• Few would disagree that direct examination of a specimen
with Gram stain is one of the most valuable procedures performed by the microbiology laboratory. • The Gram stain result rapidly provides information that is used by the clinician for selecting appropriate antimicrobial therapy; it also helps the laboratory technologist assess the quality of the specimen and the extent to which certain organisms recovered in culture will be worked up. • To prepare a smear for staining, an aliquot of the most purulent or bloody portion of the specimen is placed on a clean microscopic slide in a manner that provides both thick and thin areas. • For sterile body fluids, a centrifuge may be used to concentrate the specimen by 10 to 100 times. (We have to use the sediment to see the microbes) • The material on the slide is allowed to air-dry, is fixed with methanol or gentle heat, and then is stained with Gram stain reagents (crystal violet, Gram iodine, alcohol, and safranin). • Organisms that have a gram-positive cell wall will resist decolorization with methanol and will retain the purple color of the crystal violet; organisms that have a gram-negative cell wall will be decolorized and will stain red with safranin counterstain. Media
• Demonstration of many bacteria on Gram stain that do not grow out
in culture may indicate unusual organisms that require more specialized media or the inability of laboratory personnel to recognize certain colonial types in culture, or it could suggest a false- positive Gram stain result caused by contamination of reagents or collection materials, such as swabs, or incorrect interpretation of Gram stain results. • Gram stain results could indicate the need to inoculate additional media for a specific specimen. • For example, finding many gram-negative coccobacilli in a respiratory specimen could indicate the need for a chocolate plate to recover Haemophilus spp. which would not be recovered on a blood agar plate. CULTURE TECHNIQUES • Media for culture are selected to provide the optimal conditions for growth of pathogens commonly encountered at a particular site or in a particular type of specimen. • Consideration is given to special growth requirements of bacteria associated with a given type of infection or to the necessity of selecting certain pathogenic bacteria from a mixed population of indigenous (local) flora. • Therefore, the media chosen may include selective and differential media, in addition to standard enrichment agar. • Blood-supplemented agar is a good general growth medium and can be used to demonstrate the hemolytic action of colonies on the red blood cells. • Antibiotics or chemicals can be added to create a selective medium such as colistin–nalidixic acid (CNA) agar or phenylethyl alcohol agar, both of which are used to inhibit the growth of gram-negative bacilli, while permitting gram-positive bacteria to grow. • Heating the blood to make chocolate agar and adding vitamin supplements creates an enriched medium with available hemin (X factor) and nicotinamide adenine dinucleotide (V factor) for the isolation of Haemophilus spp. and other fastidious bacteria. • Gram-negative bacilli may be separated from gram-positive bacilli by using bile salts and dye in a medium such as MacConkey’s agar, which additionally divides the colonies into lactose-positive and lactose negative colonies, thus making it both selective and differential. • Bacterial cultures are generally incubated at 37 - 35°C and are examined initially after 18 to 24 hours of incubation. • For recovery of anaerobes, inoculated media should be placed into an anaerobic environment as quickly as possible. Several types of anaerobic culture systems are available. • There is an anaerobic jar, in which water is added to a CO2 and hydrogen (H2) generator package, and oxygen (O2) is catalytically converted to water with palladium-coated alumina pellets contained in a lid chamber. • The blood agar- is an enriched medium in sheep whole blood supplements the basic nutrients. • The chocolate agar-is enriched with lysed sheep red blood cells, which turn brown and give the medium the color for which it is named. • selective media are used to eliminate the large number of inappropriate bacteria in these specimens. 1. Sodium azide-select- for gram Positive bacteria over gram Negative bacteria 2. Bile salts- select- for gram Negative enteric bacteria, and inhibit gram Negative mucosal and most gram Positive bacteria 3. Colistan- inhibit the growth of many gram Negative bacteria • Examples of selective media is MacConkey agar ( contains bile), that selects for the Enterobacteriaceae CNA agar ( contains colistin), that select for Staphylococci and Streptococci. • CNA agar Base is prepared with the same formula as Columbia Agar Base with the addition of 10 mg/L colistin and 15 mg/L nalidixic acid to inhibit the growth of gram-negative bacteria and to support the growth of staphylococci, hemolytic streptococci and enterococci MEDICALLY IMPORTANT BACTERIA GRAM-POSITIVE COCCI Staphylococcus Characteristics • Staphylococci are catalase-positive and catalase negative spherical cocci that often appear in grape-like clusters in stained smears. • They grow well on any peptone-containing nutrient medium under aerobic and anaerobic conditions and may produce hemolysis of various species of animal blood cells and yellow or orange pigment on certain types of agar. • Growth of staphylococci is readily detected on blood agar plates or in various types of nutrient broth. • A selective medium for the isolation of S. aureus is one containing 7.5% to 10% sodium chloride (NaCl) with mannitol. • S. aureus is differentiated from other species of staphylococci principally by its production of coagulases, which are capable of clotting plasma. • Staphylococci produce coagulase in two forms. One is bound to the cell wall, the other is as free coagulase. • Bound coagulase acts directly on the fibrinogen which causes