Colony forming unit (CFU): refers to individual colonies of bacteria, yeast or mold. A colony of bacteria or yeast refers to a mass of individual cells of the same organism, growing together. For molds, a colony is a group of hyphae (filaments) of the same mold growign together. CFUs are used as a measure of the number of microorganisms present in or on surface of a sample. To determine the number of CFUs, a sample is preapred and spread uniformaly on a surface of an agar plate and then incubated. The colonies that form are counted. 

CFU/mL=(# colonies)*dilution factor)/(volume plated in mL). So if you pipete 100 ul of the bacterial onto a plate with a 10 x 5 dilution, your CFU/mL = (400 * 10 to the 5th)(0.1 mL)=4*10 to the 8th) CFU/mL

Experiment; Determining CFUs (Legionella)

(1) spread your plates that have special agar for Lp growth on table. Do in triplicates for each sample.  Label the plates (time: eg. 24 hrs), (same #: eg., 1, 2…), (immature vs. mature DC, eg, i vs m), 

1 24 i LF only         2 24 i                1 24 PA only    2 24 PA only

1 24 i   LF only      2 24 i                1 24 PA only     2 24 PA only

1 24 i LF                2 24 i                1 24 PA only    2 24 PA only

(2) Stack plates under hood

1 24 10e1     1 24 PA only  ….

(3) Obtain polysterine glass tubes with plastic tops (kept in cabinet next to pcr). set up in groups as with plates in blue holder

(4) obtain 0.2% Saponin in water (lyses cells and releases bacterium from the cells). Use the small filter to refilter the saponin into a well plate

(5) add 100 ul to 100 ul of your samples. This will make [finial] saparin 0.1 %.   Time this for 10 min

Stop Reaction

(1) place HBSS into a 50 ml tube

(2) unscrew your polysterine tubes and place 800 ul of HBSS into each.

(3) after 10 min reaction time is up, transfer well of plate to polysterine tube (3 at a time, 100 ul)

(4) If need to you can do a dilution. Would put 800 ul of HBSS into 2nd set of polysterine tubes and take 100 ul of the 1st set and put into this set. This will be 10e2 dilution.

Streak Plates

(1) obtain bunsen burner (light one glass) and alcohol 95% ETOH.

(2) take sample polysterine tube, vortex and put 100 ul into plate

(3) light glass rod tip under burner, let cool slightly, steak.

(1) incubate plates for 3 -4 days to let bacterial grow.

(1) get w black agar plates (Jimmy makes it); (use 2 plates just in case 1 does not grow)  label LP on back of plate with Date and strain #. The vials are in last shelve of freezer (use Lp past through thioglycolated AJ Mqs. Swabs are in needles and swab drawer. 

(2) Dip sterile swab into vial (or pour entire contents of vial into plate if culturing only 24 hours. then use swab to spread it around on plate and use same swab to streak other plate) 

(3) streak (long) onto agar

(4) culture for 48 hours upside down (place in lower incubator).

(5) clean up area and use UV light for 10-15 min.

Introduction:

Legionella pneumophila is an intracellular gram-negative bacillus that causes acue febrile pneumonia (Legionnaires’ disease).

Legionella is composed of 48 different species and 70 different serotypes, although L. pneumophila accounts for the vast majority of huma infections (>90%), with other species such as Legionella longbeachae, Legionella bozmanii, and Legionella micdadei being isolated for less commonly.

The incidence of Legionella infections is not know precisely, but it is estimated that from 6% to 15% of all pneumonias requiring hospitalization are due to these organsisms.

Legionella appear pleomorphic, thin,. They do not stain well with common reagents but can be stained with Deiterle silver stain. They cause Pontiac fever as well as a severe form of pneumonia called Legionnaires’ disease. Legionella are facultative intracellular parasites, capable of multiplication in alveolar macrophages and monocytes.

Serogroups: 

Legionella pneumophila is the most common pathogenic species, with 15 serogroups described. L pneumophila serogroup 1 accounts for most culture confirmed cases of legionellosis. Legionella pneumophila serogroup 1 is estimated to cause 70% to 90% of reported human legionellosis.  Non-serogroup 1 causes only about 7% of legionellosis cases. L. pneumophila serogroups 1, 3, 4, and 6 cause most human infections. (In general, the DNA relatedness between strains within the same species is 70% o r more. Strains that are closely related by DNA hybridization to a previously recognized species, but that fail to react to antisera against that species, are considered to be new serogroups.)

Transmission:

Pulmonary infection with L. pneumophila usually occurs by direct inhalation of contaminated aerosols and aspiration. The bacteria are commonly present in natural bodies of water such as lakes and streams, air conditioning cooling towers and condensers. Lp is ubiquitous in the acquatic environment, where the bacterium invades and replicates with protozoa.

In most cases, L. pneumophila pneumonia is attributed to inhaling contaiminated aerosols produced by cooling towers, showeres and nebulizers. Aspiration is also a possible mechnism of transmission.

The immunosuppressed are at greatest risk for the disease. Treatment with corticosteroids is a particular risk factor for nosocomial acquisition of legionellosis and possibly because of this, infection with Legionella appears to have a predilection for trasplantation recipients.

Hot tubs can be a source of Legionella growth and transmission when they are inadequately maintained and operated. See CDC

 Pathology:

Mononuclear phagocytes in the respiratory tract (i.e., alveolar macrophages) are regarded as a critical component of the first-line host defense against Legionella infection. In addition to the innate immune response to the microorganism, the importance of adoptive cellular immunity, especially that mediated by CD4+ T cells is underscored by the clinical observation that Legionnaires’ disease is ofted contracted by individuals with depressed cell-mediated immunity, including transplant recipients, patients receiving corticosteroids, and patients suffering from AIDS. 

How Lp avoids Immune Response: (Santic 2005, Infect. Immun. 73: 3166). The Dot/Icm TFSS is essential for integrity of the phagosome. The Dot/Icm system secretes some unknown factor.

Virulence Factors: Virulence facts include –several adhesions called MOMP and MIP, –factors that inhibit phagosome lysosome fusion and –mannose containing surface proteins that trigger cytokine production. 

How Cultures: Legionellae are fastidious organisims that are not readily recovered from routine diagnostic media. The medium most commonly used for isolation of legionellae is buffered charcoal yeast extract (BCYE) agar. 

Diagnosis is by –antigen detection (ELISA, RIA, etc), –serology (antibody titers of 256 or higher are evidence) –as well as morphology and culture characteristics above. 

Upon entry into host cells, Legionella modulates phagosome transport to prevent the formation of degradative phagolysosome. As they are transported, phagosomes containing Legionella associate with vesicles exiting the ER and are converted into a ribosome-lined organelle that supports intracellular replication. The ability of Legionella to evade transport to lysosomes and create an ER derived vacuole requires a bacterial protein secretion apparatus encoded by the dot and icm genes. The Dot-Icm secretion system injects bacterial proteins into eukaryotic host cells during infection that direct transport of Legionella-containing phagosomes to the ER. Legionella mutants defective in this dot/icm encoded secretion system do not replicate intracellularly because they are unable to modulate phagosome transport and reside in conventional phagosomes that are rapidly transported to lysosomes.

Cell Entry: uses  to enter the cell.

Involvement of apoptosis: Molmeret et al, 2004, Cell Microbiol, 6:33. Cleaveage of Rabaptin-5 may involve preventing of fusion.  (Abu-Zant et al, 2005, Infect. Immun. 73:5339) There is robust activation of caspase 3, but no apoptosis, suggesting blocking of apoptosis. Even with inducement of of apoptosis with staurosporine, infected cells are resistant to apoptosis. By microarray, showed anti-apoptotic genes induced in anti-apoptotic genes.At early in stage infection. apoptosis delayed (12-14 hours) during stage Lp needs to get out of cell. This corresponds with upregulation of anti-apoptotic genes.

Animal Models: 

A/J mice strains Lp grows well but all other mice strains clear Lp. For example, Legionella infected macrophages of BALB/c mice (growth restrictive), but not in the macrophages of A/J mice (growth-permissive).

See also Enterobacteriaceae 

Clinically relevant Gram-negative bacteria include Salmonella, Shigella, Escherichia, Klebsiella, Enterobacter, Seratia, Proteus, Morganella, Providencia, yersinia, Neisseria, Moraxella (Branhamella), the Acinetobacter and other less common genera. 

Gram-negative bacillis account for many nosocomial pneumonias, cinluding fatal ones. Gram negative bacillary pneumonias typically occur in infants, the elderly, or other immunocompormised hosts. The most important pathogen is Klebsiella pneumoniae, which causes Friedlander’s pneumonia. Other Gram negative pathogens associated with pneumonia include Pseudomonas aeruginosa, Escherichia coli, Enterobacter sp., Proteus sp., Serratia marcescens, and Acinetobacter.

