Section I– Microbiology By Nada Sajet

Wound infections and rare cause of

septicemia

P.damsela

V. fluvialis Gastroenteritis

V. furnissii Rarely associated with human infections

Grimontia hollisa Gastroenteritis; rare cause of septicemia

Rare cause of septicemia; involvement in

gastroenteritis

is uncertain

V. metschnikovii

V. mimicus Gastroenteritis; rare cause of ear infection

Wound infections and septicemia;

involvement in

gastroenteritis is uncertain

V. vulnificus

Gastroenteritis, wound infections,

bacteremia, and

miscellaneous other infections, including

endocarditis,

meningitis, pneumonia, conjunctivitis, and

osteomyelitis

Aeromonas spp. produce various toxins and

factors, but their specific role in virulence is

uncertain

Aeromonas spp.

Rare but dangerous infection. Begins with

cellulitis or

lymphadenitis and can rapidly progress to

systemic

infection with abscess formation in various

organs and

septic shock

Endotoxin, adhesins, invasins and cytolytic

proteins have been described.

Chromobacterium

violaceum

C. violaceum is not associated with gastrointestinal infections, but acquisition of this organism by

contamination of wounds can lead to fulminant, life-threatening systemic infections.

Laboratory diagnosis

Specimen collection and transport

Because no special considerations are required for isolation of these genera. However, stool specimens

suspected of containing Vibrio spp. should be collected and transported only in Cary-Blair medium.

 Buffered glycerol saline is not acceptable, because glycerol is toxic for vibrios. Feces is preferable, but rectal

swabs are acceptable during the acute phase of diarrheal illness.

Specimen processing

No special considerations are required for processing of the organisms .

Direct detection methods

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 V. cholerae toxin can be detected in stool using an enzyme linked immunosorbent assay (ELISA) or a

commercially available latex agglutination test but these tests are not widely used in the United States.

Microscopically, vibrios are gram-negative, straight or slightly curved rods (Figure 1).

When stool specimen from patients with cholera are examined using darkfield microscopy, the bacilli exhibit

characteristic rapid darting or shooting-star motility. However, direct microscopic examination of stools by any

method is not commonly used for laboratory diagnosis of enteric bacterial infections.

Aeromonas spp. are gram-negative, straight rods with rounded ends or coccobacilli. No molecular or serologic

methods are available for direct detection of Aeromonas spp. Cells of C. violaceum are slightly curved, medium

to long, gram-negative rods with rounded ends. A polymerase chain reaction (PCR) amplification assay has

been developed for identification of C. violaceum.

Cultivation:

Media of Choice

Stool cultures for Vibrio spp. are plated on the selective medium thiosulfate citrate bile salts sucrose (TCBS)

agar.

TCBS contains 1% sodium chloride, bile salts that inhibit the growth of gram-positive organisms, and sucrose

forthe differentiation of the various Vibrio spp. Bromothymol blue and thymol blue pH indicators are added to

the medium. The high pH of the medium (8.6) inhibits

the growth of other intestinal flora. Although some Vibrio spp. grow very poorly on this medium, those that

grow well produce either yellow or green colonies, depending on whether they are able to ferment sucrose

(which produces yellow colonies).

Figure 1 Gram stain of Vibrio parahaemolyticus

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 Alkaline peptone water (pH 8.4) may be used as an enrichment broth for obtaining growth of vibrios from

stool. After inoculation,the broth is incubated for 5 to 8 hours at 35°C and then subcultured to TCBS.

Chromogenic Vibrio agar, which was developed for the recovery of Vibrio parahaemolyticus from seafood,

supports the growth of other Vibrio spp. Colonies on this agar range from white to pale blue and violet.

Aeromonas spp. are indistinguishable from Yersinia enterocolitica on modified cefsulodin-irgasan-novobiocin

(CIN) agar (4 μg/mL of cefsulodin); therefore, it is important to perform an oxidase test to differentiate the two

genera. Aeromonas agar is a relatively new alternative medium that uses D-xylose as a differential

characteristic.

These organisms typically grow on a variety of differential and selective agars used for the identification of

enteric pathogens. They are also beta-hemolytic on blood agar.

C. violaceum grows on most routine laboratory media. The colonies may be beta-hemolytic and have an

almond like odor. Most strains produce violacein, an ethanolsoluble violet pigment.