clumping of the staphylococcal cells, while free coagulase acts on the prothrombin. • many laboratories are now utilizing polymerase chain reaction (PCR) assays for detection of S. aureus in nasal swabs and directly from positive blood culture bottles. Many of these assays enable detection of methicillin-resistant S. aureus (MRSA). Clinical Manifestations and Pathogenesis • S. aureus may be present among the indigenous flora of the skin, eye, upper respiratory tract, gastrointestinal tract, urethra, vagina. • Therefore, infection may arise from an endogenous or an exogenous source. • Factors of importance in the development of infection due to S. aureus include breaks in the integrity of mucosal and cutaneous surfaces, the presence of foreign bodies or implants, prior viral diseases, antecedent antimicrobial therapy, and underlying diseases with defects in cellular or humoral immunity. Infections caused by S. aureus may affect multiple organ systems. Among the most common are those involving the skin , such as impetigo, folliculitis, mastitis, and infection of surgical wounds. S. aureus is among the leading causes of bacteremia in hospitalized patients, and it may cause endocarditis, particularly in persons with heart disease and in intravenous drug users. S. aureus is the most common cause of spinal epidural abscess and intracranial phlebitis, and brain abscesses, typically following trauma. Meningitis caused by S. aureus is uncommon and generally follows head trauma or a neurosurgical procedure. • S. aureus is responsible for many cases of osteomyelitis, is the most common cause of septic arthritis in prepubertal children, and is occasionally responsible for septic arthritis in adults. • S. aureus is an infrequent cause of community-acquired pneumonia but a common cause of nosocomial pneumonia, which usually follows aspiration of endogenous nasopharyngeal organisms. • Predisposing factors include infection with measles or influenza A virus, cystic fibrosis, and immune deficiency. Urinary tract infections caused by S. aureus are rare, but cases of pyelonephritis and intrarenal and perirenal abscesses can be found. Several factors play a role in the virulence of S. aureus. The polysaccharide capsule, if present, has antiphagocytic properties. Cell wall peptidoglycans have endotoxin-like activity, stimulating the release of cytokines by macrophages, activation of complement. Protein A, an immunologically active substance in the cell wall, has antiphagocytic properties that are based on its ability to immunoglobulin IgG. Other surface proteins may play an important role in the ability of staphylococci to colonize host tissues. The significant involvement of superantigens of S. aureus in sepsis, endocarditis, and acute kidney injury (abscess) has been elucidated. S. aureus produces numerous toxins. The exotoxin TSST-1 is responsible for toxic shock syndrome, and enterotoxins A to E are responsible for staphylococcal food poisoning. The exfoliative toxins—epidermolytic toxins A and B—cause skin erythema and separation, as seen in scalded skin syndrome. Various enzymes are also produced, including protease, lipase, and hyaluronidase, all of which destroy tissue. Scalded skin syndrome occurs in infants infected with a strain of S. aureus producing exfoliative toxin. The illness begins abruptly with erythema, followed in 2 to 3 days by the formation of flaccid bullae, which slough, leaving denuded areas that eventually resolve completely. Staphylococcal food poisoning, characterized by nausea, vomiting, abdominal cramps, and diarrhea, occurs 1 to 6 hours after ingestion of foods contaminated with preformed staphylococcal enterotoxin, which is usually introduced into the food product by food handlers who prepare and/or serve the food. Toxic shock syndrome (TSS) is primarily the result of a superantigen- mediated cytokine storm and M protein-mediated neutrophil activation, resulting in the release of mediators leading to respiratory failure, vascular leakage, and shock. The illness is most common in women 15 to 25 years of age who use tampons during menstruation, but it also may occur in nonmenstruating individuals, including women in the postpartum period, male or female patients with a surgical wound or other focal infection, and individuals who have had a surgical procedure in the nose or sinuses. Toxic shock syndrome begins abruptly with fever, myalgias, vomiting, and diarrhea, followed by hypotension, hypovolemic shock, and an erythematous rash that frequently involves the palms and soles and flakes in 1 to 2 weeks. The diagnosis is clinical; isolation of S. aureus from any site is not required. Over the past 10 to 15 years, cases of community-acquired infection with S. aureus that are oxacillin resistant (CA- MRSA) have become more common. In these isolates, a toxin referred to as leukocidin toxin, which has rarely been associated with hospital-acquired strains of S. aureus, has been found. leukocidin has been shown to be responsible for necrotizing skin and soft tissue infections and has been infrequently demonstrated to cause a necrotizing and occasionally fatal pneumonia. HA-MRSA strains are usually resistant to all antibiotics except the glycopeptides, such as vancomycin. S. aureus bacteremia can be caused by community and nosocomial strains of S. aureus. A recent study compared the risk factors, morbidity, and mortality due to bacteremia related to health care–associated MRSA. Patients with MRSA bacteremia were likely to have diabetes mellitus, chronic liver disease, and HIV infection. Coagulase-negative species • Infections caused by coagulase-negative species (CoNS) of Staphylococcus usually occur in association with foreign bodies, especially implanted prosthetic valves, joints, and shunts. • Isolates of CoNS are usually considered less pathogenic than S. aureus, although that varies among the species and strains. • The presence of biofilms and antibiotic resistance of