Bacteriology.pdf

Staphylococcus and Streptococcus Dr. Bwalya Daka-Lalusha Common characteristics Among the most pathogenic bacteria for human Both are Gram positives Staphylococcus – form clumps Streptococcus-grow in chains Staphylococci Morphology Gram-positive cocci in clusters (grapelike)...

Staphylococcus and Streptococcus Dr. Bwalya Daka-Lalusha Common characteristics Among the most pathogenic bacteria for human Both are Gram positives Staphylococcus – form clumps Streptococcus-grow in chains Staphylococci Morphology Gram-positive cocci in clusters (grapelike) Non-motile, non-spore forming, non-capsulated They are aerobic organisms pH 7.4-7.6 Species include S. aureus, S.epidermidis, S. saprophyticus Staph. Aureus Beta- hemolytic yellow colonies of gram-positive cocci in clusters on Blood agar. Catalase + Coagulase + Ferments mannitol on mannitol salt agar Reservoir: N/flora on mucosal and on skin Transmission: spread via hands, sneezing, surgical wounds etc Predisposing factors Surgery or break in skin. Pts with severe neutropenia, cystic fibrosis pts, IV drug users in general have more s.aureus on their skin than s. epidermidis Factors for survival Peptidoglycan :gives rigidity to the cell wall, activates the complement. Teichoic acid- major antigenic component , it protects the cell from compliment mediated opsonization. Protein A- has chemotactic, antiphagocytic and anti complimentary action. Toxins and enzymes Toxins and enzymes Staph aureus produce a number of toxin and extra cellular enzymes Toxins 1. Cytolytic toxin (Hemolysin and Leucocidins) 2. Enterotoxin A-E (food poisoning) 3. Toxic shock syndrome toxin- TSST-1 ( fatal multi-system dz with fever, hypotension, Myalgia, vomiting, Diarrhea, erythematous rashes) 4. Exfoliative (Epidermolytic toxin)- scalded skin syndrome (SSS) Enzymes Coagulase –convert fibrinogen to a fibrin clot Lipases Hyaluronidase Nuclease Staphylococcal Disease Skin and soft tissue infxns Foliculitis Furancles (Boils) Wound infxn Breast abscess Carbuncles Cellulitis Styes Impetigo Toxic shock syndrome Soft tissue injuries Infections Musculoskeletal infxns Osteomyelitis Infective arthritis Respiratory infxns Tonsilitis Pharyngitis Sinusitis Otitis Lung abscess Empyema Pneumonia INFXN…… CNS INFECTIONS Brain abscess Meningitis Others UTI Bacteriemia, Pyemia, Endocarditis, Ssepticemia TOXIN ASSOCIATED DZ. Food poisoning Toxic shock syndrome Scalded Skin syndrome Diagnosis Specimens – pus swabs, CSF, Blood culture, urine , sputum Culture – blood agar Gram stain TX- Abscess- incision and drainage, antibiotics ( aminoglycosides and macrolide) Streptococcus Morphology Gram positive cocci in chains or pairs Catalase negative Non-spore forming, non motile Facultative anaerobes Hemolysis – varies with the species (Beta, gamma, alpha) Streptococcus are grouped using known antibodies to the cell wall carbohydrates (Lancefield grp A-G) S. Pyogenes Group A Streptococcus Beta-hemolytic inhibited by bacitracin on B.A PYR+ Reservoir: – human throat and skin Transmission: spread by respiratory droplets or direct contact Pathogenesis Hyaluronic acid capsule- non-immunologic Inhibits phagocytic uptake M-protein: Virulence factor, anti-phagocytic ( the protein is used to type Grp A strep) M12 strains are associated with acute glomerulitis Toxins Streptolysin O- immunogenic, hemolysin and cytolyisin Streptolysin S- hemolysin, cytolysin Exotoxins A-C – pyogenic or erythrogenic exotoxins , cause fever and the rash of scarlet fever. Superantigens – activate helper T cell by bridging T-cell receptors and MHC class II markers without processed antigens Enzymes (spreading factors) Streptokinase – breaks down fibrin clot Streptococcal DNase – liquefies pus and extends the lesion Hyaluronidase- hydrolyses the ground substance of the connective tissue, imp to spread cellulitis. S.Pyogens infxns lab work up swab (pus, throat etc)-M/C/S Blood culture Serology – ASO titers ( antibodies to streptolysin O) ELISA Tx- based on sensitivity patterns (beta lactam drugs, macrolides) Streptococcus agalactiae (GBS) Beta hemolytic Bacitracin resistant CAMP test positive Resv: human vagina(15-25%), GIT Transmission: vertical to newborns Pathogenesis Capsule Beta hemolysin and CAMP factor Disease: neonatal sepsis, meningitis TX- antibiotics (penicillin, ampicillin) S. Pneumoniae alpha hemolytic Optochin sensitive on blood agar Lancet shaped diplocococci (short chains) Lysed by bile Reservior – human upper respiratory tract Transmission: resp droplets Pathogenesis Polysaccharide Capsule is the major virulence factor Ig A protease Pneumolysin O :hemolysin/cytolysin - damages respiratory epi. - inhibits leucocyte resp. burst and inhibits classical complement. Diseases Pneumonia Meningitis Otitis media Sinusitis in children Lab work up: M/C/S Serology – agglutination tests TX and prevention Antibiotics –penicillins, carbapenems, vancomycin Peadiatric vaccine- PCV ( pneumococcal conjugate vaccine) Adults ( PPV- pneumococcal polysaccharide vaccine) Strep. Viridans Alpha hemolytic Resistant to optochin Resv: oropharynx ( normal Flora) Transmission: endogenous Pathogenesis Dextran (biofilm)- mediated adherence onto tooth enamel or damaged heart valves and to each other DZ;- dental carries- S.mutans dextran mediated adherence glues the oral flora onto teeth forming plaques and causing carries Subacute infective endocarditis TX- antibiotics (pen G) THANK YOU Haemophilus Dr. Daka-Lalusha Genus features Small Gram-negative, pleomorphic rod (coccobacilli) Requires growth factors to grow - factor X- Hemin and factor V (Nicotinamide Adenine Dinucleotide ) for growth on blood agar. Grows well on chocolate agar. 2 species of medical importance Heamophilus influenzae Heamophilus ducreyi Haemophilus influenzae Distinguishing features Encapsulated (some species do not have a capsule) Capsulated are divided in 6 serotypes (a-f) Gram –neg rods (coccobacillus) Fastidious – requires factors X and V Epidemiology Strictly human pathogen No known animal or environmental reserviour Nasopharygeal flora of 20-80% of healthy persons Spread is via respiratory droplets Pathogenesis Only encapsulated are invasive Use pili and adhesins to bind epithelial cells. Virulence factors – polysaccharide capsule is able to evade the immune system ( antiphagocytic- confers resistance to C3b deposition in the ) Ig A protease Lipooligosaccharides (LOS) serve as endotoxin Diseases Diseases Meningitis acute purulent meningitis may follow sinusitis or otitis media. Acute epiglottitis this is a dramatic infection in which the inflamed epiglottis and surrounding tissues obstruct the airway. Pts present with fever, sore throat, inspiratory stridor, inflamed swollen cherry-red epiglottis with protrudes in the airway Other infections –Ottitis media, cellulitis, bacteremia, Pneumonia etc Diagnosis Clinical picture Gram stain Blood or CSF culture on chocolate media PCR Antigen detection of the capsule Treatment Antibiotics – usually 3rd gen cephalosporins (Ceftriaxone IV) Prevention Vaccines- conjugate capsular polysaccharide- protein vaccine Polyribitol capsule conjugated to protein vaccine Haemophilus ducreyi Reservoir – human genitals Transmission – direct contact /sexual Diseases- Chancroid Genital ulcers ( enhance the risk of HIV ) Diagnosis –very difficult TX – antibiotics – azithromycin, ceftriaxone, vancomycin Thank you  PSEPSEUDOMONAS UDOMONAS PSEUDOMONAS PTM4310/3520 DR CHILESHE DAVID 05/12/2023 DR CHILESHE DAVID 1 Introduction The genus Pseudomonas originally consisted of a large heterogeneous collection of non-fermentative bacteria that were grouped together because of their morphologic similarity. Pseudomonas aeruginosa is the most important species and the one discussed here. Members of the genus are found in soil, decaying organic matter, vegetation, and water. It is uncommon for carriage to persist in humans as part of the normal microbial flora, except in hospitalized patients and ambulatory, immunocompromised hosts. 05/12/2023 DR CHILESHE DAVID 2 Physiology and Structure Pseudomonas species are usually motile, straight or slightly curved, gram-negative rods typically arranged in pairs. The organisms utilize carbohydrates through aerobic respiration, with oxygen the terminal electron acceptor. Although described as obligate aerobes, they can grow anaerobically using nitrate or arginine as an alternate electron acceptor. They are oxidase and catalase positive. 05/12/2023 DR CHILESHE DAVID 3 Physiology and Structure….. Some strains appear mucoid because of the abundance of a polysaccharide capsule. These strains are particularly common in patients with cystic fibrosis (CF). Some species produce diffusible pigments (e.g., pyocyanin [blue], pyoverdin [yellow-green], pyorubin [reddish-brown]) that give them a characteristic appearance in culture and simplify the preliminary identification. 05/12/2023 DR CHILESHE DAVID 4 Pathogenesis and Immunity P. aeruginosa has many virulence factors, including adhesins, toxins, and enzymes. The delivery system used by Pseudomonas, the type III secretion system, is particularly effective in injecting toxins into the host cell. Adhesins At least four surface components of P. aeruginosa facilitate this adherence: (1) flagella, (2) pili, (3) lipopolysaccharide (LPS), and (4) alginate. 05/12/2023 DR CHILESHE DAVID 5 Pathogenesis and Immunity…… Flagella and pili also mediate motility in P. aeruginosa, and the lipid A component of LPS is responsible for endotoxin activity. Alginate is a mucoid exopolysaccharide that forms a prominent capsule on the bacterial surface and protects the organism from phagocytosis and antibiotic killing. Secreted Toxins and Enzymes Exotoxin A (ETA) is believed to be one of the most important virulence factors produced by pathogenic strains of P. aeruginosa. This toxin disrupts protein synthesis by blocking peptide chain elongation in eukaryotic cells. 05/12/2023 DR CHILESHE DAVID 6 Pathogenesis and Immunity…… A blue pigment, pyocyanin, produced by P. aeruginosa catalyses the production of superoxide and hydrogen peroxide, toxic forms of oxygen. Pyocyanin also stimulates interleukin (IL)-8 release, leading to enhanced attraction of neutrophils. A yellow-green pigment, pyoverdin, is a siderophore that binds iron for use in metabolism. Pyoverdin also regulates secretion of other virulence factors including ETA. 05/12/2023 DR CHILESHE DAVID 7 Pathogenesis and Immunity…… Two elastases, LasA (serine protease) and LasB (zinc metalloprotease), act synergistically to degrade elastin, resulting in damage to elastin-containing tissues. These enzymes can also degrade complement components and inhibit neutrophil chemotaxis and function, leading to further spread and tissue damage in acute infections. Similar to the elastases, alkaline protease contributes to tissue destruction and spread of P. aeruginosa. It also interferes with the host immune response. Phospholipase C is a heat-labile hemolysin that breaks down lipids and lecithin, facilitating tissue destruction. 