Evaluation of Presurgical Skin Preparation Agents in African Clawed Frogs (Xenopus Laevis)

Evaluation of Presurgical Skin Preparation Agents in African Clawed Frogs (Xenopus laevis)

Blythe H Philips,1,2 Marcus J Crim,4,5 F Claire Hankenson,6 Earl K Steffen,4 Peter S Klein,3 Angela K Brice,1,2 and Anthony J Carty1,*

Despite the routine collection of oocytes from African clawed frogs (Xenopus laevis) for use in research, few studies have evaluated methods for preparing their skin for surgery. We evaluated 3 skin preparatory agents by examining their antibac- terial efficacy and the gross and microscopic appearance ofXenopus skin after exposure. Frogs (n = 14) were sedated and treated (contact time, 10 min) with 0.9% sterile NaCl on one-half of the ventrum and with 0.5% povidone–iodine or 0.75% chlorhexidine on the other half. Bacterial cultures were obtained before and after skin treatment; bacteria were identified by mass spectrometry. To assess inflammation and degenerative changes, the incision sites were photographed and biopsied at 0, 1, and 7 d after surgery. We isolated at least 22 genera of bacteria from the skin of our frog population (mean ± SE, 5.21 ± 0.82 genera per frog). Iodine (2.00 ± 0.44 genera) and chlorhexidine (0.29 ± 0.76 genera) both had greater antimicrobial activity than did saline. Skin erythema did not correlate with treatment group. Histologic evidence of epidermal degeneration and necrosis was greater on days 1 and 7 after chlorhexidine treatment than after iodine or saline. In addition, frogs treated with chlorhexidine had a higher incidence of clinical illness associated with the exposure site. In summary, although chlorhexidine has adequate antimicrobial activity against organisms on X. laevis skin, it leads to skin damage and subsequent clinical complications. We therefore do not recommend chlorhexidine as a preoperative preparation agent in Xenopus.

Abbreviations: MALDI–TOF MS, matrix-assisted laser desorption–ionization time-of-flight mass spectrometry

Female African clawed frogs (Xenopus laevis) are commonly might improve the quality of oocytes and postoperative health used in biomedical research, largely due to the abundance and of the donor frog.18 Citing amphibians’ own robust natural de- accessibility of their large oocytes which are used to study fenses, copious flushing with physiologic saline is occasionally various aspects of development, physiology, toxicology, and offered as an alternative to application of an antiseptic.22 Lastly, molecular biology.7,38 Many of these studies require the surgi- although differences among antiseptics—including effects on cal collection of early-stage oocytes.38,45 Despite the frequency wound healing and the potential for surgical site infections— of this procedure at many institutions, little information in the are recognized in mammals,14,26,33,42 comparatively little is literature describes the optimal method by which to cleanse known about this topic as it regards amphibians. Because frog skin prior to surgery for oocyte harvest. amphibian skin is well documented to heal very differently Amphibian skin is highly biologically active and is involved than does mammalian skin,1,19 additional investigation in this in osmoregulation, thermoregulation, reproduction, respiration, area is needed. and protection from predators and microbes.10,38,55 Because frog Among the sources that recommend presurgical skin antisep- skin performs so many critical homeostatic functions, mechani- tic use in amphibians, the agents most commonly suggested for cal or chemical damage of the skin (for example, as might be use in Xenopus spp. are dilute chlorhexidine and dilute povi- sustained by presurgical antiseptic application) is more likely done–iodine.21,38 However, cases of presumed chlorhexidine13,49 to result in systemic consequences than are exposures of a and iodine47 toxicity in amphibians have been reported, and similar nature in mammals.10,40 Furthermore, the antimicrobial other sources have argued against the use of these agents for peptides, natural microbiota, and mucus layer produced by that reason.21,22,24,40,49,56 One study comparing topical povi- amphibian skin provide essential endogenous defenses against done–iodine with 3 other topical antiseptic agents (ethacridine bacterial and fungal invasion.25,31,44,51 For these reasons, the lactate, potassium permanganate, and benzalkonium chloride) veterinary community has debated whether application of in X. laevis found that iodine was well-tolerated and was the presurgical antiseptic preparations is necessary or potentially only agent that provided good antimicrobial activity without harmful for these species.22 Moreover, some evidence suggests inciting an obvious inflammatory response.18 No similar study that inadequate preparation of the surgical site negatively affects has been performed to evaluate topical chlorhexidine, and the the quality of Xenopus oocytes retrieved by means of surgical question of whether eliminating bacterial microbiota at the harvest and that treatment of the skin with a topical antiseptic surgical site is possible or even necessary in frogs remains unresolved.22 Received: 15 Oct 2014. Revision requested: 03 Dec 2014. Accepted: 10 Feb 2015. We evaluated 3 commonly used presurgical skin preparatory 1University Laboratory Animal Resources, 2Department of Pathobiology, School of Vet- agents (0.75% chlorhexidine, 0.5% povidone–iodine, and 0.9% erinary Medicine, and 3Department of Cell and Molecular Biology, School of Medicine, sterile NaCl) to determine which, if any, maximally decreased University of Pennsylvania, Philadelphia, Pennsylvania; 4IDEXX BioResearch, Columbia, Missouri; 5Department of Veterinary Pathobiology, College of Veterinary Medicine, Uni- the bacterial load at the site of surgery in X. laevis. Concurrently, versity of Missouri, Columbia, Missouri; and 6University Laboratory Animal Resources, we recorded gross, microscopic, and clinical observations to Michigan State University, East Lansing, Michigan. investigate whether treatment with these agents resulted in *Corresponding author. Email: [email protected]

