Capra hircus coreanae). The present study assessed the prevalence, risk factors, and co-infectivity of TBPs in ticks parasitizing NKGs. In total, 107 hard ticks, including Haemaphysalis longicornis, Ixodes nipponensis, and Haemaphysalis flava, were obtained from NKGs in South Korea between 2016 and 2019. In 40 tested tick pools, genes for four TBPs, namely Coxiella-like endosymbiont , Candidatus Rickettsia longicornii (45.0%), Anaplasma bovis (2.5%), and Theileria luwenshuni (5.0%) were detected. Ehrlichia, Bartonella spp., and severe fever with thrombocytopenia syndrome virus were not detected. To our knowledge, this is the first study to report CLE and T. luwenshuni in H. flava ticks in South Korea. Considering the high prevalence of Candidatus R. longicornii in ticks parasitizing NKGs, there is a possibility of its transmission from ticks to animals and humans. NKG ticks might be maintenance hosts for TBPs, and we recommend evaluation of the potential public health threat posed by TBP-infected ticks.Tick-borne pathogens (TBPs) are considered zoonotic re-emerging pathogens, with ticks playing important roles in their transmission and ecology. Previous studies in South Korea have examined TBPs residing in ticks; however, there is no phylogenetic information on TBPs in ticks parasitizing native Korean goat (NKG;  Ticks are considered the main arthropod vectors for infectious disease agents, and they are related to important veterinary and medical health issues . NumerouThe control of transmission of zoonotic pathogens with regard to vertebrate hosts and ticks, which are related in a continually changing environment, is usually difficult because of habitat distributions, zoonotic/domestic hosts, and transmission cycles in regions with coexistence of pastured domestic animals and wild animals . The gloCapra hircus coreanae) is an individual breed of goat primarily raised for natural health supplements and meat in South Korea, and it is known to be an essential natural reservoir host of several TBPs that influence human, wildlife, and domestic animal populations [Understanding the ecology of local tick species and recognizing TBPs have high health significance. There is a rapidly increasing number of reservoir-adapted pathogens among various species of ticks, which are known or suspected vectors of zoonotic TBPs. Previous studies have investigated the presence of TBPs in South Korea by assessing ticks and cattle, using molecular methods ,11,12,13ulations ,15. As NAnaplasma, Ehrlichia, and Rickettsia), Bartonella spp., Coxiella burnetii, piroplasms (Babesia and Theileria), and severe fever with thrombocytopenia syndrome virus (SFTSV), among ticks from NKGs in South Korea.In this study, TBP surveillance was conducted to detect tick species parasitizing goats, and to identify related tick-borne zoonotic pathogens possibly negatively influencing medical health and veterinary medicine in South Korea. The study assessed the prevalence, risk factors, and co-infectivity of TBPs, including rickettsiae  from NKGs. In the tick samples, we used universal primers for the mitochondrial cytochrome c oxidase subunit I (cox1) gene to amplify 710-bp fragments. The cox1 gene was obtained and sequenced from representative ticks to eliminate potential issues in identification, especially among nymphs and larvae. In this study, the cox1 gene sequences were divided into three groups according to nucleotide identity. The three groups shared close genetic relationships with H. longicornis (98.7–100% nucleotide identity), H. flava (98.8–100% nucleotide identity), and I. nipponensis (99.2–100% nucleotide identity). A phylogenetic tree was created according to the cox1 genes of several ticks deposited in GenBank, and the collected ticks were divided into three clades related to the following three species , H. flava , and I. nipponensis  (We obtained 107 ticks (40 tick pools) involving two genera and three species ( species : H. long0 pools) .Anaplasma bovis , Coxiella-like endosymbiont (CLE) , and unidentified Rickettsia  were detected in the ticks  was detected in the ticks (Rickettsia citrate synthase (gltA) . The 16S rRNA sequences of he ticks . Additiohe ticks . Among tT. luwenshuni, CLE, and unidentified Rickettsia, and one (2.5%) was co-infected with A. bovis and T. luwenshuni. Ehrlichia spp., Bartonella spp., and SFTSV were not detected in this study.With regard to infection, one (2.5%) tick was co-infected with H. longicornis nymph was positive for A. bovis; one (6.7%) H. longicornis nymph and one (25.0%) H. flava adult were positive for T. luwenshuni; one (6.7%) H. longicornis nymph and one (25.0%) H. flava adult were positive for CLE; and eight (53.3%) H. longicornis nymphs and 10 (100%) H. longicornis adults were positive for unidentified Rickettsia.With regard to each pathogen, one (6.7%) A. bovis , MN842273–MN842274 (H. flava), MN842275–MN842278 (H. longicornis), MN836550 (A. bovis), MN833312–MN833313 (T. luwenshuni), MN836551–MN836552 (CLE), MN836547–MN836549 (unidentified Rickettsia 16S rRNA), and MN842268–MN842270 (unidentified Rickettsia gltA).The identified representative sequences were submitted to GenBank. The accession numbers are as follows: MN842271–MN842272 (H. longicornis (62.5%), H. flava (25.0%), and I. nipponensis (12.5%) in NKGs, and the most common species was H. longicornis. These findings are similar to the results of a previous South Korean study, which found 569 H. longicornis organisms  from 27 goat farms [The present study found cox1 sequences from collected H. longicornis showed 97.1–99.6% nucleotide identity with known cox1 sequences of H. longicornis  from Korean water deer [H. longicornis ticks  [H. longicornis ticks  from goats in North Korea [Anaplasma spp. have been detected in NKGs  [H. longicornis nymph from an NKG was positive for A. bovis.Several TBPs, including ivestock . In natud humans . A. boviountries . A. bovinditions . A. boviter deer , and in th Korea . Moreove%, 7/39) . In the T. luwenshuni mainly infects small ruminants and is transmitted by H. longicornis [T. luwenshuni has been previously identified in roe deer , and H. longicornis  [T. luwenshuni (one H. longicornis nymph and one H. flava adult). To our knowledge, the present study is the first to report the presence of T. luwenshuni in H. flava ticks in Korea.Theileriosis, which is a tick-borne hemoprotozoan disease, can affect domestic animals, most frequently sheep and cattle in tropical and sub-tropical areas, and it causes high economic loss . T. luwegicornis . In Sout3 pools) , and in er 100%, /23, and C. burnetii and is a zoonosis, has a global distribution, and it can result in a severe condition in animals and humans [C. burnetii, and some of the organisms might behave like symbionts that are engaged in complex interactions with ticks. It is considered that CLE are closely related to, but genetically distinct from, C. burnetii, suggesting extensive diversity within Coxiella. Through multilocus sequencing, Coxiella has been divided into at least four extremely divergent genetic clades (A to D), and C. burnetii has been categorized within clade A [C. burnetii was previously identified in NKGs  [H. longicornis ticks  from horses [H. longicornis nymph and one H. flava adult). To our knowledge, the present study is the first to report the presence of CLE in H. flava ticks in Korea. However, in this study, C. burnetii was not detected. Additional investigations are needed to evaluate the prevalence of infection involving C. burnetii or CLE among ticks and NKGs.Q fever, which is caused by d humans . The bio clade A . In Sout 57/597) , and CLERickettsia have been divided into various groups, including the typhus group, spotted fever group (SFG), Rickettsia canadensis group, and Rickettsia bellii group [Candidatus status was detected. A possibly novel SFG rickettsia classified into the putative new subgroup “Candidatus Rickettsia longicornii” was identified in the species H. longicornis  in South Korea [H. longicornis in Japan, China, and South Korea have unknown pathogenicity and uncertain taxonomic statuses that are likely associated with Candidatus R. longicornii or a closely connected species [Candidatus Rickettsia jingxinensis from H. longicornis  in China [Rickettsia species were found to be the same species by phylogenetic analysis involving several genes [Rickettsia spp. in H. longicornis were Candidatus R. longicornii  in South Korea [Candidatus R. jingxinensis  in China [Rickettsia spp. (45.0%) were detected in eight H. longicornis nymphs and 10 H. longicornis adults, and the pathogen was identified as Candidatus R. longicornii by phylogenetic analysis. Further surveys are needed to identify other novel Rickettsia spp. in ticks and their host animals.Organisms from the genus ii group . Obligatii group . The sigii group . In thisth Korea . These s species . The othin China . The twoal genes . In a prth Korea  and Candin China . MoreoveThe bootstrap support values need to be analyzed carefully. As a limitation in this study, some bootstrap values were low. This could be due to using small amplification fragments for phylogeny. Thus, an additional study is needed to analyze longer fragments of genes in phylogeny for better presentation of data.Candidatus R. longicornii. To the best of our knowledge, this is the first report of CLE and T. luwenshuni in H. flava ticks from South Korea. Additionally, there is a high prevalence of Candidatus R. longicornii in ticks parasitizing NKGs. Both local inhabitants of NKG farms and animals are at high risk of exposure to coxiellosis, anaplasmosis, theileriosis, and rickettsiosis. The present findings extend our knowledge of the distribution and probable vector spectrum of TBPs and suggest that ticks are a potential reservoir for TBP transmission to other animals and humans via bites. Investigation of the tick infestation risk is important, especially from the zoonotic and public health perspectives. These data associated with a high TBP prevalence in ticks indicate the requirement for further studies on the impacts of tick-borne diseases in animals and humans. Better understanding of local major tick species and thorough TBP characterization are essential to public health. Limited information is available about the TBPs of ticks from small ruminants, such as NKG, in South Korea. Although limited numbers of ticks were collected from goats in this study, tick abundance and distribution patterns were similar to those in previous studies [H. longicornis, followed by H. flava and I. nipponensis. Thus, we believe that the present findings extend our knowledge of the distribution and probable vector spectrum of TBPs.In conclusion, four TBPs were identified in ticks obtained from NKGs in South Korea, including zoonotic pathogens of CLE and  studies ,17, showIn 2018, the total number of NKGs reared in South Korea was reported to be 542,744 from 14,644 farms . NKGs ar® ProFi Taq PCR Premix Kit . Molecular identification of tick species was conducted by amplifying the sequence of the cox1 gene using specific primers [A commercial DNeasy Blood & Tissue Kit  was used according to the provided instructions for genomic DNA extraction from the ticks. The extracted DNA was kept at −20 °C. Polymerase chain reaction (PCR) amplification was conducted using the AccuPower primers .® Rickettsiales 3-Plex PCR Kit (Bioneer) for the detection of rickettsiae 16S rRNA. For species identification, positive samples were then amplified further. For Rickettsia spp., positive samples were confirmed by PCR that targeted gltA [Coxiella (including C. burnetii and CLE) [Bartonella spp. [®Babesia & Theileria PCR Kit (Bioneer) to detect piroplasm 18S rRNA. Positive samples were then re-amplified by PCR using primers designed from the common sequence of the 18S rRNA gene of some piroplasm species [The ticks were then screened for several TBPs with primers specific to each pathogen. The presence of rickettsiae was initially assessed by PCR with a commercial AccuPowerted gltA . Multipland CLE) . Additiolla spp. . The S slla spp. . Piropla species .All primers and amplification conditions used for the detection of TBPs in ticks from NKGs in the present study are described in Escherichia coli DH5α-competent cells , and the cells were incubated at 37 °C overnight. Plasmid DNA was extracted using the Plasmid Miniprep Kit (Qiagen), according to the provided instructions.Agarose gel extraction kits (Qiagen) were used to purify positive PCR products. The purified products were introduced into pGEM-T Easy vectors , according to the provided instructions. The ligated products were then transformed into Sequencing was performed using recombinant plasmid clones. The sequences were analyzed and aligned with the multiple sequence alignment CLUSTAL Omega (v. 1.2.2) program, and the alignments were corrected with BioEdit (v. 7.0.4). Phylogeny was assessed with the maximum likelihood method and the Kimura 2-parameter distance model in MEGA (v. 5.0). Sequence analysis involved a similarity matrix. For tree stability, bootstrap analysis with 1,000 replicates was performed.