All of the genera considered in this lecture grow well on 5% sheep blood, chocolate, and MacConkey agars.

They also grow well in the broth of blood culture systems and in thioglycollate or brain-heart infusion broths.

Incubation Conditions and Duration

 These organisms produce detectable growth on 5% sheep blood and chocolate agars when incubated at 35°C

in carbon dioxide or ambient air for a minimum of 24 hours. MacConkey and TCBS agars only should be

incubated at 35°C in ambient air. The typical violet pigment of C. violaceum colonies (Figure 2) is optimally

produced when cultures are incubated at room temperature (22°C).

Figure 2 Colonies of Chromo bacterium violaceum on DNase agar. Note violet pigment

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Organism Medium Appearance

Large, round, raised, opaque; most pathogenic strains are beta-hemolytic except A.

caviae, which is usually nonhemolytic Both NLF and LF

BA

Mac

Aeromonas spp.

Round, smooth, convex, some strains are beta-hemolytic; most

colonies appear black or very dark purple; cultures smell of ammonium cyanide

(almond-like) NLF

BA

Mac

Chromobacterium

violaceum

Medium to large, smooth, opaque, iridescent with a greenish hue; V. cholerae, V.

fluvialis, and V. mimicus can be beta-hemolytic NLF except V. vulnificus, which may

be LF

BA

Mac

Vibrio spp. and

Grimontia

hollisae

Medium to large, smooth, opaque, iridescent with a greenish hue; may be betahemolytic NLF

BA

Mac

P. damsela

BA, 5% sheep blood agar; Mac, MacConkey agar; LF, lactose fermenter,

NLF, non–lactose fermenter.

Colonial Appearance

 Table 3 describes the colonial appearance and other distinguishing characteristics (e.g., hemolysis and odor)

of each genus on 5% sheep blood and MacConkey agars. The appearance of Vibrio spp. on TCBS is shown in

Figure ( 3).

Table( 3 ) Colonial Appearance and Characteristic

Figure 3 Colonies of Vibrio cholerae (A) and V. parahaemolyticus (B) on TCBS agar.

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Approach to identification

The colonies of these genera resemble those of the family Enterobacteriaceae but can be distinguished notably

by their positive oxidase test result (except for V. metschnikovii, which is oxidase negative). The oxidase test

must be performed from 5% sheep blood or another medium without a fermentable sugar (e.g., lactose in

MacConkey agar or sucrose in TCBS), because fermentation of a

carbohydrate results in acidification of the medium, and a false-negative result may occur if the surrounding pH

is below 5.1. Likewise, if the violet pigment of a suspected C. violaceum isolate interferes with performance of

the oxidase test, the organism should be grown under anaerobic conditions (where it cannot produce pigment)

and retested.

The reliability of commercial identification systems has not been widely validated for identification of these

organisms, although most are listed in the databases of several systems. The API 20E system (bioMérieux,

St.Louis, Missouri) is one of the best for vibrios. Because the inoculum is prepared in 0.85% saline, the amount

of salt often is enough to allow growth of the halophilic (salt-loving) organism.

The ability of most commercial identification systems to accurately identify Aeromonas organisms to the

species level is limited and uncertain, and with some kits, difficulty arises in separating Aeromonas spp. from

Vibrio spp. Therefore, identification of potential pathogens should be confirmed using conventional

biochemical tests or serotyping. show several characteristics that can be used to presumptively group Vibrio

spp., Aeromonas spp., and Chromobacterium violaceum

Figure 4 String test used to differentiate Vibrio spp. (positive( from

Aeromonas spp. and P. shigelloides (negative).

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Comments Regarding Specific Organisms

 V. cholerae and Vibrio mimicus are the only Vibrio spp. That do not require salt for growth. Therefore, a key

test for distinguishing the halophilic species from V. cholerae, V. mimicus, and Aeromonas spp. is growth in

nutrient broth with 6% salt. Furthermore, the addition of 1% NaCl to conventional biochemical tests is

recommended to allow growth of halophilic species.

The string test can be used to differentiate Vibrio spp. from Aeromonas spp. In this test, organisms are

emulsified in 0.5% sodium deoxycholate, which lyses Vibrio cells, but not those of Aeromonas spp. Cell lysis

releases DNA, which can be pulled up into a string with an inoculating loop Figure ( 4).

