Antibacterial selection for complete bacterial cure after surgical management of canine pyometra.
Pyometra is a common disorder of middle and old age intact bitches with absence of pregnancy. Progressive thickening of endometrium by repeated exposure of estrogen and cystic endometrial hyperplasia (CEH complex) in successive heat cycles by progesterone making the bed favourable for pathogens. The cervix remains open during pro-estrus and estrus allowing normal flora present in vagina to ascend into uterus. The bacterial colonies in thickened uterine lining are not expelled out as they would be in a healthy bitch and antibiotics cannot diffuse into the sea of pus in bacteria filled uterus (Watts et al, 1996). In closed and open pyometra endotoxemia may cause either septic shock following systemic inflammatory response syndromes (SIRS) in 61% of pyometra (Hagman et al., 2009) or chronic polysystemic diseases of renal, hepatic, cardiovascular, pulmonary and sometimes cerebral dysfunction (Stone, 2003; Baluch et al., 2007). The treatment of choice has traditionally been ovariohysterectomy (Hardy and Osborne, 1974), but some bitches are severely affected to survive (Wheaton et al., 1 987). Symptomatic treatment may render temporary relief but recurrences and changes of antibiotics several times invite antibiotic resistance and death. Identification of causative organism by isolation and selection of antibiotics of choice by antibiotic sensitivity test (AST) were the objectives of this work for pre and post-operative management of 20 bitches affected with pyometra during surgical treatment by ovariohysterectomy.
Materials and Methods
The bitches (n=20) with clinical symptom of anorexia, polydypsia, polyurea, vomition, vaginal oozing of pus presented for treatment and were tentatively diagnosed for pyometra and included in the study. The bitches were subsequently confirmed for pyometra by positive ultrasonogram, radiography and high blood urea nitrogen (BUN), creatinine, aspartate aminotransferase (AST) or alanine transaminase (ALT) values (Table 2). Material for isolation and antibiotic sensitivity test of bacteria were the pus samples collected from deep vaginal discharge after cleaning vaginal superficial area with rectified sprit in case of open and after ovariohysterectomy operation in closed pyometra.
The causative organisms from pus samples were isolated and identified by morphology and biochemical tests i.e. catalase, oxidase (disc method), indole, methyl-red, voges-proskauer, citrate utilization and urease. (Quinn et al.,1994). All isolates identified were subjected to antibiotic sensitivity test (AST) by disc diffusion method to different antibiotics (Bauer et al., 1966). The antibiotics used for AST were Ciprofloxacin, Gentamicin, Amikacin, Ceftazidime, Ceftriaxone, Ceftriaxone +Tazobactum, Sparfloxacin, Amoxicillin + Clavulanic acid, Cefepime + Tazobactam, Enrofloxacin, Oxacillin, Cefoxitin and Linezolid.
Result and Discussion
The pyometra affected bitches in present study were aged 8.54 [+ or -] 0.77 years. The most affected breed was Labrador (35%) and least affected was GSD (5%) and Mongrel (5%) in contrast to findings of Egenvall et al. (2001). This breed variation might be due to availability of breeds in a particular area. Before the age of 10 years, about 24.0% of bitches suffered from pyometra (Egenvall et al., 2001). The 60% (12/20) of bitches had open pyometra and rest were affected with closed pyometra. The majority bitches (60%) of population of this study which suffered from pyometra were nulliparous (Table 1).
Stoppage of supportive therapy causes enhancement of endotoxin (ET) production and results to increased biomarkers for liver, kidney and heart (Hagman et al., 2006). Increase of kidney biomarkers means magnitude of either acute or chronic kidney damage. Kidney can no longer retain water in body, urine gets more diluted, the dog drinks more water to compensate the loss as evidenced by polydypsia in most pyometra cases. Kidney cells do not regenerate. Following Slatter (2003), the bitches with high creatinine values were first rehydrated intravenously with Ringer's lactate (10ml/kg/hour), then Dopamine @ 0.6[micro]g/kg/mm was administered followed by diuretics, Frusemide. This helped to flush out system and toxin, decreasing BUN and creatinine values. Here ovariohysterectomy (OHE) for pyometra was not delayed more than 48-72 hours of pre-operative supportive therapy in general and in emergency cases of closed pyometra in particular within 12-24 hours of that therapy as also practiced by Melih et al. (2012).