Salmonella spp. were multidrug resistant. Sixteen serotypes were detected, but only serotype Infantis confirmed reduced susceptibility to colistin. Seven colistin resistant Salmonella Infantis were studied in detail using the WGS approach. Three sequence types were identified corresponding to different epizootiology region. The isolate from the Province of Vojvodina 3842 and isolates from Jagodina (92 and 821) are represented by the sequence type ST413 and ST11, respectively. Four isolates from Kraljevo are ST32, a common S. Infantis sequence type in humans, poultry and food. The fosfomycin resistance gene fosA7 in isolate 3842 and the vgaA gene in isolate 8418/2948 encoding resistance to pleuromutilins were reported for the first time in serovar Infantis. The changes in relative expression of the phoP/Q, mgrB and pmrA/B genes were detected. Single nucleotide polymorphisms of the pmrB gene, including transitions Val164Gly or Val164Met, and Arg92Pro are described. Analyses of quinolone resistance determining region revealed substitutions Ser83Tyr in GyrA protein and Thr57Ser and Ser80Arg in ParC protein. Based on WGS data, there are two major clusters among analyzed Salmonella Infantis isolates from central Serbia.The antimicrobial susceptibility testing was conducted on 174 single isolates from poultry farms in Serbia and it was determined that seven  Salmonella Infantis has been ranked among five most prevalent infection-causing serovars in humans [S. Enteritidis, S. Infantis serovar tends to develop multidrug resistant phenotype more often. It has been recognized as a persistent clone, causing long lasting contamination at poultry farms in Europe and Japan [S. Infantis epidemic clone was identified in a rehabilitation center of an oncology clinic in Germany, indicating that poultry and/or kitchen contamination may present a possible reservoir of infection [S. Infantis isolates in Israel [Salmonella in humans by facilitating clonal competition [S. Infantis strains carrying large conjugative plasmid with tetA gene and integron 1 were identified in Hungary. These isolates belong to various, but closely related pulsotypes, including the new B2 type established in humans and poultry hosts, not only in Hungary, but also in other European countries [S. Infantis isolates from the USA, Latin America, Japan, and Russia as well [S. Infantis is an important serovar in human medicine and food producing animals frequently serve as a vehicle of transmission. This serovar is difficult to eradicate at poultry farms due to lack of serovar specific vaccines. Not only that S. Infantis have the ability to develop multiple mechanisms of antimicrobial resistance, but some strains produce extended-spectrum-β-lactamase enzymes [S. Infantis isolates from broilers in Italy [S. Infantis isolates from humans, poultry, and food from Serbia from 2008–2011 were resistant to ciprofloxacin due to mutations in gyrA and parC genes. At the time, the resistance to colistin was not investigated [Salmonella enterica isolates from poultry farms and determination of colistin minimal inhibitory concentration was included in the study. In addition, the whole genome sequencing (WGS) was used for in-depth analysis of resistance and virulence genes in colistin-resistant S. Infantis strains, as well as for establishing genetic relatedness between those strains.As reported by the World Health Organization (WHO), salmonellosis is taking third place among foodborne diseases in humans, causing death. In European Union, n humans . Unlike nd Japan ,3. In adnfection . Such con Israel . It was petition . Multidrountries ,8. The m as well . Therefo enzymes ,11,12,13in Italy . Since cstigated . TherefoSalmonella spp. isolates from poultry farms in Serbia. S. Infantis is a well-established clone in poultry flocks all around the world due to extremely persistent infections in poultry and contamination of the environment [Due to public health significance of resistance to polymixins in Gram-negative bacteria, a research was undertaken to evaluate resistance to antibiotics, including colistin, in ironment ,5,16. OnSalmonella spp. isolates. Those were six isolates of serovars Infantis, and one isolate of serovar Stanley, serovar Kotbus and Typhimurium. Multidrug resistance in Salmonella spp. is often detected in well-established worldwide clones such as S. Typhimurium definite phage type 104 (STDT104) or S. Kentucky, sequence type 198, which are frequently resistant to ciprofloxacin [S. Typhimurium isolates was resistant to multiple antibiotics . It is interesting to note that S. Typhimurium isolates from poultry in Serbia are susceptible to antibiotics and that this serovar is rarely detected in other food-producing animals. The lack of surveillance and monitoring of antimicrobial resistance in veterinary medicine in Serbia (presently) or the use of less sensitive methods for Salmonella detection in some veterinary laboratories could explain the low numbers of S. Typhimurium isolates in food-producing animals. Out of the seven S. Infantis isolates included in the WGS analysis four were both multidrug-resistant and with reduced susceptibility to colistin (MICs from 4 µg/mL to >16 µg/mL), and three were susceptible to 14 antibiotics but were resistant to colistin only. Therefore, the acquired resistance to colistin may be easily overlooked in isolates susceptible to some other antibiotics.Antimicrobial resistance profiles of isolates respective to resistotypes included in this study are listed in floxacin ,18. HoweS. Infantis isolates with reduced susceptibility to colistin were sequenced using Illumina HiSeq 2500 platform. The contig dataset was used to determine functional analysis. MLST analysis of seven S. Infantis colistin-resistant isolates resulted in identification of three sequence types from Serbian poultry farms, ST32 , ST11 (isolates 92 and 821) and ST413 (isolate 3842)  . It was Salmonella spp. [pmrA/pmrB genes induced by colistin reduce the negative charge of the LPS, which results in lower susceptibility to this antibiotic in Salmonella enterica [mcr1 gene. This was first discovered during routine monitoring in China by Liu et al. (2015) [Escherichia coli isolates from food-producing animals. The plasmid-mediated the mcr1 gene was transferable to other bacterial species. Further investigation revealed mobile resistance to colistin in E. coli isolates from patients as well. After the initial report from China, several new variants of the mcr1 gene (mrc1-mcr9) were further identified all over the world [mcr gene can become a transposon-mediated gene or may also be found in the chromosome of E. coli [Due to the growing problem of antibiotic resistance in Gram negative bacteria, polymyxin E (colistin) and polymyxin B are used as valuable antibiotics for the therapy of complicated infections in humans. However, bacteria may develop acquired resistance to polymyxins utilizing different levels of resistance, mostly toward adaptive mechanisms . Among dlla spp. . Cationienterica . Another. (2015)  in commehe world . These dhe world . In addi E. coli . The ovepmrB gene, resulting in Val164Met (isolates 9181/1 and 9520/2), Val164Gly (isolate 1371/1), and Arg92Pro (isolate 8418/2948) amino acid substitutions. Mutations in the phoPQ, pmrA, and mgrB genes were not found in the analyzed genomes , plasmid mediated resistance (PMQR) and antibiotic efflux driven by efflux pumps. Analysis of quinolone resistance-determining region revealed mutations in the gyrA and parC genes of all ST32 isolates and in the parC gene for ST413 isolate. Four isolates of ST32 group  had mutation in the gyrA gene, resulting in Ser83Tyr amino acid transitions. Mutation resulting in Thr57Ser transition was found in the parC gene of five isolates from both ST32 and ST413 groups  and Ser80Arg was found only in the parC gene of one isolate (14.29%). Mutations in gyrB and parE genes were not found in the analyzed genomes  Iaa confers resistance to tobramycin, kanamycin and amikacin and this gene was found in S. Infantis isolates from this paper  or by tetA and tetK genes (8418/2948 and 9520/2).The aminoglycoside acetyltransferase gene is paper . Previouificance . Resistaate 3842 . This geidelberg . The proablished . The vgaus ST398 ,35. Thislla spp. , but in e et al. . ResistaS. Infantis ST11 (isolate 92). pMLST profile was IncF RST, with FIB_22 and FIIS_1 alleles identified. FIB_22 allele was identified within NODE_36 of isolate 92 genomic sequence, which shared 98.91% of identity with plasmid pSJUTF10978  of S. Enteriditis strain SJTUF10978. FIIS_1 allele was identified within NODE_64 of isolate 92 genomic sequence, which shared 100% identity with plasmid pPT1-1  of S. Enteriditis strain PT-1. However, replicon typing PCR developed by Caratolli et al., [S. Infantis isolates 9520/2, 9181/1, 8418/2948, and 1371/1 possess IncP plasmids while isolate 821 has plasmid IncFIIA. In addition, the PCR replicon typing system confirmed the existence of the IncFIIA plasmid in isolate No. 92. This result could indicate the high prevalence of extrachromosomal DNA in our isolates, and thus the possibility of horizontal gene transfer potential and history. In the recent research of McMillan et al. [Salmonella spp. isolates had at least one plasmid and that many of them carried significant number of resistance genes. All of the isolates assessed in their research were from the NARMS isolate collection obtained from food animals and a few S. Heidelberg strains from humansPlasmid contigs were found only in  et al.,  has reven et al. , it was In silico search for virulence determinants revealed presence of nonfimbrial and fimbrial adherence determinants, genes for magnesium uptake, phoPQ, macrophage inducible genes (mig-5 or mig-14), TTSS encoded by SPI-1, SPI-2, and both TTSS-1 and TTSS-2 translocated effectors. Besides those general factors, the rck serum resistance factor, spvABR and ACE T6SS were present in genomes of isolates belonging to sequence type ST11 , involved in host tropism and pathogenicity. This new strain has replaced classical S. Infantis within a few years in Israel and, surprisingly, the large plasmid increased the fitness of the strain [S. Infantis with the endemic multiresistant plasmid pSI54/04 was also detected in Hungary, but the plasmid transfer to pre-emergent strain did not increase virulence in one day old chicken after challenge experiments [Salmonella serovars is the Typhi colonization factor Tcf due to the role of this fimbria in pathogenicity. In S. Infantis, Tcf is responsible for increased colonizing capacity of mouse intestine, while this gene expression was lower in S. Swarzengrund and S. Heidelberg compared to S. Infantis [S. Infantis from this paper also possess Tcf virulence factors and five isolates  possess adherence factor K88, suggesting that those properties may play a role in persistent intestinal infection of poultry in Serbia as well  harbor a variety of virulence genes that are required for interaction with hosts and they are differently arranged through the genome of Salmonella. These pathogenicity islands carry genes that are involved in host invasion, systemic infection, replication within macrophages, macrophage apoptosis mechanism, and they