E. faecium strains found in healthy feedlot beef cattle and those causing extraintestinal infections in humans is not yet fully understood. This study used whole-genome sequencing to analyse the antimicrobial resistance profile of E. faecium isolated from beef cattle (n = 59) at a single feedlot and compared them to previously reported Australian isolates obtained from pig (n = 60) and meat chicken caecal samples (n = 8), as well as human sepsis cases (n = 302). The E. faecium isolated from beef cattle and other food animal sources neither carried vanA/vanB responsible for vancomycin nor possessed gyrA/parC and liaR/liaS gene mutations associated with high-level fluoroquinolone and daptomycin resistance, respectively. A small proportion (7.6%) of human isolates clustered with beef cattle and pig isolates, including a few isolates belonging to the same sequence types ST22 , ST32 (eight beef cattle and one human isolate), and ST327 (two beef cattle and one human isolate), suggesting common origins. This provides further evidence that these clonal lineages may have broader host range but are unrelated to the typical hospital-adapted human strains belonging to clonal complex 17, significant proportions of which contain vanA/vanB and liaR/liaS. Additionally, none of the human isolates belonging to these STs contained resistance genes to WHO critically important antimicrobials. The results confirm that most E. faecium isolated from beef cattle in this study do not pose a significant risk for resistance to critically important antimicrobials and are not associated with current human septic infections. The extent of similarity between  Enterococcus spp. such as Enterococcus faecalis strain Symbioflor 1 are used as probiotics to treat diarrhoea, improve immunity, and provide other health benefits [Enterococcus spp. such as Enterococcus faecium are among the most important causes of hospital-acquired infections in humans, including endocarditis, sepsis, urinary tract, and central nervous system infection [E. faecium or Enterococcus faecalis [E. faecium and E. faecalis possess additional virulence factors that have contributed to their pathogenesis and clinical significance [Enterococci are ubiquitous Gram-positive commensal bacteria that encompass more than 50 species found in various environments, such as the gastrointestinal tract of animals and humans, as well as the hospital environment . Some Enbenefits ,3. Howevnfection . More thificance . Some ofificance . E. faecium are also intrinsically resistant to various antimicrobial classes, including aminoglycosides, cephalosporins, and trimethoprim-sulfamethoxazole [E. faecium is known for its efficiency in recruiting and exchanging AMR determinants through mutation or horizontal transfer of ARGs located on mobile genetic elements (MGEs), which can be readily transferred between E. faecium lineages [E. faecium is also capable of acquiring plasmid-mediated resistance determinants from other Enterococcus species [E. faecium to methicillin-resistant Staphylococcus aureus (MRSA) via broad host range plasmids is a major concern [E. faecium isolates [E. faecium is the most common cause of hospital-acquired infections compared to other species of enterococci [E. faecium isolates have developed resistance to last-resort antimicrobials, such as daptomycin, linezolid, tigecycline, and vancomycin, which could have dire consequences for public health in the future [E. faecium isolates worldwide, the vanA and vanB vancomycin resistance genotypes are the most prevalent ARG variants [hoxazole ,8,9. E. lineages . E. faec species . Moreove concern . Plasmid concern . The preisolates . Multidrerococci . In addie future ,17,18. Avariants . E. faecium sequence types (STs), together with their correlation with specific hospital-infection-associated AMR determinants and virulence factors. Australia represents a unique study site to explore the host specificity of animal-origin E. faecium isolates given its isolation, quarantine restrictions including a ban on importation of live food-producing animals, and conservative regulation of antimicrobial use in both human and animal health [Whilst some genetic similarity has been found among enterococci isolated from animals and those causing human infections , other sl health . As a cal health ,25. As al health ,27,28. Enterococcus spp. prevalence, with E. faecium infrequently isolated from entry samples , but then becoming the most prevalent species in exit samples . The interplay between diet, management practices, and microbial ecology is a potential factor that could contribute to the observed shift in Enterococcus spp. [Enterococcus prevalence is noteworthy as it highlights the importance of monitoring alterations in microbial communities, where these changes can have implications for both animal health and food safety. Further genomic interrogation is now required to determine the genetic similarity of these cattle-origin E. faecium isolates compared to those detected in other food animals and humans in Australia as well as to examine capacity for cross-host species transmission and potential spread via the food chain or the environment. Therefore, this study aimed to conduct further genetic analysis on the beef cattle E. faecium isolates through comparing them to isolates obtained from healthy meat chickens and pigs at slaughter as well as human clinical blood sepsis isolates in Australia described in previous studies [In our previous study, we investigated the frequency of AMR in enterococci from faecal samples collected at the entrance to and exit from a beef feedlot in South Australia . While tcus spp. ,31. This studies ,29. E. faecium from beef cattle were analysed via WGS and compared with commensal isolates from healthy slaughter age pigs (n = 60), meat chickens (n = 8), and a large collection of human clinical sepsis isolates (n = 302). SNP analysis of the beef cattle, chicken, and pig isolates confirmed that they formed two main clades (Clade 1 and Clade 2). Clade 2 was subsequently divided into two subclades (Clade 2a and Clade 2b). Clade 2b contained a notable monophyletic subclade (Clade 2ba) that was composed entirely of more closely related human isolates  and efaAfm (adherence) were most prevalent across all sources of isolates. Among the beef cattle isolates, acm was found in 56/59 (95.0%) and efaAfm in 58/59 (98.3%) , cylA , cylL , cylM , espfm , and hylEfm . The human E. faecium isolates located in STs 32, 22, and 372 also did not contain espfm and hylEfm markers typically exclusive to hospital-associated strains, but carried both acm and efaAfm.Out of the eight  (98.3%) . The remaadA5, aadD, aph(2”)-Ia, aph(2”)-Ie, aph(3’)-III, aac(6’)-aph(2”), aac(6’)-Ii, ant(6)-Ia, ant(9)-Ia), ß-lactams , fluoroquinolones , folate pathway inhibitor (dfrG), glycopeptides , lipopeptides , macrolides/lincosamides, and/or streptogramins (MLS) , erm(B), erm(T), lnu(B), lnu(G), lsa(E), msr(C), vatE), phenicols , and tetracyclines (tet(45), tet(L), tet(M), tet(O), tet(S), tet(W)) were identified in the entire collection of 429 isolates  are associated with multidrug efflux pumps and other transporters, which may potentially lead to resistance against multiple antimicrobials. The number of ARGs identified in each isolate ranged from one to ten. Human E. faecium isolates possessed the highest mean number of ARGs per isolate (n = 8) compared to pig and chicken  and beef cattle isolates (n = 3).Heat maps were generated based on the identification of genes from the ResFinder and CARD databases . Overallisolates . Among tE. faecium isolate collection, including those imparting resistance to aminoglycosides (aac(6′)Ii, and ant(6)-Ia), ß-lactams (pbp5), fluoroquinolones (efmA), MLS , vat(E), msr(C), and erm(B)), and tetracyclines (tet(45), tet(L), tet(M), and tet(S)) (aac (6’)-Ii  and the chromosomally encoded msr(C) , eatAv , pbp5 , and efmA , which are responsible for resistance to aminoglycosides, MLS, β-lactams, and fluoroquinolones, respectively. A total of 13 ARGs were identified in the beef cattle  tet(S)) . The mosE. faecium isolates, none of the E. faecium isolates from beef cattle were found to possess gyrA or parC mutations associated with high-level fluoroquinolone resistance. However, some did possess the ABC transporter gene efmA, which potentially confers low-level resistance as a fluoroquinolone efflux pump . Overall, 43 (72.9%) of beef cattle isolates contained at least one plasmid, and 2 isolates harboured six plasmids. A significant number of beef cattle isolates  did not possess any plasmids compared to human  and pig isolates . Plasmid replicon rep1 (broad host range) was predominantly found in beef cattle  and pig isolates , but of comparatively lower prevalence in human isolates , while rep2 (narrow host range) was more commonly found in human (238/302 78.8%) and pig isolates  but was present in a much smaller proportion  of beef cattle isolates. Highly diverse sets of plasmids were identified among the is cases .vanA (found only in isolates from humans) was mostly linked to three plasmid replicons: rep2 , repUS15 , and rep11a . Most E. faecium isolates carrying the streptogramin resistance gene vatE  were associated with the plasmid replicons rep2  and rep17  (E. faecium ARGs known to be plasmid-mediated were cross tabulated and compared according to isolate source (beef cattle vs. human), clear differences in matches were apparent between the two host sources, apart from repUS15, which was highly prevalent in both groups of isolates and often associated with aac(6’)-Ii, erm(B), and tet(M) ARGs  . When pl(M) ARGs . E. faecium, can cause serious nosocomial infections, including endocarditis, sepsis, and urinary tract infections in humans [E. faecium isolated from both animal production and human hospital settings can provide important information about the evolution and spread of resistance and the relative impact of food and/or environmental transmission compared to direct human-to-human contact. Through detailed whole bacterial genome bioinformatic analyses, this study focused on AMR, plasmid, and virulence factor profiles identified in E. faecium isolated from the gastrointestinal tracts of healthy slaughter-age beef cattle from a single feedlot in Australia and compared these with previous data established for similar pig [Enterococci are commonly found as commensals in the gastrointestinal tract of animals and humans, but some species, such as n humans ,32. Reguilar pig  and meatilar pig  togetherilar pig . E. faecium isolates from healthy slaughter-age beef cattle, pig, and meat chicken sources (found in Clades 1–2b) were genetically distinct from most human clinical isolates, which formed a monophyletic clade (Clade 2ba). Second, none of the animal-origin isolates from beef cattle, pig, and meat chicken sources possessed ARGs or mutations responsible for high-MIC-level resistance to three critically important antimicrobials  typically