Isolation of bacteria from pus samples
Escherichia coli and Staphylococcus spp. were presumptively identified from 9 (50%) and 5 (28%) samples respectively, whereas from 4 (22%) samples both of organisms were isolated (Fig.3). This finding corroborates with Bigliardi et al. (2004). No isolates could be traced from rest 2 samples. All presumptive E. coli isolates showed characteristic pink coloured colony in MacConkey agar and metallic sheen in EMB agar. Staphylococcus spp. showed characteristics yellow/white colony on mannitol salt agar.
i. Biochemical identification of isolates
All Escherichia coli isolates were gram negative small rods (Fig. 1) and showed standard results of biochemical test like catalase (+ve), oxidase (-ve), indole-methyl red-voges proskauer-citrate (++--) and urease (-ve). Staphylococcus spp. isolates were gram positive cocci arranged in grape like clusters (Fig. 2) and showed standard results of biochemical test like catalase (+ve), oxidase (-ve), indole (-ve), methyl red (+ve) and urease (+ve).
ii. Antibiogram of Isolates
All 13 (9+4) E. coli and 9 (5+4) Staphylococcus spp. isolated were tested for their sensitivity (Table 3 and 4). In case of E. coli, resistance was observed most frequently to Ceftazidime (76.92%), Co-trimoxazole (69.23%), Amoxi-clav (53.84%) and Sparfloxacillin (46.15 %) and less frequently to Ceftriaxone (15.38%), Ciprofloxacin (30.77%) and Gentamicin (30.77%). No resistance was observed in case of Cefepime + Tazobactam and Amikacin (Table 3). None of isolates were sensitive to Co-trimoxazole (Table 3). Multidrug resistance was observed in 76.92% of isolates. The Staphylococcus isolates were resistant to Gentamicin (88.89 %), Ceftazidime (77.78%), Co-trimoxazole (66.67%) and less commonly to Ciprofloxacin (22.22%), Amoxi-clav (22.22%) and Sparfloxacillin (22.22 %), whereas, none of the isolates were sensitive to Linazolid and Oxacillin (Table 4). In case of Cefepime + Tazobactam, none of Staphylococcus isolates were sensitive, but 66.67% of isolates were of intermediate susceptibility. Multidrug resistance was detected in 66.67% Staphylococcus isolates.
Many gram negative (E. coli, Proteus spp., Salmonella spp., Pseudomonas spp., Klebsiella spp. etc) and gram positive (Staph. aureus, Staph. pyogenes, Corynebacterium spp., Enterococcus spp. etc.) bacteria have been isolated from cases of canine pyometra (Shambulingappa and Manegar, 2010 ; Coggan et al., 2008), but E. coli has been identifies as major cause of clinical cases of canine pyometra in different parts of world (Bassessar et al., 2013; Hagman and Kuhn, 2002; Roy et al., 2009 Silva et al., 2004 Dhaliwal et al. (1998) and was isolated in our study also. Ciprofloxacin and Gentamicin sensitive Staphylococcus spp. has also been found in a similar study conducted by Maity et al. (2009). Mixed infection with Escherichia coli and Staphylococcus spp. was identified in 4 (20%) cases in our study. This is in support of the previous study by Bassessar et al. (2013) where they found mixed infection of Escherichia coli and Staphylococcus spp. in two cases of canine pyometra. Higher percentage (71.43%) of resistance in E. coli isolates from pyometra against Amoxicillin-Clavulanic acid was also reported by Maity et al. (2009). Ciprofloxacin and Gentamicin has been recommended as most effective antibiotic in cases of pyometra (Bassessar et al., 2013; Silva et al., 2004; Murugavel et al., 2001; Roy et al., 2009, Shambulingappa and Manegar, 2010, Maity et al., 2009; Coggan et al., 2008; Oluoch et al., 2001). However, the percentage of sensitive isolates varies from study to study. No resistance was