788 Skin preparation agents in Xenopus laevis

unwanted tissue effects, including opportunistic infection, were anesthetized with tricaine methanesulfonate (MS222, 1 g/L inflammation, and epidermal degeneration, any or all of which buffered with 1 g/L NaHCO3; Argent Chemical Laboratories, would contraindicate the use of that product in this species. Redmond, WA) by immersion.30 After loss of righting reflex, Whereas the bacterial pathogens of rodents are well char- frogs were placed on a moist, clean paper towel in dorsal re- acterized and generally readily identifiable in laboratories, cumbency. The skin of the frog’s caudal ventrum was swabbed the pathogens of aquatic animal models are relatively poorly with a sterile culturette (BBL CultureSwab Plus, Becton Dick- characterized, do not necessarily grow under standard culture inson, Sparks, MD) to establish baseline bacterial microbiota. conditions, and often pose considerable problems in terms of A ruler was placed along midline to divide the frog’s ventrum identification according to conventional microbiologic tech- into 2 halves and was left on the frog as a visual guide for the niques. Because X. laevis are ectotherms that are typically housed remainder of the procedure. One half of the ventrum was rinsed at approximately 21 to 22 °C,22 some bacterial species that colo- with a steady stream of 0.9% sodium chloride (n = 14; Baxter, nize Xenopus skin grow poorly or not at all at 37 °C. Therefore, Deerfield, IL) from a 20-mL syringe through an 18-gauge needle bacterial culture for amphibians must often be performed at a for approximately 5 s, and the contralateral side was rinsed decreased temperature and for a prolonged period of time,8 thus similarly with a steady stream of either 0.75% chlorhexidine complicating or delaying organism identification in laboratories gluconate solution (n = 7, Phoenix, St. Joseph, MO) or 0.5% povi- optimized for identifying mammalian pathogens. In addition, done–iodine solution (n = 7, Aplicare, Meriden, CT), with care variability in biochemical metabolic test results among different to prevent the solution from crossing midline. The saline-rinsed strains or isolates of diverse or poorly characterized bacterial side was cultured after following rinsing by swabbing the center taxa substantially complicates definitive bacterial identification of the treated area with another sterile culturette. After 10 min by using traditional biochemical tests. of contact time, the antiseptic (chlorhexidine or iodine)-treated Matrix-assisted laser desorption–ionization time-of-flight side of the frog was rinsed with sterile saline and was cultured mass spectrometry (MALDI–TOF MS) is a state-of-the-art15 as described. The antiseptic treatment choice and side of the spectrophometric technique that identifies bacteria according ventrum to be treated were randomized by frog. Culturettes to unique molecular fingerprints of 2- to 20-kDa proteins, which were placed in Amies transport medium (BBL CultureSwab are highly abundant in bacterial cells. This technique is being Plus, Becton Dickinson, Sparks, MD) without charcoal and were used increasingly for bacterial identification in basic research submitted to IDEXX BioResearch (Columbia, MO) for aerobic and human medical diagnostic applications,32 has recently culture and identification. emerged as a diagnostic platform for laboratory animals, and Bacterial culture and identification. Culture swabs were can improve the precision and rapidity with which laboratories aseptically removed from the transport medium, swab heads distinguish organisms that have been historically difficult to were removed from the shafts by using sterile scissors, placed identify.2 MALDI–TOF MS has been used to study antimicrobial into sterile 2-mL microcentrifuge tubes containing 200 μL of compounds produced by frog skin41,54 and for identification sterile PBS, and then vortexed. A 100-μL of this solution was of pathogenic bacteria in ornamental fish35 and aquaculture subsequently plated onto BBL Trypticase Soy Agar with 5% systems,48 but to our knowledge the use of MALDI–TOF MS sheep blood (TSA II; Becton Dickinson). The contents of a to evaluate the microbiota of frogs used in biomedical research sterile disposable calibrated (10 μL) inoculating loop (Becton has not previously been reported. Although the pathogens of Dickinson) from each sample were streaked onto a second aquatic species are underrepresented in the available spectral plate of BBL Trypticase Soy Agar with 5% sheep blood (TSA II). databases, spectra are available for aquatic bacteria that are Cultures were incubated at 26 °C. Colonies of each colony type known to be human pathogens17,28 or that are important agents were counted for each swab (or estimated when there were more linked to seafood spoilage.3-6 than 50 colonies of a colony type), and isolates were categorized as exhibiting light, moderate, or heavy growth according to the number of colonies for each colony type. Total colony counts Materials and Methods (in cfu) were estimated by adding the number of colonies of all Animals. All procedures performed for this study were ap- colony types from each swab and reflect the number of cfu per proved by the IACUC of the University of Pennsylvania and 100 μL or, when the colonies were too numerous to count on were carried-out within an AAALAC-accredited facility under the initial plate, 10× the number of cfu per 10 μL. controlled temperature and humidity with a 12:12-h light:dark One or more representative colonies of each colony type was cycle. Naïve, healthy, adult, purpose-bred female X. laevis (n = selected for proteomic analysis by MALDI–TOF by using a 16, Nasco, Fort Atkinson, WI), were maintained at a density of mass spectrometer (Microflex, Bruker Daltronics, Billerica, MA) no more than 1 frog per 4 L of water in an automated 400-gal with flexControl software (Bruker Daltronics). Briefly, a sterile continuous flow recirculating system with 10% water changes wooden toothpick was used to harvest a small bacterial sample daily (Pentair Aquatic Habitats, Apopka, FL). Input water was from the apex of the colony, corresponding to approximately 106 filtered through a water purification system (Type 2 Elix, Merck bacteria; the bacterial sample was distributed directly within Millipore, Darmstadt, Germany), adjusted to 1600 μS with an etched circle (diameter, 3 mm) on a stainless steel target marine salt, and maintained at pH 7.4 and a temperature of 18 with a 96-spot configuration (Bruker Daltronics). Each spot to 20 °C. Conductivity, pH, and temperature were monitored on the target was overlaid with 1 μL of HCCA matrix (Bruker continuously and adjusted automatically to maintain these Daltronics), a saturated solution of α-cyano-4-hydroxycinnamic parameters. Water underwent mechanical filtration, charcoal acid in 50% acetonitrile, 2.5% trifluoroacetic acid, and allowed filtration, UV irradiation, and biofiltration. Frogs were fed to air dry at room temperature. The target was placed in the exclusively a standard diet of Nasco frog brittle twice weekly spectrometer under vacuum, and each spot was excited in (Nasco, Fort Atkinson, WI). sequence by an automated series of laser shots; this process Antimicrobial efficacy. To evaluate the antimicrobial efficacy gently ionized and desorbed microbial proteins, which migrate of 0.75% chlorhexidine and 0.5% povidone–iodine as compared a fixed distance in an electric field to a charged detector. The with 0.9% saline, frogs (n = 14) were fasted for 24 h and then TOF of microbial proteins to the detector is a direct function of

