Antimicrobial Original Research Paper

An unpredicted side effect of prophylactic antibiotic use Cemal Alper Kemalog˘lu1, Galip Kemali Gu¨nay2, Duygu Perc¸in3, Kemal Deniz4 1

Medical Doctor, 2Department of Plastic, Reconstructive and Esthetic Surgery, Erciyes University, Kayseri, Turkey, 3Department of Microbiology, Erciyes University, Kayseri, Turkey, 4Department of Pathology, Erciyes University, Kayseri, Turkey Introduction and Objective: We investigated the effects of cefazolin sodium (CS), on wound healing in rats. Material and Method: Forty rats in which an incisional wound model was created by removing two skin strips (461 cm), and suturing the wound edges, were included. Four groups were formed in a randomized way with each having 10 rats. The Control group (Group 1) received 1 cc 0.9% NaCl twice daily, whereas Group 2 received single-dose preoperative 30 mg/kg CS, Group 3 received single-dose preoperative 30 mg/kg CS followed by the same dose for three postoperative days, and Group 4 received single-dose preoperative 30 mg/kg CS followed by the same dose for seven postoperative days (via i.p. route). On the first day of the study, as the wound was created, a skin strip of 161 cm area was collected for bacteriologic examination. On the third day, specimens were acquired from the incision for histopathologic examination. The rats were sacrificed on the seventh day and more specimens were gained for histopathologic, tensiometric, and bacteriologic tests. Result: Group 4 demonstrated disrupted normal skin flora; reduced inflammatory cell density, fibroblastic activity, and collagen density; and decreased wound tensile strength. The histopathologic findings with Groups 2 and 3 were as same as with Group 4 and wound tensile strength showed no significant difference compared with the Control group. Group 2 revealed no significant difference compared with the Control group with regard to all parameters. Discussion: Seven-day CS therapy had a negative effect on wound healing and changed the normal skin flora.

Keywords: Surgical prophylaxis, Cefazolin sodium, Wound healing

Introduction and Objective Wound healing is the entire complicated and interrelated chain of events exhibited in response to a traumatic tissue damage that includes cellular, physiologic, and biochemical processes. In this reaction, any delay or negative event leads to failure of or delayed wound closure. During the First World War and the following years, the importance of wound site cultures and the direct influence of the type and quantity of bacteria growing in the culture have been understood.1,2 Application of improper postoperative antibiotherapy protocols is directly associated with the occurrence of elevated bacterial resistance, side effects, and higher cost.3 In the literature, the necessity of prophylaxis and its duration in clean surgical wounds have been examined by comparative studies and no significant difference has been found between prophylaxis and control groups.4–6 Although prophylactic antibiotic Correspondence to: Cemal Alper Kemalog˘lu, MD, Sanayi Mah. Atatu¨rk Bulvarı Hastane Cad. No:78 38010 Kocasinan, Kayseri, Turkey. Email: [email protected]

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ß 2014 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947813Y.0000000131

therapy for clean surgical wounds recommended single preoperative dose, many surgeons continued postoperative 7 days prophylaxis.7–9 Despite such frequent use of antibiotics on surgical wounds, the number of studies focussing on the effects of antibiotics on wound healing is limited.10–12 To our knowledge, there is no study in the literature that examines the impact of cefazolin on skin wounds. In our study, we aimed to reveal the histopathologic, bacteriologic, and mechanic associations between the systemic application of cefazolin sodium (CS) and cutaneous wound healing using a rat incisional wound model.

Material and Method Forty Wistar-Albino rats weighing between 250 and 400 g are used. Following the achievement of intraperitoneal anaesthesia with ketamin and xylazin, dorsal hairs of the rats were clipped. The rats were fixated at facedown position and cleaned with saline. By using no.15 scalpel and sterile gloves, two skin strips of 461 cm area, parallel and at least 2 cm distant from each other, were removed from the

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in a spiral fashion by a 100 ml pipette. After keeping them in the sterilizer at 35uC for 24 hours, the number of colonies was counted. The number of colonies was converted into the quantity of bacteria per gram and recorded in CFU (colony forming unit).13 Gr(z) Staphylococci were identified by API Rapid ID 32 Staph (BioMerieux, Lyon, France) kit and Gr (z) Streptococci were identified by API Rapid ID 32 Strep (BioMerieux) kit.

