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CASE REPORT

Lethal septic shock after dental scaling in a healthy dog due to Ochrobactrum anthropi-contaminated propofol P. Franci, G. Dotto, A. Cattai and D. Pasotto Department of Animal Medicine, Production and Health, University of Padua, Viale dell’Università, 16 - 35020, Legnaro (Padua), Italy

A 10-year-old, 6-kg male Yorkshire terrier dog was scheduled for routine dental cleaning. No significant problem was observed either during anaesthesia, which was induced with propofol, or during recovery. However, 2 hours after discharge, the dog’s owner returned to the clinic, complaining that the animal was lethargic and had had bloody diarrhoea. On physical examination the dog was depressed, dyspnoeic, tachycardic and hypoglycaemic. Despite supportive treatment, the dog deteriorated and died within a few hours. A presumed diagnosis of sepsis was confirmed by laboratory testing. Bacteriological and molecular examinations of both premortem blood samples and the anaesthetic, highlighted the presence of Ochrobactrum anthropi, an opportunistic pathogen usually associated with immunocompromised hosts with indwelling medical devices. To the authors’ knowledge, this is the first case of sepsis in a healthy dog due to contamination of an anaesthetic solution by O. anthropi, suggesting a potential role of this microorganism as an emerging pathogen. Journal of Small Animal Practice (2014) DOI: 10.1111/jsap.12284 Accepted: 31 August 2014

INTRODUCTION Propofol is one of the most popular anaesthesia-induction agents both in human and veterinary medicine. It has been shown to promote microbial growth, especially of some Gram-negative bacteria (Crowther et al. 1999). The proliferation of such bacteria is usually associated with endotoxin release, and contaminated lipid-based solutions, such as propofol, especially if maintained at room temperature, can contain endotoxins at high concentrations. The administration of highly contaminated drugs may cause severe sequelae in the patient. Ochrobactrum anthropi is an opportunistic, environmental Gram-negative bacillus rarely associated with the development of severe life-threatening infections. It has been found mainly in hospital water sources and in synthetic materials, including antiseptic solutions, vials and tubing kits. Although clinical infections are rare, in the last few decades the increasing number of severe local and systemic infections in debilitated, immunocompromised human patients associated with contamination of Dr. Franci and Dr. Dotto contributed equally to this work. Journal of Small Animal Practice © 2014 British Small Animal Veterinary Association

indwelling medical devices suggests that this genus might represent an emerging pathogen (Scholz et al. 2008). This is the first report of sepsis due to O. anthropi-contaminated propofol in a dog.

CASE HISTORY A 10-year-old, 6-kg male Yorkshire terrier dog was scheduled for routine dental cleaning. The patient was classified as American Society of Anaesthesiologists (ASA) status I, based on physical examination and basic haematology and biochemistry. Thirty minutes after the administration of intramuscular (im) pre-anaesthetic medication of 0·002 mg/kg dexmedetomidine (Dexdomitor; Pfizer, Italy) and 0·2 mg/kg methadone, (Eptadone; Zambon, Italy), a 22 G catheter was inserted into the right cephalic vein. Anaesthesia was induced with 4 mg/kg propofol (Propofol Kabi 1%; Fresenius Kabi, Italy) and maintained with isoflurane in oxygen (delivered via a breathing system, Mapleson C), following oro-tracheal intubation. Ringer’s solution was infused intravenously (iv) at 10 mL/kg/hour. During anaesthesia, the dog was kept warm using a heating pad and the 1