Alcaligenes

Alcaligenes Faecalis: are gram-negative, rod shaped, motile, non-nitrate reducing, oxidase and catalase positive, alpha hemolytic, and citrate positive ogligate anaerobe commonly found in the environment such as the soil and water. On sheep blood agar the colonies are umbonate with an opaque center and a spreading edge. The pathogen is generally considered non-pathogenic but opportunistic infection does occur, often in the form of a urinary infection. 

Acinetobacteria

Acinetobacter is a group of bacteria (germs) commonly found in the environment, like in soil and water. While there are many types, the most common cause of infections is Acinetobacter baumannii, which accounts for most Acinetobacter infections in humans. See CDC

Acinetobacter baumannii can cause infections in the blood, urinary tract, and lungs (pneumonia), or in wounds in other parts of the body. It can also “colonize” or live in a patient without causing infections or symptoms, especially in respiratory secretions (sputum) or open wounds.

Bacteroidetes

Bacteroides fragilis is a prominent member of the gut microbiota within the phyla Bacteroidetes. This commensal bacterium produces unique capsular polysaccharides processed by antigen-presenting cells and activates CD4+ T cells to secrete inflammatory cytokines. Bacteroides fragilis is a gram-negative, anaerobic, rod-shaped bacterium. Bacteroides fragilis is a normal inhabitant of the human gastrointestinal tract, respiratory tract, and female urogenital tract. Bacteroides fragilis can cause opportunistic infections in humans, such as intra-abdominal abscesses and bacteremia.

Among the anaerobic pathogenic bacteria causing human infections, Bacteroides fragillis is the most fequent isolate, and multi-drug resistant strains are on the rise accounting for most treatment fialures. Metronidazone (MTZ) remains the antibiotic of choice for teh management of infections casued by anaerobes and resistance to MTZ is generally still low, however, resistant strains have been reported in regional surfvey studies. (Hoffman, “new funcitons of pirin proteins and a 2-ketoglutarate: Ferredoxin oxidoreductase ortholog in Bacterioids fragilis metabolism and their impact on antimicrobial susceptibility to metronidazole and amixicile” DOI: 10.1002, mbo3.1429 (July 2024). 

Bordetella

Bordetella are strictly aerobic, gram-negative nonmotile, slow growing, coccobacilli. One species, B. pertussis is the agent responsible for “whooping cough.” Virulence factors include –pertussis toxin (PT), –pertactin and filamentous hemagglutinin which are surface proteins for attachment to cells, and –the endotoxin LPS. Infants are at greatest risk (with 70% mortality rate). Because they are fastidious, culture can be difficult. Laboratory diagnosis can be by use of “cough plate” technique. Treatment includes erythromycin. There is a new vaccine which contains the PT and filamentous hemagglutinin toxoid.

Burkholderia

Burkholderia is a genus genus refers to a group of virtually ubiquitous Gram-negative, obligately aerobic, rod-shaped bacteria that are motile by means of single or multiple polar flagella, with the exception of Burkholderia mallei, which is nonmotile. The genus includes both animal and plant pathogens as well as some environmentally important species. For example, B. xenovorans is known for being catalase positive and its ability to degrade chloroganic pesticides and polychlorinated biphenyls. 

Due to their antibiotic resistance and the high motality rate from their associated diseases, B. mallei and B. pseudomallei are considered to be potential biological ware agents. 

Burkholderia pseudomallei: causes meioidosis, the third most common casue of death from infectious diseases in northeast Thailand. B. pseudomallei is commonly found in soil and water in southeast Asia and northeast Auralia. Melioidosis patietns were reported to be infected by inhalation or percutaneous inoculation from contaiminated muddy soil or stagnant water in endemic location, resulting in pneumonia and sepsis and upt to 40% of treated pateints died with this infecition. (Am J Trop Med Hyg. 2014, Mar 5; 90(3); 480-485). 

Burkholderia multivorans is a member of the Burkholderia cepacia complex, a group of water-related, Gram-negative opportunistic bacteria commonly found in soil and water. Outbreaks of these organisms have been associated with contaminated medications, aqueous medical products, and medical devices and are of clinical importance because these organisms can be antibiotic-resistant and can lead to severe infections, especially among immunocompromised and critically ill patients. During 2021–2022, contaminated ice and water from ice machines were linked to 23 Burkholderia multivorans cases at two southern California hospitals.  See CDC article

Chlamydia

Chlamydia are obligate intracellular bacteria (and require ATP from the host). Unlike other gram negative bacterial, they lack a  between the inner and outer membranes but do possess LPS. They are pleomorphic in that they take on different shapes such as rod like as well as coccus. Chlamydiae replicate via a unique growth cycle that is initatied by attachment of an infectious elementary body (EB) to microvilli of susceptible cells. Within 6-8 hours after entering the cell the EBs reorganize into metabolically active reticulate bodies (RB). The EB is rigid and resistant to disruption whereas the RB is fragile. 

Upon infection, chlamydiae have been found to interfere with cellular functions in various ways. One such way is the modulation of the host cell’s apoptosis machinery.

• C. trachomatis is the most frequent STD in the U.S. and a major cause of infertility in women, as well as the major cause of preventable blindness. Inoculation is through a break in the skin or membrane. Infections include PID in women as with N. gonorrhoeae, conjunctiva, and lymphogranuloma venereum (LGV) (male homosexuals at risk). C. trachomatis is a gram negative bacterium which causes which causes the STD Chlamydia. it is the most common STD which can be cured. Most people with the infection have no symptoms and do not seek testing. Chlamydia is most common among young people. Two-thirds of new chlamydial infections occur among youth aged 15-24 years. In 2021, chlamydia rates for African Americans/Blacks were six times that of Whites. Chlamydia is also common among MSM. Among MSM screened for rectal chlamydial infection, positivity ranges from 3.0% to 10.5%. Chlamydia spreads through vaginal, anal, or oral sex with someone with the infection. Semen does not have to be present to get or spread the infection. Some refer to chlamydia as a “silent” infection. This is because most people with the infection have no symptoms or abnormal physical exam findings. Studies find that the proportion of people with chlamydia who develop symptoms vary by setting and study methodology. 

  Anyone with the following genital symptoms should not have sex until they see a healthcare provider:

  • A discharge
  • A burning sensation when peeing
  • Unusual sores, or a rash

  Routine screening is not necessary for men. However, consider screening sexually active young men in clinical settings with a high prevalence of chlamydia. This can include adolescent clinics, correctional facilities, and STD clinics. Consider this when resources permit and do not hinder screening efforts in women. Screen sexually active MSM who have insertive intercourse for urethral chlamydial infection. Also screen MSM who have receptive anal intercourse for rectal infection at least yearly. Screening for pharyngeal infection is not recommended. MSM, including those with HIV, should receive more frequent chlamydia screening at 3- to 6-month intervals, if risk behaviors persist or if they or their sexual partners have multiple partners.To diagnose genital chlamydia in women using a NAAT, vaginal swabs are the optimal specimen. Urine is the specimen of choice for men. Urine is an effective alternative specimen type for women. See CDC

• C. pneumoniae causes a range of respiratory infections, more commonly upper airway infections but also pneumonia. Immunopathogenesis from inflammation is a hallmark of Chlamydia-induced disease. Chlamydial infections in vivo typically result in chronic inflammation characterized cellularly by the presence of activated monocytes and macrophages. At sites of chlamydial infections pro-inflammatory cytokines (IL-1?, IL-6, TNF?) and TH1 associated cytokines (IFNy and IL-12) have been identified. Promotion of any or all of these responses could be evoked by chronic infection as well as by chlamydial products such as LPS, heat shock proteins, and outer membrane proteins.

Chlamydia pneumonia has been linked to alzheimer’s disease.

• C psittaci causes psittacosis, an important and sometimes fatal respiratory disease usually cuased by close contact with infected birds.

Haemophilus

Haemophilus are gram negative bacilli that are parasites present on mucous membrane of humans and animal species. 

Haemophilus influenzae are small, gram negative, non spore forming, non-motile, aerobic anaerobes or facultative anaerobes. Virulence factors include –capsule (polyribitol phosphate (PRP), –pili which include adhesins, –LPS which leads to inflammation, –capsule and –outer membrane proteins IgA proteases.

Klebsiella pneumonia:

Klebsiella pneumonia is a gram-negative, non-motile, encapsulated, lactose fermenting, facultative anaerobic, rod shaped bacterum.The colonies are mucoid, raised and shiny. 

Renckens Blood, 109(4), 2007) disclose that plasminogen activator inhibitor type-1 (PAI-1) overexpression in the lung markedly improved host defense against Klebsiella pneumonia and sepsis., demonstrating that PAI-1 is essential for host defense against severe Gram-negative pneumonia. 

Mycoplasma

Mycoplasma are a genus of bacteria and represent the smallest known cell with a diameter of about 10.1 microm (um). They lack a cell wall and as such, are unaffected by many common antiboiotics such as penicillin. Several species are pathogenic in humans, including M. genitalium, which is beleived to be involved in pelvic inflammatory diseases, and M. pneumoniae which is an important cause of atypical pneumonia and other respitory disorders. 