Histopathologic examination

Figure 1 HE-stained histopathologic view of incisional wounds in Controls (A) and in Group 4 (B) on the seventh day (inflammatory cell density in Controls is shown with red arrow, reduced inflammatory cell density in Group 4).

interscapular skin area (Fig. 1). The incisional wound model was generated by suturing the incisions by 3/0 silk with 10 mm intervals. A 161 cm area of the skin strips was stored for analysing bacterial load and the type of bacteria. Thereafter, the study population was split into four groups, each including 10 rats. Group 1 received 1 cc 0.9% NaCl twice daily, whereas Group 2 received single-dose preoperative 30 mg/kg CS and Group 3 received single-dose preoperative 30 mg/kg CS followed by the same dose divided into two equal doses daily for three postoperative days. Groups 2 and 3 did not get any normal saline after cefazolin administration until they were sacrificed. Group 4 received single-dose preoperative 30 mg/kg CS followed by the same dose divided into two equal doses daily for seven postoperative days (via i.p. route). All animals were sacrificed on the seventh day.

Measuring bacterial load and identification of bacterial type The 161 cm portion of the 461 cm skin strips obtained while inducing a wound, and other skin specimens of 161 cm that were acquired from the midline of the left incision with a no.15 scalpel, were put into sterile urine cups. The specimens were sent to the Bacteriology Laboratory within 2 hours and weighed with precision balance. Each specimen weighed 1 g. The tissue specimens were placed into sterile mortars and pestles including 1 cc serum physiologic (SP) per 1 g of tissue. Following the vortexing, they were put into blood agar and spread

Full-thickness skin specimens of 161 cm were obtained from the right incisional wound on the third day and from the left incisional wound on the seventh day for histopathologic examination. In order to study wound tensile strength on the seventh day on the left wound and to cause no unintentional wound dehiscence on the third day, histopathologic specimens were acquired from the right incisional wound. The preparations were dyed with haematoxylin and eosin, and examined under light microscopy. The tissues were evaluated with regard to inflammatory cell infiltration, fibroblastic activity, and collagen density as follows: (0), none; (1) low; (2), moderate; and (3), high.

Tensile strength (MPa N/mm2) and rupture force (N) After sacrificing the rats on the seventh day, tensile strength and rupture force were measured from the dorsal incision of the rats and two skin strips of 361 cm were obtained. Prior to measuring the rupture force, the skin thickness at the middle of the incision and the width of the skin strip over the incision were measured by a calliper and the surface area of the sections was calculated. The acquired specimens were studied within 2 hours by an Instron tensiometer (TT-CM Model, InstronEngCooperation, MA, USA). After two measurements of tensile strength and rupture force, mean values of those measurements were used.

Statistical method Tensile strength, intergroup bacterial load on the first day, and histopathologic data on the third and the seventh days were analysed by Kruskal–Wallis test, whereas intragroup bacterial load on the first and the seventh days was evaluated by t test. P,0.05 was recognized as statistically significant.

Results Tensile strength (MPa N/mm2) and rupture force (N) in incisional wounds Group 4 demonstrated a significantly reduced tensile strength and rupture force compared with the Control group (P50.004). However, there was no significant difference between Groups 1, 2, and 3. The results are summarized in Table 1.

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Figure 2 Seventh day inflammatory cell density scoring level (mean and standard deviation).

Bacterial load and identification of bacterial type None of the groups exhibited a difference on the first day with regard to bacterial load; however, when intragroup bacterial load per gram was measured on the first and the seventh days, Groups 3 and 4 were found to have a significantly elevated bacterial load on the seventh day, whereas Groups 1 and 2 did not show such a significant increase between day 1 and 7. Regarding the identification of bacterial type, the skin specimens obtained at the first day showed growth of Staphylococcus cohnii in all groups, whereas at 7 days, Groups 1 and 2 exhibited growth of S. cohnii, Group 3 showed mainly growth of S. cohnii and Enterococcus hirae in patches, and Group 4 showed growth of E. hirae. The distribution of bacterial load is shown in Table 2.

Histopathologic findings Inflammatory cell density Inflammatory cell density was lower in Groups 3 and 4 compared with the other groups both on the third and the seventh days (P50.02, P50.004, respectively) (Figs. 1 and 2).

Figure 3 Seventh day fibroblastic activity scoring level (mean and standard deviation).

Fibroblastic activity There was no difference between the groups on the third day with regard to fibroblastic activity (P50.08), whereas Groups 3 and 4 demonstrated lower fibroblastic activity on the seventh day compared with the other groups (P50.01) (Fig. 3). Collagen density Groups 3 and 4 showed lower collagen density on the seventh day compared with the other groups (P50.01). Furthermore, Group 4 displayed disrupted and delayed collagen configuration and organization compared with the other groups (Figs. 4 and 5).

