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
Feline Blood-Types
[vc_row][vc_column][vc_column_text] Feline Blood-Types Andy Pachikerl, Ph.D Introduction Like humans, cats have blood grouping. However, cats do not have the blood-type O positive. The blood type classification of cats, however, is currently based on the AB system, but like dogs, there are other antigens besides the AB system , such as the Mik blood type. The blood type of cats is composed of mainly A, B, and AB. Type A is the most common, type B is rarer, and type AB is rarest. About 95% of domestic cats are type A blood, and some varieties such as exotic short-haired cats, British short-haired cats, Persian cats, and Scottish folds have a higher percentage of type B blood. As mentioned, the blood-type of cats is mainly A, B, or AB. Peculiarly for AB type, other blood types have innate antibodies. Unlike dogs, cats have antibodies against “non-self” or foreign erythrocytes that can cause lethal immuno -reaction. Therefore, cats cannot obtain a “wrong” blood of different blood types. Before any blood transfusion clinically, cat blood typing is extremely important. Incompatibility of blood type can lead to fatal acute hemolysis reaction, particularly, the blood of a type A cat was given to a type B cat. The anti-type B antibodies found in type A cats have weaker affinity towards each other, causing a mild immune response. However, type B cats have a strong affiliated anti-type A antibody, which can cause a strong immune response. Once type B cat transfuses A-type blood, the red blood cells are rapidly destroyed,resulting in intravascular hemolysis. As little as 1 ml of type A cat blood, it is enough to cause a serious immune reaction in type B cat and then causes absolute lethality. Keep in mind that blood typing is not only extremely vital prior a blood transfusion, but also for cat breeding! Neonatal isoerythrolysis (NI) occurs when a mommy cat with type B blood gives birth to kittens with type A or AB blood and breast-feed them with a high chance of having antigens of type A blood antibodies in the milk, which can cause a severe hemolysis reaction in the kittens. There are no obvious clinical signs to severe hemolytic anemia, but only subtle symptoms such as hemoglobinuria and jaundice. Therefore, we must pay attention to the blood type of the parent before breeding. Despite the best of efforts to prevent them, transfusion reactions may still happen. Depending on the severity, therapy can include glucocorticoids, epinephrine, IV fluids, and discontinuing the transfusion. Fever is usually mild, requiring no treatment. Furosemide should be administered if volume overload occurs. The blood product can be warmed to no more than 37 ° C if hypothermia occurs. Crossmatching blood is the best means of preventing immune-mediated transfusion reactions even if the blood type is known for both cats. It is also imperative blood be collected and administered as aseptically as possible and cats receiving blood products are monitored carefully. The distribution of feline blood types varies by geographic region and breed (Table 1) 1-2. Type-A is the most common type among most cats. There is, however, geographic variation in the prevalence of type-B domestic shorthaired cats. Over 10% of the domestic shorthair cats in Australia, Italy, France and India are type- B. Breed distribution does not vary as much by location because of the international exchange of breeding cats. Over 30% of British Shorthair cats, Cornish and Devon Rex cats, and Turkish Angora or Vans have type-B blood. In contrast, Siamese and related breeds are almost exclusively type-A. Ragdoll cats appear to be unique regarding blood types. Approximately 3.2% of Ragdoll cats are discordant for blood group when genotyping is compared to serology, necessitating further investigation in this breed. Table 1: Selected Blood Type A and B Frequencies in Cats (ignoring AB blood types) The AB blood type is very rare while the frequency of the MiK blood type is unknown. The presence of red blood cell antigens in addition to the AB group may explain why transfusion compatibility is not guaranteed by blood typing; crossmatching is recommended prior to any transfusion 3. Breeding queens, along with blood donors and, if possible, blood recipients should be blood typed. Feline blood-typing methods There are various methods are that can be used to determine blood type, both in a laboratory and veterinarian clinic. Usually in a diagnostic laboratory, they would use various serological methods by adding reagents to samples of blood and observe for any agglutination reactions marking a positive result. In addition, genetic testing is now available to identify blood types A and B using buccal swabs, although it cannot distinguish between A and AB blood groups. In veterinarian clinics, testing may be performed using a card typing system (BIOGUARD® Feline blood -typing kit, New Taipei City, Taiwan and Rapid Vet-H®, Flemington, NJ). If the card-typing system is used, type-AB and type-B results should be confirmed by a referral laboratory as some cross-reactions have been known to occur. Recently,there was an introduction to an alternative novel method for blood typing ie using the gel column agglutination test (DiaMed-Vet® feline typing gel, DiaMed, Switzerland). This test is easier to interpret than the card method, although it requires a specially designed centrifuge that may be cost-prohibitive in some settings. An evaluation of various blood typing kits and methods revealed that accuracy of blood type must be high and working hand in hand with time efficiency. A complete comparison of kits and methods for blood typing is as follow.An evaluation of various blood typing kits and methods revealed that accuracy of blood type must be high and working hand in hand with time efficiency. A complete comparison of kits and methods for blood typing is as follow.An evaluation of various blood typing kits and methods revealed that accuracy of blood type must be high and working hand in hand with time efficiency. A complete comparison of kits and methods for blood typing is
Antibiotic Resistance: Horizontal Gene Transfer
