Isolation of Pathogenic Aerobic Bacteria from the Blood of Septicaemic Neonatal Calves and the Susceptibility of Isolates to Various Antibiotics

Article — Artikel

Isolation of pathogenic aerobic bacteria from the blood of septicaemic neonatal calves and the susceptibility of isolates to various antibiotics

E Kireçcia*, Y Ozkanlarb, M S Aktasb, M H Uyanikc and H Yazgic

the samples following the antisepsis ABSTRACT procedure to prevent possible bacterial An automated blood culture system (BACTEC 9240) was used for the isolation of aerobic contamination risk. Ten millilitres of bacteria from the blood of septicaemic neonatal calves. Blood samples were collected from whole blood was drawn from the jugular 90 clinically septicaemic and 20 healthy neonatal calves and inoculated into blood culture vein of each calf with an 18-gauge needle bottles. There were 89 significant isolates from 90 positive blood cultures using the BACTEC into a sterile disposable syringe and then system. Escherichia coli was the most common pathogen detected accounting for 56 (63 %) infused into a blood culture bottle12. β out of 89 isolates. The other pathogens were -haemolytic streptococci (15.7 %), Staphylococ- The BACTEC 9240 blood culture ‘kits’ cus aureus (10.1 %), Klebsiella sp. (5.6 %) and Corynebacterium sp. (5.6 %). All isolates showed a each contained an aerobic blood culture susceptibility rate of 100 % to enrofloxacin, cefepim, cefoperazone/sulbactam, imipenem bottle, a needle, a syringe and an instruc- and meropenem while some of them were ranged from 75 to 91.7 % susceptible to amoxicillin/clavulanic acid, ampicillin/sulbactam, gentamicin and cephalosporins. tion brochure (BD BACTEC Aerobic, Becton Dickinson). Isopropyl alcohol Keywords : antimicrobial susceptibility,BACTEC blood culture, neonatal calves, septicaemia. was poured on the rubber septum of the Kireçci E, Ozkanlar Y, Aktas M S,Uyanik M H, Yazgi H Isolation of pathogenic aerobic bottles for decontamination and the bacteria from the blood of septicaemic neonatal calves and the susceptibility of isolates to blood samples were inoculated directly various antibiotics. Journal of the South African Veterinary Association (2010) 81(2): 110–113 into the bottles after exchanging the Õ (En.). School of Health, Kahramanmara Sutcuimam University, 46050, K.Maras, Turkey. needle used for venipuncture with a new sterile needle according to the manufac- turers’ recommendations. Inoculated bot- INTRODUCTION culture systems using infrared spectro- tles were immediately transported to the