present in human isolates. Third, a small proportion  of human clinical isolates were found clustered with some of the animal isolates (including some belonging to the same ST) within Clades 1–2b, but none of these human isolates possessed ARGs to critically important antimicrobials. These results confirm that, as previously determined for Australian pig and poultry E. faecium isolates [E. faecium (or their ARGs) to humans via the food chain or through the environment with corresponding minimal impact to human health. Evidence for this conclusion can be found in the limited crossover of animal vs. human STs (the majority of beef cattle isolates were located in Clade 1) and the differences in virulence genes and ARG arrays observed, particularly to the critically important antimicrobials . Furthermore, far more plasmid replicons were observed in the E. faecium isolated from humans compared to beef cattle isolates. The study had three major findings. First, phylogenetic analysis confirmed that the isolates , beef caE. faecium isolates from beef cattle in this study revealed significant diversity in their STs with most located in Clade 1. Only three primarily beef-cattle-origin STs  contained either one or two E. faecium isolates obtained from human sepsis cases. This is in agreement with a previous report that clinical E. faecium isolates causing infections in humans tend to belong to different sub-lineages than those found in animals, food, and the environment [E. faecium ST17, a clonal lineage responsible for hospital-acquired infections throughout the globe, was only identified among human isolates in the present study . Compared to other strains in the collection, ST17 E. faecium were more likely to exhibit high-level resistance to aminoglycosides, beta-lactams, fluoroquinolones, and tetracycline, which correlated with the possession of aac (6’)-Ii, tet (L), and tet (M) genes, as well as mutations in gyrA, parC, and pbp5, as documented in a previous international study [vanB was detected in the hospital-derived ST17 human isolates . This study found that the human isolates carrying the van operon were not clustered in the same evolutionary clade as the beef, chicken, and pig isolates. However, the ST22 and ST32 isolates shared the same ARG types, including plasmid-mediated genes responsible for resistance to aminoglycosides (aac(6’)-I); the chromosomally encoded ampicillin resistance mutation (pbp5); low-level fluoroquinolone resistance (efmA); and macrolide, lincosamide, and streptogramin ARGs (msr(C) and eatAv in ST327 isolates). It has been previously reported that ST32 and ST22 share the same ARG profiles, yet they are not typical clones associated with vancomycin resistance nor do they have point mutations in gyrA and parC responsible for high-level fluoroquinolone resistance, indicating a limited human health risk [The MLST analysis of ironment . For exaal study . Additiolth risk ,36,37. BE. faecium infections in humans [E. faecium isolates was comparatively lower compared to human and pig isolates. Some beef E. faecium isolates carried the ermB gene , which encodes an enzyme responsible for methylating the 23S rRNA component of the bacterial ribosome, whilst nearly all contained the msr(C) gene , which encodes an efflux pump, resulting in resistance to macrolides and streptogramin B , and most contained the eatAV gene which encodes an ABC transporter and encodes resistance to lincosamide, pleuromutlins, and streptogramin A [vatE gene responsible for streptogramin A  resistance. Possession of either ermB or eatAV with msr(C) genes is associated with resistance to quinupristin–dalfopristin, a combination of streptogramin A and B [msr(C) in the beef cattle isolates may also be related to the common use of macrolides as first-line treatments for bovine respiratory disease in Australian feedlots [Enterococci are rapidly evolving to become resistant to various classes of antimicrobials, including fluoroquinolones, glycopeptides, lipopeptides, oxazolidinones, and streptogramins ,39,40. Wn humans . The curgramin A . Further A and B . Whilst  A and B , the prefeedlots . As virgfeedlots ,47. vanA has been reported in E. faecium isolated from animals and animal-derived food products [VanA-positive enterococci were isolated from food animals in England in 1993 [E. faecium has been shown to be associated with the use of avoparcin for growth promotion in food animals [vanA and vanB were found in 21.5% and 28.5% of human isolates, respectively. However, no vancomycin ARGs were found in any of the beef, chicken, and pig isolates. In another study in Australia using phenotypic methods, enterococci isolated from cattle were sensitive to a number of medically important antimicrobials, such as daptomycin, linezolid, tigecycline, and vancomycin [E. faecium isolated from Australian healthy slaughter-age beef cattle, pigs, and meat chickens are mutually exclusive from the source of the circulating vancomycin-resistant strains of clinical significance in the public health sector [In recent decades, the vancomycin resistance gene products . VanA-po in 1993 . Since t in 1993 ,51,52. T animals ,54. Howe animals . In thisncomycin . The finE. faecium isolated from food-producing animals harboured known resistance genes for daptomycin. However, 23 (7.6%) of the human isolates showed mutations in the genes responsible for daptomycin resistance (liaR and liaS). Contrary to this, some studies have shown that daptomycin resistance does not always result from liaFSR mutations. Mutations in chromosomal genes cls and gdpD, which encode cardiolipin synthase and glycerophosphoryl diester phosphodiesterase, respectively, have been linked to daptomycin resistance [cls gene. In our previously published study, we detected daptomycin resistance in E. faecium isolates obtained from both entry  and exit  beef cattle faecal samples using a phenotypic method, but the underlying mechanism behind this resistance could not be determined [Daptomycin is another antimicrobial used as a last-resort antimicrobial to treat vancomycin-resistant enterococci . Howeversistance . In thistermined . These ftermined ,58, incltermined . E. faecium isolates is increasing globally, posing a significant threat to public health. In recent years, efflux pumps have played a crucial role in the MDR status of various bacteria [efmA gene efflux pump, which encodes resistance to fluoroquinolones and macrolides, was observed in E. faecium isolated from beef (35.6%), chicken (100%), human (92.4%), and pig (56.7%) samples. Efflux pumps are known to contribute to intrinsic and acquired resistance to antimicrobials used to treat infectious diseases [parC) and DNA gyrase (gyrA), as previously reported [E. faecium isolates obtained from beef cattle. The prevalence of fluoroquinolone resistance among bacteria . In thisdiseases . Howeverreported . In thisEnterococcus spp. [vanA and vatE genes, responsible for resistance to vancomycin and quinupristin-dalfopristin, respectively, were mostly associated with narrow host range rep-2 plasmid replicons. By comparison, the most commonly observed plasmids in E. faecium isolates from beef cattle were repUS15 (67.8%) and broad host range rep1 (55.9%), with only a relatively minor proportion carrying rep2 (8.5%). Other international studies have also shown the strong association between carriage of rep2 plasmid replicons and resistance to glycopeptides in enterococci isolated from both humans and food-producing animals [E. faecium isolates (together with pig and meat chicken isolates) pose limited risk to human health from horizontal gene transfer.The potential for AMR to be transferred to other bacteria exists through several means, with plasmids serving as one of the primary mechanisms for this transfer to occur . Plasmidcus spp. . Among t animals ,65. In sE. faecium isolates, with the acm and efaAfm genes being the most commonly detected among all isolates regardless of source and espfm only detected in human isolates. Both acm and efaAfm were prevalent in over 95% of E. faecium isolates from Australian beef cattle, similar to the results obtained for chicken and pig isolates. In human E. faecium isolates, the virulence genes espfm (18.2%) and hylEfm (27.1%) were also commonly observed in addition to acm and efaAfm. The hylEfm gene, which is responsible for intestinal colonization, has been detected in ampicillin-resistant E. faecium ST17 and vancomycin-resistant E. faecium (VREF) strains in hospitals worldwide [E. faecium found in food-producing animals differ from those found in human isolates. Enterococci can carry various virulence genes, including those involved in biofilm formation, adhesion, and invasion of host cells . This storldwide ,68,69. TE. faecium isolates from meat chickens, pigs, and humans were obtained from a universal database as secondary data. Second, the beef cattle isolates were obtained as part of longitudinal study comparing entry and exit samples from a single feedlot, which may not be sufficient to generalize the results to the wider population. Ideally, this study could now be replicated in a wider range of feedlots. Third, the E. faecium isolates from food animals obtained during surveillance were compared with pathogenic isolates from human sepsis cases, and future studies could also examine human carriage isolates. Finally, due to the use of short-read sequencing data, it was not possible to determine the AMR profile and virulence factor composition of each plasmid replicon. In the future, the scope of this study could be expanded to include a range of farms and geographical locations.This study has some limitations that should be taken into account. One major limitation is that the E. faecium isolates from beef cattle were whole-genome sequenced and assembled using a previously described method (BioProject PRJNA879912) [E. faecium isolates from healthy pigs (accession number PRJNA639902) [E. faecium complete genome (NCBI Assembly Accession: CP003583.1). The software Snippy v4.6.0  was used to call core SNPs, i.e., SNPs that can be determined in all isolates. A maximum likelihood (ML) tree was constructed with RAxML v8.2.10 using the model GTRCAT, and a rapid bootstrap analysis with 100 bootstraps for the best-scoring ML tree was undertaken [https://itol.embl.de/, accessed on 23 October 2022) for display [The A879912) . The obtA639902)  and meatA639902)  from slaA639902)  from Ausdertaken . This wadertaken . Multilodertaken . The fin display . A heat www.genomicepidemiology.org, accessed on 12 October 2022) was used to identify acquired ARGs in the WGS data, using a threshold of 98.0% identity (ID). ARGs were also predicted using the Antibiotic Resistance Genes Database (ARDB) and the Comprehensive Antibiotic Resistance Database (CARD) in addition to ResFinder [The detection of virulence genes was carried out using VirulenceFinder 2.0 ,75. To iesFinder . To deteesFinder . Categorical measured traits including ARGs, the presence of plasmid replicons, and virulence factors were converted into a numerical code, with 1 indicating presence and 0 indicating absence. Proportions of AMR, MDR, ARGs, plasmid replicons or virulence factors were calculated using R Statistical Package version 4.0.0. Resistance profiles were categorised as MDR if the isolate exhibited resistance to one or more antimicrobials in three or more antimicrobial classes . AMR andE. faecium isolates obtained from beef cattle at a single feedlot with those obtained from different animal hosts (pigs and meat chickens) and humans within Australia. Beef cattle, meat chicken, and pig-source E. faecium isolates were found to be of limited risk and largely unrelated to human-source isolates. The strict regulation of antimicrobials used in food animals is likely the reason for the low level of AMR found in food animals in Australia. The higher levels of AMR observed in human isolates may be because they are clinical isolates exposed to more antimicrobials than the surveillance isolates from food animals included in this study. This research approach helps understand the general trend and spread of AMR E. faecium in different sectors. The continued use of antimicrobial agents in health care and agriculture settings inevitably leads to the emergence of AMR. This study compared the prevalence of virulence genes, ARGs, and plasmid replicon types identified among