observed in case of Cefepime + Tazobactam and Amikacin (Table 3) in present study. Similarly, Oluoch et al. (2001) found 85.9% sensitivity of E. coli isolates to amikacin in different types of infections in dogs including the genitor urinary tract. Multidrug resistance was observed in 76.92% (10/13) isolates which was contradictory to findings by Agostinho et al. (2014) and Siqueira et al. (2009). Higher prevalence of multidrug resistant E. coli is of major concern considering the treatment of the patients and public health importance of the organisms. In this study, sensitivity to Enrofloxacin was observed in 44.45% (4/9) of Staphylococcus isolates, but other studies have reported higher efficacy of Enrofloxacin against Staphylococcus spp (Bassessar et al., 2013; Maity et al., 2009). Higher sensitivity of Staphylococcus isolates to Ceftriaxone + Tazobactum in support of the previous observation by Maity et al. (2009). Invitro and in-vivo situations are different. After getting the above result selection of antibiotic was judicious. Linazolide (100% sensitive against staphylococcus), a macrolide is not easily available. Penicillin is good for staphylococcus but it binds with pus protein in vivo and may cause allergy and shock. Cefepime + Tazobactum (100% sensitive against E.coli) is not commonly available in the market. Amikacin (62% sensitive against E. coli) is nephrotoxic and cannot work in acidic media of pus (Huber,1982). As Ceftriaxone and Tazobactum combination showed appreciable sensitivity (approx.70%) against both E.coli and Staph. aureus, this was preferred combination for pre or post operative administration.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
High levels of pre operative biochemical values approach normalcy by stabilization of patients before ovariohysterectomy to reduce peri and post operative anaesthetic complications. Multi-drug resistant E.coli (pre-dominantly) along with Staphylococcus spp. are main causative organism of pyometra combination of antibiotics is better to use of single to reduce bacterial load.
We are thankful to Dr. Tapas Kumar Sar, Senior Assistant Professor, Veterinary Pharmacology for his continuous advice and to Dr. Achiwta Kr. Mohanty, Ph.D. Scholar, Veterinary Microbiology for his active help duirng this study.
Agostinho, J.M.A., Souza, A., Schocken-Iturrino, R.P., Beraldo, L.G., Borges, C.A., Avila, F.A. and Marin, J.M. (2014). Escherichia coli strains isolated from the uteri horn, mouth and rectum of bitches suffering from pyometra-virulence factors, antimicrobial susceptibilities, and clonal relationships among strains. International J. Microbiol. 40 : 1 -8.
Baluch, A., Janoo, A., Lam, K., Hoover, J. and Kaye, A. (2007). Septic shock review and anesthetic consideration. M.E.J. Anaesth. 19: 71-86.
Bassessar, V., Verma, Y. and Swamy, M. (2013). Antibiogram of bacterial species isolated from canine pyometra. Vet World. 6: 546-49.
Bauer, A.W., Kirby, W.M., Sherris, J.C. and Turck, M. (1966). Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol. 45: 493-96.
Bigliardi, E., Parmigiani, E., Cavirani, S., Luppi, A., Bonati, L. and Corradi, A. (2004). Ultrasonography and cystic endometrial hyperplasia - pyometra complex in the bitch. Reprod Domestic Anim. 39: 136-40.
Coggan, J.A., Priscilla, A.M., de Oliveira, C.M., Faustino, M., Moreno, A.M. and Benites, N.R. (2008). Microbiolgical and histopathological aspects of canine pyometra. Brazilian J. Microbiol. 39: 477-83.
Dhaliwal, GK., Wray, C. and Noakes, D.E. (1998). Uterine bacterial flora and uterine lesions in bitches with cystic endometrial hyperplasia (pyometra). Vet. Rec. 143: 659-61.
