Staphylococcus aureus (S. aureus) infections, particularly methicillin-resistant Staphylococcus aureus (MRSA) in humans and animals, have become a significant concern globally. The present study aimed to determine the prevalence and antibiogram of S. aureus isolated from animal handlers in Peninsular Malaysia. Furthermore, the genotypic characteristics of S. aureus isolates were also investigated. Nasal and oral swab samples were collected from 423 animal handlers in Peninsular Malaysia. The antibiogram profiles of S. aureus against 18 antibiotics were established using a Kirby–Bauer test. The genotypic profile of S. aureus, including the presence of antimicrobial resistance (AMR), virulence genes and spa genotypes, was investigated using molecular techniques. The overall carriage rate of S. aureus, MRSA and MDRSA was 30.5%, 1.2% and 19.4%, respectively. S. aureus was highly resistant against penicillin (72.3%) and amoxicillin (52.3%). Meanwhile, gentamicin and linezolid were fully effective against all the isolated S. aureus from animal handlers. It was observed that animal handlers with close exposure to poultry were more likely to carry S. aureus that is resistant to tetracycline and erythromycin. S. aureus isolates harboured tetracycline resistance , erythromycin resistance  and immune evasion cluster (IEC) genes . Seventeen different spa types were detected among the 30 isolates of MDRSA, with t189 (16.7%) and t4171 (16.7%) being the predominant spa type, suggesting wide genetic diversity of the MDRSA isolates. The present study demonstrated the prevalence of S. aureus strains, including MRSA and MDRSA with various antimicrobial resistance and genetic profiles from animal handlers in Peninsular Malaysia. Staphylococcus aureus (S. aureus) is an opportunistic, zoonotic pathogen that is capable of causing a wide array of infections, ranging from mild skin infections to life-threatening bacteremia in both humans and animals [S. aureus, especially MRSA infections, has always been a significant problem in both medical settings and the community, due to its ability to show multiple resistance to various antibiotics, especially towards beta-lactam antibiotics [S. aureus strains, such as livestock-associated MRSA (LA-MRSA) in animals, animal handlers such as veterinarians and farmers are equally at risk of being colonized and infected by AMR strains originating from animals [S. aureus among pig farmers ranged from 26.1% to 57.0% [S. aureus and MRSA among farmers and veterinarians as compared to individuals without contact with pigs, indicating that constant exposure to animals may be a risk for S. aureus colonization [S. aureus, tetracycline-resistant S. aureus (TRSA), multidrug-resistant S. aureus (MDRSA) and livestock-associated S. aureus (LA-SA) than individuals that lacked exposure [S. aureus and MRSA from animal origin to the human community [ animals ,2. Recen animals . The issibiotics . However animals ,6,7. Preto 57.0% ,9. Meanwto 57.0% ,9. Nonetnization . In addiexposure . Moreoveommunity ,11.S. aureus, particularly from strains that have acquired antimicrobial resistance (AMR) genes [S. aureus had acquired resistance to multiple antibiotics (multidrug resistance) through the acquisition of AMR genes that respond to different groups of antibiotics [S. aureus when the mecA gene was found to be responsible for the resistant trait against beta-lactam antibiotics, including methicillin, penicillin, oxacillin and cefoxitin [S. aureus (MSSA) was frequently reported to be harboring AMR genes from other groups of antibiotics simultaneously, including tetracyclines and macrolides [S. aureus is also capable of producing multiple virulence factors that increase the pathogenicity of the bacteria [scn, chp, sak, sea and sep. Past studies reported that IEC genes were frequently found in S. aureus lineages well adapted to humans, but not in livestock-associated S. aureus and LA-MRSA strains [S. aureus is Panton–Valentine leukocidin (PVL) encoded by lukF/lukS-PV gene, a bi-component pore-forming toxin capable of causing the destruction and lysis of leukocytes [tst gene, is one of the most important pyrogenic toxin superantigens (PTSAgs) produced by S. aureus that may lead to high fever, diffuse erythematous rash, desquamation of the skin after onset and hypotension [eta and etb genes are responsible for skin and soft tissue infections such as Staphylococcal scalded skin syndrome (SSSS) [S. aureus AMR surveillance studies were mainly conducted in patients, medical staff or students with constant exposure to the medical settings [S. aureus (MSSA) isolates [S. aureus in relatively healthy human populations, particularly in Malaysian animal handlers. Until 2018, the prevalence rate of MRSA among S. aureus isolated from human patients and university students ranged from 15.0% to 28.1% [S. aureus from animal handlers in Malaysia remain scarce. This creates knowledge gaps on the data concerning antimicrobial resistance and the genotypic profile of Malaysian MSSA isolates. Realizing this knowledge gap, the present study was carried out with the aim to determine the carriage rate, phenotypic antimicrobial resistance profile and genotypic characteristics of S. aureus isolated from animal handlers in Peninsular Malaysia.In the last d-ecade, there have been reports of the increasing epidemiological risk of R) genes ,12,13. Pibiotics . This isefoxitin ,16. In acrolides ,17,18. Ibacteria . These vbacteria ,19. Five strains ,21. Anototension . Meanwhie (SSSS) . In Malasettings . Furtherisolates . As a reto 28.1% ,14. In aS. aureus via polymerase chain reaction (PCR) screening of the nuc gene and considered to be S. aureus positive. These 155 isolates originated from 129 individuals; thus, the overall prevalence rate of S. aureus is 30.5% . Eighty-three S. aureus isolates originated from nasal  while 72 isolates originated from oral swab samples . Twenty  individuals were carrying S. aureus in both the nasal and oral parts. Further PCR assay revealed that six  S. aureus isolates were mecA gene positive, indicating the presence of MRSA among five animal handlers . Five of the MRSA isolates were from nasal  while only one was from oral swab  samples. The S. aureus and MRSA carriage rates of veterinarians, pet owners and animal farmers are shown in S. aureus and MRSA in categorical variables (p > 0.05).Among the 846 swab samples, 155, or 18.3% , swabs were found harbouring S. aureus was summarized in S. aureus isolates showed resistance against one or more antibiotics. The findings also revealed that S. aureus showed the highest resistance rate towards penicillin  and amoxicillin . Meanwhile, MRSA isolates showed the highest resistance rate against penicillin , cefoxitin , amoxicillin  and chloramphenicol . Gentamicin and linezolid were fully effective (100% susceptible) against all the isolated S. aureus from animal handlers. In addition, no significant differences were observed when comparing the antibiotic resistance profile of the S. aureus according to categories of animal handlers . However, individuals with constant contact with poultry  were more likely to carry S. aureus that showed resistance against tetracycline as compared to ruminants  and pet animals . Similarly, S. aureus from animal handlers with constant contact with poultry  showed a higher resistance rate against erythromycin as compared to ruminants  and pet animals  handlers. Meanwhile, individuals with constant exposure to ruminant animals  were less likely to carry S. aureus resistant to penicillin compared to poultry handlers , pet owners  and aquatic animal handlers . In the present study, 19.4%  of S. aureus isolates were categorised as multidrug resistant as they showed resistance to antibiotics from three different groups of antibiotics. The MDRSA prevalence rates of veterinarians, pet owners and animal farmers were summarized in S. aureus isolates have an MARI value of 0.2 and above. The relatedness of the S. aureus isolates based on their phenotypic antibiotic resistance pattern were displayed in the dendrogram  of the isolates carried the tetK genes, four  carried the tetL genes while only one  isolate was harbouring tetM gene. In addition, the prevalence rate of ermA, ermB, ermC and msrA genes were 1.3% (2/155), 1.3% (2/155), 4.5% (7/155) and 3.2% (5/155), respectively. Both vanA and mecC genes were not detected among the isolated S. aureus.In the current study, 12  of the S. aureus collected from animal handlers contained at least one of the five IEC genes . Furthermore, three out of six MRSA isolates were found harbouring IEC genes. The scn genes were detected in 53 isolates , while the occurrence rates of sak and chp genes were 30.3% (47/155) and 8.4% (13/155), respectively. A low number of sea  and sep  genes were detected from two different S. aureus isolates. No IEC genes were found among the 71 (68 MSSA and 3 MRSA) isolates. Fifty S. aureus isolates with the presence of scn were later categorised into IEC types. The predominant IEC type in this study was type H (21 isolates), as shown in S. aureus isolates.In regard to virulence genes, 44.5% (69/155) of S. aureus (MDRSA) isolates (5 MRSA and 25 MDR-MSSA) were characterized using spa typing (n = 2), t3293 (n = 2), t4171 (n = 1), and t3080 (n = 1). Based on Thirty of the multidrug-resistant a typing . SeventeS. aureus strains, especially MRSA, has rapidly changed around the globe [S. aureus and MRSA in different communities is highly required to obtain the latest data regarding the prevalence, antibiogram profile and genotypic characteristics of these bacteria. Moreover, the antibiogram profiles obtained in this study can be used to better understand the AMR status of S. aureus in a relatively healthy community and as a reference to select the best antibiotics for the successful treatment of S. aureus and MRSA infection in Malaysian animal handlers.Since its first discovery, information regarding he globe . Thus, cS. aureus among animal handlers reported in this study is within the range of the reported 20% to 30% nasal carriage rate in the United States and European countries [S. aureus carriage rate among European countries, with the Hungarian participants having a significantly lower S. aureus prevalence rate (12.1%) as compared to a 29.4% carriage rate among participants from Sweden [S. aureus among local individuals with constant exposure to animals such as farmers, pet owners and veterinarians need to be updated. Furthermore, the sample sizes of animal handlers in the previous studies were relatively small (less than 100 participants), with more emphasis on MRSA isolates [S. aureus samples were isolated from Malaysia poultry farm workers, with none of the isolates being mecA positive [The prevalence rate of ountries . Howeverm Sweden . In Malaisolates ,28. In 2positive . Anotherpositive . The reapositive .S. aureus from animal handlers showed resistance against 18 different antibiotics, with 19.4% (30/155) of S. aureus isolates categorised as multidrug resistant. This finding is similar to the 19.4% MDRSA prevalence rate among individuals with close contact with livestock [S. aureus from animal handlers was highly resistant to penicillin (72.3%). This finding is lower than the 90% to 100% penicillin resistance rate among S. aureus from hospital and university students in Malaysia [S. aureus isolates were fully sensitive towards linezolid and gentamicin, suggesting that these antibiotics can be used to treat persistent S. aureus infections among animal handlers. Nonetheless, as clinical tests were not carried out in the study, the effectiveness of these antibiotics against S. aureus infections remains inconclusive. Further MAR index assessment revealed that 16.7% S. aureus had an index value of 0.2 and above. MAR index values of 0.2 and above have been used to differentiate low- and high-risk regions where antibiotics are overused, thus suggesting that 