789 Vol 54, No 6 Journal of the American Association for Laboratory Animal Science November 2015 the mass:charge ratio (m/z) of each protein.11 For each bacte- on the contralateral side) or 7 d (n = 6, 3 frogs treated with rial sample, as many as or more than 100 peaks are detected in chlorhexidine and 3 with iodine; all frogs were treated with the predetermined range of maximal discrimination with an saline as a control on the contralateral side) after incision. In m/z between 2000 and 20,000 Da, which together comprise addition, skin biopsies were harvested, one from each side of a spectrum that serves as a molecular fingerprint of the most the ventrum, from 2 frogs that had not undergone prior skin abundant proteins for each bacterial sample (Figure 1). To ob- incisions and were treated only with a 0.9% sterile saline rinse, tain genus- and species-level identification, automated analysis to control for tissue-level effects due to the incision itself (4 by MALDI BioTyper software (Bruker Daltronics) compared untreated, unincised biopsies total). the spectra for each isolate to an integrated reference database Skin biopsy procedure. For each frog, gentle traction was ap- of spectra that includes 6137 bacterial strains representing 2290 plied to the previously placed nylon sutures, and a sterile size bacterial species. A logarithmic score from 0 to 3 is generated 15 scalpel blade was used to make a 1- to 2-mm incision in the based on matching the peaks of each database spectrum to the skin adjacent to the prior incision. Iris scissors were introduced unknown spectrum, matching the peaks of the unknown spec- into this space and were used to gently excise a 0.7 × 0.5 cm oval trum to the database spectrum, and the correlation of matched of skin, which encompassed the previous surgical site. Hemo- peak intensities. stasis was achieved by applying gentle manual pressure to the Gross skin reaction. To evaluate whether application of each incision site. After biopsy, skin was closed with 3-0 nylon50 and preparatory agent was associated with a gross skin reaction bupivacaine was applied, as previously described.34,37 Frogs or adverse effects postsurgically, we took photographs of the were recovered in shallow, fresh, system water before they were treated sites for gross scoring at various stages of the study. returned to their home tanks and were monitored daily for 5 d Each frog was photographed 1) immediately after loss of the after biopsy for adverse events or illness. righting reflex (that is, prior to the application of any agent; n Tissue samples were fixed in 10% neutral buffered formalin = 14), 2) immediately after the creation of a 0.5-cm skin incision (Richard-Allan Scientific, San Diego, CA), processed, embedded, (described following; n = 14), and 3) at either 24 h (n = 8, 4 frogs sectioned, and stained with hematoxylin and eosin for micro- treated with chlorhexidine and 4 with iodine) or 7 d (n = 6, 3 scopic evaluation. Samples were randomized and evaluated frogs treated with chlorhexidine and 3 with iodine) after skin by a board-certified veterinary pathologist (AKB), blinded to incision. Photographs were taken from approximately 30 cm experimental treatments, for evidence of tissue reaction, such above the frog by using a camera (PowerShot SD450, Canon, as inflammation, including inflammatory infiltrate, fibrin, and Tokyo, Japan) held perpendicular to the frog’s ventrum as po- edema, and epidermal degeneration and necrosis. Each sam- sitioned on the procedure table. ple was assigned a score for lesion severity from 0 (absent to Skin incision procedure. Frogs were anesthetized and treated minimal) to 3 (severe) for each category (Figure 3). After these with saline and either 0.75% chlorhexidine solution or 0.5% described surgeries, the experimental plan was for all frogs to povidone–iodine solution as described. After the contact times recover and remain in the colony for potential enrollment in a listed previously and after confirming by using firm pinch of a nonsurgical protocol. toe on the right hindlimb that the frog was at a surgical plane of Statistical analysis. Differences between time points and treat- anesthesia, a 0.5-cm skin incision was made on either side of the ment groups were evaluated by using a generalized linear model frog’s ventrum by a surgeon (BHP) wearing powder-free latex that accounted for the correlation of repeated measures over gloves and using a size 15 scalpel blade and aseptic technique.21 time and at 2 sides of the same frog by using the generalized Hemostasis was achieved by applying gentle manual pressure estimating equations. Data are reported as means with standard to the surgical sites, and the incisions were closed with 3 simple error. Two-sided P values of less than 0.05 were considered to be interrupted 3-0 nylon sutures.50 The incision sites were photo- statistically significant. All statistical analyses were performed graphed as described. Bupivacaine (1 mg/kg; 0.25%, Hospira, by using SAS (version 9.3, SAS Institute, Cary, NC). Lake Forest, IL) was applied to the tip of a sterile cotton-tipped applicator, which was used to swab the incision site to provide 34,37 Results postoperative analgesia; the frog was returned to a shallow Antimicrobial efficacy. At least 38 bacterial isolates represent- dish containing fresh system water for recovery prior to its re- ing 22 different genera were cultured from the skin of the frogs turn to the home tank. Frogs were monitored daily for 5 d after in this study (Table 1); 26 isolates were gram-negative, and 9 surgery for signs of adverse reactions or illness, including poor (Enterococcus malodoratus, 4 Micrococcus isolates, 3 Rhodococcus appetite, skin reaction, excessive mucus production, buoyancy isolates, and an unidentified organism) were gram-positive. abnormalities, and dysecdysis. Medical treatment decisions Five of the gram-negative genera identified in the current study were made in conjunction with the facility veterinarian, and (Aeromonas, Pseudomonas, Citrobacter, Chryseobacterium, and Fla- exclusion criteria were determined such that if frogs did not vobacterium) have been implicated as opportunistic pathogens in respond to treatment, they were euthanized. cases of dermatosepticemia in amphibians.16,29,43 The estimated 2 Photographs were cropped to display an approximately 2-cm total number of colonies from iodine- or chlorhexidine-treated area around each incision and were paired with the baseline skin was lower than that for the paired pretreatment skin swab image for each frog. Photograph order was randomized, and for all frogs. The number of colonies recovered from iodine- photographs were evaluated by a scorer blinded to treatment treated skin swabs was, on average, 91% lower (n = 7) than that (FCH), who scored each incision for erythema (from 0 [none] for paired pretreatment swabs. Similarly, the total number of to 3 [severe]) relative to baseline (Figure 2). colonies from chlorhexidine-treated skin swabs was an average Microscopic skin reaction. To evaluate frogs for possible mi- of 98% lower (n = 7) than the total number of CFU from the croscopic skin reactions or infections associated with treatment paired pretreatment swabs. For 13 of 14 frogs, the total number with these agents, frogs that had received skin incisions again of colonies recovered from the swab of saline-treated skin was were anesthetized by MS222 immersion to undergo surgical lower than the total number of colonies from the pretreatment site biopsies at 24 h (n = 8, 4 frogs treated with chlorhexidine swab from the same frog, averaging 70% fewer colonies across and 4 with iodine; all frogs were treated with saline as a control all frogs (n = 14).

