Xenopus laevis as an infection model for human pathogenic bacteria

Xenopus laevis as an infection model for human pathogenic bacteria | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Xenopus laevis as an infection model for human pathogenic bacteria Ayano Kuriu , View ORCID Profile Kazuya Ishikawa , Kohsuke Tsuchiya , View ORCID Profile Kazuyuki Furuta , View ORCID Profile Chikara Kaito doi: https://doi.org/10.1101/2025.03.10.642459 Ayano Kuriu 1 Division of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kazuya Ishikawa 1 Division of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kazuya Ishikawa Kohsuke Tsuchiya 2 Division of Immunology and Molecular Biology, Cancer Research Institute, Kanazawa University Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kazuyuki Furuta 1 Division of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kazuyuki Furuta Chikara Kaito 1 Division of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Chikara Kaito For correspondence: ckaito{at}okayama-u.ac.jp Abstract Full Text Info/History Metrics Preview PDF Abstract Animal infection models are essential for understanding bacterial pathogenicity and corresponding host immune responses. In this study, we investigated whether juvenile Xenopus laevis could be used as an infection model for human pathogenic bacteria. Xenopus frogs succumbed to intraperitoneal injection containing the human pathogenic bacteria Staphylococcus aureus , Pseudomonas aeruginosa , and Listeria monocytogenes . In contrast, non-pathogenic bacteria Bacillus subtilis and Escherichia coli did not induce mortality in Xenopus frogs. The administration of appropriate antibiotics suppressed mortality caused by S. aureus and P. aeruginosa . Strains lacking the agr locus, cvfA ( rny ) gene, or hemolysin genes in S. aureus , LIPI-1-deleted mutant of L. monocytogenes , which attenuate virulence within mammals, exhibited reduced virulence in Xenopus frogs compared to their respective wild-type counterparts. Bacterial distribution analysis revealed that S. aureus persisted the blood, liver, heart, and muscles of Xenopus frogs until death. These results suggested that intraperitoneal injection of human pathogenic bacteria induces sepsis-like symptoms in Xenopus frogs supporting their use as a valuable animal model for evaluating antimicrobial efficacy and identifying virulence genes in various human pathogenic bacteria. Introduction According to a 2019 report, 13.6% of global deaths were associated with infections caused by bacterial pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa ( 1 ). S. aureus is a Gram-positive pathogenic bacterium that resides in the nasal cavity of approximately 30% of healthy adults and causes pneumonia, osteomyelitis, and endocarditis ( 1 ). Drug-resistant S. aureus , known as methicillin resistant S. aureus (MRSA), is spreading worldwide, necessitating the development of new treatments ( 2 ). P. aeruginosa is a bacterium commonly found in environments such as water and soil and in the intestinal tract of healthy humans. However, during infection, it can cause urinary tract infections, sepsis, and other diseases. Most antibiotics are ineffective against infections caused by multidrug-resistant P. aeruginosa ( 3 ). Listeria monocytogenes is found in river water and animal intestinal tracts and causes meningitis, septicemia, and fetal septic granuloma in foodborne infections ( 4 ). In the United States, approximately 20% of all foodborne deaths are attributed to L. monocytogenes infections ( 5 ). To establish treatment strategies for these bacterial infections, understanding bacterial pathogenic mechanisms and host immune system to counter the bacterial infection is crucial. Mammalian animal models such as mice have traditionally been used to elucidate bacterial and host factors involved in the bacterial infection process. These models have the advantage of having immune systems similar to those of humans; however, ethical and high-cost issues limit the use of large numbers. Recently, a global movement has been initiated to reduce animal testing and use alternative methods, as evidenced by the 2013 EU ban on the sale of cosmetics tested on animals ( 6 ). Alternatives to animal testing, including in vitro and in silico experiments and use of cultured mammalian cells, have attracted attention ( 7 , 8 ); however, they fail to reproduce the complex interactions occurring in animal bodies. Animal infection models remain necessary to investigate bacterial-host interactions and disease progression leading to death. To avoid ethical and cost problems associated with mammals, invertebrates, such as nematodes ( 9 ), fruit flies ( 10 ), silkworms ( 11 ), honey worms ( 12 ), and crickets ( 13 ), as well as vertebrate fish like zebrafish ( 14 , 15 ), are being used as infection models ( 16 , 17 ). These animals allow large-scale testing of bacterial pathogenicity and antimicrobial agents. However, invertebrates lack antibody-mediated adaptive immune mechanisms, making it impossible to analyze bacterial interactions with the adaptive immune system. In addition, their open vasculature, unlike vertebrates, uniquely affects the pathways for bacterial spread via body fluids. While zebrafish possess immune systems relatively similar to those of humans ( 14 , 15 ), their respiratory system, which relies on gill breathing, differs significantly from that of humans. Establishing a non-mammalian infection model with new advantages is expected to advance research on infectious diseases. Xenopus laevis , an amphibian vertebrate, has been widely used in developmental biology for several decades ( 18 ). X. laevis harbors organs, organ arrangements, and circulatory systems similar to those of humans ( 19 ). Moreover, its immune system closely resembles that of humans and possesses key factors of innate and acquired immunity, including macrophages, neutrophils, B cells, T cells, immunoglobulins, complement system, and antimicrobial peptides ( 20 , 21 ). These characteristics suggest that X. laevis could be useful for analyzing bacterial dissemination and immune interaction comparable to those in humans. The entire genome sequence of X. laevis has been completed ( 22 ), and genome editing using TALEN and CRISPR Cas9 is now possible ( 23 – 25 ). Xenopus frog can be kept at a high densities in aquariums without aeration because of their lung breathing ( 26 ). Fifty juvenile Xenopus frogs, each approximately 3 cm in length, can be maintained in about 10 L of water. In addition, they can be easily injected using human clinical tuberculin syringes without anesthetic treatment, as ice cold anesthesia is effective. These features provide marked advantages in experiments that require large number of animals to test using injections. Xenopus frogs have been used as an infection model for Mycobacterium marinum ( 19 ), which infects various aquatic organisms. However, they have not yet been employed as infection models for human pathogenic bacteria. Furthermore, no reports have demonstrated the use of Xenopus frogs to evaluate bacterial virulence genes or antimicrobial agent efficacy. In this study, we aimed to establish a Xenopus frog infection model for human pathogenic bacteria. Animal infection models should be effective for evaluating bacterial virulence genes. The agr locus ( 27 ) and srtA ( 28 ) and cvfA ( rny ) genes ( 29 ) encode important S. aureus virulence factors. The agr locus encodes quorum sensing-related proteins and regulates toxin and cell surface protein expression ( 30 ). The srtA gene is required to anchor cell wall proteins involved in bacterial adhesion to host cells ( 31 ). The cvfA gene encodes RNase Y, which is essential for hemolysin production ( 29 , 32 , 33 ). Similarly, Listeria Pathogenicity Island 1 (LIPI-1) region in L. monocytogenes ( 34 ) encodes prfA , plcA , hly , mpl , actA , and