the species appears to be associated with bacteremia, whereas specific adhesion capabilities of the CNS were associated with prosthetic joint infections. • CoNS are one of the most commo organisms associated with CSF infections; they are rarely involved in urinary tract infections, pneumonia, or skin and soft tissue infections. More than 20 species of CoNS are known, of which S. epidermidis is the species most frequently involved in such infections. S. saprophyticus is an important cause of bacteriuria, particularly among sexually active young women. S. hemolyticus, reported to rank second in frequency to S. epidermidis in clinical specimens, can be resistant to vancomycin. • More than 90% of staphylococci are resistant to penicillin due to inducible plasmid-encoded β-lactamase. • When β-lactamase is positive by either, isolates should be reported as resistant to penicillin. • Resistance to the penicillinase-resistant methicillin, oxacillin, nafcillin- occurs in up to 80% of coagulase-negative staphylococci and in more than 50% of isolates of hospital-acquired S. aureus. • Clindamycin may be used to treat staphylococcal infection.. For coagulase negative staphylococci - using oxacillin. • Several assays have been developed for rapid detection of oxacillin resistance. • These include nucleic acid amplification, nucleic acid probe assays and latex agglutination assays. Chromogenic media specific for the detection of MRSA require overnight incubation but allow easy detection of specific-colored colonies. Those commercially available are MRSASelect (bioRad Laboratories, Redmond, Wash.), CHROMagar MRSA (BD, Sparks, Md.), Brilliance MRSA (Oxoid, Thermofisher Scientific, Lenexa, Kan.), and MRSA-ID • Three molecular assays that can detect MRSA directly in clinical specimens (nasal swabs, blood cultures, and other) within 90 minutes (are GeneOhm Both Gene-OHM). • MRSA in clinical specimens and from positive blood cultures. • Although oxacillin-resistant staphylococci may appear to be susceptible to cephalosporins, they should be considered resistant to all β-lactam agents (penicillins, cephalosporins, and carbapenems). • Hospital-acquired strains usually are resistant to many non–β- lactam antibiotics as well. • Many of the CA-MRSA strains still remain susceptible to most non–β-lactam antibiotics . Vancomycin resistance, although rarely seen in S. aureus, is a serious issue that laboratories need to be aware of and should screen for. Because the latter have not been uniformly detected in automated systems for susceptibility testing, the Centers for Disease Control and Prevention (CDC) recommends that all S. aureus isolates tested on an automated instrument should also be tested by a vancomycin screen assay. Streptococcus and Enterococcus • Streptococci are catalase-negative, gram-positive, shaped cocci, often seen in pairs or chains. • They are facultatively anaerobic. • Streptococci can be broadly classified according to the hemolytic reaction on blood agar. • Those strains that completely hemolyze the red cells around their colonies are called β-hemolytic Streptococci • Important members of this group include Streptococcus pyogenes (group A) and Streptococcus agalactiae (group B). • Gram stain of S. pyogenes (group A streptococcus) in a specimen from abscess material on the arm of a patient with cellulitis. • Those gram-positive cocci in chains that produce partial hemolysis (cause “greening” of the agar) are α-hemolytic. • An important member of this group is S. pneumoniae. Streptococci that do not hemolyze blood are γ-hemolytic. • An important member of this group is Streptococcus bovis complex. Some S. agalactiae may also be γ-hemolytic. • Most of the remainder of the α- and γ-hemolytic streptococci are collectively called viridans streptococci, including Streptococcus mutans, Streptococcus sanguis, Streptococcus mitis, Streptococcus salivarius, and Streptococcus anginosus. Members of the genus Enterococcus • They are facultatively anaerobic. Most enterococci are α- or γ- hemolytic on blood agar, but some may exhibit β-hemolysis. • The most common species are yellow motile strains of the enterococci, usually nonpathogenic, include Enterococcus casseliflavus and Enterococcus gallinarum. • These latter two species are usually intrinsically vancomycin resistant, and it is important to differentiate them. Other genera of catalase-negative gram-positive cocci that may be isolated from clinical specimens. These organisms are considered to have low virulence potential and generally are pathogenic only in the immunocompromised host. However, some of these isolates may be confused with viridans streptococci, in particular, and their differentiation is important because of their lower virulence and their potential for vancomycin resistance. Further differentiation should be considered if a vancomycin- resistant viridans streptococcus is thought to be clinically relevant. One of the most common clinical manifestations of group A streptococci is pharyngitis. This may be accompanied by scarlet fever, a punctate exanthem overlying diffuse erythema that usually first appears on the neck or upper chest, becomes generalized, and then desquamates. Skin infections caused by group A streptococcus include cellulitis, erysipelas, and pyoderma. Acute rheumatic fever, characterized by carditis, polyarthritis, erythema marginatum, chorea, and subcutaneous nodules, may occur 1 to 5 weeks after group A streptococcal pharyngitis. Acute glomerulonephritis may develop 10 days to 3 weeks after group A streptococcal pharyngitis or pyoderma. Beginning in the late 1980s, serious group A streptococcal clinical syndromes, including necrotizing fasciitis, myositis, malignant scarlet fever, bacteremia, and toxic shock–like syndrome, began to be seen with increasing frequency. These have been associated with high morbidity rates and mortality rates of up to 30% or more. The reason for this increase is not completely