Klebsiella pneumoniae (K. pneumoniae) is the second leading cause of community-acquired and hospital-acquired gram-negative bloodstream infection (BSI). This study aimed to assess the epidemiological and microbial-resistance characteristics and clinical factors associated with K. pneumoniae BSI in Saudi Arabia.K. pneumoniae isolates diagnosed between January 2019 and January 2020 at King Fahad Medical City, Riyadh, Saudi Arabia were evaluated retrospectively. Clinical records of the patients were collected and analysed statistically.Data of 152 K. pneumoniae BSI were identified. Adult patients (66.4%) were at a higher risk of developing the infection than paediatric patients (33.6%). The rate of infection was slightly higher in women than in men. Neurological disorders were the predominant underlying conditions for the acquisition of K. pneumoniae BSI, at all ages. Most of the deceased patients were adults with multi-organ dysfunction. Klebsiella pneumoniae showed disturbing resistance to amoxicillin-clavulanate and cefuroxime (72.4%), ceftazidime (67.8), cephalothin (76.3%), and to Carbapenems (36.1%).In total, 152 cases of K. pneumoniae BSI was seen not only at the patient level, but also at the community level, and was related to multi-drug resistant infection. These findings provide a better understanding of microbial resistance and its association with patient clinical outcomes.The impact of  Klebsiella pneumoniae, a normal commensal organism living on human mucosal surfaces, including the gastrointestinal tract and oropharynx, can invade other tissues and cause severe infections [Escherichia coli [fections . It is thia coli . BSI canhia coli .K. pneumoniae [Several investigations have indicated that diabetes, hepatobiliary illness, and neoplastic disease were associated as risk factors to around 50% of community-acquired eumoniae , 5 Lin Yeumoniae .K. pneumoniae has become clinically significant owing to its increased antibiotic resistance, propensity to develop antibiotic resistance, and ability to produce serious outcomes [K. pneumoniae is one of the main factors responsible for nosocomial infections, including pneumonia, urinary tract infections, and bloodstream infections (BSIs); its production of extended-spectrum β-lactamases (ESBL) and carbapenemases causes high mortality rates (40–50%), primarily among critically ill and immunocompromised patients [K. pneumoniae from all available antimicrobials [Over the past two decades, outcomes , 8. Moreoutcomes . Multidrpatients . The emecrobials , 12.K. pneumoniae is a global health concern listed by the World Health Organization (WHO) as a critical priority. Unfortunately, in Saudi Arabia, research findings regarding K. pneumoniae infections are consistent with the global emergence of drug-resistant strains [K. pneumoniae in Saudi Arabia [K. pneumoniae has been reported [K. pneumoniae represented (33%) of the MDR/XDR cultures [The increasing prevalence of multidrug-resistant  strains , 14. Thei Arabia , 16, andreported . Furthercultures .K. pneumoniae bacteraemia cases at the King Fahad Medical City (KFMC) in Riyadh, Saudi Arabia. The primary objectives were to evaluate the epidemiological patterns, determine the antimicrobial resistance profiles of the isolates, and clarify their association with patients’ clinical outcomes.Such reports emphasise the necessity of continuous monitoring of the antimicrobial resistance of bacterial isolates, as this can be used to provide guidance to clinicians in the application of empirical treatment. This retrospective study investigated Klebsiella pneumoniae isolates from blood clinical samples were analysed. The clinical history of 152 patients was included in this study.This retrospective study was conducted over one year (January 2019 to January 2020) at KFMC, which has a capacity of 1200 beds. A total of 152 Klebsiella pneumoniae were collected from blood . The following were the included collection categories: (A) age (paediatric or adult). (B) Ward or clinic to which the patient was admitted . (C) blood sample source and location or site; and (D) bacterial-resistance category , and Carbapenem-Resistant strains). Any growth other than that of K. pneumoniae was excluded from the study. Clinical history was collected from the KFMC database for paediatric and adult patients admitted to ICU. The clinical history collected for ICU patients included different criteria: (1) if present, the type of co-infection; (2) exposure to carbapenem or other antibiotics in the past 14–30 days; (3) renal dialysis at isolation or not; (4) mechanical ventilation or not; (5) chronic diseases such as diabetes mellitus (DM), hypertension, renal disease, or malignancy; (6) presence of clinical symptoms such as fever, gastrointestinal tract (GIT) symptoms, or respiratory symptoms; (7) presence of wound or urinary tract infection; (8) presence of bacteraemia or septicaemia; and (9) clinical outcomes and additional notes, if available.In total, 152 samples of Klebsiella species, using a Phoenix BD instrument  for full identification and sensitivity testing. We included only patients whose isolates were definitively identified as K. pneumoniae. Antimicrobial sensitivity testing (AST) was performed for the following antibiotics: ampicillin (AMP), amoxicillin-clavulanate (AMC), piperacillin-tazobactam (TZP), imipenem (IPM), meropenem (MER), ertapenem (ETP), cephalothin (CEF), cefuroxime (CXM), ceftazidime (CTZ), cefoxitin (FOX), cefepime (CFPM), cefotaxime (CTX), ceftriaxone (CRO), ciprofloxacin (CIP), levofloxacin (LVX), gentamicin (GM), amikacin (AMK), tigecycline (TGC), colistin (COL), and trimethoprim-sulfamethoxazole (TMP-SMX). Susceptibility was classified as follows: susceptible, intermediate, or resistant. Confirmation of resistant isolates was performed using Microbroth dilution. Additional tests of disc diffusion or gradient diffusion (Etest) methods were performed using Mueller-Hinton agar which were then incubated in ambient air at 35 ℃ for 16–20 h. For interpretation, CLSI M100 Interpretive Document for Enterobacterales. EUCAST was used for the interpretation of tigecycline activity.All isolates were presumptively identified as n) and percentages. P < 0.05 was considered statistically significant. Relative risk (RR) was computed to demonstrate how much the risk variables raised the risk of mortality following a study by Hafiz et al. (2022) [The data were analysed using GraphPad Prism version 9.3.1. Descriptive analysis, using contingency tables and graphs, was used to illustrate the following data: age divisions, sex, ward/clinic, sample source, and sample site. The descriptive data are expressed as absolute numbers (. (2022) .This project was approved by the institutional review board (IRB) of KFMC. Consent was obtained from KFMC according to the ICH GCP ethical code . Informed consent was obtained from all the participants and from the legal guardians of the participants who were below 16 years of age.K. pneumoniae isolated from the central or peripheral venous catheter. Approximately two-thirds (66%) of the study population were aged > 15 years. Females were fairly probable as males to be infected with K. pneumoniae BSI. Among the incident K. pneumoniae bloodstream infections, 53 isolates were classified as ESBL strains, 55 as Carbapenem-Resistant strains, and 44 as susceptible strains. More than half of the K. pneumoniae isolates originated from critical care wards, such as ER and ICU wards . However, septic shock was significantly more frequent in adult patients (P = 0.0092). In adult patients, K. pneumoniae BSI and comorbidities, such as diabetes mellitus, hypertension, malignancy, chronic kidney disease, and ischaemic heart disease, were significantly associated . Notably, paediatric patients with acute respiratory distress syndrome were slightly more vulnerable to K. pneumoniae BSI than adult patients (P = 0.040).Univariate analysis was conducted to compare clinical manifestations between paediatric and adult patients with ns Table . PaediatK. pneumoniae bloodstream infection. Table K. pneumoniae BSI (n = 148), the overall mortality rate was 32.4% (48/148 patients). Univariate analysis revealed many risk factors associated with mortality (ranked from highest to lowest significance): mechanical ventilation, multi-organ dysfunction, septic shock, gastrointestinal infection, chronic kidney disease, carbapenem resistance, age > 15 years, ischaemic heart disease, and hypertension . The risk of mortality was 34% higher in adult patients and approximately 50% lower in paediatric patients . Patients with multi-organ dysfunction were at a substantially high risk of death from K. pneumoniae BSI . In addition, the susceptible K. pneumoniae strains were significantly isolated from non-ICU patients compared to the ICU patients (P = 0.0108). Carbapenem resistance, mechanical ventilation, respiratory infection, multi-organ dysfunction, mortality, ischemic heart disease, and septic shock were significantly higher among ICU patients than non-ICU patients .A univariate analysis was conducted to compare the clinical characteristics and risk factors of patients with Klebsiella pneumoniae showed alarming resistance with XXXore than 60% of isolates were resistant to AMC as well as most of the cephalosporins . Approximately half of the isolates were resistant to TMP-SMX. They exhibited lower resistance to the aminoglycosides (GM and AMK) than to the β-lactam antibiotics. Carbapenem-Resistant strains accounted for approximately 36% of all isolates. Interestingly, one-third of