Egenvall, A., Hedhammar, A, Bonnett, B.N. and Olsson, P. (2001). Breed risk of pyometra in insured dogs in Sweden. J. Vet Internal Med. 15: 530-38.
Fransson, B.A. (2003). Systemic inflammatory response in canine pyometra. Doctoral dissertation. Acta Universitatis Agriculturae Sueciae Veterinaria, Sweden.
Gobello, C., Castex, G., Klima, L., Rodriuez, R. and Corrada, Y. (2003). A study of two protocols combining aglepristone and cloprostenol to treat open cervix pyometra in the bitch. Theriogenology. 60: 901-08.
Hagman, R. and Kuhn, I. (2002). Escherichia coli strains isolated from the uterus and the urinary bladder of bitches suffering from pyometra: comparison by restriction enzyme digestion and pulsed- field gel electrophoresis. Vet. Microbiol. 84: 143- 53.
Hagman, R., Kindahl, H. and Lagerstedf, A.S. (2006). Pyometra in bitches induces elevated plasma endotoxin and prostaglandin F2 metabolite levelsm. Acta. Vet. Scand. 47 : 55-68.
Hagman, R., Reezigt, B., Ledin, H.B. and Karlstam, E. (2009). Blood lactate Levels in 31 female dogs with pyometra. Acta Vet. Scand. 51: 2
Hardy., R.M. and Osborne., C.A. (1974). Canine pyometra: pathophysiology, diagnosis and treatment of uterine and extra-uterine lesions. J. Am Anim Hosp Assoc. 10: 245-267.
Huber, W.G. (1982). Aminoglycosides and other antibiotic agents. In: Joknes Veterinary Pharmacology and Therapeutics. 5th Edn., Iowa State University Press, Kalyani Publisher Indian Print, p. 748-771.
Maity, S., Sarkar, S. and Saha, T. (2009). Bacteriology and antibiogram of canine pyometra. Indian Vet. J. 86: 896-97.
Melih, U., Cagatay, T., Mehmet, C., Ahmet, G.S., Adem, S., Funda, A. and Tulay, B. (2012). Optimum timing for operation in bitches with pyometra related to endotoxemia. Turk. J. Vet. Anim. Sci. 36: 35-42.
Murugavel, K., Senthil Kumar, P. and Alphonse, R.M.D. (2001). Pyometra in Doberman bitch. Indian J. Anim. Reprod. 22: 91-9
Oluoch, A.O., Kim, C., Weisiger, R.M., Koo, H.Y, Siegel, A.M., Campbell, K.L., Burke, T.J., Mckiernan, B.C. (2001). E. coli isolates from canine pyometra. In: Non enteric Escherichia coli isolates from dogs: 674 cases (1990-1998). J. Am. Vet. Med. Ass. 218: 381-84.
Quinn, PJ., Cartar, M.E., Markey, B.K., Carter, G.R. (1994). Clinical Veterinary Microbiology,3rd edition, Eolfe publications,UK,pp.21-36.
Roy, S.K., Das, B. and Batabyal, K. (2009). Antibiogram of pathogenic Escherichia coli isolated from canine pyometra cases. J. Interacademicia. 13: 481-83.
Shambulingappa, B.E. and Anand Manegar, G. (2010). Aerobic bacterial flora and antibiogram profile in canine metritis. Indian Vet. J. 87: 556-558. robic bacterial flora in canine abortions. Vet World. 3: 111-12.
Silva, L.B.G., Castro Junior, I.F., Cunha, A.P., Mota, R.A., Silva, K.P.C. and Pinheiro Junior, J.W. (2004). Aetiological and therapeutic study of pyometra in bitches from the metropolitan region of Recife, Pernambuco State, Brazil. A Hora Veterinaria. 24: 37-39.
Siqueira, A.K., Ribeiro, M.G. and Leite, D.D.S. (2009). Virulence factors in Escherichia coli strains isolated from urinary tract infection and pyometra cases and from feces of healthy dogs. Res Vet Sci. 86: 206-10.