16.7% of the S. aureus isolates from Malaysian animal handlers were exposed to environments with high antibiotic use and high selective pressure [S. aureus strains that exhibit phenotypic resistance traits among local animal handlers remains uncertain. It is well known that repeated therapeutic and indiscriminate usage of antibiotics will cause the development of antimicrobial resistance in microorganisms [S. aureus with AMR traits according to the usage of antibiotics within a three-month period, suggesting short-term antibiotic usage alone was unlikely to cause the emergence of AMR. However, it is possible that these participants were colonized by S. aureus with AMR traits through direct contact with infected animals or humans with a prolonged antibiotic usage history, environmental agents and animal food products contaminated with AMR bacteria [S. aureus from individuals with exposure to poultry were found to have a higher resistance rate against tetracycline and erythromycin as compared to other animal species, suggesting there might be a relationship between these two variables. Previous research has indicated that individuals that were frequently exposed to live poultry were more likely to carry livestock-associated S. aureus strains, such as LA-MRSA [S. aureus with erythromycin- and tetracycline-resistant traits may have originated from the poultry. However, as no samples were collected from the animals, it is uncertain that the S. aureus from poultry origins may be presented with the similar antibiotic resistance pattern. Thus, it would be beneficial for future studies to collect samples from various sources  over a longer period to determine the possible origin of AMR bacteria. In most of the cases, phenotypic antibiotic resistance is encoded by the various antimicrobial resistance genes found within the bacteria. Thus, it is coherent to speculate that S. aureus isolates resistant against these antibiotics may harbour antimicrobial resistance genes that encode for defensive mechanisms, including the production of defensive enzymes, reduced permeability, enzymatic inactivation, efflux pumps or ribosomal protection [mecA, tetK, tetL, tetM, ermA, ermB, ermC and msrA genes were detected. The tetK and tetL genes are responsible for the tetracycline efflux pump system, while tetM is involved in ribosomal protection [ermA, ermB and ermC genes are responsible for the ribosomal binding site modification to protect the S. aureus against erythromycin antibiotics, while the msrA gene encodes for an ATP-dependent efflux pump [S. aureus isolates in this study displayed a higher prevalence of ermC . This finding is in agreement with a study in South Africa where the ermC gene is the most frequently found macrolide–lincosamide resistance gene in pigs [In the current study, Malaysia . Meanwhiicillin 7.3%. Thisth 19.4% /155 of Srganisms . In the bacteria ,32,33,34 LA-MRSA . In Mala LA-MRSA . Hence, otection . In the otection . Meanwhilux pump . The S.  in pigs . Nonethe in pigs ,38.S. aureus to adapt and survive in the human host. IEC has been shown to play an essential role in the disruption or inhibition of the normal function of the human immune system, as well as causing food poisoning [S. aureus in human hosts. In addition, the absence of scn gene in S. aureus is considered to be a marker and strong indicator for livestock-derived strains [S. aureus isolates showed that 44.5% (69/155) of isolates collected from animal handlers were found to contain at least one of the IEC genes, with the predominant IEC type in this study being Type H. This finding is different compared to other studies that recorded a high prevalence of IEC genes in clinical S. aureus (>75%) isolates, with the predominant IEC type being Type B [S. aureus from animals [S. aureus from animal sources in Malaysia should be monitored closely. Nonetheless, most of the S. aureus samples were generally susceptible to the tested antibiotics and lacked virulence factors, suggesting the isolates were of less pathogenic significance to humans [Immune evasion virulence factor is one of the many vital factors that enable oisoning . Thus, t strains ,19. The g Type B ,39. Howe animals . Furthero humans .S. aureus is important to understand the dissemination and epidemiology of bacteria. In the present study, spa typing was used to identify the genotypes and determine the relatedness of the isolated MDRSA. According to a study in 2018, the most prominent spa types among clinical S. aureus isolates in the Asia region were t307 and t002 [spa type in clinical isolates in Malaysia was t307 [spa types identified were t189  and 4171 . The presence of MRSA and MSSA t189 was previously reported among S. aureus isolated from hospital patients, university students and poultry farm workers in Malaysia, suggesting the widespread of this genotype among S. aureus in local settings [S. aureus t189 was also reported as the prominent genotype in clinical isolates from the Netherlands, Italy, Korea, China and Taiwan [spa type t4171 was only reported in clinical MSSA isolates and MSSA/MRSA isolates from swine in Malaysia [spa types, such as t548, t050, t3239, t084, t095, t3080, t9531, t605, t550, and t714, detected in this study were reported for the first time in Malaysia. Even though some of the isolates shared the similar spa types, their antimicrobial resistance and virulence gene profiles are different, showing that the MDR trait may not be specific to certain strains or locations. Thus, continuous surveillance of S. aureus in various communities and environments should be carried out in order to have a more holistic insight on the distribution and emergence of AMR strains. Future genotyping study of local MDRSA strains such as MRSA t4171 using multilocus sequence typing (MLST) and whole genome sequencing (WGS) techniques should be carried out to provide data for better understanding and comparison with international MSSA/MRSA strains. Furthermore, policies and programs that promote the responsible usage of antimicrobials as well as good bacterial infection prevention and control measures among animal handlers in Peninsular Malaysia should be strengthened to prevent the emergence of AMR in local animals.Molecular typing of the and t002 . Meanwhiwas t307 . The presettings ,42. MoreS. aureus carriage rate of 30% was used to determine the maximum sample size. Informed consent was obtained from all recruited volunteers included in the study. In the present study, 846 swab samples  were collected from 423 animal handlers in various states of Malaysia  from the period of September 2019 to December 2019. In the present study, an animal handler was defined as an individual with frequent exposure to domestic livestock and companion animals, such as animal farmers (n = 343), pet owners (n = 70) and veterinarians (n = 10). Nasal swab samples were collected by inserting the swab (approximately 2 cm) into the nostril. The swab was rotated several times against the nasal wall and the procedure was repeated in the other nostril using the same swab. Meanwhile, oral swabs were taken by swabbing the back of the throat, roof of the mouth and cheek for several seconds. Swab samples were carefully placed into the falcon tubes containing transport medium and kept in a cooler box at around 4 °C and sent to the Universiti Sultan Zainal Abidin (UniSZA) Microbiology laboratory for further analysis. The swab samples were then streaked onto mannitol salt agar  and incubated at 37 °C for up to 48 h. Yellow colonies with yellow zones growing on mannitol salt agar were considered to be putative S. aureus colonies. The putative S. aureus colonies were then sub-cultured on nutrient agar  supplemented with 6.5% sodium chloride (NaCl) and incubated at 37 °C for 24 h. Putative S. aureus colonies were then subjected to DNA extraction using the heat lysis method, where the bacterial colonies were suspended in 120 µL distilled water and boiled for 5 min at 95 °C [S. aureus and MRSA. The primers used for the detection of S. aureus and MRSA were nuc (S. aureus species-specific gene) and methicillin-resistant genes (mecA and mecC), respectively, as shown in w/v) agarose gel stained with GelRed Nucleic Acid Gel Stain  and gel electrophoresis was run in 1X tris-borate-EDTA (TBE) running buffer  at 80 V for 2 h. The gels were visualized using Fujifilm LAS-4000 gel documentation system . Bacteria isolates that showed the presence of DNA bands at 278 bp (nuc gene) were considered to be S. aureus. isolates that displayed that the presence of both nuc (278 bp) and mecA (533 bp) were positively confirmed as MRSA. Meanwhile, bacterial isolates that carried both nuc (278 bp) and mecC (138 bp) genes were identified as LA-MRSA. Bacterial isolates with the presence of nuc genes only were referred to as methicillin-susceptible S. aureus (MSSA). S. aureus ATCC 700699 was used as the control strain, and were used as the positive control for nuc and mecA genes detection.The sample size of animal handlers was determined using GraphPad StatMate© software (5% absolute precision and 95% level of confidence) for cross-sectional and random sampling. The expected ers n = 3, pet ownS. aureus isolates was determined using the Kirby–Bauer test on Mueller–Hinton agar , according to the recommendations given by the Clinical and Laboratory Standards Institute (CLSI). The AST was performed using 18 antibiotics as listed in S. aureus ATCC 700699 was used as the control strain. S. aureus isolates that showed resistance against three or more categories of antibiotics were classified as multidrug-resistant S. aureus (MDRSA) [The antibiotic susceptibility of tetK, tetL, tetM and tetO), erythromycin-resistant , and vancomycin-resistant genes (vanA) as mentioned in scn, chp, sak, sea and sep), exfoliative toxins , PVL toxin (pvl) and TSST-1 (tst) were also investigated [S. aureus isolates were later classified into eight different IEC types according to the combination of genes detected. The presence of the scn gene was mandatory for the consideration of the IEC types [All MRSA and MDRSA were further screened using PCR to detect the presence of different antimicrobial resistance genes, including tetracycline-resistant  and spa-1517R (5′-GCTTTTGCAATGTCATTTACTG-3′) [spa type of S. aureus isolates. The DNA sequence data of S. aureus isolate with unknown spa repeat and type were uploaded to Ridom Spa Server website  for the assignment of new spa type.Thirty isolates of non-repetitive MDRSA (both MRSA and MDR-MSSA) were further investigated using ACTG-3′) . The PCRS. aureus and MRSA as well as different antimicrobial and virulence genes was counted and presented in percentages (%). The 95% confidence interval (CI) of the occurrence rates of S. aureus, MRSA and MDRSA among the animal handlers was calculated. Categorical data were analysed using Chi-square or Fisher’s exact test , with the 95% confidence interval (p < 0.05) being set to indicate the significant difference. The antibiotic resistance percentage was calculated as the proportion of isolates tested that had an inhibition zone below the respective antibiotic breakpoint. The relationships between the antibiotic exposure and overall antibiotic resistance of the isolated S. aureus were assessed via the multiple antimicrobial resistance index (MARI). The MARI was calculated as the proportion of antibiotics to which S. aureus isolate was phenotypically resistant.The occurrence rate of the S. aureus (30.5%) and MRSA (1.2%) of animal handlers from Peninsular Malaysia. The findings also showed that S. aureus isolates were highly resistant against penicillin and amoxicillin while showing full susceptibility against gentamicin and linezolid. Subsequently, 19.4% of S. aureus isolates were categorized as MDRSA, demonstrating the low prevalence of S. aureus with multiple drug-resistant traits among Malaysian animal handlers. Nonetheless, different numbers of antimicrobial resistance  and IEC  genes were detected among the S. aureus isolates, raising concern regarding the further emergence of AMR bacteria among animal handlers through the dissemination of antibiotic-resistant genes. Furthermore, molecular typing showed the presence of seventeen different spa genotypes were detected among the MDRSA, with t189 (16.7%) and t2174 (16.7%) been the prominent genotypes. The findings from this study suggest the emergence of MDRSA with great genetic diversity and antimicrobial resistance profiles among animal handlers from different locations of Peninsular Malaysia.The present study revealed the carriage rate of