carry genes that encode effector proteins of type III secretion system T3SS and T2SS, which are important for pathogenicity [genicity ,43.Salmonella Infantis is presented in S. Infanits from this paper as well  and only one cas gene in isolate 3842 (Cas3_0_IE) . However, 8 s3_0_IE) .S. Infantis isolates revealed that the analyzed genomes are phylogenetically related, as they share a high number of common genes . Transcription of the mgrB gene was downregulated in isolates belonging to ST11 (821 and 92) and one ST32 isolate (9181/1), but upregulated in two isolates belonging to ST32 (8418/2948 and 9520/2), as well as ST413 (3842)  while expression of the pmrB gene was upregulated in isolates 92 and 1371/1  A. The led 1371/1 B. AlthouSalmonella enterica isolates. Samples were collected during official routine monitoring program, as stated in the Official Gazette of the Republic of Serbia, No. 36: The rulebook for early detection, diagnostics, prevention, suppresion and eradication of particulate Salmonella serotypes in poultry flocks. Therefore, the samples were overshoes or feces collected from poultry farms. The following serovars were identified through the study: S. Infantis were represented by 51 isolates, S. Enteritidis by 78 isolates, S. Typhimurium by 15 isolates, S. Tennessee by 6 isolates, S. Seftenberg by 5 isolates, S. Mbandaka by 3 isolates, S. Newport, S. Hadar, S. Kottbus, and S. Yoruba by 2 isolates while S. Bovismorbificans, S. Nitra, S. Stanleyville, S. Virchow, S. Napoli, S. Stanley and S. Thompson were represented by 1 isolate each. One isolate was typed as group E (O:19). Except for S. Infantis, and S. Enteritidis, all other serovars were sent to the Institute of Public Health of Serbia, Reference Laboratory for Salmonella, Shigella, Vibrio cholere, and Yersinia enterocolitica, Belgrade, Serbia for serological identification. Results of antimicrobial susceptibility testing including determination of colistin MIC are provided in One hundred and seventy-four single isolates from poultry farms in Serbia were included in the study. Five veterinary institutes from the north to the south of the country participated by providing Escherichia coli ATCC 25922 was included in the study, each day when antimicrobial susceptibility testing was performed. Multiresistance was designated if isolates were resistant to three or more antibiotics of different classes [S. Enteritidis were susceptible to antibiotics included in antimicrobial susceptibility testing.Susceptibility testing was done by disk diffusion method. Interpretation of the results was performed according to recommendation of the Clinical and Laboratory Standards Institute CLSI (documents number M07-A10 and M100-S25) ,48. The  classes . S. EnteE. coli ATCC 25922 and E. coli NCTC 13846 were included on each plate. The interpretation of MIC values for ATCC 25922 control strain recommended by European Committee on Antimicrobial Susceptibility Testing (EUCAST) range from 0.25–2 mg/L. The NCTC 13846 results in MIC of 4 mg/L. The MIC analysis of Salmonella spp. were interpreted according to EU Directive 2013/652/EU [S. Enteritidis was excluded from the MIC analysis because clinical breakpoint of colistin for this serovar is not established [The minimal inhibitory concentration (MIC) analyses were done according to Gwozdzinski et al. (2018)  by broth3/652/EU , which rablished .S. Infantis isolates was sequenced using Illumina HiSeq  and the quality of each genome was checked using FastQC [http://www.mgc.ac.cn/VFs/main.htm) [https://card.mcmaster.ca/), ResFinder 3.1 (www.genomicepidemiology.org). In addition, presence of resistance-conferring mutations in QRDR and pmrAB, phoPQ, and mgrB genes was confirmed using DNA Strider with corresponding genes of S. Infantis 1326/28 (GenBank GCA_000953495.1), which were used as a negative control. Plasmidome (PlasmidFinder 2.0), plasmid MLST (pMLST 2.0), multilocus sequence typing (MLST 2.0) and the presence of Salmonella pathogenicity islands (SPIfinder 1.0) were analyzed using databases of the Center for Genomic Epidemiology (www.genomicepidemiology.org). Additionally, CRISPRs and cas genes were detected using the CRISPRCasFinder program (https://crisprcas.i2bc.paris-saclay.fr/CrisprCasFinder/Index). Draft genome sequences of seven S. Infantis isolates have been deposited at the NCBI GenBank database under accession numbers JAAGKW000000000-JAAGLB000000000 and JAALLI000000000.Genomic DNA of seven g FastQC . Genome ain.htm) . GeneticS. Infantis isolates. Genome sequences were firstly annotated using Prokka (version 1.13) and obtained annotated GFF files were further clustered by Roary (version 3.13.0) into core genes (selection threshold for hard core genes was presence in >99% of the isolates and for soft core genes threshold was presence in 95–99% of isolates) and accessory genes . Phylogenetic tree was created by Roary and visualized by Phandango. Genomic diversity among seven S. Infantis isolates and identification of strain specific features were determined using BPGA tool.Pan-genome analysis approach was used in order to compare genomes of seven S. Infantis isolates were incubated in Luria–Bertani (LB) broth supplemented with 2 µg/mL of colistin sulfate (Sigma-Aldrich) at 37 °C with shaking overnight. The same overnight cultures were used for isolation of DNA for genome sequencing and for further RNA isolation experiments. RNA was isolated from those overnight cultures diluted in the fresh LB broth supplemented with colistin sulfate (2 µg/mL) after reaching OD600 value of 0.5. RT-qPCR was used for determination of listed genes transcription level: phoP, phoQ, mgrB, pmrA, and pmrB. Primers used in RT-qPCR are listed in gyrB gene using the ΔΔCT method (relative). The obtained values were then normalized against results for colistin-susceptible S. Infantis isolate 9060. RT-qPCR experiments were done in triplicate.Isolation of the total RNA from bacterial cells, DNase I treatment and reverse transcription were done as previously reported  In briefS. Infantis isolate 9060. Values at p < 0.05 were considered to be statistically significant.All results are represented as mean values ± standard deviations. One way ANOVA, followed by Tuckey’s post hoc test was used to compare differences in results obtained for colistin-resistant isolates and colistin-susceptible S. Infantis was not detected, perhaps due to the more responsible use of enrofloxacin antibiotic in the past few years. However, the resistance to nalidixic acid is still worrying, indicating that prudent use of antibiotics must continue in poultry industry in Serbia. Fluctuations in phoPQ, pmrAB, and mgrB mRNA levels that are usually associated with colistin-resistance phenotype were described. Additionally, mutations leading to amino acid susbtitutions were found within the pmrB gene. WGS approach has helped identify the peculiar but important genetic differences among S. Infantis, which will help with future epidemiological studies worldwide.In this paper, resistance to fluoroquinolones in


Systomus and recently many new species were described under this genus. Systomus sarana subnasutus is considered a valid subspecies of S. sarana sarana although revisions have been done by some researchers. We employed a combination of morpho-meristics and molecular tools  to resolve the two species. Three morpho-meristic characters, head length/maxillary barbel length (HL/MxBL), Lateral Line Scales (LLSs) as well as two truss-based characters, had discernible variation between the two taxa. The sequence analysis (2353 nucleotides) depicted a separate clad of S. sarana subnasutus with high bootstrap support. The findings from combined use of morphology, meristics and mitogenes were concordant. The corroborative results suggest the possibility of two different species. The results suggest to adopt suitable management measures, accordingly.Taxonomic ambiguity exists in genus  Systomus  is an economically important group, comprising of 19 fish species native to tropical Asia  considered it as a valid subspecies. Dahanukar  tree was generated with 1000 replications (Saitou and Nei To ascertain the inter-relatedness of the two subspecies of p < .05) differences between the two taxa. However, discriminant analysis of significant variables demonstrated three variables namely, head length/maxillary barbel length (HL/MxBL), lateral line scales (LLSs) and vertebrae counts (VCs). Significant (p < .05) Fisher’s distances between the groups were observed.The ANOVA revealed eight morphometric ratios and four meristic characters to exhibit significant (p < .05) at 57 transformed morphometric characters, out of a total 90. PCA extracted 10 principal components accounting for 92.2% of the total variation. HL/MxBL, LLSs, VCs, eye diameter and distance between pectoral fin origin to operculum differentiated the two Systomus species.The ANOVA revealed that the samples of two species differed significantly (S. sarana sarana (19) and S. sarana subnasutus (18), revealed six haplotypes, with two and four haplotypes were found, respectively  from two species, ectively . A totalS. sarana subnasutus and S. sarana sarana were 2.4% and 2.5%, respectively (Systomus (Sahayadria) group was presented when Indian major carp species were used as outgroups. However, the polyphyletic clustering was obvious for Systomus and Sahayadria genera. The topology is similar to our own work (under review) which supported the distinctiveness of three taxa  did not exhibit significant differences. However, the average genetic distance between the ectively . NJ treeree taxa . FurtherS. sarana sarana (n = 15) and S. sarana subnasutus (n = 18). The genetic distance was 0.18% within taxa, while 0.5% observed between individuals of S. sarana sarana and S. sarana subnasutus. Analysis of Cytochrome b (1141 bp) presented 1090 conserved sites, whereas 51 were variable ones. Pairwise genetic distance between S. sarana sarana and S. sarana subnasutus was 3.73%, whereas different populations of both the species showed low genetic distance (0.89% and 0.35%), respectively. The NJ tree highlighted similar tree topologies with high bootstrap (71–100)  revealed two haplotypes, each from (71–100)  for bothPuntius has been considered one of the largest group among subfamily cyprininae. This subfamily is represented by over 120 valid species, widely distributed in south and south-east Asia. With all these revisions, the species of genus Puntius , Sahyadria and Pethia. Sarana group of genus Systomus is a complex consisting of S. sarana sarana and its three valid subspecies viz. S. sarana spilurus, S. sarana subnasutus and S. sarana orphoides.Previously, several researchers demonstrated the taxonomic status of both species Menon . The genHamilton  along wiTraditional morphology is often used for species identification by gathering and analysing, data from large sample sizes, though molecular markers are proven to be more robust in reconstructing phylogenies. The combined use of the morphological characters and mitochondrial data provides a strong framework for discriminating species, compared to the use of morphological characters alone. In the present investigation, we used molecular evidences  to corroborate the taxonomic status of two species.S. sarana sarana differs significantly from S. sarana subnasutus. Earlier, Jayaram (Puntius reported that in P. sarana sarana (Hamilton), LLSs are 30–34 with mostly 31 or 32; and all populations are distributed north of the Krishna river system in Southern India, while its subspecies, P. sarana subnasutus , the LLSs range 28–31 and all populations were distributed in Krishna river (and south to it) in the Peninsular India. In P. sarana sarana contains a dark blotch on lateral line before base of caudal fin which is distinguishable with the similar blotch at 24th scale in P. sarana subnasutus (Jayaram The overall results from this study indicated that  Jayaram , in his  Jayaram .S. sarana sarana, eye diameter is larger than S. sarana subnasutus. Similarly, distance between pectoral fin origin to operculum is larger in S. sarana sarana compared to S. sarana subnasutus. In this study, truss morphometry also yielded concordant results. Truss-based differentiation has been used in several fish species (Cavalcanti et al. S. sarana sarana and S. sarana subnasutus on the basis of NJ phylogram which was supported by high bootstrap values in all three mitochondrial markers. The pattern of clustering demonstrated their separate existence.In S. sarana subnasutus is a valid species and distinct from the S. sarana sarana. However, taxonomic re-description is required for elevating from subspecies to species level. The findings are important for conservation and management of resources.Conclusively, with the molecular evidence corroborating to the morphological inferences, this study strongly advocates that there is probability that