Discussion The present study was conducted to find out whether CS, the most commonly used prophylactic antibiotic agent in Turkey and across the world, has an impact on wound healing or not. Knowing the impact of CS on wound healing in prophylactic use bears importance with regard to evaluating the duration of prophylaxis, benefit/side effect, and cost. In the literature, prophylactic antibiotic use is recommended to be performed according to the surgical wound classification outlined in 1964.8 All aesthetic surgeries

Table 1 Tensile strength and rupture force values of the incisional wounds on the seventh day

Groups

Rupture force (N)

Tensile strength (N/mm2)

Average6SD, Medyan (min–max)

Average6SD, Medyan (min–max)

Group 1 (n510)

5.58461.449 SD 5.22 (4.14–8.18) 3.65961.405 SD 3.21 (2.14–6.57) 3.72661.327 SD 3.55 (2.28–5.45) 2.86260.980 SD 3.08 (1.07–4.56)

Group 2 (n510) Group 3 (n510) Group 4 (n510)

0.2760.07 SD 0.26 (0.20–0.40) 0.1760.07 SD 0.16 (0.12–0.32) 0.1860.06 SD 0.17 (0.11–0.27) 0.1360.04 SD 0.15 (0.05–0.22)

Table 2 Comparison of bacterial load per gram on the first and the seventh days Groups Group Group Group Group

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1 2 3 4

Bacterial load day 1 (CFU/g) (average6SD)

Bacterial load day 7 (CFU/g) (average6SD)

P value

6.4610266.76102 2.1610261.66102 1.2610361.36103 3.5610264.56102

3610363.76103 4.6610267.66102 5.6610362.96103 1.6610367.56102

0.123 0.922 ,0.001 ,0.001

(n510) (n510) (n510) (n510)

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Figure 4 Seventh day collagen density scoring level (mean and standard deviation).

and most of plastic and reconstructive operations involve clean wounds. The infection rate for clean surgical wounds in plastic surgery is 1.8%.14,15 In the literature, there are many studies indicating that prophylactic antibiotic use is unnecessary, based on the infection rates of clean surgical wounds.4–6 However, clinical applications suggest otherwise. In the study by Perrotti et al., most of the surgeons were found to use prophylactic antibiotics in aesthetic operations (clean surgical wounds) and the duration of prophylaxis was observed to be up to 7 days.7 The number of studies investigating the effects of antibiotics over wound healing was limited in the past. Among those, Zimmermann et al. reported that systemic ampicillin, gentamicin, and rolitetracycline reduced wound tensile strength of cutaneous wound healing in rats.10 Borden et al. showed that seven-day postoperative metronidazole therapy impaired fascial healing in rats but did not affect the skin healing.11 Scher et al. conducted the only study about the association between CS and wound healing wherein seven-day CS therapy in rats was shown to impair fascial healing.12 The authors investigated only wound tensile strength in their study and therefore had difficulty in explaining the underlying mechanisms of the results. The most reliable explanation appears to be the influence of bacterial load and type of bacteria on wound healing, and the negative effect of applied antibiotics on the process. The bacteria induced into the wound site have been shown to have a positive influence on the wound healing process.16–18 The authors associated these results with increased interferon release from macrophages due to the effect of bacteria on some special proteins on the cell wall and the stronger stimulation of fibroplasia. In our study, seven-day CS therapy was observed to impair the normal skin flora of rats. Although the prevalent bacterium of the normal skin flora on the

Figure 5 HE-stained histopathologic view of incisional wounds in Controls (A) and in Group 4 (B) on the seventh day. Red arrow shows disrupted and delayed collagen configuration and organization.

first day was S. cohnii, it was E. hirae in Group 4 at the end of the seventh day. These findings were due to the natural resistance of Enterokoks to penicilin and cephalosporins.19 Groups 3 and 4 had lower inflammatory cell density on the third and the seventh days compared with the Control group. This negative impact of CS on the inflammatory stage may stem directly from its anti-inflammatory effect or from the inhibition of its role in inflammatory cell chemotaxis by the normal flora; further studies are required in order to understand this mechanism. In this study, although the fibroblastic activity levels had no significant difference in Groups 3 and 4 compared with others on the third day, it was significantly lower in Groups 3 and 4 than in others on the seventh day. Similarly, collagen density was significantly reduced in the groups receiving CS therapy on the third and the seventh days. In these groups wherein the inflammatory response was weak, fibroblastic activity and collagen density displayed a decrease. Another important result of our study was the impaired collagen configuration and organization in Group 4. We believe that the flora changes along with the decreased fibroblast density and inflammatory response, and delays collagen organization. Group 4 had significantly lower wound tensile strength. We showed that impaired wound healing was associated with reduced inflammatory response and disrupted collagen organization. The reduced inflammatory response was not in synch with the bacterial quantity. Groups 3 and 4 exhibited elevated bacterial load per gram.