05/12/2023 DR CHILESHE DAVID 8 Pathogenesis and Immunity…… Exoenzymes S and T are extracellular toxins which facilitates bacterial spread, tissue invasion, and necrosis. This cytotoxicity is mediated by actin rearrangement. Antibiotic Resistance P. aeruginosa is intrinsically resistant to many antibiotics and can acquire resistance to additional antibiotics through horizontal transfer of resistance genes and mutations. A third form of resistance, adaptive resistance, is induced when Pseudomonas is exposed to environmental stimuli or specific antibiotics. In vitro susceptibility tests can identify resistance due to intrinsic and acquired mechanisms but would likely not be able to predict adaptive resistance, underlying the limitations of these lab tests. 05/12/2023 DR CHILESHE DAVID 9 Clinical Diseases Pulmonary infections: range from mild irritation of the bronchi (tracheobronchitis) to necrosis of the lung parenchyma (necrotizing bronchopneumonia). Primary skin infections: opportunistic infections of existing wounds (e.g., burns) to localized infections of hair follicles (e.g., associated with immersion in contaminated waters such as hot tubs). Urinary tract infections: opportunistic infections in patients with indwelling urinary catheters and following exposure to broad-spectrum antibiotics (selects for these antibiotic-resistant bacteria). 05/12/2023 DR CHILESHE DAVID 10 Clinical Diseases …… Ear infections: can range from mild irritation of external ear (“swimmer’s ear”) to invasive destruction of cranial bones adjacent to the infected ear (malignant external otitis). Eye infections: opportunistic infections of mildly damaged corneas. Bacteraemia: dissemination of bacteria from primary infection (e.g., pulmonary) to other organs and tissues; can be characterized by necrotic skin lesions (ecthyma gangrenosum). Endocarditis: is uncommon and is primarily seen in intravenous drug abusers. 05/12/2023 DR CHILESHE DAVID 11 Clinical Diseases …… Figure Gram stain of Pseudomonas aeruginosa Figure showing Pseudomonas aeruginosa surrounded by mucoid capsular material in pigment production cystic fibrosis patient 05/12/2023 DR CHILESHE DAVID 12 Laboratory Diagnosis Microscopy : Observation of thin gram-negative rods arranged singly and in pairs is suggestive of Pseudomonas but not definitive. Culture :Because Pseudomonas has simple nutritional requirements, the bacteria are readily recovered on common isolation media such as blood agar and MacConkey agar. Identification: Rapid biochemical tests are sufficient for the preliminary identification of these isolates. P. aeruginosa grows rapidly and has flat colonies with a spreading border, β-hemolysis, a green pigmentation and a characteristic sweet, grapelike odor. 05/12/2023 DR CHILESHE DAVID 13 Treatment, Prevention, and Control The antimicrobial therapy for Pseudomonas infections is frustrating because (1) the bacteria are typically resistant to most antibiotics and (2) the infected patient with compromised host defenses cannot augment the antibiotic activity. A combination of active antibiotics is generally required for therapy to be successful in patients with serious infections. Attempts to eliminate Pseudomonas from the hospital environment are practically useless given the ubiquitous presence of the organism in water supplies. 05/12/2023 DR CHILESHE DAVID 14  THE END 05/12/2023 DR CHILESHE DAVID 15 MYCOPLASMA PTM4310/3520 DR CHILESHE DAVID Introduction The order Mycoplasmatales is subdivided into four genera: Eperythrozoon, Haemobartonella, Mycoplasma, and Ureaplasma. The most clinically significant genera are Mycoplasma (124 species) and Ureaplasma (7 species), and the most important species is Mycoplasma pneumoniae(also called Eaton agent ). Other commonly isolated pathogens include Mycoplasma genitalium, Mycoplasma hominis, and Ureaplasma urealyticum 05/12/2023 2 Physiology and Structure Mycoplasma and Ureaplasma organisms are the smallest free-living bacteria. They are unique among bacteria because they do not have a cell wall and their cell membrane contains sterols. In contrast, other cell wall–deficient bacteria (called L forms) do not have sterols in their cell membrane and can form cell walls under the appropriate growth conditions. Absence of the cell wall renders the mycoplasmas resistant to antibiotics that interfere with synthesis of the cell wall. 05/12/2023 3 Physiology and Structure….. The mycoplasmas form pleomorphic shapes.Many can pass through the 0.45-µm filters used to remove bacteria from solutions, which was why the mycoplasmas were originally thought to be viruses. However, the organisms divide by binary fission (typical of all bacteria), grow on artificial cell-free media, and contain both RNA and DNA. Mycoplasmas are facultatively anaerobic (except M. pneumoniae, which is a strict aerobe), and require exogenous sterols. The mycoplasmas grow slowly, with a generation time of 1 to 16 hours, and most form small colonies that are difficult to detect without extended incubation. 05/12/2023 4 Pathogenesis and Immunity M. pneumoniae is an extracellular pathogen that adheres to the respiratory epithelium by means of a specialized attachment structure that forms at one end of the cell. The structure consists of a complex of adhesion proteins, with the P1 adhesin the most important. The adhesions interact specifically with sialated glycoprotein receptors at the base of cilia on the epithelial cell surface (and on the surface of erythrocytes). Ciliostasis then occurs, after which first the cilia, then the ciliated epithelial cells, are destroyed. 05/12/2023 5 Pathogenesis and Immunity….. Loss of these cells interferes with the normal clearance of the upper airways and permits the bacteria to spread to the lower respiratory tract. This process is responsible for the persistent cough present in patients with symptomatic disease. M. pneumoniae functions as a superantigen, stimulating inflammatory cells to migrate to the site of infection and release cytokines, initially tumor necrosis factor-α and interleukin (IL)-1 and later IL-6. A number of Mycoplasma species are able to rapidly change expression of surface lipoproteins, which is believed to be important for evading the host immune response and establishing persistent or chronic infections. 05/12/2023 6 Epidemiology M. pneumoniae is a strict human pathogen. Respiratory disease (e.g., tracheobronchitis, pneumonia) caused by M. pneumoniae occurs worldwide throughout the year, with no consistent increase in seasonal activity. Epidemic disease occurs every 4 to 8 years. Disease is most common in schoolage children and young adults (ages 5 to 15 years), although all age groups are susceptible. M. pneumoniae disease is not a reportable disease, and reliable diagnostic tests are not widely available, so the true incidence is not known. 05/12/2023 7 Epidemiology…… M. pneumoniae is not part of the normal mucosa flora of humans; however, prolonged carriage can occur following symptomatic disease. Infants, particularly females, are colonized with M. hominis, M. genitalium, and Ureaplasma species, with Ureaplasma organisms being isolated most often. carriage of these mycoplasmas usually does not persist, a small proportion of prepubertal children remains colonized. 05/12/2023 8 Epidemiology…… The incidence of genital mycoplasmas rises after puberty, corresponding to sexual activity. Approximately 15% of sexually active men and women are colonized with M. hominis, and 45% to 75% are colonized with Ureaplasma. The incidence of carriage in adults who are sexually inactive is no greater than that in prepubertal children 05/12/2023 9 Clinical Diseases Exposure to M. pneumoniae typically results in asymptomatic carriage. The most common clinical presentation of M. pneumoniae infection is tracheobronchitis. Low-grade fever, malaise, headache, and a dry, non-productive cough develop 2 to 3 weeks after exposure. Acute pharyngitis may also be present. Symptoms gradually worsen over the next few days and can persist for 2 weeks or longer. 05/12/2023 10 Clinical Diseases….. The bronchial passages primarily become infiltrated with lymphocytes and plasma cells. Pneumonia (referred to as primary atypical pneumonia or walking pneumonia) can also develop, with a patchy bronchopneumonia seen on chest radiographs that is typically more impressive than the physical findings. Myalgias and gastrointestinal tract symptoms are uncommon. Secondary complications include neurologic abnormalities, pericarditis, hemolytic anemia, arthritis, and mucocutaneous lesions. 05/12/2023 11 Clinical Diseases….. The genitourinary tract is colonized with other Mycoplasma species and Ureaplasma, it is difficult to determine the role of these organisms in disease in individual patients. It is generally accepted that M. genitalium can cause nongonococcal urethritis (NGU) and pelvic inflammatory disease. U. urealyticum can cause NGU, pyelonephritis, and spontaneous abortion or premature birth. M. hominis can cause pyelonephritis, postpartum fevers, and systemic infections in immunocompromised patients. 05/12/2023 12 Table Showing Diagnostic Tests for Mycoplasma Infections 05/12/2023 13 Treatment, Prevention, and Control Erythromycin, tetracyclines (particularly doxycycline), and fluoroquinolones are equally effective in treating M. pneumoniae infections. The prevention of Mycoplasma disease is problematic. M. pneumoniae infections are spread by close contact. Isolation is impractical, however, because patients are typically infectious for a prolonged period, even while receiving appropriate antibiotics. 05/12/2023 14 Treatment, Prevention, and Control Inactivated vaccines and attenuated live vaccines have also proved disappointing. The protective immunity conferred by infection has been low. Infections with M. hominis, M. genitalium, and Ureaplasma are transmitted by sexual contact. Therefore these diseases can be prevented by avoidance of unprotected sexual activity 05/12/2023 15 THE END 05/12/2023 16 BACTERIODES AND ABSCESSES MULENGA NONDE LECTURE OUTLINE 1. INTRODUCTION 2. EPIDIEMIOLOGY 3. MAIN CHARACTERISTISCS 4. PATHOGENESIS 5. CLINICAL DISEASES 6. DIAGNOSIS 7. TREATMENT INTRODUCTION The genus Bacteroides is composed of more than 90 species and subspecies GIT is full of anaerobes, there are more anaerobes than aerobes Most do not cause infection but Bacteriodes is the culprit These bacteria are common in the human body and are the most frequent members of the intestinal microbiota Most important disease causing species of the Genus is Bacteriodes fragilis EPIDEMIOLGY Anaerobes colonize the human body in large numbers (functioning to stabilize the resident bacterial flora) These normal protective organisms produce disease only when they move from their endogenous homes to normally sterile sites Endogenous infections are characterized by the presence of a polymicrobial mixture of organisms. B. fragilis is commonly associated with intra abdominal and genital infections.  