Metabolic diseases are an increasing problem in society with the brain-metabolic axis as a master regulator of the human body for sustaining homeostasis under metabolic stress. However, metabolic inflammation and disease will trigger sustained activation of the hypothalamic-pituitary-adrenal axis. In this study, we investigated the role of metabolic stress on progenitor cells in the hypothalamic-pituitary-adrenal axis.In vitro, we applied insulin and leptin to murine progenitor cells isolated from the pituitary and adrenal cortex and examined the role of these hormones on proliferation and differentiation. In vivo, we investigated two different mouse models of metabolic disease, obesity in leptin-deficient ob/ob mice and obesity achieved via feeding with a high-fat diet.Npy and Agrp changed in both the hypothalamus and adrenal.Insulin was shown to lead to enhanced proliferation and differentiation of both pituitary and adrenocortical progenitors. No alterations in the progenitors were noted in our chronic metabolic stress models. However, hyperactivation of the hypothalamic-pituitary-adrenal axis was observed and the expression of the appetite-regulating genes It is well-known that chronic stress and stress hormones such as glucocorticoids can induce metabolic changes including obesity and diabetes. In this article, we show for the first time that this might be based on an early sensitization of stem cells of the hypothalamic-pituitary-adrenal axis. Thus, pituitary and adrenal progenitor cells exposed to high levels of insulin are metabolically primed to a hyper-functional state leading to enhanced hormone production. Likewise, obese animals exhibit a hyperactive hypothalamic-pituitary-adrenal axis leading to adrenal hyperplasia. This might explain how stress in early life can increase the risk for developing metabolic syndrome in adulthood. •Insulin enhances proliferation and differentiation of adrenocortical and pituitary progenitors.•Obesity leads to hyperactivation and priming of the HPA axis.•Obesity leads to overexpression of appetite-regulating genes in the hypothalamus.•Obesity leads to a decrease in the expression of appetite-regulating genes in the adrenal gland. DysreTo regulate body weight, the hypothalamus must interpret and integrate incoming signals such as levels of glucose, free fatty acids, and amino acids but also hormones such as leptin and insulin from the adipose tissue and pancreas, respectively. The hypothalamus then synthesizes and secretes corticotropin-releasing hormone (CRH), which regulates pituitary adrenocorticotropic hormone (ACTH) secretion, leading to glucocorticoid release from the adrenal cortex . In addiPomc (among others encoding ACTH), Npy, and Lep . W. W38]. Wnditions . Leptin-cultures B. Howevenchanged B.Figure 3.2Cyp11b1), Cyp11a1, and Nr5a1 (gene encoding SF1). Leptin treatment had no effect on the expression of differentiation markers compared to the control  cells maintained the expression of eYFP irrespective of Nestin expression, allowing lineage tracing of Nestin (+) cells. After further 7 days in proliferative conditions with treatment, the culture conditions were changed to induce differentiation. After 7 days in differentiation conditions, cells were fixed and immunostained for the zG marker RGS4 [We next investigated whether Nestin (+) cells treated with insulin preferentially differentiate into cells in the zG or zona fasciculata (zF). We isolated adrenocortical cells from ker RGS4 . The perThese results indicated that under basal conditions, adrenocortical Nestin (+) cells were primarily non-committed and slowly differentiated into aldosterone- (zG) and corticosterone-producing (zF) cells. Conversely, high levels of insulin led to enhanced differentiation, particularly into zG cells, accompanied by higher steroid secretion. Not all the RGS4 (+) cells were positive for YFP. Therefore, it must be considered that absolute recombination was not achieved or that other stem/progenitor cells were also able to differentiate into RGS4 (+) cells.3.3in vitro under clonogenic stem/progenitor-promoting conditions, which only allow uncommitted cells to survive [in vitro in medium containing different concentrations of insulin and leptin. We did not observe any significant differences in the number of colonies  cells remained positive for Nestin  cells migrate centripetally from the adrenal capsule toward the adreno-medullary boundary, becoming positive for the steroidogenic markers StAR, CYP11A1, and CYP11B2 . To deter Nestin . Similar/ob mice , indicat3.7ob/ob mice, we carried out immunofluorescence staining with antibodies against Ki67. We did not observe any difference in the percentage of Ki67(+) cells. Nestin (+) pituitary progenitors might represent a subpopulation of SOX2 (+) stem cells, which also reside in the anterior lobe and play an important role in long-term physiological maintenance of the adult pituitary gland [ob/ob animals. Moreover, the appearance and localization of SOX2 (+) cells were unchanged  cells in both the adrenal cortex and anterior lobe of the pituitary gland. We used concentrations of insulin that were very high compared to physiological concentrations [in vitro, in which we did not observe any effect on pituitary stem cells at low  concentrations of insulin or leptin. At physiological concentrations, insulin activates the insulin receptor, leading to the activation of the PI3K/Akt pathway. At higher concentrations, signaling can also act via IGF receptors [In the present study, we have shown that trations  as it watrations . Furthertrations . The samtrations ,62. Thiseceptors , suggesteceptors ,64. IGF-eceptors .Nes in the proliferation cultures. However, lineage tracing revealed that adrenocortical Nestin (+) cells showed an enhanced differentiation into particularly zG cells in the presence of insulin , as their morbid obesity and T2D are similar to those observed in humans, making them a good model without any interfering factors [ob/ob females [ob/ob mice, since all of the steroidogenic genes were upregulated in these animals. Conversely, in the severely diseased 10-month-old obese mice, a downregulation of Star and Cyp11b2 was detected. We inconsistently observed an expansion of the adrenal cortex after 10 months of progressive obesity. A possible explanation might be chronic inflammation, which typically accompanies symptoms of obesity, especially in long-lasting manifestations [A T2D model that has been widely studied is  factors . Althoug factors . Various factors . However females . Our obsstations ,75. Therstations ,24.in vitro and in vivo [Both our systems were chronic stress models running over several months. This might explain why we did not observe any significant changes in the gene expression of stem/progenitor cells at the end of the experiments. This was supported by our previous restraint stress studies, in which we observed an extended proliferation of progenitors in short stress experiments, but after a resting period, homeostasis was re-achieved ,43. To r in vivo .ob/ob mice. Consistent with our restraint stress model, we ascertained higher Npy and Agrp expression levels that consequently may lead to increased stress-induced food intake and therefore an increased vulnerability to developing metabolic disorders. AgRP is likely to have an autocrine/paracrine role in the adrenal glands, with adrenal-derived AgRP being regulated by glucocorticoids and blocking the induction of corticosterone secretion by alpha-melanocyte-stimulating hormone [ob/ob mice and 20-week HFD model showed lower expression of Npy and Agrp in adrenal glands. Furthermore, both metabolic and restraint stress led to an attenuated expression of Agrp in adrenal glands, suggesting shared underlying adaptive stress response mechanisms. This emphasizes the complex interplay between nutritional status in mammals and stress hormone regulation. Not only internal stressors, such as factors regulating feeding behavior, are capable of shaping the endocrine stress axis, but an inverse effect also exists. This at least in part explains the shared biology of obesity and maladaptive processes in the endocrine stress response [In general, stress is defined as a challenge to an individual's homeostasis . The bid hormone ,78. Bothresponse .5In conclusion, we have shown that insulin activates stem cells of the HPA axis by enhancing their proliferation and differentiation, leading to augmented hormone production. We have also demonstrated that animal models of chronic metabolic stress lead to hyperactivation of the HPA axis. We recently introduced the concept of stress-inducible stem cells and discussed if stress affects stem/progenitor cells through epigenetic modifications whereby they will be predisposed to adult disease . OngoingM.W., H.M., C.L.A., S.R.B., and C.S. designed this study. M.W., I.B., L.D.S., A. Sa., P.S., H.B., and M.P. conducted the research. M.W., I.B., L.D.S., A. Sa., A. Sc., A.J.F.K., S.R.B, and C.S. analyzed the data. M.W. and C.S. wrote the paper. S.R.B. and C.S. supervised the study.We thank Uta Lehnert and Linda Friedrich for technical assistance. Erwin Weiss from the FACS facility at the CMCB Technology Platform at TU Dresden is thanked for his assistance with the cell sorting.10.13039/501100001659Deutsche Forschungsgemeinschaft  project no. 314061271, TRR 205/1: “The Adrenal: Central Relay in Health and Disease” and project no. 288034826, IRTG 2251: “Immunological and Cellular Strategies in Metabolic Disease”.This work was supported by the The authors declare no competing interests.