Slatter, D. (2003). In: Anesthesia for Veterinary Technicians, 4th edn, WB Saunders, Philadelphia, pp. 2516-20.
Stone, E.A. (2003). Ovary and Uterus. Chap. 12 In: Textbook of Small Animal Surgery, edited by Saunders' D.S. 3rd edn, Philadelphia, pp. 1489-93.
Watts, J.R., Wright, P.J. and Whithear, K.C. (1996). Uterine, cervical and vaginal microflora of the normal bitch throughout the reproductive cycle. J. Small Anim Prac. 37: 54-60.
(1.) Post Graduate Scholar
(2.) Assistant Professor & Head and Corresponding author. E-mail: firstname.lastname@example.org
(3.) Assistant Professor, Department of Veterinary Microbiology
(4.) Associate Professor, Department of Veterinary Biochemistry
(5.) Associate Professor
Toufique Ahamed (1), Asit Kr.Maji (2), Indranil Samanta (3), S. Batabyal (4) and Debaki Ghosh (5)
Department of Veterinary Surgery and Radiology
Faculty of Veterinary and Animal sciences
West Bengal University of Animal and Fishery Sciences (WBUAFS)
37, K.B. Sarani
Kolkata--700037 (West Bengal)
Table 1: Details of bitches included in the study Sr. Age B. wt. Whelping Last No. (yrs.) (kg.) status Breed parturition 1 8% 32 Once Labrador 5 yrs. ago 2 5 37 Once Golden Retriever 3 yrs. ago 3 11 29 Nulliparous German Shepherd -- 4 8 33 Once Golden Retriever 6 yrs. ago 5 12 9 Twice Spitz 5 yrs. ago 6 5 34 Nulliparous Labrador -- 7 6 1/2 37 Nulliparous Labrador -- 8 11 1/2 46 Twice Labrador 6 yrs. ago 9 7 27 Nulliparous Cross breed -- 10 5% 19 Once Mongrel 3 yrs. ago 11 9 39 Nulliparous Labrador -- 12 5 33 Nulliparous Golden Retriever -- 13 13 13 Twice Spitz 8 yrs. ago 14 8% 40 Nulliparous Labrador -- 15 9 11 Nulliparous Spitz -- 16 6% 38 Once Labrador 4 yrs. ago 17 12 9 Nulliparous German Spitz -- 18 8 42 Twice Golden Retriever 3 yrs. ago 19 7 12 Nulliparous Spitz -- 20 6% 34 Nulliparous Golden Retriever -- Sr. No. Diagnosis 1 Open pyometra 2 Open pyometra 3 Open pyometra 4 Closed pyometra 5 Closed pyometra 6 Open pyometra 7 Open pyometra 8 Closed pyometra 9 Open pyometra 10 Open pyometra 11 Closed pyometra 12 Open pyometra 13 Closed pyometra 14 Open pyometra 15 Open pyometra 16 Closed pyometra 17 Open pyometra 18 Closed pyometra 19 Open pyometra 20 Closed pyometra Table 2: Initial mean values of haematological and biochemical parameters Parameter Mean [+ or -] S.E. Haemoglobin (Hb) 13.66 [+ or -] 0.58 Total Leucocytic Count 15,110 [+ or -] 3026.16 (TLC) Neutrophil (N) 66.6 [+ or -] 1.06 Eosinophil (E) 4.7 [+ or -] 0.3 Basophil (B) 0 [+ or -] 0 Lymphocyte (L) 25.7 [+ or -] 1.04 Monocyte (M) 3 [+ or -] 0.149 Glucose (GLU) 92.4 [+ or -] 2.59 Blood Urea Nitrogen (BUN) 19.88 [+ or -] 4.91 Creatinine (CN) 2.4 [+ or -] 0.52 Alanine Transaminase(ALT) 