Clinical Bacteriology MLS 411 Finals 2022-2023 PDF

CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE PART 1.A...

CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE PART 1.A THE FAMILY ENTEROBACTERIACEAE o Yersinia - Generally are GENERAL CHARACTERISTICS NM @37 °C, but M at RT Often referred as ENTERICS - o That is why it is imperative Consists of a large number of that a motility medium that diverse organisms (Frequently does not exhibit a positive encountered in the lab as motility after incubation at causative agents of infections 37 °C overnight should be re-incubated at room in humans) temperature. So that Yersinia species, if present o Enterics because majority would be able to demonstrate its motility of them resigned in the gastrointestinal tract of Motile members have peritrichous flagella except: humans and animals o Tatumella ptyseos which has polar flagella Normal flora of the GI tract of animals and humans All members grow luxuriantly on CHOC and BAP as Gram negative bacilli or coccobacilli moist, smooth, gray, shiny, entire, convex and o Gram negative because they are able to retain opaque colonies. the secondary stain “safranin” o TAKE NOTE: However, their growth in these two o Appear as bacilli media do not have any diagnostic significance Associated in infections almost throughout the body. since members of the family Enterobacteriaceae Ubiquitous share the same growth characteristic and o Found everywhere therefore cannot be differentiated using CHOC or All Enterobacteriaceae are: BAP o Facultative anaerobe, non-spore-former Klebsiella and sometimes Enterobacter produce ▪ Can grow in the presence or absence of air mucoid colonies ▪ Recall: there are only two pathogenic genera o The mucoid colonies they produce is due to the that are able to form spores: genus presence of a polysaccharide capsule on their Clostridium, genus Bacillus surface o Glucose fermenters ▪ That is why if you inoculate a member of the family in Triple Sugar Iron (TSI) agar or in Kligler’s Iron Agar (KIA) they will always attack glucose in 6-12 hours of incubation because all of them are able to ferment glucose ▪ However, not all of them have the capability to ferment lactose or sucrose. Therefore, they can be differentiated through their ability or inability to ferment those other 2 sugars Beta-hemolysis is seen in some strains. o Cytochrome oxidase negative o However, we cannot utilize chocolate agar and ▪ Except: genus Plesiomonas blood agar media to differentiate members of the the only cytochrome oxidase positive family Enterobacteriaceae member of the family Enterobacteriaceae All are catalase positive except for one group of o All reduce nitrates to nitrites except: Erwinia, Shigella species and Xenorhabdus. Pantoea agglomerans, Photorhabdus, and SELECTIVE AND DIFFERENTIAL MEDIA Xenorhabdus ▪ Almost all posses the enzyme nitrate In the laboratory, the isolation of enteric bacteria from reductase except for the mentioned genera clinical specimens is primarily done with the use of above selective and differential media All members are motile except: Selective media – contain inhibitors that suppress o Klebsiella the growth of other bacteria particularly inhibitors that o Shigella NM @ 37 °C & RT suppress the growth of gram-positive bacteria. Therefore, making the media able to support the growth of the gram-negative enteric bacteria RMT - 2024 1 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE PART 1.A Differential media – it provides avenues for an ANTIGENS - substances (proteins) present on the organism to exhibit their specific and unique surface of the bacteria that react with specific biochemical characteristics that can be used in the antibodies laboratory to differentiate them ANTIBODIES (ANTI-SERA) - proteins that react to Primary culture media used in the presumptive antigens. identification of enteric organisms in the laboratory. o Illustration: o Presumptive only because by looking at the o Red arrow – surface colonies of the organism on selective and of bacterium differential media, we cannot just simply point out o Yellow box – antigens the presence of a particular species enteric (proteins in nature); bacteria as many bacteria share the same colony they are capable of characteristics reacting to antibodies EMB, MAC, XLD, HEA, SSA o Purple box – antibodies; specific to antigens o MAC ANTIGENIC VARIATIONS – refers to the variations ▪ contains crystal violet and bile salts that in the antigenic structures of antigens. inhibit gram-positive bacteria o You need to have another antigen that would ▪ it has lactose that helps differentiate if the work or react with these type of antigens organism is able or unable to ferment lactose o This is where antisera come in; because we can ▪ if they are unable to ferment lactose rapidly, use different anti-sera with different specificities we can visualize that through the use of pH according to the type of antigens and its variance indicator incorporated in the media that we wanted to determine o These variations of antigens (on the surface of bacteria) would also mean the presence of a different subtype or different species based on the differences on their antigenic structure Note: 1. Salmonella/Escherichia/Shigella species may look the same under the microscope, but they can be separated into different many serotypes according to the variations of the antigens found on their surface. 2. Serotyping is determined by the agglutination of bacteria with specific antisera to identify variants of O and H antigens. NOTE: 1. For the culture media, especially for the primary isolation of Enterobacteriaceae, Please read Appendix A of Mahon, C. R., Lehman, D. C., & Manuselis, G. (2018). Textbook of diagnostic microbiology-e-book. Elsevier Health Sciences. 2. A huge percentage of the post quiz for Enterobacteriaceae will cover the principle, procedure of inoculation, incubation requirement, and interpretation of these culture media and also in their correlation with the results of the biochemical If there is agglutination, it means that the reagent tests for enteric bacteria. antibodies are able to positively react to that particular SEROTYPING (SEROGROUPING) OF antigen to which that antibody is specific to. That ENTEROBACTERIACEAE would indicate the presence of that particular SEROTYPING - a serologic test that determines serotype that you are looking. species/subtypes of bacteria based on the antigens present on its surface. RMT - 2024 2 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE PART 1.A ANTIGENIC STRUCTURES USED FOR Heat labile polysaccharide found in encapsulated IDENTIFICATION THROUGH SEROTYPING strains. Covers the O antigen (must be removed to detect the O antigen!!) o Hinders the detection of O antigen To remove the K antigen – the organism must be boiled for 30 minutes. Klebsiella, Salmonella, and some strains of E. coli have K antigens. Virulence antigen for Salmonella is categorized as K antigens. SEROGROUPS OF Escherichia coli BASED ON ITS O AND H ANTIGENS H ANTIGEN – FLAGELLAR ANTIGEN An antigen found in the flagellum of motile members Protein in nature Heat labile o Can easily be destroyed through heating Used in the serotyping between species of Salmonella and other motile bacteria. O ANTIGEN – SOMATIC ANTIGEN Heat stable (withstand heating), located in the cell wall All of these are E. coli Lipopolysaccharide of the cell wall composing the Under the microscope: Look the same (gram- endotoxin portion negative bacilli) o Lipopolysaccharide – primary component of Detecting the variations of O and H antigen using gram-negative bacteria’s cell wall specific antisera – identify the serotypes of E. coli ▪ Referred as the O Antigen o O156:H47 – use antisera specific for this There are over 160 types of O antigen for E. coli and serotype specific types are associated with diseases. ROUTE OF INFECTIONS o Variation of O antigen – used to serotype different Since members of Enterobacteriaceae are mostly types of bacteria found as inhabitants GI tract, infections are o E.g., 160 types of O antigen for E. coli – different associated with lapses in personal hygiene via: types of antibodies can be used as reagents to o Fecal-oral route – Most Common! detect the presence of different O antigens ▪ When a person defecates indiscriminately ▪ Different subtypes of E. coli can also be and contaminates water or food, and another identified based on the different O antigens person consumes it, the disease is ▪ Serotypes of E. coli according to variations of transmitted. the O antigen – associated with diseases o Poor sanitation in impoverished countries Shigella have been grouped into four serotypes ▪ Lack of sanitary comfort rooms based on the O antigen: serotypes A, B, C, and D o Colonization of the skin and respiratory tract of 60 types of O antigens exist for Salmonella hospitalized patients (nosocomial) K ANTIGEN – CAPSULAR ANTIGEN/ENVELOPE Common types of infections include: ANTIGEN o UTI, Gastroenteritis, Septicemia Refers to the capsular antigen/envelope antigen Other severe infections include: Only found in those bacteria that possess a o Meningitis and systemic infections associated polysaccharide capsule with Enterobacteriaceae RMT - 2024 3 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE PART 1.A FACTORS AFFECTING VIRULENCE Primary pathogens Virulence – the degree of pathogenicity o Enteric bacteria that are harmful or able to cause Degree of virulence – influenced by virulence factors disease in healthy and immunosuppressed Virulence factors – components of the bacterial cell individuals (surface components or secretions produced by o Home immune status is irrelevant organisms) Escherichia coli (COLON BACILLUS) The organisms ability to cause disease is influenced Comprises the normal microbiota that reside in the GI by their ability to: tract of humans and animals o Adhere to host cells Previously considered as a harmless member of the ▪ Mediated by the lipopolysaccharide or pili or colon biota fimbriae First described by Theodore Escherich in 1885 o Colonize body surfaces Associated with wide range of clinical syndromes o Release certain toxins (endotoxins and Primary marker of fecal contamination in water exotoxins) o E. coli is found in water microbiological analysis. ▪ Endotoxin – lipid portion of the LPS anchored If the water sample tested positive for E. coli, it's beneath the outer membrane, requiring the unsafe to drink because E. coli and other lysing of the bacterial cell before it can be pathogenic Enterobacteriaceae may be present. released o Why E. coli serves as a marker for fecal Endotoxemia – heightened immune contamination? response ▪ Since it is part of the normal microbiota of the Stimulate macrophages to release pro- colon of humans and animals inflammatory cytokines, turning o What makes it significant despite the fact that it is inflammation harmful instead of generally harmless? beneficial. ▪ As a marker, the presence of E. coli in water Inflammation – double-edged sword does not only mean there is only E. coly, but ▪ Exotoxins – proteins or enzymes secreted by rather the presence of E. coli in water also the bacteria means the presence of other pathogens o Invade deeper tissues Closely related to Shigella in terms of DNA ▪ Some bacteria possess virulence factors, homology but can be easily differentiated through allowing them to invade cells and deeper lactose fermentation: tissues resulting to severe diseases o E. coli is lactose fermenter (dysentery) o Shigella is non-lactose fermenter o Ability to transfer plasmids carrying antibiotic CHARACTERISTICS resistance genes Motile (peritrichous flagella), ▪ Can be transferred through conjugation possess an adhesive fimbriae (mediated by pili) and sex pili ▪ Cause another bacterium to acquire the o Fimbriae – adhere and resistance genes → antibiotic resistant colonize host cells, bacteria particularly the intestine ▪ Antimicrobial resistance among o Sex pili – conjugation, and attachment to host Enterobacteriaceae is still a continuous cells problem Has O, H and K antigens TWO BROAD CATEGORIES OF GENERAL LABORATORY FINDINGS ENTEROBACTERIACEAE Fermenter of: Glucose, lactose, trehalose and xylose Opportunistic pathogens o All members of the family are glucose fermenter o Harmless Enteric bacteria, especially among Can produce indole from tryptophan healthy population, but can become harmful if Further ferments glucose in the MR pathway opportunities arise (e.g., immunosuppression, o Can degrade pyruvic acid further in the methyl red and prolonged hospitalization, resulting in the pathway, producing more mixed acids colonization of bacteria) RMT - 2024 4 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE PART 1.A o Presence of mixed acids further acidify the methyl o Serves as a protective mechanism against red medium, dropping the pH < 4.4 colonization and disease initiation o Adding methyl red (indicator) it will turn the o Patients suffering from achlorhydria – higher risk medium red, which is a positive reaction compared to normal persons. Negative for H2S, DNAse, Urease and PAD ▪ Achlorhydria – refers to lack of HCl Cannot utilize citrate as source of carbon PATHOPHYSIOLOGY ENTEROTOXIGENIC E. coli (ETEC) Toxigenic – indicates that the primary equivalence factor of the strain is its ability to produce toxins Associated with diarrhea of infants (major cause!) and adults in tropical and subtropical areas (its includes the Philippines) Most common cause of diarrheal disease commonly referred as TRAVELLER’S DIARRHEA/TOURIST’S, DIARRHEA/ “MONTEZUMA’S REVENGE” o Tourists coming from not a tropical country and visited a tropical country could acquire ETEC strains if they consumed a contaminated water. Before they are able to produce any of these two o Montezuma is an Aztec ruler in Mexico. In his toxins (heat-labile and heat-stable), there should be time, diarrhea associated with ETEC strain is a colonization of ETEC in the small intestine mediated common occurrence. Those who are not from by fimbriae Mexico would suffer diarrhea. Additionally, it was Releases toxins in the small intestine also coined as Montezuma’s revenge because an o Heat – Labile (LT) toxin outbreak of diarrhea occurred following his defeat ▪ Similar in action to cholera toxin with a Spanish emperor. ▪ Has A and B portion – [refer to the image o Thus, this disease is acquired by an ingestion of above] This is the heat-labile toxin [boxed in contaminated food and water. red]. It has one A subunit and five B subunits. Acquired