Staphylococcal enterotoxins are the most common cause of foodborne intoxications  and cause a wide range of diseases. With at least six variants staphylococcal enterotoxin C (SEC) stands out as particularly diverse amongst the 25 known staphylococcal enterotoxins. Some variants present unique and even host-specific features. Here, we review the role of SEC in human and animal health with a particular focus on its role as a causative agent for foodborne intoxications. We highlight structural features unique to SEC and its variants, particularly, the emetic and superantigen activity, as well as the roles of SEC in mastitis and in dairy products. Information about the genetic organization as well as regulatory mechanisms including the accessory gene regulator and food-related stressors are provided. Staphylococcus aureus in food and cause intoxication upon ingestion. In the EU alone, 114 cases of foodborne outbreaks were attributed to these heat-stable SEs in 2018 [Foodborne illnesses are one of the world’s leading health issues. They are estimated to cause 420,000 deaths every year and generate health costs and economic losses in the range of 110 billion USD worldwide [ in 2018 ,4.S. aureus during growth, alongside a number of other virulence factors. The organism can not only cause food intoxications but also various infectious diseases including toxic shock syndrome (TSS) [lZ) have been described, excluding variants and TSST-1 (formerly SEF), but new types are frequently discovered [l) [lZ are termed "new enterotoxins". Historically, classical toxins were demonstrated to be emetically active in rhesus monkey feeding assays, while new toxins were either not emetic or had not been tested in monkeys [SEs are produced by me (TSS) ,6,7. Stame (TSS) . Staphylme (TSS) ,10 and tme (TSS)  and foodme (TSS) ,13 have me (TSS) . So far,scovered . Non-emeered [l) . These pered [l) ,17,18. Tered [l) ,20, shorered [l) ,22. All  monkeys ,23. Howe monkeys , and new monkeys ,26. Addi monkeys ,28. So fbovine and SECovine show the exceptional ability of S. aureus to adapt to different niches. SEC is by far the most-frequently-isolated toxin in animals suffering from mastitis [S. aureus infection. [S. aureus or its toxins into the food-chain. In addition to TSS, mastitis, and food intoxication SEC seems to play a critical role in the development of infective endocarditis and atopic dermatitis [Originally, SEs were differentiated by immunological methods, whereas today it is recommended to identify the relationship of new toxins via sequence homology  . SEC takmastitis ,32,33,34fection. . There afection. . Contamirmatitis ,37. Othermatitis .Staphylococcus species can produce SEC and variants such as SECcanine [S. aureus SECs. The great variety of SEs and their genomic location further complicates the intricately-intertwined regulatory pathways of S. aureus virulence factors. Previously, SEs in general [An overview of different SEC variants and their relationships is given in ECcanine , this re general ,16,40,41 general ,43 have S. aureus survives very poorly in gastric juice [S. aureus intestinal carriage rate ranges from 8–31% in the healthy population [S. aureus.A striking feature of SEs is their emetic activity. It provokes vomiting, the key symptom in SFP. Some SEs, but not SEC also cause diarrhea . The exaic juice . Still, pulation . It is tS. aureus growth. The pathogen is usually introduced by food handlers and produces SEs if conditions allow bacterial growth in the food matrix [S. aureus but SEs remain emetically active [S. aureus virulence factors [S. aureus [Most SFP outbreaks are attributed to SEA, although there might be some bias since enterotoxins other than SEA–SEE cannot be detected using commercial kits. Many strains produce multiple toxins and it is often unclear which enterotoxin was the cause for SFP. Synergistic effects of different toxins are also likely. Some outbreaks could be traced back to SEC presumably being the only SE involved ,99,100. d matrix . Most cad matrix . Due to y active . SEs arey active . Once SEy active . This pr factors . SEA was factors ,105. How factors . The rol. aureus . The inv. aureus . The bas1 [2 [3 [2 was also tested in the house musk shrew [Whether SEC variants have a different emetic potential is unclear. SEC1 , SEC2 [21 [2 [3  have beesk shrew . TherefoS. aureus was found with a prevalence of up to 71% [Intoxications caused by SEs are the most common cause of food poisoning after consumption of raw milk or products made thereof. SEC and SEA are the most-commonly-occurring SEs in milk and dairy products ,111,112.p to 71% . This lip to 71% .S. aureus was grown in milk the expression of SEC was significantly reduced. Downregulation of the agr system (see chapter 5) likely contributed to the observed reduction, but other factors are also expected to be involved [bovine in milk and during cheesemaking. S. aureus numbers increased during cheesemaking but did generally not reach the >108 CFU/mL required for SECbovine detection in this food matrix. The influence of the added starter culture was not investigated in detail [Weissella paramesenteroides GIR16L4 or Lactobacillus rhamnosus D1 or both together used as starter cultures on the expression of S. aureus SEC was examined. The starter cultures were not able to reduce S. aureus growth, but they influenced toxin expression in some strains [S. aureus and therefore influence agr-regulated SEs. In another trial, Staphylococcus vitulinus was used as a starter culture to successfully inhibit SEC-producing S. aureus growth in a barbeque cheese production facility [The effect of milk on SE production has been investigated experimentally. When involved . The effinvolved  and highinvolved . The amon detail . Recentl strains . Lactic facility .All SEs are classified as SAgs. In contrast to normal antigens, they bind to MHCII in a location adjacent to the peptide groove. They thus stimulate T-cell receptors (TCRs) in a non-specific way by cross-bridging them with major histocompatibility complex class II (MHCII) on antigen-presenting cells (APC). The resulting global overstimulation of T-cells interferes with immune system functions that normally counteract bacterial infections . In infeS. aureus [The exact location of MHCII binding varies, depending on the SE. Group II antigens, including SEC, bind to the low-affinity α-chain . The res. aureus .1–3: C1: 3.2, 6.4, 6.9, 12, 15.1; C2: 12, 13, 14, 15, 17, 20; and C3: 5.1, 12 [bovine has been shown to specifically activate the Vβ repertoire of cattle [ovine strains showed a particularly-strong response in cattle peripheral blood mononuclear cells (PBMCs) [S. aureus to adapt to different host species [1 was able to still induce T-cell proliferation, but SEC2 and SEC3 were not. A few specific amino acids near the NH2-terminus seem to be responsible for this difference [TCR binding is specific to the variable region Vβ of the receptor ,122 Fig. This re 5.1, 12 ,124,125.f cattle . Further (PBMCs) . This ad species . Even infference . HoweverAnother factor adding to the immunostimulatory properties of SEs is their ability to co-bind CD28 on T-cells . This haOne of the consequences of the overshooting T-cell proliferation is potentially fatal toxic shock syndrome (TSS). TSS was originally attributed to toxic shock syndrome toxin (TSST-1) and the use of high-absorbing tampons . HoweverAll SEs share almost identical structural features, although their amino acid sequences vary. In addition, they are structurally related to streptococcal SAgs such as streptococcal pyrogenic exotoxins (SPEs). They are water-soluble peptides with a length of 220–270 aa and have a molecular weight of ~22–29 kDa . Mature 2 and SEC4 are almost identical with only two aa differences, while SEC3 and SECovine differ the most with 18 aa. Sequence similarity amongst SEC variants is at least 93%  present in all emetic SEs and absent in some weakly- or non-emetic types has been implicated in the toxin’s emetic activity (in the N-terminus between β4 and β5). Structural analyses in SEC have confirmed the important role of the disulphide loop in emesis. Substitution of the two cysteines to alanine in SEC did not . This wa did not . These fMutational analysis in SEA revealed that emetic and superantigenic properties can be attributed to different regions of the peptide, hence separating emesis and superantigenic activity as two different functions of SEs ,145. In sec) are generally located on a S. aureus pathogenicity islands (SaPIs), but plasmids carrying sec have also been described [S. aureus evolution via horizontal gene transfer. They derived from prophages and require helper phages for transmission [sec gene has been described in SaPIbov1, SaPIn1/m1, SaPImw2, SaPIov1, and SePI1. Moreover a BLAST search resulted in SaPINuSaα2, SaPITokyo, and SaPIbov5  [Genes coding for SEC  agr) that uses autoinducing peptides (AIP). It acts on toxin transcription indirectly via RNAIII that represses the repressor of toxins (rot) [B, and SaeRS may play a role in regulation of SEC when environmental factors change  ,151,152.s change  [45,153,pression . In a lapression . In an opression .sec mRNA and SEC protein levels were considerably reduced [In conclusion, under both glucose and NaCl stress  reduced ,149,155.S. aureus occurrence by CFU/gram food, regardless of the respective toxin expression, reflect these shortcomings.Many challenges arise from the great variety of SEs. Firstly, new SEs cannot yet be detected by commercial assays. Therefore, reports on the frequency of SEs associated with intoxication and disease are likely biased towards classical SEs. Secondly, the influence of the food matrix and host factors can influence toxin expression in strain-specific, toxin-specific, and even variant-specific ways. Additionally, the genetic location of SEs may play a role in genetic regulation. Nonetheless, many studies do not account for these aspects. Legislative documents limiting For SEC variants in particular, it remains unknown whether genetic location or external stressors result in variant-specific responses in genetic regulation. Furthermore, although agr undoubtedly plays an important role in SEC regulation, additional regulatory elements should be considered in future studies. Whether SEC variants differ in their effects on human and animal health has not been resolved. The different Vβ-specificities of SEC variants likely trigger slightly different superantigenic responses. Hence, some variants might be more relevant in acute diseases while others could be implicated with chronic or persistent progressions. Additionally, variants have never been compared directly with respect to their emetic activity. Therefore, correlations between variants and emetic dosage cannot be provided.In conclusion, aspects such as toxin variants, strain-specific responses, and influences of external stressors should be considered when investigating SEC expression in the future, and results should not be generalized. Taking all of these factors into consideration will contribute to predicting and maintaining high food safety standards and improving human and animal health.