790 Skin preparation agents in Xenopus laevis

Figure 1. Representative MALDI–TOF spectra of bacteria isolated from frog 1 in this study. (A) Aeromonas sp. (B) Chryseobacterium sp. (C) Cit- robacter sp. (D) Flavobacterium sp. (E) Microbacterium phyllospaera. (F) Microbacterium sp. (G) Micrococcus luteus. (H) Shewanella putrefaciens. (I) Unidentified gram-negative species. (J) Unidentified gram-positive species.

Overall, frogs had the greatest number of bacterial genera on Citrobacter freundii and Chryseobacterium sp. were both their skin at baseline (5.21± 0.82; Figure 4) and treatment with cultured from most frogs prior to treatment (9 of 14 frogs for saline was not associated with a significant reduction in bacte- Citrobacter freundii and 10 of 14 frogs for Chryseobacterium sp.), rial genera (4.21 ± 0.41); fewer genera were isolated from frogs and neither was recoverable after treatment with either iodine treated with 0.5% povidone–iodine (2.00± 0.44, P = 0.0001) or or chlorhexidine. Flavobacterium sp. was cultured from 4 of 14 0.75% chlorhexidine solution (0.29 ± 0.76, P < 0.0001). Iodine- frogs at baseline; 3 of these frogs were in the chlorhexidine treated frogs had fewer genera isolated from their skin than group, and 1 was in the iodine group. In addition, 1 of the 3 did saline-treated frogs (P = 0.0002). Chlorhexidine-treated chlorhexidine-treated frogs that grew Flavobacterium at baseline frogs had fewer genera isolated from their skin than did saline- still had Flavobacterium present after treatment. Even though treated frogs (P < 0.0001) and povidone–iodine-treated frogs (P Flavobacterium was cultured from only 1 frog in the iodine group = 0.0004). After treatment with chlorhexidine, one frog still had at baseline, it was cultured from 3 frogs after treatment with identifiable bacteria present at the site of culture, whereas only povidone–iodine. Aeromonas sp. was isolated from 9 of the 14 one frog did not have identifiable bacteria after treatment with frogs at baseline and was still present on 2 of 4 frogs treated with iodine (Figure 5). iodine and 1 of 5 frogs treated with chlorhexidine. Pseudomonas

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Figure 2. Images used to score frogs for gross erythema after surgery. Photographs were cropped to display an approximately 2-cm2 area around each incision and were paired with the baseline image for each frog. Photographs were evaluated by an observer who was blinded to treatment group and who scored each incision from 0 (none) to 3 (severe) for erythema relative to baseline. (A) Skin of frog 2 at 1 day after surgery, score of 0 (right side, treated with saline). (B) Skin of frog 10 immediately after surgery, score of 1 (right side, treated with chlorhexidine). (C) Skin of frog 9 at 7 d after surgery, score of 2 (right side, treated with chlorhexidine). (D) Skin of frog 2 immediately after surgery, score of 3 (left side, treated with chlorhexidine). sp. was isolated from 11 of the 14 frogs at baseline; 2 of 6 frogs surgery (0.86 ± 0.34) than did saline-treated frogs (P = 0.005) or were still positive for Pseudomonas after treatment with iodine, chlorhexidine-treated frogs (P = 0.01) at that time point. There whereas no Pseudomonas was isolated from the 5 frogs treated were no significant differences between treatment groups at 1 with chlorhexidine. or 7 d after surgery. Gross skin reaction. Saline-treated frogs had higher gross Microscopic skin reaction. Histologic inflammation score. erythema skin scores immediately after surgery (1.71 ± 0.22) Saline-treated frogs had higher histologic inflammation scores and at 7 d after surgery (2.17 ± 0.31) than they did on day 1 on day 7 relative to day 1 after surgery (2.33 ± 0.11 compared after surgery (0.88 ± 0.23; P = 0.01 and P = 0.0002, respectively) with 1.31 ± 0.16, respectively; P = 0.002; Figure 7 A . Similarly, Figure 6 . Chlorhexidine-treated frogs had higher gross skin iodine-treated frogs had higher histologic inflammation scores erythema scores immediately following surgery than they did 1 d on day 7 relative to day 1 (2.67 ± 0.17 compared with 1.38 ± 0.24, after surgery (1.86 ± 0.26 and 0.75 ± 0.25, respectively; P = 0.008). P = 0.02). Iodine-treated frogs had higher histologic inflamma- There were no significant differences between gross erythema tion scores on day 7 than did saline-treated frogs (P = 0.046). scores in iodine-treated frogs between time points. Iodine- Chlorhexidine-treated frogs had higher histologic inflamma- treated frogs had lower gross erythema scores immediately after tion scores on day 1 (1.88 ± 0.38) than did saline-treated frogs