the isolates were resistant to tigecycline, a third were moderately sensitive, and a third were sensitive. Only isolates that were Carbapenem-Resistant were reported to have colistin resistance (6.6%), whereas isolates that were susceptible, ESBL, and Carbapenem-Resistant were reported to have tigecycline resistance.Figure Klebsiella pneumoniae BSIs are associated with high mortality rates worldwide. The emergence of antibiotic-resistant K. pneumoniae complicates the management of infections caused by these bacteria. This study aimed to evaluate the epidemiology, resistance profiles, and clinical outcomes of K. pneumoniae BSIs. The infection rate was slightly higher in females (52%) than in males (48%). The ICU and ER had the highest prevalence of K. pneumoniae BSI. ICUs are considered factories that create, amplify, and disseminate antibiotic resistance [Klebsiella pneumoniae BSIs in patients admitted to the ICU were significantly associated with multiple risk factors including carbapenem resistance, mechanical ventilation, respiratory infection, multi-organ dysfunction, ischemic heart disease, and septic shock were significantly higher among ICU patients than non-ICU patients. In a recent study by Wang et al. (2023), age over 70 years, admission to ICU, and urinary tract infection were found to be the risk factors for Carbapenem-resistant and ESBL-KP-resistance [K. pneumoniae were ICU admission, respiratory failure, admission from the Emergency, and imipenem use [sistance , 21. Thesistance . In anotet al. 20, age oveKlebsiella spp. (27.9%) bloodstream infection in ICU patients with poor outcomes [In addition, the mortality rate was higher among ICU patients and contributed to 45.9% of the death rate. This finding is supportive of the EUROBACT-2 international cohort study on epidemiology and outcomes of hospital-acquired bloodstream infections in ICU patients which revealed predominant outcomes .K. pneumoniae BSI development [K. pneumoniae BSIs was greater for adults than paediatric patients and was high for those with multi-organ failure. The higher mortality rate can be attributed to the greater virulence of the carbapenemase-producing strains, inappropriate antibiotic therapy, the greater toxicity and reduced effectiveness of antibiotics, and severe underlying diseases such as DM and chronic kidney disease [Various hospital-based studies have suggested multiple comorbidities, including DM, biliary disease, and liver disease, as risk factors for elopment , 26. Herelopment , which relopment , 28. Her disease .E. coli and K. pneumoniae. It has been estimated that resistance to fluoroquinolones and β-lactam antibiotics, including carbapenems, cephalosporins, and penicillins, is responsible for more than 70% of deaths attributable to antimicrobial resistance [K. pneumoniae pose a global public health concern. In Saudi Arabia, the rate of K. pneumoniae resistance has increased substantially in the last few years, reaching 100% resistance in some regions [Antimicrobial resistance is one of the most urgent public health concerns worldwide. Based on a comprehensive global analysis, it caused 1.27 million deaths in 2019, more than those caused by HIV/AIDS or malaria , and it sistance . The eme regions . We beli regions . Further regions , 35.K. pneumoniae was second only to E. coli as a cause of BSI. In the current study, 34.87% of the K. pneumoniae isolates produced ESBL, whereas this rate was 21.3% for the Makkah region (western Saudi Arabia) [K. pneumoniae in Saudi Arabia is presumably related to factors such as antibiotic prescription rates and resistance-related reporting. The rapid development of resistance is associated with membrane permeability, efflux pump activity, and β-lactamase production [ESBLs, produced primarily by gram-negative bacteria, mediate resistance to a wide range of β-lactam antibiotics, including extended-spectrum cephalosporins and the monobactam aztreonam. Most ESBL-encoding genes are carried by mobile genetic elements, facilitating the spread of resistance genes among bacteria. Several national and international studies have reported an increase in the prevalence of ESBL production among clinical isolates, reaching approximately 74% in some countries –38. A st Arabia) . Similar Arabia) . This reoduction .Our findings revealed high resistance (64–76%) to cephalosporins. Overall, our observed cephalosporin resistance rates were lower than those previously reported for Saudi Arabia: resistance to third- and fourth-generation cephalosporins increased substantially from 2011 to 2021, reaching 84.9%, 85.1% and 85.8% for ceftazidime, cefotaxime, and cefepime, respectively .K. pneumoniae is the most common of the carbapenem-resistant Enterobacteriaceae found in hospitalised patients [K. pneumoniae has significantly increased in Saudi Arabia: K. pneumoniae resistance to imipenem increased from 6.6% to 2011 to 59.9% in 2021, suggesting an increase in the consumption of antibiotics [In this study, 36.18% of the isolates were Carbapenem-Resistant strains. Resistance to carbapenems is a major public health problem, as they are the last line of drugs for treating severe bacterial infections caused by ESBL-producing gram-negative bacteria. In Saudi Arabia, patients . Unfortuibiotics .K. pneumoniae was the second most common pathogen after E. coli, responsible for the most deaths attributable to antimicrobial resistance, causing > 600,000 antimicrobial resistance-associated deaths globally. In our study, mortality rates were higher for patients infected with ESBL- and Carbapenem-Resistant strains K. pneumoniae than for those infected with susceptible K. pneumoniae strains. BSI caused by ESBL-producing K. pneumoniae isolates were associated with 33.3% mortality, whereas higher mortality (52.1%) was associated with BSIs caused by Carbapenem-Resistant K. pneumoniae (52.1%), consistent with prior national studies [K. pneumoniae was responsible for 55,700 deaths in 2019, followed by carbapenem-resistant Acinetobacter baumannii. Higher mortality is associated with infections caused by carbapenem-resistant K. pneumoniae than with those caused by carbapenem-sensitive K. pneumoniae [K. pneumoniae BSI was responsible for 41% of in-hospital mortality, compared to 79% in those infected with Carbapenem-Resistant K. pneumoniae [In 2019,  studies , 44. Gloeumoniae . A case-eumoniae .K. pneumoniae.Our sample size was small, comprising only data collected over one year at KFMC. Using a larger sample will increase the precision of our findings with regard to statistical analysis. Additionally, further analysis is essential to examine the effects of the COVID-19 pandemic on the resistance profile of K. pneumoniae isolates with a significant incidence of resistance were found in the institution. These results help shed light on microbial resistance and its relation to patient clinical outcomes. The high mortality rate among ICU patients is significantly associated with several risk factors, highlighting the importance of further investigating each factor and revising policies that might decrease the probability of such an outcome. Additionally, the observed resistance to colistin and tigecycline is concerning, particularly considering that a similar recent local study found a high prevalence of tigecycline resistance in ESBL-producing K. pneumoniae [eumoniae . This ma


Staphylococcus aureus, Cronobacter sakazakii, Shigella spp., Campylobacter jejuni, and Campylobacter coli were detected and identified in 47.5%, 20%, 45%, 5%, and 30% of the sweet samples, respectively. We found S. aureus and Shigella spp. were the most prevalent bacterial pathogens. S. aureus was found to be the most frequent pathogenic bacteria profiled in these samples. We also found a significant correlation between the presence of C. coli and Cr. sakazakii. We detected the blaSHV resistance gene in 97.5% of the sweet samples; however, blaTEM was detected in only one sample (2.5%). Conclusions: Regarding these results, we suggest preventive strategies such as implementing automation of food processing; monitoring the personal hygiene and health of food handlers, and testing regularly for antibiotic resistance in raw materials and products.Background: This study aimed to investigate the prevalences of some important antibiotic-resistance genes (ARGs) and foodborne bacterial pathogens in sweet samples collected from local markets in Iran. Methods: Forty sweet samples were collected. Foodborne pathogens and ARGs were detected in the sweet samples by conventional and multiplex PCR assays using species-specific primers. Results:  Foodborne diseases are defined as intestinal or extraintestinal disorders resulting from ingesting or consuming contaminated water, food, or food products . FoodborCampylobacter spp., pathogenic Escherichia coli (E. coli), non-typhoidal Salmonella spp., and Shigella spp.; however, S. typhi, enteropathogenic E. coli, enterotoxigenic E. coli, Vibrio cholerae, and Campylobacter spp. are the major hazards considering the global number of deaths caused by bacterial foodborne pathogens. Also, other bacterial agents, such as Listeria monocytogenes (L. monocytogenes), Clostridium botulinum, Brucella spp., Bacillus cereus (B. cereus), Staphylococcus aureus (S. aureus), and Clostridium perfringens (Cl. perfringens), have been considered as other major causes of foodborne diseases worldwide [Regarding the global number of foodborne illness cases, the major foodborne hazards are orldwide . bla genes, have been most frequently detected in food samples.On the other hand, resistance to different classes of antibiotics has been regarded as one of the most important challenges in public health and food safety. Foods and foodborne pathogens are the main routes of transmission of ARGs to the human gut microbiota, leading to intestinal drug-resistant infections ,6. Beta-The safety level of foods also depends on the microbial quality of the raw materials used to produce these products. Different foodborne pathogens and antibiotic-resistance genes (ARGs) have been detected in the final products ,7,8. FooS. aureus strains and toxins are usually isolated from and detected in sweet products since the initial contamination sources of these bacteria are the mucous membranes and skin of humans [f humans . S. aureus has mostly been known as one of the most important food safety challenges and public health concerns, causing intoxication, vomiting, and diarrhea in humans as a foodborne pathogen via the secretion of a heat-stable toxin, alpha-toxin [Campylobacter spp., Shigella spp., and Cronobacter sakazakii (Cr. sakazakii), as a new emerging foodborne pathogen, have been isolated from low-moisture food products such as sweets [ha-toxin . Other ks sweets ,15. Thess sweets ,13,14,15C. jejuni, C. coli, Shigella spp., Cr. sakazakii, and S. aureus, and ARGs, in sweet samples.Investigation of the prevalences of bacterial pathogens and ARGs in low-moisture food products, especially