plcB ( 35 , 36 ). Hemolysin LLO ( hly ) ( 37 ) and phospholipase C (PLC) ( plcA and plcB ) ( 38 ) facilitate L. monocytogenes escape from phagosomes or autophagy ( 35 , 39 ). These virulence genes of S. aureus and L. monocytogenes are crucial for bacterial pathogenicity in animal models such as mice ( 40 – 43 ), rabbits ( 44 ), crickets ( 13 ), silkworms ( 28 , 42 ), and nematodes ( 45 , 46 ). Therefore, these bacterial genes are suitable for examining the effectiveness of novel animal infection models to evaluate bacterial virulence. Animal infection models should also be effective for assessing the efficacy of antimicrobial agents. In this study, methicillin-susceptible and methicillin-resistant S. aureus strains, and drug-susceptible and multidrug-resistant P. aeruginosa strains were used to evaluate the efficacy of clinically used antimicrobial agents. Results Intraperitoneal injection of S. aureus can kill Xenopus frogs To determine whether Xenopus frogs are susceptible to human pathogenic bacteria, bacterial suspensions of S. aureus were intraperitoneally injected into the Xenopus frogs. The frogs died within 24 h post-injection and their entire body exhibited reddening ( Fig. 1A ). Download figure Open in new tab Fig. 1. Intraperitoneal injection of S. aureus is lethal to Xenopus frogs. (A) Intraperitoneal injection of frogs is shown (left panel). Frogs injected with S. aureus NCTC8325-4 strain (2.8 × 10 8 CFU/frog) were dead within 24 h post-injection (right panel). Frogs injected with PBS survived (center panel). (B) Time-course survival analysis of frogs intraperitoneally injected with varying doses of S. aureus NCTC8325-4 strain. Five frogs were injected per sample, and survival data were pooled from four independent experiments (n=20). ns: p > 0.05, **: p < 0.01, ****: p < 0.0001. (C) Dose-response survival curve of frogs injected with live or heat-killed S. aureus NCTC8325-4. Overnight culture of S. aureus was autoclaved at 121°C for 20 min before use as heat-killed bacteria. Five frogs were injected per sample and LD 50 was determined from survival curve pooled from three independent experiment data. To assess whether mortality was dependent on the number of injected bacteria, frogs were injected with varying concentrations of S. aureus suspension, and their survival rate was observed over time. Mortality was observed between 12 and 36 h after injection, which increased with higher bacterial concentrations ( Fig. 1B ). These results indicated that S. aureus infection leads to frog death in a dose-dependent manner. To confirm that the death of frogs post-injection was caused by live bacteria, we examined whether heat-killed S. aureus exhibited similar lethal activity. The survival rate of frogs injected with live S. aureus decreased depending on the number of bacteria at 24 h post-injection, whereas that of frogs injected with heat-killed S. aureus did not decrease at 24 h post-injection ( Fig. 1C ). The median lethal dose (LD 50 ) of live and heat-killed S. aureus were 3.2 × 10 7 and 7.1 × 10 9 CFU/frog, respectively. These results suggested that frogs died owing to the biological activity of viable S. aureus , such as bacterial growth and toxin production. Various human pathogenic bacteria can kill Xenopus frogs To determine whether the frog-killing ability is conserved among human pathogenic bacteria other than S. aureus NCTC8325-4, we examined the virulence of other S. aureus strains, P. aeruginosa , and L. monocytogenes against Xenopus frogs. Intraperitoneal injection of MRSA (MRSA8), P. aeruginosa strain PAO1, multidrug-resistant P. aeruginosa (BAA-2114), and L. monocytogenes strain EGD caused frog death based on the bacterial number ( Fig. 2A , 2B , 2C , 2D ), similar to S. aureus NCTC8325-4 ( Fig. 1C ). Additionally, we examined whether laboratory strains of non-pathogenic bacteria such as Escherichia coli ( 47 ) and Bacillus subtilis ( 48 ) induces mortality. Intraperitoneal injections of E. coli BW25113 and B. subtilis 168 trpC2 showed little killing activity to frog within the tested bacterial concentrations ( Fig. 2E , 2F ). LD 50 calculated from survival curves showcased that P. aeruginosa BAA-2114 had the highest LD 50 of 1.8 × 10 9 CFU/frog, whereas S. aureus NCTC8325-4 and L. monocytogenes EGD exhibited lower LD 50 values (< 4.3 × 10 7 CFU/frog) ( Fig. 2G ). LD 50 values of E. coli BW25113 and B. subtilis 168 trpC2 were not determined, as they exceeded 3.3 × 10 9 and 1.6 × 10 9 CFU/frog, respectively ( Fig. 2G ). These results suggested that nonpathogenic bacteria have limited lethality in frogs, whereas human pathogenic bacteria demonstrate frog-killing ability, which varies among bacterial species and strains. Download figure Open in new tab Fig. 2. Various species of human pathogenic bacteria induce mortality in Xenopus frogs. (A–F) Dose-response survival curves of frogs injected intraperitoneally with S. aureus MRSA8 (A), P. aeruginosa PAO1 (B), P. aeruginosa BAA-2114 (C), L. monocytogenes EGD (D), E. coli BW25113 (E), and B. subtilis 168trpC2 (F) generated from the survival rate at 24 h post-injection. Five frogs were injected with three different concentrations of bacterial suspensions in a single experiment, and survival data obtained from three independent experiments are shown. (G) Based on the survival data (A–F), logistic regression analysis was performed using the statistical software R to determine LD 50 . Error bars indicate standard error. LD 50 for NCTC8325-4 was calculated from the survival data in Fig. 1C . Evaluation of antibacterial drug efficacy in Xenopus To determine whether Xenopus frogs can serve as a model for evaluating antimicrobial efficacy, we examined the therapeutic effects of antimicrobials on frog death induced by human pathogenic bacteria. First, the toxicities of kanamycin (KM), oxacillin (OX), vancomycin (VCM), ciprofloxacin (CPFX), and ceftazidime (CAZ) were tested, which confirmed that none of the tested antibiotics caused frog mortality at the antimicrobial concentrations used in this study ( Table S1 ). Antimicrobials were administered immediately after bacterial injection, and frogs were considered recovered if their survival rate was greater than 50% at 120 h post-infection. MRSA8, an MRSA strain of S. aureus , is resistant to oxacillin (β-lactam) and kanamycin (aminoglycoside), but remains sensitive to vancomycin ( 11 , 13 , 49 ). BAA-2114 is a multidrug-resistant P. aeruginosa isolated from the sputum samples of patients with cystic fibrosis ( 50 ), which displays resistance to kanamycin, ceftazidime (β-lactam), and ciprofloxacin (fluoroquinolones) ( 54 – 56 ). Frogs injected with NCTC8325-4, a methicillin-susceptible strain of S. aureus , without antibacterial injections exhibited a survival rate of less than 10%, whereas administration of KM, OX, or VCM after injecting bacterial suspension resulted in survival rates of > 50% at 120 h post-infection ( Fig. 3A ). Frogs injected with S. aureus MRSA8 had 0% survival at 120 h post-infection, whereas those injected with VCM after MRSA8 achieved a 100% survival rate in the same time ( Fig. 3B ). In contrast, injecting KM or OX following MRSA8 resulted in nearly 0% survival at 120 h post-infection ( Fig. 3B ). These results indicated that NCTC8325-4 infection in frogs can be treated with KM, OX, and VCM, whereas MRSA8 infection respond only to VCM. Download figure Open in new tab Fig. 3. Antimicrobial agents inhibit bacterial infections in Xenopus frogs Therapeutic effects of kanamycin (KM), oxacillin (OX), and vancomycin (VCM) on frogs injected with S. aureus NCTC8325-4 (A) and MRSA8 (B) were examined. Similarly, the effects of kanamycin (KM), ciprofloxacin (CPFX), and ceftazidime (CAZ) on frogs infected with P. aeruginosa PAO1 (C) and BAA-2114 (D) were examined. Immediately after intraperitoneal injection of bacterial suspensions, frogs were