Enterococcus hirae are common in animals, with instances of transmission to humans being rare. Further, few cases have been reported in humans because of the difficulty in identifying the bacteria. Herein, we report a case of pyelonephritis caused by E. hirae bacteremia and conduct a literature review on E. hirae bacteremia.Infections caused by E. hirae, and pyelonephritis was diagnosed. The patient was treated successfully with intravenous ampicillin followed by oral linezolid for a total of three weeks.A 57-year-old male patient with alcoholic cirrhosis and neurogenic bladder presented with fever and chills that had persisted for 3 days. Physical examination revealed tenderness of the right costovertebral angle. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) of the patient’s blood and urine samples revealed the presence of E. hirae bacteremia is frequently reported in urinary tract infections, biliary tract infections, and infective endocarditis and is more likely to occur in patients with diabetes, liver cirrhosis, and chronic kidney disease. However, mortality is not common because of the high antimicrobial susceptibility of E. hirae. With the advancements in MALDI-TOF MS, the number of reports of E. hirae infections has also increased, and clinicians need to consider E. hirae as a possible causative pathogen of urinary tract infections in patients with known risk factors.The literature review we conducted revealed that  Enterococcus hirae primarily causes zoonosis ) OR ("Enterococcus hirae"[TW]) OR (hirae[TIAB])) AND ((Bacteremia[MH]) OR (bacteremia*[TIAB] OR bacteraemia*[TIAB]))) OR ((("Enterococcus hirae"[Mesh]) OR ("Enterococcus hirae"[TW]) OR (hirae[TIAB])) AND Humans[MH]), and the Embase database using the keywords (('bacteremia'/exp OR 'gram negative sepsis'/exp OR bacteraemia* OR bacteremia*) AND ('enterococcus hirae'/exp OR hirae)) OR (('enterococcus hirae'/exp OR hirae) AND [humans]/lim). PubMed and Embase searches generated 170 and 229 articles, respectively. Of these, 158 and 218 articles from PubMed and Embase, respectively, were excluded because they were not case reports  . HE. hirae infection recurred during treatment [Enterococcus spp. with 516 cases of infective endocarditis caused by Enterococcus spp. collected prospectively from 35 centers in Spain, recurrence was significantly higher in cases of infective endocarditis caused by Enterococcus spp. (3.5% vs. 1.7%) [E. hirae urinary tract infections with no recurrences or deaths.Three cases of reatment , 7, 21, s. 1.7%) . There wE. hirae to antimicrobial agents is similar to that of E. faecalis, which is susceptible to penicillin. Table E. hirae infections in humans. Although some reports have reported high resistance to gentamicin [. The relatively low mortality and antimicrobial resistance suggest that E. hirae is more similar to E. faecalis than E. faecium. In the present case, the patient could not tolerate ampicillin due to drug allergy and was successfully treated with linezolid after confirming susceptibility. Resistance to clindamycin and gentamicin has been reported repeatedly, and the possibility of resistance should be considered when these drugs are used. The accumulation of human clinical data is warranted to generate an accurate evaluation.The susceptibility of ntamicin , of the Enterococcus spp. compared with the gold standard rpoA gene sequencing method for the identification of bacteria of environmental origin. The occurrence of Enterococcus spp., including E. hirae, in wild birds was correctly identified by MALDI-TOF MS [E. hirae may have been underdiagnosed because of the limitations of the diagnostic method [E. hirae since 2015 following the advent of MALDI-TOF MS.Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was developed in the 1980s and was accurate in 80–95% of bacterial isolates . SpeciesI-TOF MS . Before c method . This reEnterococcus hirae is a newly recognized causative pathogen of urinary tract infections, especially in patients with underlying diseases. Clinical data such as risk factors, clinical manifestations, and antimicrobial susceptibility are lacking, and more cases should be accumulated following accurate identification.E. hirae infections reported has increased following the development of MALDI-TOF MS. Although E. hirae may have a low virulence, as do other enterococci, clinicians need to consider E. hirae as a causative pathogen of urinary tract infection.In summary, the number of


ConVarT incorporates annotations (including phenotypic and pathogenic) into variants, and these previously unexploited phenotypic MatchVars from mice and C. elegans can give clues about the functional consequence of human genetic variants. Our analysis shows that many phenotypic variants in different genes from mice and C. elegans, so far, have no counterparts in humans, and thus, can be useful resources when evaluating a relationship between a new human mutation and a disease.The availability of genetic variants, together with phenotypic annotations from model organisms, facilitates comparing these variants with equivalent variants in humans. However, existing databases and search tools do not make it easy to scan for equivalent variants, namely ‘matching variants’ (MatchVars) between humans and other organisms. Therefore, we developed an integrated search engine called ConVarT ( The progress of the genomics field is accelerating with the aid of next-generation sequencing technologies, most recently exemplified by the launch of the Exome Aggregation Consortium (ExAC) and Genome Aggregation Database (gnomAD). These genomic databases were gathered from >140 000 randomly selected individuals, providing large-scale collections of human genetic variants . Humans Though examining the functional effects of variants is an efficient strategy for categorizing them as being either pathological or benign, a considerable workload and financial demand are limiting factors for such experimental efforts . In thisCongruent clinical Variation Visualization Tool) to search for equivalent variants  between human and non-human species. As a proof of concept, we matched amino acid-changing variants from humans with variants from mice and the nematode Caenorhabditis elegans and generated a list of equivalent variants called matching variants (MatchVar) between human and mice or C. elegans. Furthermore, we integrated available annotations  for variants into the database, typically making it easy for users to search for MatchVars with phenotypic data. Through the use of MatchVars associated with functional data, our comprehensive database can provide an additional layer of proof for the functional interpretation of human variants.Here, we develop an integrated search tool and database called ConVarT (C. elegans and Mutagenetix for mice) containing non-naturally occurring variants and phenotype data for C. elegans and mice enables us to equate them with human genetic variants from gnomAD, Clinical Variants (ClinVar), COSMIC and dbSNP  of all combinations of these protein sequences to ensure alignment of the corresponding amino acid positions. The schematic workflow of ConVarT is presented in Figure C. elegans into corresponding amino acid positions in each gene. We restricted our comparisons to amino acid-changing variants, as amino acids are more conserved than non-amino acid-changing variants, and protein function is likely to be affected by an alteration in the corresponding amino acids. For visualization of MSAs together with variants from humans, mice, and C. elegans, we used in-house software . We have named these equivalent variants ‘matching variants’ (MatchVars)  to facilitate consistent and rapid visualization of MatchVars and variant annotations on MSAs together with human PTMs and protein domains. ConVarT offers a protein sequence similarity index based on the matching residues for three species below part of MSAs so that the researchers utilize this section to gain more insight into the conservation of the sequence in the model organism compared to humans.Our analysis revealed that many equivalent amino acid substitutions exist between humans and these two organisms Figure . For exa) Figure . For a v4 Figure . FinallyS. cerevisiae, who have yet to be incorporated into ConVarT.ConVarT reduces the time needed to find the phenotypic and unknown significance of MatchVars in the genomes of three organisms from hours to seconds. A comprehensive list of identifiers that can be used to search for genes on ConVarT can be found in C. elegans overlapping with pathogenic variants of ClinVar is calculated to be significantly higher , which is consistent with the equality of pathogenic with phenotypic variants. For phenotypic MatchVars from mice, the mouse phenotypic MatchVars/ClinVar pathogenic variants ratio is slightly higher than the ratio of the mouse phenotypic MatchVars/ClinVar benign variants   in C. elegans KAT-1 (Human ACAT1) was identified as phenotypic (short life span) in 2010. The p.G388E variant (NP 000010) in human ACAT1 was pathogenic seven years later, resulting in human acetyl-CoA acetyltransferase deficiency  , and variant of uncertain significance (VUS) make up the majority of these SNVs . Expecte) Figure  and D. Cficiency ,26. We pD Figure . Second,) Figure –D  encoding tubulin beta chain  were implicated in displaying resistance to benzimidazole (BZ) and were labeled as putative loss-of-function (LoF) variants  are the MatchVars of p.M257I and p.F167Y (variant IDs: COSM3929966 and COSM6451925) in human TUBB4B (NP_006079.1), respectively, predicted to be pathogenic by the FATHMM . In. InC. elC. elegans. ConVarT was designed with the goal of expanding and incorporating variants from other organisms, such as Zebrafish and Drosophila. ConVarT recently added 24 658 526 human variants from TOPMed to its database . The. TheC. eC. elegans F42G8.19 (ceph-41) is the most likely ortholog of human CEP41, C. elegans F42G8.19 does not emerge as an ortholog of human CEP41 based on orthology searches from C. elegans gene orthology database  ,31–32. Idatabase ,35.ftp://ftp.ncbi.nlm.nih.gov/pub/clinvar/ (ClinVar), http://gnomad.broadinstitute.org/ (Aggregation Database (gnomAD v2.03), https://cancer.sanger.ac.uk/download (COSMIC), https://ftp.ncbi.nih.gov/snp/ (dbSNP) and https://www.phosphosite.org/Supplemental_Files (PhosphoSitePlus) (https://mutagenetix.utsouthwestern.edu/about.cfm) and Wormbase generously shared variants and phenotypic data for Mouse and C. elegans, respectively (https://pb.apf.edu.au/phenbank/homePage.html).The human variants and organisms specific variants were downloaded from the following resources:  gnomAD v.03, httpitePlus) ,19–20,24ectively ,18. We oC. elegans, D. melanogaster, and frog, and used ClustalW v2.1 pairwise alignment to perform 2,379,397 distinct combinations of multiple sequence alignments (MSAs) of these protein sequences  on GitHub.The recently developed MSA visualization tool was used to co-visualize MSAs along with annotations (variants and phenotypic data) . We consAdditional materials and methods are available in the supplementary file.https://github.com/thekaplanlab.All codes used for generating data, websites, and figures are accessible from gkab939_Supplemental_FilesClick here for additional data file.