sweet samples, is strongly limited. On the other hand, there are also limited studies regarding the correlations among the presence of different types of foodborne pathogens in food samples . Therefow/w), including 4 different subtypes and 3 various brands, were purchased and collected from local markets in Qazvin and Tehran cities, Iran , between January and April 2022. Samples were immediately transported aseptically in cool boxes containing ice packs to the central microbiology laboratory at the university. Forty sweet samples  at 30 °C for 10 min. All samples were diluted 1:10 with phosphate-buffered saline and homogenized again with a stomacher for 5 min at 30 °C . All sam−1 by dilution with the addition of sterilized nuclease-free water. The DNA samples were kept at −20 °C for further molecular analysis.The total DNA in the prepared and diluted sweet samples was extracted by using a SinaClon commercial tissue total DNA extraction kit  according to the manufacturer’s instructions. The instructions of the kit have been optimized for total DNA extraction from food samples. The quantity and quality of the extracted total DNA were assessed by using a NanoDrop spectrophotometer model 1000 . The optical density ratios of 260/280 of all DNA samples were observed in the range of 1.5–1.8, indicating the high purity of the extracted DNA. The concentrations of all extracted total DNA were adjusted to 50 ng. μLC. jejuni, C. coli, Shigella spp., Cr. Sakazakii, and S. aureus by conventional PCR methods (cPCR) using species-specific primers, as shown in S. aureus in sweet samples was performed by detecting the spa gene in DNA templates according to the method and specific primers previously described and used by Larsen et al. (2008). The spa gene encodes Staphylococcal protein A in S. aureus strains. Detection of this genetic element proves species-specifically the presence of S. aureus in clinical, food, and environmental samples [In the present study, we detected and identified five different foodborne bacterial pathogens, including  samples . Cr. sakazakii was detected in the samples according to the cPCR previously used to detect the ompA gene (outer membrane protein virulence factor encoding gene) in powdered infant formula samples by Pakbin et al. (2022) [. (2022) . Shigella spp. in each sample, the invasion plasmid antigen encoding gene (ipaH) was detected by a PCR assay as previously described by Pakbin et al. (2021) [To identify and detect . (2021) . C. coli and C. jejuni strains were identified in sweet samples by detecting the cadF and hipO species-specific genes, as described and employed before by Nafarrate et al. (2021) [. (2021) . −1), 1 µL of each specific primer (10 mM. μL−1), and nuclease-free sterilized water up to the final reaction volume of 20 µL. PCR mixes were subjected to initial denaturation at 95 °C for 5 min, 40 cycles of amplification , and a final elongation step at 72 °C for 5 min [w/v agarose gel containing DNA safe-stain . The PCR products were electrophoresed on agarose gels were evaluated and recorded by using a UV transillumination and gel documentation system . Positive and negative samples were used as quality controls for detecting each foodborne pathogen in this study. DNA templates extracted from reference strains, including S. aureus (ATCC 25923), C. jejuni (ATCC 33291), C. coli (ATCC 43478), and Sh. sonnei (ATCC 29031), were used as the positive controls, and sterilized distilled water was used as the negative control in this study.PCR assays were carried out with 10 µL of PCR 2X master mix , 2 µL of DNA template , Escherichia coli (ATCC 25922), and Staphylococcus aureus (ATCC 25923) harboring all of these ARGs were included and used as quality controls in this study.Beta-lactamase resistance genes, including p < 0.05 and one degree of freedom. Fisher`s exact test was used to evaluate significant differences (p < 0.05) between the prevalences of different bacterial pathogens, the prevalences of different ARGs, and different bacterial pathogen profiles. All statistical analyses were performed by using SPSS software version 22.0.1 . All evaluations and measurements were carried out in triplicate.Correlations among the different foodborne bacterial pathogen prevalences in the sweet samples were evaluated by using Pearson’s Chi-square test, with a significant difference defined as S. aureus (n = 19), Cr. sakazakii (n = 8), Shigella spp. (n = 18), C. jejuni (n = 2), and C. coli (n = 12) in 47.5%, 20.0%, 45.0%, 5.0%, and 30.0% of the sweet samples (N = 40), respectively. Among these bacterial pathogens, S. aureus and Shigella spp. significantly (p < 0.05) showed the highest prevalences compared to the other pathogens. Also, the results in this study illustrated that C. coli was significantly (p < 0.05) more often detected than C. jejuni.In the present study, we detected some important foodborne pathogens in sweet samples and evaluated the bacterial profiles and correlations between the presence of these pathogens in the collected samples. This study detected S. aureus single bacterial profile was the significantly (p < 0.05) most prevalent profile in this study. A double bacterial profile, including Shigella spp. and C. coli, was significantly (p < 0.05) the most common non-single pathogenic bacterial profile detected in these samples.The profiles of the foodborne bacterial pathogens detected in sweet samples in this study are shown in p < 0.05) positive correlation (0.335) was between the presence of Cr. sakazakii and C. coli in the sweet samples. Significant correlations were not observed between the presence of other bacterial pathogens in this study.We measured the possible correlations between the presence of different pathogens with Pearson’s Chi-square test in this study. blaSHV gene was detected in 39 out of 40 sweet samples (97.5%); however, the blaTEM gene was only detected in one sample (2.5%). blaOXA, blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, and blaCTX-M-9 resistance genes were detected in 25%, 37.5%, 37.5%, and 20% of the sweet samples, respectively.This study detected beta-lactam resistance genes in total DNA extracted from the sweet samples by using cPCR. The Foodborne bacterial pathogens are the main causes of intestinal  and some extraintestinal illnesses around the world . RegardiS. aureus and Shigella spp. prevalences in these samples. We detected at least one of these pathogens in 85% of the samples. S. aureus was also found to be the most prevalent profile detected in this study. S. aureus is an opportunistic pathogen and commensal colonizing the human mucous membranes and skin [S. aureus strains occurs causing gastrointestinal disorders caused by staphylococcal toxins [S. aureus in 45 out of 212 ready-to-eat samples (21.2%) collected in Vietnam [S. aureus [S. aureus in some food samples produced in Iran, including meat, dairy, and sweet products, and they detected S. aureus in 29.1% of the sweet samples [S. aureus in 2.67% of the pastry sweets produced in Iran [S. aureus in sweet products [The relatively small sample size (N = 40) could be considered this study’s main limitation. We observed the highest and skin . Since sl toxins . Houng e Vietnam . Kim et . aureus . Mahfooz samples . Hassani in Iran . Using aproducts ,23.Shigella is a highly infectious foodborne pathogen belonging to the Enterobacteriaceae family. The main reservoir of this pathogen is the human intestinal system, and it causes mild to severe diarrhea in humans as a facultative intracellular pathogen. Shigella species are transmitted to foods via fecally contaminated water or human feces under deplorable hygienic and sanitation conditions [Shigella is predictable [Shigella contamination in retail raw food samples in Pakistan. They reported that transmission via raw foods, hospital waste, and unhygienic food handling are the main risk factors for Shigella food contamination. This is the first study to report significantly higher levels of Shigella contamination in sweet samples; however, several other studies previously reported a high prevalence rate of S. aureus contamination in sweet and confectionery samples, as we also observed in the present study. The high prevalence rate of Shigella spp. indicates that the health and hygienic practices of personnel and food handlers during the manufacturing process of traditional sweet products should be considered more than other potential risk factors affecting the microbial quality of the products [Shigella spp. and S. aureus in traditional foods [ipaH gene is also present in entero-invasive E. coli, indicating that these strains of E. coli might also be detected in this study in addition to Shigella spp. [nditions . Regardidictable . Nisa etproducts ,29,30,31lla spp. .Cr. sakazakii is a Gram-negative foodborne pathogen belonging to the Enterobacteriaceae family. It causes severe intestinal and extraintestinal diseases , and is associated with high mortality in infants and neonates [Cr. sakazakii in sweet samples. Transmission of this pathogen to foods commonly occurs via contaminated raw food materials and insufficient thermal processing during food preparation [Cr. sakazakii in food samples (Sunshik) produced in Korea [Cr. sakazakii in 7.14% of pastry sweet samples. Regular microbiological monitoring of raw food materials, especially in terms of detecting Cr. sakazakii by rapid methods, is the main strategy for protection against contamination with this foodborne pathogen [neonates . This neneonates ,11. In tparation . Kim et in Korea . Hassanipathogen .Campylobacter is another foodborne pathogen associated with enterocolitis in humans. According to the global burden of diarrheal diseases, gastrointestinal disorders caused by this pathogen are very prevalent [Campylobacter species, and poultry products are also known as the main source of contamination with this pathogen in other foods [C. jejuni and C. coli are the prevalent species of this pathogen, frequently isolated from food products formulated with eggs. Egg whites and yolks are mainly used in the formulation of sweets; therefore, the presence of Campylobacter species in sweets is highly probable [Campylobacter in sweet samples; however, the prevalence of C. coli was significantly more than that of C. jejuni. We also found a high correlation between the presence of C. coli and Cr. sakazakii in sweet samples. Consequently, the lack of thermal processing can also be considered a risk factor [Campylobacter species [revalent . Poultryer foods . C. jejuprobable . In thisk factor . Systemi species ,39. To produce sweet products with a high level of microbial quality and safety; automation of food processing, improving the personal hygiene of food handlers, and microbiological monitoring of raw food materials can be considered effective and practical strategies . NotablyCl. perfringens, B. cereus, and L. monocytogenes, and also some non-bacterial  foodborne pathogens, have also been detected and