via ingestion of contaminated food and Some literatures, it is written as A5B. Those water toxins that have A and B moiety—B subunit MAJOR CONTRIBUTING FACTORS binds to receptors and host cells. On the o Poor hygiene – it is a fecal-oral route other hand, A subunit is the enzymatically o Reduced availability of sources of potable water active portion of the toxin. This is also o Inadequate sanitation responsible for the catalytic reaction that 106 – 1010 (1000000 – 10000000000) organisms is creates the toxin effect. the infective dose of organisms in o Heat – stable (ST) toxin immunocompetent individuals. LT toxin (heat labile) – consists of 2 subunits, one A o ETEC is an opportunistic pathogen because it subunit and five B subunit. requires a high number of organisms before it can B subunit binds to the GM1 ganglioside receptor, the elicit an infection even to healthy individuals. same receptor used by cholera toxin to bind on the Stomach acidity epithelial cells in the small intestine. RMT - 2024 5 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE PART 1.A o Epithelial cells, the small intestine [boxed in blue], Isolation of lactose fermenting organisms on selective express on its surface the GM2 ganglioside and differential media – In MAC, the organism would receptor. This is the receptor to which the B appear pink because of lactose-fermentation and in subunit of LT toxin binds to the whole toxin. EMB, the organism would create a green-metallic The whole toxin is endocytosed by the epithelial sheen due to vigorous acid production. These cells of the intestine reactions are presumptive test. This does not surely o The entire toxin is endocytosis by the epithelial indicate the presence of E. coli. There are other cell and contained in a vesicle and the bacteria that exhibit the same color reaction— transported into Golgi apparatus. The A subunit Citrobacter and Enterobacter spp. dissociates from the B subunit. Biochemical tests – definitive identification of the The vesicle that contains the whole toxin transports organism the toxin to the golgi apparatus Multiplex PCR – to amplify gene specific for E. coli. o [refer to the boxed in In the Golgi apparatus, the A subunit dissociates from the B subunit. The A subunit moves across the membrane of the vacuole and interact with the G proteins found in the basolateral membrane of intestinal cell. o This G proteins regulate adenylate side planes. In the golgi apparatus, the A subunit dissociates from the B subunit and it moves across the membrane of the vacuole and interact with a membrane protein (Gs) that regulates adenylate cyclase Adenylate cyclase enzyme catalyzes the Lactose fermenting colonies as PRESUMPTIVE transformation of adenosine triphosphate (ATP) to IDENTIFICATION for Escherichia coli cyclic adenosine monophosphate (cAMP). NOTE: The presence of colonies on EMB that exhibit a The effect of this interaction is an increase in cyclic “green metallic sheen” does not definitively indicate adenosine monophosphate (cAMP) level that presence of Escherichia coli since some Citrobacter and Enterobacter species are also able to The increased levels of cAMP stimulate the intestinal produce the same type of colony characteristic on EMB. cells to hyper secrete fluids and electrolytes into the ENTEROINVASIVE E. coli (EIEC) lumen of the intestine. This results in watery diarrhea (similar to The primary virulence factor of the organism is its ability to invade the intestinal cell. cholera). Associated with dysentery (watery diarrhea with Diarrhea associated with ETEC is not bloody. It does blood, leukocytes, and mucus) similar to Shigella not even contain leukocytes or even mucus because spp. the disease pathophysiology does not involve invasion. There is no invasion because there is no Occur in adults and children damage or inflammation that occurred in the small Direct person-to-person transmission via fecal oral intestine. route – primary mode of transmission HEAT – STABLE (ST) TOXIN Clinical infection characterized by: fever, severe The heat-stable toxin after it is released from ETEC— abdominal cramps, malaise, and watery diarrhea. Binds to the transmembrane guanylate peptide PATHOPHYSIOLOGY cyclase receptor found on the surface of the intestine. Enteroinvasive E. coli has the ability to invade the Binding results in an increased accumulation of intestinal cell. Its ability to invade is mediated by cyclic guanosine monophsophate (cGMP) which bacterial invasion, which is coded by the plasmid or a also causes hyper-secretion of fluid and electrolytes. gene that codes for the ability of Entero-invasive E. This results in watery diarrhea coli to invade intestinal walls. DIAGNOSIS Upon binding of Enteroinvasive E. coli to the M cell of Characteristic symptoms such as watery diarrhea, the Peyer patches, the M cell will endocytose the abdominal cramps, and nausea organism and contain it in a vacuole and transported into the basement membrane of the intestine. Once in RMT - 2024 6 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE PART 1.A the basement membrane, the organism is For example, a patient with dysentery submits a stool phagocytosed by macrophages. sample to the laboratory and a gram (-) bacteria is Once they are inside the vacuole, EIEC would lyse isolated, to confirm if it is invasive or not, then we can this phagocytic vacuole, so that they will be able to perform Sereny Test. replicate in the cytoplasm of the phagocytosis— HOW DOES THE TEST GO? invasion. Also, since the organism has a plasmid A suspension of isolated bacterium is prepared, and encoded gene, the capacity of undergo cell-to-cell a drop of that suspension is put into the conjunctiva invasion or movement. This movement is facilitated of the guinea pig. by actin tails. If there is conjunctivitis or redness of the conjunctiva Plasmid mediated bacterial invasion (pInv genes) - into the part where the suspension is dropped, it is a mediate the colonization of EIEC into the colonic positive result for Sereny Test epithelium SIMILARITIES OF EIEC WITH Shigella EIEC lyses phagocytic vacuole and replicate into Both are gram negative, facultative anaerobic and the cell cytoplasm – invasion nonspore-forming bacilli During the infection of intestinal cells, there is what Non-motile (EIEC is a strain of E. coli that is non- we called actin filament formulation, as a result of the motile; Most E. coli are motile) intestinal cell destruction. This actin-filaments would non-lactose fermenters (Generally, E. coli are lactose propel EIEC from a cell into adjacent cell, allowing it fermenters except for EIEC) to further spread. This further facilitates its invasion Negative for lysine decarboxylation into the adjacent intestinal cell. Cross reactions between O antigens (This makes the Movement within the cytoplasm and into the adjacent identification difficult because of similar O antigens) cells is regulated by formation of actin filaments Take note that EIEC and Shigella spp. are somehow o This destroys colonic epithelium cells and results difficult to differentiate because these 2 organisms in inflammation that could progress to colonic share almost similar characteristics. ulceration. DIFFERENTIATION IS DONE THROUGH: ▪ Blood and mucus in the stool of