However, this is still unclear for the IRLC species. Here, we aimed to investigate the relationships between repeat content and the degree of genomic rearrangements in plastomes of Medicago and its relatives Trigonella and Melilotus, which are nested firmly within the IRLC.Although plastomes are highly conserved with respect to gene content and order in most photosynthetic angiosperms, extensive genomic rearrangements have been reported in Fabaceae, particularly within the inverted repeat lacking clade (IRLC) of Papilionoideae. Two hypotheses, i.e.Melilotus dentata) in addition to the previous first reported cases in Medicago minima. We also conducted comparative genomic analysis to evaluate plastome evolution. Our results indicated that the overall repeat content is positively correlated with the degree of genomic rearrangements. Some of the genomic rearrangements were found to be directly linked with repetitive sequences. Tandem repeated sequences have been detected in the three genes with accelerated substitution rates  and their length variation could be explained by the insertions of tandem repeats. The repeat contents of the three localized hypermutation regions around these three genes with accelerated substitution rates are also significantly higher than that of the remaining plastome sequences.We detected abundant repetitive elements and extensive genomic rearrangements in the 75 newly assembled plastomes of 20 species, including gene loss, intron loss and gain, pseudogenization, tRNA duplication, inversion, and a second independent IR gain (IR ~ 15 kb in Our results suggest that IR reemergence in the IRLC species does not ensure their plastome stability. Instead, repeat-mediated illegitimate recombination is the major mechanism leading to genome instability, a pattern in agreement with recent findings in other angiosperm lineages. The plastome data generated herein provide valuable genomic resources for further investigating the plastome evolution in legumes.The online version contains supplementary material available at 10.1186/s12870-021-03202-3. Pelargonium transvaalense (Geraniaceae) [Erodium (Geraniaceae) [Carnegiea gigantean (Cactaceae) [Tahina spectabilis (Arecaceae) [In photosynthetic angiosperms, plastid genomes (plastomes) are highly conserved in gene content and structure and exhibit quadripartite structure with a pair of inverted repeats (IRs), which separate large single copy (LSC) and small single copy (SSC) regions , 2. The niaceae) , where tniaceae) , 9, Carnctaceae) , Tahina ecaceae) , the Putecaceae)  and the ecaceae) .Erodium [Pelargonium [Plantago [Erodium [Pelargonium, Plantago, and Silene have different levels of add substitution rates compared with the SC genes, which result from a mixture of locus-specific, lineage-specific, and IR-dependent effects [Although IR loss seems to be more common than previously thought, the presence of IR across angiosperms is still predominant, suggesting its functional importance in angiosperms. Early findings, which suggest that lineages lacking the IR have undergone more frequent genomic rearrangement than those that have retained the IR, support the hypothesis that IR plays a role in stabilizing plastome structure –15. HoweErodium , Pelargoargonium , and PlaPlantago  showed t[Erodium . Other f[Erodium  have als[Erodium –19. Howe effects , 17. Inc effects , 21, andrpl22, infA, substitution of rps16, losses or pseudogenization of accD and ycf4, IR reemergence, and losses of two clpP introns during their evolution [Passiflora plastomes [Lathyrus, in which c. 1.5 kb localized hypermutation region around ycf4 was caused by repeated DNA breakage and repair [Medicago minima [Previous studies have suggested that some species of the IRLC have acquired dramatic variations in plastome structures, including abundant inversions, mutation hotspots, gene transfers of volution , 22–24. lastomes –28. Illelastomes , 30. Desd repair . In addio minima , 31. NotMedicago (M.) L. and its relatives Trigonella (T.) L. and Melilotus Miller, all belonging to the tribe Trifolieae, which is nested firmly within the IRLC. Species of these three genera are very important legume forage with significant ecological and economic values, including the widely cultivated major forage crop species M. sativa, the legume model species M. truncatula and the widely cultivated medicinal species T. foenum-graecum. Choi et al. [Medicago species and one Trigonella species and revealed modest structural variations among them, but their discussion focused mainly on the IR reemergence in M. minima. Here, we took advantage of whole-genome resequencing data and assembled the plastomes of 75 individuals representing 20 species in the three genera. We aimed to characterize plastome structural variations of the 20 species at multiple individual levels and investigate the correlations between structural variations and repetitive elements.In this study, we focus on i et al.  completeM. orthoceras) to 142,713 bp (Melilotus dentata), and numbers of unique annotated genes from 110 to 111  suggested the presence of a large inverted repeat , which contained 10 coding genes, including ycf1, the conserved four rRNA genes  and five tRNA genes  in the seed plants  between the two IR copies in Melilotus dentata 02 and Melilotus dentata 03  in M. minima 01, duplicate of trnN-GUU in some individuals of Melilotus officinalis and Melilotus alba  in the Melilotus clade. Two kinds of duplicated tRNA were present in three Melilotus and two Medicago species. The tRNA duplicates were confirmed by read mapping as shown in Fig. clpP, atpF and rpoC1 in M. lupulina and M. minima was consistent with previous findings [clpP was lost in all the 20 species, consistent with previous studies [rpoC1 was shared by six Medicago species  was unique to M. polymorpha, and others were shared by multiple species, mostly by closely related species. For inversions, the large ones (> 30 kb) occurred mainly in two groups: the Trigonella clade and the clade containing M. platycarpos, M. ruthenica, and M. archiducis-nocolai (section Platycarpae). For the remaining inversions, two were unique to T. emodi, two were present in the Melilotus clade, and one was shared by M. lupulina and M. minima.The plastome structural variations, including three IR regainba Table , and twoula Fig. . Most offindings . The int studies , 32. We ies Fig. . After vM. orthoceras to 571 pairs in Melilotus indicus. The second abundant repeat type was palindromic repeats ranging from 17 pairs in M. edgeworthii, M. truncatula 02, and T. cachemiriana to 47 pairs in M. truncatula 01. Then a pair of reverse repeats in M. polymorpha, M. edgeworthii, M. monantha, and Melilotus dentata to 50 pairs in Melilotus officinalis. Lastly, the complimentary repeats were infrequent among the species ranging from 1 to 7 while others did not have such as M. polymorpha, M. truncatula, M. sativa, M. archiducis-nicolai, and M. falcata. A total of 38–110 tandem repeats were recognized across the 75 plastomes with their sizes ranging from 2033 to 5951 bp , ranging in size from 2550 to 7315 bp Table . The mosbp Table .M. orthoceras) to 8179 bp (Melilotus indicus) repetitive sequences across the 75 plastomes, accounting from 2.98 to 6.39% of their full plastomes, respectively  in T. cachemiriana had a pair of 41 bp inverted repeats flanked to its two endpoints  in M. ruthenica and the 36 kb inversion (petN—rps18) in M. archiducis-nicolai, M. ruthenica and M. platycarpos  and 16 highly divergent non-coding regions (π > 0.1)  Fig. .Fig. 4CP < 0.0001) in the substitution rates (dN and dS) of clpP, accD, and ycf1 compared to matK and rbcL for all the comparisons within both IRLC taxa and our own 20 species (pink branch)  to 2190 bp (M. minima); clpP from 582 bp  to 684 bp (M. ruthenica) and ycf1 from 4893 bp  to 5352 bp (M. lupulina), while the length of the other two genes did not show much variation (1512 or 1521 bp in matK and 1428 bp in rbcL)   than that of the remaining plastome sequences  Table . To inveL) Table . As expeces Fig. . TherefoErodium (Geraniaceae) [Carnegiea gigantea [M. minima in the IRLC [Melilotus dentata, in which ~ 15 kb of a large inverted repeat is regained, containing 10 coding sequences compared to the ~ 17 in the typical IR of angiosperms. Choi et al. [M. minima occurs via synthesis-dependent strand annealing or the formation and resolution of Holiday junctions during recombination-dependent DNA repair. Both processes need to be mediated by repeats. Choi et al. [rps12_5’ and between trnN-GUU and ycf1, and a series of tandem repeats between trnN-GUU and ycf1) retained in Medicago suffruticosa, which is closely related to M. lupulina and M. minima. The position of repeats in M. suffruticosa flanked the endpoints corresponding to the novel IRs in M. minima suggests that their ancestor may have experienced repeat-mediated perturbations, which may have caused IR regain in the two species. Repeat-mediated and recombination-dependent replication play a significant role during the IR reemergence [Melilotus dentata and its relatives, which may be due to incomplete sampling in our study. Dynamic characteristics of IR boundary migration process [M. minima and Melilotus dentata supports the hypothesis that the novel IRs are quite possible to expand continuously and include more adjacent canonical IR genes gradually [The existence of a pair of inverted repeats is a feature of plastomes throughout the 400 million years of land plant evolution  with theniaceae) , 14, Pinniaceae) , and Cargigantea . In addithe IRLC , we repoi et al.  speculati et al.  detectedmergence . However process  and the radually . A deepeclpP and ycf1 have been reported as rapidly evolving genes in Papilionoids [clpP is also accelerated in Mimosoid [ycf4 is accelerated in most legumes especially in the tribe Fabeae [accD in Jasminum [Silene [Plantago [matK and rbcL  complex, thus it is essential for plant viability [Gene-specific rate acceleration has been frequently reported in plastome evolution. Among legumes, lionoids . The genMimosoid  and ycf4e Fabeae , 39. AccJasminum , Silene  [Silene , and PlaPlantago  has alsobcL Fig. -S13. Theynthesis  and clpPprotease . For ycfiability . Notablyrpl22 and infA found in the 20 species and also in other legumes [rps16 in the 20 species could be explained by gene substitution as suggested for M. truncatula [Although the gene content and order of plastome is highly conserved in most seed plants , extensi legumes  and almo legumes  have beeuncatula .M. minima and Melilotus dentata, IR > 9 kb) compared with other species  should es  and reported a second independent IR gain in Melilotus dentata for the IRLC species. We detected abundant repetitive elements and extensive genomic rearrangements in these plastomes. Notably, we found that the overall repeat content is positively correlated with the degree of genomic rearrangements. Moreover, the overall repeat contents within localized hypermutation regions around genes with accelerated substitution rates were also significantly higher than that of the remaining plastome sequences. Our findings highlight the role of repetitive sequences in affecting plastome stability in the IRLC species. The plastome data generated herein provide valuable genomic resources for further investigating the plastome evolution in legumes.In this study, we completed 75 plastomes representing 14 species of Medicago and its relatives Trigonella and Melilotus, including 14 Medicago, 2 Trigonella, and 4 Melilotus species. Our sampling included 19 species of the three genera that are distributed in China and the model species of