identified in low-moisture sweet and confectionary products at relatively lower prevalence levels. However, bacterial pathogens are still considered the leading human health-threatening concern in these products [Other foodborne pathogens, such as products ,15,37,41blaTEM, blaSHV, blaOXA, blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, and blaCTX-M-9 in sweet samples. Beta-lactamases are the main enzymes responsible for the resistance of Gram-negative bacteria, such as Cronobacter, Campylobacter, and Shigella spp., against the beta-lactam antibiotics [blaSHV gene as the most prevalent ARG in low-moisture food samples. The blaSHV gene, which was the dominant beta-lactam resistance gene in the sweet samples in this study, encodes resistance against amoxicillin and amoxicillin-clavulanic antibiotics [tetW, tetT, and tetA46 genes in these samples are associated with resistance to antibiotics belonging to the class of tetracyclines. They also detected the erm41 and tlrC genes associated with resistance against the antibiotic class of macrolides [In this study, we detected beta-lactam resistance genes, including ibiotics . Pakbin ibiotics . There acrolides . RegardiS. aureus and Shigella spp. S. aureus was the most frequent pathogenic bacteria in these profiles. We also found a significant correlation between the presence of C. coli and Cr. sakazakii in sweet samples. The blaSHV gene was detected most frequently in sweet samples; adversely, the blaTEM gene was only detected in one sample. Considering these results, we suggest automation of food processing, improving the personal hygiene of workers, evaluation of microbial contamination of raw materials, and regular monitoring of ARGs in raw materials and the final products to produce safe sweet products with higher levels of microbial quality. This is the first study to report a high prevalence rate of Shigella spp. in traditional sweet products; consequently, preventive strategies regarding the health and hygiene of the manufacturing personnel are suggested to be more considered than other strategies during the production of traditional sweets. Comprehensive epidemiological studies concerning the prevalences of different foodborne bacterial, fungal, and viral pathogens in various sweet types are also highly recommended to be implemented in the future.We found that sweet samples were significantly more contaminated with


Candida auris is a newly emerging multidrug-resistant fungal pathogen considered to be a serious global health threat. Due to diagnostic challenges, there is no precise estimate for the prevalence rate of this pathogen in Iran. Since 2019, only six culture-proven C. auris cases have been reported from Iran, of which, five belonged to clade V and one to clade I. Herein, we report a case of otomycosis due to C. auris from 2017 in a 78-year-old man with diabetes mellitus type II without an epidemiological link to other cases or travel history. Short tandem repeat genotyping and whole genome sequencing (WGS) analysis revealed that this isolate belonged to clade I of C. auris (South Asian Clade). The WGS single nucleotide polymorphism calling demonstrated that the C. auris isolate from 2017 is not related to a previously reported clade I isolate from Iran. The presence of this retrospectively recognized clade I isolate also suggests an early introduction from other regions or an autochthonous presence. Although the majority of reported C. auris isolates worldwide are resistant to fluconazole and, to a lesser extent, to echinocandins and amphotericin B, the reported clade I isolate from Iran was susceptible to all antifungal drugs. Candida auris is a newly described emerging multidrug-resistant yeast which is regarded as a serious global health threat [C. auris have pointed to environmental and/or animal niches [C. auris infections [C. auris has unique abilities such as long-term persistence on inanimate surfaces and protracted cross-transmission between patients [C. auris isolates have been shown to be resistant to or have reduced susceptibility to registered antifungal drugs [C. auris isolates have been allocated to five geographical clades (I to V): the South Asian, East Asian, African, South American, and Iranian clade, respectively [C. auris clades evolved seemingly simultaneously at independent geographical locations and display both genetic and phenotypic diversity [C. auris otomycosis of a patient in Bushehr, Iran, compare this with earlier reported cases, and performed susceptibility testing on the isolate.h threat ,2,3,4. Th threat , followeh threat  and a glh threat ,8. The rl niches ,10,11. Cfections . Comparepatients . Furtheral drugs . Based oectively , while aectively . These diversity ,17. Herealbicans Candida species. The patient was managed with topical empirical therapy with betamethasone 1% for the itching and inflammation of the external ear canal, as well as hydrogen peroxide 1% and ciprofloxacin 0.3%. The patient completely improved after one week with this regimen and did not receive specific antifungal therapy.In April 2017, a 78-year-old man with discharge from and itching of the external ear canal was admitted to an ear, nose, and throat specialist in Bushehr, a coastal city at the Persian Gulf, Iran. He had diabetes mellitus type II for more than 10 years as the only predisposing factor. With the clinical suspicion of a fungal infection, the ear discharge was submitted for investigation. Round and oval cells were observed with microscopic examination with 10% potassium hydroxide (KOH). The specimen was cultured on Sabouraud’s dextrose agar supplemented with chloramphenicol , and incubated at 35 °C for 48 h. White-to-cream, smooth, yeast-like colonies were germ tube-negative and thus identified as non-C. auris. For DNA isolation, the isolate was resuspended in 700 µL MagNA Pure bacteria lysis buffer and MagNA Lyser green beads and lysed with the MagNA Lyser system . Subsequent DNA extraction and purification was performed with the MagNA Pure 96 instrument and the MagNA Pure DNA and Viral NA Small Volume kit (Roche Diagnostics) [Retrospective molecular identification was performed 5 years later with ITS rDNA sequencing ,19 and mnostics) ,21. The nostics) . In addinostics) . For whonostics) . All rawC. auris clades showed that MRL32 was phylogenetically distinct from other Iranian C. auris isolates belonging to the local clade V, clustered with the South Asian clade (I), but had a different genotype than the previously reported Iranian clade I isolate (CSF1020)  .To determine the genetic distance between both Iranian clade I isolates, WGS single nucleotide polymorphism (SNP) calling was performed on both isolates and compared with other clades . The WGSThe minimum inhibitory concentrations of MRL32 were as follows: amphotericin B (1 μg/mL), fluconazole (8 μg/mL), itraconazole (0.5 μg/mL), posaconazole (0.25 μg/mL), voriconazole (0.125 μg/mL), isavuconazole (0.125 μg/mL), miconazole (4 μg/mL), caspofungin (0.25 μg/mL), anidulafungin (0.063 μg/mL), and micafungin (0.125 μg/mL). While the previously reported clade I fluconazole-resistant isolate (CSF1020) from Iran grouped with other fluconazole-resistant isolates from India, the fluconazole-susceptible isolate MRL32 did not group with any fluconazole-resistant isolates. In agreement with the phenotypic susceptibility testing, the visual alignment inspection with JBrowse of the resistance associated genes ERG11, TAC1b, and FKS1 showed the absence of unique mutations in MRL32 when compared to the resistant isolates. The previously reported fluconazole-resistant isolate from Iran was found to harbor the ERG11Y132F substitution.Candida auris cases are depicted in The patient characteristics and diagnostic details of the present case and previously published Iranian C. auris is an emerging multidrug-resistant pathogen becoming a global threat due to its potential for nosocomial spreading [C. auris among COVID-19 patients having DM was 52.9% [C. auris infection among COVID-19 patients was 5.7%. DM is among the main predisposing factors of C. auris infection. Here, we report a retrospectively detected case from 2017 in a 78-year-old man with otitis externa caused by a susceptible C. auris. Up until now, the prevalence of C. auris in Iran was low [C. auris. However, diagnostic allele-specific PCR, STR typing, or whole-genome sequencing (WGS) are necessary for categorizing the geographical clades [C. auris from 2017 was actually from clade I. Retrospective studies from France also identified an early clade I introduction in 2007 [C. auris isolates from clade V had a similar susceptibility pattern as this clade I isolate from 2017 [preading  during bpreading ,29. A syas 52.9%  and that was low ,25,26,27l clades ,20,30. W in 2007 . The reprom 2017 ,25,26,27C. auris in Iran. The geographical distribution of C. auris cases in Iran is as follows: two cases from Babol in the north of Iran [C. auris isolate from Iran in 2021 was from an invasive infection linked to travel to the Indian subcontinent and was fluconazole-resistant [Our case is the sixth reported case of  of Iran ,24, one  of Iran , one cas of Iran , one cas of Iran , and the of Iran . The antesistant . Geneticesistant .C. auris in Iran. Otitis externa was the most common presentation of C. auris (three out of six) in Iran. The case from Shiraz had epidermal dysplasia, while the Tehran case involved meningitis with C. auris in a 30-month-old male infant [C. auris cases. Among the patients with clade V isolates, none traveled abroad until the diagnosis of the infection, while the 30-month-old male infant from Tehran with clade I was hospitalized in Pakistan  between the isolates, it can be concluded that the introduction of the second clade I isolate was independent from the first isolate. The first isolate displayed low SNP differences when compared to other clinical-fluconazole resistant isolates, as is frequently observed for C. auris , and alsC. auris . As therC. auris .C. auris cases in Iran. We found a second clade I isolate in Iran, which was not related to the previous clade I isolate. The addition of this and earlier reports to the literature may motivate researchers to focus on the detection of this pathogen.To conclude, misdiagnosis and the application of conventional diagnostic methods lead to a lack of data about