understood but appears to be related to changes in the prevalence of organisms having an enhanced virulence potential. S. pyogenes produces numerous virulence factors. One of the most important is the antiphagocytic cell wall M protein. Antibodies against the specific M protein confer lifelong type-specific immunity; however, because more than 60 M protein types exist, infection with a group A streptococcus possessing a different M protein may occur. Another important cell wall component is lipoteichoic acid, which permits bacterial adherence to the respiratory epithelium. S. pyogenes also elaborates about 20 extracellular products, including enzymes (streptolysins, hyaluronidase, streptokinase, Streptolysin O, an antigenic, oxygen-labile enzyme, produces subsurface hemolysis on blood agar plates; streptolysin S, a nonantigenic, oxygen-stable enzyme, produces surface hemolysis. Neither streptolysin has a proven role in the pathogenesis of human disease. Streptokinase promotes fibrinolytic activity by converting plasminogen to plasmin, and hyaluronidase may enhance the spread of the organism through connective tissue. The pathogenesis of acute rheumatic fever is not fully understood. Certain M protein types of S. pyogenes may be rheumatogenic. • The presence of complexes of immunoglobulin and the C3 component of complement along the sheaths of cardiac myofibers from individuals with rheumatic carditis suggests that myocarditis results from the production of antibodies directed against a streptococcal cell wall M protein that cross-reacts with myocardial tissue. Renal damage in acute glomerulonephritis is caused by deposits of circulating streptococcal–antistreptococcal immune complexes in the • glomeruli and subsequent activation of complement. • Cell-mediated reactions to an altered glomerular basement membrane or activation of the alternate complement pathway also may be involved. • Isolates of S. pyogenes have been linked to toxic shock syndrome, with a clinical picture very similar to S. aureus; streptococcal superantigens (Sags) are thought to be responsible for the toxic shock–like syndrome caused by strains of S. pyogenes . The most common infections caused by Group B streptococci (GBS) are neonatal sepsis, pneumonia, and meningitis. Colonization of the maternal genital tract is associated with colonization of infants and risk of neonatal disease, with early-onset infections occurring within the first few days after delivery and late-onset infections appearing after 1 week of age. • To reduce the incidence of neonatal disease, the CDC published specific guidelines to facilitate early identification and treatment of women colonized with GBS and identification and treatment of neonates at risk for developing disease. • All pregnant women at 35 to 37 weeks of gestation should have vaginal/rectal specimens collected and processed for detection of GBS. • If urine is positive, screening of vaginal/rectal cultures may not be necessary. Molecular tests that can rapidly identify the presence of GBS are available and are used in some laboratories at the time of labor and delivery. • Group B streptococcal infections in adults include postpartum endometritis, urinary tract infection, bacteremia, skin and soft tissue infections, pneumonia, endocarditis, meningitis, arthritis, and osteomyelitis. Infections caused by S. pneumoniae include pneumonia, meningitis (especially in infants and the elderly), spontaneous bacteremia (in persons who do not have a spleen), otitis, sinusitis, and spontaneous peritonitis. S. pneumoniae is seen in the normal flora of the upper respiratory tract of 25% to 50% of preschool children, 36% of primary school-age children, and nearly 20% of adults, termed carriers. Its spread is enhanced by upper respiratory tract infections and crowding. • Pneumonia may develop when the host immune defenses are impaired. • Most cases are endogenous, following aspiration of oral secretions containing normal flora that includes S. pneumoniae. Person-to-person transmission during epidemics occurs by droplet aerosols. Vaccines designed to protect against infection by pneumococci of many of the predominant capsular polysaccharide types are available. The CDC recommends that infants receive the 13-valent conjugated vaccine starting at age 2 months. It also is recommended that adults 65 years of age and older receive both the 13-valent conjugated vaccine and the 23-valent polysaccharide vaccine. • Immunosuppressed patients of any age should receive both vaccines . Bacterial endocarditis is the most common infection caused by viridans streptococci; others include abscesses in the brain or liver, bacteremia, and dental caries. The milleri streptococci complex (S. constellatus, S. intermedius, and S. anginosus) consists of the most common viridans streptococci responsible for liver, spleen, and brain abscesses; they often are more susceptible to antibiotics than other strains of viridans streptococci, although resistance to penicillin is increasingly being recognized. Streptococcus bovis S. bovis bacteremia has been associated withmalignancies of the gastrointestinal tract. S. bovis is now recognized as a complex of seven strains and/or subspecies. Biochemical differentiation remains difficult within the species. • Enterococci are not highly pathogenic; however, they are a common cause of urinary tract infection in hospitalized persons. • They may also cause endocarditis, bacteremia, and wound infection. Vancomycin-resistant enterococci offer a greater potential for infection, especially in immunocompromised patients and patients with implanted foreign devices. • Urinary tract infections are most common, but rare cases of more serious infection, including bacteremia, are seen. Laboratory Diagnosis • Streptococci grow well on blood or chocolate agar. Blood agar is preferred because the hemolytic properties of the organism can be assessed. • When culturing vaginal/rectal swabs from