patients with Their infective dose dysentery caused by Invasive E. coli. o EIEC – has higher infective dose (106) Subsequent inflammation would in the o Shigella – as few as 10 organisms recruitment of polymorphonuclear leukocytes Acetate or mucate utilization as carbon source – EIEC into the site of injury. Therefore, the stool of is positive (EIEC is able to utilize acetate or mucate patients with dysentery that is characterized as carbon source while Shigella spp. cannot) by the presence of polymorphonuclear ENTEROPATHOGENIC E. coli (EPEC) leukocytes—blood and mucus Non-toxigenic and non-invasive strain that causes Fecal – oral route (Transmission of Disease) diarrhea Manifestation: Pathophysiology is based on its adhesive properties o Fever, severe abdominal cramps, malaise, and Major cause of infant diarrhea in impoverished watery diarrhea (with blood and along with it are countries mucus and white blood cells) Characterized by watery diarrhea with large amounts o Clinical symptoms similar to Shigella spp. (Must of mucus but no blood! be differentiated from Shigella species) PATHOPGYSIOLOGY (EPEC) Virulence Test: Sereny Test Infection is initiated by EPEC attachment to the What you see on the epithelial cells of the small intestine with subsequent side is Sereny Test. effacement (destruction) of the microvillus It is not a test that (attachment/effacement). differentiates Shigella Bundle-forming pili (BFP) - mediates the initial from EIEC. aggregation of EPEC that leads to the formation of Instead, it determines if micro-colonies on the epithelial cell surface. the isolated bacteria from the patient’s stool sample is EPEC is non-toxigenic and non-invasive, but its invasive or not (Test for Invasiveness) adhesiveness contributes to its virulence. The organism has Bundle-forming pili that facilitates the RMT - 2024 7 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE PART 1.A organism’s attachment to the microvilli of the As the organism attaches, (Red Circle) it has its type intestine. It also causes microcolony infection. 3 secretion system that allows the passage of The presence of microcolony infection on the surface proteins (Green Circle). These proteins come from of the intestinal cells result in the effacement or the bacterium, and it passes through Type III destruction of epithelial cells in the intestine. Secretion (Blue Box). The Tir comes from the bacterium, and it passes through the type 3 secretion system and will attach onto the surface of the host cell and this serves as the receptor for bacterial adhesin, intimin. Intimin is found on the surface of the EPEC. EspF will act on the tight junctions of the intestinal barrier and will cause it to lose its tight junctions allowing the leakage of water and electrolytes into the Gut Lumen resulting in diarrhea. And also, EspF stimulates the intestinal cells to apoptosis (programmed cell death). ENTEROHEMORRHAGIC E. coli (EEC) Causes hemorrhagic diarrhea and colitis o The word hemorrhagic, therefore the symptoms associated is bloody diarrhea and colitis EEC strain 0157: H7 -associated with hemorrhagic diarrhea, colitis, and hemolytic uremic syndrome (HUS) Classic symptoms - watery diarrhea that progresses Also, the attachment of the EPEC unto the surface of to bloody diarrhea with abdominal cramps, low grade the microvilli would allow it to actively secrete proteins fever or an absence of fever. through a Type 3 secretion. Stool contains no leukocytes - distinguishes it o Translocated intimin receptor (Tir) – inserted with dysentery caused by Shigella or EEC into the host’s epithelial cell membrane and infections functions as a receptor for an outer membrane Risk factors - consumption of processed meats such bacterial adhesin, intimin. as undercooked hamburgers served at fast food o Binding of intimin to Tir – results in restaurants, unpasteurized dairy products and apple polymerization of actin and accumulation of cider, bean sprouts, and spinach. cytoskeletal elements beneath the attached PATHOPGYSIOLOGY: HEMORHAGIC DIARRHEA bacteria, loss of cell surface integrity, and death. AND COLITIS o Escherichia coli secreted protein F (EspF) - causes loss of intestinal barrier function through disruption of tight junctions and induces host cell death through apoptosis. RMT - 2024 8 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE PART 1.A EHEC O157:H7 (a.k.a STEC) produces two toxins: The A1 fragment of the B subunit binds to the 28s verotoxin 1 and verotoxin 2 rRNA and causes cessation of protein synthesis. o Verotoxin 1 –phage encoded toxin identical to the Once protein synthesis of the intestinal cell is halted, shiga toxin (Stx) produced by Shigella this would lead to the damage of the intestinal cell dysenteriae type 1. It produces damage to Vero leading to colitis and hemorrhagic diarrhea and Cells (African Green Monkey kidney cells). eventually cell death. o Biologically similar to, but immunologically PATHOPHYSIOLOGY: HEMOLYTIC UREMIC similar from both Stx and verotoxin 1 SYNDROME (HUS) For its pathophysiology, EHEC O157:H7 (aka STEC) produces 2 toxins that are similar to the toxins produced by shigella dysenteriae and those toxins are known as verotoxin 1 and verotoxin 2. Verotoxin 1 is a phage encoded toxin which is identical to the shiga toxin which is produced by the shigella dysenteriae and damages vero cells (African green monkey cells). The verotoxin 1 is similar in terms of function with verotoxin 2 but in terms of immunologic mechanism, Renal endothelial cells express high concentrations they are different because verotoxin 1 is cross of GB3 receptor reactive with antibodies to shigella toxin indicating Endothelial cells appear to be particularly important antigenic similarities while verotoxin 2 is not cross target cells for Shiga toxins. reactive and therefore indicates the lack of antigenic The toxin is transported retrograde in membrane- similarities. Verotoxin 1 and verotoxin 2 share the bound vesicles to the endoplasmic reticulum. same biologic function. They only differ in terms of As it reaches the kidney, the toxin binds with the GB3 immunologic characteristics. receptor present on the endothelial cells. The A Verotoxins and Shiga toxins are similar in quaternary subunit halts protein synthesis of the endothelial cells, structure to the heat-labile toxin of ETEC; they are resulting to cell death of the endothelial cell composed of a pentamer of receptor-binding B The A subunit induces depuration in ribosomal RNA; subunits that are noncovalently bound to a single this leads to cessation of protein synthesis and death enzymatic A subunit. of the cell. o Verotoxins and shiga toxins are similar in Affected tissue – endothelial cell quaternary structure to the heat-labile toxin of Since there is already damage of the endothelial cell ETEC. As it is also composed of the A and B due to cessation of protein synthesis, platelets will subunit to which, the B subunit binds to the begin to adhere. The adherence of platelets to the specific glycolipid receptor found on the surface damage epithelial cell is facilitated by the von of the intestinal walls and endothelial cells called Willebrand factor Globotriaosylceramide(Gb3). Shiga-toxin binding to proteins on the surface of B subunit of the