Staphylococcus epidermidis and Staphylococcus capitis can be found on the skin of virtually every human being. Here, we applied a culture-independent amplicon sequencing approach to identify staphylococcal species on the skin of healthy human individuals. While S. epidermidis and S. capitis were found as primary residents of back skin, surprisingly, the third most abundant member was Staphylococcus saccharolyticus, a relatively unstudied species. A search of skin metagenomic datasets detected sequences identical to the genome of S. saccharolyticus in diverse skin sites, including the back, forehead, and elbow pit. Although described as a slow-growing anaerobic species, a re-evaluation of its growth behavior showed that S. saccharolyticus can grow under oxic conditions, and, in particular, in a CO2-rich atmosphere. We argue here that S. saccharolyticus was largely overlooked in previous culture-dependent and -independent studies, due to its requirement for fastidious growth conditions and the lack of reference genome sequences, respectively. Future studies are needed to unravel the microbiology and host-interacting properties of S. saccharolyticus and its role as a prevalent skin colonizer.Coagulase-negative staphylococcal species constitute an important part of the human skin microbiota. In particular, facultative anaerobic species such as  In recent years, new discoveries regarding the composition and functionality of the human skin microbiota have been made, that have enabled a more comprehensive description of this ecosystem ,2,3. StuStaphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus hominis occupy many human skin sites S. saccharolyticus within available metagenomes deposited in the Sequence Read Archive (SRA) was initially assessed using the online tool at www.searchsra.org to provide a broad overview of datasets with matches. A more detailed investigation of the level of representation of S. saccharolyticus in existing human skin and other human-associated metagenomic datasets  was then conducted by mapping pooled sequencing reads from metagenomic datasets against the S. saccharolyticus genome sequence . Sequencing reads were obtained from the SRA and processed using Geneious Prime 2020 to remove low quality reads  and duplicates (Dedupe from BBTools), with default parameters. The resulting collections of high-quality reads were mapped against the genome sequences of S. saccharolyticus 05B0362, Cutibacterium acnes strain ATCC 6919 (NZ_CP023676.1) and Staphylococcus epidermidis strain ATCC 14990 (NZ_CP035288.1) using the Geneious Prime 2020 map to reference tool with the following criteria: 100% identity; no gaps or mismatches; maximum ambiguity = 1. For each metagenomic dataset mapped, the total number of reads mapped to each reference genome was normalized by the total size of the dataset, to provide reads mapped per megabase DNA. The presence of sequences similar to S. saccharolyticus-specific gene as a query to search metagenomic datasets derived from back skin [hya gene encoding a hyaluronate lysase was chosen (locus tag DMB78_01130 in the genome of strain 05B0362), due to its low average nucleotide identify with hya genes from other staphylococcal species. The search was performed as an SRA nucleotide BLAST. The gene was considered as being present when the coverage exceeded 40%.A more targeted search strategy was also applied using an ack skin . The hyaS. saccharolyticus (strain DVP5-16-4677) was grown on Fastidious anaerobic agar (FAA) plates  and incubated anaerobically at 37 °C for 4 days. For liquid growth, brain-heart infusion-yeast broth supplemented with 0.05% (w/vol) cysteine (BHCY broth) was used. The following growth conditions were evaluated and performed at 37 °C: anoxic conditions , and oxic conditions with and without CO2-supplementation . Optical density (OD) data at 600 nm was determined until the stationary growth phase. tuf gene fragment was applied on swab material derived from the upper back of 19 healthy volunteers, to determine the diversity and relative abundance of staphylococcal species. In total, twelve different staphylococcal species were identified . The four most abundant staphylococcal species identified were S. epidermidis (average abundance 34.0%), S. capitis (26.6%), S. saccharolyticus (20.5%), and S. hominis (6.5%) (S. saccharolyticus was identified in 8 out of the 19 samples (42%) tested and, if present, was a dominant species, comprising a minimum of 10.9% and a maximum of 90.4% of the total reads (Amplicon sequencing based on a entified . The majs (6.5%) B. S. sacal reads C.S. saccharolyticus was detected in 8 out of 19 back skin samples tested, we next decided to determine the prevalence and distribution of sequences with similarity to S. saccharolyticus within existing skin metagenomic datasets, using two different search strategies. First, sequencing reads from all skin metagenomes derived from back, forehead, and armpit samples (n = 43) [S. saccharolyticus genome with high stringency . As a comparison, we also mapped the same reads to the genomes of two bacterial species known to be abundant on human skin, Cutibacterium acnes and S. epidermidis. Reads mapping to the S. saccharolyticus genome were found in all 43 skin metagenomic datasets searched  identified as carrying sequences with similarity to S. saccharolyticus, using the SRA metagenomic search tool. Sequencing reads from these human-associated metagenomic datasets were also found to map to all bacterial genomes assessed, but at magnitudes of order lower levels than observed for human skin datasets. Given that (n = 43) , availabsearched . The perS. saccharolyticus. Using this approach, we found high-stringency matches (reads with 100% identity) within three of the twelve volunteers at multiple time points . Instead, it grows on Trypticase soy yeast (TSY) agar plates supplemented with 0.5% Tween-80 or FAA plates when incubated for 72 to 96 h under anaerobic conditions (data not shown). Not much is known about its growth in broth. Thus, we recorded the growth of S. saccharolyticus in brain-heart infusion-yeast broth supplemented with 0.05% cysteine (BHCY medium). Different incubation conditions were applied, including anaerobic and aerobic conditions and the supplementation with CO2 (2-enriched conditions (approx. 6% CO2 and 15% O2), compared to anaerobic conditions. Growth yields were reduced under atmospheric conditions. Our data indicate that with CO2 . ResultsS. epidermidis was found to be the most abundant staphylococcal species on human back skin, followed by S. capitis. Surprisingly, S. saccharolyticus ranked third. This species has previously not been reported to be abundant on human skin. However, two studies from 1978 reported the presence of S. saccharolyticus, formerly named Peptococcus saccharolyticus, in forehead and armpit skin samples [S. saccharolyticus. The organism grew on TSY (supplemented with 0.5% Tween-80) agar plates after 4–7 days of incubation, with preference for anaerobic conditions. Identification (and differentiation from other CoNS) was based on cell and colony morphology, anaerobic growth preference, and a weak catalase activity. Biochemically, S. saccharolyticus cannot produce lactic acid from glucose (in contrast to other staphylococci); it can ferment glucose, fructose, and glycerol, but not maltose. Interestingly, Evans et al. [S. epidermidis might outcompete S. saccharolyticus on (standard) agar plates. This could explain why S. saccharolyticus was overlooked in culture-dependent studies. The skin microbiome affects the health-state of our skin. Understanding the bacterial composition on non-diseased skin is therefore of importance. Here, we focused on the staphylococcal composition of the human back skin. Similar to most previous studies ,2, S. ep samples ,18. In ts et al.  stated ts et al. ,18. In aS. saccharolyticus was previously not detected in culture-independent studies, which are nowadays more frequently conducted. Many culture-independent studies are carried out using 16S rRNA gene amplicon sequencing, which relies on sufficient differences in the 16S rRNA gene to distinguish species. However, the 16S rRNA gene of S. saccharolyticus does not carry many single nucleotide polymorphisms (SNPs) that can easily distinguish it from other CoNS, namely S. capitis [S. saccharolyticus can be indistinguishable from S. capitis.However, this does not explain why  capitis . Thus, dS. saccharolyticus, no such reference genome was available before March 2019. Adding to the confusion, three genomes assigned to S. saccharolyticus were publicly available in GenBank before 2019, but these were wrongly classified as S. saccharolyticus, and actually belong to S. capitis, as previously noted [S. capitis. Besides the genomes of seven S. saccharolyticus strains that have been sequenced during our previous study [S. saccharolyticus (ATCC 14953/NCTC 11807) has been sequenced by two independent teams (WGS projects UHDZ01 and RXWW01), resulting in nearly identical genome sequences. Taken together, before March 2019 there was no correct reference genome sequence of S. saccharolyticus available; this has been now resolved. Thus, current and future shotgun sequencing studies should be able to identify S. saccharolyticus correctly. In recent years, shotgun sequencing was employed to identify the skin metagenome ,2. Howevly noted . They weus study , the typtuf gene fragment, with primers that were designed by Martineau et al. [tuf gene, encoding the elongation factor Tu, is a highly conserved gene in all staphylococcal species. We modified this method by choosing different amplification primers, since we noticed that the reverse primer designed by Martineau et al. [tuf gene sequence in the genome of S. saccharolyticus. It is thus likely that the primers of Martineau et al. [tuf gene of S. saccharolyticus, or only with reduced efficiency. Here, we employed an amplicon sequencing method that is a modification from an existing method by Strube et al. . The metu et al. . The tufu et al.  has two u et al.  do not aS. saccharolyticus has a preference for anaerobic growth conditions [2-rich atmosphere is comparable to the growth under anaerobic conditions. A mechanistic explanation of the effect of CO2 on bacterial growth was recently published by Fan et al. [2 and the CO2-dependency of small colony variants of S. aureus, whose growth defect can be compensated by increased CO2/bicarbonate supplementation. They found that staphylococci employ a CO2-concentrating mechanism that enables them to grow at atmospheric CO2 levels. More specifically, they found that the system MpsAB is crucial for S. aureus growth at atmospheric CO2 levels. From a set of carefully designed experiments, they concluded that the MpsAB system represents a dissolved inorganic carbon transporter, or bicarbonate concentrating system, which creates an elevated concentration of intracellular bicarbonate. Consequently, a staphylococcal species without a functional MpsAB system would only grow poorly at atmospheric CO2 levels, and would rely on increased CO2 concentrations for accelerated growth. As we previously noted that the genome of S. saccharolyticus contains over 300 frameshift mutations [mpsAB genes. Indeed, the mpsAB genes in the genome of S. saccharolyticus are frameshifted, and thus the MpsAB system is most likely non-functional in S. saccharolyticus. In conclusion, the lack of a functional CO2-concentrating system in S. saccharolyticus is a likely explanation for its insufficient growth under atmospheric conditions. This can be compensated by providing increased CO2 concentrations.Previous studies reported that nditions ,18. Heren et al. . The aututations , we checS. saccharolyticus look like? In this regard, an interesting feature of S. saccharolyticus is the presence of a hyaluronate lyase (Hya), which is absent in other human-associated skin-resident CoNS. Closest homologs of the hya gene of S. saccharolyticus are present in two animal-associated staphylococci: Staphylococcus agnetis and Staphylococcus hyicus. S. agnetis is associated with lameness in broiler chickens, and S. hyicus causes skin diseases, such as exudative dermatitis in piglets [hya, possibly from an animal-associated staphylococcal species, contributed to a lifestyle switch of S. saccharolyticus. Hyaluronate lyases degrade hyaluronic acid, a major polysaccharide of the extracellular matrix of tissues [S. saccharolyticus is likely better equipped to penetrate and propagate in deeper layers of the epidermis. We further speculate that a strong host association in deeper layers might have been established, where S. saccharolyticus would have access to a range of host-derived compounds including amino acids and cofactors. This in turn would render bacterial genes to synthesize such compounds dispensable. As a consequence, genome