MAIN CHARACTERISTICS Gram negative cell wall surrounded by a polysaccharide capsule. Cell wall contains Lipopolysaccharide but has little to no endotoxin activity Pleomorphic rod shape Anaerobic Stimulated by bile able to grow on bile esculin agar Gentamicin resistant Grows rapidly in cultures, grows on blood agar Cont’d B. fragilis Toxin heat-labile zinc metalloprotease toxin i) Chloride secretion and fluid loss ii) IL – 8 secretion CLINICAL DISEASES INTRA ABDOMINAL INFECTIONS GYNECOLOGIC INFECTIONS GATROENTERITIS INTRA ABDOMINAL INFECTIONS Once there is a leakage of bacteria, immediate host response is mounted Much of the contaminating bacteria are removed by the diaphragmatic lymphatics If the peritoneal defenses are unable to eradicate spilled intestinal content, an abscess usually develops. ABSCESS FORMATION Bacterial contamination triggers an increase in vascular permeability, resulting in the influx of plasma and fibrin deposition Bacteria that are adherent to or near the peritoneal mesothelium become trapped in fibrin matrices that develop to mark out the necrotic material and residual bacteria Cont’d These areas become surrounded by a thick, fibrous collagen containing capsule. Inside the capsule are live and dead white blood cells, bacteria, and cell debris. In general, these abscesses resemble those caused by staphylococci Inside peritoneal abscesses, the dominant B. fragilis is often accompanied not only by facultative anaerobes but also by other strict anaerobes, such as clostridia or anaerobic streptococci Cont’d Intra-abdominal abscesses exact a high toll on the host because they can extend to nearby sites and cause necrosis of adjacent tissue They serve as a focus of bacterial contamination from which organisms can enter the bloodstream. The resulting bacteremia may produce septic shock or cause metastatic infections and abscesses at distant sites. Liver abscesses caused by Bacteroides fragilis Synergistic polymicrobial infection involving Bacteroides fragilis and other anaerobes. GYNECOLOGIC INFECTIONS Mixtures of anaerobes are often responsible for causing infections of the female genital tract Examples of these include pelvic inflammatory disease, abscesses, endometritis, surgical wound infections Variety of anaerobes can be isolated in patients with these infections B. fragilis is commonly responsible for abscess formation in uterus and genital tract. GASTEROENTERISTIS Strains of enterotoxin-producing B. fragilis can produce a self-limiting watery diarrhea. Majority of infections have been observed in children younger than 5 years, although disease has also been reported in adults. DIAGNOSIS Intra-abdominal abscesses should be suspected in patients with prior history of appendicitis, diverticulitis, abdominal surgery patients, or other predisposing conditions who present with abdominal pain Various imaging procedures are used to confirm and localize the abscesses. Laboratory Diagnosis Culture Specimens should be collected and transported to the laboratory in an oxygen-free system, promptly inoculated onto specific media for the recovery of anaerobes, and incubated in an anaerobic environment Sequence analysis of species-specific genes (e.g., 16S ribosomal RNA gene) is a reliable but time consuming and expensive approach. Growth of Bacteroides fragilis on Bacteroides bile esculin agar. TREATMENT Antibiotic therapy combined with surgical intervention is the main approach for managing serious anaerobic infections B. fragilis group and other anaerobes produce β-lactamases. The best against these gram-negative anaerobic rods is metronidazole, carbapenems (e.g., imipenem,meropenem), and β- lactam–β-lactamase inhibitors (e.g., piperacillin-tazobactam). Bordetella pertussis MULENGA NONDE  Lecture Outline 1. General characteristics 2. Bacteriology 3. Epidemiology 4. Pathogenesis 5. Immunity 6. Manifestations 7. Diagnosis 8. Treatment and prevention  1. General characteristics 8 species are currently recognized B. Pertussis is the most important Species similar to B. pertussis may cause mild whooping cough Bordetella is an extremely small (0.2 to 0.5 × 1 μm), strictly aerobic, gram-negative coccobacillus Has: – Lipopolysaccharide – Outer membrane proteins (Pertactin ;which is an adhesisn) – LOS – Pilli  1. General characterisitcs Very susceptible to environmental changes and survives only briefly outside the human respiratory tract Has Filamentous HemAgglutinin (FHA) FHA also stimulate cytokine release and interferes with TH1 responses  2. Extracellular Products A. Pertussis toxin (PT): Major virulence factor It is an A-B toxin with a single Enzymatic subunit and 5 binding subunits Binding subunits attach to carbohydrate moieties and the active subunit is internalized Enzymatic subunit ADP-ribosylates a G-protein that affects adenylate cyclase activity This inactivates adenylate cyclase and disrupts multiple intracellular signaling pathways Results include: Insulinemia Lymphocytosis Histamine sensitization  2. Extracellular Products B. Other Toxins: 1. Adenylate Cyclase (AC): Pore-forming Enters host cell and catalyzes ATP –cAMP This inhibits i. leukocyte chemotaxis ii. Phagocytosis and killing This toxin is important for the initial protection of bacteria during early stages of disease  2. Extracellular Products B. Other Toxins: 2. Tracheal Cytotoxin (TCT): Fragment of peptidoglycan generated during cell wall synthesis It is directly toxic to ciliated tracheal epithelial cells causing their death Little or no effect on non-ciliated cells  3. Epidemiology Worldwide distribution, more than 90% in developing countries, children younger than 1 year are at greatest risk Human reservoir host Disease is spread by airborne, person to person by infectious aerosols and is highly contagious Animals may be carriers Asymptomatic carriers are rare except in an outbreak Immunization reduces disease prevalence and age distribution (Infants and adults beginning late adolescence )  4. Pathogenesis When introduced into the respiratory tract, it has remarkable tropism for ciliated cells attaching to cilia itself Adherence mediated by: FHA, Pilli, Pertactin, and binding subunits of PT When attached they immobilize the cilia and progressively destroy the ciliated cells and extrude them from the epithelial boarder The local injury is primarily due to the tracheal toxin The result is an epithelium devoid of the ciliary blanket to prevent substances from going to the lower tract Persistent coughing is the result Organism does not invade deeper tissues  4. Pathogenesis ❑Virulence factors: In addition to local effects on epithelial cells, the other virulence factors contribute in a considerable way The combined action of PT and AC on neutrophils, macrophages, and lymphocytes creates paralysis and even death of these immune effector cells Systemic manifestations such as lymphocytosis, histamine sensitization and insulin secretion are due to the action of circulating PT absorbed at the primary infection site.  5. Manifestations After an incubation period of 7-10 days, pertussis follows a long course consisting of three overlapping stages: 1. Catarrhal phase: Primary feature is profuse and mucoid rhinorrhea persisting for 1-2 weeks Malaise, fever, sneezing, and may anorexia Disease is most communicable at this stage  5. Manifestations 2. Paroxysmal Phase: The presence of persistent cough marks the transition from catarrhal and paroxysmal coughing phase At this stage episodes of paroxysmal cough persist up to 50x a day for 2 – 4 weeks The characteristic inspiratory whoop follows a series of coughs as air is rapidly drawn through the glottis Vomiting frequently follows the whoop The combination of mucoid secretions, whooping cough and vomiting produces a miserable exhausted child barely able to breath Apnea may follow such episodes especially in infants Marked lymphocytosis reaches its peak at this time  5. Manifestations 3. Convalescent Phase: Lasts 3-4 weeks Frequency and severity of the paroxysmal coughing and other features fade gradually Partially immune persons and infants younger than 6 months may not show all typical features of pertussis  5. Manifestations Complications: Pneumonia especially in superinfections with S. pneumoniae Atelectasis is common, confirmed by radiologic examination Convulsions, Subconjuctival or Cerebral bleeding are related to the venous pressure effect during paroxysmal coughing and anoxia produced by inadequate ventilation and apneic spells  6. Diagnosis Isolation of B. pertussis from nasopharyngeal swab or secretions Greatest chance of isolation in specimens collected in catarrhal and early paroxysmal stages Culture (Bordet-gengou) 3-7 days, hemolytic, transparent Direct Fluorescent Antibody Rapid diagnosis  7. Treatment Once paroxysmal stage has been reached, treatment is primarily supportive Antimicrobial therapy is important in early stages and to limit spread to other susceptible hosts Macrolides are preferred for both treatment and prophylaxis Erythromycin is most effective  8. Prevention Active immunization DPT Acellular vaccines ie DTaP – PT – FHA  Treponema pallidum E. M Mumbula The University of Zambia, School of Medicine Department of Pathology and Microbiology  Outline 1. General characteristics 2. Bacteriology 3. Epidemiology 4. Pathogenesis 5. Immunity 6. Manifestation 7. Diagnosis 8. Treatment and Prevention  Objectives By the end of this lecture students should be able to; 1. Understand the bacteriological characteristics of Treponema pallidum 2. Describe the pathogenesis of treponema pallidum 3. Distinguish among primary, secondary, latent, tertiary and congenital syphilis 4. Know the diagnosis and treatment of syphilis  1. General characteristics Treponema pallidum is a slim spirochete which causes syphilis It has regular spirals whose wavelength and amplitude resemble and cockscrew It is readily seen by immunoflourescence, or darkfield microscopy, Live cells show characteristic rotating motility with sudden 90-degrees angle flexions It is extremely susceptible to any deviation from physiologic conditions It dies rapidly on drying, and it is killed by a wide range of disinfectants and detergents  1. General characteristics It is less studied because the organism cannot be grown on artificial culture media, multiplies only a few generations in cell cultures, and is difficult to subculture It grows very slowly and produces few definitive products or structures The sluggish growth is felt to be due to lack of enzymes that detoxify oxygen species, and lack of sufficient energy producing cycles The structure shares G-ve structure but lacks LPS and contains few proteins  2. Epidemiology It is an exclusively human pathogen under natural conditions Infection is acquired from direct sexual contact with a person having active primary or secondary syphilitic lesions Less commonly, the disease may be spread through nongenital contact with a lession eg lips, Transplacental transmission possible within approximately the first 3 years of maternal infection Late disease is not infectious  2. Epidemiology The incidence of new primary and secondary syphilis in developed countries is minimal World-wild