pregnant women for group B streptococci, specimens should first be inoculated to a selective broth, such as Lim (Lysine, Indole, Motility Medium) (Formula :Dextrose, Peptone, L-Tryptophan.) • or carrot broth, or on to selective agar, such as Granada agar, to enrich for this organism. Tests that may be used in the clinical microbiology laboratory to presumptively name the β-hemolytic species of Streptococcus. More than 99% of isolates of group A streptococcus are susceptible to bacitracin. All isolates of group A streptococcus and more than 99% of isolates of Enterococcus are PYRase positive. Pyrrolidonyl arylamidase (PYR), also known as pyrrolidonyl aminopeptidase, is a bacterial enzyme. Testing by rapid PYR methods. • PYR (Pyrrolidonyl Aminopeptidase) Test is used for the detection of pyrolidonyl arylamidase (also called pyrrolidonyl aminopeptidase) activity in Streptococcus pyogenes (group A strep), Enterococcus spp., some coagulase-negative staphylococci, and some Enterobacteriaceae. • It is also known as PYR (L-pyrrolidonyl-β-naphthylamide) which serve as a substrate for the detection of pyrrolidonyl peptidase. • A nucleic acid probe (Gen-Probe, San Diego) is also available for direct detection of group A streptococcus on throat swabs. • Group A streptococcal antigen may be detected directly in throat swab specimens by using commercial kits designed to generate a rapid result. • These tests are highly specific but, given their low sensitivity, which varies among studies from 31% to 95% , a negative antigen test in children should be followed by culture or probe. • More recently, clinical guidelines recommended that negative antigen assays in adults need not be followed up with culture. • Serologic tests to detect streptolysin O and DNase B antibodies in acute and convalescent serum samples are used primarily to diagnose acute rheumatic fever and acute glomerulonephritis following infection with group A streptococcus. • Latex agglutination assays are available for direct detection of group B streptococcus (as well as S. pneumoniae, some serotypes of N. meningitidis, E. coli, and H. influenzae type b) in CSF, serum, and urine. These assays have been shown to have sensitivities equivalent to or lower than a Gram stain and may give false-positive results; hence, they do not in general provide additional useful information above that provided by the CSF Gram stain. The rapid bacterial antigen tests are much more expensive and labor intensive than Gram stain; most laboratories no longer offer these tests or strictly limit their use. There are no vancomycin-resistant streptococci, but occasionally a • “viridans”-like isolate is reported as vancomycin resistant. • Usually this is an enterococcus, but it could also be a member of some more uncommon genera such as Leuconostoc or Pediococcus. If vancomycin resistance has been demonstrated, it would be important to differentiate the intrinsically vancomycin-resistant Leuconostoc and Pediococcus from enterococci that have acquired vancomycin resistance. Bacillus Characteristics Members of this genus are strictly aerobic or facultatively anaerobic, rodshaped, spore-forming, gram-positive, and catalase-positive organisms. With the notable exception of Bacillus anthracis, they are usually motile by means of lateral or peritrichous flagella. Some strains stain gram-negatively and, because of their variable oxidase reactions, can be confused with gramnegative bacilli. The most reliable diagnostic characteristic of the genus is spore formation, which occurs optimally and on a variety of media under aerobic conditions at 25° to 30°C. In Gram-stained smears, endospores are detectable by the presence of unstained defects or holes within the cell. The spores themselves can be stained by any of several methods. • Of the numerous species of Bacillus, B. anthracis is the only one that is uniformly and highly pathogenic. Great care must be exercised when handling material suspected of harboring this species. Work should be in biological safety cabinets by gloved, gowned, masked, and personnel; work surfaces must be disinfected with 5% hypochlorite or 5% phenol; and all supplies, materials, and equipment must be decontaminated. • Because B. anthracis spores have been used as a means of bioterrorism, cultures containing suspect B. anthracis should be handled only by reference or public health laboratories. • Bacillus anthracis is the only bacterium with a capsules composed of protein. • This capsule prevents Phagosytosis • Humans are exposed to the spores of bacillus anthracis during direct contact with infected animals or soil • Human-to-human transmission never been reported. is most common in Africa and central and southern Asia. • The pathogenicity of B anthracis depends on two virulence factors: a poly-y- D-glutamic acid polypeptide capsule, which protects it from phagocytosis. • The toxin consists of three proteins: protective antigen (PA) , lethal factor (LF) , and edema factor (EF) . • Three forms of anthrax are recognized: cutaneous, inhalation, and intestinal. • Inhalation anthrax • Inhaled anthrax is the most dangerous form of this infection, but also the rarest. • It causes symptoms that start out like the flu. These symptoms include fever, chest discomfort, malaise (weakness), tiredness, and dry cough. • The signs of illness appear as early as 48 hours after the spores of the bacteria have been inhaled. • If the symptoms are not treated quickly, the infection can rapidly turn into a severe infection similar to pneumonia, and Meningitis. • Fortunately, early treatment decreases the risk of death. Intestinal anthrax
• When a person breathe in anthrax spores, they can develop