intraperitoneally administered with 50 µL of 2 mg/mL antimicrobial agent (100 µg/frog) or PBS, and survival was measured every 12 h. Five frogs were used per agent, and survival data were pooled from three independent experiments (n=15). Significant differences between PBS-- and antimicrobial-injected groups were determined using log-rank test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Frogs injected with P. aeruginosa PAO1 without antimicrobials showed 0% survival at 120 h post-infection, while those treated with KM, CPFX, or CAZ following PAO1 administration, showed survival rates of > 50% ( Fig. 3C ). Similarly, frogs injected with P. aeruginosa BAA-2114 without antimicrobial agents was 0% at 120 h post infection, whereas those injected with KM, CPFX, or CAZ after BAA-2114 showed less than 50% survival rates in the same timeframe ( Fig. 3D ). These findings suggested that PAO1 infections in frogs can be effectively treated with KM, CPFX, or CAZ, whereas BAA-2114 infections are refractory to these antimicrobial agents. Evaluation of bacterial virulence genes To determine whether the virulence genes of pathogenic bacteria could be evaluated using Xenopus frogs, virulence gene deletion mutants of S. aureus and L. monocytogenes were intraperitoneally injected into Xenopus frogs, and their viability was measured 24 h post-infection. S. aureus NCTC8325-4 mutants with deleted agr locus ( 27 ), srtA ( 28 ), and cvfA ( 29 ) killed frogs in a dose-dependent manner ( Fig. 4A ). The LD 50 values of agr - and cvfA -deleted mutants were more than 10-fold higher than those of the wild-type strain ( Fig. 4A ). In contrast, the LD 50 of srtA -deleted mutant was nearly the same as that of the wild-type strain ( Fig. 4A ). These results suggested that agr and cvfA contribute to S. aureus virulence against Xenopus frogs, while srtA does not. Download figure Open in new tab Fig. 4. Deletion of bacterial virulence genes reduces bacterial killing activity against Xenopus frogs. (A) Comparison of the frog-killing abilities of S. aureus NCTC8325-4 strain (WT) and strains with deleted agr , srtA , and cvfA . (B) Comparison of the frog-killing abilities of NCTC8325-4 WT and strains with deleted agr , hla , hlb , and hla / hlb . (C) Comparison of the frog-killing ability of L. monocytogenes EGD (WT) and a strain with deleted of LIPI-1. The data for L. monocytogenes EGD WT are the same as those presented in Fig. 2A . For all panels, bacterial suspensions were injected intraperitoneally into frogs, and survival was measured at 24 h post-injection. Five frogs were injected with each of three different concentrations of bacterial suspensions per strain in a single experiment. Dose-response survival curves were generated using logistic regression based on data from three independent experiments, and LD50 and standard error were calculated. Significant differences between gene-deleted and WT strains were determined using the likelihood ratio test (**: p < 0.01, ***: p < 0.001, ****: p < 0.0001). Error bars represent standard error. Given that the agr- and cvfA -deleted mutants attenuated virulence against Xenopus frogs and agr and cvfA are required for hemolysin production ( 30 , 57 ), we hypothesized that the reduced hemolysin production in these mutants may contribute to their attenuated virulence. To verify this, we assessed the virulence of hemolysin gene deletion mutants in Xenopus frogs. Deletion mutants of hla (encoding α-hemolysin) and hlb (encoding β-hemolysin) are known to reduce virulence in mammals ( 28 , 44 ). The LD 50 values of hla - and hlb -deleted mutants were higher than those of the wild-type strain ( Fig. 4B ). In addition, the LD 50 of the hla / hlb double deletion mutant exceeded 2.4 × 10 9 CFU/frog, which was higher than the LD 50 values of mutants harboring either deletion ( Fig. 4B ). These results suggested that hla and hlb act in an additive manner to kill Xenopus frogs. Xenopus frogs injected with the LIPI-1-deleted mutant of L. monocytogenes EGD showed minimal mortality at 24 h post-infection ( Fig. 4C ). The LD 50 of wild-type strain was 3.6 × 10 7 CFU/frog, whereas that of the LIPI-1-deleted mutant was greater than 2.8 × 10 9 CFU/frog ( Fig. 2G , 4C ). These results suggested that the LIPI-1 locus of L. monocytogenes is essential for its virulence against Xenopus frogs. Intraperitoneal injection of S. aureus into Xenopus frogs causes bacterial dissemination We examined the visceral morphology of Xenopus frogs intraperitoneally injected with S. aureus NCTC8325-4 and compared it with that of non-injected frogs ( Fig. 5A ). The stomachs of non-injected frogs were pale pink, and the muscles were soft and movable. In contrast, the stomachs of injected frogs, examined immediately after death at 7 h post-injection, were red and deformed, and rigor mortis had set in, with hard and immobile muscles ( Fig. 5A ). In addition, blood obtained by amputating the upper legs of non-injected frogs was dark red, whereas that of frogs immediately after death appeared light red. Download figure Open in new tab Fig. 5. Bacteria disseminate from intraperitoneal cavity to whole body in Xenopus frogs. (A) Photograph of a Xenopus frog with its abdomen open. The left shows a non-injected frog. The right shows a frog that died 7 h after intraperitoneal injection of S. aureus NCTC8325-4 strain (8.6 × 10 8 CFU/frog). (B) S. aureus NCTC8325-4 strain (WT) or NCTC8325-4 strain carrying pND50-gmkp1-lux was injected intraperitoneally into frog, and the abdomen was opened 7 h post-injection to observe luminescence. Bright field (left), luminescence (center), and merged images (right) are displayed. Scale bar: 10 mm. The bacterial dose administered was 8.6 × 10 8 CFU/frog. (C) Xenopus frogs were intraperitoneally injected with S. aureus NCTC8325-4 strain (8.6 × 10 8 CFU/frog), and bacterial CFUs were quantified in blood and organs at 30 min, 4 h, and 7 h post-injection. At each time point, three animals were analyzed in a single experiment, and two independent experiments were performed. Mean CFU count from six frogs at each time point is represented by a solid horizontal bar, with the error bar indicating standard error. Open symbols indicate data from PBS-injected control frogs (n=2). To investigate the distribution of S. aureus in Xenopus frogs after intraperitoneal injection, frogs were intraperitoneally injected with an S. aureus strain expressing the lux operon, which enables a luminescence reaction without the need for external substrates. Seven h post-infection, luminescence was observed at the periphery of the abdominal cavity ( Fig. 5B ). Conversely, no luminescence was detected in frogs administered with S. aureus strain lacking the lux operon ( Fig. 5B ). These results suggested that S. aureus intraperitoneally injected into Xenopus frogs disseminated throughout the abdominal cavity. To further investigate the systemic distribution of S. aureus in Xenopus , Xenopus frogs were intraperitoneally injected with S. aureus and dissected at 30 min, 4 h, and 7 h post-injection to quantify bacterial counts in the blood and various organs (stomach, leg muscle, heart, and liver). S. aureus were detected at all time points and in all examined organs, with bacterial numbers maintained from 30 min to 7 h post-infection ( Fig. 5C ). In contrast, S. aureus were not detected in the organs of the frogs injected with PBS ( Fig. 5C ). Therefore, all detected S. aureus were derived from the intraperitoneally injected bacteria. Notably, S. aureus were observed in the blood, and in the leg muscles that does not directly contact the abdominal cavity, suggesting that intraperitoneally injected S. aureus spread throughout the body via blood. Discussion This study demonstrated that Xenopus frogs are susceptible to intraperitoneal injection of the human pathogenic bacteria, S. aureus , P. aeruginosa , and