decay would be accelerated, aiming at a slimmer, less energy-consuming lifestyle that is adapted to an oxygen-depleted niche, i.e., the epidermis below the stratum corneum. Many questions remain, e.g., regarding the preferred niche of S. saccharolyticus on human skin and its evolutionary history that can be regarded as an example of reductive evolution, indicative of the massive genome decay . Genomic piglets ,28. It i tissues . In the  tissues . Thus, aS. saccharolyticus in human disease. The microorganism has been described as the etiologic agent of infective endocarditis, empyema and bone and joint infections such as shoulder synovitis and vertebral osteomyelitis [S. saccharolyticus in implant-associated infections, such as prosthetic valve endocarditis [S. saccharolyticus was identified from tissue biopsies [S. saccharolyticus is widespread on human skin, as our study results suggest, one would expect to see more reports regarding the potential disease association of this bacterium, as seen for example for other skin-resident CoNS, such as S. epidermidis and S. capitis. As outlined above in detail, we hypothesize that mainly due to the fastidious growth conditions, S. saccharolyticus was overlooked in numerous disease cases, in particular in implant-associated infections, as such infections are often caused by skin-derived bacteria, including CoNS. In several cases, S. saccharolyticus has been identified, but was labeled as contaminant [S. saccharolyticus in disease requires thorough investigations to exclude skin-derived contamination of the biopsy material or contamination during subsequent sample processing steps. Carefully designed and executed future studies are needed to elucidate the etiology and frequency of S. saccharolyticus in human disease.Another open question remains regarding the clinical significance of these findings. At present, only few studies, mainly case reports, have reported the involvement of myelitis ,34,35,36myelitis . A few rcarditis  and we rbiopsies . If S. staminant . As alson = 19) and the focus on back skin. Moreover, only relatively young participants were investigated in this study. Appropriate skin sampling methods have previously been discussed [S. saccharolyticus.The study has some limitations, most importantly the small sample size (iscussed ; due to S. saccharolyticus is relatively often found in human skin samples, as judged from a culture-independent amplicon sequencing approach. When present, the organism can comprise a major part of the staphylococcal skin population, and is found in several different skin sites. It has yet to be shown in the future if skin that is primarily colonized with S. saccharolyticus has distinguishable features from skin colonized with S. epidermidis or S. capitis. In conclusion, here we found that the coagulase-negative species


The microbiota plays an important role in host health. Although rubidium (Rb) has been used to study its effects on depression and cancers, the interaction between microbial commensals and Rb is still unexplored. To gain the knowledge of the relationship between Rb and microbes, 51 mice receiving RbCl-based treatment and 13 untreated mice were evaluated for their characteristics and bacterial microbiome changes.Rikenellaceae, Alistipes, Clostridium XlVa and sulfate-reducing bacteria including Deltaproteobacteria, Desulfovibrionales, Desulfovibrionaceae and Desulfovibrio, but significantly inhibited the abundances of Tenericutes, Mollicutes, Anaeroplasmatales, Anaeroplasmataceae and Anaeroplasma lineages. With regarding to the archaea, we only observed two less richness archaea Sulfolobus and Acidiplasma at the genus level.The 16S ribosomal RNA gene sequencing of fecal microbiota showed that RbCl generally maintained fecal microbial community diversity, while the shifts in fecal microbial composition were apparent after RbCl exposure. RbCl significantly enhanced the abundances of Changes of fecal microbes may in part contribute to the anticancer or anti-depressant effects of RbCl. These findings further validate that the microbiome could be a target for therapeutic intervention.The online version contains supplementary material available at 10.1186/s12866-021-02095-4. Rubdium (Rb) is found in air, soil, water and organisms, and is a less studied alkali metal element and can efficiently transfer to the human body through the food chain (soil-plant-human) . Since t+ levels in the body [Researchers found that many cancers were caused by the changes of Rbthe body , 9. Somethe body , 10. Thethe body , 12. Latthe body . AlthougFusobacterium nucleatum, Enterococcus faecalis, Bacteroides fragilis, and Escherichia coli, etc., were enriched in the stool of CRC patients and promoted the development of CRC [Firmicutes and Bacteroidetes, might be involved in the occurrence and the development of depression [Lactobacillus and Bifdobacterium were reported to be beneficial to the treatment of depression [The microbiome is a dynamic ecological community which mainly includes bacteria, archaea, fungi and viruses . There it of CRC . Some repression . Meanwhipression , 23.However, to our best knowledge, no work has been reported on the effect of chemical element Rb on the microbiome, and whether Rb inhibits tumor and depression thorough changing the community composition of microbiome is still not clarified. Therefore, the present study was to investigate the relationship between the addition of rubidium chloride (RbCl) and composition of fecal microbiome in order to further understand the mechanism of Rb against cancer and neurological disease from the perspective of fecal microbial community.To better understand the effects of RbCl on mice, we conducted a follow-up study of 64 mice and recorded body weights and multiple organ weights of each mouse. Changes of the body weights in all the groups were shown in Fig. P = 0.9401; Sobs, P = 0.5239; Ace, P = 0.7497; Shannon, P = 0.5082; Simpson, P = 0.5401), middle-dose  and high-dose groups . In addition, to assess the effect of the different treatments on the assembly of bacterial communities, we compared the β-diversity (between-samples diversity) using Bray Curtis distances and performed constrained principal coordinate analysis (CPCoA). This analysis revealed a clear differentiation of samples belonging to the control, low-dose, middle-dose, and high-dose groups that explained as much as 6.62% of the overall variance of the data . Thus, the above results showed that RbCl did not affect the diversity and richness of the fecal microbial community in general. However, it altered the structure of the fecal bacterial community, reflected in changes in fecal microbial composition.From the results of 16S rRNA gene sequencing, we obtained a total of 1,481,388 high-quality reads for 64 fecal samples of four groups, which could be clustered into 486 OTUs. Figure Firmicutes was the dominant phylum with average abundances of 51.03, 50.18, 47.15 and 43.73% in control, low-dose, middle-dose and high-dose groups, respectively  and Actinobacteria  were observed in control, low-dose, middle-dose and high-dose groups, respectively (Table. SActinobacteria and depletion of Tenericutes (P < 0.01) were correlated with high doses of RbCl.All OTUs were clustered into 12 phyla, 19 classes, 27 orders, 44 families, 92 genera. The venn diagram  than those of the control , middle-dose (P = 0.0014) and high-dose groups (P = 0.0022) . The relative proportion of Anaeroplasmatales was significantly increased in control group (P < 0.05) , middle-dose (P = 0.0219) and high-dose groups (P = 0.0033) than control group  Fig. a, while P = 0.021 and high. Others families with relative abundance less than 5% were observed in Table. SAnaeroplasmataceae was found significantly higher in control group (P < 0.05) than three experimental groups  , middle-dose (P < 0.0054) and high-dose groups (P < 0.0033)  Fig. b. Beside33) Fig. c.Fig. 1Bacteroides and Helicobacter  . We could also get this information from heat map . The relative abundances of Anaeroplasma  and Desulfovibrio  were significantly different in various treatment groups. We observed an increase in the proportion of Desulfovibrio in RbCl treatment mice. The abundances of Alistipes  and Clostridium XlVa  were significantly higher in all the treatments than those of the control.Figure  Table. S. Figure Deltaproteobacteria, Desulfovibrionales, Desulfovibrionaceae, Desulfovibrio, Rikenellaceae, Alistipes and Clostridium XlVa. The control group was enriched for Tenericutes, Mollicutes, Anaeroplasmatales, Anaeroplasmataceae and Anaeroplasma.The LEfSe with default parameters was used to identify significant differences in relative abundances of fecal microbiota between the RbCl groups and control group. LEfSe analysis further confirmed enrichment microbes in different groups Fig. a and b. Crenarchaeota and Euryarchaeota  and Ferroplasmaceae (the average abundances were 0–0.01%) were observed (Table. S). The relative abundance of Sulfolobaceae increased in middle-dose group compared with the control group  patients as compared to healthy controls [Helicobacter contributed to the pathogenesis of GC. Exactly, RbCl did not change proportion of Helicobacter.In our study, we observed that RbCl maintained the abundances of n et al.  reportedecreased , 32. In t tissue –35. In d changed , 37, whi changed . Therefocrobiota –41. Propthy rats , 43. Altdiseases , 44. In i et al.  reporteduborders . Major dbacteria . Yang etbacteria  reportedive rods . Changesdiabetes , 50. In ncreased . The Baccontrols . It was Tenericutes, Mollicutes, Anaeroplasmatales, Anaeroplasmataceae and Anaeroplasma lineages. Yang et al. [Tenericutes significantly decreased in the depression mice. RbCl did not improve reduction of Tenericutes, which was consistent with reports. A previous animal study demonstrated that antidepressant drug (R)-ketamine and (S)-ketamine also did not improve the reduced proportion of Tenericutes [Tenericutes [Mollicutes were significant pathogens in human disease. A study also found that some Mollicutes were associated with diseases [Mollicutes were significantly lower after treatment with RbCl. The reduction of Mollicutes could decrease the pathogenesis of depression and cancers. However, one study has reported a significant reduction in the relative abundance of Mollicutes in MDD patients [Mollicutes in depression was unclear, further studies on the relationship between depression and Mollicutes are needed. Anaeroplasmatales is an order of Mollicutes bacteria which do not have a cell wall [Anaeroplasmatales significantly increased in depression group. In addition, ketamine, known to induce antidepressant effects, significantly reduced the abundance of Anaeroplasmatales [Anaeroplasmatales were significantly lower in RbCl groups. Anaeroplasmataceae, which belongs to Class Mollicutes and Order Anaeroplasmatales, is strictly anaerobic wall-less bacteria [Anaeroplasmataceae significantly increased in depression group [Anaeroplasmataceae significantly increased in patients with Crohn’s disease localized in the colon (CCD), but significantly decreased in patients with ulcerative colitis (UC) [Anaeroplasmataceae. The reduction of Anaeroplasmataceae could decrease the pathogenesis of depression. In the study of colon cancer, Zeng et al. [Anaeroplasma increased in the HFD-azoxymethane (AOM) group. The Anaeroplasma is a gram-negative bacterium, which belongs to Mollicutes class, Tenericutes phylum. Anaeroplasma was opportunistic pathogens which elicited various host immune responses in numerous human diseases including colon cancer [We also found RbCl significantly inhibited the abundances of g et al.  reportedericutes . Additioericutes . Tully eericutes  reporteddiseases . It was patients . As the ell wall . Song etell wall  found Ansmatales . Exactlybacteria . The abuon group . Moreovetis (UC) . Interesg et al.  found thn cancer , 61. IntDeltaproteobacteria, Desulfovibrionales, Desulfovibrionaceae and Desulfovibrio were significantly higher in RbCl groups than control group. Deltaproteobacteria belonging to Proteobacteria is sulfate-reducing bacteria [2S) produced by SRB was a product of sulfate reduction [2S could lead to chronic inflammation and imbalance between cellular proliferation, apoptosis and differentiation by damaging the intestinal epithelium [Deltaproteobacteria was possibly associated with CRC [Deltaproteobacteria was commonly pathogenic bacteria in the intestine. Desulfovibrionales, belonging to Deltaproteobacteria, is also a sulfate-reducing bacteria that can reduce sulfur to produce hydrogen sulfide (H2S) [Desulfovibrionaceae, which was the main biological source of hydrogen sulfate (H2S), involved in a wide range of physiological processes by influencing cellular signaling pathways and sulfhydration of target proteins [Desulfovibrionaceae