792 Skin preparation agents in Xenopus laevis

higher epidermal degeneration and necrosis scores on day 1 following surgery than did biopsies from saline-rinsed, unincised control frogs (P < 0.0001). Biopsies from chlorhexidine-treated frogs that underwent skin incisions had higher epidermal degeneration and necrosis scores at both day 1 (3.00 ± 0.00) and day 7 (2.00 ± 0.00) when compared with saline-rinsed, unincised control frogs (1.25 ± 0.25; P < 0.0001 and P = 0.0005, respectively). Chlorhexidine-treated frogs had higher epidermal degeneration and necrosis scores on day 1 (P = 0.005) and day 7 (P = 0.01) than saline-treated frogs at both time points. Chlorhexidine-treated frogs had higher epidermal degeneration and necrosis scores on day 1 (P = 0.005) and day 7 (P = 0.01) when compared with iodine-treated frogs at day 1 (2.00 ± 0.41) and day 7 (0.33 ± 0.33). Clinical observations. All frogs recovered uneventfully for the first few days after surgery, and the majority of the frogs remained healthy throughout the experimental protocol. How- ever, 3 of the 7 chlorhexidine-treated frogs developed unilateral erythematous flank lesions approximately 3 to 4 d after surgery. Skin lesions were not associated with the location of the surgical incision, but all 3 cases developed on the side of the frog that had been treated with chlorhexidine. Of the 3 frogs, one had a mild lesion that resolved without intervention, and another had a moderate ulcerated skin lesion that healed with 10 d of daily baths in 100 mM marine salts (Figure 8 A). The third frog had a large, erythematous skin lesion that encompassed the majority of the left flank. Skin cytology from the day that the lesion was identified revealed numerous bacterial rods but no fungi; a superficial skin scrape showed no evidence of cutaneous nematodes. The frog was started on enrofloxacin (10 mg/kg topically daily) and daily baths in 100 mM marine salts, but the lesion progressed. At 2 d after the start of treatment, the area surrounding the erythematous skin became devitalized, and the frog was euthanized (Figure 8 B). A necropsy was performed. Heart blood culture grew Aeromonas hydrophila. Microscopic examination of the skin showed multi- focal, extensive erosions and ulcers with abundant superficial gram-negative bacilli adhered to the ulcerated surface (Figure 9 A and B). Areas of full-thickness necrosis occasionally contained clusters of superficial fungal hyphae. Within the kidneys, many glomerular capillaries contained uniform, eosinophilic hyaline material. This material stained dark blue with phosphotungstic acid hematoxylin, indicative of microthrombi within the glomerular capillaries (Figure 9 C and D).

Figure 3. Representative skin biopsies from Xenopus laevis for micro- Discussion scopic skin reaction scoring. Hematoxylin and eosin stain. (A) Biopsy The appropriate technique with which to prepare amphib- from a control frog (no skin incision, rinsed with saline). Inflammation score: 0 (none); epidermal degeneration and necrosis score: 1 (mild). ian skin for surgery and whether presurgical antiseptic use is 18,21,22,24,38,40 Magnification, 10×. (B) Biopsy from frog 9, at the site treated with necessary remain a debate in the literature. The goal chlorhexidine on the right ventrum. Inflammation score: 3 (severe). of the current study was to evaluate 2 of the most commonly Magnification, 10×. (C) Biopsy from frog 10 at the site treated with used presurgical skin antiseptics, 0.75% chlorhexidine and 0.5% chlorhexidine on the left ventrum. Epidermal degeneration and necro- povidone–iodine compared with 0.9% sterile saline, for safety sis score: 3 (severe). Magnification, 10× (inset, 40×). and efficacy in X. laevis. Through identification with MALDI– TOF MS, at least 38 different bacterial isolates, representing 22 (P = 0.02). All biopsies from frogs that underwent skin incisions genera, were obtained from the skin of the frogs used in this had higher histologic inflammation scores at both time points study. Of these, 5—Aeromonas, Chryseobacterium, Citrobacter, and with all treatments than biopsies from saline-rinsed, un- Flavobacterium, and Pseudomonas—were genera that have incised control frogs (P < 0.0001 for saline, chlorhexidine, and historically been associated with potential for clinical disease, iodine on day 7; P < 0.0001 for saline and chlorhexidine on day specifically dermatosepticemia, in amphibians.16,23,29,36,39,43 Most 1; P = 0.0002 for iodine on day 1). of the isolates were gram-negative rods and therefore consistent Histologic epidermal degeneration and necrosis score. Saline- with much of what is known about the normal microbiota of treated frogs had higher epidermal degeneration and necrosis amphibians and their environment.16,29 scores on day 1 relative to day 7 (2.50 ± 0.19 compared with For this study, we considered the number of bacterial genera 0.50 ± 0.22; P = 0.01; Figure 7 B). Biopsies from saline- and isolated from each frog in addition to the estimated total colony chlorhexidine-treated frogs that underwent skin incisions had count from each frog before and after treatment to address the