inhalation anthrax. • People who work in places such as wool mills, butchery, and tanneries may breathe spores when working with infected animals or contaminated animal products • Inhalation anthrax starts primarily in the lymph nodes of the chest before spreading throughout the body, ultimately causing severe breathing problems and shock. • Very high mortality associated with pulmonary anthrax, it is felt to be an ideal candidate for biological terrorism and warfare. • This fear came to United Stases in October 2001 when 22 cases of anthrax were identified. • This initial cases were discovered in postal workers in New Jersey and Washington DC. Three letters were found which contained anthrax spores. • 11 affected individuals developed cutaneous anthrax and recovered with appropriate therapy. • Of 9 cases of intestinal anthrax 4 died. • 2 cases of inhalation anthrax both patient died. • Inhalation of anthrax spores can lead to acute bronchopneumonia, mediastinitis, and septicemia (“woolsorter’s disease”). The mortality in recognized cases of this form of disease is nearly 100%. • Intestinal anthrax follows the ingestion of contaminated food and is manifested by nausea, vomiting, and diarrhea. In some cases, gastrointestinal bleeding is followed by prostration, shock, and death. Septicemia can occur in all three of anthrax and may lead to a fatal, purulent meningitis. • More recently, a fourth type of anthrax, injectional anthrax, has been recognized in heroin addicts, resulting in severe soft tissue infections with an increased risk of shock and higher level of mortality than is associated with cutaneous infections Cutaneous anthrax
• Cutaneous anthrax is much less dangerous than the inhaled form
When infection occurs on the skin develops symptoms like spider bite. Within a day or two, develop that usually turn black in the center. • Usually, people with cutaneous anthrax feel only mildly ill. Early antibiotic treatment has good result. • If cutaneous anthrax is not treated, the bacteria may get into the bloodstream and cause more serious symptoms and disease • In its cutaneous form, anthrax produces a small, red, macular lesion that progresses to a vesicle and finally to necrosis with formation of a characteristic black eschar. Regional lymphadenopathy and septicemia may occur. • Mortality in untreated cases with this form of disease is approximately 20%. Laboratory Diagnosis • In cases of suspected cutaneous anthrax, vesicle fluid and material under the edge of the eschar should be collected with a swab for smear and culture. • For suspected inhalation anthrax, sputum should be collected for and culture. • Cultures of stool should be considered in the intestinal form. Smears and cultures consisting of CSF should be requested in suspected meningitis. In the septicemic stage, blood cultures should be performed. • Finding large, boxcar-shaped gram-positive cells in smears of any of these specimens should raise suspicion for the diagnosis. • Fluorescent, available in some state health laboratories and at the CDC,provide a rapid presumptive diagnosis. • Species of Bacillus grow well on sheep blood agar. Colonies of B. anthracis are usually flat, with an irregular margin (“Medusa head”), appear with a ground glass surface, and are usually nonhemolytic. Gram-negative bacteria • Pseudomonas aierugnosa • Pseudomonas aeruginosa is from bacterial family Pseudomonadaceae. The family includes only members of the genus Pseudomonas. • Pseudomonas aierugnosa has become increasingly recognized as an emerging opportunistic pathogen of clinics. • Several different epidemiological studies - as a nosocomial pathogen The ability of Pseudomonas aeruginosa to invade tissues depends production of extracellular enzymes and toxins that break down physical barriers and damage host cells, as well as resistance to phagocytosis and the host immune defenses. • Pseudomonas aeruginosa produces other soluble protein it is a cytotoxin or leukocidin. • Two extracellular proteases : elastase and alkaline protease. • Elastase has several activities that relate to virulence. The cytotoxin is a pore-forming protein. It was originally named leukocidin because of its effect on leukocytes. Of the two hemolysins, one is a phospholipase and the other is a lecithinase. • They appear to act to break down lipids and lecithin. • The cytotoxin and hemolysins contribute to invasion through their cytotoxic effects on neutrophils, lymphocytes and other eukaryotic cell. • Elastase disrupts the respiratory epithelium and interferes with ciliary function. • Alkaline protease interferes with fibrin formation and lyse it. • Together, elastase and alkaline protease destroy the ground substance of the cornea and other supporting structures composed of fibrin and elastin. • Elastase and alkaline protease together are also reported to cause the inactivation of gamma interferon (IFN) and tumor necrosis factor (TNF). • Pseudomonas pigment is probably a determinant of virulence for the pathogen. • The blue pigment, pyocyanin, impairs the normal function of human nasal cilia, disrupts the respiratory epithelium, and cause a proinflammatory effect on phagocytes. • A pyocyanin is produced under low-iron conditions to isolate iron from the environment for the growth of the pathogen. • It can play a role in invasion if it extracts iron from the host to permit bacterial growth in a relatively iron-limited environment. • Serious Pseudomonas infections usually occur in people in the hospital and/or with weakened immune systems. • Infections of the blood, pneumonia, and infections following surgery can lead to severe illness and death in these people. • Pseudomonas aeruginosa causes bacteremia primarily in immunocompromised patients, with AIDS, neutropenia, diabetes , and severe burns. • Most Pseudomonas bacteremia is acquired in hospitals and nursing homes. • Pseudomonas accounts for about 25 % of all hospital acquired Gram-negative bacteremia. • Pseudomonas aeruginosa causes meningitis and brain abscesses. • Pseudomonas aeruginosa can produce disease in any part of the gastrointestinal tract from the oropharynx to the rectum. MYCOBACTERIUM TUBERCULOSIS COMPLEX • The tubercle bacilli that make up the