syphilis remains a major public health problem with an expected 12 million new cases annually Evidence shows that syphilitic lesions are portals for HIV transmission  3. Pathogenesis The spirochete reaches subepithelial tissue via unapparent breaks in the skin or by passage between epithelial cells of mucous membranes It is aided by an adhesin that binds to fibronectin and elements of the extracellular matrix In the submucosa, it multiplies slowly, stimulating little initial tissue reaction This is relatively due to scantiness of antigens in the T. pallidum outer membrane In experimental infections, the organism spreads from primary site to the bloodstream within minutes and are established in distant sites within hours  3. Pathogenesis As lesions develop, the basic pathologic finding is endarteritis The small arterioles show swelling and proliferation of their endothelial cells This reduces or obstructs blood supply accounting for the necrotic ulceration in primary lesions and other sites Dense granulomatous cuffs of lymphocytes, monocytes, and plasma cells surround the vessels No evidence shows that injury is due to toxins or other products of T. pallidum  3. Pathogenesis The primary lesions heal spontaneously, but the bacteria has disseminated to other sites through lymph nodes and bloodstream The disease is silent until secondary stage develops, and then is silent again with entry into latency Mechanisms by which it evades immunity at this stage is not known The organisms found in secondary lesions are antigenicaly similar to those found in primary chancre  3. Pathogenesis It is postulated that the low antigenic determinants in the outer membrane and the slow growth rate of the organism help in its little trigger of the immune system Injury is often due to prolonged delayed type hypersensitivity  4. Immunity Immunity develops slowly and incompletely Immunity to reinfection does not appear until early latency Host response may clear most, but not all treponems Mechanisms are not clear, but appear to involve both Humoral and cell-mediated immunity In reinfection, there is appearance of antitreponemal antibodies which are able to immobilize and kill the bacteria Cell-mediated immunity is more dominant in syphilitic lesions with T cells, and macrophages present Activated macrophages play an important role in clearance of treponems variable T cell suppression may link to the stages of syphilis In the immunocompromised, syphilis is more aggressive  5. Manifestations A. Primary Syphilis: ▪ The primary syphilitic lesion is a papule that evolves into an ulcer at the site of infection ▪ This appears on external genitalia or the cervix ▪ The ulcer becomes indurated and ulcerates but remains painless, though slightly sensitive to touch ▪ The fully developed ulcer with a firm base and a raised margins is called a chancre ▪ Firm, painless enlargement of the regional lymph nodes usually develops within a week of the primary lesion and may persist for a month ▪ Incubation period until the appearance of chancre is about 3 weeks ▪ It heals spontaneously after 4-6 weeks  5. Manifestations B. Secondary Syphilis: ▪ Develops about 2-8 weeks after the appearance of the chancre ▪ The primary lesion has usually healed, but may still be present ▪ Characterized by ▪ symmetrical mucocutaneous macopapulary rash ▪ Generalized nontender lymph node enlargement ▪ Fever ▪ Malaise ▪ Other manifestations of systemic infection  5. Manifestations B. Secondary Syphilis: ▪ Skin lesions are distributed on the trunk and extremities often including palms, soles, and face ▪ About 1/3 of pts may develop painless warty erosions called Condylomata lata ▪ The erosions usually develop in warm moist sites such as perineum and genitals ▪ All lesions of secondary syphilis have spirochetes and are highly infectious ▪ They resolve spontaneously after a few days to many weeks ▪ But infection resolves only in about 1/3. remaining 2/3 go into latency  5. Manifestations C. Latent Syphilis: ▪ This is the stage in which no clinical manifestations are present ▪ Continued infection is evidenced by serologic tests ▪ In the first few years, latency may be interrupted by less severe relapses of secondary syphilis ▪ In late latent syphilis (>4yrs), relapses cease and pt becomes resistant to reinfection ▪ Transmission to others is only possible from relapses, transfusion or other contacts with blood products ▪ Mothers may transmit to fetus in latency ▪ About1/3 of untreated pts do not progress beyond this stage  5. Manifestations D. Tertiary Syphilis: ▪ Manifestations may appear as early as 5yrs after infection, but characteristically occur 15-20 yrs later ▪ Manifestations depend on the site involved: most important are neural and cardiovascular  5. Manifestations D. Tertiary Syphilis: ▪ Neurosyphilis: ▪ Damage is produced by meningovasculitis and degenerative parenchymal changes in virtually any part of the nervous system ▪ The most common entity is a chronic meningitis with fever, headache, increased cells and protein in the CSF ▪ Cortical degeneration of the brain causes mental changes ranging from decreased memory to hallucinations ▪ In the spinal cord, demyelination of posterior columns, dorsal roots and dorsal root ganglia, produces a syndrome called Tabes dorsalis which includes ataxia and loss of sensation ▪ Most advanced CNS findings include combinations of neurologic deficits and behavioral disturbances called paresis (Personality, Affect, Reflexes, Eyes, Sensorium, Intellect, Speech)  5. Manifestations D. Tertiary Syphilis: ▪ Cardiovascullar syphilis: ▪ Due to artaritis involving the vasa vasorum of the aorta and causing medial necrosis and loss of elastic fibres ▪ The usual result is dilatation of the aorta and aortic valve rings ▪ This in turn leads to aneurisms of the ascending and transverse segments of the aorta or aortic valve incompetence ▪ The expanding aneurism can produce pressure to necrosis of adjuscent structures or even rapture ▪ A localised granulomatous reaction called Gumma may be found in skin, joints, or other organs  5. Manifestations E. Congenital Syphilis: ▪ Fetuses are susceptible to syphilis only after the 4th month of gestation ▪ Adequate treatment of the mother before that time prevents fetal damage ▪ Routine serological testing is done early in pregnancy in high risk groups and repeated in the last trimester ▪ Untreated maternal infection may result in fetal loss or congenital syphilis which is analogous to secondary syphilis in adults ▪ Child may have rhinitis and papulary rash ▪ Bone involvement produces characteristic changes in the entire skeletal system ▪ May have anemia, thrombocytopenia, and liver failure  6. Diagnosis A. Microscopy: ▪ T. pallidum can be seen in dark field microscopy in primary and secondary lesions ▪ The observed characteristics are cocksrew morphology and motility ▪ A negative result does not exclude syphilis  6. Diagnosis B. Serologic Tests: ▪ Most used method of diagnosis ▪ Detects antibodies directed against either lipid or specific treponemal antigens  6. Diagnosis C. Non treponaemal tests: ▪ They measure antibodies against cardiolipin, a lipid complex ▪ Anticardiolipin antibody (Reagin) is detected by Rapid plasma Reagin (RPR) and Venereal Disease Research Laboratory (VDRL) ▪ Results are +ve in the early stages of primary lesions except in immunocompromised, and slowly wane in later stages if disease ▪ In neural syphilis VDRL test on CSF may be positive when serum reverts to negative ▪ These test are nonspecific; so may have false positives in autoimmune diseases or involving substantial tissue or liver damage; such as SLE, viral hepatitis, infectious mononucleosis, and malaria. Occasionally in pregnancy and HIV ▪ They are cheap and preferred for screening ▪ Test are valuable for monitoring treatment  6. Diagnosis D. Treponaemal tests: ▪ They detect antibody specific for T. pallidum ▪ They are considerably more specific than cardilipin-based tests ▪ Their primary role is to confirm positive RPR and VDRL tests ▪ They are not useful in screening or after therapy because once positive, they are always positive (except in immunocompromised)  7. Treatment and Prevention ▪ They are sensitive to penicillins in all stages ▪ Those hypersensitive to penicillin are treated with doxycyclin ▪ Safe sex practices are the most effective preventive measure Enteric Bacteria – Watery & Bloody Diarrhea  Outline Introdution to enteric bacteria Watery diarrhoea causing bacteria E.coli Bloody diarrhoea causing bacteria Shigella Salmonella E.coli Campylobacter  Enteric Bacteria Enteric bacteria are a group of bacteria that primarily inhabit the intestines of humans and animals. They play a significant role in the gastrointestinal (GI) system and can have both beneficial and harmful effects on the host Diarrhoea – abnormal faecal discharge characterised by frequent and fluid stool Resulting disease of the intestines involving increased fluid and electrolyte loss Diarrhoea can either be watery or bloody  Watery diarrhoe Caused by the loss of electrolytes and fluids from small intestine  E.coli Gram negative Bacilli Reseviour – gut Transmission – consumption of contaminated water or food e.g undercooked meat products and raw milk  E.coli Subtypes of E. coli causing watery diarrhoea; 1. Enterotoxigenic E. coli (ETEC) - produces two toxins that cause watery diarrhoea. Common cause of traveller's diarrhoea  Pathogenesis Microbe overcomes the host defenses untill it reaches the small intestines colonization of the small bowel by organisms that survived their passage across the stomach These strains produce one or both enterotoxins, called LT and ST These toxins act by changing the net fluid transport in the gut from absorption to secretion. LT is structurally identical to cholera toxin and activates the adenylate cyclase–cAMP system  E.coli 2. Enteropathogenic E. coli (EPEC) causes watery diarrhoea. A common cause of diarrhoea outbreaks in nurseries  Pathogenesis Diarrhoea caused by disruption of the microvilli surface and alteration of host cellular signalling processes organisms adhere to target epithelial cells with help of adhensins-intimin Effacement of microvilli which disrupts disrupts their normal function and leads fluid loss and to inflammation leading to malabsorption of nutrients and osmotic imbalance in the gut leads to increased secretion of fluid and electrolytes into the intestinal lumen. The excess fluid cannot be adequately absorbed by the damaged intestinal lining, resulting in watery diarrhoea.  E.coli Symptoms- abdominal cramps and diarrhoea, which may be bloody. Fever and vomiting may also occur. Most patients recover within 10 days, in a few cases the disease may become life-threatening Diagnosis – stool culture Treatment – fluid replacement  Bloody diarrhoea Infections of the intestinal tract leading to local tissue damage or inflammation The blood is a trace of an invasion of bowel tissue Transmission- faecal–oral route Spread and multiplications - Shigella and Salmonella invade intestinal cells and enter the lamina propria. Shigella can spread directly from cell to cell. EHEC colonizes the surface of the mucosa.  