increased in animal models of metabolic syndrome. Desulfovibrio could also produce H2S by reducing sulfate [2S derived from Desulfovibrio was associated with gastrointestinal disorders, such as UC, Crohn’s disease, and irritable bowel syndrome [Desulfovibrio produced metabolites such as secondary bile acids, which may catalyze the formation of colorectal cancer. However, it should be noted that the proportions of sulfate-reducing bacteria were promoted by RbCl. RbCl led to the enrichment of sulfate-reducing bacteria which could cause inflammation directly or indirectly in mice. It was likely that RbCl used as antigen in healthy mice which could elicit immune responses.Expressions of sulfate-reducing bacteria (SRB) including bacteria . Hydrogeeduction . H2S couithelium . Reportswith CRC , 66. Jinwith CRC  reportedde (H2S) . Desulfoproteins , 69. Zhaproteins  reported sulfate . H2S dersyndrome . Besidessyndrome  also repRikenellaceae, Alistipes and Clostridium XlVa. Wu et al. [Rikenellaceae decreased in the colitis-associated colorectal cancer (CAC) group compared with control group. Alkadhi et al. [Rikenellaceae reduced in CAC mice. In addition, the report found that Rikenellaceae was overrepresented in healthy control subjects [Rikenellaceae increased in the present study. Therefore, the increase in Rikenellaceae abundance could accelerate the antitumor efficacy of RbCl. Alistipes, which belongs to Bacteroidetes, is present in the human intestinal tract [Alistipes was indole-positive and may thus influence tryptophan availability [Alistipes. As tryptophan was also the precursor of serotonin, enrichment of Alistipes might affect serotonergic system by interfering with tryptophan metabolism. Clostridium XlVa, belonging to Firmicutes phylum, produces short-chain fatty acids (SCFAs) [Clostridium XlVa. The increase in abundance Clostridium XlVa could alleviate the pathogenesis of depression and cancers. Clostridium XlVa was significantly lower in CRC patients than healthy subjects [Clostridium XIVa was overrepresented in healthy control subjects [In addition, RbCl significantly increased the abundances of u et al.  found thi et al.  also repsubjects . Followial tract . Alistiplability . In our  (SCFAs) . The SCF (SCFAs) . SCFAs c (SCFAs) –81. RbClsubjects . Clostrisubjects , 82.Crenarchaeota, Thermoprotei, Sulfolobales, Sulfolobaceae and Sulfolobus lineages increased in middle-dose RbCl groups. Crenarchaeota was originally considered to grow in habitats characterized by high temperature, high salinity, or an extreme pH. Later studies found that Crenarchaeota also seem to occur ubiquitously in temperate or cold aquatic [Crenarchaeota in intestinal tracts was reported by Friedrich et al. [Crenarchaeota was present in the microbiota of the human digestive ecosystem. Thermoprotei, the crenarchaeal class, consists solely of obligate thermophiles. Thermophiles were well-known for participating in rampant lateral gene transfer (LGT) [Thermoprotei mostly occurred in the marine environment [Thermoprotei was observed to have an appreciably higher representation in healthy child [Thermoprotei was promoted by middle-dose RbCl in this study. Sulfolobales, a monophyletic group within the Crenarchaeota, is thermophilic sulfur-metabolizing archaea [Sulfolobales was present in human feces sample [Sulfolobaceae is composed of extreme thermoacidophiles that are found in terrestrial environments [Sulfolobaceae could produce bacteriocin, which played an important role in microbial interaction or microbe-environment interactions, and therefore improved their adaptation in extreme environments [Sulfolobaceae promoted by middle-dose RbCl may be beneficial in combating disease-related adverse environments. The genera Sulfolobus, which belongs to Sulfologaceae, grows at low pH (2–3) and high temperature (70–85 °C) [Sulfolobus offered many obvious advantages for industrial applications [Sulfolobus was able to reduce ferric iron when growing on elemental sulfur as an energy source [Regarding the composition of archaea, the abundances of  aquatic  and terr aquatic . The preh et al. . In addih et al.  suggesteer (LGT) , 88. It ironment . Howeverhy child . Interes archaea . The reps sample . The famronments . The Sulronments . Enrichm0–85 °C) , 95. Theications , 97. In y source .Euryarchaeota, Thermoplasmata, Thermoplasmatales, Ferroplasmaceae, Acidiplasma lineages. Euryarchaeota, one of the four major divisions of archaea, contributed substantially to global energy cycling [Euryarchaeota was detected in marine picoplankton [Methanobrevibacter smithii, which belonged to Euryarchaeota phylum, was a major archaeal player in human gut system [M. smithii was probably involved in inflammatory bowel disease (or Crohn’s disease), irritable bowel syndrome, colorectal cancer, and obesity [Methanobrevibacter oralis, belonging to Euryarchaeota phylum, was the predominating methanogenic species in the oral cavity [M. oralis was identified in apical periodontitis [Euryarchaeota might play key roles for human health and disease. However, the proportions of Euryarchaeota did not significantly change after RbCl treatment. Thermoplasmata was affiliated with Euryarchaeota phylum. Auguet et al. [Thermoplasmata represented important component of soil microbial communities. In the human body, Li et al. [Thermoplasmata was not the predominant archaeons in the subgingival dental plaque and Thermoplasmata was closely correlated with chronic periodontitis. Following RbCl treatment, the abundance of Thermoplasmata did not significantly change. Horz et al. [Thermoplasmatales existed in the human oral cavity. He et al. [Thermoplasmatales was also observed in healthy subjects, but the abundance of Thermoplasmatales increased in individuals with periodontitis. It was possible that enrichment of Thermoplasmatales contributed to the pathogenesis of periodontitis. Exactly, RbCl did not improve the enrichment of Thermoplasmatales. The Ferroplasmaceae is represented by cell wall-deficient, acidophilic, facultatively anaerobic and iron-oxidizing archaea [. As iron oxidizers, the family Ferroplasmaceae may contribute to the cycle of iron and sulfur [Ferroplasmaceae was involved in the pathogenesis of diseases through oxidizing iron. Thus, further studies on the relationships between diseases and Ferroplasmaceae are needed. Interestingly, RbCl did not significantly change the abundance of Ferroplasmaceae. Acidiplasma, which belongs to the family Ferroplasmaceae, order Thermoplasmatales, phylum Euryarchaeota, is a novel acidophilic, cell-wall-less archaeon [Acidiplasma included two species, namely Acidiplasma aeolicum and Acidiplasma cupricumulans [Acidiplasma aeolicum and Acidiplasma cupricumulans were isolated from the hydrothermal pool located on Vulcano Island  and chalcocite/copper-containing heaps (Myanmar), respectively [Acidiplasma and diseases. In our results, Acidiplasma was observed in stool samples and its abundances were not significantly changed by RbCl.Furthermore, RbCl maintained the abundances of archaea  cycling . Euryarcplankton , 101 andplankton . Methanot system . A few s obesity , 105. Mel cavity . M. oraldontitis . Therefot et al.  showed ti et al.  found thz et al.  found the et al.  reported archaea . As irond sulfur . It was archaeon . The gencumulans . Acidiplectively . It shouClostridium XlVa, SCFAs producing bacteria, was significantly promoted by RbCl. SCFAs could promote colonic 5-HT production [Alistipes promoted by RbCl might disrupt the intestinal serotonergic system by affecting tryptophan metabolism. Therefore, the microbes might partly promote the anticancer and anti-depressant effects of RbCl via brain-gut-microbiota axis.Some reports found Rb could be used as anticancer and anti-depressant drugs. The mechanisms of Rb against cancer and neurological disease remain unclear. Microbiota may participate in the pathogenesis of depression through the brain-gut-microbiota axis . Serotonoduction , 118. EnIn summary, our results revealed RbCl significantly altered fecal microbial composition. RbCl maintained the abundances of dominant bacteria. However, RbCl significantly altered the abundances of less richness microbes. Changes in fecal microbes might in part contribute to the anticancer or anti-depressant effects of RbCl. Clearly, further functional analysis of the role of specific fecal microorganisms and their interactions with brain-gut-microbiota axis is expected.n = 13), and the other three groups were divided into low-dose (n = 17), middle-dose (n = 17), and high-dose group (n = 17) according to the different RbCl dosage (20 mg/L (0.17 mmol/L), 50 mg/L (0.41 mmol/L), 100 mg/L (0.83 mmol/L), respectively). Five or four mice were randomly placed in each mouse cage. The mice of the above experimental groups were intragastrically administered of RbCl in 0.2 mL twice per day for 6 consecutive weeks. During this period, the mice were weighed weekly.Three-week old male Swiss mice used as experimental animals (license number SCXK (Xiang) 2016–0002) were purchased and raised in the Laboratory Animal Science Department (LASD) of Central South University with Specific Pathogen Free (SPF) level environment. The living environment of the mice was of constant temperature (20 ± 2 °C), constant humidity (50 ± 10%), and free access to water and food. Mice were strictly controlled in normal biological rhythms and the light and dark environments were 12 h, respectively. All animal experiments in this study were approved by the Animal Breeding and Committee of the Department of Laboratory Animal Science of Central South University and were strictly evaluated in accordance with the Regulations on Animal Management of Central South University. The mice were kept in the LASD for a week without any treatment to adapt to the environment. Sixty-four mice were randomly assigned into four groups: one was blank control group which was intervened with normal saline  in the ultra-clean workbench. The kidneys, heart, lungs, pancreatic, spleen, stomach and liver were rapidly excised from mouse and weighed.Each fecal sample (approximately 0.2 g) was used for total gut microbiome DNA extraction with QIAGEN QIAamp kit. Extractions were performed according to specific operating instructions. The extracted total genomic DNA was detected by agarose gel electrophoresis and qualified DNA samples were used in subsequent experiments. PCR amplification and library preparation were performed using 515 F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) primers to target the V4 region of the 16S rRNA gene. The PCR products for each sample were subjected to electrophoresis at a voltage of 100 V for about 1 h using a 2% agarose gel. The target band was recovered by tapping under UV light, and E.Z.N.A.TM Gel Purification Kit  was used for product purification. The purified product was quantified using a Nanodrop spectrophotometer . Illumina MiSeq  sequencing required the library constructed from the mixture of 200 ng of each purified product.http://zhoulab5.rccc.ou.edu/) at University of Oklahoma. The resulting sequences were further filtered based on quality score and sequence length. To merge the paired-end reads into full-length amplicon sequence, the FLASH software tool was used based on overlapping bases. The sequences were clustered into operational taxonomic units (OTU) at or above 97% identity. According to previous reports, OTUs reaching 97% similarity were used to analyze alpha diversity (Shannon and Simpson), and richness  [The MiSeq sequencing data were analyzed using the Galaxy pipeline developed by Prof. Zhou’s lab (nd Sobs) , 120.http://www.ehbio.com).IBM SPSS Statistics 19.0 software was used for statistical analysis. Since comparison was performed between two groups , Student T-test was applied for detecting significant differences in specific measured parameters. All values were expressed as the mean ± standard deviation (SD). Probability values of less than 0.05 were considered to show a statistical significance. Microbiota community diversity and richness were analyzed using vegan package and R software (version 3.5.1). LEFSe , CPCoA  and Heatmap plot were performed on ehbio BioPharm platform .Additional file 4. Statistical analysis of relative abundance of fecal archaea (order).Additional file 5. Statistical analysis of relative abundance of fecal archaea (family).Additional file 6. Statistical analysis of relative abundance of fecal archaea (genus).Additional file 7. Relative abundances of all microorganisms at the phylum level.Additional file 8. Relative abundances of all microorganisms at the class level.Additional file 9. Relative abundances of all microorganisms at the order level.Additional file 10. Relative abundances of all microorganisms at the family level.Additional file 11. Relative abundances of all microorganisms at the genus level.