Mycoplasma penetrans: is pathogenic species infecting humans, typically be penetration into cells of the urogenital and respitatory tracts. 

Neisseria

Neisseria are aerobic, gram-negative cocci which occur in pairs. They are not motile nor do they have endospores. They are oxidase and catalase positive. 

Neisseria gonorrhoeae bacterium causes gonorrhea. N. gonorrhoeae infects the mucous membranes of the reproductive tract, including the cervix, uterus, and fallopian tubes in women, and the urethra in women and men. N. gonorrhoeae can also infect the mucous membranes of the mouth, throat, eyes, and rectum. Gonorrhea is a very common infectious disease. CDC estimates that approximately 1.6 million new gonococcal infections occurred in the United States in 2018, and more than half occur among young people aged 15-24. Gonorrhea is transmitted through sexual contact with the penis, vagina, mouth, or anus of an infected partner. Ejaculation does not have to occur for gonorrhea to be transmitted or acquired. Gonorrhea can also be spread perinatally from mother to baby during childbirth. Many men with gonorrhea are asymptomatic. When present, signs and symptoms of urethral infection in men include dysuria or a white, yellow, or green urethral discharge that usually appears one to fourteen days after infection. Most women with gonorrhea are asymptomatic. Untreated gonorrhea can cause serious and permanent health problems in both women and men. Urogenital gonorrhea can be diagnosed by testing urine, urethral (for men), or endocervical or vaginal (for women) specimens using nucleic acid amplification testing (NAAT). See CDC 

–Compared to other STDs: Bacterium Treponema Pallidum causes syphilis, and Neisseria Gonorrhoeae Bacterium causes gonorrhoea. Syphilis and gonorrhoea transmission can both be minimised through use of condomsm as they reduce skin-to-skin contact and help to stop bodily fluids from the anus, mouth, penis or front hole from being shared. Syphilis and gonorrhoea are both transmitted by sex; however, their modes of transmission are different. Syphilis transmission most commonly occurs by skin-to-skin contact with sores (known as “chancres”), usually found on your genitals, arse, mouth, lips or skin. Gonorrhoea is primarily transmitted during sex by infected fluids such as semen, precum and front hole fluids. See ACON Chlamydia is more common than gonorrhea. According to a 2017 report, over 1.7 million cases of chlamydia were reported in the United States, while just over 550,000 cases of gonorrhea were documented. With chlamydia, symptoms may not appear for a few weeks after you’ve contracted the infection. And with gonorrhea, people who have female anatomy may never experience any symptoms at all or may only show mild symptoms, while people who have male anatomy are more likely to have symptoms that are more severe. See Healthline

–Treatment: Neisseria gonorrhea is showing resistance to mutliple classes of antibiotics. It exhigits significant variation in the antigens on its surface proteins and can easil alter its surface structure during infection. Limma Tech Biologics is developing a vaccine against N. gonorrhea that is in preclinical testing. To target the issue of surface variability, the candidate contains multiple immunogenic antigens that are invariable or conserved. 

Neisseria meningtidis: causes meningococcal disease, which is a rare but life-threatning illness that reqire prompot antibiotic treatment as wll as antibiotic prophylaxis for close contacts.

–Transmission/Prevention: N. meningitidis is transmitted person-to-person via respiratory droplets and oropharyngeal secretions. Asymptomatic persons can carry N. meningitids and transmit the bacteria to otehrs, potentially cuasing illness among susceptible persons. Outbreaks can occur in conjunciton with large gathering. (McNamara, “Cases of Meningococcal disease assocaited with travel to Saudi Arabia for Umrah Pilgrimage–United States, United Kingdom, and France, 2024” Mobidity and Mortality Weekly Report, vol 73, No. 22, 2024).  

Large meningococcal disease outbreaks assocaited with Hajj and Umrah were reproted in 1987, 1992, and 2000-2001. Since 2002, Saudi Arabia has required documentation of either a quadrivalent eningococcal 9MenACWY) polysaccharide vaccine within the alst 3 years or a MenACWY conjugate vacine within the last 5 years and adminsitered greater or equal to 10 days before arriveal for all pilgrims age 1 year or mroe enteirng the country. 

Prophylaxis with rifampin, ceftriaxone, or azithromycin should be preferentially considered instead of ciprofloxacin for close contacts of pateints with Saudi Arabia travel assocaited cases. McNamara, “Cases of Meningococcal disease assocaited with travel to Saudi Arabia for Umrah Pilgrimage–United States, United Kingdom, and France, 2024” Mobidity and Mortality Weekly Report, vol 73, No. 22, 2024).

–Treatment:

First line options for prophylaxis are ifampin, ciprofloxacin and ceftriaxone; azithromycin can also be used in areas with ciprofloxacin-resistant strains. Historically, antibotic resistance in N. meningitidis has been uncommon in the U.S. However, in 2020, CDC idnetified 100 ciprofloxacin and penicillin resistant N. meningitidis serogroup Y (Nmy) isoaltes form cases occurring in 2019 and 2020. (CDC, “Selection of antibiotics as prophylaxis for clsoe contacts of patients with Meningococcal Disease in areas with ciproloxacin resistance -United States, 2024”. 

Proteus

Proteus vulgaris: are rod-shaped which inhabit the intestinal tracts of humans and animals. They are opportunistic and known to cause urinary track infection and wound infections. It ferments glucose and amygdalin, but does not ferment mannitol or lactose. 

Pseudomonas

Pseudomonas aeruginosa are gram – rods, aerobes (do not ferment glucose), Lactose negative (so will not form pinkish red colonies on a MacConkey Agar as will Lac+ bacteria), oxidase positive opportunistic pathogens (cause chronic colonization in cystic fibrosis patients) which may be pignmented. They are very stable organisms that are seen around hospitals and tend to get into catheters, IV lines, etc. In healthy people they tend to be limited to puncture wounds, swimmer’s ear and contact lens infections. Virulence factors include –endotoxins, –exotoxin A and –some may have a capsule (alginate).

Infections with P. aeruginosa are critical in ventilated and poly-traumatized patients and these bacteria cause frequent and chronic pulmonary infections in patients with cystic fibrosis. Kirschnek discloses that annexin II is a novel receptor for P. aeruginosa and that incubation of the bacteria with recobminant, soluble annexin II prevents internalization of P. aeurginosa into human epithelial cells. Typically, individuals infected with STEC develop abdominal pain and mostly bloody diarrhea (hemorrhagic colitis) within 2-5 days, following exposure. Althought the illness sually resoves without sequelae, hemolytic uremic syndrome (HUS) can occur several days following the onset of bloody diarrhea in 5-10 % of susceptible individuals, particulalry children and the elderly. HUS, characterized by hemolytic anemial, thrombocytopenia, acute renal damage, and various degress of central nervous system complications, can result in death or chronic, irreversible renal dysfunction. STEC produce one or two genetically and antigenically distinct exotoxins designated Shiga toxin 1 (Stx1) an Stx2, of which Stx2 is the primary virulence factor for HUS.  (Biochemical and Biophysical Reserch Communications, 327 (2005) 900-906).  

See CDC

Rickettsiae

The Rickettsiae consists of aerobic, gram-negative bacilli that are obligate intracellular parasites. Treatment of the diseases below is with tetracycline.

Rickettsia rickettsii are the agent of Rocky Mountain spotted fever (RMSF). They are found in the cytoplasm in triad. Ticks are their vector and rodents, dogs and ticks are their reservoir. Detection if with direct IFA, PCR or retrospective serology. Treatment is with tetracyclines.

Coxiella burnettii causes Q fever. A common symptom is chronic endocarditis. The reservoir are animals but transmission is via inhalation rather than vectors like ticks. The organisms is environmentally resistant and can survive in soil for years. It is found inside cell vacuoles rather than the cytoplasms as with rickettsii.

Ehrlichia cahffeensis is the agent of erhlichiosis which is similar to RMSF but without a rash. Treatment is with tetracylcine.

Rickettsia typhi is a small, aerobic, obligate intracellular, rod shaped gram negative bacterium. It belongs to the typhus group of the Rickettsia genus, along with R. prowazekii. 

–Fleaborne typhus (also known as murine typhus), a widely distributed vectorborne zoonosis caused by Rickettsia typhi, is a moderately severe, but infrequently fatal illness; among patients who receive doxycycline, the case-fatality rate is <1%. Fleaborne typhus is transmitted from infected fleas by inoculation of flea feces into the flea bite site, a skin abrasion, or mucous membranes. (Alarcon)

Salmonella

Salmonella are a genus of rod shaped non-spore forming bacteria.

Salmonella tryphimurium: are predominantly found in the intestinal lumen which cause gasteroenteritis in humans and other animals. When the bacteria cells enter epithelial cells lining the intestine, they cause damage to the microvilli on the surface of the cell. This causes a rush of white blood cells into the mucosa which upsets the ratios between absorption and secretion and leads to diarrhea. 