Shigella The genus Shigella consists of four species distinguished serologically by the O antigen of their lipopolysaccharide (LPS) Species are named S. dysenteriae (serogroup A), S. flexneri (group B) S. boydii (group C), and S. sonnei (group D Gram-negative rods, nonmotile Reservoir - human colon only (no animal carriers) Few infective dose Incubation period – 1-4 days  Pathogenesis These organisms are relatively acid resistant during certain phases of their growth When exposed to acid, the organisms survive but are less able to invade cells Once they reach the small bowel (an alkaline or neutral environment) and begin to grow again, their acid resistance is repressed, and the invasive phenotype is restored Invade M cells and epithelial cells Release endotoxin that triggers inflammation Cytotoxin kills intestinal epithelial and endothelial cells Result is surface erosion in the gut wall, or an ulcer  Shigella Symptoms lower abdominal cramps, tenesmus, diarrhoea first watery then bloody, shallow ulcers Severity depends on the age of patient and the strain; S. dysenteriae type 1 with toxin most severe Diagnosis-isolation from stool during illness and culture on selective media Treatment Mild cases: fluid and electrolyte replacement only Severe cases: antibiotics Prevention - proper sanitation (sewage, clean drinking water, hand washing)  E.coli Subtypes of E. coli causing bloody diarrhoea; 1. Enterohaemorrhagic E. coli (EHEC) Produce verotoxin identical to shiga (shigella) toxin Attach to the mucosa of the large intestine Produced toxin has direct effect on intestinal epithelium resulting in diarrhoea 2. Enteroinvasive E. Coli (EIEC) causes bloody or non-bloody diarrhoea  Salmonella Gram negative bacilli Non-acid fast Non-capsulated Non-sporing Aerobes and facultatively anaerobic Characterized by O, H and Vi antigens 2 species; S. enterica and S.bongori Medically important is the Salmonella enterica serovar Typhimurium = Salmonella Typhi  Salmonella Motile with the help of peritrichous flagella Catalase positive Oxidase negative Natural reservoirs - domestic and wild animals Humans also serve as a natural host Transmission fecal oral route Once the meat is contaminated consumption of contaminated poultry and meat products  Virulence factors Ability to invade cells A complete lipopolysaccharide coat Replicate intracellularly Salmonella cytolethial distending toxin (S-CDT) After being ingested it needs to encounter barriers of immune system that is the gastric acid in the stomach.  Pathogenesis After being ingested it encounters barriers of immune system that is the gastric acid in the stomach It binds to intestinal epithelial cell with use of fimbriae and invades mucosa Invasion of the mucosa causes the epithelial cells to synthesize and release proinflammatory cytokines ( IL-1, IL-6, IL-8) that may be responsible for damage to the intestine Symptoms; fever, chills, abdominal pain, leukocytosis, and diarrhea. Stool may contain polymorphonuclear leukocytes, blood, and mucus.  Diagnosis and treatment Specimen; blood, stool and urine for culture examination Hematological tests, Biochemical tests, Slide Agglutination test (Widal test), Serological test Media used include Xylose Lysine Deoxycholate (XLD), Salmonella-Shigella (SS) as selective medias 1st week culture culture of blood 2nd week and 3rd week serum can be used for antigen detection by widal test, also stool and urine Treatment; Repleninshipng body fluids and electrolytes, Hospitalized use IV fluids, Antibiotics including Ciproloxacin Azithromycin and Ceftriaxone  Campylobacter Genus: Campylobacter Campylobacter spp. are curved or S-shaped Gram-negative rods Oxidase positive Non-spore forming They are microaerophilic and thermophilic – growing well at 42OC  Campylobacter Among the most common causes of diarrhoea C. jejuni, C. coli, C. fetus, C. lari, C. helveticus, and C. hyointestinali  Campylobacter Resevoir: intestinal tracts of humans, cattle, sheep, dogs, cats, poultry Transmission: Infections are acquired by consumption of contaminated food - poultry, milk or water. Oral fecal route About 80% cases of all human campylobacteriosis are due to consumption of contaminated poultry Incubation period: 2-11 days  Virulence factors Flagella – responsible for its characteric corkscrew motility Adhesion proteins Serine protease Cytolethal distended toxin  Pathogenesis Low infectious dose (500-800 organisms) Invades mucosa of the colon Produces a thermo labile enterotoxin ,CDT, which causes cytotoxicity CDT also triggers secretion of IL-8 as an immune response Apoptosis and thus leading to inflammatory diarrhoea (bloody watery)  Symptoms and diagnosis Symptoms Bloody diarrhoea, fever, and abdominal cramps Generally self-limiting in 3 - 5 days but may last longer Diagnosis Culture on Campylobacter or Skirrow agar at 42°C Immunoassays such as ELISA and FAT Molecular techniques such as PCR  Treatment Mostly supportive via fluid and electrolyte replacement Erythromycin, fluoroquinolones, penicillin resistant Urinary Tract Infections  UTI Lower UTI Upper UTI cystitis Pyelonephritis 2  UTI May also be classified as 1. Isolated UTI a single episode of lower tract infection occurs frequently in females and is rarely complicated. 2. Recurrent UTI is >2 infections in 6 months, or 3 within 12 months. 3  Predisposing causes of urinary tract infection Incomplete emptying of the bladder, bladder outflow obstruction, bladder diverticulum, neurogenic bladder A calculus, foreign body or neoplasm. Incomplete emptying of the upper tract, dilatation of the ureters associated with pregnancy, or vesicoureteric reflux VUR. 4  Oestrogen deficiency, which may give rise to lowered local resistance. Colonisation of the perineal skin by strains of Escherichia coli expressing molecules that facilitate adherence to mucosa Diabetes. Immunosuppression 5  Acute cystitis urinary infection of the lower urinary tract, principally the bladder. Acute cystitis ♀ > ♂. The primary mode of infection is ascending from the periurethral / vaginal and fecal flora. The diagnosis is made clinically. 6  PRESENTATION There is frequent voiding of small volumes, dysuria, urgency, suprapubic pain, hematuria. 50% of women will have an episode of cystitis in their lifetime – Fever and systemic symptoms are rare. 7  INVESTIGATIONS Urinalysis WBCs in the urine & hematuria may be present. Urine culture is required to confirm the diagnosis & identify the causative organism. 8  RADIOGRAPHIC IMAGING In uncomplicated infection of the bladder, radiologic evaluation is often not necessary recurrent UTI, Fever, UTI in children U\S VCUG 9  Treatment Management for acute cystitis consists of a short course of oral antibiotics. –TMP SMX –Nitrofurantoin –Quinolones Short oral course 3-5 d ♀ Days for ♂ & child 7 d 10  Honeymoon Cystitis Is the term for a UTI that often occurs after sexual activity. Sexual activity can push infecting bacteria into the urethra resulting in an infection. 11  Recurrent UTI may be due to – Reinfection (i.e., infection by a different bacteria) or – Bacterial persistence (infection by the same organism). Bacterial persistence is caused by – the presence of bacteria within calculi – antimicrobial resistance, – patient noncompliance with therapy 12  Recurrent UTI 1. Bacterial persistence presence of bacteria within a site in the urinary tract, leads to repeat episodes of infection. Such sites include urolithiasis anywhere in the urinary tract, chronic bacterial prostatitis, obstructed or atrophic kidney 13 The recurrent UTIs will not resolve until this underlying problem has been addressed and corrected. e.g. surgical removal of the infected source (such as urinary calculi) 14  Recurrent UTI 2. Reinfections usually occur after a prolonged interval (months) from the previous infection and are often caused by a different organism than the previous infecting bacterium 15 Pyelonephritis 16 Definition It is Bacterial infection of the renal pelvis, tubules and interstitial tissue of one or both kidneys potentially organ- and/or life-threatening infection that characteristically causes some scarring of the kidney with each infection and may lead to significant damage to the kidney Pathophysiology and aetiology Infection usually ascends from the urethra most bacterial causes bowel organisms eg Ecoli (70-80%) Hospital-acquired infections may be due to coliforms and enterococci. Haematogenous spread is rare eg Staph aureus Frequently due to ureterovesical reflux 17  53-72 18-57 Causes of UTI's Outpatients Inpatients (%) (%) Escherichia coli 53-72 18-57 Coagulase negative 2-8 2-13 Staphylococcus Klebsiella 6-12 6-15 Proteus 4-6 4-8 Morganella 3-4 5-6 Enterococcus 2-12 7-16 Staphylococcus 2 2-4 aureus Staphylococcus 0-2 0.4 saprophyticus Pseudomonas 0-4 1-11 Candida 3-8 2-26 18 Complicated UTI Etiology (%) Escherichia coli 21 – 54 Klebsiella pneumoniae 1.9 – 17 Enterobacter species 1.9 – 9.6 Citrobacter species 4.7 – 6.1 Proteus mirabilis 0.9 – 9.6 Providencia species 18 Pseudomonas aeruginosa 2 – 19 Enterococci species 6.1 – 23 19  Microbiology of Community-Acquired Urinary Tract Infection Dysuria-Pyuria Sydrome in Females Children Adults More frequent. Escherichia coli. Escherichia coli. Staphylococus saprophyticus (young, sexually active patient) Less Frequent. Other Enterobacteriaceae. Other Enterobacteriaceae. Enterococci. Enterococci. Streptococcus agalactiae Other Community-Acquired Infection Children Adults More frequent. Escherichia coli. Escherichia coli Less Frequent. Other Enterobacteriaceae. Other Enterobacteriaceae. Enterococci. Enterococci Microbiology of Nosocomial Acquired Urinary Tract Infection in Children or Adult Catheter-Associated Short-Term (< 30 –d) Catheterization More frequent. Escherichia coli Less Frequent. Other Enterobacteriaceae. Pseudomonas Spp.. Staphylococcus epidermidis Catheter-Associated Long-Term (> 30 –d) Catheterization More frequent. Providencia stuartii. Pseudomonas Spp.. Escherichia coli. Other Enterobacteriaceae Less Frequent. Staphylococcus epidermidis  Pyelonephritis may be acute or chronic Pathology Kidneys enlarge Interstitial infiltration of inflammatory cells Abscesses on the capsule and at corticomedullary junction Result in destruction of tubules and the glomeruli When chronic, kidneys become scarred, contracted and nonfunctioning 22  Pathogenesis Rectal and/or vaginal reservoirs Colonization of perianal area Bacterial migration to perivaginal area Bacteria ascend through urethra to bladder Intercourse may contribute urethral colonization and ascending infection 23  Clinical Manifestations of acute pyelonephritis Symptoms develop rapidly (38°C Flank pain and Nausea/vomiting Renal angle tenderness Confusion in elderly Leukocytosis Pyuria Bacteriuria In addition symptoms of lower tract involvement Dysuria Frequency 24  Risk factors Mechanical: – Structural abnormalities to the kidneys and the urinary tract vesicoureteral reflux (VUR) especially