Antimicrobial Susceptibility Testing
[vc_row][vc_column][vc_column_text] Antimicrobial Susceptibility Testing Robert Lo, Ph.D, D.V.M Antimicrobial compounds, including both naturally and chemically synthesized compounds, have been one of the most important inventions to combat infections. The first commercial antibiotic, penicillin, was accidentally identified of by Alexander Fleming in 1928. Lots of antibiotics with different mechanisms of antimicrobial activity have been discovered or synthesized after the discovery of penicillin. Currently, antibiotics are classified into different groups based on their mechanism of antimicrobial activity. The main groups are: inhibit cell wall synthesis (β-lactams and glycopeptides), depolarize cell membrane (lipopeptides), inhibit protein synthesis (aminoglycosamides, chloramphenicol, lincosamides, macrolides, oxazolidinones, streptogramins, and tetracyclines), inhibit nucleic acid synthesis (Quinolones), inhibit fatty acid synthesis (platensimycin), and inhibit metabolic pathway (sulfonamides and trimethoprim). Antibiotics have saved millions of lives worldwide from diseases and infections once considered life threatening and fatal. With so many groups of antibiotics to treat pathogens, it seems that antibiotics would win the battles against the infection. In fact, antibiotics were also widely used not only in the healthcare industry but also in food and animal industries because of their versatile nature. However, bacterial pathogens have their own ways, antibiotic resistance, to fight with antibiotics and even win the battles. Currently, antibiotic-resistant bacterial pathogens are a global health epidemic, spreading at a rapid rate. A recent report on the casualties related to antibiotic resistance by the world health organization (WHO) depicted an alarming 700,000 lives per year currently, and predicts a disturbing 10 million/year by 2050, ensuring that antibiotic resistance will be the most prevalent cause of death (Brogan and Mossialos, 2016). This epidemic is accelerated by widespread misuse of antibiotics in clinics and agriculture over the last few decades, allowing bacteria to evolve and develop means of resistance (Laxminarayan et al, 2013; Van Boeckel et al, 2014). The clinical microbiology laboratory serves as a valuable ally to clinicians in the diagnosis and treatment of infectious diseases via the isolation of bacteria to confirm susceptibility to chosen empirical antimicrobial agents, or to detect resistance in individual bacterial isolates. Through the use of in vitro antimicrobial susceptibility testing (AST), the laboratory can specifically determine which antibiotics effectively inhibit the growth of a given bacterial isolate, allowing for targeted therapy. Antimicrobial resistance is a growing concern in both community and health care settings; as such, decisions surrounding empirical antibiotic treatment are becoming more complicated, and the importance of routine antimicrobial susceptibility testing to guide therapeutic decisions has increased. Currently, AST is usually performed in a clinical microbiology lab, which necessitates transportation of the patient samples from the healthcare provider to the lab. Susceptibility testing requires a pure culture of the offending pathogen, a process which may take several days. This delay prolongs the time to diagnosis of resistant bacteria and decisions for appropriate and effective antibiotic therapy. Delays in timely administration of appropriate therapeutics lead to increased patient mortality, poor clinical outcomes (Daniels, 2011), and use of broad-spectrum antibiotics, the latter of which promotes antibiotic resistance. Every hour of delay in administrating the targeted antibiotics to septic shock patients, decreases their chances of survival by 7.6% (Puskarich et al, 2011). To survive this evolutionary war against bacteria, obtaining rapid AST results to determine the Minimum inhibitory concentration (MIC) values are of high priority in any clinical setting. MICs of various antimicrobial susceptibility testing (AST) are categorized by various international agencies. These MIC guidelines determine whether an antibiotic is susceptible or not. MIC is defined as the lowest concentration of antibiotic required preventing visible growth of a microorganism in a agar or broth dilution susceptibility test and is used to determine if the infected pathogen is susceptible or resistant to an antibiotic (Bauer et al, 2014; Jorgensen and Ferraro, 2009; Wiegand et al, 2008). A breakpoint is defined as the concentration of an antibiotic that enables interpretation of AST to define isolates as susceptible, intermediate, or resistant (Humphrieset al, 2016; Wiegand et al, 2008). The Clinical and Laboratory Standards Institute (CLSI) provides the most popular guidelines, which are based on pharmacokinetic–pharmacodynamic (PK-PD) properties and mechanisms of resistance [9]. Most European countries follow the MIC cut-offs based on PK-PD properties, and the epidemiological MIC cut-offs (ECOFFS) as