Shigella

Shigella is a genus of gram negative rod shaped facultative anaerobic nonspore forming bacteria. Shigella causes bacillary dysentery which is a serious illness characterized by abdominal cramps, nausea, fever and a bloody and mucus diarrhea. Shigella are classified into four species based on their antigen characteristics. Shigella species are related to E. coli. In fact, enteroinvasive E. coli are strains that have the ability to cause dysentery using the same method as Shigella. Despite their relatedness, Shigella could always be spearated form E. coli based on their physiological and biochemical characteristics. Most of the E. coli strains are motile, lysine decarboxylase (LDC) positive,, form gas from D-glucose and are indol positive. In contrast, Shigella strains are always non-motile, always LDC negative and never form gas from D-glucose, except Shigella flexneri serotype 6, which is indol negative. 

Shiga first described Shigella as Bacillus dysenteriae in 1898. He named it Bacillus because it seemed to be related to Vacillus coli, which is now called E. coli. In the 1940s, Ewing proposed to classify four species in the new genus, Shigella, namely S. dysenteriae, S. flexneri, S. boydii and S. sonnei, based on the antigen characteristics of those species.

Shiga toxin (Stx) producing E. coli (particularly E. coli O157:H7): 

Shiga toxin producing Escherichia coli (STEC) 0157.H7 is an enteric illness that can cause hemolytic uremic syndrom (HUS), a severe, life threatening condition which affects the kidneys; young children (ages <5) are among the most susceptible to HUS. (CDC, “Shiga Toxin-Producing Excherichia coli 0157:H7 illness outbreak associated with untreated, pressurized, municipal irrigation water -Utah, 2023” (May 9, 2024).

Shiga toxin producing E coli have occurred with increasing fequency. Within the US alone, it is estimated that annually there are about 100k of STEC infection, of which some 73k are due to infection with O157:H7. The most common sources of infction are food and water contaminated with animal and human effluents, of which cattle are considered the primary animal reservoir. Infection via contacted with infected individuals, however, is also important. (Tzipori, Clinical Microbiology Reviews, Oct. 2004, p. 926-941).

Durng July-september 2023, an outbreak of Shiga toxin producing Escherichia coli 0157:H7 illness among children in city A Utah, casued 13 confirmed illnesses, 7 pateints were hospitalized, including two with hemolytic uremic syndrome. Pulbic health officials linked the illnesses to utnreated, pressureized, municpal irrigation water. 12 of 13 ill chidren reported playing in or drinking this water. Microbial source tracking identified birds and ruminants as potential sources of fecal contamination of the water. Investigators submitted samples of bird feces to the Utah Public Health Laboratory for STEC 0157.H7 culture and submitted all other environmental sampels to CDC for cultrue of sTEC 0157:H7, followed by WGS of confirmed STEC 0157:H7 isolates. (CDC, “Shiga Toxin-Producing Excherichia coli 0157:H7 illness outbreak associated with untreated, pressurized, municipal irrigation water -Utah, 2023” (May 9, 2024). 

Gram – Bacilli

• N. meningitides are gram-negative diplococci and are aerobic or facultative anaerobes. They are both catalase and oxidase positive. Virulence factors include –encapsulation, –hemolysis which lyse RBCs, –toxins, –pili which allows N. meningitidis to colonize the nasopharynx. N. meningitidis is subdividied into serogroups (A,B,C,Y and W135). The nasopharyngeal rates are highest in school-age children. Meningococcus bacteria are spread through the exchange of respiratory and throat secretions like spit (e.g., by living in close quarters, kissing). Meningococcal disease can be treated with antibiotics, but quick medical attention is extremely important. Keeping up to date with recommended vaccines is the best defense against meningococcal disease. There are vaccines to each of these serogroups. See CDC for more information

• N. gonorrhoeae are diplococci which cause pelvic inflammatory disease in women and can cause infertility if left untreated. They are easily transmitted from person to person and is the second most commonly reported STD in the US. It is found only in humans and its reservoir is asymptomatically infected women. Their virulence factors include — a capsule which helps them evade phagocytosis (unlike N. meningitidis), –pili composed of pillins that have antigen variation (facilitates lack of immunity) and –additional proteins in the outer membrane which facilitate attachment to mucosal cells. 

• Shigella flexneri: can cause diarrhea in humans which can typically be treated with antibiotics although some strains have become resistant. 

• S. marcescens: is a rod-shapped gram negative bacteria which is a facultative anaerobe. It is an opportunistic bacteria and commonly involved in hospital acquired infections, particularly catheter assocaited bacteremia., urinary tract infections, and wound infections. It is responsible for about 1.4% of hosptial aquired infections in the US. It is also abundant in the environment and prefers damp conditions. For example, it is commonly found growing in bathrooms, especially on tile grout, shower curtains, toilet water lines, and basins. It is pink or pink to orange. It feeds off phopsphorous containing items or fatty substances such as soap and shampoo residue. It can also be found in dirt. and subgingiviral biofilm of teeth. It produces a a reddish-organe trippyrole pigment. Rinsing and drying surfaces after use can prevent its establishment by removing its food surfaces and making its environment less hospital. Complete erradication of the bacterial is difficult but can be accomplished with by a bleach based disinfectant. 

Other Zoonotic Diseases

Zoonoses is the transmission of disease from animals to human. Bacteria which cause zoonotic disease are often highly virulent and are difficult to culture or diagnose (cause “fevers of unkown origin” FOU). 

• Francisella tularensis is the causative agent of Tularemia otherwise known as “rabbit, tick or deerfly fever”. It is a small gram negative coccobacillus, strictly aerobe (nonfermenter), facultative intracellular pathogen. Collection and processing of specimens for its isolation is extremely hazardous because it can penetrate through the skin and mucous membrane due to its small size or be inhaled if aerosols are produced. 

• Brucella genus consists of several species including B. abortus. They are gram negative, aerobic coccobacillus. They are nonmotile, non capsulated facultative intracellular pathogens (can survive in macrophages). Symptoms include an “undulant” or intermittent fevor. Treatment is by way of tetracylcine. 

• Bartonella are gram negative rods obligate intercellular bacteria (rickettsia-like). They include several species including B henselae which causes “cat-scratch disease” as well as bacillary angiomatosis (BA) which affects immuncompromised patients (like those with AIDS and causes lesions similar to Kaposi’s sarcoma). They respond to antibiotics. 

Gram – Spirochetes

Spirochetes have a gram negative cell envelope but no LPS. They are typically thin and not visible with gram stain. The first one below Treponema pallidum is an STD whereas the other two are zoonotic.

Treponema pallidum

1. Treponema pallidum is the agent for syphilis. It is a thin and long spirochete that can not be seen using standard microscopic techniques. Instead, diagnosis is by darkfield microscopy or serology. It is sensitive to penicillin but more difficult to treat in patients with HIV infections. (See Syphillis outline)

Borrelia Burgdorferi

Borrelia burgdorferi is the agent for lyme disease. They are larger than other spirochetes. Reservoirs for these bacteria include rodents, deer, etc. Vectors are ticks. 

Leptospira interrogans

Leptospira interrogans are aerobic, motile and can be cultivated in the laboratory. Reservoirs include dogs, cattle, rodents, etc (making control difficult). They are transmitted by exposure to contaminated water or infected animals and include a flu-like illness. Treatment is with penicillin or tetracycline.

Companies: TB Alliance 

Tuberculosis (TB), caused by members of the Mycobacterium tuberculosis complex, is one of the most common human infectious diseases, causing three million deaths a year world-wide. While the disease is associated with impoverished economicconditions, TB is on the rise in many industrialized nations. The spread in TB is due to immigration, the emergence of drug resistant strains, and the AIDS epidemic. 

Agent of the disease:

Mycobacterium tuberculosis is the main TB causing micro-organisms in human. The mycobacteria include species in the genus . These small, non-motile, rod-shaped bacilli are obligate aerobes which grow most successfully in tissues with a high oxygen content, such as the lungs. They are distinguished by a complex, lipid rich cell envelope possessing large amounts of mycolic acid. The presence of mycolic acid makes the bacteria very difficult to stain, which is responsible for their characterization acid-fast bacilli.

The zoonotic disease bovin TB is caused by a closely related M. bovis. It can pose a significant threat to human health and can be responsible for up to 10% of human TB cases. Thus, both human and bovine TB should be targeted for efficient control strategy.  CDC regulates nonhuman primate importation and quarantine under the Public Health Service Act (42 US Code 264). All NHP entering the US must be imported by CDC registered facilites and are required to undergo quarantine and TB ttesting under 42 CFR 71.53.  Importers are required to submit samples from any NHP that dies or is euthranized druing quarantine and is suspected to have tB for confirmatory culture. nonhman primate (NHP) iomportation and quarantine under the Public Health Service Act (42 US Code 264). ALL NHP entering the US must be imported by CDC registered facilities and are requried to undergo quarantine and tuberculosis testing under 42 Code of Federal Regulations Seciton 71.53. Importted NHP must have at least three negative TB skin tests. (Swisher “Outbreak of Mycobacterium oregulates CDC rynomolgus Macagues imported form Southeast Asia -United States, February – May 023).