Septicaemia is an important sequel to photometry to monitor CO2 production medical microbiology laboratory at bacterial infection that causes a high by growing bacteria with a fluorescence Ataturk University for culture. All bottles mortality. It is most likely to be present in sensor19. This system detects blood-borne were loaded into the instrument in the colostrum-deprived neonatal calves kept organisms rapidly and accurately. It mini- computer-assigned positions and incu- under poor shelter and hygienic condi- mises the incubation time, contamination bated for 5 days with continuous agita- tions6,11. Common clinical signs of septi- risk and false negative rate14,21. Although it tion. The system used infrared spectro- caemia are diarrhoea, dehydration, seems to be a relatively expensive method, photometry to monitor carbon dioxide, lethargy, hypothermia, lack of suckling the system may reduce the cost of treat- produced by microorganisms on a contin- reflex and recumbency in 1–7-day-old ment through early recognition of the uous basis. The bottles were removed calves. Because clinical signs are non- infection that enables effective manage- from the instrument when a signal alarm specific, early diagnosis of septicaemia is ment. Antimicrobial susceptibility results was obtained. An aliquot of the blood- difficult. Rapid and reliable culture with for the isolates using an accurate method, broth mixture was then aspirated asepti- subsequent identification of pathogens such as the BACTEC 9240 system, will cally using a syringe for initial Gram stain- from the blood is vital so that effective help to determine a suitable drug treat- ing. Based on Gram-staining results, antimicrobial therapy can be provided. A ment programme. aliquots from the bottles were sub- bacteriological culture of blood is neces- The purpose of the study was to detect cultured on 5 % sheep blood agar (Merck, sary to confirm a diagnosis of septicae- aerobic pathogenic bacteria in the blood Germany) and MacConkey agar (Merck, mia11,23. Automated blood culture systems of septicaemic neonatal calves with the Germany) plates and incubated aerobi- are commonly used to identify infectious BACTEC 9240 blood culture system, and cally. Microorganisms isolated from posi- agents causing septicaemia in human to determine the antimicrobial drug tive cultures were identified using con- medicine9,13,15. In veterinary medicine, susceptibility of isolates. ventional biochemical tests20. however, conventional blood culture methods are used in neonatal calves with MATERIALS AND METHODS Susceptibility testing septicaemia7. Oxoid Signal18 and BACTEC Sampling and processing In vitro antimicrobial susceptibility test- blood culture systems3 have rarely been Blood samples were collected from 90 ing of 89 isolates from septicaemic calves employed in the veterinary field. The septicaemic and 20 healthy (control) neo- was carried out using the disk diffusion BACTEC 9240 system is one of the new natal calves aged 0–7 days. The septi- method according to the Clinical and Lab- automated continuous BACTEC blood caemic calves were referred to the Faculty oratory Standards Institute2. Antibiotic aSchool of Health, KahramanmaraÕ Sutcuimam Univer- of Veterinary Medicine’s clinics of inter- susceptibility tests were carried out using sity, 46050, K.Maras, Turkey. nal medicine in Erzurum in eastern Tur- the Kirby-Bauer disk diffusion method on bDepartment of Internal Medicine, Faculty of Veterinary Medicine, Ataturk University, Erzurum, Turkey. key. Prior to venipuncture, hair was Mueller-Hinton agar (Merck, Germany) 1 cDepartment of Microbiology and Clinical Microbiology, shaved and the skin disinfected by scrub- plates . Bacterial isolates were tested Faculty of Medicine, Ataturk University, Erzurum, Turkey. bing with 70 % ethyl alcohol and apply- against the following antimicrobial drugs: *Author for correspondence. E-mail: [email protected] ing 10 % povidone iodine for 1 minute. An cefoxitin, cefaclor, ceftazidime, ceftriaxone, Received: March 2010. Accepted: June 2010. experienced veterinary clinician collected cephazolin, cephalotin, cefuroxime,