determined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (Wiegand et al, 2008). These numbers provide valuable information to physicians to determine the appropriate targeted antibiotic to be administered to the patient. It is important to note here that just having either bacterial identification or AST alone, will not yield clinically significant reports for patient treatment. The combined results from bacterial identification and AST are imperative to meaningfully determine the right antibiotic choice for that particular pathogen (Marschal et al, 2017). Here, we summarize some of the current phenotypic methods, discuss the emerging technologies, and provide scientific opinions on future AST technologies. Disk Diffusion Disc diffusion or the Kirby–Bauer test is one of the classic microbiology techniques, and it is still very commonly used (Bauer et al, 1966; Clinical and Laboratory Standards Institute, 2009; Jorgensen and Turnidge, 2007). Because of convenience, efficiency, and cost, the disc diffusion method is probably the most widely used method for determining antimicrobial resistance around the world. Commercially-prepared, fixed concentration, paper antibiotic disks are placed on the inoculated agar surface (Figure 1). Plates are incubated for 16–24 h at 35°C prior to determination of results. The diameter of the zone of clearance around the disc is measured and compared to the CLSI reference table to determine if the organism is susceptible, intermediate or resistant against the antibiotic agents tested (Reller et al, 2009). This method can test multiple drugs or concentrations on a single agar plate but only yields qualitative results since it doesn’t determine the MIC values which is of high clinical significance for effective patient treatment. Figure 1. Disk diffusion, demonstrating of inhibition zones. Broth Dilution One of the earliest antimicrobial susceptibility testing methods was the macrobroth or tube-dilution method (Ericsson and Sherris, 1971). This procedure involved preparing two-fold dilutions
Canine Parvovirus
Canine Parvovirus CHINESE EDITION IS WRITTEN BY DR. WANG, SHIH-HAO / ENGLISH EDITION IS TRANSLATED AND EDITED BY DR. LIN, WEN-YANG (WESLEY) Abstract The canine parvovirus (CPV) is a common, acute, high morbidity and high morality virus that mainly infect canine population. This virus possess highly survival rate for 5 weeks in the natural environment. It is highly contagious and easily transmitting among canine population by the fecal-oral route through contacting contaminated feces. CPV usually attack digestive system. Sometimes it may induce myocarditis among canine and cause sudden death. All ages, sexes and breeds of dogs could be susceptible to CPV, especially puppies. Clinical sighs of infected dogs may include fever, lethargy, continuous vomiting, continuous diarrhea, stinky viscous diarrhea with blood, dehydration and abdominal pain etc. Canine show signs of the disease would usually die within 3 to 5 days. There are no specific drugs for curing CPV until now. Supportive care such as consuming water-electrolyte fluid is the only present solution to maintain physiological function and relieve symptoms. The infected canine should have medical care as soon as possible; otherwise, more severe conditions like acute dehydration, hypovolemic shock, bacterial infections and death will occur. Infection prevention measures include environmental disinfection and routine vaccines. Pathogens The canine parvovirus (CPV) is an ssDNA virus, which belongs to the species carnivore protoparvovirus 1 within the genus protoparvovirus in the family parvovirus (parvoviridae). CPV is 98% identical to feline panleukopenia virus (FPLV) with variant in six coding nucleotide of structural proteins VP2: 3025, 3065, 3094, 3753, 4477, 4498 that makes CPV-2 infect canine host instead of replicating in cats. Two types of canine parvovirus were discovered – canine minute virus (CPV1) and CPV2, both can attack canine population and canidae family such as raccoons, wolves and foxes. Canine parvovirus may be susceptible to cats without pathogenic, and it is an inapparent infection. CPV2 could stably survive in feces for 5 months with ideal condition. Furthermore, CPV-2a, CPV-2b and CPV-2c type viruses have been isolated and sequenced from animals. Other than targeting on canine, large cats are susceptible to CPV-2a, CPV-2b. CPV-2c type viruses have high prevalence on infecting leopard cats. Figure 1. Model of CPV evolution showing VP2 amino acid differences between each virus and indicating the virus host ranges. (Karla M. Stucker, Virus Evolution In A Novel Host: Studies Of Host Adaptation By Canine Parvovirus, Published in 2010) Epidemiology In 1978, a novel infectious canine disease was firstly occurring in the east coast of America. Within 12 months, scientists identified CPV-2 as the aetiological key of severe symptoms among canine. Due to characters of highly contagious and potential environmental resistance, CPV-2 spread swiftly over entire USA, European countries, Australia and Asia. In 1978, canine parvovirus also invade among