 A Vibrio static test using 0/129 (2,4-diamino-6,7-diisopropylpteridine)–impregnated disks also has been used

to separate vibrios (susceptible) from other Oxidase positive glucose fermenters (resistant) and to differentiate

V. cholerae O1 and non-O1 (susceptible) from other Vibrio spp. (resistant). However, recent strains of V.

cholerae O139 have demonstrated resistance, so the dependability of this test is questionable.

Serotyping should be performed immediately to further characterize V. cholerae isolates. Toxigenic strains of

serogroup O1 and O139 can be involved in cholera epidemics. Strains that do not type in either antiserum are

identified as non-O1. Although typing sera are commercially available, isolates of V. cholerae are usually sent

to a reference laboratory for serotyping.

Identification of V. cholerae or V. vulnificus should be reported immediately because of the life-threatening

nature of these organisms.

Aeromonas spp. and C. violaceum can be identified using the characteristics. Aeromonas spp. identified in

clinical specimens should be identified as A. hydrophilia, A. caviae complex, or A. veronii complex.

Pigmented strains of C. violaceum are so distinctive that a presumptive identification can be made based on

colonial appearance, oxidase, and Gram staining. Nonpigmented strains (approximately 9% of isolates) may be

differentiated from Pseudomonas, Burkholderia, Brevundimonas, and Ralstonia organisms based on glucose

fermentation and a positive test result for indole. Negative lysine and ornithine reactions are useful criteria for

distinguishing C. violaceum from Plesiomonas shigelloides. Failure to ferment either maltose or mannitol also

differentiates C. violaceum from Aeromonas spp.

Serodiagnosis

 Agglutination, vibriocidal, or antitoxin tests are available for diagnosing cholera using acute and convalescent

sera. However, these methods are most commonly used for epidemiologic purposes. Serodiagnostic techniques

are not generally used for laboratory diagnosis of infections caused by the other organisms discussed in this

lecture.

Antimicrobial susceptibility testing and therapy:

 Two components of the management of patients with cholera are rehydration and antimicrobial therapy

(Table4). Antimicrobials reduce the severity of the illness and shorten the duration of organism shedding. The

drug of choice for cholera is tetracycline or doxycycline; however, resistance to these agents is known, and the

use of other agents, such as chloramphenicol, ampicillin, or trimethoprim-sulfamethoxazole, may be necessary.

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The Clinical and Laboratory Standards Institute (CLSI) has established methods for testing for V. cholerae, and

the CLSI document should be consulted for this purpose.

 The need for antimicrobial intervention for gastrointestinal infections caused by other Vibrio spp. and

Aeromonas spp. is less clear. However, extraintestinal infections with these organisms and with C. violaceum

can be lifethreatening, and directed therapy is required.

 C. violaceum is often resistant to β-lactams and colistin. Antimicrobial agents with potential activity are

listed, where appropriate, in Table 4. It is important to note these organisms’ ability to show resistance to

therapeutic agents; especially noteworthy is the ability of Aeromonas spp. to produce various beta-lactamases.

Potential Resistance to

Therapeutic Options

Species Therapeutic Options

Adequate rehydration plus

antibiotics. Recommended agents

include tetracycline or doxycycline;

alternatives include

trimethoprimsulfamethoxazole,

erythromycin,

chloramphenicol, and quinolones

Adequate rehydration plus

antibiotics. Recommended agents

include tetracycline or doxycycline;

alternatives include trimethoprimsulfamethoxazole,

erythromycin,

chloramphenicol, and quinolones

Vibrio cholerae

Similar to resistance reported

for V. cholerae

No definitive guidelines. For

gastroenteritis, therapy may not

be needed; for wound infections

and septicemia, potentially active

agents include tetracycline,

chloramphenicol, nalidixic acid,

most cephalosporins, and

quinolones

Other Vibrio spp.

Capable of producing various

beta-lactamases that

mediate resistance to

penicillins and certain

cephalosporins

No definitive guidelines. For

gastroenteritis, therapy may not

be needed; for soft tissue

infections and septicemia,

potentially active agents include

ceftriaxone, cefotaxime,

ceftazidime, imipenem,

aztreonam, amoxicillinclavulanate,

quinolones, and

trimethoprim-sulfamethoxazole

Aeromonas spp.