110 0038-2809 Tydskr.S.Afr.vet.Ver. (2010) 81(2): 110–113 cefoperazone, cefotaxime, cefepim, imi- Table 1: Bacterial isolates recovered from blood cultures. penem piperacillin/tazobactam, amoxicillin/clavulanic acid, ampicillin/ Bacterial species or group No. of isolates Percentage of isolates sulbactam, meropenem, cefoperazone/ sulbactam, ticarcillin/clavulanic acid, Aerobic Gram-negative bacilli Escherichia coli 56 63.0 enrofloxacin, and trimethoprim/sulpha- Klebsiella sp. 5 5.6 metaxazole (Oxoid). The antimicrobial susceptibilities were expressed as the per- Aerobic Gram-positive organisms β centage of isolates that were susceptible. -Haemolytic streptococci 14 15.7 Staphylococcus aureus 9 10.1 Corynebacterium sp. 5 5.6 RESULTS The calves included in the study showed Total 89 100 clinical signs of septicaemia, namely lack of suckling reflex, lethargy, weakness, anorexia, recumbency, hypothermia/ Table 2: Bacterial species of blood culture yield by the day of incubation. hyperthermia, and/or clinical shock and diarrhoea. Of these symptoms, diarrhoea, Bacterial species or group No. of isolates recovered dehydration and hypothermia were the Day 0 Day 1 Day 2 Day 3 most prominent. The BACTEC system detected growth Escherichia coli 20 31 5 0 of bacteria in 89 of 90 aerobic bottles. A Klebsiella sp. 0 5 0 0 β single organism was isolated from each -Haemolytic streptococci 0 9 0 5 Staphylococcus aureus 50 40 sample of the septicaemic group. Esche- Corynebacterium sp. 0 0 5 0 richia coli was the most common pathogen accounting for 56 (63 %) out of 90 Total 25 45 14 5 isolates. The other pathogens detected were 14 β-haemolytic streptococci (15.7 %), the world, resulting in economic losses in In the veterinary field, conventional 9 Staphylococcus aureus (10.1 %), 5 Klebsiella herd production. The isolation of micro- blood culture systems have infrequently sp. (5.6 %) and 5 Corynebacterium sp. (5.6 %) organisms directly from blood can be a been used in diagnosis of septicaemia in using standard procedures (Table 1). guide to accurate diagnosis and effective animals6,7. Oxoid Signal blood culture and Eighty-nine blood culture bottles were treatment in these cases4. Rapid, auto- BACTEC systems have also been investi- positive within 3 days of incubation: 25 mated and reliable blood culture systems gated in other studies3,18. The BACTEC on day 0 (28.1 %), 45 on the 1st day have been developed and are routinely system is now used not just for humans, (50.6 %), 14 on the 2nd (15.7 %), and 5 on used in human medicine. Of these sys- but also for cattle. It has provided a the 3rd (5.6 %). The species are given in tems, the BACTEC system is frequently positive diagnosis of Johne’s disease Table 2. used. In human medicine it has been caused by Mycobacterium paratuberculosis Coagulase-negative staphylococci shown that the BACTEC 9240 automated in cattle and the system was found to be (CoNS), as contaminant bacteria, were blood culture system is more effective faster than standard methods3. isolated in only 2 of 20 samples in the con- than conventional methods14. Its main ad- Although the objective of this study was trol group from healthy calves. No bacte- vantages over previous generations of not a validation of the BACTEC system, it ria were isolated from the remaining blood culture instruments include full enabled the 9240 automated blood cul- 18 samples. automation, quicker detection of blood ture system to be evaluated as to whether The results of antimicrobial drug pathogens, and improved laboratory it will be a practical method for the diag- susceptibilities of the bacterial isolates are workflow because it is not labour-inten- nosis of bacterial septicaemia in naturally shown in Table 3. β-Haemolytic strepto- sive17. affected neonatal septicaemic calves. To cocci and Corynebacterium sp. were found With conventional methods, isolates the authors’ knowledge, this system has to be susceptible to all antibiotics tested. needed to be checked for growth of not previously been evaluated in neona- All the isolates showed a 100 % sensitivity bacteria during culturing and passaged if tal calves. to enrofloxacin, cefepim, cefoperazone/ necessary, which is time-consuming and The calves recruited in the study were sulbactam, piperacillin/tazobactam, carries the risk of contamination. The clinically diagnosed as septicaemic. Most ticarcillin/clavulanic acid, imipenem and number of passages should be restricted of these calves showed apathy, lack of meropenem. Susceptibility rates to other to increase medium reliability and suckling reflex, dehydration and diarr- antibiotics ranged from 75 to 91.7 % to method sensitivity in processing of blood hoea. In a previous study the authors amoxicillin/clavulanic acid, trimetho- cultures. The BACTEC system shakes the evaluated a clinical score of calves in prim/sulphamethoxazole, ampicillin/ blood culture bottles periodically and terms of general attitude, hydration sulbactam and gentamicin. Susceptibility sounds an alarm when bacterial growth is status and appearance of scleral vessels, to cephalosporins was 100 % to cefepim, detected. This saves time and decreases and found a prevalence of 31 % for 91.7 % to cephalotin, cephazolin, cefu- the consumption of medium and the risk bacteraemia8. Accurate identification of roxime, ceftriaxone, cefoperazone and of contamination. Compared with con- the pathogens in their bloodstream was cefotaxime and 83.4 % to cefoxitin, ventional systems (i.e. BACTEC enrich- necessary for confirmation of the diagno- cefaclor and ceftazidime. ment broth and Oxoid Signal blood sis. The pathogenic bacteria isolated were culture) the BACTEC system was deter- identified as S. aureus, Streptococcus sp. DISCUSSION mined to be most sensitive and time-effi- and species of the Enterobacteriaceae. In Bloodstream infections such as septi- cient, having a low rate of false negatives this study the microorganisms isolated caemia and bacteraemia result in the for the isolation of microorganisms in cul- were regarded as pathogens owing to the death of young farm animals throughout ture of blood and sterile body fluids5,22,24. severe clinical signs manifested by the

0038-2809 Jl S.Afr.vet.Ass. (2010) 81(2): 110–113 111 Table 3: In vitro antimicrobial drug susceptibilities of bacterial isolates recovered from blood cultures.

Antimicrobial drug % Susceptibility E. coli Klebsiella sp. b-haemolytic S. aureus Corynebacterium sp. streptococci