L. monocytogenes and lead to mortality. Furthermore, clinically effective antimicrobial agents suppressed frog death infected with S. aureus and P. aeruginosa , while bacterial mutant strains with deleted virulence genes in S. aureus and L. monocytogenes exhibited attenuated virulence. This study is the first to suggest that Xenopus frogs are a viable animal infection model for evaluating both the efficacy of antimicrobial agents and virulence gene function of human pathogenic bacteria. This study showed that S. aureus disseminated into the blood and organs of Xenopus frogs following intraperitoneal injection, resulting in systemic infections. In humans, intraperitoneal infections caused by laparotomy or intestinal obstruction can progress to sepsis ( 58 , 59 ). In addition, intraperitoneally injected bacteria have been detected in the blood and liver of mouse infection model ( 60 , 61 ). These observations suggested that the intraperitoneal bacterial infection in Xenopus frogs can simulate bacterial sepsis in mammals, where intraperitoneally infected bacteria disseminate throughout the body. In this study, we found that hla , hlb , as well as agr and cvfA , which regulate the transcriptional expression of hla and hlb , are required for the lethality of S. aureus in Xenopus frogs ( Fig. 4A , 4B ). S. aureus strains lacking hla and hlb showcase reduced virulence in rabbits ( 44 ). In human alveolar epithelial cells, α-hemolysin interacts with the host cell surface protein ADAM10 and forms a heptameric β-barrel structure, creating pores in the cell membrane ( 62 ). ADAM10 knockout in mouse alveolar epithelial cells confers resistance to lethal pneumonia caused by S. aureus ( 60 ). β-Hemolysin is a neutral sphingomyelinase that degrades sphingomyelin in cell membranes ( 64 ). According to Xenbase, a database of X. laevis and X. tropicalis , X. laevis has ADAM10 (target of α-hemolysin) and a sphingomyelin synthase (target of β-hemolysin), suggesting that α-hemolysin and β-hemolysin may exert similar effects in X. laevis as in mammals. These findings support the use of Xenopus frogs for analyzing bacterial virulence mechanisms mediated by α- and β-hemolysins. During intraperitoneal infection in Xenopus frogs, the srtA -deleted mutant in this study showed virulence comparable to that of the wild-type S. aureus ( Fig. 4A ). In rats and mice, the pathogenicity of srtA -deleted mutant is lower than that of the wild-type strain administered via various methods, including intraperitoneal injection ( 62 – 64 ). Similarly, in silkworms and crickets, the srtA -deleted mutant is less virulent than the wild-type strain in systemic infection models induced by intrahemolymph injection ( 13 , 28 ). In contrast, in the nematode infection model, where bacteria were fed to the nematodes, srtA -deleted mutant displayed similar pathogenicity to that of the wild-type strain ( 68 , 69 ). As srtA deficiency attenuates virulence in mice following intraperitoneal injection ( 65 ), this suggests that the non-attenuated virulence of srtA -deleted mutant observed in Xenopus frogs is not owing to the infection route, but rather to host animal-specific differences. The srtA gene is involved in anchoring more than 10 different cell wall adhesion proteins ( 70 , 71 ). The molecular mechanisms underlying why cell adhesion by these cell wall proteins is not required for intraperitoneal infection in Xenopus frogs need to be analyzed in the future. The mortality of Xenopus frogs caused by drug-susceptible and drug-resistant strains of S. aureus and drug-susceptible strains of P. aeruginosa was suppressed by the administration of appropriate antimicrobial agents. However, multidrug resistant P. aeruginosa remained lethal despite antimicrobial treatment ( Fig. 3 ). Hence, Xenopus frogs could prove useful for evaluating antibiotic efficacy against sepsis caused by S. aureus and P. aeruginosa . Xenopus juvenile frogs are smaller than mice, which enables the evaluation and exploration of antimicrobial agents using small drug doses. In this study, antimicrobials were intraperitoneally injected immediately after injecting bacterial suspensions. Future studies should evaluate the efficacy of other antimicrobial administration routes used in clinical practice, such as oral and subcutaneous administration. The Xenopus infection model has potential for identifying and analyzing key human pathogenic bacterial genes involved in sepsis progression and evaluating therapeutic interventions. Materials and Methods Ethics statement This study was conducted in strict accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, 2006. All mouse protocols complied with the Regulations for Animal Care and Use of Okayama University and were approved by the Animal Use Committee at Okayama University (approval number: OKU-2024557). Xenopus laevis X. laevis juvenile frogs were purchased from a breeding company (Xenopus Yoshoku Kyozai, Ibaraki, Japan). The frogs were 3–4 cm in size and weighed approximately 2.5 g. Fifty frogs were housed in a 13.6 L (37 cm W × 22 cm D × 25 cm H) aquarium, which was half-filled with water. The aquarium was maintained at a room temperature of 22°C. The frogs were fed with dried goldfish food (Cat. 4971453050347, Itosui, Tokyo, Japan) every two days. The rearing water was replaced with fresh dechlorinated water once every two days ( 72 ). Bacterial strains All bacterial strains used in this study are listed in Table 1 . S. aureus gene deletion mutants for agr locus ( 73 ), srtA ( 28 ), cvfA ( 29 ), hla ( 28 ), hlb ( 28 ), and hla / hlb ( 28 ) were constructed in our previous studies. We confirmed the deletion of the respective genes by PCR, using genomic DNA as the template and oligonucleotide primers ( Table 2 ). L. monocytogenes mutant strain lacking LIPI-1 was constructed according to a previously described method ( 74 ). The downstream regions of prfA and plcB were amplified via PCR and ligated into pHS-MCS. L. monocytogenes EGD strain was electroporated with the obtained plasmids, resulting in a LIPI-1-deleted mutant. The genomic DNA of L. monocytogenes was extracted using the QIAamp Blood Mini Kit (QIAGEN,Venlo, Netherlands) as previously described ( 75 ), and LIPI-1 deletion was confirmed through PCR. View this table: View inline View popup Download powerpoint Table 1. List of the bacterial strains and plasmids used View this table: View inline View popup Download powerpoint Table 2. Primers used in this study Preparation of bacterial solution A single S. aureus colony on tryptic soy agar was inoculated into 5 mL of tryptic soy broth medium and aerobically incubated at 37°C overnight. The overnight culture was centrifuged at 10,400 × g for 10 min, and the bacterial pellet was resuspended in phosphate-buffered saline (PBS) and diluted to prepare bacterial solutions at appropriate concentrations. The same procedure was performed for P. aeruginosa and L. monocytogenes , using Lysogeny broth and brain heart infusion media, respectively. In multiple independent experiments, the optical density at 600 nm (OD 600 ) of overnight cultures was measured, and a calibration curve was generated to determine the relationship between CFU and OD 600 . This calibration curve was used to estimate the CFU injected into frogs based on the OD 600 of overnight cultures in the infection experiments. Preparation of antimicrobial solutions Antimicrobial solutions were prepared as follows: 10 mg each of kanamycin monosulfate (TCI, Tokyo, Japan), oxacillin (SIGMA, St. Louis, MO), vancomycin hydrochloride (FUJIFILM, Tokyo, Japan), and ceftazidime hydrate (LKT, St. Paul, MN) were dissolved in 5 mL PBS to achieve a concentration of 2 mg mL −1 . Ciprofloxacin hydrochloride monohydrate (TCI, Tokyo, Japan) was dissolved in Milli-Q water to a concentration of 2 mg mL −1 . All antimicrobial solutions were stored at -30°C and thawed before