Activity of penicillins is

variable; activity of firstand

No definitive guidelines. Potentially

active agents include cefotaxime,

Chromobacterium

violaceum

Table 4 Antimicrobial Therapy and Susceptibility Testing

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second-generation

cephalosporins is poor

ceftazidime, imipenem, and

aminoglycosides

*Validated testing methods include standard methods recommended by the Clinical and Laboratory

Standards Institute (CLSI) and commercial methods

approved by the U.S. Food and Drug Administration (FDA).

Prevention:

 No cholera vaccine is available in the United States. Two oral vaccines are available outside the United States

although the World Health Organization no longer recommends immunization for travel to or from

cholerainfected areas. Individuals who have recently shared food and drink with a patient with cholera (e.g.,

household contacts) should be given chemoprophylaxis with tetracycline, doxycycline, or trimethoprimsulfamethoxazole. However, mass chemoprophylaxis during epidemics is not indicated.

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Lecture Six

Genus Campylobacter

Campylobacter jejuni and C. coli can colonies the intestinal tract of most mammals and birds and are the most

frequently isolated Campylobacter species in humans with gastro-enteritis. Transmission from animals to

humans is mainly through consumption and handling of animal food products but also direct contact with

colonized animals may contribute to human Campylobacteriosis.

The fecal contamination of meat (especially poultry meat) during processing is

Considered to be a major source of human food-borne disease.

In humans, extra intestinal infections, including bacteremia, Guillain–Barré syndrome, reactive arthritis, and

abortion could occur as sequelae of infection.

Detection of intestinal colonization is based on the isolation of the organism from feces, rectal swabs and / or

caecal contents.

Alternatively, their high motility can be exploited using filtration techniques for isolation. Preliminary

confirmation of isolates can be made by light microscopy. The organisms in the log growth phase are short and

S-shaped in appearance, while coccoid forms predominate in older cultures.

Under phase-contrast microscopy the organisms have a characteristic rapid corkscrew-like motility. Phenotypic

identification is based on reactions under different growth conditions. Biochemical and molecular tests can be

used to confirm various Campylobacter species. Polymerase chain reaction assays also can be used for the

direct detection of C. jejuni and C. coli.

In 1991 a revision of the taxonomy and nomenclature of the genus Campylobacter was proposed. According to

Bergey’s Manual, the genus Campylobacter comprises sixteen species and six subspecies. More recently, two

additional species have been added.

Members of the genus are typically gram-negative, non-spore-forming, S-shaped or spiral shaped bacteria (0.2–

0.8 μm wide and 0.5–5 μm long), with single polar flagella at one or both ends, conferring a characteristic

corkscrew-like motility. These bacteria require micro aerobic conditions, but some strains also grow aerobically

or anaerobically. They neither ferment nor oxidise carbohydrates. Some species, particularly C. jejuni, C. coli

and C. lari, are thermophilic, growing optimally at 42°C. They can colonies mucosal surfaces, usually the

intestinal tract, of most mammalian and avian species tested. The species C. jejuni includes two subspecies (C.

jejuni subsp. jejuni and C. jejuni subsp. doylei) that can be discriminated on the basis of several phenotypic

tests (nitrate reduction, selenite reduction, sodium fluoride, and safranine) and growth at 42°C (subsp. doylei

does not grow at 42°C). Subspecies jejuni is much more frequently isolated then subspecies doylei.

Passive filtration

Passive filtration, a method developed by Steele and McDermott for the need for selective media; thus it is very

useful for the isolation of antimicrobial-sensitive Campylobacter species.

The method does not use expensive selective media; it may be used in laboratories with fewer resources.

For passive filtration, feces are mixed with phosphorylate buffered saline (approximately 1/10 dilution) to

produce a suspension. Approximately 100 μl of this suspension are then carefully layered on to a 0.45 or 0.65

μm filter, which has been previously placed on top of a non-selective blood agar plate. Care must be taken not

to allow the inoculum to spill over the edge of the filter. The bacteria are allowed to migrate through the filter

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for 30–45 minutes at 37°C or room temperature. The filter is then removed, the fluid that has passed through

the filter is spread with a sterile glass or plastic spreader, and the plate is incubated micro aerobically at 42°C

(Micro aerobic atmospheres of 5–10% oxygen, 5–10% carbon dioxide are required for optimal growth).