canine in Taiwan. Therefore, CPV caused large scale of canine death at the early stage of pandemic. By the establishment and development of CPV vaccine, global wide spreading of CPV has been rarely happen today. However, canine parvovirus still widely exists in domestic dogs and wild canidae. It became one of the canine endemic disease. Pathogenesis Incubation period of CPV-2 lasts 4 to 5 days. The virus mostly attacks rapidly dividing cells especially lymphopoietic tissues, the bone marrow, crypt epithelia of the jejunum, ileum and (in young dogs under 4 weeks old) myocardial cells. Rottweilers, black Labrador Retrievers, Doberman Pinschers, and American Pit Bull Terriers are more susceptible than other species; once they are infected, would suffer severer conditions. Besides, CPV-2 take the major place to affect canine and wild canids. After entering into hosts’ body, CPV-2 firstly replicates in oropharynx lymphoid tissues, mesenteric lymph nodes and thymus gland, then spreading to other lymph nodes, lung, liver, kidney and rapidly dividing tissues (e.g. bone marrow, intestinal epithelial cell and myocardial cell) by the blood stream. 4 to 5 days after, clinical sighs like diarrhea, vomiting, lymphopenia, anorexia, depression, dehydration, hypothermia, thrombocytopenia and neutropenia would appear. Severe dehydration and hypovolemic shock may happen due to lose large amount of fluid and protein by vomiting and diarrhea. Transmission Fecal-oral route is the main transmission pathway of CPV-2. Large amount of virus would be detected in feces of infected canine within 1 to 2 weeks of acute phase. An infected pregnant canine could transmit virus to fetus through placenta. Fomites include contaminated shoes, cages, food bowls and other utensils could serve as CPV transmitting objects also. Clinical forms There are four clinical forms according to distinct signs and lesions: enteric, myocardial, systemic infection and inapparent Infection. A. Enteric form : It is known that CPV-2 caused enteritis symptoms. This form infect host with low virus titers (around 100 TCID50). Symptoms in initial stage are sopor, loss of appetite, acute diarrhea, vomiting, dehydration, slight elevated body temperature, frailty and acting like in extreme pain. Severity of illness vary according to the age of canine, healthy condition, infectious dose of the virus, and other pathogens in intestine and so on. Typical signs of CPV induced enteritis and its course include loss of appetite, sopor, fever (39.5℃-41.5℃) within 48 hours follow vomiting. 6 to 24 hour after vomiting follow watery stool in yellow or white color, mucus stool or bloody stool with stench in severe cases. Due to consistent diarrhea and vomiting, dogs suffer worsen dehydrated condition. Common clinical pathologic examination consist assessing dehydrated condition and significant decreasing of white blood cell of dogs (400 to 3000 /μL). B. Myocardial form: This form only appear in puppies around 3 to 12 weeks of age. Major cases show pups’ age under 8 weeks. Mortality rate is extremely high with myocardial form (almost up to 100%). Clinical signs include irregular breathing, cardiac arrhythmia. Collapse, hard breathing may happen to acute cases follow death within 30 minutes. Most cases would die within 2 days. The subacute form would also die from hypoplastic heart syndrome within 60 days. Nevertheless female adult canine acquire antibodies against myocardial form by vaccination or infection, puppies may
Peritonitis in Canine
[vc_row][vc_column][vc_column_text] Peritonitis in Canine Andy Pachikerl, Ph.D Introduction: Peritonitis is the inflammation of the peritoneum, which is a silk-like membrane that lines the inner abdominal wall of mammalian bodies and covers the organs within the abdomen, and it is usually due to a bacterial or fungal infection. Peritonitis typically results in rupture (perforation) in the abdomen or causes other medical conditions. In canine or dogs this condition is not that different compared to other mammals, which is the peritoneum of the abdominal cavity, becomes inflamed. In canines, this normally occurs because of an injury by physical trauma, disease, a stomach ulcer, or other problems (Latimer, et al., 2019). The most common cause of peritonitis in canine is actually bacterial infection that moves to the abdomen from an external wound or from perforation of an internal organ. An affected dog may seem to be well, then suddenly become ill. The condition is usually painful, and most dogs will show signs of discomfort when they are been touched on the abdomen (Kine, et al., 2019). Classification and Etiology Peritonitis in dogs are classified in various ways, but there are two main methods of identification are (1) localized or diffuse and (2) primary, secondary, or tertiary. Localized septic peritonitis occurs when a small amount of contamination, whether bacterial or fungal is confined. The contamination usually originates from an intraabdominal organ due to secondary surgery or an underlying disease process, such as gastrointestinal (GI) perforation