use. Intraperitoneal injections Xenopus frogs were anesthetized by placing them on crushed ice for 2 min. A 50 µL bacterial solution was injected into the abdominal cavity of X. laevis , just above the left leg, using a 27G needle (outer diameter) of 3/4-inch length (Terumo, Tokyo, Japan) ( Fig. 1A ) attached to a 1 mL tuberculin syringe (Terumo, Tokyo, Japan). Five animals per sample were injected, placed in a 1 L beaker containing 500 mL water, and kept at 27°C in the dark. The frogs recovered from cold anesthesia and begin swimming immediately after being placed in the beaker. To exclude the possibility of death due to low-temperature anesthesia or injection-related stress, a negative control group injected with PBS was prepared for all experiments. Calculation of LD 50 Three different concentrations of bacterial solutions were employed for each experiment. Overnight cultures were centrifuged at 10,400 × g for 10 min, the bacterial pellet was resuspended in PBS, and a three-fold dilution series was prepared using PBS. Five animals were injected per sample, and survival rates were recorded at 24 h post-infection. The experiment was performed thrice, and the survival rate data and injected CFU were plotted on a graph. A dose-response survival curve was created using logistic regression using the statistical software R to obtain LD 50 and standard error. Survival data for each experiment are presented in Table S1 . Luminescence measurement DNA fragments containing lux operon were amplified by PCR using genomic DNA from S. aureus Xen29 ( 76 ) as template and a primer pair ( Table 2 ). The amplified DNA was inserted into the BamHI and PstI recognition sites of a plasmid carrying the gmk promoter region ( 77 ), resulting in pND50- gmk p1-lux. S. aureus RN4220 strain was transformed with pND50- gmk p1-lux through electroporation, and the plasmid was subsequently transferred into NCTC8325-4 strain using phage 80α via transduction. The NCTC8325-4 strain carrying pND50- gmk p1-lux was intraperitoneally injected into Xenopus frogs. At 7 h post-injection, the abdomen was opened and chemiluminescence was measured using a CCD camera (iBright 1500, Thermo Fisher, Waltham, MA). The camera was configured to an exposure time of 10 min, zoom 3.0, and focus 390. Three images were captured per animal under the same conditions, and the average function of ImageJ was used to compute the mean luminescence intensity from the three photographs. Measurement of bacterial count in organs Xenopus frogs were intraperitoneally injected with S. aureus NCTC8325-4. At 30 min, 4 h, and 7 h post-injection, the frogs were anesthetized by immersing them in 2 g L −1 ethyl m-aminobenzoate methanesulfonate solution (Nacalai Tesque, Kyoto, Japan) for 5 min. The right upper leg was amputated, and blood was collected. Muscles of the right upper leg, stomach, heart, and liver were excised. To remove residual blood, the organs other than blood were washed with PBS, weighed, and then homogenized in 400 µL PBS using TissueLyser II (QIAGEN) at 25 Hz for 1.5 min. A 10-fold dilution series of blood and organ homogenates were prepared using 0.9% NaCl, Approximately 5 µL of each dilution was spread onto mannitol salt agar plates, a selective medium for S. aureus , and incubated at 37°C overnight. The colonies were counted according to a previously defined method ( 78 ). Bacterial numbers per gram of blood and organ were calculated based on colony counts and dilution ratios. Statistical Analysis The log-rank (Mantel-Cox) test was performed using GraphPad Prism software (ver. 10.2.3) to determine significant differences in the time-survival curves. A dose-response survival curve was generated via logistic regression to plot frog survival against CFU injected into frogs using R for Windows (ver. 4.3.3). The LD 50 and standard error were estimated using the glm function from the MASS library ( 79 ). A likelihood ratio test was conducted using the anova function to determine significant differences between the two dose-response survival curves. The R code used for these analyses is registered in the GitHub repository (URL: https://doi.org/10.5281/zenodo.14978657 , doi: 10.5281/zenodo.14978657). Acknowledgements This study was supported by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (Grants 22K14892, 23K24131, 23K06130, 24K01760, 24K21872). We thank the National BioResource Project-E. coli (National Institute of Genetics, Japan) for providing the E. coli BW25113 strain and BGSC (Bacillus Genetic Stock Center) for providing B. subtilis 168 strain. References 1. ↵ Ikuta KS , Swetschinski LR , Aguilar GR , Sharara F , Mestrovic T , Gray AP , Weaver ND , Wool EE , Han C , Hayoon AG , Aali A , Abate SM , Abbasi-Kangevari M , Abbasi-Kangevari Z , Abd-Elsalam S , Abebe G , Abedi A , Abhari AP , Abidi H , Aboagye RG , Absalan A , Ali HA , Acuna JM , Adane TD , Addo IY , Adegboye OA , Adnan M , Adnani QES , Afzal MS , Afzal S , Aghdam ZB , Ahinkorah BO , Ahmad A , Ahmad AR , Ahmad R , Ahmad S , Ahmad S , Ahmadi S , Ahmed A , Ahmed H , Ahmed JQ , Rashid TA , Ajami M , Aji B , Akbarzadeh-Khiavi M , Akunna CJ , Hamad HA , Alahdab F , Al-Aly Z , Aldeyab MA , Aleman AV , Alhalaiqa FAN , Alhassan RK , Ali BA , Ali L , Ali SS , Alimohamadi Y , Alipour V , Alizadeh A , Aljunid SM , Allel K , Almustanyir S , Ameyaw EK , Amit AML , Anandavelane N , Ancuceanu R , Andrei CL , Andrei T , Anggraini D , Ansar A , Anyasodor AE , Arabloo J , Aravkin AY , Areda D , Aripov T , Artamonov AA , Arulappan J , Aruleba RT , Asaduzzaman M , Ashraf T , Athari SS , Atlaw D , Attia S , Ausloos M , Awoke T , Quintanilla BPA , Ayana TM , Azadnajafabad S , Jafari AA , B DB , Badar M , Badiye AD , Baghcheghi N , Bagherieh S , Baig AA , Banerjee I , Barac A , Bardhan M , Barone-Adesi F , Barqawi HJ , Barrow A , Baskaran P , Basu S , Batiha A-MM , Bedi N , Belete MA , Belgaumi UI , Bender RG , Bhandari B , Bhandari D , Bhardwaj P , Bhaskar S , Bhattacharyya K , Bhattarai S , Bitaraf S , Buonsenso D , Butt ZA , Santos FLC dos , Cai J , Calina D , Camargos P , Cámera LA , Cárdenas R , Cevik M , Chadwick J , Charan J , Chaurasia A , Ching PR , Choudhari SG , Chowdhury EK , Chowdhury FR , Chu D-T , Chukwu IS , Dadras O , Dagnaw FT , Dai X , Das S , Dastiridou A , Debela SA , Demisse FW , Demissie S , Dereje D , Derese M , Desai HD , Dessalegn FN , Dessalegni SAA , Desye B , Dhaduk K , Dhimal M , Dhingra S , Diao N , Diaz D , Djalalinia S , Dodangeh M , Dongarwar D , Dora BT , Dorostkar F , Dsouza HL , Dubljanin E , Dunachie SJ , Durojaiye OC , Edinur HA , Ejigu HB , Ekholuenetale M , Ekundayo TC , El-Abid H , Elhadi M , Elmonem MA , Emami A , Bain LE , Enyew DB , Erkhembayar R , Eshrati B , Etaee F , Fagbamigbe AF , Falahi S , Fallahzadeh A , Faraon EJA , Fatehizadeh A , Fekadu G , Fernandes JC , Ferrari A , Fetensa G , Filip I , Fischer F , Foroutan M , Gaal PA , Gadanya MA , Gaidhane AM , Ganesan B , Gebrehiwot M , Ghanbari R , Nour MG , Ghashghaee A , Gholamrezanezhad A , Gholizadeh A , Golechha M , Goleij P , Golinelli D , Goodridge A , Gunawardane DA , Guo Y , Gupta RD , Gupta S , Gupta VB , Gupta VK , Guta A , Habibzadeh P , Avval AH , Halwani R , Hanif A , Hannan MA , Harapan H , Hassan S , Hassankhani H , Hayat K , Heibati B , Heidari G , Heidari M , Heidari-Soureshjani R , Herteliu C , Heyi DZ , Hezam K , Hoogar P , Horita N , Hossain MM , Hosseinzadeh M , Hostiuc M , Hostiuc S , Hoveidamanesh S , Huang J , Hussain S , Hussein NR , Ibitoye SE , Ilesanmi OS , Ilic IM , Ilic MD , Imam MT , Immurana M , Inbaraj LR , Iradukunda A , Ismail NE , Iwu CCD , Iwu CJ , J LM , Jakovljevic M , Jamshidi E , Javaheri T , Javanmardi F , Javidnia J , Jayapal SK , Jayarajah U , Jebai R , Jha RP , Joo T , Joseph N , Joukar F , Jozwiak JJ , Kacimi SEO , Kadashetti V , Kalankesh LR , Kalhor R , Kamal VK , Kandel H , Kapoor N , Karkhah S , Kassa BG , Kassebaum NJ , Katoto