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main cardiovascular and respiratory parameters were monitored (electrical activity of the heart, heart rate, haemoglobin saturation with oxygen, lung resistance and dynamic compliance, pulse rate, blood pressure, respiratory rate and concentration or partial pressure of CO2 in the respiratory gases). A short phase of mild arterial blood hypotension (blood pressure was 55 mmHg) occurred immediately after induction but resolved spontaneously. Immediately after the induction of anaesthesia, preventive antimicrobial therapy was established with an im injection of 8·75 mg/kg amoxicillin and clavulanic acid (AMC; Synulox Injection; Pfizer, Italy). No other problems were observed during anaesthesia and recovery. The patient was discharged when it was able to walk, approximately 1 hour after the end of anaesthesia. Two hours after discharge, the dog’s owner returned to the clinic, complaining that the dog had become lethargic and had had bloody diarrhoea. The dog’s signs included depressed mentation, dyspnoea (30 to 40 breaths/minute), tachycardia (200 beats/ minute) and hypoglycaemia [2·22 mmol/L (reference interval 3·8 to 7·4)]. The mucous membranes appeared congested and the femoral pulses were weak, while the body temperature was within the normal range. Although supportive therapy was established with the administration of 20 mL/kg/hour of Ringer’s lactate solution iv, to which 20 mmol/L potassium chloride and 10% glucose were supplemented, the blood glucose concentration failed to increase. Over the next hour, the dog deteriorated and died 3 hours later. On the basis of the data available, a presumed diagnosis of sepsis was made. Bacteriological examination of both pre-mortem blood samples and the vials/anaesthetic was performed. One millilitre of each sample – blood (in K3EDTA) and propofol – was plated onto Columbia agar supplemented with 5% sheep blood (bioMérieux, Marcy l’Etoile, France), and incubated in both aerobic and anaerobic conditions at 37°C for 5 days. The API 20 NE system (bioMérieux, Marcy l’Etoile, France) identified O. anthropi (code 1240345, 99·9% ID) from the biochemical profile of the colonies grown from both the blood samples and the propofol. The bacterial identification of the two strains (i.e. that isolated from the drug solution and from the bloodstream), was confirmed after amplification and nucleotide sequencing of a portion of the 16S rRNA gene as previously described (Bergamans et al. 1996). Comparative analyses of nucleotide sequences with those published in the National Centre for Biotechnology Information database were performed (www.ncbi.nlm.nih.gov/blast/Blast. cgi) and they showed 99% homology with an O. anthropi strain isolated from human gastric biopsies (GenBank accession No. AB841137.1). The antimicrobial susceptibility of these isolates was assessed by the agar disk diffusion method, according to the Clinical and Laboratory Standards Institute for Brucella spp. (CLSI 2006), testing the following 10 antimicrobials (OXOID, Basingstoke, UK): 20/10 µg amoxicillin and clavulanic acid (AMC), 30 µg ceftazidime (CAZ), 30 µg cefotaxime (CTX), 10 µg imipenem (IPM) 15 µg erythromycin (E), 5 µg ciprofloxacin (CIP), 5 µg enrofloxacin (ENO), 10 µg gentamicin (CN), 30 µg tetracycline 2

(TE) and 1·25/23·75 µg trimethoprim/sulphamethoxazole (SXT). The isolates were susceptible in vitro to all the antimicrobials tested except for the β-lactams.

DISCUSSION From the evidence presented it is likely that this dog died of sepsis after the administration of O. anthropi-contaminated propofol. Propofol is one of the most popular anaesthesiainduction agents both in human and veterinary medicine. This anaesthetic, which is virtually insoluble in water, has been shown to promote the growth of bacteria (Crowther et al. 1999). Despite its widespread use in veterinary anaesthesia, to the best of the authors’ knowledge, this is the first report that describes severe sequelae resulting from contaminated propofol administration in an animal. It is well known that the proliferation of Gram-negative bacteria is usually associated with the release of endotoxins. Contaminated lipid-based solutions, such as propofol, especially if maintained at room temperature, can contain endotoxins at high concentrations (Jarvis & Highsmith 1984). Although dogs are usually relatively resistant to the effects of bacterial endotoxins (Poli-de-Figueiredo et al. 2008), the presence of such molecules can exert direct toxic effects on the microvascular endothelium of the host and, consequently, on the activation of the immune system and on the coagulation pathways. Indeed, several experimental models of septic shock in animals are based on parenteral administration of endotoxin at sub-lethal or lethal doses. A bolus injection of endotoxin quickly causes a pronounced increase in pro-inflammatory cytokines, usually transient, as described in animals and human volunteers. These animals develop acute cardiovascular depression, raised oxygen consumption and acidaemia. Cardiovascular depression, however, tends to partially recover a few minutes after the injection (Remick & Ward 2005). In this case, the referring veterinarian reported that the ampoule of propofol used to induce anaesthesia was opened the day before, left at room temperature during the day and then stored in the fridge overnight. Factors that might affect the sterility of medications contained in multiple-dose vials include the number of withdrawals made from the vial, the sterility of the techniques employed by the personnel, injection of environmental air into the vial during extraction, duration of use and the storage conditions of the container (temperature, sun exposure, etc.), and whether preservatives are present in the vial (Plott et al. 1990). Given these considerations, the contamination of the anaesthetic solution could have occurred as a result of repeated withdrawals with accidentally contaminated devices (i.e. syringes or after the needle made contact with the contaminated outer surface of the vial plug before withdrawing the drug). Therefore, aliquoting the anaesthetic into single doses immediately after opening, together with proper hygiene practice during all anaesthetic and surgical procedures, could reduce the probability of microbiological contamination. Moreover, the incorrect storage of the anaesthetic at room temperature over several hours may have facilitated the bacterial growth. As reported by Crowther Journal of Small Animal Practice © 2014 British Small Animal Veterinary Association