in young children, calculi urinary tract catheterisation nephrostomy pregnancy neurogenic bladder (e.g. due to spinal cord damage, spina bifida or multiple sclerosis) and prostate disease (e.g. benign prostatic hyperplasia) in men bladder tumours urethral strictures Constitutional: – diabetes mellitus, immunocompromised states  Diagnosis Is not always straightforward A number of studies using immunochemical markers have shown that many women, who initially present with lower tract symptoms, actually have pyelonephritis The extremes of age, the presentation may be so atypical (feeding difficulty or fever) In the elderly presentation may be mental status change or fever 26  Laboratory Diagnosis of pyelonephritis Urinalysis ❑ 10 WBC/hpf is the usual upper limit of normal ❑Positive nitrate dipstick test result for bacteriuria is only moderately reliable; false-negative results are common ❑Urine culture and sensitivity ❑Blood culture 27 Laboratory Diagnosis of UTIs Causes of UTIs Specimen collection. Suprapubic aspiration Midstream Catheter Urine Bag Transportation Within 2 hours after collection Unless refrigerated Or in a preservative Specimen processing plated using calibrated loops for the semiquantitative method. Provide information regarding the number of cfu/mL, as well as providing isolated colonies for identification and susceptibility testing. blood agar and MacConkey's agar Cultures should be incubated overnight at 35°C–37°C in ambient air before being read. Detection of bacteriuria by urine microscopy. Gram staining Centrifuged Uncentrifuged Direct wet preparation Leukocytes RBCs Critical Points: Collection Time to processing Interpretation of results Significant bacteriuria Insignificant No growth Sensitivity results  Mechanism of Action and Resistance Siame Amon MBCHB Students AUGUST, 2023 Objectives of At the end of this lecture, students should understand the following: the lecture 1. Different classes of antibiotics 2. Mechanisms of action of different antibiotic classes 3. Mechanisms of resistance of different antibiotic classes History of Antibiotic Development Selective Toxicity Is the ability to suppress or kill an infecting microbe without injury to the host. Selective toxicity is achievable because the drug accumulates in a microbe at a higher level than in human cells. The drug has a specific action on cellular structures or biochemical processes that are unique to the microbe or more harmful to the microbe. Understanding selective toxicity has made antimicrobial drugs safe and effective for managing infection in humans. Selective Toxicity § Inhibition of cell wall synthesis e.g. by Penicillins, cephalosporins & vancomycin üHuman cells do not have a cell wall, so these drugs are specific only for bacteria. They will kill or stop replication of the bacteria without damaging the host. § Inhibition of protein synthesis e.g. by Tetracycline, aminoglycosides, chloramphenicol, erythromycin üSelective toxicity relies on the fact that the bacterial ribosome differs in size to the human ribosome üThe bacterial ribosome is smaller (70S) than the mammalian ribosome (80S) and is composed of 50S and 30S subunits (as compared to 60S and 40S subunits in human). § Inhibition of nucleic acid synthesis ü Affect microbial specific enzymes, e.g. DNA dependent RNA polymerase.  Selective Toxicity contd.. § Antimetabolites ü Affect the metabolism of the organism by having a negative effect on some vital metabolite. ü Humans are unable to synthesize foliate and so must get it from the food, whereas bacteria must make their own. Hence, inhibition of foliate metabolism can hinder bacterial growth. e.g. Trimethoprim ü Folate-derived cofactors are essential for the synthesis of purines and pyrimidines (precursors of RNA and DNA) and other compounds necessary for cellular growth and replication. Therefore, in the absence of folate, cells cannot grow or divide. To synthesize the critical folate derivative, tetrahydrofolic acid, humans must first obtain preformed folate in the form of folic acid as a vitamin from the diet. In contrast, many bacteria are impermeable to folic acid and other folates and, therefore, must rely on their ability to synthesize folate de novo. The sulfonamides (sulfa drugs) are a family of antibiotics that inhibit this de novo synthesis of folate. Carbapenem antibiotics were developed from the carbapenem thienamycin, a naturally derived product of Streptomyces cattleya.  Mechanism of action of Aminoglycosides § They passively diffuse via porin channels across the cell wall of the bacteria. § The drug is then transported across the cell membrane into the cytoplasm by active transport which require energy and O2- It bind irreversibly to the 30s unit of the ribosome and distorts the reading frame. § Protein synthesis can still continue, but distortion results in either misense or nonsense codons leading to the wrong amino acid being used and premature termination. § The proteins produced by the bacteria which are required in maintaining cell integrity and functions are going to be non-functional ones. Gyrase is involved primarily in supporting nascent chain elongation during replication of the chromosome, whereas topoisomerase IV separates the topologically linked daughter chromosomes during the terminal stage of DNA replication Mechanism of Action of Tetracyclines § Tetracyclines enter microorganisms in part by passive diffusion (through cell wall) and in part by an energy-dependent process (active transport through cell membrane). § Susceptible cells concentrate the drug intracellularly § Once inside the cell, tetracyclines bind reversibly to the 30S subunit of the bacterial ribosome, blocking the binding of aminoacyl-tRNA to the acceptor site on the mRNA-ribosome complex. This prevents addition of amino acids to the growing peptide. Antibiotic Resistance Defined as micro-organisms that are not inhibited by usually achievable systemic concentration of an antimicrobial agent with normal dosage schedule and / or fall in the minimum inhibitory concentration (MIC) range. Some microorganisms may 'born' resistant, some 'achieve' resistance by mutation or some have resistance 'thrust upon them' by plasmids 5. Alterration of target metabolic pathway  Representation of different types of efflux pumps in gram- positive and gram-negative bacteria The five major families of efflux pumps are: ATP-binding cassette (ABC) superfamily, Major Facilitator Superfamily (MFS), Multidrug and Toxic-compound Extrusion (MATE) family, Small Multidrug Resistance (SMR) family and the Resistance Nodulation Division (RND) family. Source: Piddock LJ. Nat Rev Microbiol. 2006;4(8):629–36 Strategies to combact AMR INFECTIONS OF THE DIGESTIVE SYSTEM Miss Chinoya Introduction A garden of unsurpassed variety and complexity in health or disease Normal stomach is an effective sterilization chamber, limiting entry of microbes to s/intestines and beyond → provision of nonspecific protection Vs many enteric pathogens  Normal GIT flora goes up to 400+ distinct spp of bacteria, fungi, and protozoa, living in a symbiotic relationship with the host: From host - The host provides room while the bacteria helps the host in various ways: 1. Conversion of unabsorbable glucose to absorbable organic acids 2. Supply of essential vitamin K 3. Reabsorption and conservation of estrogens and androgens excreted in bile 4. Resistance to colonization by invading pathogens  INFECTIONS Vary from Most prevalent (dental caries) to fairly common (diarrheas and food poisoning) And may cause unusual opportunistic infections of the immune compromised pts diarrheal diseases are the far greatest manifestation of infection. Infections of this system range in severity from: >> asymptomatic or silent (polio) to mild (diarrhea), to life-threatening loss of fluid and electrolytes (cholera) and severe mucosal ulceration (bacillary dysentery)  MODES OF MICROBIAL ENTRY Fecal-oral route Contaminated food, water or by vectors POSSIBLE DAMAGE DUE TO COLONISATION OF GIT Several signs and symptoms are indicative of GIT related infections: 1) Pharmacological Action * Some bacterial toxins alter normal intestinal function without causing lasting damage to target cells → enterotoxins of V. cholerae or some E.coli which provoke copious watery diarrhea reduces absorptive capacity of s/intestines, sending more fluid to colony - overwhelming it, resulting in "overflow" diarrhea, leading to dehydration and loss of electrolytes 2. Local Inflammation * May result as a consequence of microbial invasion * Often local invasion spreads to contiguous tissue and beyond * The mouth is often affected, usually in the gums from normal gingival flora (periodontitis) * In the intestines, infections can cause inflammation may result in dysentery 3. Deep Tissue Invasion Certain organisms are able to spread to adjacent tissues and to enter the blood stream or lymph worm ---- strongyloides protozoa --- entamoeba bacterium --- salmonella 4. Perforation Injury to mucosal epithelia may result in spillage of normal flora into sterile areas, often with serious consequences: * rapture of appendix may lead to peritonitis * perforation of esophagus may lead to mediastinitis.  DISEASES OF THE PRINCIPLE SITES OF THE GIT 1. MOUTH Main microbial port of entry as it is constant interaction with food, fluid and fingers. - Specific defenses of the mouth include: * nonpathogenic resident flora (resist by space and metabolic inhibitors) * mechanical action of saliva and tongue * enzymes and secretions (lysozyme and IgA) H. Pylori causing oral sores by migration affecting gums. 2. STOMACH Mostly sterile with > Shigella; Salmonella; Campylobacter; Yersinia; E. coli o Diarrhea of the large intestines is often characterized by presence blood, mucus and pus, oConsequences include rectal prolapse, toxic megacolon.  