due to a foreign body. Diffuse peritonitis arises from either a larger amount of contamination or a failure to control localized septic peritonitis. Primary septic peritonitis is spontaneous, and it is the infection of the peritoneal cavity with no specific intraperitoneal source of infection detected during surgery or necropsy. This type of peritonitis is more common in cats rather than dogs, with 14% of cats with septic peritonitis having primary septic peritonitis in one study (Costello, et al., 2004; Odonez & Puyana, 2006; Culp, et al., 2009). Primary septic peritonitis is usually monomicrobial, whereas secondary septic peritonitis is often polymicrobial (Mueller, et al., 2001). In one study (Mueller, et al., 2001), bacteria cultured from patients with primary peritonitis were gram positive in 80% of dogs and in 60% of cats. It is postulated that primary septic peritonitis may result from hematogenous or lymphogenous bacterial spread, transmural bacterial migration from the GI tract, or bacterial spread from the oviducts (Culp, et al., 2009; Enberg, et al., 2006). Secondary septic peritonitis is a consequence of an underlying primary disease process and is the most common cause of septic peritonitis in dogs and cats (Mueller, et al., 2001). There are many possible causes of secondary septic peritonitis in animals; the most common are loss of integrity of the GI tract (53% to 75% of cases), foreign-body penetration, perforating ulcers (Figure 1) and surgical wound dehiscence (Mueller, et al., 2001; Costello, et al., 2004). It is the upmost recommendation that canines showing peritonitis signs should seek medical help from a veterinarian for a proper diagnosis and treatment, as it can be a life-threatening condition. Figure 1: Septic peritonitis secondary to jejunal rupture from a perforating ulcer (arrow). Symptoms of Peritonitis in Dogs In most cases, the symptoms of peritonitis in canines are easy to recognize. A dog may seem fine, then suddenly become very ill the following day. They will almost certainly show signs of pain when their abdomen is been touched (DeClue, et al., 2011). Canine that has been injured or wounded seemed fine but suddenly develop the following symptoms the next day then one may consider seeking a veterinarian right away. Fever: – normal body temperature in canine ranges from 99.5 – 102.5 ° F, whereas a body temperature of at least 103.5 ° F (39.7 ° C) can be considered as fever. Vomiting Diarrhoea Black stools Anorexia Lethargy Weakness Abdominal pain Taking unusual positions to relieve pain Low blood pressure Increased heart rate Increased respiration rate Low body temperature Pale gums Jaundice: – Jaundice in canines refers to a build-up of yellow pigment in the blood and tissue, which causes a yellow discoloration in the skin, gums, and eyes. Swelling in the abdomen Ascites: – Ascites in canines is an abnormal build-up of fluid in the abdomen. It is also called abdominal effusion. Arrhythmia: – Arrhythmia in canines is an abnormality in the rhythm of the heart, which can include the speed, strength, or regularity of heart beats. There are cases when peritonitis could become severely complicated by gut microbiota of the dog. These can lead to changes in the dog’s micro flora forever. Such a case is shown as follow. Case Presentation: An 11‐year‐old intact male Poodle was brought to Oklahoma State University, Center for Veterinary Health Sciences (OSU‐CVHS), with a record of vomiting, abdominal distention, increased serum activities of alkaline phosphatase, γ‐glutamyl transferase, and hyperbilirubinemia. The dog had received amoxicillin and clindamycin (dosage and administration route unknown) prescribed by a referred veterinarian. An abdominal ultrasonographic examination revealed a moderate amount of peritoneal fluid, widespread vascular mineralization, and a markedly thickened and irregular gallbladder wall. The abdominal fluid had a total nucleated cell count (TNCC) of 51,000/μL (CELL‐DYN 3500 analyzer; Abbott Diagnostics, Abbott Park, IL, USA), and a total protein of 5.6 g/dL via refractometry. Cytologic examination of direct smears of the abdominal fluid stained with an aqueous Romanowsky stain (Hematek 2000; Siemens Healthcare Diagnostics, Deerfield, IL, USA) demonstrated marked pyogranulomatous inflammation with abundant golden‐to‐dark green pigment, consistent with bile, present extracellularly and within macrophages. Cytologic diagnosis was bile peritonitis. No infectious organisms were identified, and successive aerobic and anaerobic bacterial cultures were negative. The dog was intravenously dosed with ampicillin/sulbactam and enrofloxacin. Exploratory abdominal laparotomy revealed a ruptured gall bladder and a cholecystectomy and duodenotomy were performed. Following surgery, the effusion persisted, despite antimicrobial therapy, and the dog’s condition began to deteriorate. Abdominal fluid collected 6 days postsurgery had a TNCC of 96,600/μL, and a total protein of 5.3 g/dL. Cytologic examination was shown to have pyogranulomatous inflammation with several yeast organisms