiology Hub, Kericho , US Naval Medical Research Unit No. 2 , US Naval Medical Research Unit No. 3 , and US Naval Medical Research Unit No. 6 . Collection dates ranged from 2006 to 2013 , Mastern = 46 genes), aminoglycosides (n = 42), macrolides (n = 27), lincosamides (n = 22), streptogramins (n = 18), quaternary amines (n = 2), ansamycins (n = 1), diaminopyrimidines (n = 28), antimicrobial peptides (n = 1), tetracyclines (n = 38), phenicols (n = 10), glycopeptides (n = 12), platensimycin/platensin (n = 1), fluoroquinolones (n = 4), sulfonamides (n = 3). Many of the macrolide, lincosamide, and streptogramin ARDs overlap in specificity. Samples with > 85–90% gene sequence identity to the reference sequence can successfully hybridize to the microarray, allowing a broader variety of ARDs—i.e., families of genes—to be detected. However, the microarray is unable to distinguish between these similar genes. A gene was deemed present if at least 50% of its representative probes had signals above the 95% probe threshold  or if ≥ 70% of its probes had signals above either of two less stringent thresholds  [With the exception of Cambodia, all subsequent steps were performed in NRL facilities  as previously described ; all ste + 2 SD) ,113. Usi + 2 SD) ,44.intI1 and intI2 [qacEΔ1 + sul1 or aadA1/A2 + sat2 + dfrA1, respectively. Retrospective analysis of samples from Cambodia could not be performed.Where sufficient sample was available, PCR amplifications confirming the presence of nd intI2  were pert-tests and analysis of variance (ANOVA) when normally distributed and using Mann Whitney rank sum test when not normally distributed. Chi-square tests were used to compare binomial proportions (presence/absence) in independent samples (2 × n contingency tables). P < 0.05 indicates significance.Statistical comparisons between populations were performed using two-tailed This pilot study—using DNA preparations from 130 human diarrheal isolates—demonstrated that a standardized methodology could be used to provide molecular surveillance for over 200 AMR genes. We detected a wide variety of ARDs amongst the tested isolates and observed some generalized trends, although the small sample size limited our ability to make many conclusions with sufficient statistical power. Overall, we detected the greatest numbers and diversity of ARDs in isolates from Egypt and the lowest numbers and diversity in isolates from Cambodia. Though we could not make any conclusions regarding the phenotypes based on the ARDs detected, this preliminary study serves as a valuable starting point for more detailed follow-on investigations. The present study’s findings suggest that the implementation of molecular detection platforms with sufficient breadth—when used in conjunction with complementary techniques—have the ability to improve surveillance efforts that seek to monitor the migration and evolution of MDR organisms over time and space.