Medicago, i.e. Medicago truncatula. For M. truncatula, plastomes were assembled using whole-genome resequencing data, which were downloaded from NCBI . For Medicago sativa, seeds were germinated in the greenhouse and emergent leaves from a single plant from each accession were collected. For other species, leaves were collected from the wild (Table http://www.iplant.cn/foc/) for the nomenclature system in this study, but treated T. arcuata and T. cancellata as M. arcuata and M. cancellata, respectively, based on a recent study [Trifolium subterraneum (NC_011828.1) as an outgroup  as seed. The rbcL sequence of Trigonella foenum-graecum voucher I.S. (GenBank accession: NC_042857.1) was used as seed for Trigonella cachemiriana and Trigonella emodi. For Medicago polymorpha, M. truncatula, M. sativa, M. lupulina, M. minima, and M. falcata, their seeds were provided by their own rbcL sequences . The rbcL sequence of M. falcata (GenBank accession: NC_032066.1) was used as seed for other Medicago species. We left other parameters as the default values (see NOVOPlasty README.md). We annotated the assembled plastomes using GeSeq [ycf1 in the four Melilotus species and T. emodi, and two extra sequences inserted within accD in M. falcata when comparing with the sequences of the two genes in other species. In order to validate whether these insertions were introns or not, we downloaded transcriptomic data of M. falcata (SRR1823822), Melilotus albus (SRR5115455), and Trigonella foenum-graecum L. (SRR8281660) from NCBI. For the other three Melilotus species and T. emodi, transcriptomic data are not available and the validation of ycf1 in these species was based on the transcriptomic data of Melilotus albus and Trigonella foenum-graecum, respectively. We assembled these transcriptomes to get fasta sequences using trinityrnaseq-Trinity-v2.8.5 [http://transdecoder.sf.net) and aligned the coding sequence (CDS) with our gene sequences (both exons and introns) via ncbi-blast-2.10.0+ [ycf1 was true in T. emodi  but false in the four Melilotus species. In addition, the intron gain of accD in M. falcata was true (it does have two introns) but the intron boundaries were different from the original result. The visual images of the annotations of all species were generated by OGDRAW v.1.3.1 [https://chlorobox.mpimp-golm.mpg.de/OGDraw.html). All the plastomes of the 75 individuals were newly assembled and deposited in the Genome Warehouse of CNCB-NGDC (under BioProject accession PRJCA005341).The total genomic DNA was extracted using the modified CTAB procedure from the dried leaves , which wng GeSeq  with MPIng GeSeq . For conng GeSeq . In addiy-v2.8.5 , then co-2.10.0+ . In this v.1.3.1   among the newly assembled plastomes of 20 species that were estimated using progressiveMauve v.2.4.0 . In progWe calculated the repeat content of all the 75 plastomes. Tandem Repeats Finder v.4.09  was usedM. falcata as a reference. To detect the sequence divergence and determine highly divergent regions of the 20 species, we used the python script “get_annotated_regions_from_gb.py”  [https://github.com/Kinggerm/PersonalUtilities/) [Because structural variations among individuals within each species were almost the same (see Results), we selected 20 individuals representing all the 20 species   to automlities/) . We usedlities/)  to concalities/) .Medicago and its relatives Trigonella and Melilotus, we selected the same 20 individuals as mentioned above and used Trifolium subterraneum (NC_011828.1) as an outgroup. The coding sequences (CDS) of 73 protein-coding genes (PCGs) shared across the 21 species  [https://github.com/Kinggerm/PersonalUtilities/) [To reconstruct the phylogenetic relationships among es Table  were extlities/) . Each relities/) . Then welities/)  to concalities/) . Molecullities/)  with 100accD, clpP, ycf1, rbcL, and matK. We then downloaded the sequences of these five genes of the IRLC species from NCBI (taxon names and accession numbers are listed in Table Nonsynonymous (dN) and synonymous (dS) substitution rates were calculated using “yn00” from the PAML4.8 package  for fiveAdditional file 1 : Table S1. The plastome assembly, annotation information, and distributions of genomic rearrangements for the 75 individuals. Table S2. Detailed information of repeat content for the 75 individuals. Repeat content for the three IR regained plastomes were calculated using only one IR copy. Table S3. Repeats mediated tRNA duplicates. For dispersed repeats, F: forward repeat; C: complement repeat; P: palindromic repeat; R: reverse repeat, and the numbers after the colon represent length of dispersed repeats. For tandem repeats, the numbers before the colon represent length of tandem repeats, the content after the colon represent unit size × copy number. Table S4. Repeats around endpoints of inversions. For dispersed repeats, F: forward repeat; C: complement repeat; P: palindromic repeat; R: reverse repeat, and the numbers after the colon represent length of dispersed repeats. For tandem repeats, the numbers before the colon represent length of tandem repeats, the content after the colon represent unit size × copy number. Palindromic repeat (P) are marked in red. Table S5. Palindromic repeat sequences around endpoints of inversions. Table S6. Repetitive DNA in the acquired introns. Table S7. Nucleotide diversity (pi) for different genes, intergenic regions, and datasets. PC, plastid coding regions; PN, plastid noncoding regions; PCN, the whole plastome. Eight highly divergent coding regions (π > 0.04) and 16 highly divergent non-coding regions (π > 0.1) are marked in red. Table S8. Sequence divergence in accD among the IRLC species. Table S9. Sequence divergence in clpP among the IRLC species. Table S10. Sequence divergence in ycf1 among the IRLC species. Table S11. Sequence divergence in matK among the IRLC species. Table S12. Sequence divergence in rbcL among the IRLC species. Table S13. Information of repetitive elements for the coding sequence (CDS) of three genes  with accelerated substitution rates and two relatively conserved genes (matK and rbcL). There is no coding sequence (CDS) in accD for M. polymorpha because it is a pseudogene (truncated sequence). Table S14. Percent repetitive DNA of the three localized hypermutation regions around the three genes  with accelerated substitution rates and the remaining plastome sequences. Table S15. Locations of the 75 individuals representing 20 Medicago, Trigonella, and Melilotus species. The individuals for which were planted in laboratory are marked by asterisks. Plastomes of Medicago truncatula were assembled from whole-genome resequencing data downloaded from NCBI (SRR1524305 and SRR965443). The outgroup was downloaded from NCBI (NC_011828.1). The individuals for which were chosen as the representatives of each species are marked in red. Table S16. The 73 protein-coding genes (CDS) shared across 21 taxa included in the phylogenetic analysis. Table S17. Taxa included in the synonymous and nonsynonymous divergence analyses of accD, clpP, ycf1, matK, and rbcL. (√) adopt in analysis, (−) not available in NCBI and not adopt in analysis.Additional file 2 : Figure S1. Plastome maps for the 20 Medicago, Trigonella, and Melilotus species. Genes shown outside the circle are transcribed clockwise and those inside are transcribed counter clockwise. Genes belonging to different functional groups are color-coded. The dark gray area in the inner circle indicates GC content and the thick black line shows the extent of different regions. LSC: large single copy; SSC: small single copy; IRA: inverted repeat A; IRB: inverted repeat B. The two areas enclosed by red boxes in (d) indicate small IRs in Medicago lupulina. In (k), red arrows No. 1 and 2 outside the circle point to the breakpoints of the inverted region involving all genes from ycf1 to rpl20. Gene order in the M. truncatula 02 ptDNA between the arrows is in the reverse orientation and its length is ~ 44-kb . Below the map are shown the alignments of 24-bp incomplete inverted repeats in the inversion endpoints in the M. truncatula 02 and cognate sequences in M. truncatula 01. Figure S2. IRs alignment within the four Melilotus dentata individuals and the repetitive elements of indel regions in Melilotus dentata 02 and Melilotus dentata 03. Yellow bar represents protein coding gene; red bar represents rRNA gene; pink bar represents tRNA gene. Numerals above indicate nucleotide positions within the repeat alignment indicate the length of an indel within the IR. Mismatches are indicated by colored blocks and identical bases are gray. Mean pairwise identity over all pairs in each alignment column is indicated by the histogram: green 100%. IRA: inverted repeat A, IRB: inverted repeat B. Different repetitive elements were marked with different colored boxes. Figure S3. Mauve (Multiple Alignment of Conserved Genomic Sequence with Rearrangements) alignment of the plastomes of the 20 species using plastome of Wisteria floribunda as a reference. Figure S4. Confirmation of tRNA duplication in five species. Plastome sequences were mapped to themselves which contain all copies of the replicated tRNA (lower) and plastome sequences which contain a single copy of the replicated tRNA (upper). The scale at the left reports the depth of sequences, which is indicated graphically by the blue histogram. Figure S5. The degree of genomic rearrangements shows significant positive correlation with (a) dispersed repeats and (b) tandem repeats. Figure S6. The mechanism of tRNA duplication detected in five species. Both (a) and (b) contain a tandem repeat and two forward repeats that duplicate trnV-GAC twice. The three species in (c) contain a tandem repeat that duplicates the trnN-GUU gene. Thick black lines represent double stranded DNA. Pink boxes represent gene sequences, red and blue boxes represent tandem repeats, green boxes represent forward repeats and numbers indicate the length of repeats. Figure S7. Repeats around endpoints of all inversions. Red boxes represent regions of inversion, orange boxes represent protein-coding genes, and pink boxes represent tRNA genes. Black lines above double stranded DNA represent tandem repeats. Black lines below double stranded DNA represent dispersed repeats. The palindromic repeats are indicated by red lines. Figure S8. Repetitive DNA in the acquired introns. Yellow boxes represent exons. The lines between the yellow boxes represent acquired introns. The red content represents repetitive sequences. Figure S9. Comparison of the 20 plastomes using the annotation of Medicago falcata as a reference. The vertical scale indicates the percentage of identity, ranging from 50 to 100%. The horizontal axis indicates the coordinates within the plastomes. Genomic regions are color-coded as conserved non-coding sequences (CNS), exons, and tRNA or rRNA. Figure S10. Significance test for nonsynonymous (dN) (upper) and synonymous (dS) (lower) substitution rates of clpP, accD, and ycf1 compared to rbcL for all the comparisons within both IRLC taxa we have found (left) and our own 20 species (right). ****, P < 0.0001 (T-test). Detailed information can be found in Tables Figure S11. Synonymous and nonsynonymous divergence in the IRLC species for five chloroplast genes: matK (a), rbcL (b), accD (c), clpP (d), and ycf1 (e). Shown are dN (left) and dS (right) trees resulting from a codon-based maximum likelihood (ML) analyses using RAxML, rooted using three Robinioid sequences (non-IRLC species): Lotus japonicus, Sesbania grandiflora, and Robinia pseudoacacia. The species are in the same order from top to bottom in the dN and dS trees of each gene.