PD , Keykhaei M , Khajuria H , Khan A , Khan IA , Khan M , Khan MN , Khan MA , Khatatbeh MM , Khater MM , Kashani HRK , Khubchandani J , Kim H , Kim MS , Kimokoti RW , Kissoon N , Kochhar S , Kompani F , Kosen S , Koul PA , Laxminarayana SLK , Lopez FK , Krishan K , Krishnamoorthy V , Kulkarni V , Kumar N , Kurmi OP , Kuttikkattu A , Kyu HH , Lal DK , Lám J , Landires I , Lasrado S , Lee S , Lenzi J , Lewycka S , Li S , Lim SS , Liu W , Lodha R , Loftus MJ , Lohiya A , Lorenzovici L , Lotfi M , Mahmoodpoor A , Mahmoud MA , Mahmoudi R , Majeed A , Majidpoor J , Makki A , Mamo GA , Manla Y , Martorell M , Matei CN , McManigal B , Nasab EM , Mehrotra R , Melese A , Mendoza-Cano O , Menezes RG , Mentis A-FA , Micha G , Michalek IM , Sá ACMGN de , Kostova NM , Mir SA , Mirghafourvand M , Mirmoeeni S , Mirrakhimov EM , Mirza-Aghazadeh-Attari M , Misganaw AS , Misganaw A , Misra S , Mohammadi E , Mohammadi M , Mohammadian-Hafshejani A , Mohammed S , Mohan S , Mohseni M , Mokdad AH , Momtazmanesh S , Monasta L , Moore CE , Moradi M , Sarabi MM , Morrison SD , Motaghinejad M , Isfahani HM , Khaneghah AM , Mousavi-Aghdas SA , Mubarik S , Mulita F , Mulu GBB , Munro SB , Muthupandian S , Nair TS , Naqvi AA , Narang H , Natto ZS , Naveed M , Nayak BP , Naz S , Negoi I , Nejadghaderi SA , Kandel SN , Ngwa CH , Niazi RK , Sá ATN de , Noroozi N , Nouraei H , Nowroozi A , Nuñez-Samudio V , Nutor JJ , Nzoputam CI , Nzoputam OJ , Oancea B , Obaidur RM , Ojha VA , Okekunle AP , Okonji OC , Olagunju AT , Olusanya BO , Bali AO , Omer E , Otstavnov N , Oumer B , A MP , Padubidri JR , Pakshir K , Palicz T , Pana A , Pardhan S , Paredes JL , Parekh U , Park E-C , Park S , Pathak A , Paudel R , Paudel U , Pawar S , Toroudi HP , Peng M , Pensato U , Pepito VCF , Pereira M , Peres MFP , Perico N , Petcu I-R , Piracha ZZ , Podder I , Pokhrel N , Poluru R , Postma MJ , Pourtaheri N , Prashant A , Qattea I , Rabiee M , Rabiee N , Radfar A , Raeghi S , Rafiei S , Raghav PR , Rahbarnia L , Rahimi-Movaghar V , Rahman M , Rahman MA , Rahmani AM , Rahmanian V , Ram P , Ranjha MMAN , Rao SJ , Rashidi M-M , Rasul A , Ratan ZA , Rawaf S , Rawassizadeh R , Razeghinia MS , Redwan EMM , Regasa MT , Remuzzi G , Reta MA , Rezaei N , Rezapour A , Riad A , Ripon RK , Rudd KE , Saddik B , Sadeghian S , Saeed U , Safaei M , Safary A , Safi SZ , Sahebazzamani M , Sahebkar A , Sahoo H , Salahi S , Salahi S , Salari H , Salehi S , Kafil HS , Samy AM , Sanadgol N , Sankararaman S , Sanmarchi F , Sathian B , Sawhney M , Saya GK , Senthilkumaran S , Seylani A , Shah PA , Shaikh MA , Shaker E , Shakhmardanov MZ , Sharew MM , Sharifi-Razavi A , Sharma P , Sheikhi RA , Sheikhy A , Shetty PH , Shigematsu M , Shin JI , Shirzad-Aski H , Shivakumar KM , Shobeiri P , Shorofi SA , Shrestha S , Sibhat MM , Sidemo NB , Sikder MK , Silva LMLR , Singh JA , Singh P , Singh S , Siraj MS , Siwal SS , Skryabin VY , Skryabina AA , Socea B , Solomon DD , Song Y , Sreeramareddy CT , Suleman M , Abdulkader RS , Sultana S , Szócska M , Tabatabaeizadeh S-A , Tabish M , Taheri M , Taki E , Tan K-K , Tandukar S , Tat NY , Tat VY , Tefera BN , Tefera YM , Temesgen G , Temsah M-H , Tharwat S , Thiyagarajan A , Tleyjeh II , Troeger CE , Umapathi KK , Upadhyay E , Tahbaz SV , Valdez PR , Eynde JV den , Doorn HR van , Vaziri S , Verras G-I , Viswanathan H , Vo B , Waris A , Wassie GT , Wickramasinghe ND , Yaghoubi S , Yahya GATY , Jabbari SHY , Yigit A , Yiğit V , Yon DK , Yonemoto N , Zahir M , Zaman BA , Zaman SB , Zangiabadian M , Zare I , Zastrozhin MS , Zhang Z-J , Zheng P , Zhong C , Zoladl M , Zumla A , Hay SI , Dolecek C , Sartorius B , Murray CJL , Naghavi M . 2022 . Global mortality associated with 33 bacterial pathogens in 2019: a systematic analysis for the Global Burden of Disease Study 2019 . The Lancet 400 : 2221 – 2248 . OpenUrl 2. ↵ Turner NA , Sharma-Kuinkel BK , Maskarinec SA , Eichenberger EM , Shah PP , Carugati M , Holland TL , Fowler VG . 2019 . Methicillin-resistant Staphylococcus aureus: an overview of basic and clinical research . Nat Rev Microbiol 17 : 203 – 218 . OpenUrl CrossRef PubMed 3. ↵ Pachori P , Gothalwal R , Gandhi P . 2019 . Emergence of antibiotic resistance Pseudomonas aeruginosa in intensive care unit; a critical review . Genes & Diseases 6 : 109 – 119 . OpenUrl CrossRef PubMed 4. ↵ Schlech WF . 2019 . Epidemiology and Clinical Manifestations of Listeria monocytogenes Infection . Microbiology Spectrum 7 : doi: 10.1128/microbiolspec.gpp3-0014-2018 . OpenUrl CrossRef 5. ↵ Scallan E , Hoekstra RM , Angulo FJ , Tauxe RV , Widdowson M-A , Roy SL , Jones JL , Griffin PM . 2011 . Foodborne Illness Acquired in the United States—Major Pathogens . Emerg Infect Dis 17 : 7 – 15 . OpenUrl CrossRef PubMed Web of Science 6. ↵ Petetta F , Ciccocioppo R . 2021 . Public perception of laboratory animal testing: Historical, philosophical, and ethical view . Addiction Biology 26 : e12991 . OpenUrl CrossRef PubMed 7. ↵ Štampar M , Žegura B. 2024 . In vitro hepatic 3D cell models and their application in genetic toxicology: A systematic review . Mutation Research - Genetic Toxicology and Environmental Mutagenesis 900 : 503835 . OpenUrl CrossRef 8. ↵ Jean-Quartier C , Jeanquartier F , Jurisica I , Holzinger A . 2018 . In silico cancer research towards 3R . BMC Cancer 18 : 408 . OpenUrl CrossRef PubMed 9. ↵ Garsin DA , Sifri CD , Mylonakis E , Qin X , Singh KV , Murray BE , Calderwood SB , Ausubel FM . 2001 . A simple model host for identifying Gram-positive virulence factors . Proceedings of the National Academy of Sciences 98 : 10892 – 10897 . OpenUrl Abstract / FREE Full Text 10. ↵ Needham AJ , Kibart M , Crossley H , Ingham PW , Foster SJ . 2004 . Drosophila melanogaster as a model host for Staphylococcus aureus infection . Microbiology 150 : 2347 – 2355 . OpenUrl CrossRef PubMed Web of Science 11. ↵ Kaito C , Akimitsu N , Watanabe H , Sekimizu K . 2002 . Silkworm larvae as an animal model of bacterial infection pathogenic to humans . Microb Pathog 32 : 183 – 190 . OpenUrl CrossRef PubMed Web of Science 12. ↵ Desbois AP , Coote PJ . 2011 . Wax moth larva (Galleria mellonella): an in vivo model for assessing the efficacy of antistaphylococcal agents . Journal of Antimicrobial Chemotherapy 66 : 1785 – 1790 . OpenUrl CrossRef PubMed Web of Science 13. ↵ Kochi Y , Miyashita A , Tsuchiya K , Mitsuyama M , Sekimizu K , Kaito C . 2016 . A human pathogenic bacterial infection model using the two-spotted cricket, Gryllus bimaculatus . FEMS Microbiol Lett 363 : fnw163 . OpenUrl CrossRef PubMed 14. ↵ Prajsnar TK , Cunliffe VT , Foster SJ , Renshaw SA . 2008 . A novel vertebrate model of Staphylococcus aureus infection reveals phagocyte-dependent resistance of zebrafish to non-host specialized pathogens . Cellular Microbiology 10 : 2312 – 2325 . OpenUrl CrossRef PubMed 15. ↵ Clatworthy AE , Lee JS-W , Leibman M , Kostun Z , Davidson AJ , Hung DT . 2009 . Pseudomonas aeruginosa infection of zebrafish involves both host and pathogen determinants . Infect Immun 77 : 1293 – 1303 . OpenUrl Abstract / FREE Full Text 16. ↵ Animal infection models using non-mammals - Kaito - 2020 - Microbiology and Immunology - Wiley Online Library . https://onlinelibrary.wiley.com/doi/10.1111/1348-0421.12834 . Retrieved 16 December 2024 . 17. ↵ Hu C , Yang W . 2023 . Alternatives to animal models to study bacterial infections . Folia Microbiol 68 : 703 – 739 . OpenUrl CrossRef 18. ↵ Bouwmeester T . 2001 . The Spemann-Mangold organizer: the control of fate specification and morphogenetic rearrangements during gastrulation in Xenopus . Int J Dev Biol 45 : 251 – 258 . OpenUrl CrossRef PubMed 19. ↵ Rhoo KH , Edholm E-S , Forzán MJ , Khan A , Waddle AW , Pavelka MS , Robert J . 2019 . Distinct Host-Mycobacterial Pathogen Interactions between Resistant Adult and Tolerant Tadpole Life Stages of Xenopus laevis . J Immunol 203 : 2679 – 2688 . OpenUrl Abstract / FREE Full Text 20. ↵ Robert J , Ohta Y . 