Specimen Types

Fresh stool specimens or rectal swabs from patients suspected to be infected with Campylobacter species, or

meat.

Stool specimens, swabs, and food samples should not be older than 24 to 48 hours. Swabs must be inserted into

appropriate transport media (e.g., Cary Blair medium). If not processed immediately, store specimens in

transport media at 4 to 8° C; avoid exposure to oxygen.

Samples Processing

Streak the specimen for dilution as soon as possible after it is received in the laboratory onto Butzler or

Skirrow. Meat or other foods should first be minced or homogenized and then inoculated directly or after

suspension in a small amount of peptone broth onto the medium.

If material is being cultured directly from a swab, roll the swab over a small area of the surface at the edge;

then streak from this inoculated area.

Incubate inoculated plates, protected from light, at 35 ± 2 ° C or 42 ±2° C in a reduced oxygen, increased CO2

(micro aerobic) atmosphere. The incubation at 42° C results in better selectivity, but is inhibitory to

Campylobacter jejuni subsp. doylei and a variety of other species.

The micro aerobic atmosphere can be achieved by using Campy gaspak. An incubation period of 2 to 3 days is

usually sufficient, but 5 to 7 days of incubation were shown to increase the isolation rates.

Cultivation Media

Campylobacter agar known as Butzler plus Campylobacter agar known as Skirrow; are 2 selective media for

the isolation of Campylobacter species from clinical specimens.

The genus Campylobacter includes important pathogens causing intestinal infections such as diarrhea. In rural

areas and in less developed countries, Campylobacters are at least as frequent as Salmonella as intestinal

pathogens.

The most frequently isolated species is Campylobacter jejuni subsp. jejuni, whereas C. coli and C. lari are

rarer.

Butzler, in 1973, developed a selective medium containing five antimicrobials. Skirrow, in 1977, reported a

selective culture medium containing three antimicrobial agents.

In Butzler, meat extract and peptone provide the nutrients, and sodium chloride maintains the osmotic stability.

Novobiocin and colistin inhibit gram negative enteric bacteria; cephazolin and bacitracin inhibit gram positive

bacteria. Cycloheximide inhibits many fungi.

In Skirrow, heart infusion, casein peptone, and yeast extract provide nutrients, and sodium chloride maintains

the osmotic stability. Vancomycin inhibits gram positives, and trimethoprime and polymyxin B inhibit many

gram negative organisms. Lysed horse blood provides nutrients and heme for bacterial catalase.

Isolate Confirmation

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A pure culture is required for confirmatory tests, but a preliminary confirmation can be obtained by direct

microscopic examination of suspect colony material.

1- Identification on solid medium: on Skirrow or other blood-containing agars, characteristic

Campylobacter colonies are slightly pink, round, convex, smooth and shiny, with a regular edge. On

charcoal-based media, the characteristic colonies are grayish, flat and moistened, with a tendency to

spread, and may have a metal sheen.

2- Microscopic examination of morphology and motility: material from a suspect colony is suspended in

saline and evaluated, preferably by a phase-contrast microscope, for characteristic, spiral or curved

slender rods with a corkscrew-like motility. Older cultures show less motile coccoid forms.

3- Detection of oxidase: take material from a suspect colony and place it on to a filter paper moistened

with oxidase reagent. The appearance of a violet or deep blue color within 10 seconds is a positive

reaction.

4- Micro aerobic growth at 25°C: Inoculate the pure culture on to a non-selective blood agar plate and

incubate at 25°C in a micro aerobic atmosphere for 48 hours.

5- Aerobic growth at 41.5°C, inoculate the pure culture on to a non-selective blood agar plate and

incubate at 41.5°C in an aerobic atmosphere for 48 hours.

6- Latex agglutination tests for confirmation of pure cultures of C. jejuni / C. coli (often also including C.

lari) are commercially available.

Helicobacter pylorus

Helicobacter pylorus (H. pylori) is a spiral-shaped bacterium that is found in the gastric mucous layer or

adherent to the epithelial lining of the stomach.

H. pylori cause more than 90% of duodenal ulcers and up to 80% of gastric ulcers. Spicy food, acid, stress, and

lifestyle were considered the major causes of ulcers.

Majority of patients were given long-term medications, such as H2 blockers, and more recently, proton pump

inhibitors, without a chance for permanent cure.