Mycobacteriumtuberculosis genomics, virulence, and pathophysiology within theframework of Koch postulates. More recently, the advent of “-omics” approacheshas broadened our appreciation of how “commensal” microbes have coevolved withtheir host and have a central role in shaping health and susceptibility todisease. It is now clear that there is a diverse repertoire of interactionsbetween the microbiota and host immune responses that can either sustain ordisrupt homeostasis. In the context of the global efforts to combatting TB, suchfindings and knowledge have raised important questions: Does microbiomecomposition indicate or determine susceptibility or resistance toM. tuberculosis infection? Is thedevelopment of active disease or latent infection upon M.tuberculosis exposure influenced by the microbiome? Doesmicrobiome composition influence TB therapy outcome and risk of reinfection withM. tuberculosis? Can the microbiome beactively managed to reduce risk of M.tuberculosis infection or recurrence of TB? Here, weexplore these questions with a particular focus on microbiome-immuneinteractions that may affect TB susceptibility, manifestation and progression,the long-term implications of anti-TB therapy, as well as the potential of thehost microbiome as target for clinical manipulation.Tuberculosis (TB) remains an infectious disease of global significance and aleading cause of death in low- and middle-income countries. Significant efforthas been directed towards understanding  Mycobacterium (M.)tuberculosis. In the majority of individuals,M. tuberculosis infection is thought to resultin clinically asymptomatic latent tuberculosis infection (LTBI). There is currentlyno standardized test to confirm the presence of viable M.tuberculosis in individuals with LTBI, and diagnosis is largelybased on immunological tests that indicate antigen experience ; IFNγ release assays (IGRA) detecting reactivity of CD4+ T cells toM. tuberculosis-specific antigens in wholeblood). Of note, there are reports of individuals showing no signs of antigenexperience or active TB disease in settings of repeated high exposure andtransmission of M. tuberculosis. While it isdifficult to determine how these “resisters” may be protected from productiveinfection with M. tuberculosis, a range of innateand adaptive immune mechanisms governed by genetic and epigenetic factors, as wellas antigen experience may contribute [M.tuberculosis [M. tuberculosis. The review ofhistoric data suggested that the risk for active TB beyond 2 years after exposuredeclines sharply, arguing that reactivation of LTBI might be a much less commonevent than currently believed and that active TB later in life might result fromre-exposure rather than reactivation [Tuberculosis (TB) persists as one of the top 10 causes of death in the world, withcurrently an estimated 1.4 million deaths annually [ntribute . It is crculosis , with a tivation .First-line anti-TB antibiotics isoniazid, pyrazinamide, and ethambutol arenarrow-spectrum, showing little or no activity outside the mycobacterial genus, but areThe colonization of the host by microorganisms begins within minutes of birth orhatching. There is a gradual succession in the diversity and density of thesecommunities, influenced by a myriad of genetic, environmental, and behavioral inputs,23. DuriBy removing the obligatory step of microbial cultivation, a much greater appreciationof the structural and functional dynamics of these communities in the context ofhealth and disease has been developed. In addition to the oral cavity, themicrobiota of the large intestine is the most studied compartment of the “humanmicrobiome” . UntilrM. tuberculosis-infected host in animal modelshas been the subject of significant efforts are reported, and relative abundances of Proteobacteria, which, when remarked upon,are increased in M. tuberculosis-infectedindividuals  across different bacterialtaxa. Thus, future studies should aim to combine longitudinal microbiome analyseswith transcriptome and metabolome profiling to establish whether changes in therelative abundance of any taxa translate into biologically meaningful changes in theconcentrations of immunomodulatory metabolites, and other molecules, at local anddistant tissue sites.Characterization of the microbiome composition of TB patients and the efforts  and has ividuals ,46.Phylividuals . Membersividuals . Butyratividuals –51, albeividuals . Duringnfection . Given tcrobiota . ImportaPrevotella, Bulleidia, andAtopobium) [M. tuberculosis infection [Streptococcus andPrevotella) in patients without M.tuberculosis [M. tuberculosis. This highlights potentialchallenges for the precise annotation of the TB-associated lung microbiota whenusing 16S rRNA gene profiling [M. tuberculosis, revealed increased microbialdiversity early after infection (1 month), with the relative abundances ofAggregatibacter, Streptococcus, andStaphylococcus genera elevated by 4 months post infection, andthe relative abundances of members of the Lachnospiraceae family being significantlydecreased [M. tuberculosis infection in macaques [M. tuberculosis infection, andwhether these result from microbial interactions within the niche, or as aconsequence of mucosal  immune responses to M.tuberculosis infection. As the importance of microbiomecomposition of the respiratory tract for susceptibility to infections is emerging[As reflected in opobium)  needs toopobium) ,57. Alteopobium) . The larnfection . The relrculosis  contrastrofiling . Neverthecreased . The magmacaques . Indeed,macaques –64. For emerging, constraemerging,67.M.tuberculosis is one of the most significant constraints toimproving the clinical management of TB [M. tuberculosis infection, the results to dateindicate that TB antibiotics have a long-lasting impact on the gut microbiomecomposition [The phenotypic and genetics basis of drug resistance in nt of TB . Treatment of TB . Antibioposition –13,42–44Clostridioides difficileinfection often results in long-term failure of antibiotics to control thisinfection [M. tuberculosis [M. tuberculosis-derived epitopes that showedsimilarities with microbiota species [M.tuberculosis infection [M. tuberculosis infection and the quality ofthe immune response. Understanding of how prolonged antibiotic use affectspredisposition to recurrent TB and/or reinfection is an important area of futureinvestment. Notwithstanding the limits of current studies , anti-TB antibiotic regimens exert selective pressure andreorganization of the gut and/or lung microbiota with profound and long-lastingeffects. Knowledge of the functional implications of these alterations via thegut-lung axis on host immune response are emerging. The following sections examinethe physiological and metabolic cues arising from the gut (and lung) microbiota withimplications for host susceptibility or resistance to the clinical manifestations ofM. tuberculosis infection.It is increasingly appreciated that commensal bacteria can confer a form ofcolonization resistance against nonresident species including pathogens, viacompetition for metabolic and/or spatial niches, as well as their production ofbioactive molecules that can directly inhibit/suppress the growth of susceptiblemicrobes . The susnfection , and thirculosis ,71. Inde species . In a monfection , alludinM. tuberculosis infection[Bioactive metabolites are a key element of the crosstalk between the host and itsmicrobial collective. Such metabolites arise from microbial metabolism  as well as microbe-facilitated modulation of host- or dietary-derivedmetabolites  . Signifinfection.M. tuberculosis entryinto the human host is transmission via aerosol droplets. The size ofM. tuberculosis-containing dropletsallows entry into the alveoli of the lower respiratory tract where thebacteria encounter respiratory epithelium, alveolar macrophages, andresident microbiota. The roles of alveolar epithelial cells in the hostdefense against M. tuberculosis areincompletely understood. M. tuberculosishas been found in cells of the alveolar epithelium in humans, and infectedalveolar epithelial cells in vitro in some but not all studies [M. tuberculosis [M. tuberculosis entry. Pulmonaryepithelial cellular defense mechanisms are directly responsive tomicrobiota-derived SCFAs [M. tuberculosis [M.tuberculosis, and bacterial pathogens more generally.Moreover, microfold (M) cells in the upper respiratory tract have beensuggested to act as entry points for M.tuberculosis across the epithelial barrier intomucosa-associated lymphoid tissues, which may result in extrapulmonarymanifestation of M. tuberculosis  [M. tuberculosis virulence factor EsxAand scavenger receptor B1 on M cells in the airway epithelium [M. tuberculosis infectionin the antibiotic-naïve or antibiotic-experienced host.The main route of  studies –80. Tranrculosis . While trculosis –85,it ied SCFAs . Whetherrculosis ,88, is snary TB) ,90. Thisithelium . With miithelium , microbiHost-microbiota interactions are critical in governing tissue homeostasis atsites of close contact as well as distant sites. Yet, microbial dysbiosisand compromised but barrier functions, e.g., in the context of chronicinflammation and antibiotics treatment, have been implicated in inflammationand metabolic dysfunction at distant sites. This is driven at least in partthrough innate immune activation of macrophages and other innate immunecells by microbiota-derived bacterial products such as LPS ,93–96 F. Some stM.tuberculosis, and bacterial interference withmacrophage antimicrobial defense mechanisms enable intracellular persistenceand replication [M. tuberculosis survival have beenimplicated in facilitating the exponential intracellular replication ofM. tuberculosis within alveolarmacrophages for several days post infection [M. tuberculosis infection [M. tuberculosis after a course oftreatment with the TB antibiotics isoniazid and pyrazinamide for 8 weeksresulted in slightly higher lung bacterial burden. This was accompanied byan altered phenotype of alveolar macrophages, including diminished MHCIIexpression, TNF and IL-1β production, as well as cellular respiration andATP production [M.tuberculosis replication in ex vivo cultures. Theauthors linked functional dysbiosis to these outcomes, which were reversedby fecal microbiota transfer (FMT). It is interesting to note that theantibiotic-driven phenotypic alteration of alveolar macrophages was notinducible in in vitro culture in the presence of isoniazid and pyrazinamidebut required the in vivo tissue context [M. tuberculosis-induced IFNγproduction and promoted IFNγ-driven macrophage antimicrobial defensemechanisms [M.tuberculosis infection with implications for developingactive disease or LTBI.Macrophages are major host cells for intracellular lication . The immnfection . The airnfection ,103, incnfection . Infectioduction .Alveola context , suggestchanisms .Thus, pM. tuberculosis infection iscritically dependent on intact IL-12 and IFNγ signaling, and IFNγ-mediatedprotection is largely attributed to adaptive T cell responses [Microbial products, including metabolites, distinctly guide development andfunctions of innate and innate-like lymphocytes. Conversely, thelocalization of innate and innate-like lymphoid cells to mucosal sitesdirects the composition and spatial distribution of the microbiota . SCFAs sesponses . HoweverBased on their cytokine expression profiles, ILCs are categorized into group1, including natural killer (NK) cells and noncytotoxic type 1 ILCs , group 2 (IL-4/5/13), and group 3 (IL-17/22) .+ NK cells in the lunghas also been associated with LTBI in nonhuman primates [+ T cellresponses, and lyse mycobacteria-infected monocytes, macrophages, as well asregulatory T cells expanded in the presence of mycobacterial antigens [M. tuberculosis orMycobacterium bovis bacille Calmette-Guérin (BCG)[−/− γc−/−) resulted in higher susceptibilityto M. tuberculosis infection compared to Tand B cell deficiency (RAG2−/−), which was attributed toIL-12-driven IFNγ production by innate lymphocytes [M.tuberculosis early during infection have emerged,specifically in the formation of inducible bronchus-associated lymphoidtissue (iBALT) [M. tuberculosis infection [IFNγ-expressing NK cells have been described to accumulate in the pleuralfluid of patients with TB pleurisy . Individprimates . In contprimates .NK cellantigens –117. Patantigens , which iin (BCG),119. Train (BCG), suggestphocytes . More re (iBALT) ,which infection .M. tuberculosis shapedby the metabolic capacity of the microbiome , natural killer T cells (NKT), and γδ T cells recognize microbiallyderived nonpeptide antigens via semi-invariant T cell receptors (TCRs)resulting in cytokine production and/or cytotoxic activity. Among these,MAIT cell development has been closely linked to the presence of themicrobiota driven by thymic presentation of bacteria-derived antigen –135, altM.tuberculosis-infected nonhuman primates have shown onlylimited accumulation in infected lung tissue [M. tuberculosisinfection compared to wild-type mice [M. tuberculosis infection resulted inexpansion of MAIT cells but did not affect M.tuberculosis burden in the lung [+ Tcell priming in mesenteric lymph nodes [M.tuberculosis infection conferred some protection in thelung dependent on IL-17A, but not TNF or IFNγ. A possible interpretation ofthese observations is that the microenvironment and/or activation status ofMAIT cells at the time of stimulation skews their cytokine profile towardsregulatory or inflammatory functions [M. tuberculosis infection, and that itappears to be important to determine whether the timing of their engagementin the context of