2009 . Comparative and developmental study of the immune system in Xenopus . Developmental Dynamics 238 : 1249 – 1270 . OpenUrl CrossRef PubMed Web of Science 21. ↵ Ruiz VL , Robert J . 2023 . The amphibian immune system . Philosophical Transactions of the Royal Society B: Biological Sciences 378 : 20220123 . OpenUrl CrossRef PubMed 22. ↵ Genome evolution in the allotetraploid frog Xenopus laevis | Nature . https://www.nature.com/articles/nature19840 . Retrieved 23 January 2025 . 23. ↵ Suzuki KT , Isoyama Y , Kashiwagi K , Sakuma T , Ochiai H , Sakamoto N , Furuno N , Kashiwagi A , Yamamoto T . 2013 . High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos . Biology Open 2 : 448 – 452 . OpenUrl Abstract / FREE Full Text 24. Wang F , Shi Z , Cui Y , Guo X , Shi Y-B , Chen Y . 2015 . Targeted gene disruption in Xenopus laevis using CRISPR/Cas9 . Cell & Bioscience 5 : 15 . OpenUrl CrossRef PubMed 25. ↵ Bhattacharya D , Marfo CA , Li D , Lane M , Khokha MK . 2015 . CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus . Developmental Biology 408 : 196 – 204 . OpenUrl CrossRef PubMed 26. ↵ Brett SS , Shelton G . 1979 . Ventilatory Mechanisms of the Amphibian, Xenopus Laevis; The Role of the Buccal Force Pump . Journal of Experimental Biology 80 : 251 – 269 . OpenUrl Abstract / FREE Full Text 27. ↵ Vandenesch F , Kornblum J , Novick RP . 1991 . A temporal signal, independent of agr, is required for hla but not spa transcription in Staphylococcus aureus . J Bacteriol 173 : 6313 – 6320 . OpenUrl Abstract / FREE Full Text 28. ↵ Miyazaki S , Matsumoto Y , Sekimizu K , Kaito C . 2012 . Evaluation of Staphylococcus aureus virulence factors using a silkworm model . FEMS Microbiology Letters 326 : 116 – 124 . OpenUrl CrossRef PubMed Web of Science 29. ↵ Nagata M , Kaito C , Sekimizu K . 2008 . Phosphodiesterase Activity of CvfA Is Required for Virulence in Staphylococcus aureus * . Journal of Biological Chemistry 283 : 2176 – 2184 . OpenUrl Abstract / FREE Full Text 30. ↵ Novick RP . 2003 . Autoinduction and signal transduction in the regulation of staphylococcal virulence . Molecular Microbiology 48 : 1429 – 1449 . OpenUrl CrossRef PubMed Web of Science 31. ↵ Mazmanian SK , Ton-That H , Schneewind O . 2001 . Sortase-catalysed anchoring of surface proteins to the cell wall of Staphylococcus aureus . Molecular Microbiology 40 : 1049 – 1057 . OpenUrl CrossRef PubMed Web of Science 32. ↵ CvfA Protein and Polynucleotide Phosphorylase Act in an Opposing Manner to Regulate Staphylococcus aureus Virulence - ScienceDirect . https://www.sciencedirect.com/science/article/pii/S0021925820523405?via%3Dihub . Retrieved 29 July 2024 . 33. ↵ Khemici V , Prados J , Linder P , Redder P . 2015 . Decay-Initiating Endoribonucleolytic Cleavage by RNase Y Is Kept under Tight Control via Sequence Preference and Sub-cellular Localisation . PLoS Genet 11 : e1005577 . OpenUrl CrossRef PubMed 34. ↵ Wiktorczyk-Kapischke N , Skowron K , Wałecka-Zacharska E . 2023 . Genomic and pathogenicity islands of Listeria monocytogenes—overview of selected aspects . Front Mol Biosci 10 . 35. ↵ Comparison of listeriolysin O and phospholipases PlcA and PlcB activities, and initial intracellular growth capability among food and clinical strains of Listeria monocytogenes | Journal of Applied Microbiology | Oxford Academic . https://academic.oup.com/jambio/article/124/3/899/6714327?login=true . Retrieved 29 July 2024 . 36. ↵ Hadjilouka A , Paramithiotis S , Drosinos EH . 2018 . Genetic Analysis of the Listeria Pathogenicity Island 1 of Listeria monocytogenes 1/2a and 4b Isolates . Curr Microbiol 75 : 857 – 865 . OpenUrl CrossRef PubMed 37. ↵ Gaillard JL , Berche P , Mounier J , Richard S , Sansonetti P . 1987 . In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2 . Infect Immun 55 : 2822 – 2829 . OpenUrl Abstract / FREE Full Text 38. ↵ Requirement of the Listeria monocytogenes Broad-Range Phospholipase PC-PLC during Infection of Human Epithelial Cells | Journal of Bacteriology . https://journals.asm.org/doi/10.1128/jb.185.21.6295-6307.2003 . Retrieved 29 July 2024 . 39. ↵ Listeria monocytogenes Evades Killing by Autophagy During Colonization of Host Cells: Autophagy : Vol 3 , No 5 . https://www.tandfonline.com/doi/abs/10.4161/auto.4450 . Retrieved 29 July 2024 . 40. ↵ Heyer G , Saba S , Adamo R , Rush W , Soong G , Cheung A , Prince A . 2002 . Staphylococcus aureus agr and sarA Functions Are Required for Invasive Infection but Not Inflammatory Responses in the Lung . Infect Immun 70 : 127 – 133 . OpenUrl Abstract / FREE Full Text 41. On the role of Staphylococcus aureus sortase and sortase-catalyzed surface protein anchoring in murine septic arthritis - PubMed . https://pubmed.ncbi.nlm.nih.gov/11992276/ . Retrieved 8 July 2024 . 42. ↵ Kaito C , Kurokawa K , Matsumoto Y , Terao Y , Kawabata S , Hamada S , Sekimizu K . 2005 . Silkworm pathogenic bacteria infection model for identification of novel virulence genes . Mol Microbiol 56 : 934 – 944 . OpenUrl CrossRef PubMed Web of Science 43. ↵ Roles of Listeria monocytogenes virulence factors in survival: virulence factors distinct from listeriolysin are needed for the organism to survive an early neutrophil-mediated host defense mechanism | Infection and Immunity . https://journals.asm.org/doi/10.1128/iai.60.3.951-957.1992?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed . Retrieved 23 January 2025 . 44. ↵ O’Callaghan RJ , Callegan MC , Moreau JM , Green LC , Foster TJ , Hartford OM , Engel LS , Hill JM . 1997 . Specific roles of alpha-toxin and beta-toxin during Staphylococcus aureus corneal infection . Infection and Immunity 65 : 1571 – 1578 . OpenUrl Abstract / FREE Full Text 45. ↵ Sifri CD , Begun J , Ausubel FM , Calderwood SB . 2003 . Caenorhabditis elegans as a Model Host for Staphylococcus aureus Pathogenesis . Infection and Immunity 71 : 2208 – 2217 . OpenUrl Abstract / FREE Full Text 46. ↵ Neuhaus K , Satorhelyi P , Schauer K , Scherer S , Fuchs TM . 2013 . Acid shock of Listeria monocytogenes at low environmental temperatures induces prfA, epithelial cell invasion, and lethality towards Caenorhabditis elegans . BMC Genomics 14 : 285 . OpenUrl CrossRef PubMed 47. ↵ Datsenko KA , Wanner BL . 2000 . One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products . Proceedings of the National Academy of Sciences 97 : 6640 – 6645 . OpenUrl Abstract / FREE Full Text 48. ↵ Zeigler DR , Prágai Z , Rodriguez S , Chevreux B , Muffler A , Albert T , Bai R , Wyss M , Perkins JB . 2008 . The Origins of 168, W23, and Other Bacillus subtilis Legacy Strains . J Bacteriol 190 : 6983 – 6995 . OpenUrl Abstract / FREE Full Text 49. ↵ Akimitsu N , Hamamoto H , Inoue R , Shoji M , Akamine A , Takemori K , Hamasaki N , Sekimizu K . 1999 . Increase in Resistance of Methicillin-ResistantStaphylococcus aureus to β-Lactams Caused by Mutations Conferring Resistance to Benzalkonium Chloride, a Disinfectant Widely Used in Hospitals . Antimicrobial Agents and Chemotherapy 43 : 3042 – 3043 . OpenUrl FREE Full Text 50. ↵ Masadeh MM , Alzoubi KH , Masadeh MM , Aburashed ZO . 2021 . Metformin as a Potential Adjuvant Antimicrobial Agent Against Multidrug Resistant Bacteria . Clin Pharmacol 13 : 83 – 90 . OpenUrl 51. Meletis G , Bagkeri M , Meletis G , Bagkeri M . 2013 . Pseudomonas aeruginosa: Multi-Drug-Resistance Development and Treatment OptionsInfection Control . IntechOpen . 52. Dabbousi AA , Dabboussi F , Hamze M , Osman M , Kassem II . 2022 . The Emergence and Dissemination of Multidrug Resistant Pseudomonas aeruginosa in Lebanon: Current Status and Challenges during the Economic Crisis . 5. Antibiotics 11 : 687 . OpenUrl 53. Kunz Coyne AJ , El Ghali A , Holger D , Rebold N , Rybak MJ . 2022 . Therapeutic Strategies for Emerging Multidrug-Resistant Pseudomonas aeruginosa . Infect Dis Ther 11 : 661 – 682 . OpenUrl CrossRef PubMed 54. ↵ Queck SY , Jameson-Lee M , Villaruz AE , Bach T-HL , Khan BA , Sturdevant DE , Ricklefs SM , Li M , Otto M . 