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Table 1. Bacteria isolated from Xenopus laevis skin by using aerobic culture techniques and identified by using MALDI–TOF mass spectrometry % of frogs positive for this Genus organism Species identified in this genus Gram-negative organisms Aeromonasa 92.9 Unidentified spp. Pseudomonasa 92.9 P. koreensis, P. xanthomarina, unidentified spp. Citrobactera 85.7 C. freundii, unidentified sp. Chryseobacteriuma 78.6 Unidentified sp(p). Flavobacteriuma 64.3 Unidentified sp(p). Shewanella 57.1 S. putrefaciens, unidentified sp. Psychrobacter 50.0 Unidentified sp(p). Acinetobacter 42.9 A. tjernbergiae, unidentified sp(p). Fluviicola 14.3 Unidentified sp(p). Hydrogenophaga 14.3 H. palleroni Stenotrophomonas 14.3 Unidentified sp(p). Acidovorax 7.1 A. temperans Bosea 7.1 Unidentified sp. Delftia 7.1 D. acidovorans Erythrobacter 7.1 E. citreus, E. vulgaris Pseudoxanthomonas 7.1 P. mexicana, P. japonensis, P. ginsengisoli Rhizobium 7.1 R. radiobacter Sphingobacterium 7.1 S. faecium Unidentified gram-negative organisms 71.4 Gram-positive organisms Rhodococcus 21.4 R. erythropolis, R. fascians, unidentified sp. Microbacterium 14.3 M. phyllosphaerae, M. schleferi/M. lacticum Enterococcus 7.1 E. malodoratus Micrococcus 7.1 M. luteus Unidentified gram-positive organisms 21.4 sp., one isolate of the genus was identified; spp., multiple isolated of the genus were obtained; sp(p), whether one or multiple isolates were obtained could not be determined definitively aOrganism has been implicated in the pathogenesis of dermatosepticemia in amphibians.

question of antimicrobial efficacy. Overall, 0.75% chlorhexidine gluconate solution had superior antimicrobial activity in comparison with 0.5% povidone–iodine and 0.9% NaCl. After treatment with chlorhexidine for 10 min of contact time, only 1 of 7 frogs still had detectable bacterial growth at the surgical site. However, chlorhexidine treatment was often associated with clinical illness, specifically skin erythema, and, in one case, dermatosepticemia. In contrast, only 1 of the 7 iodine-treated frogs became free of detectable viable bacteria. There was no statistical difference between the number of genera isolated from sterile saline-treated frogs and their baseline values. Figure 4. Mean number of bacterial genera isolated from frogs at base- The bacterial genera that were isolated and that are as- line (Pre; n = 14) and after topical treatment with 0.9% sterile saline (n sociated with dermatosepticemia in amphibians (Aeromonas, = 14), 0.5% povidone–iodine (Iodine; n = 7), or 0.75% chlorhexidine solution (Chlorhex; n = 7). Error bars represent standard error. Chryseobacterium, Citrobacter, Flavobacterium, and Pseudomonas) are generally considered opportunistic organisms, as they can be found on healthy frogs as well as ill animals. When disease treated with iodine and 20% of those treated with chlorhexidine. occurs, it is usually accompanied by stress, injury, immunosup- Similarly Pseudomonas spp. were isolated from the majority of 16,23,29,36,39,43 pression, or disruption of the mucus layer. In our frogs before antiseptic treatment; these organisms were still study, these opportunistic bacteria had variable susceptibility present after treatment with iodine, whereas they were consist- patterns regarding the antiseptics we evaluated. Both Citrobac- ently eliminated after treatment with chlorhexidine. This result ter freundii and Chryseobacterium sp. were cultured from most suggests that these Pseudomonas species were more susceptible frogs at baseline, but neither was recovered after treatment with to chlorhexidine relative to iodine at the concentrations and either iodine or chlorhexidine, suggesting that these 2 organ- contact times that we used in this study. In contrast, Flavobac- isms are highly susceptible to both antiseptics. Aeromonas sp. terium sp. was cultured from 4 frogs at baseline and remained was variably susceptible to both agents; it was isolated from after antiseptic treatment in all of these animals (3 were treated most frogs at baseline and was still present in half of the frogs with iodine; 1 was treated with chlorhexidine), suggesting that

794 Skin preparation agents in Xenopus laevis

Figure 5. Number of bacterial isolates identified from each frog at baseline and immediately after treatment (10 min contact time) with (A) 0.9% sterile saline or 0.5% povidone–iodine or (B) 0.9% sterile saline or 0.75% chlorhexidine.

Figure 6. Mean gross erythema scores for frogs treated topically with 0.9% sterile saline (day 0, n = 14; day 1, n = 8; day 7, n = 6), 0.5% povidone–iodine (Iodine; day 0, n = 7; day 1, n = 4; day 7, n = 3), and 0.75% chlorhexidine solution (Chlorhex; day 0, n = 7; day 1, n = 4; day 7, n = 3). Scores were assigned as follows: 0, no erythema; 1, mild; 2, moderate; and 3, severe. Error bars represent standard error.