M. tuberculosis complex (MTBC) include Mycobacterium tuberculosis, Mycobacterium bovis, M. bovis Bacille Calmette-Guérin strain (BCG), • Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium africanum, Mycobacterium microti, and Mycobacterium canettii. The species that most commonly infects humans is M. tuberculosis. M. bovis, which infects many warm-blooded animals (e.g., cattle, dogs, cats, pigs, badgers, deer, elk, and some birds) as well as primates and humans, is responsible for a small fraction of tuberculosis (TB) cases today. M. bovis BCG is used for vaccine purposes in many countries outside the United States. It also is administered intravesically to induce immunostimulation against bladder cancer, and in such situations rarely may cause disseminated disease. M. africanum causes TB in humans in tropical Africa but has been reported elsewhere, including the United States, primarily in persons who had lived in Africa. • According to the World Health Organization, the global burden of TB has declined slowly over the past several years, but it remains high. 2012, there were an estimated 8.6 million (range, 8.3 million to 9.0 million) incident cases of worldwide. Of these, 0.5 million occurred in children, 2.9 million in women, and 1.1 million in persons infected with HIV. An estimated 450,000 of these cases were multidrug resistant (MDR- TB), defined as resistance to rifampin or any two of the other primary antituberculous drugs (discussed later in the chapter). 58% of the total TB cases occurred in Southeast Asia and Western Pacific regions, 28% in the African region, 8% in the Eastern Mediterranean region, 4% in the European region, and 3% in the region of the Americas. The five countries with the highest number of cases were India, China, South Africa, Indonesia, and Pakistan. The lowest rates were in the United States, Canada, Western Europe, Japan, Australia, and New Zealand. An estimated 1.3 million people died from TB in 2012, of which 320,000 were infected with HIV and 170,000 had MDR-TB. Tubercle bacilli are transmitted primarily via inhalation of dried residues of small infected droplets (1 to 10 µm in diameter). Infection also may be acquired by direct inoculation of abraded skin— an event most likely to occur when pathologists or other laboratory personnel handle infected tissues. In addition, M. bovis may be transmitted from cattle to humans by drinking contaminated raw milk or by respiratory exposure to live infected cattle or their carcasses, from humans to cattle via exposure to urine from persons with urinary tract infections due to M. bovis, and from cattle and wild reservoirs to cattle probably by respiratory secretions. The most important source of infection is an undiagnosed person with cavitary (and sputum smear–positive) pulmonary disease. The risk for active pulmonary disease is low after one exposure to the organism but increases under conditions of stress or in a closed environment where repeated exposures to the organism occur. persons who become infected with M. tuberculosis do not develop active disease. The lifetime for active disease is 5% to 10% for immunocompetent persons. For persons infected with both MTBC and HIV, in contrast, the risk for developing TB is 7% to 10% per year. Increased risk Persons at increased risk for MTBC infection are close contacts of those with known or suspected active TB, foreign-born persons from an area with a high incidence of active TB (e.g., Asia, Africa), persons who visit a country with a high prevalence of active TB, residents and employees of whose clients are at increased risk for active TB (e.g., correctional facilities, homeless shelters), health care workers who care for patients at increased risk for active TB, and persons who abuse drugs or alcohol. The usual host response to infection with MTBC is activation of the cell-mediated immune system. During primary (initial) infection, inhaled bacilli travel to the alveolar spaces, where they are ingested by resident macrophages. These macrophages are unable to kill the tubercle bacilli, which multiply intracellularly during the first several days after infection. Macrophages infected with mycobacteria migrate to regional tracheobronchial lymph nodes and present sensitizing antigen(s) to immunocompetent T cells, or they enter to blood and travel back to the lungs and to organs such as lymph nodes, kidneys, epiphyseal areas of the long bones, vertebral bodies, and meninges, where bacilli continue to multiply until the cellular immune response is activated. Immunocompetent T cells migrate from regional lymph nodes to the site of infection in the lung. There they release chemotactic, migration inhibitory, and mitogenic cytokines, which stimulate recruitment of blood derived monocytes and lymphocytes, macrophage and lymphocyte division, and macrophage activation. Activated macrophages produce cytokines such as interleukin- 1, interferon-γ, and tumor necrosis factor, which stimulate or regulate other components of the immune system, properties that help control infection. • The cytokines and lytic enzymes released by the macrophages also contribute to concomitant local tissue destruction. • Over time, the activated T cell population declines and is replaced by long-lived memory immune T cells, which protect against reinfection with MTBC and provide some cross-protection against infection with other mycobacteria. The primary focus of pulmonary infection, called the Ghon lesion, is usually subjacent to the pleura in the lower part of the upper lobes or in the upper part of the lower lobes of one lung, corresponding to areas of the lung that receive the greatest volume of air. Lesions(tubercles) are well-circumscribed, 1- to 2-cm–diameter areas of grayish consolidation with soft to necrotic centers. Similar-appearing tubercles typically are found in the regional tracheobronchial lymph nodes. Over time, these lesions are replaced by hyalinized fibrous tissue