2008 . RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus . Mol Cell 32 : 150 – 158 . OpenUrl CrossRef PubMed Web of Science 55. Lopez N , Kobayashi L , Coimbra R . 2011 . A Comprehensive review of abdominal infections . World J Emerg Surg 6 : 7 . OpenUrl CrossRef PubMed 56. ↵ Arun Kumar RV , Channabasappa SM . 2016 . A retrospective cohort study of perioperative prognostic factors associated with intra-abdominal sepsis . Anesth Essays Res 10 : 50 – 53 . OpenUrl CrossRef PubMed 57. ↵ Knapp S , Vos AF de , Florquin S , Golenbock DT , Poll T van der . 2003 . Lipopolysaccharide Binding Protein Is an Essential Component of the Innate Immune Response to Escherichia coli Peritonitis in Mice . Infection and Immunity 71 : 6747 . OpenUrl Abstract / FREE Full Text 58. ↵ Rauch S , DeDent AC , Kim HK , Bubeck Wardenburg J , Missiakas DM , Schneewind O . 2012 . Abscess Formation and Alpha-Hemolysin Induced Toxicity in a Mouse Model of Staphylococcus aureus Peritoneal Infection . Infect Immun 80 : 3721 – 3732 . OpenUrl Abstract / FREE Full Text 59. ↵ Wilke GA , Wardenburg JB . 2010 . Role of a disintegrin and metalloprotease 10 in Staphylococcus aureus α-hemolysin–mediated cellular injury . Proceedings of the National Academy of Sciences 107 : 13473 – 13478 . OpenUrl Abstract / FREE Full Text 60. ↵ Inoshima I , Inoshima N , Wilke GA , Powers ME , Frank KM , Wang Y , Wardenburg JB . 2011 . A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice . Nat Med 17 : 1310 – 1314 . OpenUrl CrossRef PubMed 61. ↵ Huseby M , Shi K , Brown CK , Digre J , Mengistu F , Seo KS , Bohach GA , Schlievert PM , Ohlendorf DH , Earhart CA . 2007 . Structure and Biological Activities of Beta Toxin from Staphylococcus aureus . J Bacteriol 189 : 8719 – 8726 . OpenUrl Abstract / FREE Full Text 62. ↵ Weiss WJ , Lenoy E , Murphy T , Tardio L , Burgio P , Projan SJ , Schneewind O , Alksne L . 2004 . Effect of srtA and srtB gene expression on the virulence of Staphylococcus aureus in animal models of infection . Journal of Antimicrobial Chemotherapy 53 : 480 – 486 . OpenUrl CrossRef PubMed Web of Science 63. Wang L , Bi C , Wang T , Xiang H , Chen F , Hu J , Liu B , Cai H , Zhong X , Deng X , Wang D . 2015 . A coagulase-negative and non-haemolytic strain of Staphylococcus aureus for investigating the roles of SrtA in a murine model of bloodstream infection . Pathogens and Disease 73 : ftv042 . OpenUrl CrossRef PubMed 64. ↵ Cheng AG , Kim HK , Burts ML , Krausz T , Schneewind O , Missiakas DM . 2009 . Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues . The FASEB Journal 23 : 3393 – 3404 . OpenUrl CrossRef PubMed Web of Science 65. ↵ Bae T , Banger AK , Wallace A , Glass EM , Åslund F , Schneewind O , Missiakas DM . 2004 . Staphylococcus aureus virulence genes identified by bursa aurealis mutagenesis and nematode killing . Proceedings of the National Academy of Sciences 101 : 12312 – 12317 . OpenUrl Abstract / FREE Full Text 66. Sibbald MJJB . 2010 . The Staphylococcus aureus secretome. s.n., Groningen . 67. Mazmanian SK , Liu G , Ton-That H , Schneewind O . 1999 . Staphylococcus aureus Sortase, an Enzyme that Anchors Surface Proteins to the Cell Wall . Science 285 : 760 – 763 . OpenUrl Abstract / FREE Full Text 68. ↵ Mazmanian SK , Liu G , Jensen ER , Lenoy E , Schneewind O . 2000 . Staphylococcus aureus sortase mutants defective in the display of surface proteins and in the pathogenesis of animal infections . Proceedings of the National Academy of Sciences 97 : 5510 – 5515 . OpenUrl Abstract / FREE Full Text 69. ↵ Böswald LF , Matzek D , Popper B . 2022 . Current Feeding Practice of Xenopus laevis in a Laboratory Setting . 9. Animals 12 : 1163 . OpenUrl 70. ↵ Matsumoto Y , Kaito C , Morishita D , Kurokawa K , Sekimizu K . 2007 . Regulation of Exoprotein Gene Expression by the Staphylococcus aureus cvfB Gene . Infect Immun 75 : 1964 – 1972 . OpenUrl Abstract / FREE Full Text 71. ↵ Tsuchiya K , Hara H , Kawamura I , Nomura T , Yamamoto T , Daim S , Dewamitta SR , Shen Y , Fang R , Mitsuyama M . 2010 . Involvement of Absent in Melanoma 2 in Inflammasome Activation in Macrophages Infected with Listeria monocytogenes . The Journal of Immunology 185 : 1186 – 1195 . OpenUrl CrossRef PubMed 72. ↵ Lake FB , Van Overbeek LS , Baars JJP , Koomen J , Abee T , Den Besten HMW . 2021 . Genomic characteristics of Listeria monocytogenes isolated during mushroom (Agaricus bisporus) production and processing . International Journal of Food Microbiology 360 : 109438 . OpenUrl CrossRef PubMed 73. ↵ Kadurugamuwa JL , Sin L , Albert E , Yu J , Francis K , DeBoer M , Rubin M , Bellinger-Kawahara C , Parr , TR , Contag PR . 2003 . Direct Continuous Method for Monitoring Biofilm Infection in a Mouse Model . Infection and Immunity 71 : 882 – 890 . OpenUrl Abstract / FREE Full Text 74. ↵ Kaito C , Hirano T , Omae Y , Sekimizu K . 2011 . Digestion of extracellular DNA is required for giant colony formation of Staphylococcus aureus . Microbial Pathogenesis 51 : 142 – 148 . OpenUrl CrossRef PubMed 75. ↵ Uneme M , Ishikawa K , Furuta K , Yamashita A , Kaito C . 2024 . Overexpression of the flagellar motor protein MotB sensitizes Bacillus subtilis to aminoglycosides in a motility-independent manner . PLOS ONE 19 : e0300634 . OpenUrl CrossRef PubMed 76. ↵ Venables WN , Ripley BD . 2002 . Modern Applied Statistics with S. Springer, New York, NY . http://link.springer.com/10.1007/978-0-387-21706-2 . Retrieved 23 January 2025. 77. ↵ Novick R . 1967 . Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus . Virology 33 : 155 – 166 . OpenUrl CrossRef PubMed Web of Science 78. ↵ Holloway BW . 1955 . Genetic Recombination in Pseudomonas aeruginosa . Microbiology 13 : 572 – 581 . OpenUrl CrossRef PubMed Web of Science 79. ↵ Mitsuyama M , Takeya K , Nomoto K , Shimotori S . 1978 . Three Phases of Phagocyte Contribution to Resistance against Listeria monocytogenes . Microbiology 106 : 165 – 171 . OpenUrl CrossRef PubMed Web of Science 80. Yamagishi J , Kojima T , Oyamada Y , Fujimoto K , Hattori H , Nakamura S , Inoue M . 1996 . Alterations in the DNA topoisomerase IV grlA gene responsible for quinolone resistance in Staphylococcus aureus . Antimicrob Agents Chemother 40 : 1157 – 1163 . OpenUrl Abstract / FREE Full Text 81. Hara H , Kawamura I , Nomura T , Tominaga T , Tsuchiya K , Mitsuyama M . 2007 . Cytolysin-Dependent Escape of the Bacterium from the Phagosome Is Required but Not Sufficient for Induction of the Th1 Immune Response against Listeria monocytogenes Infection: Distinct Role of Listeriolysin O Determined by Cytolysin Gene Replacement . Infection and Immunity 75 : 3791 – 3801 . OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted March 11, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Xenopus laevis as an infection model for human pathogenic bacteria Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Xenopus laevis as an infection model for human pathogenic bacteria Ayano Kuriu , Kazuya Ishikawa , Kohsuke Tsuchiya , Kazuyuki Furuta , Chikara Kaito bioRxiv 2025.03.10.642459; doi: https://doi.org/10.1101/2025.03.10.642459 Share This Article: Copy Citation Tools Xenopus laevis as an infection model for human pathogenic bacteria Ayano Kuriu , Kazuya Ishikawa , Kohsuke Tsuchiya , Kazuyuki Furuta , Chikara Kaito bioRxiv 2025.03.10.642459; doi: https://doi.org/10.1101/2025.03.10.642459 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42003) Biophysics (21470) Cancer Biology (18624) Cell Biology (25533) Clinical Trials (138) Developmental Biology (13390) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40432) Molecular Biology (17200) Neuroscience (88681) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4828) Physiology (7653) Plant Biology (15161) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9826) Zoology (2271)