Prevention/Transmission:

Transmission: M. tuberculosis is transmitted usually by airborne droplets which must penetrate deep into the respiratory tree. Predisposing factors which can influence the onset of clinical disease include HIV infection , diabetes, smoking, alcoholism, malnutrition, and overcrowded living conditions. Clinical tuberuculosis invariably starts with the pulmonary form of the disease; however, the pathogen can subsequently disseminate via the circulatory or lymphatic systems and multiply in extrapulmonary host sites such as the skin, lymph nodes, central nervous system, genitourinary tract, and skeleton. 

Diagnosis:

Mantoux tuberculosis skin test (TEST): This test involves injecting a small amoutn of TB protein derivative into a pateint’s forearm and then observing the injection site 48-72 hours after the injection. A positive TEST test indicates that a patient has been infected with TB. A sptum culture — collecting and culturing phelgm from the upper resperiatory tract — is used to determine whether an infected patient actually has TB as distinguished from a latent infection. An ID injection of tuberculin, purified protein derivative (PPD) of the cell wall stimulates pre-primed CD4-helper cells at the injection site that secrete  and lead to a positive result. PPD skin conversion occurs about 4 weeks after exposure. A few people with active TB convert to negative (anergy) through an unknown mechanism. Immunization results in positivity.

ESAT-6 protines: Oxford Immunotec Ltd has developed a TB test using a unique protein called ESAT-6 that is produced by M. tuberculosis. The test uses specified concentrations of eight peptides that are components of ESAT0-6 which are contacted with a population of T cells form the hoest in vitro and detecting an IFN-gamma secretion from the T cells. 

rpoB gene: Scientists from Roche adn the Mayo Foundation for Medical Education and Research sequenced the rpoB gene from MTB and discovered that the gene contains eleven position specific signature nucleotidesthat are only present in MTB but not in other bacteria. These signature nucleotides can thus be used to identify MTB. See US Patent No: 5,643,723

QFT-Plus is a major scientific advance over the 100-year-old TB skin test (sometimes called Mantoux, tuberculin skin test, TST or PPD). QFT-Plus uses four unique blood collection tubes that enable immediate exposure of viable blood lymphocytes (immune cells) to highly specific TB antigens and test controls coated on the inner surface of the tubes. Exposure to these TB antigens causes lymphocytes (specifically CD4 and CD8 T cells) to produce a quantifiable small molecule called Interferon-γ (IFN-γ). Interferon-gamma production is correlated to the presence or absence of TB infection, and this IFN-γ response is measured in a laboratory to aid in the diagnosis of TB infection. see Qiagen

Interferon-Gamma Release Assays (IGRAs): Quest sells an IGRA whole-blood test that detects the immuen system’s response to M. tuberculosis. M. tuberculosis or TB bacterail usually attack the lungs. However, TB bacteria can attack any par to fthe boyd such as the spine, kidneys and brain. In fact, no everyone that becomes infected with TB will become sick. As a result, two TB related conditions exist: latent TB infection (LTBI) and TB disease. This test does not differential between the two. LTBI is a carrier state of TB that can last for weeks or years before developing into TB disease. Active TB is when TB overwhilm a person’s immune syste and symptoms start to appear. This test can be a first step in determing if one has latent TB or TB disease. Further tesitng may be required if the blood test is positive. 

Pathology:

Following engulfment by alveolar macrophages, M. tuberculosis replicate freely in the cell because they evade phagocytic destruction by inhibiting phagolysosome fusion. In a healthy adult exposed to low numbers of bacteria, the TH1 response and collections of activated macrophages (called granulomas) appear early enough to stop infection. But viable bacteria may remain with potential for future reactivation. 

Symptoms:

Symptoms of the disease include fever, coughing, bloody sputum. 

Prevention/Vaccination: 

There is only one licensed vaccine M. bovis Bacillus Calmette-Guerin (BCG) with variable efficacy (WO 2005/023867). It has been distributed since the 1920 and more than 3 billion people have received the vaccine. BCG vaccination, however, remains a matter of debate due to safety aspects, loss of sensitivity to tuberculin as a diagnostic reagent, and varying efficacy (form 0 to 85%) in different BCG vaccine trials. 

Several TB subunit vaccines have been developed, mainly based on M. tuberculosis secreted components such as early secretory antigenic target, ESAT-6 and the antigen 85B (Ag85B). Both antigens have an impressive track record of studies. ESAT-6 and Ag85B are common to both M. tuberculosis and M. bovis. 

(Floss, J. Biomedicine and Biotechnology, 2010, article ID 27434) discloses a fusion between elastin-like peptide (ELP) and Ag85B and ESAT-6 which are produced in plants. Mice and piglets immunized with the TBAg-ELP fusion exhibited exhibited a mycobacterial specific immune response with no side effects. The ain reactivity of the TBAg-ELP induced antibodies against Ag85B. 

(Olsen, Infect Immun, 69(5), 2773-8, 2001) discloses a TB subunit vaccine based on fusion proteins of ESAT-6 and antigen 85. The fusion proteins were adminsitered to mice in the adjuvant combination dimethyl dioctadecylammonium bromide-monophosphoryl lipid A such that a strong dose dependent immune response was induced to both single components as well as to the fusion proteins.

Treatment:

Treatment: Treatment requires at least 2 agents like streptomycin and and is more than 90% successful even in AIDS patients. Almost 10% of new M. tuberculosis patients in the US show resistance to at least one of the first line antituberculosis drugs (isoniazid (INH), pyrazinamide (PZA), rifampin (RIF), ethambutol (EMB) and streptomycin (STR) with about 2-3% of cases resistant to both INH and RIF. The term multi-drug-resistant tuberculosis (MDR-TB) is sometimes used to refer to tuberculosis which is resistant to at least isoniazid and rifampicin, the two most common anti-TB drugs. 

Jolles (WO2005/023867) discloses treating subjects with IVIG following infection with M. tuberculosis have significantly lower colony counts in the lungs and spleen.

Research Models:

The two key tasks for an antimycobacterial regimen are the prevention of the emergence of drug-resistant strains (early bactericidal activity) and the eradication of one or more, slowly metabolizing populations of bacilli that cause relapse (sterilizating activity). Only rifampin appears active in both these respect. Traditional in vitro measures of antimicobial activity predict the efficacy of drugs for the prevention of the emergence of drug resistance, not the sterilizing activity of antituberculous drugs. Although widely used, in vitro tests or the activity of drug comibnations do not appear to predict the results of multidrug treatment regimens. Thus although more expensive and labrious than in vitro assays, animal models have been the best prelicnical predictor of the efficacy of single drugs and multidrug regimens for tuberculosis (Burman, Am J Med Sci 1997, 313(6), 355-363).

Introdcution to Upper Respiratory Tract Diseases casued by S. pyogenes

The most serious cases of pharyngitis (inflammation of the throat) are casued by Streptococcus pyogenes, a group A streptococcus. S. pyogenes is a gram positive occus that grows in chains, does nto form endospores, is nonmotile and forms capsules and slim layers. It is a facultative anaerobe that ferments a variety of sugars. 

Untreated streptococcal throat infections can occasionaly result in serious complications. The psot streptococcal conditions can be caused by the presence of an extra toxin (as in scarlet fever) or by the deposition of antigen-antibody complexes in the body (as in glomerulonepthritis). 

About 30% of sore throats may be caused by S. pyogenes, resulting in several million cases each year. Most transmission of S. pyogenes is via respiratory droplets or direct contact with mucus secretions. Thsi bacterium is carried as normal bioa by 15% of the population, but transmsiion fromt his reservoir is less likely than from a person who is experiencing active disease because of the higher number of bacteria prsent in the disease condition. 

Humans are the only significant reservoir of S. pyogenes. 

More than 80 serotypes of S. pyogenes exist, and thus people can experience multiple infections throught their lives becasue immunity is eserotype specific. 

Streptococcus pyogenes is sensitive to a minute concentration of bacitracin. This serves for a rpaid, immunologic test for diagnosis of group A infections., the bacitracin disc test. Any zone of inhibition around the B disc is interpreseted as a presumptive indication of this species. 

The antibiotic choice for S. pyogenes is penicillin. 

Rheumatic Fever

Cause: Rheumatic fever is an illness caused from complications from untreated or inadequately treated Group A Streptococcus.

Who is at Risk? Most outbreaks of scarlet fever occur under conditions of impoverished overcrowding where access to antibiotics is limited. Since children are more at risk for strep throat, they are also more at risk for scarlet fever. The peak age is 6-20 years.

Mechanism/Virulence: Scarlet fever is believed to be caused when the host’s immunologic response (antibodies) to the bacterial antigens cross react with target organs. In particular, Rheumatic fever is thought to be due to an immunologic cross reaction between the streptococcal M protein and heart muslce. The lymphocyte clones activated by the M protein react with an epitope on the heart muscle. 