Ficus species, F. hispida and F. heterostyla, based on wide and intensive population sampling across Indo-Burma ranges, using chloroplast  and nuclear microsatellite (nSSR) markers, as well as ecological niche modeling.Understanding biodiversity patterns and their underlying mechanisms is of interest to ecologists, biogeographers and conservationists and is critically important for conservation efforts. The Indo-Burma hotspot features high species diversity and endemism, yet it also faces significant threats and biodiversity losses; however, few studies have explored the genetic structure and underlying mechanisms of Indo-Burmese species. Here, we conducted a comparative phylogeographic analysis of two closely related dioecious F. hispida showed slightly higher chloroplast diversity but lower nuclear diversity than F. heterostyla. Low-altitude mountainous areas of northern Indo-Burma were revealed to have high genetic diversity and high habitat suitability, suggesting potential climate refugia and conservation priority areas. Strong phylogeographic structure and a marked east‒west differentiation pattern were observed in both species, due to the interactions between biotic and abiotic factors. Interspecific dissimilarities at fine-scale genetic structure and asynchronized historical dynamics of east‒west differentiation between species were also detected, which were attributed to different species-specific traits.The results indicated large numbers of population-specific cpDNA haplotypes and nSSR alleles in the two species. We confirm hypothesized predictions that interactions between biotic and abiotic factors largely determine the patterns of genetic diversity and phylogeographic structure of Indo-Burmese plants. The east‒west genetic differentiation pattern observed in two targeted figs can be generalized to some other Indo-Burmese plants. The results and findings of this work will contribute to the conservation of Indo-Burmese biodiversity and facilitate targeted conservation efforts for different species.The online version contains supplementary material available at 10.1186/s12870-023-04324-6. Patterns of biodiversity and their underlying mechanisms are among the central issues in ecology and biogeography and provide core knowledge for conservation –4. The d2 Indo-Burma hotspot supports ca. 13,500 plant species, of which approximately 7,000 were estimated to be endemic to this hotspot  mya  to 72.0 5.8] mya . Here, 5D considering the frequency of mutations [FS [SSD) and Harpending’s raggedness index  were edex , Last Glacial Maximum (LGM: ca. 21 kya before present), Mid-Holocene (MIH: ca. 5 kya before present), present (1970–2000) and future (2070). We recently performed ENM analysis of F. heterostyla [F. hispida across Indo-Burma were obtained from the GBIF , the CVH  and our field expeditions. The 19 bioclimatic variables corresponding to the five focal periods were downloaded from the WorldClim database (http://www.worldclim.org). Pairwise correlations of the 19 variables were tested to avoid variable multicollinearity and model overfitting. Variables with Pearson correlation coefficients of |r| ≤ 0.8 were used for subsequent analyses. 75% of the occurrence records were used as training data, and 25% were used as test data in 10 replications. MaxEnt outputs represent the habitat suitability, ranging from 0.0 to 1.0, in Cloglog format. The accuracy of each model prediction was evaluated statistically using the area under the receiver operating characteristic (ROC) curve [F. hispida. The map of ecoregions was derived principally from patterns of rainfall, temperature, geological history, broad vegetative patterns and expert opinion on community distributions [ENM was carried out in MaxEnt 3.4.1 , 141 to erostyla . In totaC) curve . The AUCC) curve . The impibutions , 145.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Penicillium, Aspergillus, Cladosporium, Fusarium, and Schizophyllum, which could have important implications in carbon and nitrogen cycling in this isolated environment. In order to determine the diversity and unknown metabolic capabilities of fungi in deep-sea sediments, their genomes need to be fully analyzed. In this study, two Penicillium species were isolated from South Pacific Gyre sediment enrichments during Integrated Ocean Drilling Program Expedition 329. The inner gyre has very limited productivity, organic carbon, and nutrients.Marine deep subsurface sediments were once thought to be devoid of eukaryotic life, but advances in molecular technology have unlocked the presence and activity of well-known closely related terrestrial and marine fungi. Commonly detected fungi in deep marine sediment environments includes Penicillium species using Illumina HiSeq and PacBio sequencing technologies. Single-copy homologues within the genomes were compared to other closely related genomes using OrthoMCL and maximum-likelihood estimation, which showed that these genomes were novel species within the genus Penicillium. We propose to name isolate SPG-F1 as Penicillium pacificasedimenti sp. nov. and SPG-F15 as Penicillium pacificagyrus sp. nov. The resulting genome sizes were 32.6 Mbp and 36.4 Mbp, respectively, and both genomes were greater than 98% complete as determined by the presence of complete single-copy orthologs. The transposable elements for each genome were 4.87% for P. pacificasedimenti and 10.68% for P. pacificagyrus. A total of 12,271 genes were predicted in the P. pacificasedimenti genome and 12,568 genes in P. pacificagyrus. Both isolates contained genes known to be involved in the degradation of recalcitrant carbon, amino acids, and lignin-derived carbon.Here, we present high-quality genomes of two proposed novel Penicillium isolates from deep marine sediments, which will be useful for future studies of marine subsurface fungal diversity and function. Furthermore, these genomes shed light on the potential impact fungi in marine sediments and the subseafloor could have on global carbon and nitrogen biogeochemical cycles and how they may be persisting in the most energy-limited sedimentary biosphere.Our results provide the first constructed genomes of novel The online version contains supplementary material available at 10.1186/s12864-023-09320-6. Recent surveys of microbial life in the marine subsurface have discovered fungi as a common community member alongside prokaryotes, thanks to advancements in high-throughput sequencing and culturing techniques –5. HowevAspergillus, Penicillium, Fusarium, Cryptococcus, and Schizophyllum  < 20) . For PacDe novo hybrid assembly of the Illumina and PacBio reads was performed using Unicycler . Unicyclgff3_merge and maker2zff. The zff files were used to train SNAP [fathom, forge, and hmm-assembler.pl. The HMM file was incorporated into MAKER2 and gene predictions were ran for a second time. The accessory scripts fasta_merge and gff3_merge were used to combine the final gene predictions into a gff3 file and a fasta protein file that would be used for phylogenomics and functional gene annotation.A MAKER2 pipeline was used for annotation without RNA-seq data on small eukaryotic genomes . The pipain SNAP  using thPenicillium isolates were clustered and compared with single-copy homologues from other Penicillium and Aspergillus genomes downloaded from NCBI and JGI .Single-copy homologues from both –3) [Metabolic pathways were determined using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Automatic Annotation Server (KAAS) , 31 with–3)  to searc–3) .Additional file 1: Supplementary Table 1. Genome accessions for Fig. Supplementary Table 2. Additional substrate and description information for CAZy annotations in Fig.


Arachis hypogaea (peanut) is a leading oil and protein-providing crop with a major food source in many countries. It is mostly grown in tropical regions and is largely affected by abiotic and biotic stresses. Cysteine-rich receptor-like kinases (CRKs) is a family of transmembrane proteins that play important roles in regulating stress-signaling and defense mechanisms, enabling plants to tolerate stress conditions. However, almost no information is available regarding this gene family in Arachis hypogaea and its progenitors. This study conducts a pangenome-wide investigation of A. hypogaea and its two progenitors, A. duranensis and A. ipaensis CRK genes . The gene structure, conserved motif patterns, phylogenetic history, chromosomal distribution, and duplication were studied in detail, showing the intraspecies structural conservation and evolutionary patterns. Promoter cis-elements, protein–protein interactions, GO enrichment, and miRNA targets were also predicted, showing their potential functional conservation. Their expression in salt and drought stresses was also comprehensively studied. The CRKs identified were divided into three groups, phylogenetically. The expansion of this gene family in peanuts was caused by both types of duplication: tandem and segmental. Furthermore, positive as well as negative selection pressure directed the duplication process. The peanut CRK genes were also enriched in hormones, light, development, and stress-related elements. MicroRNA (miRNA) also targeted the AhCRK genes, which suggests the regulatory association of miRNAs in the expression of these genes. Transcriptome datasets showed that AhCRKs have varying expression levels under different abiotic stress conditions. Furthermore, the multi-stress responsiveness of the AhCRK genes was evaluated using a machine learning-based method, Random Forest (RF) classifier. The 3D structures of AhCRKs were also predicted. Our study can be utilized in developing a detailed understanding of the stress regulatory mechanisms of the CRK gene family in peanuts and its further studies to improve the genetic makeup of peanuts to thrive better under stress conditions. Plants are exposed to a great number of biotic as well as abiotic stresses throughout their life. These include salinity, drought, cold, and pathogens. This has led to the evolution of several mechanisms in the immune system that helps them survive during these stresses. One of these mechanisms is receptor-like kinases (RLKs), which belong to transmembrane proteins, which sense external signals and send them to the intracellular environment . In thisArabidopsis thaliana. Two copies of the domain of unknown function 26 , which contains four conserved cysteines, make up the protein’s extracellular region. These three cysteines together make up the motif C-8X-C-2X-C, which may play a role in the production of disulfide bridges that might be used to regulate thiol redox , in response for extracellular ROS production. CRK2’s activity is vital for ROS bursts in response to elicitors, impacting defense against pathogens. Phosphorylation of RBOHD’s C-terminal enhances ROS production, suggesting an evolutionarily conserved mechanism, highlighting CRK2’s role in regulating ROS in response to microbial patterns  form a major group of RLKs having more than 40 members in ol redox . CRKs arpatterns . In CRKspatterns . Severalls of SA .Arabidopsis, higher expression of CRK1, also known as AtCBK3, results in an increased thermotolerance (CRK3 and cytosolic glutamine synthetase (GLN1), which mobilized nitrogen during leaf senescence, while CRK1 and CRK5 conferred drought stress tolerance by negatively regulating ABA signaling , whereas A. hypogaea is an allotetraploid  resulting from the hybridization occurrence between two diploid species, followed by polyploidization  and Arachis ipaensis  contributed to the hybridization . The protein