These medications relieve ulcer-related symptoms, heal gastric mucosal inflammation, and may heal the ulcer,

but they do not treat the infection.

When acid suppression is removed, the majority of ulcers, particularly those caused by H. pylori, recur. So

most ulcers which caused by H. pylori, appropriate antibiotic regimens can successfully eradicate the infection

in most patients, with complete resolution of mucosal inflammation and a minimal chance for recurrence of

ulcers; approximately 2/3 of the world’s population is infected with H. pylori.

H. pylori are more prevalent among adults and lower socioeconomic groups.

Most persons who are infected with H. pylori never suffer any symptoms related to the infection; however, H.

pylori cause chronic active, chronic persistent and atrophic gastritis in adults and children. Infection with H.

pylori also causes

duodenal and gastric ulcers.

Infected persons have a 2- to 6-fold increased risk of developing gastric cancer and mucosal associatedlymphoid-type lymphoma compared with their uninfected counterparts.

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The role of H. pylori in non-ulcer dyspepsia remains unclear. Peptic ulcer disease at some point in their

lifetime. Each year there are 500,000 to 850,000 new cases of peptic ulcer disease and more than one million

ulcer-related hospitalizations.

The pain typically occurs when the stomach is empty, between meals and in the early morning hours, but it can

also occur at other times.

It may last from minutes to hours and may be relieved by eating or by taking antacids.

Less common ulcer symptoms include nausea, vomiting, and loss of appetite. Bleeding can also occur;

prolonged bleeding may cause anemia leading to weakness and fatigue.

Persons with active gastric or duodenal ulcers or documented history of ulcers should be tested for H. pylori,

and if found to be infected, they should be treated.

Several methods may be used to diagnose H. pylori infection. Serological tests that measure specific H. pylori

IgG antibodies can determine if a person has been infected. The sensitivity and specificity of these assays range

from 80% to 95% depending upon the assay used.

Other diagnostic method is the breath test. In this test, the patient is given either 13C or 14C-labeled urea to

drink. H. pylori metabolize the urea rapidly, and the labeled carbon is absorbed. This labeled carbon can then be

measured as CO2 in the patient’s expired breath to determine whether H. pylori is present. The sensitivity and

specificity of the breath test ranges from 94% to 98%.

During endoscopy, biopsy specimens of the stomach and duodenum are obtained and the diagnosis of H. pylori

can be made by several methods:

1. The biopsy urease test - a colorimetric test based on the ability of H. pylori to produce urease; it

provides rapid testing at the time of biopsy.

2. Histological identification of organisms - considered the gold standard of diagnostic tests.

3. Culture of biopsy specimens for H. pylori, which requires an experienced laboratory and is necessary

when antimicrobial susceptibility testing is desired.

Therapy for H. pylori infection consists of 10 days to 2 weeks of one or two effective antibiotics, such as

amoxicillin, tetracycline (not to be used for children <12 yrs.), metronidazole, or clarithromycin, plus either

ranitidine bismuth citrate, bismuth subsalicylate, or a proton pump inhibitor.

Acid suppression by the H2 blocker or proton pump inhibitor in conjunction with the antibiotics helps in relieve

ulcer-related symptoms (i.e., abdominal pain, nausea), helps heal gastric mucosal inflammation, and may

enhance efficacy of the antibiotics against H. pylori at the gastric mucosal surface.

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Lecture Seven

Haemophilus

is a genus of Gram-negative, pleomorphic, coccobacilli bacteria belonging to the Pasteurellaceae family. While

Haemophilus bacteria are typically small coccobacilli, they are categorized as pleomorphic bacteria because of

the wide range of shapes they occasionally assume.

All Haemophilus are nonmotile, they are minute in size, H. influenzae measuring 0.3 micrometer across and up

to 2 micrometers long.

The genus includes commensal organisms along with some significant pathogenic species such as H. influenzae

a cause of sepsis and bacterial meningitis in young children and H. ducreyi, the causative agent of chancroid.

All members are either aerobic or facultative anaerobic.

Members of the Haemophilus genus will not grow on blood agar plates as all species require at least one of the

following blood factors for growth: hemin (factor X) and/or nicotinamide adenine dinucleotide (factor V).