Streptococci display numerous surface antigens. Specialized polysaccharides on the surface of the cell wall help to protect the bacterium from being dissolved by the lysozyme of the host. Lipoteichoic acid, LTA, contribues to the adherence of S. pyrogens to epithelial cells in the pharynx. A spiky curae protection called M protein contributes to virulence by resisting phagocytosis and possibly by contributing to adherence. A capsule made of hyaluronic acid (HA) is formed by most S. pyogenes strains. It probably contribues to teh bacterium’s adhesiveness. 

Group A streptococci owe some of their virulence to the effects of hemolysins called streptolysins which casue beta-hemolysis of sheep blood agar. These hemolysins (streptolysin O and S) injure many cells and tissues, including leukocytes and liver and heart muscle. 

Symptoms: include fever, arthritis in large joints, electrical changes in the heart, shortness of breath due to heart failure, skin rashes and subcutaneous nodules. Other symptoms include arthritis in multiple joings and appearance of nodules over bondy surfaces just under the skin. 

Treatment: includes oral antibiotics (e.g., penicillin). Prevention of reorrurence is usually necessary (injection every 3 weeks for up to 5 years or even more.

Scarlet Fever

Cause: Scarlet fever is a disease caused by exotoxin released by Streptococcus pyogenes.

Mechanism/Virulence: The S. pyorgenes strain is itself infected with a bacteriophage which gives the streptococcus the ability to produce erythrogenic toxin. 

A key toxin in the development of scarlet fever is erythrogenic toxin which is responsbiel for the bright red rash typical of thsi disease, and also induces fever by acting upon the temperature regulatory center in the brain. 

Symptoms include sore throat, high fever, bright red tongue and a rash which appears 12-28 hours after the fever. The rash is sandpaper like, most often on the neck, chest, elbows, inner surfaces of the thighs. Immune complications include rheumatic fever above.

Who is at Risk? Scalrlet fever most often affects school-age children and was a source of great suffering in the U.S. in the early part of the 20th centure. The disease can have a fatality rate of up to 95%. Although the disease had all but disappeared int he last centruy, a resurgence in cases in the UK and in parts of Asia has been documented. 

Streptococcus pyogenes (Group A Streptococcu)s is the etiologic agent responsible for many conditions related to streptococcal infections. It is more commonly known as Group A Strep and has more than 6,000 recognized subtypes, with new strains constantly arising. The most common form of infection involves a colonization of the pharyrix known as strep throat. cause pharyngitis, scarlet fever, and toxic shock. 

Biochemical and Morphological Characteristics

They tend to be short chains in clinical samples with longer chains in liquid media. They are beta hemolytic and contain an outer capsule. In the pre-antibiotic era severe infections due to group A ß-haemolytic streptococcus were major causes of morbidity and mortality. 

Diagnosis and Symptoms 

The clinical features of group A sreptococcal pneumonia include sudden high fever, pleuritic chest pain, chills, caugh, sputum and dyspnoea. The most typical feature suggesting the diagnosis of group A streptococcal pneumonia is the rapid accumulation of a plueral effusion. In view of the emergence of more virulent group A streptococcal infections, it is important to initiate early and appropriate antibiotic therapy for common uncomplicated streptococcal infections such as pharyngitis, tonsillitis, impetigo and cellulitis.

Mechanisms of pathology:

Secretion of Enzymes:

–Endoglycosidases: S. pyogenes secretes two enzymes shwoing remarkable specificity for IgG; EndoS and IdeS. 

(i) EndoS efficiently and specifically hydrolyzes the functionally important N-linked glycan of IgG and treatment with EndoS abrogates the pathogenic activity of IgG in mouse models of autoimmune disease.

 (ii) IdeS or streptococcal Mac-1 is a secreted cysteine proteinase that specifically cleaves the heavy chain of IgG. Due to its early and sustained expression during growth and its highly specific proteolytic activity, IdeS is a tailor-made defense against Fc mediated phagocytic killing. In one study, the enzyme was found to block the development of IgG-induced arthritis. Johansson (“IdeS: a bacterial proteolytic enzyme with therapeutic potential” 2008) showed that this enzyme in vitro efficiently cleaves IgG in human whole blood, removes IgG from the blood circulation of rabbits without any side effices and cures mice from lethal IgG induced thrombocytopenia.

Staphytlococcus aureus (S. aureus) is the most virulent Staphylococcus species and causes a variety of infections in humans, both localized and systemic. Most commonly, S. aureus causes a localized skin infection after a skin break or wound. Another common site of entry is the respiratory system, and Staphylococcal pneumonia is a frequent complication of influenza. More serious illnesses may result whn S. aureus enters the blood stream including osteomyelitis, endocarditis, sepsis and meninitis, among others. S. aurues is a major cause of nosomial (hosptial aqcquired) infections. 

S. aureus produces several protenal virulence factors such as alpha, beta, gamma and delta toxins, toxic shock syndrome toxin, enterotoxins, leucocidin, proteases, coagulase and clumping factor. The emergence of antibiotic-resistant forms of S. aureus (e.g., MRSA) is a major problem.

The surface of most strains is coated with protein A that binds the Fc region of IgG. Teichoic acids are bound to both peptidoglycan layer and cytoplasmic membrane. 

Biochemical, Morphological and Structural Characteristics

S. Aureus are alpha hemolytic and destroy red blood cells. They are facultative anaerobic, coccal bacteria. They appear as grape-like clusters (because they reproduce asexually by binary fission, and the two daughter cells remain attached) and have large, golden-yellow colonies, often with hemolysis when grown on blood agar plates. 

Protein A of Staphylococcus aureus (“SpA): refers to a 42 kda multi-domain protein isolated from S. aureus. SpA is bound to the bacterial cell wall via its carboxy-terminal cell wall binding region, referred to as the X domain. At the amino-terminal region, it includes 5 immunoglobulin binding domains, referred to as E, D,A,B and C. Each of these domains contains about 58 amino acid residues and they share 675-90% amino acid sequence identity. SpA is widely used for antibody purification due to its high affinity for immunoglobulins. For more on SpA as an affinity ligand for purification of antibodies see “Protein Purification”. 

Pathogenesis

S. aureus oes much of its pathogenic process to proteins such as Surface protein A (SpA) anchored to its cell well. These adhesins facilitate evasion of th host immune response and host colonisation by binding to plasma proteins and host endothelial cells. SpA is anchored to the bacterial cell surface via an LPXTG motif, which is typical of microbial surface components that recognize adhsive matrix molecules. The binding activity of SpA acts to cloak the bacerial cell with IgG, thus blocking any interaction with Fc receptors on nuetrophils and hingering phagocytosis (Atkins, Molecular Immunology 45, 2008, 1600-1611). 

S. aureus causes disease by either production of toxin or direct invasion and destruction of tissue. Some toxins produced include exfoliative toxin which causes staphylococcal scalded skin syndrome (SSSS), toxic shock syndrome (TSS) and enterotoxins which are heat resistant toxins that cause food poisoning. 

Entry/Colonisation

Adehsion to tissues is required for bacterial colnisation to occur. For this purpose, S aureus express surface adhesins which interact with host matrix proteins such as fibronectin, collagen, etc. In addition, Staphylococci are able to bind several serum proteins, such as IgG, possibly masking the bacteria from the immune system of the host. 

The most studied receptor in S aureus is protein A, a cell wall associated protein, which binds to the Fc and the Fab regions of IgG from several species. The fact that protein A binds so well has been taken advantage of for the purification of IgG (see “biotechnology”, “protein purification” and “affinity A” under “chromatography”). 

Treatment

S aureus are usually treatment with penicillins or cephalosporins. Methicillin resistant S. aureus are resistant to all beta-lactams, however. Only vancomycin is effective.(a characteristic that can be used to distinguish them from S. epidermidis which are gamma hemolytic)

USDA (food safety) 

Introduction

The genus Clostridium is comprised of gram-positive, anaerobic, spore-forming bacilli. The natural habitat of these organisms is the environment and the interstinal tracts of humans and other animals. Indeed, clostridia are ubiquitous; they are commonly found in soil, dust, sewage, marine sediments, decaying vegetation, and mud. Despite the identiciation of about 100 species of Clostridium, only a small number have been recognized as etiologic agents of medical and veterinary importance. Nonetheless, these species are associated with very serious diseases, including botulism, tetanus, anaerobic cellulitis, gas gangrene, bacteremia, pseudomembranous colitis, and clostridial gastroenteritis. As virtually all of these species have been siolated form fecal samples of apparently healthy persons, some of these isolates may be transient, rather than permanent residuents of the colonic flora (US20040115215). 

Botulism is caused by a neurotoxin produced by a member of the genus Clostridium, usually Clostridium botulinum. Organisms classified in this species produce neurotoxins distinguished by their serologic properties into toxin types A, B, C, D, E, and F. 