infection is beneficial or detrimental to immune responsesthat control mycobacterial infections. Of note, a genetic polymorphism inMR1 has been associated with TB susceptibility andmanifestation in humans [M.tuberculosis exposure are associated with protectionfrom productive infection [Peripheral blood MAIT cell numbers are significantly diminished in TBpatients –146 and g tissue . Observag tissue ,149,150.ype mice . Exogenothe lung ,152, desph nodes . On the unctions . Whetherunctions . Such inunctions . Thus, en humans , and hounfection ,154.Finnfection  might benfection ,156. Whe+ T cells are critical in the host control ofM. tuberculosis infection, withcontributions of CD8+ T and B lymphocytes increasinglyappreciated. Inflammatory circuits, e.g., driven by IL-12/IFNγ, TNF, andIL-17, are central to controlling M.tuberculosis, yet tight regulation of these immuneeffector mechanisms, e.g., by regulatory T (Treg) cells andIL-10, is essential for preventing severe pathology and poor pathogencontrol [M.tuberculosis infection [CD4control . Withthcontrol , there infection ,158.M.tuberculosis infection. A recent study in a smallcohort of patients with active TB (prior to treatment commencement), LTBIand healthy controls reported a positive correlation between the abundanceof Coriobacteriaceae in fecal samples of LTBI individuals withM. tuberculosis antigen-specific IFNγresponses in peripheral blood [M.tuberculosis. Specifically, impaired host control ofM. tuberculosis in mice exposed tobroad-spectrum antibiotics exposure was associated with decreasedproportions of IFNγ+ and TNF+ CD4+ T cellsalongside an increased percentage FoxP3-positive Treg cells in the spleen[M. tuberculosis lung burden at the onsetof the chronic phase of infection, which was associated with impairedantimicrobial defense by alveolar macrophages, without impact on thepercentages of TB antigen-specific T cells [M.tuberculosis control by the host [There is evidence suggesting that microbiota composition licenses T cellfunctions critical to controlling al blood . Observae spleen. Incont T cells . In boththe host ,160. Thethe host .Klebsiellaaeromobilis, Klebsiella pneumoniae, andBilophila wadsworthia [M.tuberculosis-specific T cell responses towardsincreased effector functions  and whether this translatesinto benefits for the host in controlling M.tuberculosis might offer opportunities for targetedintervention. This might encompass promotion of a specific microbiotacomposition but could equally be explored for metabolic capacities of themicrobiota that define host immune functions. Microbial products andmetabolites, in particular SCFAs, have been established as key mediators ofimmune-modulatory functions of the microbiota [There are examples of microbiota species that have been suggested to poisethe host towards Th1 responses, including sworthia ,163. Defcrobiota . In thisM. tuberculosisantigen-specific IFNγ and IL-17A production and elevated IL-10 production ofin vitro cultured human peripheral blood mononuclearcells (PBMCs) [FOXP3 expression and Treg differentiation [IL12b,Nos2) by macrophages and dendritic cells [M.tuberculosis. If present at the time ofM. tuberculosis encounter, this mayrepresent a risk factor for successful infection and progression to activeTB. Support for this hypothesis may be drawn from a recent study in a cohortof HIV+ healthy individuals undergoing antiretroviral therapy(ART) in a high-TB incidence environment. Individuals undergoing ART arecharacterized by SCFA-producing microbiota in their lower airways, and inthis cohort, SCFA serum concentrations positively correlated with elevatedrisk of subsequently developing TB, as well as induction ofFoxP3+ Tregs in PPD-stimulated cultures of BAL lymphocytes[Prevotella in the lower airways [M.tuberculosis infection. This might seemcounterintuitive when considering the decrease in the relative abundance ofRuminococcaceae and/or Lachnospiraceae described in some studies  ,166.Thintiation ,168. Addntiation , inhibitic cells ,171, as ic cells . Thus, tphocytes. Elevate airways . These c studies . HoweverM. tuberculosis infection and TBdisease.Mucosal and systemic antibody responses are directly shaped by themicrobiome. Exploration of these microbiota-immune interactions has largelyfocused on the gut microbiota, a critical regulator of gut immunoglobulin A(IgA) production ,174.MicM. tuberculosis infection, andrelative abundance of memory B cells, plasma blasts, and plasma cells hasbeen correlated with TB disease state . M.tubesponses –186. B creported .M. tuberculosis infection in theimmune-competent host elicits robust antibody responses against diversemycobacterial protein and oligosaccharide antigens [M.tuberculosis control by host cells [M.tuberculosis restriction by infected human and mousemacrophages associated with enhanced phagocytosis and delivery tophagolysosomal compartments [M. tuberculosis infection [M.tuberculosis infection in light of reports that passivetransfer of purified, mycobacteria-specific IgA diminished bacterial burdenin infected lungs [M.tuberculosis are highly heterogenous and influenced bycomplex interactions of a number of factors, including age, state ofinfection (active TB disease or LTBI), and immune competency . With the fundamental contributions of the microbiota to shapinglocal airway mucosal as well as systemic antibody responses [M. tuberculosis infection. Thedesign of future studies needs to include considerations on the impact ofsystemic and mucosal antigen exposure on antibody repertoire [M.tuberculosis-specific antibodies may be further definedby distinct glycosylation profiles characteristic to disease state, i.e.,active versus latent TB [M. tuberculosis. Acomprehensive view of B cell functionality, beyond antibody responses, inthis context will further enhance understanding of cellular drivers of localinflammatory responses [antigens . Recent st cells ,190 haveartments –194.Mornfection .Antibionfection . Thisobed lungs –198.Theesponses ,199, it atent TB . It willatent TB  and whetesponses ,187,macesponses , neutropesponses ,201, andesponses ,203.M.tuberculosis (re)infection, protective immune responses, anddisease manifestation, the questions arising now center on how this knowledge mighttranslate into therapeutic or preventative measures. Areas of focus includeopportunities at the gene product , organismal, FMTs), and dietary level ofinterventions to correct microbial dysbiosis or specifically deliver functionalcapabilities that reshape host immune responses and resilience toM. tuberculosis infection and/or recurrence.With the notion that the larger collective of “commensal microorganisms” may,directly and indirectly, shape host susceptibility to M. tuberculosis,which raises the possibility of defining a gut microbiota that reduces hostsusceptibility to M. tuberculosis infection and TBdisease manifestation [Strategies that promote the introduction and/or restoration of a “beneficial”microbiota, such as dietary interventions or defined probiotic formulations mayprove to be an effective strategy to complement TB treatment, in particular incorrecting the long-lasting dysbiosis that occurs as consequence of prolonged TBantibiotics regimens. Moreover, gut microbiota composition prior to infection hasbeen found to correlate with disease manifestation in nonhuman primatesexperimentally infected with estation .Gut micestation . Proteinestation –208.Bifidobacterium spp. as an adjunct therapy withconventional TB antibiotics are reported to restore and maintain what is considereda “healthy microbiome” [Lactobacillus acidophilus, Lactobacilluscasei, Lactobacillus rhamnosus, Lactobacillusbulgaricus, Bifidobacterium breve,Bifidobacterium longum, and Streptococcusthermophilus) combined with supplementation of vitamins B1,B6, and B12 increased serum concentrations of IFNγ andIL-12, compared to the control group receiving only anti-TB antibiotics and vitaminB6 [Probiotics such as robiome” –211. AltaminB6 .WhethertaminB6  remains Mycobacterium manresensis. Indeed, formulations using thisenvironmental bacterium that is commonly found in drinking water are being exploredfor potential benefits in the treatment of TB. In a susceptible mouse model ofM. tuberculosis infection, orally administeredheat-killed M. manresensis reduced lung pathology,bacterial burden, and inflammatory responses, and in combination with TBantibiotics, expanded the life span of infected mice when compared to mice treatedonly with antibiotics [M.manresensis-based food supplement on gut microbiotacomposition, antigen-specific CD4+ T cell responses, as well as time to sputumconversion and reduction in bacterial burden (NCT03851159).Immune cross-reactivity between mycobacterial species as well as direct impact on themicrobiota are associated with beneficial effects of orally administered heat-killedibiotics .Followiibiotics ,216, a pClostridioides difficile infection, offering high therapeuticefficacy and with limited prevalence of adverse events, at least in the short term[M. tuberculosisinfection [Perhaps the most dramatic approach to “probiotic therapy” is the integration of FMTinto clinical practice. Although practiced by some cultural groups for centuries, FMT hasort term. These fort term–221. In nfection ,160 warrnfection –224.Peptostreptococcus anaerobius, has been shown to inhibit growthof M. tuberculosis, both in vitro and in vivo.This has been attributed to antagonistic effects of IPA on M.tuberculosis tryptophan biosynthesis, leading to suggestionsthat IPA per se and/or targeting the M.tuberculosis tryptophan pathway may be avenues for thediscovery of novel antimycobacterials [Lactobacillussalivarius, Streptococcus cricetus, andEnterococcus faecalis exceeds that of the TB antibioticrifampicin [M.tuberculosis, Mycobacterium kansasii,Mycobacterium smegmatis, and Mycobacteriumavium subspecies paratuberculosis [E.faecalis and ethambutol [With current limitations of probiotics and FMT, dietary interventions, definedmicrobial metabolites, and actively secreted bioactives might offer a pragmaticalternative. For example, indolepropionic acid (IPA), which is produced by bacteriataxonomically affiliated with the Clostridiales, includingcterials –228. Addcterials –232 mighcterials ,234. Thefampicin , with nirculosis ,237. Synhambutol  may offeM. tuberculosisinfection, whether modulation of SCFA production might be a target for interventionin TB requires careful consideration. With SCFA the primary microbial metabolitesreleased within the gastrointestinal tract, host evolution has favored thedevelopment of sensor-regulatory pathways linked with immune and/or metabolicpathways that can monitor and respond to alterations in these primary microbialmetabolites. In chronic diseases with characteristic gut dysbiosis , the presumptive reduction in butyrate-producingbacteria is widely considered to compromise barrier integrity, mucin production, andFoxP3+ Treg cell production [Faecalibacterium prausnitzii [Notwithstanding the notion that SCFAs poise host immune mechanisms towards apermissive environment for oduction ,240. Whioduction . Such fiusnitzii ,243 highM. tuberculosis infection or canindeed skew effector mechanisms towards improved or diminished pathogen controlrequires carefully designed prospective and longitudinal studies in large cohorts.The integration of microbiome, metagenome, and metabolome analyses, ideally in thelung as well as the gut and potentially other distant sites, alongside immunologicalcharacterization will be essential. Additionally, important confounding factors suchas nutritional status, coinfection(s), and other comorbidities [Confidence in whether the microbiome composition is associated with hostsusceptibility bidities ,244 willbidities .M.tuberculosis infection. It will be particularly important todetermine the interplay between microbiota and immune components at distinct stagesof infection and disease. Our discussions above highlight the importance ofacknowledging potential composite effects of innate and adaptive immune cellfunctions, and the multidimensional interplay between microbiota and host defensemechanisms. For example, butyrate enhanced antimicrobial defense in macrophages, thereby increasing control of extracellularand intracellular bacterial pathogens, including mycobacteria [M. tuberculosisinfection in the host at least in part through their immune-modulatory effects onadaptive immune responses. Moreover, detailed studies are required to fill currentknowledge gaps on the host interactions with viruses, fungi, and protozoa in thehuman microbiome, which likely has profound implications for shaping host responsesto infections [Candidate microbiota/microbe/metabolite approaches and functional studies in animalmodels of TB will be invaluable to further elucidate causality between microbiotacomposition, metabolic capacity, and the immune control of bacteria . Yet, SCfections ,247.M. tuberculosis infection, as indicated inmouse studies [Restoration of TB antibiotic-induced dysbiosis is an attractive and seeminglyachievable target. Nevertheless, the transition of probiotics from being dietarysupplements to an evidence-based predictive intervention in clinical settingsremains elusive ,249. Sim studies ,160, is  studies . Additio studies .Similar studies ,252. Tra studies .In summary, notwithstanding the increasing body of literature focused on establishinglinks between the microbiome and the immune control of TB, as with mostmicrobiome-focused research, the challenge at hand will be to establish causality,which would deliver solid foundations for the pursuit of targeted interventions inTB.