Lethal iv injection of contaminated propofol

et al. (1999), once contaminated, propofol supports rapid bacterial growth. That study demonstrated that Escherichia coli can heavily contaminate propofol just 6 hours after inoculum at room temperature. To date, information indicating whether propofol supports the growth of O. anthropi has not been available. O. anthropi is a ubiquitous bacillus that is closely related to Brucella species. In terms of clinical settings, it has been found in antiseptic solutions, dialysis liquids, contaminated pharmaceuticals, vials and tubing kits, and, although exposure to this bacillus may be common, clinically significant infections are rare. Recently, however, the increasing number of severe local and systemic infections in debilitated hosts associated with contamination of catheters, drains and intravenous lines suggests the emerging role of this microorganism as a pathogen. Occasionally, this bacterium has also been associated with severe human infections, such as meningitis, pneumonia, endophthalmitis, endocarditis, peritonitis and sepsis in healthy patients, and a catheter-related bloodstream infection is the most common clinical presentation (Kettaneh et al. 2003; Teyssier et al. 2005; Ozdemir et al. 2006; Naik et al. 2013). Although data concerning the clinical condition of this dog during the second hospitalization are incomplete, together with the absence of autopsy evidence (unfortunately the dog’s owner would not authorize necropsy), septic shock caused by the iv administration of a contaminated drug could explain the severity of the clinical picture described above. Indeed, according to the diagnostic criteria defining sepsis (Bone et al. 1992), tachycardia and dyspnoea, together with bloody diarrhoea and hypoglycaemia, are evidence of severe sepsis (Miller et al. 1980; Hinshaw 1985; Sibbald et al. 1993; Livingston et al. 1995; Maitra et al. 2000; Reilly et al. 2001). The propofol ampoule used in this case was opened, and probably contaminated, the day before. The manufacturer recommends that propofol should be used immediately after opening and it should be infused within 12 hours. Nonetheless, other sources of contamination cannot be excluded, such as a contaminated iv catheter, infusion tubing and/or other medical devices. Dental scaling is a well-known cause of bacteraemia. However, it is normally transient without causing sequelae in healthy patients. Moreover, O. anthropi has never been reported to cause bacteraemia after dental care. It is unlikely that a lowvirulent bacterium, such as O. anthropi, could cause severe sepsis in a healthy dog, unless it was released into the bloodstream in a high quantity. However, bacteriological culture demonstrated the presence of the same pathogen both in the drug solution and in the bloodstream, suggesting the anaesthetic as the main source of contamination and highlighting the crucial role of this bacterium in the evolution of the clinical picture. The O. anthropi isolated in this study showed resistance to β-lactams as previously described (Teyssier et al. 2005; Ozdemir et al. 2006) and this peculiarity explains the ineffectiveness of the antimicrobial prophylaxis administered to the dog. However, the rapid evolution of the clinical picture due to the iv administration of a solution heavily contaminated with bacteria and endotoxins suggests that no antimicrobial therapy would have been sufficiently effective to change the clinical course. Journal of Small Animal Practice © 2014 British Small Animal Veterinary Association