44.4 [+ or -] 1.75 Aspertase Transaminase (AST) 43.6 [+ or -] 1.50 Parameter Unit / Range Haemoglobin (Hb) gm% Total Leucocytic Count 6,300-41,100 (TLC) (x [10.sup.6]) Neutrophil (N) % Eosinophil (E) % Basophil (B) % Lymphocyte (L) % Monocyte (M) % Glucose (GLU) Gm/dl Blood Urea Nitrogen (BUN) 6.9-63 Creatinine (CN) 0.9-6.9 Alanine Transaminase(ALT) 37-53 Aspertase Transaminase (AST) 37-50 Table 3: Result of Antibiotic Sensitivity test of Escherichia coli isolates Antibiotics Symbol Conc. Sensitive (%) Gentamicin HLG 120 [micro]g 4 (30.77) Enrofloxacin EX 10 [micro]g 4 (30.77) Cefepime/Tazobactum CPT 30 [micro]g 13 (100) Amikacin AK 30 [micro]g 8 (61.54) Amoxiclav AMC 30 [micro]g 3 (23.08) Ceftazidime CAZ 30 [micro]g 0 (0) Ceftriaxone + Tazobactum CTR+TZB 30 [micro]g 10 (71.54) Ciprofloxacin CIP 5 [micro]g 5 (38.46) Co-T rimoxazole COT 25 [micro]g 0 (0) Sparfloxacin SPX 5 [micro]g 4 (30.77) Antibiotics Intermediate Resistant (%) (%) Gentamicin 5 (38.46) 4 (30.77) Enrofloxacin 4 (30.77) 5 (38.46) Cefepime/Tazobactum 0 0 Amikacin 5 (38.46) 0 Amoxiclav 3 (23.08) 7 (53.84) Ceftazidime 3 (23.08) 10 (76.92) Ceftriaxone + Tazobactum 2 (23.08) 1 (15.38) Ciprofloxacin 4 (30.77) 4 (30.77) Co-T rimoxazole 4 (30.77) 9 (69.23) Sparfloxacin 3 (23.08) 6 (46.15) Table 4: Result of Antibiotic Sensitivity test of Staphylococcus spp. isolates Antibiotics Symbol Conc. Sensitive (%) Linezolid LZ 15 [micro]g 9 (100) Gentamicin HLG 120 [micro]g 0 Enrofloxacin EX 10 [micro]g 4 (44.45) Cefepime+ Tazobactum CPT 30 [micro]g 0 (0) Oxacillin OX 1 [micro]g 6 (66.67) Amikacin AK 30 [micro]g 2 (22.22) Amoxiclav AMC 30 [micro]g 5 (55.56) Ceftazidime CAZ 30 [micro]g 0 (0) Ceftriaxone +Tazobactum CTR 30 [micro]g 5 (69.56) Ciprofloxacin CIP 5 [micro]g 4 (44.45) Co-Trimoxazole COT 25 [micro]g 0 (0) Sparfloxacin SPX 5 [micro]g 6 (66.67) Antibiotics Intermediate Resistant (%) (%) Linezolid 0 (0) 0 (0) Gentamicin 1 (11.11) 8 (89.89) Enrofloxacin 2 (22.22) 3 (33.33) Cefepime+ Tazobactum 6 (66.67) 3 (33.33) Oxacillin 3 (33.33) 0 (0) Amikacin 2 (22.22) 5 (55.56) Amoxiclav 2 (22.22) 2 (22.22) Ceftazidime 2 (22.22) 7 (77.78) Ceftriaxone +Tazobactum 1 (11.11) 3 (33.33) Ciprofloxacin 3 (33.33) 2 (22.22) Co-Trimoxazole 3 (33.33) 6 (66.67) Sparfloxacin 1 (11.11) 2 (22.22) Fig. 3: Percentage of Isolates Isolated Bacteria 5;28% Staphylococcus Sp Isolated Bacteria 9;50% E. coli Isolated Bacteria 4;22% Both Note: Table made from pie chart.
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|Title Annotation:||Clinical Article|
|Author:||Ahamed, Toufique; Kr.Maji, Asit; Samanta, Indranil; Batabyal, S.; Ghosh, Debaki|
|Date:||Jul 1, 2015|
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