[vc_row][vc_column][vc_column_text] Antibiotic Resistance: Horizontal Gene Transfer 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. Penicillin was the first natural antibiotic to be discovered from Penicillium fungus by Alexander Fleming in 1928. Following the discovery of penicillin, researchers started search for more antibiotics from soil microorganisms, including bacteria and fungi. Streptomycin produced by Streptomyces griseus was found in 1943. Most antibiotics in use today originated from the phylum Actinobacteria with nearly 80% of actinobacterial-derived antibiotics produced by soil-dwelling bacteria of the genus Streptomyces (Barka et al., 2016). 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). With so many groups of antibiotics to treat pathogens, it seems that antibiotics would win the battles against the infection. However, the marked increase of antibiotics resistance pathogens have exceeded new drug discovery, and this resistance occurs shortly after a new drug is approved for treatment. Therefore the emergence and spread of antibiotic resistance among pathogenic bacteria has raised the concerns for public health worldwide. How did antibiotics resistance pathogens appear so quickly? More importantly, how did resistance develop? Antibiotic resistance is ancient and it is expected results of the interaction of many organisms with their environment. Since most antimicrobial compounds are produced by bacteria, it is nature that those antibiotic-producing bacteria also contain self-resistance mechanisms against their own antibiotics. D’Costa et al. identified that most Streptomyces are resistant to an average of seven to eight antibiotics, including newly developed and clinically important therapeutics (D’Costa et al., 2006). In addition, it is believed that non-antibiotics producing environmental bacteria have evolved resistance mechanisms to overcome the action in order to survive via co-existence with antibiotic-producing bacteria. Researchers also identified the presence of gene-encoding resistance to β-lactams, tetracycline, and glycopeptide antibiotics in metagenome samples of 30,000-year-old permafrost (D’Costa et al., 2011). Analysis of the vancomycin resistance gene cluster in this metagenome also revealed conservation of gene sequence and synteny with modern resistance clusters in the clinic as well as protein function and structure. Resistance to antibiotics occurs either by mutation or by acquisition of resistance genes via horizontal gene transfer. It is believed that horizontal gene transfer is the most important factor in the current pandemic of antimicrobial resistance. Though the mechanisms has long been existence, the rate of antibiotic resistant gene spread and the number of resistant strains has increased tremendously over the past few decades because of selective pressure through antibiotic use. The use and misuse of antibiotics in medicine, agriculture, and aquaculture has been linked to the emergence of resistant bacteria (Cabello, 2006; Economou & Gousia, 2015). In addition, the majority of consumed antibiotics are excreted unchanged, and are then introduced into the environment directly or through waste streams (Sarmah et al, 2006). As a result, waste streams become hotspots for the spread of antimicrobial resistance because of the introduction of antibiotic selection pressure into commensals and pathogens (Graham et al., 2011). These increasing environmental exposure to antibiotics has significantly increased antibiotic resistance genes of pathogenic bacteria, especially with the tetracycline resistance gene being 15 times more abundant now than in the 1970s (Knapp et al., 2010). In addition, increasing selection pressure has accelerated bacterial horizontal gene transfer process, increasing the number of resistome elements which reside mobile DNA compared to the pre-antibiotics era (Datta and Hughes, 1983). Role of horizontal gene transfer in spread of antibiotic resistance genes Conjugation Conjugation is the process by which one bacterium transfers genetic material to another through direct contact. In conjugation, DNA is transferred from one bacterium to another. After the donor cell pulls itself close to the recipient via cell surface pili or adhesins, DNA is transferred between cells. Plasmid- mediated conjugation is considered the most prevalent method of horizontal gene transfer mechanisms in nature (Volkova et al., 2014). The acquisition of novel genes by plasmids through mobile genetic elements such as transposons (Babakhani and Oloomi., 2018) or integrons (Akrami et al., 2019), and their ability to replicate in a wide range of hosts, made them perfect vectors for the spread of AMR. Some of known plasmids-mediated antibiotic resistance gene transfer events are the spread of carbapenemase blaCTX-M extended-spectrum β-lactamases (ESBLs), which can hydrolyze β-lactams (Ramos et al., 2013), and spread of quinolone resistance gene (Robicsek et al., 2006). Transposons are found in both gram negative bacteria, including Tn1, Tn3, Tn5, Tn9, Tn10, Tn21, Tn501, Tn903, Tn1525, Tn1721, Tn2350 and Tn3926, and gram positive bacteria, including Tn551, Tn917, Tn4001, Tn4451, and Tn4003. Resistance gene cassettes most frequently associated with class 1 integrons are streptomycin-spectinomycin resistance genes and trimethoprim resistance genes. Class 1 integrins has been found in gram negative bacteria, including Escherichia, Klebsiella, Aeromonas, Enterobacter, Providencia, Mycobacterium, Burkholderia, Alcaligenes, Campylobacter, Citrobacter, Stenotrophomonas, Acinetobacter, Pseudomonas, Salmonella, Serratia, Vibrio, and Shigella (Partridge et al., 2009; Xu et al., 2011 ), and in gram positive bacteria, including Corynebacterium, Aeromonas, Staphylococcus, Streptococcus, Enterococcus, and Brevibacterium (Nandi et al., 2004; Xu et al., 2010). Class 2 integrons have been found in Salmonella, Enterobacteriaceae, Enterococcus, Acinetobacter, and Pseudomonas (Machado et al., 2008; Xu et al., 2010; Xu et al., 2011). Class 3 integrons have been reported in Escherichia, Citrobacter, Klebsiella, Salmonella, Pseudomonas, Acinetobacter, Alcaligenes, and Serratia (Rowe-Magnus et al., 2001; Ploy et al., 2003). Class 4 integrons have been linked to Pseudomonas, Vibrionaceae, Xanthomonas, Shewanella, and other proteobacteria (Clark et al., 2000; Rowe-Magnus et al., 2001). Transformation In transformation, a bacterium takes in DNA from its environment, often DNA that has been shed by other bacteria. If the DNA is in the form of a circular DNA called a plasmid, it can be copied in the receiving