Chocolate agar is an excellent Haemophilus growth medium as it allows for increased accessibility to these

factors. Alternatively, Haemophilus is sometimes cultured using the "Staph streak" technique; both

Staphylococcus and Haemophilus organisms are cultured together on a single blood agar plate. In this case,

Haemophilus colonies will frequently grow in small "satellite" colonies around the larger Staphylococcus

colonies because the metabolism of Staphylococcus produces the necessary blood factor by-products required

for Haemophilus growth

Most strains of H. influenzae are opportunistic pathogens; that is, they usually live in their host without causing

disease, but cause problems only when other factors (such as a viral infection, reduced immune function or

chronically inflamed tissues, e.g. from allergies) create an opportunity. They infect the host by sticking to the

host cell using Trimeric Auto transporter Adhesins (TAA).

Naturally acquired disease caused by H. influenzae seems to occur in humans only. In infants and young

children, H. influenzae type b (Hib) causes bacteremia, pneumonia, epiglottitis and acute bacterial meningitis.

On occasion, it causes cellulitis, osteomyelitis, and infectious arthritis.

Due to routine use of the Hib conjugate vaccine in the U.S.A. since 1990, the incidence of invasive Hib

disease has decreased to 1.3/100,000 in children. However, Hib remains a major cause of lower respiratory tract

infections in infants and children in developing countries where the vaccine is not widely used.

Unencapsulated H. influenzae strains are unaffected by the Hib vaccine and cause ear infections (otitis media),

eye infections (conjunctivitis), and sinusitis in children, and are associated with pneumonia.

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Figure shows Gram film H.influenzae in the sputum (coccobacilli)

Figure shows Haemophilus influenzae which requires X and V factors for growth. In this culture

Haemophilus has only grown around the paper disc that has been impregnated with X and V factors. There

is no bacterial growth around the discs that only contain either X or V factor

Laboratory Diagnosis

 Most Haemophilus species are normal inhabitants of upper respiratory tract of humans and other

animals.

 The species of Haemophilus that most frequently cause human infections are H. influenzae (respiratory

and invasive infections), H. aegyptius (acute conjunctivitis) and H. parainfluenzae, H. haemolyticus,

H. parahaemolyticus, H. aprophilus, H. paraphrophilus and H. segnis (abscesses and infective

endocarditis)

 These are pleomorphic Gram negative coccobacilli.

 The laboratory diagnosis of H. influenzae is based on growth and colony morphology, and cell

morphology on Gram staining.

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 These are confirmed by the haemophilic character of the genus that reflects a requirement for either or

both of the two factors called X and V.

Culture and identification for Haemophilus

 Direct Examination

 Perform Gram stain

 Gram staining shows: Gram negative pleomorphic thin rods or coccobacilli.

Culture

o Inoculate samples onto chocolate agar media

o Incubate at 37°C in aerobic atmosphere containing 3-5 % CO2 (i.e. candle jar) for 24-48 hours.

Colony morphology

On chocolate agar: large flat, colorless to gray or opaque colonies.

Colonies are 0.5 – 1mm circular, low convex, smooth, and pale grey and transparent.

With a characteristic “mouse nest” odor. No haemolysis or discoloration is seen.

Encapsulated strains appear more mucoid (watery) and non capsulated strains appear as compact

grayish colonies.

Note: Growth is enhanced on chocolate agar and satellitism around Staphylococcus aureus is seen on blood

agar.

Biochemical reactions for differentiation:

Confirmatory tests for X and V factor requirements as follow:

1. Inoculate a single suspected colony from chocolate agar onto Mueller Hinton agar plates.

2. Place commercially available X, V, and XV factor discs/strips on streaked plates.

3. Incubate plates at 37°C in 3-5 % CO2 atmosphere for 18-24 hours.

4. Observe growth around the discs and H. influenzae will only grow around the combined XV disc.

Note that if only X and V factor discs (without XV) are applied, place them at least 2 cm apart and H.

influenzae will grow between the two discs.

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Serological identification (serotyping) of Haemophilus influenzae

Slide agglutination test.

1) Agglutinating antisera for serotypes “a” to “f” are available commercially. Such sera contain

antibodies directed towards somatic antigens present in patient’s sera which result in agglutination.

2) Apply one drop of normal saline on a slide and make a homogenous suspension with a single

suspected colony of H. influenzae.

3) Add one drop of specific antiserum and mix thoroughly.