Particular Clostridium Species

Botulism: is a rare but serious illness caused by a toxin that attacks the body’s nerves and causes difficulty breathing, muscle paralysis, and even death. This toxin is made by Clostridium botulinum and sometimes Clostridium butyricum and Clostridium baratii bacteria. These bacteria can be spread by food and sometimes by other means. The bacteria that make botulinum toxin are found naturally in many places, but it’s rare for them to make people sick. These bacteria make spores, which act like protective coatings. Spores help the bacteria survive in the environment, even in extreme conditions. The spores usually do not cause people to become sick, even when they’re eaten. But under certain conditions, these spores can grow and make one of the most lethal toxins known.  See CDC

There are three types of botulism; 1) food borne, 2) infant and 3) wound. The botulinum toxin is a simple A-B toxin with a binding domain (B) and an enzymatic domain (A). There are 8 serotypes of botulinum toxin: A-H. The toxin binds to gangliosides receptor and is internalized. Each serotype cleaves a different part of the SNARE proteins that are responsible for the release of acetylcholine into the synapse from vesicles.

Botulism is a rare but serious illness caused by a toxin that attacks the body’s nerves. Botulism usually start with weakness of the muscles that control the eyes, face, mouth, and throat. This weakness may spread to the neck, arms, torso, and legs. Botulism also can weaken the muscles involved in breathing, which can lead to difficulty breathing and even death. See CDC

 –C. botulinum produces the most poisonous biological toxin known. Botulinal toxin blocks nerve transmission to the muscles, resulting in flaccid paralysis. When the toxin reaches airway and respiratory muscles, it results in respiratory failure that can cause death. C. botulinum used to be a major problem in home canning. Its spore is not killed by boiling and survives inadequate pressure sterilization. Mortality can be up to 25%. Symptoms are those common for neurotoxins like muscle weakness and paralysis.C. botulinum spores are carried by dust and are found on vegetables taken from the soil, on fresh fruits and on agricultural products such as honey. Under conditions favorable to the organism, the spores germinate to vegetative cells which produces toxin. Botulims disease may be goruped into 4 types based on the method of introduction of toxin into the bloodstream. Food borne botulims results form ingesting improperly preserved and inadequately heated food that contains botulinal toxin. There were 355 cases of food borne botulism in the US between 1976 and 1984. The death rate due to botulinal toxin is 12% and can be higher in particular risk groups. Wound induced botulism results form C. botulinum penetrating traumatized tissue and producing toxin that is absorbed into the bloostream. Since 1950, 30 cases of wound botulism have been reported. Inhalation botulism results when the toxin is inhaled.

–C. butyricum: C. butyricum is a soil inhabitant in various parts of the world, has been cultured from the stool of healthy children and adults, and is common in soured milk and cheeses. butyricum strains have been reported to be pathogenic, expressing virulence factors (i.e. toxins such as enterotoxins or botulinum neurotoxin; enzymes such as neuraminidase; adhesion molecules; and secretion of high levels of butyric acid).  See Casir 

Clostridium difficile (C. difficile) is a gram positive anaerobic bacillus. C dificile produces two main toxins: enterotoxin A (TcdA) and cytotoxin B (TcdB) which can play a role in diarrhaea. It is the leading casue of hospital acquired diarrhaea. (Pituch, Internat J Antimicrobial Agents, 33(S1), 2009, S42-S45)

C. difficile is considered one of the most difficult infections to treat. In patietns, CDI manifests in the colon and presents as abdominal cramping, colitis, and watery diarrhea. One of the biggest issues is the likelihood of reinfection. C. difficile casues about 500k cases of CDI in the US annually. Out of that population, 1 in 6 patietns will be reinfected within 8 weeks of recovery. This is known as recurrent CDI. (Ohaka, “Decreasing difficulty levels with mRNA-LNP vaccines” Tides Global, Oct 23, 2024). 

During the mid 1990s C difficile infection in acute care hosptials in the US was 30-40 cases per 100k. In 2001, this number rose to almost 50 and in 2005 was nearly three times the 1996 rate (31 per 100k). Of even greater concern are the increases in severe or fatal infection. In England, for example, C difficileinfection was listed as the primary cause of death for 599 patients in 199 and rote to 1998 in 2005 and 3393 in 2006. In addition, sporadic outbreaks have been reported in many hospitals. C dfficile infection predominantly affects elderly and frail hosptial and nursing home patients. However, a recent CDC advisory warns of the risk in populations not previously considered at risk. Metronidazole or oral vacomycin remain treatment of choice. (Kelly, NEJM, 359(18), 2008, 1932-1940)

The clincial spectrum of C difficile assocaited diase (CDAD) ranges form diarrhaea to severe life threatening psuedomembranous colitis. C difficile can be transmitted via personal contact or environmentally. C. difficile has more than 150 PCR ribotypes and 24 toxinotypes, has a pathogenicity locus (PaLoc) with genes encoding enterotoxin A (tcdA) and cytotoxin B (tcdB). (Kuijper, Clinical Microbiology and infection, 12(6), 2006, 2-18).

C difficile s also called “antibiotic associated colitis” due to disruption of intestinal flora by antibiotics which allows C. difficile to grow rapidly in unoccupied niches. Virulence factors include two toxins (A and B). Antibiotic associated pseudomenmbranous colitis results from the use of broad spectrum antibiotic agents such as clindamycin. These anti biotics cause diarrhea in about 10% of treated patietns and pseudomembranous colitis in about 1%. C. difficle causes antiotic assocaited diarrhea and almost all cases of pseudomembranous colitis. Psuedomembranous colitis results from the production of C dfficile toxin A and Toxin B in the olon. Toxin A probably caues most of the gastrointestinal symptoms because of its enterotoxic activity. The toxins may act synergistically and the initial pathology cased by toxin A allows toxin B to manifest its toxicity.

–Treatment of C. dificile:

—-Antibiotics:

Antibiotic –namely metronidazole and the glycopeptide vancomycin –are the current standard frist line therapy againt C. dificile infection.(Ohaka, “Decreasing difficulty levels with mRNA-LNP vaccines” Tides Global, Oct 23, 2024). Most patients with C. difficile associated disease are treated effectively with vancomysin or metronidazole. Other treatment includes tolevemer, a toxin binding polymer.

Although antiboiotics are generally effective, the disease frequently relapses, partly because antibotics kills not only C. difficile but also disrupt colonisation resistance of the gut microflora. Non-antibiotic strateis include probiotics, delibertate colonisation by non-toxigenic C. difficile strains, toxin binding agents, active immunisation, passive immunothepary with intravenous immunoglobulin, mAbs or bovine anti C diffcile whey concentrate and faecal transplantation. (Bauer, International J. of Antimicrobial Agents, 33(S1), 2009, S561-S56).

Between 15-30% of C. diffcile infected patients who initially respond to antimicrobial therapy experience rCDI. (Ohaka, “Decreasing difficulty levels with mRNA-LNP vaccines” Tides Global, Oct 23, 2024). 

—-IG or IGM or Both with Secretory component:

Simon (US2008/0260822) discloses treatment for C difficile using antigen specific polyclonal dimeric secretory IgA or pentameric secretory IgM. Because of the secretory component, it has resistance ot degradation in the gastrointestinal tract and can be used at lower doses. 

Simon (US 15/205359, published as US 2016/0319039) discloses treating C. difficile by adminstering a mixture of secretory IgA and secretory IgM.  In some embodiments the IgM or IgA is modified with secretory component. 

—-Vaccination against toxins A and B stimulates active immunity against C difficile in animals. Passive immunization as by serum antibodies agaisnt C difficile have shown to protect hamsters against C. difficile after oral adminsitration. Passive immunization with bovine antibodies has been prosed as a treatment for other infectious diseases of the gastrointestinal tract, such as diseases cuased by rotavius, enteropathogenic and enterotoxigenic E. coli, Vibrio cholerae and Cryptosporidium parvum. It has been reported that bovine IgG from the colostrum of cows vacinated with C. difficile toxoid protects hamsters agaisnt antibiotic assocaited ceditis. Human intravenous immunoglobuilin dervied from plasma donors has facilitated treatment in some pateints, specially patietns who lack circulating antibodies to the C difficile toxins. In vitro experiments have shown that polymeric IgA is superior to monomeric IgA and IgG in preventing C. dfficile toxin damainge to intestinal eptihelial cell monolayers. (Brown US 14.476,559). 

—-mRNA-LNP Vaccines:

Pro-Pro endopeptidase 1 (PPEP-1) is a highly conserved factor that modulates pathogen motility and adhersion through the cleavage of multiple factors on the C. difficle cell surface. PPEP-1 is a metalloprotease that plays a key role in bacterial mobility and gut colonization. It is secreted by all C. difficle strains, amking it an appealing target for such a heterogeneous family of pathogens. PPEP1 has another advantage which is that it is not present in other bacteria in the microbiome. This means that a vaccine should have a narrow-spectrum -C. difficile can be attacked without disrupting composition of the gut. As well as PPEP-1, C. difficile toxins TcdA and TcdB are targeted, further winnowing down the targets to toxigenic strains. Results showd that mRNA-LNP vaccines activate both CD4+ and CD8+ T cell resposnes, signifying an adaptive immune response. The vaccine could overcome deficits in host immunity to protect animals even after infection has occured. (Ohaka, “Decreasing difficulty levels with mRNA-LNP vaccines” Tides Global, Oct 23, 2024). 