toxin - binds to the specific glomerular endothelium and inactivating a glycolipid called Globotriaosylceramide (Gb3) metalloproteinase called von Willebrand factor- expressed on host cells particularly on the intestinal cleaving protease (VWFCP). villi and renal endothelial cells. o Metalloproteinase – supposed to regulate von Note: High concentrations of Gb3 are found on Willebrand factor so that platelets will not intestinal villi and renal endothelial cells. aggregate. However, because this o Upon binding of the B subunit, of the toxin of the metalloproteinase is already inactivated, there Gb3 receptor of the surface of the intestine, the will be uncontrolled aggregation of platelets in the toxin is internalized and cleaved into 2, and the A endothelial cells as facilitated by von Willebrand subunit dissociates with the B subunit. Once the factor B subunit is able to bind to the Gb3 receptors on o This aggregation and adhesion of platelets in the the surface of the endothelial cell, the toxin is damage endothelial cells would result in micro internalized, and A subunit dissociates from the B thrombi formation subunit and also the A subunit is cleaved into 2. RMT - 2024 9 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE PART 1.A Once the VWFCP is disabled, multimers of von o Thrombocytopenia – due to consumption of Willebrand Factor (vWF) form and initiate platelet platelets, resulting to decreased platelets over activation and cause micro thrombi formation. time o Micro thrombi formation – further narrow the o Microangiopathic hemolytic anemia – due to lumen of the kidneys’ endothelial cells lysis of RBCs as they pass through an obstructed o As it narrows, the RBCs that are passing through lumen because of the micro thrombi formation would become lyse because they are damaged o Kidney failure – due to reduced delivery of as they squeeze through the narrow endothelial oxygen by the RBCs and by the direct damage of cells (hemolysis) the toxin to the renal endothelial cells o As many RBCs are lyse it would result to anemia LABORATORY DIAGNOSIS o Since hemolysis occurs in the small blood Hemolytic diarrhea caused by EHEC strain O157:H7 vessels of the kidneys, the anemia is called is diagnosed through stool culture with serotyping microangiopathic hemolytic anemia o Serotyping using specific anti-sera The arterioles and capillaries of the body become Isolation using MAC using sorbitol instead of lactose obstructed by the resulting complexes of activated o SMAC – MAC with sorbitol platelets which have adhered to endothelium via large o All the strains of E. coli, O157:H7 does not multimeric vWF ferment sorbitol, producing colorless colonies The growing thrombi lodged in smaller vessels Serologic testing destroy red blood cells (RBCs) as they squeeze o Demonstration of a four-fold increase or greater through the narrowed blood vessels, forming in Shiga-toxin neutralizing antibody titer in schistocytes, or fragments of sheared RBCs This patients’ sera with HUS mechanism, known as microangiopathic hemolysis. Since there is uncontrolled platelet aggregation because the metalloproteinase that is supposed to inactivate the von Willebrand factor is already inactivated, there would be continued micro thrombi formation. As a result, platelet consumption increases. Over time, platelets decrease, causing severe thrombocytopenia The consumption of platelets as they adhere to the thrombi lodged in the small vessels can lead to severe thrombocytopenia Since there is already damage of the kidney’s endothelial cells and there is reduced delivery of MUG TEST (4-METHYLUMBELLIFERYL-Β-D- oxygen to the kidneys due to hemolysis, there would GLUCORONIDE) be kidney failure Based on the ability of E. coli to produce the enzyme Kidney Failure – Due to reduced blood flow leading Beta-glucuronidase that hydrolyzes 4- to Ischemia and also due to the direct damage caused Methylumbelliferyl-B-D-Glucuronide → 4- by the toxin to the renal endothelial cells methylumbelliferone. If this substance is present, it Hemolytic Uremic Syndrome – complication of EHEC would exhibit a blue fluorescence under long wave infection UV light at 366 nanometers o When infected with EHEC, the primary symptom is hemorrhagic diarrhea. HUS follows after as a complication Thus, the triad symptoms of HUS – thrombocytopenia, microangiopathic hemolytic O157:H7 does not posses beta-glucuronidase (MUG anemia, and kidney failure. test negative) EHEC O157:H7 rarely produces the enzyme, whereas 92% of other E.coli strains do produce it RMT - 2024 10 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE PART 1.A Note: Sorbitol-negative and MUG-negative red] adhesin that are similar to the bundle-forming colonies are subcultured for serotyping using anti pilus (BFP) of EPEC. serum for the detection of E.coli O157:H7 After adherence of EAEC to the surface of the intestine, mucus secretion is stimulated leading to the formation of a thick biofilm. Thick biofilm – protects the aggregated bacteria from antibiotics and phagocytic cells. Two toxins are also produced: o Enteroaggregative heat stable toxin – antigenically related to the heat-stable toxin of ETEC o Plasmid encoded toxin Both toxins induce fluid secretion Watery diarrhea with no white blood cells and red blood cells present UROPATHOGENIC E. coli Most common cause of UTI, mostly occurring among ENTEROAGGREGATIVE E. coli (EHEC) females o The enclosed proximity vehicle opening and during defecation can be the cause. If the cleaning of the anus is towards the urethral opening direction, then it increases the chance of uropathogenic E. coli contamination, knowing they are motile. Also, the urethra of females is shorter, around 1.5 to 2 inches, compared to males which is around 8 inches. Moreover, the Implicated in persistent watery diarrhea in infants in urethral opening of males is far from the anal developing countries and in travelers to these opening, countries Originate from the large intestine as resident biota, On of the few bacteria associated with chronic contaminate the urethra, and ascend into the bladder diarrhea and growth retardation in children [females] and may migrate to the kidney or prostate Characterized by their auto-agglutination in a [males] “stacked-brick arrangement” o Before a female urinates, the organism has o Their ability to form stacked-brick arrangement as already ascended to the bladder due to the short they bind to the surface of the colonic epithelium urethra of females. is also mediated by their pili o In males, it happens when an organism has able Enteroaggregative – aggregate onto the colonic to successfully colonize the urethra. The epithelium, resulting to auto-agglutination in a organism takes time to ascend into the bladder stacked-brick arrangement and urination can still flush out the organism. PATHOPHYSIOLOGY Pili – primary virulence factor which uropathogenic The “stacked-brick” arrangement is mediated by strain used to adhere to epithelial cells and not be aggregative adherence fimbriae I (AAF1) [boxed in washed off with urine flow RMT - 2024 11 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE PART 1.A Cytolysins – characterized as a hemolysin that can The presence of green metallic sheen in EMB does lyse white blood cells