and eventually calcify. • For optimal recovery of mycobacteria from clinical specimens, inoculation of both a broth and a solid medium is recommended. • because broth is more sensitive than solid media and provides more rapid detection of mycobacterial growth. • However, use of a broth system alone may be considered if data show that adding a solid medium does not enhance recovery of mycobacterial pathogens. • Two types of solid media are used for mycobacterial culture: egg-based and agar-based (Middlebrook 7H10, 7H11, and selective 7H11). These media are available in screw-cap tubes and flasks; agar-based media also are available in Petri dishes. • Gold standard for sensitivity and specificity Use of culture increases the number of TB cases found by 30–50% over smear alone • – ~10 viable bacilli/ml of sputum needed for culture • compared to at least 5000 bacilli/ml of sputum for microscopy • • Culture used for species identification, drug susceptibility testing (DST), and genotyping • Culture also used to monitor patient response to treatment • All cultures should be incubated at 37° C in an atmosphere of 5% to 10% carbon dioxide (CO2). • All cultures should be examined weekly for at least 6 weeks. • The colony appearance and growth characteristics of commonly encountered mycobacteria are outlined in. • For laboratories with a low number of requests for mycobacterial culture, the manually read MGIT tubes, which can be used to culture all specimen types except blood and urine, may be the most cost-effective option. These tubes contain modified 7H9 broth and have a fluorescent indicator embedded in silicone in the bottom. • Tubes are inoculated with bacteria and a mycobacterial growth enrichment are added, and the tubes are capped and incubated at 37°C for at least 6 weeks. Automated Systems • Continually monitor the media for detection of mycobacteria for 6 weeks • Most widely used FDA-cleared automated systems rapid detection of mycobacteria using liquid media • – Biomerieux BacT/ALERT® 3D • – Becton Dickinson BACTEC MGIT™ • – Thermo Scientific VersaTREK™ • • MGIT and VersaTrek are also FDA-cleared for • susceptibility testing of MTB MYCOBACTERIUM LEPRAE • M. leprae is the etiologic agent of leprosy, also called Hansen’s disease. • is unique among the mycobacteria by virtue of the fact that it has not been cultivated. • Useful animal models of infection are the mouse footpad model and the nine-banded armadillo. • M. leprae has been recognized in nearly every part of the world at some time. • The reported number of new cases worldwide registered each year remains between 500,000 and 700,00. • Persons born in Oceania were the population with the highest rate of leprosy in the United States. • Leprosy predominantly affects humans but is also a natural infection of wild armadillos in Louisiana, Arkansas, and Texas, and spontaneous cases have been described in monkeys. • The mechanism of transmission of M. leprae is unknown, but person-to person spread via aerosolization of organisms from the nose of a person with active lepromatous disease , which then contacts the nasal mucosa of another individual, is the privileged theory. Transmission also may occur through intact skin or via penetrating wounds, such as thorns or the bite of an arthropod. • Breast milk from lactating women with lepromatous • disease contains bacilli that may be transmitted to infants, and transplacental transmission of M. leprae is possible. • Cases of human leprosy following contact with armadillos have been reported. • sporadic cases of leprosy occur in • persons who have no known contact with human leprosy suggest that nonhuman sources of M. leprae may exist • Most persons effectively resist infection with M. leprae. Resistance depends on an effective cell-mediated immune response to M. leprae antigens, as occurs in tuberculoid leprosy. • In persons who lack specific cell mediated immunity to these antigens, bacilli multiply within macrophages, eventually resulting in widely disseminated lepromatous leprosy. • The lesions of leprosy develop after a 2- to 5-year incubation period, varying in appearance depending on the host’s immune response. Leprosy always involves peripheral nerves, almost always involves the skin, and frequently involves mucous membranes. The leprosy—lepromatous leprosy, the widespread anergic form of the disease, and tuberculoid leprosy, the localized form— stable clinically. • Lepromatous leprosy is characterized by cutaneous lesions ranging from diffuse generalized skin involvement to widespread, symmetrically distributed nodules (called lepromas) filled with organisms. • Lesions generally involve the cooler parts of the body surface: the anterior third of the eye, the nasal mucosa, and the superficial peripheral nerve trunks. In advanced disease, lesions are accompanied by sensory loss from involvement of dermal nerve fibers. Diagnosis and Treatment • Skin lesion biopsy. The skin is tested to look for skin conditions or diseases. A skin biopsy can help health care provider diagnose or rule out problems such as skin cancer or psoriasis. • Treatment • Several antibiotics are used to kill the bacteria that cause the disease. These include rifampin, clofazimine, macrolides, and minocycline. More than 1 antibiotic is often given together • A mild, less severe form of leprosy. People with this type have only one or a few patches of flat, pale-colored skin (paucibacillary leprosy). • The affected area of skin may feel numb because of nerve damage underneath. Tuberculoid leprosy is less contagious than other forms. Tuberculoid leprosy A sample of the biopsy was sent for polymerase chain reaction (PCR) for Mycobacterium leprae (M. leprae) with M. leprae-specific- repetitive-element PCR positive, M. leprae Ag 18-kDa PCR positive and GenoType Leprae-DR negative. • exudate smear microscopy was negative. • https://microbiologyinfo.com/pyr-test-principle-uses-procedure-and-r esult-interpretation/