Cephalosporins Cefoxitin 83 0 100 100 100 Cefaclor 83 100 100 100 100 Ceftazidime 83 100 100 100 100 Ceftriaxone 92 0 100 100 100 Cephazolin 92 100 100 50 100 Cephalotin 92 100 100 5 100 Cefuroxime 92 100 100 100 100 Cefoperazone 92 100 100 100 100 Cefotaxime 92 100 100 100 100 Cefepim 100 100 100 100 100 b-lactamase inhibitor combinations Amoxicillin/clavulanic acid 75 0 100 50 100 Ampicillin/sulbactam 92 100 100 100 100 Ticarcillin/clavulanic acid 100 100 100 100 100 Cefoperazone/sulbactam 100 100 100 100 100 Piperacillin/tazobactam 100 100 100 100 100 Other antibiotics Trimethoprim/sulphamethoxazole 83 100 100 100 100 Imipenem 100 100 100 100 100 Meropenem 100 100 100 100 100 Enrofloxacin 100 100 100 100 100 septicaemic calves. Coagulase negative mined in septicaemia and septic arthritis were likely to be the most effective. In pre- staphylococci (CoNS) and some Bacillus in neonatal calves using the same vious investigations, enrofloxacin has sp. occurred as contaminants24. Positive method26. In Oxoid Signal blood culture been demonstrated as effective in the blood cultures were determined in 89 of systems, E. coli (65 %) and Group D strep- treatment of E. coli-induced septicaemia 90 bottles obtained from the septicaemic tococci (11.5 %) were isolated from the in calves23. In this study, all the isolates calves. Seventy of 89 cultures showed blood of septicaemic calves and foals11, were susceptible to enrofloxacin. How- positive results within the 1st 24 hours while E. coli (56 %) and Actinobacillus sp. ever, 16.6 % of the isolates were resistant indicating the rapidity of this system. (26 %) were isolated from blood cultures in to trimethoprim/sulphamethoxazole, one Septicaemia is a common clinical problem septicaemic foals18. In human septicaemia, of the drugs most commonly used in the in exhausted neonatal ruminants. Rapid aerobic Gram-positive cocci (particularly Middle East (particularly eastern Turkey). diagnosis and treatment of septicaemia Staphylococcus sp.) were isolated most fre- Therefore E. coli antibiotic resistance in can improve the likelihood of a successful quently3,13,16. The difference in isolates the region is not surprising. outcome. Blood culture studies on septi- from humans and animals may be related Moreover, resistance to this drug caemic calves indicated that E. coli was the to different environmental living condi- among E. coli isolates from calves has most common Gram-negative bacterium tions and housing. Positive blood culture already been observed10,11. It is suggested isolated, accounting for approximately results are necessary to make a definite that the success rate of antibiotic treat- 80 % of the bacterial isolations11,23,25. These diagnosis of septicaemia. However, septi- ment can be improved by knowing the pathogens usually enter the body by caemia can progress quickly in neonatal susceptibility rate. means of contamination of the intestinal calves, therefore antibiotic treatment Although the BACTEC system may be a tract within 24 hours of parturition, result- should not be delayed until the results of relatively expensive method (compared to ing in septicaemia, death or local infec- blood cultures are obtained23. Antibiotic classic blood culture systems), the cost can tions such as meningitis, polyarthritis and therapy currently acts as the cornerstone be offset by decreasing mortality rates polyserositis in a short space of time. of treatment of septicaemic neonatal in- through early diagnosis and accurate In this study, the pathogenic species fection. Bacterial resistance to antibiotics treatment. Further detailed studies could obtained were E. coli, β-haemolytic is a common reason for a failure of treat- be undertaken to evaluate this system as a streptococci and S. aureus, in agreement ment, so appropriate antimicrobial treat- diagnostic tool for other diseases in the with previous reports. In addition, a ment to septicaemia is critical to decrease field of veterinary medicine. single organism was isolated from each of mortality rates7,23. Bacterial susceptibility the samples from septicaemic calves. The patterns should be documented to deter- CONCLUSIONS most frequent organism cultured was mine whether the microorganism is The isolation of pathogenic aerobic E. coli (63 %) in blood cultures. Other indeed susceptible to the selected anti- bacteria from blood has been achieved in organisms cultured were β-haemolytic biotic. septicaemic neonatal calves using the streptococci, S. aureus, Klebsiella sp. and In this study, antibiotic susceptibility automated BACTEC 9240 blood culture Corynebacterium sp. A previous study patterns of the isolates recovered from system. The system allows for rapid detec- similarly reported that E. coli made up blood samples from septicaemic calves tion and identification of pathogens lead- 51 % of isolates when blood from criti- were evaluated. Many of the isolates ing to an accurate diagnosis. Once the cally-ill neonatal calves was cultured in tested were sensitive to cephalosporins. pathogen is isolated, its susceptibility to the conventional way7. Furthermore, Of the cephalosporins tested, cefepim, antibiotics can be determined, ensuring E. coli and Lactococcus lactis were deter- cefotaxime, cefoperazone and cefuroxime more effective use of antibiotic treatment.

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