and inhibit phagocytosis and not necessarily mean or confirm E. coli’s presence as chemotaxis other enteric bacteria, such as Citrobacter and Aerobactin – allows uropathogenic strains to chelate Enterobacter, which also produce similar reaction. It iron is still needed to confirm E. coli’s presence through o Knowing that the urinary tract is an iron-pore biochemical test. environment, they are able to chelate iron from GENERAL BIOCHEMICAL REACTION these iron-pore sources, supplying its growth. NOTE EXTRA-INTESTINAL INFECTIONS E. coli that produces These infections happen outside the GI tract. CO2 gas as additional Meningitis – caused by E. coli (and also group B by product of CHO streptococci!) fermentation are called o Acquired in the birth canal during delivery aerogenic. o May also result if contamination of the amniotic Anaerogenic E. coli – a fluid occurs. type of E. coli that does Septicemia – originates from infections in the urinary not produce gas during tract or gastrointestinal tract fermentation. They are Encapsulated strains of E. coli (possess K1 Ag) – also lactose negative, and nonmotile and previously mostly associated with neonatal meningitis and been known as the “alkalescensdispar” group septicemia o This mentioned characteristics [non-motile and Bacteremia – E. coli is a clinically significant isolate lactose negative] also in blood cultures from adults. suggests the presence of Bacteremia in adults may result from a urogenital enteroinvasive E. coli. tract infection or from GI source. Exception to these biochemical Bacteremia is a complication of GI diseases or urinary reactions is the EIEC strain which tract infection, especially if the organism is of invasive share similar biochemical pathogen. Therefore, it can invade the bloodstream. reactions to Shigella species OTHER Escherichia species IDENTIFIFYING CHARACTERISTICS OF E. coli E. hermannii TSI: Acid slant over acid deep (A/A) Formerly called E. coli atypical or enteric group II Circling A in the butt indicates that the organism is Yellow pigmented colonies able to produce CO2 or gas as an additional by- Isolated from CSF, wounds and blood product of glucose fermentation. They are called E. vulneris aerogenic. Isolated from humans with infected wounds H 2S: Negative Half of the strains produced yellow-pigmented MacConkey: Pink-red colonies colonies EMB: Green metallic sheen E. albertii Indole: Positive Associated with diarrheal disease in children Methyl red (MR): Positive These Escherichia species are less pathogenic compared Voges-Proskauer (VP): Negative to E. coli. Citrate: Negative LABORATORY DIAGNOSIS Motility: Positive (most) Deaminase (phenylalanine): Negative Urease: Negative LDC: Positive ODC: Most strains positive ADH: Most strains negative MacConkey dry Eosin methylene XLD dry yellow ONPG: Positive pink colonies due to blue colonies with colonies Fermentation of: lactose fermentation greenish metallic Adonitol: Negative sheen L-arabinose: Positive RMT - 2024 12 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE PART 1.A Cellobiose: Negative Dulcitol: Variable Maltose: Positive D-mannitol: Positive D-mannose: Positive D-sorbitol: Positive Sucrose: Variable Trehalose: Positive D-xylose: Positive RMT - 2024 13 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE KLEBSIELLA Klebsiella pneumoniae (FRIEDLANDER’S BACILLI) GENERAL CHARACTERISTICS Most common isolated species Genus consists of several species: K. pneumoniae Has a polysaccharide capsule – DISTINCT subsp. pnuemoniae, K. oxytoca, K. planticola, K. FEATURE terrigena , K. pnuemoniae subsp. ozaenae and K. o Confers protection against phagocytosis and rhinoscleromatis, K.ornithimlytica, and K.terrigena antimicrobial absorption. Absence of motility – distinguishes Klebsiella spp., o Responsible for the moist, MUCOID, colonies from other members of the family Enterobacteriaceae o Also tests positive for Neufeld Quellung test o Lacks flagella which make them unique among (capsular swelling) the members of the family Enterobacteriaceae ▪ Nuefeld Quellung Test - One of the Usually found in the G.I tract; free living in soil, water presumptive tests that differentiates and plants Streptococcus pneumoniae from other alpha All possess a polysaccharide capsule hemolytic streptococci due to the presence of (encapsulated) polysaccharide capsule on its surface o Found on its surface; this polysaccharide capsule DISEASE ASSOCIATION is responsible for the presence of mucoid Community-Acquired Pneumonia (CAP), nosocomial colonies when these organisms are grown in o CAP - type of pneumonia acquired when you are culture media in the laboratory in a closed environment or an environment where Capable of utilizing citrate as carbon source and can a lot of people are living grow in KCN broth Opportunistic infections among newborns, elderly o They are citrate positive patients, and seriously ill patients o KCN – potassium cyanide; it contains cyanide Wound infections, UTI and bacteraemia which is toxic to bacteria Carbapenemase-producing K. pneumoniae is an ▪ The fact that Klebsiella is able to grow in the important cause of ventilator-associated pnuemonia. presence of potassium cyanide, that makes o Ventilator – machine attached to a sick person them tolerant to the toxic effects of KCN that facilitates breathing and because a tube is Unable to produce H2S inserted to a patient’s nose, that would also serve All are lactose fermenter except K. ozaenae and K. as a portal of entry for K. pneumoniae to enter the rhinoscleromatis respiratory tract o can degrade lactose in the medium and utilize Carbapenemase – these are versatile glucose as source of energy except K. ozaenae Betalactamases that can hydrolyze penicillins, and K. rhinoscleromatis cephalosporins, monobactams, and carbapenems Further ferments glucose via the VP pathway o Making them ineffective in treating K. o Or they can further degrade pyruvic acid, which is pneumoniae the product of glucose fermentation via VP Renders many beta lactam drugs inactive pathway resulting in the production of o Beta lactam drugs become ineffective antibiotics intermediate acetoin in treating infections caused by K. pneumoniae Do not produce indole (except K. oyxtoca!) o Lack enzyme tryptophanase that converts tryptophan into indole, pyruvic acid and ammonia o K. oxytoca – the only species of the genus Klebsiella that is able to produce indole from tryptophan They possess O and K antigens o But it is devoid of the H antigens because they non-motile Urease (+) and ornithine decarboxylase (-) o They are able to hydrolyze urea but unable to decarboxylate amino acid RMT - 2024 1 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE LABORATORY DIAGNOSIS Klebsiella oxytoca Gram stain – Gram negative bacilli Causative agent of antibiotic-associated Culture - large, moist, mucoid colonies on EMB, hemorrhagic colitis (AAHC). MacConkey and XLD Observed during therapy with amoxicillin-clavulanate, o That are lactose fermenting especially when amoxicillin, penicillins, or ampicillin. they are grown in EMB, MacConkey and XLD Sudden onset of bloody diarrhea a after 2 to 7 days IDENTIFICATION OF K. PEUMONIAE SUBSPECIES of treatment with the oral antibiotics (mentioned in the PNEUMONIAE previous bullet). MacConkey: Pink, mucoid colonies Antibiotic therapy disrupts the normal gut microbiota - Pink due to the presence of capsule which is and as their number is reduced, it promotes the made up of polysaccharide overgrowth of Klebsiella oxytoca with subsequent TSI: A/Ⓐ H2S – Negative production of cytotoxin called tilivallin that causes The A/Ⓐ reaction on TSI indicates that the mitotic arrest of host cells and induces apoptosis. organism on top of being a glucose fermenter is a AAHC resolves spontaneously even without lactose fermenter, and the circle in the butt reaction treatment within 2-3 days when the offending indicates that the organism is able to produce CO2 antibiotics are discontinued. as an additional byproduct of glucose fermentation Fig. 2 Description. A and therefore we could conclude that K. colonoscopy revealed pneumoniae is also an aerogenic type of bacteria red mucosa and Gas: Positive granular erosions with Indole: Negative edema from the MR: Negative proximal colon (A) to the VP: Positive distal colon (B). In the Citrate: Positive hepatic flexure area was Deaminase (phenylalanine): Negative the most severe erythema and hemorrhage, friable Motility: Negative mucosa and granular erosions with exudate (Case 1): a colonoscopy showed severe redness and Arginine: Negative hemorrhage in the proximal colon (C) to the distal Lysine: Positive colon (D) (Case 2). Ornithine: Negative Klebsiella granulomatis Urease: Weakly positive Formerly called Donovania Fermentation of: granulomatis then Adonitol: Positive Calymmatobacterium Dulcitol: Variable granulomatis before being D-mannitol: Positive reclassified as Klebsiella Salicin: Positive granulomatis D-sorbitol: Positive Causes granuloma inguinale – a granulomatous Sucrose: Positive disease affecting the genitalia and inguinal area of Growth in KCN using Moeller KCN Broth Base: both men and women who engaged in an unprotected Supplemented with potassium sexual intercourse cyanide (toxic to most bacteria), is Disease is called donovanosis useful in differentiating organisms o Due to similarities in lesions, the disease was based on their ability to grow in the once misdiagnosed as syphilis. presence of cyanide. Transmitted through unprotected sexual intercourse o Tolerant to potassium cyanide After prolonged incubation of weeks to months, (KCN) – organisms that are able to subcutaneous nodules appear on the genitalia or grow in the presence of KCN in the genital area (reddish area in the picture) o Nontolerant – those that are inhibited Used for the differentiation of the members of Enterobacteriaceae on the basis of potassium cyanide tolerance RMT - 2024 2 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE DONOVAN BODIES trachea, bronchi, middle ear, and When infected tissues in the inguinal and genital area orbit. are examined under microscope using Wright’s stain, Acquired via inhalation of Donovan bodies are observed. respiratory droplets containing Are rod-shaped, oval organisms that can be seen in the causative agents. the cytoplasm of mononuclear phagocytes or Diagnosis of rhinoscleromatis histiocytes in tissue samples from patients with depends on the identification of the granuloma inguinale. pathognomonic Mickulicz cells They appear deep purple when stained with Wright's (MCs) which is most prominent stain. during granulomatous phase. o Pathognomonic – strongly suggest or strongly indicates that the infection is caused by that particular causative agent MICKULICZ CELLS (MCs) Foamy macrophages with Illustration of a cell Actual macrophage Closed safety as numerous cytoplasmic vacuoles (mononuclear showing Donovan reference for the phagocyte) showing bodies description of containing viable and nonviable Donovan bodies Donovan bodies Klebsiella bacilli with bipolar Klebsiella ozanae densities (the polar ends of bacillus are Causative agent of chronic atrophic heavily stained) rhinitis called ozena. giving the bacteria Ozena is a disease of the nose in the appearance of a which the bony ridges and mucous closed “safety pin” membranes of the nose waste Klebsiella rhinoscleromatis away Causes rhinoscleroma a chronic granulomatous Transmitted via person-to-person contact. disease. It commonly affects the nasal cavity and nasopharynx, but it can also involve the larynx K. pneumoniae subsp. K. pneumoniae subsp. K. pneumoniae subsp. K. oxytoca Raoultella pneumoniae ozaenae rhinoscleromatis Indole - - - + + MR - + + V + VP + - - + V Urease + - - + + Lysine + V - + + Ornithine - - - - V Malonate + - + - + V. variable reaction In this table, the biochemical properties of Klebsiella ENTEROBACTER spp. GENERAL CHARACTERISTICS One striking characteristics that separate K. oxytoca Inhabits soil and from other the rest of the Klebsiella spp. is its ability water and, to a lesser to produce indole from tryptophan extent, the large K. pneumoniae subsp. pneumoniae is able to bowels of man and degrade pyruvic acid further in the VP pathway, animals resulting in the production of the intermediate product, Often confused with acetoin Klebsiella in terms of growth in MAC RMT - 2024 3 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE o Klebsiella and Enterobacter are lactose ornithine, and arginine. None of the amino fermenting bacteria. Both produce pink colonies acids present in Moeller Decarxylase medium on MAC can be decarboxylated by Pantoea o Some Enterobacter spp. possess a agglomerans. polysaccharide capsule therefore, similar to o Pantoea agglomerans HG XIII – produce a yellow Klebsiella, they also produce mucoid colonies pigment, a plant pathogen. Also share biochemical reactions with Klebsiella spp.: Fig. 19.5 Description. Mucoid, o Citrate positive yellow-pigmented colonies of o Grows in KCN (Potassium cyanide) broth Cronobacter sakazakii growing o Methyl Red test negative on brain-heart infusion agar Growth on EMB – “fisheye” Enterobacter gergoviae – found colonies in respiratory samples and is rarely isolated from Difference Klebsiella spp.: blood cultures. o Positive for ornithine Cronobacter (Enterobacter) sakazakii – produces a decarboxylase (Klebsiella yellow pigment and has been documented as a are negative) pathogen in neonates causing meningitis and o Positive for motility bacteremia often coming from powdered infant (Klebsiella are non- formula. motile) o Isolated from cultures taken from brain CHARACTERISTICS OF Enterobacter E. cancerogenus REACTION E. aerogenes E. cloacae E. geroviae E. sakazakii E. hormaechei taylorae Lysine + - + - - - Arginine - + - + + V Ornithine + + + + + + VP + + + + + + Fermentation of: Adonitol + V - - - - D-arabitol + - + + - - Lactose + + V + - - Melibiose + + + + - - Raffnose + + + + - - Sucrose + + + + - - Salicin + + + + + V Sorbitol + + - - - - Urease - V + - - + Yellow pigment - - - + - - V. variable reaction DISEASE ASSOCIATION abscesses, respiratory, and wound infections. Enterobacter cloacae and Enterobacter aerogenes Enterobacter hormaechei – isolated from human o Two common isolates from the genus. sources such as blood, wound, and sputum. o Isolated from wounds, urine, blood, and CSF Enterobacter asburiae – similar biochemically to Fig. 19.4 Description. Yellow-pigmented Pantoea Enterobacter cloacae and has been isolated from agglomerans on sheep blood agar (SBA). blood, urine, feces, sputum, and wounds. Pantoea (Enterobacter) Enterobacter cancerogenus – formerly agglomerans – gained notoriety Enterobacter taylorae has been associated with with a nationwide (U.S) osteomyelitis after traumatic wounds. outbreak of septicemia resulting from contaminated IV fluids o LOA negative. A.K.A “triple decarboxylases” negative ▪ LOA negative reaction means it doesn’t have the capacity to decarboxylate lysine, RMT - 2024 4 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE CHARACTERISTICS OF Enterobacter Serratia odorifera biogroups In terms of commonalities, all members of the genus o S. odorifera biogroup 1 – isolated are able to pathway, resulting in the production of the predominantly from the respiratory tract and is intermediate. Take note that Ornithine positive for the fermentation of sucrose and Decarboxylation is what differentiates Enterobacter raffinose, and is positive for ornithine from Klebsiella. As stated earlier, Klebsiella does not decarboxylation have the capacity to decarboxylase ornithine. o S. odorifera biogroup 2 – isolated from blood SERRATIA and CSF and is negative for the fermentation Comprise of species that are opportunistic pathogens of sucrose and raffinose, and negative for associated with outbreaks in health care settings. ornithine decarboxylation. o Serratia marcescens ▪ 50% of isolates are indole positive. o Serratia rubidea LABORATORY DIAGNOSIS o Serratia plymuthica BIOCHEMICAL REACTIONS ▪ S. marcescens, S. rubidea, TSI: A/A or K/A, H2S negative S. plymuthica – Pigment o K/A reaction happens particularly in Kligler's production at room Iron Agar to which medium lacks sucrose. temperature. “Prodigiosin” – pink-to-red in o A/A reaction happens in TSI. color o Since they are late lactose fermenters and ▪ In the ancient Church, leavened bread used sucrose fermenters, they will produce a K/A in the ritual for the last supper have always reaction in KIA, indicating glucose has been been stored at room temperature. Since it fermented. However, it is alkaline slant, acid butt, has been stored at room temperature, it because it would take time for the organism to became contaminated with Serratia ferment lactose. In TSI, it would produce A/A marcescens. So, early Christians believed reaction even if it is a late lactose fermenter due that the red color pigment is a manifestation to its sucrose fermenter characteristic. of Christ’s blood. IMVIC: - - + + o Serratia liquifaciens Urease: Negative o Serratia odorifera – emits a dirty, musty odor Lipase: Positive resembling “rotten potatoes” DNAse: Positive o Serratia ficaria Gelatinase: Positive o Serratia fonticola MaConKey: Clear colonies (Lactose negative) o Serratia entomophila o Unable to ferment lactose YET NOTE: All Serratia species except for Serratia HAFNIA fonticola are late lactose fermenters, positive for GENERAL CHARACTERISTICS ONPG. All are DNAse, Lipase, and Gelatinase Hafnia alvei (Enterobacter alvei) positive (differential for other members of the family o Resembles Enterobacter but differentiated by its Enterobacteriaceae). Serratia species are known for inability to ferment lactose, sucrose, sorbitol, their resistance to a wide range of antimicrobials. and raffinose All are sucrose fermenters. o Differentiated from Serratia because it is DNAse o Gelatinase enzyme hydrolyzes gelatin at cold and Lipase (-) temperature. Delayed positive citrate reaction – MAJOR DISEASE ASSOCIATION CHARACTERISTICS OF Hafnia Serratia marcescens – the most clinically significant o In 24 hours of incubation, the organism would species have a negative citrate reaction due to its delayed o Frequently found in hospital-acquired infections reaction. However, subsequent incubation would of the urinary tract result a positive reaction. o Bacteremic outbreaks in nurseries and cardiac Clinical significance is still questionable despite being surgery and burn units. recovered from a number of human sources, o Contamination of antiseptic solution used for joint including stools and wounds. injections has result in an epidemic of septic arthritis. RMT - 2024 5 CLINICAL BACTERIOLOGY BSMLS 3F MLS 411 | LECTURE | FINALS 2022 - 2023 ENTEROBACTERIACEAE o It cannot easily to point out Hafnia alvei as the cause of infection since they are isolated from these specimens along with other pathogenic enteric organisms. Infrequent pathogen, but has been isolated in cases of bacteremia, respiratory tract infections, and gastroenteritis. SUMMARY OF REACTIONS OF Pantoea, Hafnia, and Serratia H. S. S. REACTION Pantoea alvei marcescens liquefaciens LDC - + + + ADH - - - ODC - + + + DNase - - + + (25°C) Gelatinase - + + (22°C) Fermentation of L-arabinose + + + Adonitol - - V - Myo-inositol - + + Sucrose V - + + V. variable reaction Pantoea is a triple decarboxylase negative since it is unable to decarboxylate the three amino acids used in the carboxylation reaction. Serratia differs from the others as they are the only DNAse and gelatinase positive organisms. S. marcescens and S. liquefaciens are sucrose positive despite of late lactose fermenter characteristic. In TSI, it produces an A/A reaction. RMT - 2024 6  Opportunistic pathogens 2 Broad Categories of Primary Pathogens Enterobacteriaceae Shigella Shigella General Characteristics General Characteristics Closely related to Escherichia – both belong to the same tribe, Escherichieae Shigella sonnei – unique member of the genus Shigella – not members of the GI microbiota, and are considered as “primary pathogens” § Ability to ferment lactose slowly (LLF) producing pink colonies on MAC agar only after All species causes dysentery (bloody diarrhea with mucus, blood, and leukocytes) 48 hours of incubation Differentiate it with other members of the genus § ONPG positive Biochemical characteristics: § Non-lactose fermenters § Non-motile Shigella species – fragile and susceptible to Note: Shigella are susceptible to the acid pH of § Anaerogenic, except Shigella flexneri various effects of physical and chemical the stool. Feces suspected of containing § Do not hyrdrolyse urea § Do not produce H2S Biochemically “inert” agents: Shigella spp., must be immediately § Do not decarboxylate lysine Note: On selective and differential media used to § Disinfectants inoculated to primary culture media to § Do not use acetate or mucate as carbon source selectively isolate intestinal pathogens, Shigella appear as § High concentrations of acids and bile increase recovery of the organisms. clear, non-lactose fermenting colonies. Shigella Shigella General Characteristics Serotypes based on their O antigen All Shigella species – possess O antigens, and certain strains possess K antigens Subgroup A – S. dysenteriae (Dysentery bacillus) § Serotypes 1-10 K antigens (capsular) if present, interfere with the detection of the O antigen during § Ex. S.dysenteriae (shiga toxin) serologic grouping. Subgroup B – S. flexneri (Strong’s bacillus) § May be removed by boiling the bacterial suspension to remove heat labile K Subgroup C – S. boydii (S. ambigua) antigen and to expose the heat stable O antigen. Subgroup D – S. sonnei Non motile, do not have H antigen Shigella Shigella Mode of Transmission Disease Association Humans and other large primates are the only known Shigella species cause all t