DIARRHOEA AND DYSENTERY Diarrhea is a final common pathway of intestinal responses to many inciting agents Can be caused by some infections but also occur via noninfectious conditions. It is an increase in the daily amount of watery stool (with associated frequency) It may manifest in different forms  DIARRHEA AND DYSENTERY…… It could be considered an adaptive mechanism, dev by the body to rid itself of harmful material OR by microorganisms to ensure their transfer from one host to another! There is a teleological analogy with vomiting, which is used to rid the stomach of noxious material while dysentery is a more circumscribed term used for inflammatory disorders, mainly of the colony, ordinarily not accompanied by large increases in stool volume  TREATMENT 1. most acute diarrheas are mild and self limiting 2. oral fluid replacement is often best remedy ORT is of most importance – accelerates absorption of Na and restores normal osmolality To 1 liter of boiled cooled water, add: 1/2 teaspoon salt (3g) 1/4 teaspoon bicarbonate (1.5g) 1/4 teaspoon KCL (1.5g) tablespoons sugar (20g) 3. intervention with specific antimicrobials often dependent on severity and duration (E.coli; V.cholera and shigella). THE END SEXUALLY TRANSMITTED DISEASES Dr Daka-Lalusha  These are groups of communicable diseases in which sexual contact is the most important mode of transmission They can be viruses, bacteria, fungi or parasites. Syphilis Causative organism- Treponema Pallidum This is a Spirochaete Reservior: Man ( untreated case is infectious in the primary and secondary stages of the disease) Transmission: sexual contact via open lesions( mostly) but can also be spread congenitally (vertical transmission) Stages of Syphilis Primary syphilis Chancre at the portal of entry- firm, indurate, painless and highly infectious ulcer. Enlarged lymph nodes Spontaneously disappears without treatment after 4-6 weeks. Secondary syphilis Generalised skin rash Involvement of other parts of the body Spontaneously disappears within weeks or months followed a latent period (years). Tertiary syphilis Characterized by reappearance of symptoms Occurrence of Neurological or Cardiovascular symptoms Diagnosis Hx and clinical examination Lab test: Treponemal and non-treponemal test ( serology) Treatment – Penicillin IM, Doxycycline. Gonorrhea Caused by a bacteria Neisseria gonorrhea (Gonococcus) Gram neg intracellular diplococcus Reservoir : man Transmission: Direct sexual contact Clinical findings Acute infection In males – urethritis with a purulent discharge In females – urethritis and /or cervicitis with discharges May also present with Arthris, Pharyngitis, rectal infection, septicaemia, endocarditis or meningitis. Symptoms Diagnosis HX and Examination Lab test- M/C/S pus swab. TX: oral penicillin Chlamydia Chlamydia trachomatis : presents as - urogenital infections, Trachoma, conjunctivitis, pneumonia, lymphogranuloma venerium (LGV). C. psittaci presents as pneumonia C. pneumoniae presents as Bronchitis, sinusitis, pneumonia. Chamydia Small obligate intracellular organisms Exists as two morphological forms - Elementary body - Reticulate body Elementary bodies are small, extracellular bodies. They have a rigid outer membrane and are highly resistant. they are non-replicating, non metabolically active, but highly infectious forms. They bind to columnar epithelial cells and macrophages. Chlamydia Reticulate bodies are -larger, intracellular, -fragile membrane, - metabolically active , - replicating form, - non- infectious Chlamydia is difficult to stain and cannot grown on artificial media. Chlamydia trachomatis c. trachomatis divided into 15 serovars, these cause different diseases Trachoma (A, B, Ba, C ) Urogenital –STDs (D-K) LGV (L1,L2,L3) Trachoma Largest single cause of preventable blindness in the world Caused by serovars A, B, Ba, C Clinically presents as Chronic follicular kerato-conjunctivitis ( scarring of the conjunctival) In Neonates – inclusion conjunctivitis, which presents as a watery then mucopurulent eye discharge a5-12 days after vaginal birth. Treatment- antibiotics Urogenital manifestations Can be asymptomatic (imp in transmission) Urethral discharge ( mucopurulent discharge), epididymitis, proctitis, conjunctivitis Bleeding, dysuria , pain during intercourse, cervicitis, salpingitis etc. Complications – ectopic pregnancy, infertility, interstitial pneumonia, arthritis Lymphogranuloma venereum (LGV) 3 stages if untreated Primary stage 3-30 days after incubation Small painless papule which are self limiting Secondary stage – inguinal LN ( painful) necrosis at the LN acute , hemorrhagic proctitis Fever, myalgia and headaches LGV Tertiary stage chronic inflammatory lesions typical of chlamydial infxn Scarring of genital tract Fibrosis, lymphatic obstruction, ele[phantiasis Rectal strictures and fistulaes C. Psittaci Sporadic disease in humans Transmitted via respiratory route. Associuated with inhalation of resp. secretions , droppings of infected birds. Presents as LRTIs with hepato0-splenomegaly Symptoms include Fever, headache, malaise, dry cough, bilateral interstitial pneumonia Diagnosis Diagnosis is very difficult Hx and exam cannot distinguish it from other causes. Culture is difficult – intracellular, requires cell culture. PCR- nucleic acid tests. Treatment Doxycycline or Tetracycline Question A 35-year old woman visits her primary doctor complaining from general muscular weakness (paresis) and heart problems. The attending physician immediately admits her to the hospital. After a thorough examination and after taking a careful history, the physician discovers that this woman's problem has started several years ago. Several weeks after a sexual encounter, she develops chancre on her genitalia associated with fever. She never seek treatment, but several days later fever subsides and the rash is gone. Many weeks later, fever and rash are back, but this time her whole body is covered with rash. Again she is too scared to see a doctor and again the rashes are gone. All of these happened when she was about 27 year-old. 1. Are these episodes related? 2. Are they caused by the same bacteria? Explain your answers. 3. She tells her doctor that she may be pregnant. Is there any risk to the fetus? Explain your answer. 4. What type of treatment do you recommend?  MICROBIOLOGICAL DIAGNOSTIC PRINCIPLES MISS CHINOYA INTRODUCTION Medical microbiology is essentially concerned with diagnosis of microbial infections. What are the duties of a clinical microbiologist?  To test specimens for microorganisms To provide information about in vitro activity of antimicrobial drugs against the microorganisms when appropriate. To confirm clinical diagnosis of infectious disease with bacterial aetiology. To advise the physician. Participate in decisions regarding diagnostic studies to be performed. Give advise on type and timing of specimens to be collected. Mode of transportation and storage. Provide interpretation of lab results. LABORATORY PROCEDURES Morphological identification of the agent in specimen using light or electron microscopy. Culture isolation and identification of the agent. Detection of antigen by immunologic assays. PCR method Antibody demonstration. SPECIMEN SELECTION, COLLECTION & PROCESSING Proper specimen collection is the most important step in diagnosis of disease. And is additionally dependent on selection, timing and method of collection. The site most likely to yield the agent is most preferred. The specimen handling should favour an organisms survival and growth. GEERAL RULES TO ALL SPECIMEN Adequate quantity Representative of infectious process. Use sterile containers and observe aseptic precautions. Appropriate transportation of specimen to the laboratory and TAT. Samples ought to be take or collected before drug administration. Specimens required through operation should be enough and requires special attention. MICROBIOLOGICAL EXAMINATION & STAINS Direct examination of specimen frequently provides the most rapid indication of microbial infection. E.g. microscopic, immunologic techniques have been developed for rapid diagnosis. MACROSCOPIC EXAMINATION Colour, consistency, presence of blood. MICROSCOPIC EXAMINATION WBCs, RBCs, parasites and bacteria. STAINING METHODS Staining is of primary importance for recognition of microorganisms. Fluorescent microscopy using auramine-O Used for microscopic examination of Mtb TB MICROCOPY STAINING PROCEDURE QUALITY CONTROL With each new batch of stains, known positive smears of low positivity and known negative smears should be stained to check the stain, the technique and microscopic examination. Distilled water should be used for rinsing because tap water reduces the fluorescence. PROCEDURE Gently fix the smears with the flame or on the heating block Flood the smear with Auramine O with solution and allow staining for 15 minutes Rinse smear with distilled water and drain. Tap water contains chlorine, which may interfere with fluorescence Flood (decolorize) with 0.5% acid alcohol for 2 minutes Rinse with distilled water and drain Flood smear with potassium permanganate * and counter for minute' Time is critical with potassium permanganate because counterstaining for longer time may quench fluorescence of the acid-fast bacilli Rinse with distilled water and drain Allow smears to air dry and, do not blot dry or dry using heat. Read as soon as possible (Microscopy) or keep the smears in the dark so that fluorochrome doesn't fade *Acridine Orange may be used in place of potassium permanganate as a counterstain Giemsa stain Named after Dr Gustav Giemsa from Germany. Used for the diagnosis of malaria. PROCEDURE Pipette 3 mls of buffered water into a coupling jar Add 6 drops of giemsa stock solution Flood the slide (thick and thin) with giemsa working solution Stain for 10 minutes Wash off the slide with buffered water Air dry/dry at 370c Examine the slide using x100 CULTURE Involves the isolation and culturing of microorganisms in artificial (broth or agar)media, tissue cultures or hosts.  Solid media provides recognition of morphology of colonies od species. One can take advantage of fermentation capabilities for differential media. Culture can also be selective by incorporation of antimicrobial agents that inhibit indigenous flora and permitting the growth of specific ones resistant to the inhibitors (Thayer-Martin for N. gonorrhoeae). Containing vancomycin to inhibit G+ bacteria, colistin for G- bacilli, Bactrim for proteus species and anisomycin for fungi.  The number of bacteria in specimens is useful in determination of infection. 3+, 2+ or 1+…… Chlamydiae and viruses are cultured in cell cultures but may occasionally require inoculation into animals. Ricketsial infection is usually diagnosed serologically due to its isolation difficulty and being particularly hazardous to the individual. Some viruses cannot be isolated in cell cultures (hepatitis) and as such is dependent on detection of viral antigens or antibodies. Incubation Generally incubated at 35 to 37 degrees Celsius. Supplementation of CO₂, reduced oxygen, no oxygen depend on requirements of the microorganisms. The duration ranges from minimum of 18 to 24 hours to a maximum of 6 to 8 weeks. Interpretation Some are always considered clinically significant e.g S. dysentriae, Mtb but others are ordinarily harmless depending on site of specimen collection. Antimicrobial susceptibility Many bacteria have unpredictable susceptibility to antimicrobial agents. SERODIAGNOSIS Infection may be diagnosed by antibody response to infecting microorganism. Especially useful for those that cannot be isolated in culture e.g. EBV The technique for isolation of HIV in cell culture is demanding therefore diagnosis is by detection of antibodies to the virus. Disadvantage The lag between onset of disease and development of antibodies. Presence of antibodies may signify a past infection. Immunocompromised unable to mount antibody response. MOLECULAR TECHNIQUES Involves the use of molecular technology in diagnoses of infectious diseases through gene amplification techniques e.g. PCR. This approach has had a major applications in the detection of infectious diseases that are difficult to culture (HIV, Influenza) or those that are yet to be successfully cultured. IMMUNOLOGIC ASSAY The most common are latex particle agglutination, coagglutination, and ELISA (Enzyme-linked immunosorbent assay). THE END Bone and Joint Infections Types 1. Septic Arthritis 2. Osteomyelitis A. Septic Arthritis Infection of the joints resulting in inflammation of the synovial membrane with purulent effusion into the joint capsule. usually bacterial, 50% of cases in children 80 years 7. Chronic skin infections 8. IV drug use A. Septic Arthritis Pathogenesis: 1. Organism reaches the joint by one of the above routes, then begins an inflammatory response in the synovium (Synovitis) resulting in the exudation of fluid within the joint. 