Flavobacterium is fairly resistant to the antiseptics that were used. Although we did not identify the species of Flavobacterium that we isolated, this observation is consistent with reports in the literature that prolonged exposure to povidone–iodine is not consistently effective at eliminating surface Flavobacterium sp. Figure 7. (A) Histologic inflammation scores for skin biopsies from in salmonid eggs,27,46 and species of Flavobacterium have been frogs treated with 0.9% sterile saline (day 1, n = 8; day 7, n = 6), 0.5% found to grow in chlorhexidine solutions in hospitals.52 povidone–iodine (day 1, n = 4; day 7, n = 3), or 0.75% chlorhexidine solution (day 1, n = 4; day 7, n = 3) as compared with saline-rinsed Interestingly, both Aeromonas and Flavobacterium were isolated frogs that had not undergone surgery (control; n = 4). (B) Histologic multiple times after antiseptic treatment in instances where epidermal degeneration and necrosis scores for skin biopsies from they had not been recovered pretreatment. This result perhaps frogs treated with 0.9% sterile saline (day 1, n = 8; day 7, n = 6), 0.5% reflects differences in the bacterial microenvironment on the povidone–iodine (day 1, n = 4; day 7, n = 3), or 0.75% chlorhexidine so- frogs’ skin. Although all cultures were taken from a 2-cm2 lution (day 1, n = 4; day 7, n = 3) as compared with saline-rinsed frogs that had not undergone surgery (n = 4). For both parameters, scores region of the central ventrum of each frog, previous studies were assigned as follows: 0, none; 1, mild; 2, moderate; and 3, severe. have shown that large differences in microbial populations Error bars represent standard error. between the dorsum and ventrum of the same amphibian can be present,12 and perhaps different dermal microbial populations can cluster at various sites of the ventrum. Alternatively, the Grossly, the consistent abnormality that we observed at differences seen might reflect sampling error, given that only the surgical site during the 7 d after surgery was occasional a single swab was used on a small area of frog skin for each erythema along the incision line. In general, most frogs ex- sample. Contamination of the antiseptics used for this study perienced mild to moderate erythema after skin incision, and was highly unlikely, because fresh supplies of antiseptics and increased erythema did not reliably correlate with a particular saline were prepared daily, and all materials were handled by treatment group or day afterward. In addition, higher gross using aseptic technique. erythema scores did not correlate well with histologic evidence of skin damage. For example, the saline-treated skin of frog 3

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that were treated with chlorhexidine and that were biopsied at the 24-h postoperative time point had severe epidermal degeneration and necrosis. Chlorhexidine exposure has been associated with irritation and contact dermatitis in people, particularly infants.9,53 Perhaps the frogs in our study had a similar response to treatment with this agent. Although none of the frogs in this study developed gross or microscopic evidence of infection at their surgical sites, 3 frogs developed erythematous skin lesions along their flanks. Given that these flank lesions occurred only on chlorhexidine-treated frogs and only on the side of the body that was treated with chlorhexidine, we hypothesize that these unexpected flank lesions were associated with chlorhexidine pooling alongside the frog after the rinsing phase and as the frog lay in the surgical trough prior to incision. This situation may have resulted in unanticipated and prolonged contact time at that site, thus perhaps augmenting any potentially detrimental effects that chlorhexidine may have on the frogs’ skin. All 3 of the frogs that developed lesions had baseline cultures that were positive for Figure 8. Clinical observations of frogs on study. (A) Severe erythema organisms that are known to be opportunistic pathogens in this and mild ulceration of the right flank in frog 9, treated with 0.75% species: all 3 frogs were positive for Aeromonas sp., 1 was positive chlorhexidine on the right side of the body. (B) Severe erythema, mod- for Flavobacterium, and 2 were positive for Citrobacter, Chryseo- erate ulceration, and extensive devitalization of the skin associated bacterium, and Pseudomonas. All 3 frogs had negative cultures with the left flank in frog 13, treated with 0.75% chlorhexidine on the at the surgical site after treatment with chlorhexidine. The frog left side of the body. The blue bead sutured to the left hindlimb was used for identification. that had the most severe skin lesions and that was euthanized had a heart-blood culture positive for Aeromonas sp. This frog also had severe skin lesions and microscopic kidney changes had severe erythema (score, 3) for 1 wk after skin incision, but suggestive of glomerular microthrombi. To our knowledge, a biopsy taken at this time revealed only moderate (score, 2) glomerular microthrombosis has not previously been reported inflammation and mild (score, 1) epidermal degeneration and to occur in X. laevis, but in other species, this finding combined necrosis. In contrast, the histology of the chlorhexidine-treated with documented bacteremia is suggestive of disseminated skin of frog 10 revealed severe (score, 3) inflammation and se- intravascular coagulation secondary to sepsis.20 Additional vere (score 3) epidermal degeneration and necrosis, with only diagnostics, including clotting times and a platelet count, are mild (score, 1) gross erythema. Overall, these results indicate necessary to confirm this diagnosis, but we did not collect this that the gross appearance of the surgical site, and of erythema information, nor are reference values for these parameters well- in particular, may not be a reliable indicator of overall tissue documented in this species. We hypothesize that, for this frog, health and healing after a surgical incision in X. laevis. prolonged chlorhexidine exposure at the flank led to severe Regardless of treatment group, all frogs that underwent skin epidermal degeneration and necrosis, disruption of the frog’s incisions had higher inflammation scores when compared with natural microbiota, and potential disruption of the protective saline-rinsed, unincised control frogs. In addition, frogs in the mucus layer. This situation, combined with the potential stress saline and iodine groups had higher inflammation scores at day of anesthesia and surgery, may have led to invasion of the skin 7 relative to day 1. Inflammation is a normal part of the healing by Aeromonas, which was present on this frog’s skin at baseline, process, and these data are consistent with what is known about resulting in dermatosepticemia and disseminated intravascular skin healing in adult X. laevis, which shows that inflammatory coagulation. cells may be abundant for greater than 2 wk into the healing Overall, in light of the increased epidermal degeneration, process.1 Chlorhexidine-treated frogs had slightly higher inflam- necrosis, and clinical illness associated with topical treatment mation scores on day 1 than did saline-treated frogs, suggesting with 0.75% chlorhexidine, we do not recommend the use of this that chlorhexidine leads to a proinflammatory response on frog agent at this concentration for preoperative skin preparation in skin. The difference between treatment groups did not extend X. laevis. Both 0.5% povidone–iodine and sterile saline were well to the 7-d time point, so this proinflammatory effect likely is tolerated, although neither had antimicrobial activity equivalent significant during the only during the early postoperative pe- to that of chlorhexidine. Given the robust innate skin defenses riod. Furthermore, both of these scores (1.31 ± 0.16 for saline of Xenopus, combined with the absence of ill effects noted in and 1.88 ± 0.38 for chlorhexidine) are between mild (score, 1) animals treated with saline or dilute povidone–iodine, we sug- and moderate (score, 2), so this difference likely would not be gest that additional antimicrobials may not be necessary prior to clinically significant over time. surgery in this species. Further studies would be useful to de- There was a trend toward frogs having higher epidermal termine whether treatment with alternative antiseptic regimens, degeneration and necrosis scores after surgery on day 1 rela- such as more concentrated formulations of povidone–iodine or tive to day 7 across all 3 treatment groups. Given this finding, more dilute formulations of chlorhexidine, provide any benefit we hypothesized that frogs sustained epidermal damage to frogs and the quality of their oocytes. during surgery, but this outcome was largely resolved by a week after the procedure. However, chlorhexidine-treated Acknowledgments frogs had higher microscopic epidermal degeneration and ne- Support for this study was provided by the Office of the Senior Vice crosis scores at both 1- and 7-d after surgery, when compared Provost for Research of the University of Pennsylvania and through with iodine- and saline-treated frogs. In fact, all 4 of the frogs grant 5R25OD010986 from the National Center for Advancing Trans-

