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Antibacterial selection for complete bacterial cure after surgical management of canine pyometra.

Introduction

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]

Conclusion

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.

Acknowledgement

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.

References

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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.

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(1.) Post Graduate Scholar

(2.) Assistant Professor & Head and Corresponding author. E-mail: asit_maji07@yahoo.co.in

(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
Publication:Intas Polivet
Article Type:Report
Date:Jul 1, 2015
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