2. Joint cartilage is destroyed by inflammatory granulation tissue and lysosomal enzymes in the joint exudate. 3. Release of proteolytic enzymes (matrix metalloproteinases) from inflammatory and synovial cells, 4. cartilage, & bacteria may cause articular surface damage within 8hrs 5. Increased joint pressure may cause femoral head osteonecrosis if not relieved promptly A. Septic Arthritis Pathogenesis: 6. Outcome varies from complete healing to total destruction of joint. 7. Latter may result in a complete loss of joint movement (ankylosis). 8. The infective process can be summed up as; Serous or acute synovitis, Serofibrinous, Suppurative (purulent) arthritis A. Septic Arthritis Clinical features: a. General manifestations: constitutional symptoms and signs of acute infection b. Local manifestation: Swelling, hotness and redness Deformity with muscle spasm Restriction of all movements of the joint The joint is fixed in the position of ease A. Septic Arthritis Investigations: Blood- FBC and diff count- shows neutrophilic leucocytosis. ESR is markedly elevated. 2. Blood culture may grow the causative organism. 3. Plain X-ray- AP and lateral view; findings normal, especially in early stages Later often may reveal widening of the joint space, subluxation, or dislocation Soft tissue shadow corresponding to distended capsule due to swelling of joint. 5. Ultrasound: may be helpful to identify effusion and to guide aspiration A. Septic Arthritis Investigations: 6. Aspiration: - cell count with differential - Gram stain, culture, and sensitivity - Glucose and protein levels A septic joint aspirate will show - High WBC count (> 50,000/mm3 with >75% PMNs) - Glucose 50 mg/dl less than serum levels - High lactic acid level with infections due to gram positive cocci or gram- negative rods NB: If WBC >50,000 with >90% PMNLs suspect septic arthritis even if culture is negative. A. Septic Arthritis Complications: 1. Destruction of femoral head 2. Deformity and stiffness 3. Joint contracture 4. Hip dislocation 5. Gait deformity 6. Growth disturbance 7. Osteonecrosis 8. Pathological dislocation A. Septic Arthritis Infection of the joints resulting in inflammation of the synovial membrane with purulent effusion into the joint capsule. B. Osteomyelitis Definition: Infection of the bone, with infection or inflammation of surrounding soft tissue Acute ○steomyelitis: Infection of short-duration Characterized by suppuration (ie. abscess), but not Biofilm Often hematogenous osteomyelitis No osteonecrosis yet Systemic symptoms common B. Osteomyelitis Chronic Ostemyelitis ○ Long-lasting infection- Months to years ○ Characterized by necrotic bone and bacterial colonies in protein/polysaccharide matrix (biofilm) ○ Often no systemic symptoms B. Osteomyelitis Aetology Hematogenous seeding from remote source ○ Most common form in young children ○ Sluggish metaphyseal capillaries Contiguous spread from soft-tissue or joint infection ○ Common in older adults near arthroplasty ○ Lower extremity infections related to diabetes or vascular disease Direct inoculation from penetrating trauma or surgery ○ Common in young adults B. Osteomyelitis Pathophysiology Bacteremia seeds long bones commonly around epiphyseal endplates ○ Slow capillary metaphyseal blood flow deposit bacteria Local inflammatory response increases intramedullary pressure ○ Occludes normal blood flow causing necrosis ○ Forces infection to break through cortex and form subperiosteal abscess Erosion through periosteum compromises bone blood supply further, worsening necrosis B. Osteomyelitis Pathophysiology SAC form in center of abscess surrounded by fibrin deposits S. aureus releases coagulase and von Willebrand factor-binding protein ○Activates prothrombin ○Polymerizes fibrin network around SAC ○Protects from immune clearance ○Immune cells unable to penetrate network Bacteria binding to sequestrum release extracellular polymeric substance matrix ○Forms biofilm glycocalyx ○Reduced O2 tension and metabolic activity of bacteria ○Barrier prevents immune clearance and penetration of antibiotics B. Osteomyelitis Pathophysiology SAC forms in center of the abscess surrounded by fibrin deposits S. aureus releases coagulase and von Willebrand factor-binding protein ○Activates prothrombin ○Polymerizes fibrin network around SAC ○Protects from immune clearance ○Immune cells unable to penetrate the network Bacteria binding to sequestrum release extracellular polymeric substance matrix ○Forms biofilm glycocalyx ○Reduced O2 tension and metabolic activity of bacteria ○Barrier prevents immune clearance and penetration of antibiotics B. Osteomyelitis Pathophysiology S. aureus and epidermidis can become intracellular inside osteoblasts ○Further perpetuates chronic infection by limiting clearance by host immune system Staphylococcus surface-associated material (SAM) stimulates osteoclast activity ○release of IL-1, IL-6, TNF-α. S. aureus protein A (SpA) binds to osteoblasts affecting metabolic activity and proliferation. ○leading to osteolysis B. Osteomyelitis Pathophysiology Necrotic bone separates to form the Sequestrum ○A mixture of bone and purulence Reactive bone may form around the sequestrum, termed Involucum ○May not completely surround sequestrum ○Drainage from sequestrum may still occur through incomplete involucrum B. Osteomyelitis Pathophysiology Organisms vary based on age, type of osteomyelitis and location ○Hematogenous commonly monomicrobial ○Contiguous or direct inoculation can be monomicrobial or polymicrobial Children -S. aureus> S. pneumoniae ○Sickle cell predisposes to Salmonella but S. aureus still more common Adults -S. aureus, coag-neg Staph ○S. aureus overall most common ○Incidence of osteo increased with incidence of diabetes in population B. Osteomyelitis Clinical Features Acute ○ Pain ○ Erythema ○ Edema ○ +/- Fever (more common in pediatric) ○ Associated wound or sinus tract if direct/contiguous source Chronic ○ Generalized/systemic signs less common ○ Localized pain and edema ○ Sinus tract, non-healing ulcerations in vasculopath ○ Vertebral may present with neurological symptoms B. Osteomyelitis Diagnosis Clinical Features Laboratory ○ FBC, ESR,CRP ○ Imaging: X-ray, etc ○ Culture + Sensitivity of bone biopsy (Gold std) B. Osteomyelitis Treatment Surgery (Debridement + sequestrectomy) Bone cement with vancomycin Oral Antibiotics ○ Cephalosporins ○ Fluoroquinolones ○ Combination therapy CLOSTRIDIA MULENGA NONDE  GENERAL CHARACTERISTICS OF CLOSTRIDIUM Gram positive bacilli Anaerobic The clostridia are ubiquitous in soil, water, and sewage and are part of the normal microbial population in the gastrointestinal (GI) tracts of animals and humans. Spore forming The location of the spores varies among species They able to survive for years in the environment and return to vegetative forms when in favorable environment Cont’d Pathogenic species are virulent when Tissue oxygen is low Produce tissue destructive and neural exotoxins They are found in wound infections The deeper and more severe the wound the more prone the pt is to clostridial infections They are often acquired by contamination from foreign objects or ingested in food The remarkable ability of clostridia to cause diseases is attributed to their (1) ability to survive adverse environmental conditions through spore formation (2) rapid growth in a nutritionally enriched, oxygen-deprived environment, and (3) production of numerous histolytic toxins, enterotoxins, and neurotoxins.  There are 4 major medically important clostridial species ❑C. perfringens ❑C. botulinum ❑C. tetani ❑C. deficile Histotoxic group include: C. perfringens Neurotoxic group include: c. botulinum and c. tetani C. deficile produces enterotoxins and causes intestinal disease  the most frequent clostridial infection is minor self-limiting gastroenteritis from C. perfringens Serious clostridial infections are relatively rare, but can be fatal; eg ruptured appendix, muscle necrosis, neutropenic enterocolitis, soft tissue infection, tetanus, etc A. Clostridium perfringens They produce multiple exotoxins (A-E) with different pathogenic significance α toxin is the most important: it’s a – Phospholipase that disrupts cell membranes of many cells including erythrocyte, leukocutes and mucles θ toxin alters capillary permeability and is toxic to heart muscles. – This toxin also has pore forming ability similar to streptolysin O. A. Clostridium perfringens A. Pathogenesis: ❑Gas Gangrene: ▪ If oxidation reduction potential reduces in a wound, spores germinate and elaborate α-toxins ▪ The process passes along muscle bundles, producing rapidly spreading edema and necrosis ▪ There is devascularization creating and anaerobic environment ▪ Bacteria produce H2O2 and CO2 causing accumulation of gas ▪ Increased vascular permeability and systemic absorption of the toxin results in shock A. Pathogenesis: ❑Clostridial Food Poisoning: ▪ Spores of some strains are often particularly heat-resistant (100OC) ▪ After consuption, spores germinate and liberate enterotoxin that results in fluid outpouring ▪ The ileum is most severely invloved B. Clinical aspects: ❑Gas gangrene: ▪ Disease begins 1-4 days after injury (may start within 10hrs) ▪ Earliest finding is severe pain with a sense of heaviness or pressure ▪ Disease progresses rapidly with edema, tenderness, pallor, and discolouration and hemorrhagic bullae ▪ Gas may be apparent in tissue, but it is a late sign ▪ Systemic findings are: shock, intravascular hemolysis, hypotension, and renal failure Clostridial cellulitis B. Clinical aspects: ❑Anaerobic cellulitis: ▪ This is a clostridial infection of wounds and surrounding subcuteneous tissue in which there is marked gas formation ▪ But pain, swelling, and toxicity of gas gangrene are absent ▪ It is much less serious than gas gangrene B. Clinical aspects: ❑Food Poisoning: ▪ Incubation period 8-24 hrs ▪ Followed by nausea, abdominal pain, and diarrhoea ▪ No fever, and vomiting is rare ▪ Spontaneous recovery usually occurs in 24hrs C. Diagnosis: ❑Based ultimately on clinical observations ❑Laboratory diagnosis is adjunctive ▪ Bacteria can be isolated from contaminated wounds of pts with no clostridial disease ▪ Isolation of c. perfringens in ingested food in absence of any other organism is sufficient to confirm aeteology D. Treatment: ❑Immediate treatment of gas gangrene and endometritis is important ❑Devi