796 Skin preparation agents in Xenopus laevis

Figure 9. Microscopic images of tissues from frog 13, taken at necropsy. (A) The epidermis is ulcerated multifocally and extensively, and there is abundant hemorrhage and a marked neutrophilic dermal infiltrate extending to the serosal surface, as well as glandular degeneration and loss. Hematoxylin and eosin stain. (B) Gram-negative bacilli (arrows) were abundant within the necrotic epidermis and adhered to the exposed surface. (C) Glomerular capillaries are multifocally filled with and expanded by fibrin thrombi (arrows). Hematoxylin and eosin stain. (D) Fibrin within the glomerular capillaries stains dark blue with phosphotungstic acid hematoxylin, indicative of glomerular microthrombosis.

lational Sciences, NIH, DHHS. We thank Dr. Gui-Shuang Ying (Center 6. Bohme K, Fernandez-No IC, Barros-Velazquez J, Gallardo JM, for Preventive Ophthalmology and Biostatistics, Department of Canas B, Calo-Mata P. 2010. Comparative analysis of protein Ophthalmology, Perelman School of Medicine, University of Pennsylva- extraction methods for the identification of seafood-borne patho- nia) for statistical assistance. We would also thank the ULAR veterinary genic and spoilage bacteria by MALDI–TOF mass spectrometry. staff and members of the Klein lab for technical support. Two coauthors Anal Methods 2:1941–1947. (MJC and EKS) are employed by IDEXX BioResearch, a company that 7. Brown DD. 2004. A tribute to the Xenopus laevis oocyte and egg. J provides diagnostic testing for research animals. Biol Chem 279:45291–45299. 8. Buller N. 2004. Bacterial culture techniques, p 83–116. Bacteria from fish and other aquatic animals: a practical identification manual. References Cambridge (MA): CABI Publishing. 1. Bertolotti E, Malagoli D, Franchini A. 2013. Skin wound healing 9. Chapman AK, Aucott SW, Milstone AM. 2012. Safety of chlo- in different aged Xenopus laevis. J Morphol 274:956–964. rhexidine gluconate used for skin antisepsis in the preterm infant. 2. Biswas S, Rolain JM. 2013. Use of MALDI–TOF mass spectrometry J Perinatol 32:4–9. for identification of bacteria that are difficult to culture. J Microbiol 10. Clarke BT. 1997. The natural history of amphibian skin secretions, Methods 92:14–24. their normal functioning, and potential medical applications. Biol 3. Böhme K, Fernández-No I, Barros-Velázquez J, Gallardo J, Calo- Rev Camb Philos Soc 72:365–379. Mata P, Cañas B. 2010. Species Differentiation of seafood spoilage 11. Croxatto A, Prod’hom G, Greub G. 2012. Applications of MALDI– and pathogenic gram-negative bacteria by MALDI–TOF mass TOF mass spectrometry in clinical diagnostic microbiology. FEMS fingerprinting. J Proteome Res 9:3169–3183. Microbiol Rev 36:380–407. 4. Böhme K, Fernández-No I, Barros-Velázquez J, Gallardo J, Cañas 12. Culp C, Falkinham JI, Belden L. 2007. Identification of the natural B, Calo-Mata P. 2011. Rapid species identification of seafood spoil- bacterial microflora on the skin of eastern newts, bullfrog tadpoles, age and pathogenic gram-positive bacteria by MALDI–TOF mass and redback salamanders. Herpetologica 63:66–71. fingerprinting. Electrophoresis 32:2951–2965. 13. Darby C, Reavill DR, Schmidt RE. 2003. Fungal dermatitis in 5. Böhme K, Fernández-No I, Gallardo J, Cañas B, Calo-Mata P. captive toads (Bufo americanus) and attempted therapy. Proceed- 2011. Safety assessment of fresh and processed seafood products ings of the 2003 Conference of the Association of Reptilian and by MALDI–TOF mass fingerprinting. Food Bioprocess Technol Amphibian Veterinarians. Lawrence (KS): ARAV. 4:907–918. 14. Darouiche RO, Wall MJ Jr, Itani KM, Otterson MF, Webb AL, Carrick MM, Miller HJ, Awad SS, Crosby CT, Mosier MC,

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