sequence FASTA files of A. hypogaea, A. ipaensis, and A. duranensis were downloaded from the NCBI. NCBI command-line tool, BLAST+, was used to create a local database of these files. A BLASTp search was performed against these protein sequence databases, using Arabidopsis CRK protein sequences as queries. The resulting hits were further refined by removing duplicates and isoforms.The 44 https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (http://smart.embl-heidelberg.de/) (https://www.ebi.ac.uk/interpro/)  and protein kinase (PF00069 and PF07714) domains. For this purpose, NCBI conserved domain database (CDD) (psb.cgi) , Simple erg.de/) , and Intterpro/)  databaseterpro/)  was usedhttps://web.expasy.org/protparam/) (https://wolfpsort.hgc.jp/) , instability index (II), aliphatic index (AI), and the grand average of hydropathicity (GRAVY)] was predicted by using the ExPASy ProtParam tool (tparam/) . Subcellhgc.jp/) .A. hypogaea (AhCRKs) 36 A. duranensis (AdCRKs), 44 A. ipaensis (AiCRKs), 44 A. thaliana (AtCRKs) (K. H. vulgare (HvCRKs) (Oryzasativa (OsCRKs) (Proteusvulgaris (PvCRKs) (http://iqtree.cibiv.univie.ac.at/) (https://itol.embl.de/)  K. , 37 H. v(HvCRKs) , 36 Oryz(OsCRKs)  and 46 P(PvCRKs)  was done(PvCRKs) . A phylo.ac.at/) . Using tmbl.de/) .Arachis species was searched using the Multiple Expectation Maximization for Motif Elicitation (https://meme-suite.org/meme/tools/meme) tool  tool . A maxim TBtools .CRK genes from each of the three Arachis spp. was determined from the NCBI genomic database. The distribution of CRK genes from three Arachis species across their chromosomes was analyzed and visualized using TBtools. Duplicated pairs of AhCRK genes were identified using NCBI nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome)  , based oasthome) . This watitution . This wahttps://string-db.org/) (https://david.ncifcrf.gov/home.jsp) (miRNAsong - A tool for microRNA sponge sequence generation and testing (muni.cz))  (zhaolab.org)] (AhCRK genes using the coding sequences of AhCRKs as target sequences.Amino acid sequences of AhCRKs were subjected to the STRING database (db.org/)  to analydb.org/) . The comome.jsp) . Moreoveuni.cz))  was usedab.org)]  was usedcis-regulatory elements, 2 kb sequences upstream of the translation start site of CRK genes from each of the three Arachis species were extracted and scanned using the PlantCARE online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (For identification of e/html/) . FinallyAhCRK genes in peanut leaves under drought and salt stress were evaluated using transcriptome datasets available at the NCBI Sequence Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/sra) . The genome and annotation (GFF) files were downloaded from the genome-NCBI database (https://www.ncbi.nlm.nih.gov/genome/). Indexes of the A. hypogaea genome were built by using Bowtie2 . The AhCRK genes common to both datasets were collected. Furthermore, to access the credibility of these genes, the classification algorithm—random forest (RF) was implied in R. Assessing the performance of a model is usually subjective, which involves comparing the model’s predictions to the known values of the dependent variable in a specific dataset. DESeq normalization was applied to the salts’ count datasets for the RF classifiers, in which the genes common to both stresses were incorporated. The performance of the RF classifier was evaluated using matrices including the accuracy, the area under the receiver operating characteristic curve (AUC), specificity, and sensitivity of the data with common multi-stress-related genes.DESeq2  was applhttps://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb) (https://saves.mbi.ucla.edu) server (http://molprobity.biochem.duke.edu/)  structure of a protein is necessary for its proper functioning. Based on the expression analysis, the 3D structures of three AhCRK proteins were predicted using AlphaFold2 (2.ipynb) . The pre) server  and MolPke.edu/) . PyMOL (ke.edu/)  was usedAhCRKs, AdCRKs, and AiCRKs  and Pkinases (PFAM domains PF00069 and PF07714) domains. All the proteins from these three species contained two to four PF01657 . For Pkinase domains, some of the genes contained PF00069 (protein kinase domain) and others had PF07714 (protein tyrosine kinase domain) conserved in them  to 26,515 bp (AhCRK32). Exon and intron positions were associated and compared to increase our insights into the structural diversity among the CRK members of A. hypogaea, as well as intraspecies diversity among the members from three Arachis species. For A. hypogaea, the intron number varied from 1 (AhCRK63) to 16 (AhCRK35). Moreover, some members including AhCRK4, AhCRK42, and AhCRK65 contained no intronic regions. Members from each group contained a similar number of introns and exons.Gene length in A. duranensis, the gene length ranged from 855 bp (AdCRK17) to 19,199 bp (AdCRK9). The exon–intron pattern was also conserved among members of the same group. Group I contained zero to seven introns, whereas members of Group II contained a maximum of seven introns, and the members of Group III had introns ranging from five to eleven. For A. ipaensis, the observed gene length varied from 873 bp (AiCRK8) to 10,977 bp (AiCRK1). The members from the same group showed conservation among their gene structures, while the overall number of introns ranged from 0 (AiCRK8 and AiCRK12) to 10 (AiCRK43). This variation in the number of introns and exons might have resulted in varied gene lengths among the members from the same group, as well as intraspecies  mapped on them. Chr2, Ch7, and Chr17 had no CRK genes present in them . Only one gene AdCRK36 was present on the scaffold. A. ipaensis also followed a similar mapping pattern, with genes mapped on every chromosome except Chr7 and the highest number of AiCRKs being clustered on Chr9 (18 genes). These results show the conservation in gene position patterns on chromosomes .To evaluate the pattern of genomic distribution of  in them . In the mosomes .AhCRK, AdCRK, and AiCRK genes, and a total of 41, 14, and 20 duplicated gene pairs were found in these members, respectively. In A. hypogaea, 10 pairs of tandem and 31 pairs of segmental duplication were observed. In A. duranensis, 11 pairs of tandemly duplicated genes were found, with three pairs being segmentally duplicated. Similarly, in A. ipaensis, 13 pairs of genes resulting from tandem duplication were analyzed with seven gene pairs being segmentally duplicated. These observations are consistent with their genomic composition; for instance, A. hypogaea have double the size of the genome as that of A. duranensis and A. ipaensis¸ indicating that the AhCRK genes are dispersed on different chromosomes. Segmental supplication is the duplication of genes from one chromosome onto another; thus, the presence of more segmental duplication pairs in A. hypogaea makes sense. To analyze the evolutionary constraints of the repeated Arachis CRK genes, the Ka, Ks, and the Ka/Ks ratios of all para-homologous gene pairs were then calculated. In A. hypogaea, almost half of the gene pairs had their Ka/Ks values ranging from 0.16 to 0.99, which were smaller than 1.0, indicating significant purification selection pressure had been applied to these gene pairs. The rest of the half pairs having Ka/Ks values more than 1.0 indicated that positive selection also occurred. As a result, the divergence time of 41 duplicated pairs was between 0.67 Mya and 117.42 Mya. In A. duranensis, three gene pairs exhibited positive selection , while the rest of the duplicated pairs showed negative selection . Furthermore their divergence time ranged from 1.653 to 121.91 Mya. For A. ipaensis, eight duplicated gene pairs showed positive selection, and the rest of the 12 pairs showed purifying selection, with their divergence time ranging from 0.84 to 129.4 Mya , stress-related , hormone-related , and light-related  cis-regulatory elements were identified in each Arachis CRK members’ promoters . All these elements corresponded to gibberellin, auxin, abscisic acid, and MeJA responsiveness; endosperm and meristem expression; low-temperature responsiveness; and zein metabolism regulation. Each of the A. duranensis CRKs contain all four kinds of cis-regulatory elements. Members of each A. ipaensis CRKs contain all four types of cis-acting elements with a large number of light-related elements (GATA-motif). Moreover, AiCRK38–40 and AiCRK42 contained only light-responsive elements , cellular components (CC), and molecular functions (MF). Biological processes in which these proteins were found to be involved included responses to stimulus and defense responses. Almost all of the proteins were found to have membranes as their cellular component. Similarly, their molecular functions included kinase activity and ATP binding. Their related KEGG pathways suggest their involvement in metabolic pathways , AhCRK23 (Group III), and AhCRK57 (Group II) were found to be common in both drought and salt datasets. After the identification of these potential multi-stress responsive genes, a machine learning classifier, random forest, was implemented to evaluate the performance of these multi-stress responsive genes. To perform this task, the count data of salt stress were provided as a training dataset and multi-stress-related genes  were tested , CaCRK5 is involved in a mechanism related to the immune response against pathogens. Various elements in this gene’s promoter region contributed to this defense response , and the others were downregulated, whereas some genes showed a change in expression on different days of tolerating drought stress, including AhCRK1, AhCRK21, AhCRK38, AhCRK56, AhCRK57, and AhCRK70. This indicates their crucial roles in drought stress tolerance. Moreover, in salt stress, the expression also varied under normal and treated conditions. Some genes were highly upregulated or downregulated upon exposure to salt stress . Similar results were observed in other plants under abiotic stresses, including cold, salt . AhCRK19, AhCRK23, AhCRK56, and AhCRK69 can potentially be candidate genes for conferring tolerance against drought stress. On the other hand, AhCRK8, AhCRK23, AhCRK24¸ AhCRK56, AhCRK65, and AhCRK69 can act as potential candidate genes in providing resistance against salt stress. Machine learning approaches were utilized to evaluate the multi-stress responsiveness of these genes. Owing to their expression on both drought and salt stress, AhCRK8, AhCRK23, and AhCRK57 can be deemed candidate genes for multi-stress responsiveness. These genes are needed to be explored further and can be used in genetic engineering research to devise multi-stress-resistant and -tolerant crops. Our study will also help further investigate the functional roles of the CRK genes in peanuts.CRKs are found to have regulatory roles in plants under various abiotic and biotic stresses. This study provides not only a systematic but also a comparative analysis of