Caenorhabditis Intervention Testing Program (CITP) was founded on the principle that compounds with positive effects across a genetically diverse test-set should have an increased probability of engaging conserved biochemical pathways with mammalian translational potential. To fulfill its mandate, the CITP uses a genetic diversity panel of Caenorhabditis strains for assaying longevity effects of candidate compounds. The panel comprises 22 strains from three different species, collected globally, to achieve inter-population genetic diversity. The three represented species, C. elegans, C. briggsae, and C. tropicalis, are all sequential hermaphrodites, which simplifies experimental procedures while maximizing intra-population homogeneity. Here, we present estimates of the genetic diversity encapsulated by the constituent strains in the panel based on their most recently published and publicly available whole-genome sequences, as well as two newly generated genomic data sets. We observed average genome-wide nucleotide diversity (π) within the C. elegans (1.2e-3), C. briggsae (7.5e-3), and C. tropicalis strains (2.6e-3) greater than estimates for human populations, and comparable to that found in mouse populations. Our analysis supports the assumption that the CITP screening panel encompasses broad genetic diversity, suggesting that lifespan-extending chemicals with efficacy across the panel should be enriched for interventions that function on conserved processes that are shared across genetic backgrounds. While the diversity panel was established by the CITP for studying longevity interventions, the panel may prove useful for the broader research community when seeking broadly efficacious interventions for any phenotype with potential genetic background effects.The  Caenorhabditis elegans. The reproductive style of C. elegans makes it particularly easy to generate and maintain large populations of genetically identical individuals. In fact, the control over genetic variability helped make C. elegans the first multi-cellular organism to have its entire genome sequenced (C. elegans Sequencing Consortium 1998). Despite the success garnered using genetically homogeneous populations, it has become increasingly apparent that many of the phenotypes of interest for study are dependent on genetic background . Examples of these background-influenced phenotypes range from α-synuclein toxicity , to behavioral responses to temperature , dietary influence on lifespan and reproduction , and pharmacological efficacy . The dependence on genetic background suggests that attempts to identify core biological systems and functionality could benefit from assaying across genetic diversity to identify genetic background-independent phenotypes. One way to achieve this is to use a panel of populations with intra-population homogeneity and inter-population diversity.Model organisms have been fruitful tools for elucidating core biological principles. The power of model organism study, in part, is due to the ability to grow large populations with known genetic makeup. One of the most widely adopted genetic models is the hermaphroditic nematode Caenorhabditis Intervention Testing Program (CITP) was designed to identify anti-aging and longevity-promoting compound interventions effective in a genetically diverse set of Caenorhabditis nematode populations . To date, 55 chemical compounds have been tested for reproducible, genetic background-independent effects on longevity . The genetic diversity panel used by the CITP is composed of 22 strains from three hermaphroditic species, which facilitates maintenance of intra-population homogeneity. While the full panel is composed of 22 stains, a smaller core sub-panel of nine strains , C. briggsae , and C. tropicalis  is used in initial compound effect characterization . Here, we present estimates of the genetic diversity encapsulated by the constituent strains of the CITP panel, and core sub-panel, based on their most recently published and publicly available whole-genome sequences.With the importance of genetic background effects in mind, the C. elegans is typically isolated from cooler ecological niches than C. tropicalis , while C. briggsae is found in niches that range from cool to warm . The wide species distributions enabled the CITP to assemble a panel of strains isolated worldwide, with representatives from most continents , to project genotypes of the strains on two latent dimensions , which is consistent with previous estimates for those species . Caenorhabditis species is higher than that within human populations  and comparable to that found in mouse populations . Our analysis supports the assumption that the CITP screening panel encompasses broad genetic diversity, suggesting that lifespan-extending chemicals with efficacy across the panel should be enriched for interventions that function on conserved processes that are shared across genetic backgrounds. While the diversity panel was established by the CITP for studying longevity interventions, the panel may prove useful for the broader research community when seeking broadly efficacious interventions for any phenotype with potential genetic background effects.When establishing the genetic diversity panel for compound screening, the CITP sought strains that represented both broad geographic and genetic diversity. The three species represented in the panel are themselves globally distributed, but with ecological restrictions. For example, ntinents . We nextmensions . We thenTo generate estimates of the genetic variability among the strains in the CITP diversity panel we collected publicly available genomic data for 18 of the 22 CITP strains (see Reagents below). Because comparable Illumina-based sequencing was unavailable for N2-PD1073 and ED3092, two strains present in the core-subpanel of nine strains, we generated whole genome data for these two strains using standard protocols. In brief, we used proteinase K to digest whole worms and isolated total genomic DNA using the Genomic DNA Clean & Concentrator kit (Zymo Research). Genomic libraries were then generated using the Nextera XT DNA Library Preparation Kit . Sequencing was carried out on the Illumina NovaSeq 6000 platform by the Genomics and Cell Characterization Core Facility (GC3F) at the University of Oregon. We then used the two new genomic datasets, along with the 18 available in the NCBI database, to calculate genetic diversity estimates using our software pipeline (described below).Caenorhabditis Genetics Center (CGC), the C. elegans wild-isolates should be ordered from the Caenorhabditis elegans Natural Diversity Resource (CeNDR) . Strains unavailable from those sources  can be obtained directly from the CITP upon request. Relevant to the unavailability of PD1073 at the CGC, PD1073 is a close relative of PD1074 which is distributed by the CGC as a wild type reference strain. PD1073, PD1074, and PD1075 are three subclones of the N2-derivative strain VC2010 that were generated in the process of assembling a new N2 reference (“VC2010-1.0”) genome . All strains in the genetic diversity panel and the associated SRA read accession IDs used in this study are listed below:While most of the strains in the CITP diversity panel can be obtained from the C. elegans: strain CB4856 , ED3040 , JU1088 , JU1652 , JU775 , MY16 , QX1211 ;C. briggsae: AF16 (SRR2002620), HK104 , JU1348 (SRR1793004), JU726 (SRR1792964), NIC20 (SRR1793012), QR25 (SRR1793006);C. tropicalis: JU1373 , JU1630 (SRR12623130), NIC58 , QG131 (SRR12623047), QG834 (SRR12623046).C. briggsae strain JU1264 and C. tropicalis NIC122.No genomic data is publicly available for C. elegans N2-PD1073 and C. briggsae ED3092 strains generated in this study were deposited to the SRA database (https://www.ncbi.nlm.nih.gov/sra) under the project accession ID PRJNA773598.Raw reads for C. elegans N2-PD1073, MY16, and JU775; C. briggsae AF16, ED3092, and HK104; C. tropicalis JU1373, JU1630, and QG834.The core CITP strains are Softwareet al. 2016), and filtered and trimmed reads with Skewer v.0.2.2 . We mapped filtered reads to the C. elegans, C. briggsae,and C. tropicalis genomes obtained from the WormBase database (https://wormbase.org//) version WS280  by BWA-MEM v.0.7.17 (Li 2013) and SAMtools v.1.5 , and deduplicated with the Picard tools v.2.17.6 (Broad Institute). We called and filtered variants using GATK v.3.7  software and BEDtools v.2.25 , and estimated the nucleotide diversity using VCFtools v.0.1.15 , masking repetitive regions, indels, complex variants, and regions with too low or high coverage. We projected genotypes of the CITP strains on two latent dimensions using popVAE , and visualized them in R v.3.5 (R Core team 2018) using packages ggplot2 (Wickham 2016) and ggrepel.We estimated genetic diversity of the CITP strains using whole-genomic data for 20 out of 22 CITP strains from the panel. Reads from the SRA database were downloaded with SRA-toolkit (the SRA Toolkit Development Team). We evaluated the read quality with FastQC v.0.11.5 (Andrews 2010) and MultiQC v.1.3  and are available under the MIT license. The global map was generated in MATLAB R2021b  using the MATLAB Mapping Toolbox. The location coordinates used for the strains are included in the GitHub repository. Figure readability was improved by moving panel relative positions, updating color coding, and improving text aesthetics using Adobe Illustrator 2022 in a manner consistent with image integrity standards.