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Particularly, seven-day CS therapy was observed to cause an entire change of the flora. The association between the impact of CS on skin wounds and the duration of its use suggests that its effect may be taking place through a bacterial mechanism, rather than by a direct anti-inflammatory effect. Another possibility is direct disruption on collagen organization by CS. Further detailed studies on this subject involving analyses at the molecular level while including bacteria, toxins, and collagen should reveal the underlying mechanism clearly. We performed this study in order to explain the factors behind reduced tensile strength within the framework of the relationship between antibiotics and wound healing. In this study, we also showed that in patients with negative systemic and local factors against wound healing, continuous prophylactic antibiotic use may aggravate the wound complications.

Acknowledgements Funding: This work was supported by research fund of the Erciyes University. Conflict of interest: None. Ethical adherence: This study has been completed under the permission of Erciyes University’s 10/39 number ethical adherence.

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4 Georgiou I, Farber N, Mendes D, Winkler E. The role of antibiotics in rhinoplasty and septoplasty: a literature review. Rhinology. 2008;46(4):267–70. 5 Casaer B, Tan EK, Depoorter M. The role of antibiotic prophylaxis in abdominoplasty: a review of the infection rate in 300 cases treated without prophylaxis. Plast Reconstr Surg. 2009;123(1):42e. 6 Gravante G, Caruso R, Araco A, Cervelli V. Infections after plastic procedures: incidences, etiologies, risk factors, and antibiotic prophylaxis. Aesth Plast Surg. 2008;32(2):243– 51. 7 Perrotti JA, Castor SA, Perez PC, Zins JE. Antibiotic use in aesthetic surgery: a national survey and literature review. Plast Reconstr Surg. 2002;109(5):1685–93. 8 Hunter JG. Appropriate prophylactic antibiotic use in plastic surgery: the time has come. Plast Reconstr Surg. 2007;120(6):1732–34. 9 Lyle WG, Outlaw K, Krizek TJ, Koss N, Payne WG, Robson MC. Prophylactic antibiotics in plastic surgery: trends of use over 25 years of an evolving specialty. Aesthet Surg J. 2003;23(3):177–83. 10 Zimmermann A, Truss F. The effect of antibiotic drugs on wound-healing. Urol Res. 1974;2(2):73–7. 11 Borden EB, Sammartano RJ, Dembe C, Boley SJ. The effect of metronidazole on wound healing in rats. Surgery. 1985; 97(3):331–6. 12 Scher KS, Scott-Conner CE, Montany PF. Effect of cephalosporins on fascial healing after celiotomy. Am J Surg. 1988;155(2):361–5. 13 Garcia LS. Clinical microbiology procedures handbook. 2nd ed. Washington: ASM; 2007. p. 3.4.1–19. 14 Resnik RR, Misch C. Prophylactic antibiotic regimens in oral implantology: rationale and protocol. Implant Dent. 2008; 17(2):142–50. 15 Cruse PJ, Foord R. A five year prospective study of 23,649 surgical wounds. Arch Surg. 1973;107(2):206–10. 16 Oloumi M, Jindrak K, Weiner M, Enquist IF. The time at which infected postoperative wounds demonstrate increased strength. Surg Gynecol Obstet. 1977;145(5):702–4. 17 Levenson SM, Kan-Gruber D, Gruber C, Molnar J, Seifter E. Wound healing accelerated by Staphylococcus aurus. Arch Surg. 1983;118(3):310–20. 18 Laato M, Niinikoski J, Lundberg C, Gerdin B. Inflammatory reaction and blood flow in experimental wounds inoculated with Staphylococcus aurus. Eur Surg Res. 1988;20(1):33–8. 19 Leclercq R. Enterococci acquire new kinds of resistance. Clin Infect Dis. 1997;24(Suppl 1):80–4.

An unpredicted side effect of prophylactic antibiotic use.

We investigated the effects of cefazolin sodium (CS), on wound healing in rats...
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