C tetani is a spore forming clostridium that can become deposited in tissue through wounds. Clostridium tetani is the causative organism for the disease process known as tetanus. Clostridia are anaerobic organisms with at least 209 species and five subspecies. Clostridium tetani is one of the 4 most well-known exotoxin producing pathogens within this category. Although widespread vaccination efforts have reduced the public health threat, tetanus is a potentially fatal condition. Thus, it is important to recognize the typical clinical presentation, immediate management, and treatment of C. tetani infection. See George 

Clostridium tetani is a motile, anaerobic, spore forming bacteria (terminal spores with drum stick appearance). Vegetative cells are rod shaped, pleomorphic, and occur in pairs or short chains Footnote 1. It is Gram positive in young cultures, but becomes Gram negative upon sporulation. It is catalase and superoxide dismutase negative. It produces a potent neurotoxin tetanospasmin (TeNT), which degrades the SNARE protein required for GABA-ergic neurotransmission. 

Tetanus is caused by C. tetani, and has 4 different clinical manifestations: 1) local tetanus at the site of injury; 2) cephalic tetanus, which occurs due to head injuries or infections; 3) Generalized tetanus, which is the most common and represents 80% of the cases; 4) neonatal tetanus, which occurs in infants within 28 days of birth, due to infection of the umbilical stump. C. tetani colonizes small, non serious wounds such as a puncture wound with a splinter, and releases TeNT at the site of injury. The toxin rapidly enters the CNS through retrograde transport and blocks postsynaptic inhibition of spinal motor reflexes resulting in prolonged spasmodic contractions of the skeletal muscles. The first muscles to be affected are the neck and masseter muscles, causing rigidity of the neck and spasms of the jaw (lock jaw/trismus). See MSDS online

Because tetanus spores cannot be eliminated from the environment, and tetanus infection does not confer immunity, elimination requires ongoing active immunization with a tetanus toxoid–containing vaccine (TTCV). To protect infants from tetanus susceptibility at birth, women of reproductive age (usually 15–49 years) should be vaccinated with ≥2 doses of TTCV (TTCV2+), and immunization is recommended for undervaccinated pregnant women early in the third trimester. See MMWR

C. perfringens are ubiquitous and part of the normal flora. They produce a variety of toxins. It is a major cause of gas gangrene.

Corynebacterium: are gram-positive rods, often club-shaped, in singles, pairs (V-forms), or arranged in stacks (palisades) or irregular clusters. Some are found in the environment and many are in the normal flora (skin and mucous membranes).

–Corynebacterium diphtheriae (C. diptheriae (diphtheria)): is a non-endospore forming gram-positive club snaped bacterium.  C. diptheriae causes diptheria, a diseases which in the last 50 years has declined but has resurfaced because fewer people are getting faccinated. In Venezuela in 2017, health care was disrupted and hundreds of childen got diphteria. 

The most striking symptom of diptheria is a characteristic membrane that forms on the tonsils or pharynx. It can eventually cut of the airway, leading to death. The major virulence factor is an exotoxin encoded by a bacteriophage of C. diptheriae. Strains of the bacterium that are not lysogenized by this phage do not cause serious disease. The rlease of diptheria toxin in the blood leads to complications in distant organds, espeically myocarditis and neuritis. Neuritis affects motor nerves and may result in temperory paralysis of limbs, the soft palate and even the diaphragm.  See Wiki

Bacillus anthracis is a spore-forming bacterium that causes anthrax in humans and animals. It is a zoonaotic disease in that it is normally transmitted by handling of animal carcases. Human are an accidental host. Transmission may also be via aerosol which encounter breaks in the skin (cutaneous anthrax) or get into the lungs (pulmonary anthrax). B. anthracis is hemalysis negative and catalase positive. Sporulation does not occur in vivo and the spores can remain in the environment for years. The spores can plasmids and a chromosome. Calcium dipicolinate stabilizes the spore. 

The endospores of Bacillus anthracis are the infectious particles of anthrax. Spores are dormant bacterial morphotypes able to withstand harsh environments for decades, which contributes to their ability to be formulated and dispersed as a biological weapon. See Liu. See UCLA

Types of Anthrax: 

The least severe but most common anthrax is called  cutaneous anthrax where spores are deposited in an abrasion. Infection begins as an erythematous macule or papule which enlarges. The most severe form of anthrax is inhalation anthrax (Woolsorter’s Disease) where spores are inhaled and deposited in terminal alveoli. They are then engulfed by macrophages and transported to regional lymph nodes where they germinate. Case fatality rate is about 85% even with treatment.  Gastrointestinal anthrax is the least common anthrax and results from ingestion of contaminated meat or food where organisms or spores penetrate the intestinal mucosa. CFR is about 50%. 

The incidence (1-2 cases of cutaneous disease per year) of naturally acquired anthrax is rare in the United States. In fall 2001, intentional contamination of mail resulted in 22 cases of anthrax, of which 11 were inhalation and 11 cutaneous.

Pathology/Virulence:

The two major factors responsible for virulence are presence of the capsule and toxin production. 

Capsule:

The capsule (slime layer) is a polymer of amino acids (D-glutamate), unlike most other bacteria which have polysaccharide capsules. The cells excrete the capsule for protection and virulence. The capsule and the S-layer are compatible, but they can both be formed independently (without the presence of the other). A characteristic mucoid or “smooth” colony variant is correlated with capsule production ability. Virulent strains all form the capsule, and “rough” colony capsules are avirulent. Growth in atmospheric CO₂ cause the antiphagocytic capsule and anthrax toxin proteins to be synthesized. The nontoxic capsule has an important role in infection establishment, while the end disease phases are mediated by the toxin. See Microbe Wiki

The capsule of Bacillus anthracis, composed of poly-D-glutamic acid, serves as one of the principal virulence factors during anthrax infection. By virtue of its negative charge, the capsule is purported to inhibit host defence through inhibition of phagocytosis of the vegetative cells by macrophages. In conjunction with lethal toxin and oedema toxin, whose target cells include macrophages and neutrophils, respectively, the capsule allows virulent anthrax bacilli to grow virtually unimpeded in the infected host. See Ezzell

Exotoxins: 

Two B anthracis toxiins, lethal toxin (LT) and edema toxin (ET) are primary mediators of disease. LT and ET each consist of two components, an enzymatic activity, lethal factor (LF) and I. edema factor (EF), respectively, and a shared cofactor,  protective antigen. The protective antigen is responsible for getting the other 2 toxins into the cell. Thus if you have protection against this toxin you have immunity to the other two toxins. 

The three exotoxin compoents (PA, letal factor, and edema factor) are encoded on virulence plasmid pX01, which is 185.5 kilobase pairs (kbp) in size. The other virulence plasmid, pX02, is 95.3 kbp and codes for 3 genes (cap!, capB, and capC) associated with the synthesis of the polyglutamyl capsule, which inhibits phagocytosis of vegetative antrhax bacilli.

–Edema Toxin: consists of protective antigen (PA), which permits entry of the toxin into the host cell, and edema factor, a calmodulin-dependent adenylate cyclase that increases intracellular levels of cyclic adenosine monophospate, upsetting water homeostasis.

–Lethal toxin: consists of PA and lethal factor, a zinc metalloprotease that ininactivates mitogen-actived protein kinase in vtiro (It has been shown to cleave the N Terminus of MKKs 1, 2, 3, 4, 6, and 7 and thereby suppress phosphorylation of downstream mitogen-activated protein kinases (MAP kinases) including ERK, p38 and JNK/SAPK. ) and stimulates macrophages to release tumor necrosis factor alpha and interleukin 1beta.

Symptoms

During the first stage of inhaled anthrax illness, the symptoms are similar to influenza, including fever, coughing, sore throat, fatigue, sweating, vomiting, diarrhea, headache, nausea, chest pain, and shortness of breath. Symptoms are much more extreme in the second stage, which can result in death in 2-48 hours. 

Detection: 

Laboratory diagnosis of anthrax involves identifcation of B. anthracis microbiologically, serologically, or by use of more sensitive molecular technieques. B. anthracis forms rough gra-white colonies with characteristic “comet-tail” protrusions on sheep blood agar. It is aerobic, Gram-positive, spore-forming, and nonmotile.

Treatment: 

Treatment of B. anthracis includes penicillin, doxycycline and ciprofloxacin. Antibiotics are used against anthrax but must be administered before the bacterial load has reached 3 x 108 CFU.

Treatment of any form of anthrax infection is generally the same: aggressive antibiotics and supportive care. Intravenous ciprofloxacin or deoxycycline is recommended for treatment of anthrax, usually as part of a cocktail of antibiotics.