To the authors’ knowledge, this is the first case of septic shock due to O. anthropi in a healthy dog. These findings underline the ability of this bacterium to grow in pharmaceuticals and its potential role in developing severe life-threatening infections in healthy animal hosts. The case reported here adds to the spectrum of ways by which microorganisms can cause infections in animals and the importance of correct practice during all anaesthetic and surgical procedures to avoid severe sequelae that could cause a patient’s death. Acknowledgements We wish to thank our colleagues for referring this case. Their desire to seek the reason for the fatality described herein has allowed the writing of this paper, which might be a useful reminder for many veterinarians. Conflict of interest The authors declare that they have no competing interests. References Bergamans, A. M. C., Schellekens, J. F. P., Van Embden, J. D. A., et al. (1996) Predominance of two Bartonella henselae variants among cat-scratch disease patients in the Netherlands. Journal of Clinical Microbiology 34, 254-260 Bone, R. C., Balk, R. A., Cerra, F. B., et al. (1992) Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest 101, 644-165 Clinical and Laboratory Standards Institute. (2006) Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard. 9th edn, M2-A9, vol. 26. National Committee for Clinical Laboratory Standards, Wayne, PA, USA Crowther, J., Hrazdil, J., Jolly, D. T. et al. (1999) Growth of microorganism in propofol, thiopental, and a 1:1 mixture of propofol and thiopental. Anesthesia and Analgesia 82, 475-478 Hinshaw, L. B. (1985) Application of animal shock models to the human. Circulatory Shock 17, 205-2012 Jarvis, W. R. & Highsmith, A. K. (1984) Bacterial growth and endotoxin production in lipid emulsion. Journal of Clinical Microbiology 19, 17-20 Kettaneh, A., Weill, F. X., Poilane, I., et al. (2003) Septic shock caused by Ochrobactrum anthropi in an otherwise healthy host. Journal of Clinical Microbiology 41, 1339-1341 Livingston, D., Mosenthal, A. & Deitch, E. (1995) Sepsis and multiple organ dysfunction syndrome: a clinical-mechanistic overview. New Horizons 3, 276-287 Maitra, S. R., Wojnar, M. M. & Lang, C. H. (2000) Alterations in tissue glucose uptake during the hyperglycemic and hypoglycemic phases of sepsis. Shock 13, 379-385 Miller, S. I., Wallace, R. J. Jr, Musher, D. M., et al. (1980) Hypoglycemia as a manifestation of sepsis. The American Journal of Medicine 68, 649-654 Naik, C., Kulkarni, H., Darabi, A., et al. (2013) Ochrobactrum anthropi: a rare case of pneumonia. Journal of Infection and Chemotherapy 19, 162-165 Ozdemir, D., Soypacaci, Z., Sahin, I., et al. (2006) Ochrobactrum anthropi endocarditis and septic shock in a patient with no prosthetic valve or rheumatic heart disease: case report and review of the literature. Japanese Journal of Infectious Diseases 59, 264-265 Plott, R. T., Wagner, R. F. Jr, & Tyring, S. K. (1990) Iatrogenic contamination of multi-dose vials in simulated use: a reassessment of current patient injection technique. Archives of Dermatological Research 126, 1441-1444 Poli-de-Figueiredo, L. F., Garrido, A. G., Nakagawa, N., et al. (2008) Experimental models of sepsis and their clinical relevance. Shock 30, 53-59 Reilly, P. M., Wilkins, K. B., Fuh, K. C., et al. (2001) The mesenteric hemodynamic response to circulatory shock: an overview. Shock 15, 329-343 Remick, D. G. & Ward, P. A. (2005) Evaluation of endotoxin models for the study of sepsis. Shock 24, 7-11 Scholz, H. C., Pfeffer, M., Witte, A., et al. (2008) Specific detection and differentiation of Ochrobactrum anthropi, Ochrobactrum intermedium and Brucella spp. by a multi-primer PCR that targets the recA gene. Journal of Medical Microbiology 57, 64-71 Sibbald, W. J., Martin, C. M., Cerra, F. C., et al. (1993) The multiple organ dysfunction syndrome. In: Pulmonary and Critical Medicine. Ed R. C. Bone. Mosby-Year Book, St Louis, MO, USA. pp 1-20 Teyssier, C., Marchandin, H., Jean-Pierre, H., et al. (2005) Molecular and phenotypic features for identification of the opportunistic pathogens Ochrobactrum spp. Journal of Medical Microbiology 54, 945-953

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