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Lecture Eight

Sexually transmitted diseases (STD)

Genus Neisseria

The family Neisseriaceae includes the many genera, important one is genus Neisseria. The Neisseriae are gramnegative cocci that usually occur in pairs. Neisseria gonorrhea (gonococci) and Neisseria meningitidis

(meningococci) are pathogenic for humans and typically are found associated with or inside polymorphonuclear

cells.

Other neisseriae are normal inhabitants of the human respiratory tract, rarely if ever cause disease, and occur

extracellularly.

 N. catarrhalis (Moraxella or Branhamella catarrhalis)

 N. sicca

 N. subflava

 N. lactamica

 N. mucosa

Common or general characteristics of Neisseria are:

Gram negative diplococci, kidney shape, with flat or concave opposing edge, oxidase positive, pathogenic to

human only, Ferment carbohydrates and producing acid only, pyrogenic (produce pus), Non - hemolytic, nonmotile, non-spore forming, intracellular while non-pathogenic are extracellular, needs 48h of culturing time,

they are rapidly killed by drying, sunlight and many disinfectants, the non-pathogenic species can grow on

simple media while the pathogenic species need enriched media e.g. blood and chocolate agar, Gonococci and

meningococci are closely related with 70% DNA homology and are differentiated by few laboratory tests and

specific characteristics.

Neisseria gonorrhea (gonococcus)

Reservoir: human genital tract

Transmission: Sexual contact, birth

Sensitive to drying and cold

Gonorrhea; which differs in male than in female as in male it causes urethritis associated with yellowish

mucopurulent discharge from anterior urethra and dysuria, the infection can extend to the epididymis leading to

epididymitis and if not treated it will be complicated by fibrosis and urethral stricture.

In female the first site of infection is the cervix causing cervicitis, the infection is going to spread from cervix to

the vagina causing vaginitis and presented by vaginal discharge which is mucopurulent in nature, if not treated

it will spread upward to involve fallopian tube resulting in salpingitis plus PID (pelvic inflammatory disease)

which leads to fibrosis with tube stricture leading to infertility due to tubal damage.

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Rectal involvement will lead to proctitis; the patient presenting symptoms is purulent discharge from anus with

tensmus

Eye infection (infection of the conjunctival sac); occur in new born babies during their passage through

infected birth canal causing a condition called (ophthalmia neonatorum) which is a very serious condition and if

untreated it will lead to blindness. This case could be prevented through using antimicrobial agent as silver

nitrate and erythromycin

Throat infection (Gonococcal pharyngitis)

Sometimes Gonococcal can cause systemic infection as it disseminated by the blood stream to the distant

organs (skin, bone, joints) to gain arthritis or meningitis or endocarditis or vasculitis

Laboratory diagnosis

 Specimens: Pus and secretion are taken from the urethra, cervix, rectum, conjunctiva, throat or

synovial fluid for culture and smear.

 Smears: Gram stained smears of urethral or endocervical exudates reveal many diplococci within pus

cells. In male the finding of numerous neutrophils containing gram negative diplococci in a smear of

urethral exudates permits a provisional diagnosis of gonococcal infection and indicates that the

individual should be treated. In contrast, a positive culture is needed to diagnose gonococcal infection

in female

 Culture: Immediately after collection of pus or mucous is streaked on enriched selective media such as

Thayer – Martin which is a chocolate agar + Enrichment element +Antibiotics like colistin (against G

– ve rods); vancomycin (against G + ve), Nystatin (against fungi)

 Modified Thayer - Martin: same as the above culture medium but plus trimethoprim (inhibit proteus)

It needs 5 – 7% CO2 (candle jar, CO2 generating kit); Humid atmosphere; 35 – 37 C◦, 72 incubation hours

 Biochemical test:

I. Oxidase test (N, N, N, N, tetramethyl-P-phenylenediamine) positive for all Neisseria species,

when get contact with oxidase enzyme they will gain purple color i.e. positive oxidase test.

II. Sugar fermentation test: (important in differentiation between Neisseria spp.)

Sucrose + Maltose + Glucose is (negative + positive + positive) regarding N. meningitidis, (negative + negative

+ positive) regarding N. gonorrhea and regarding M. catarrhalis all are negative

III- Nitrate reduction into nitrite it is positive, while in Moraxella catarrhalis it is negative

 Serology: It is not of great value in the diagnosis to detect antibodies against this bacterium; because

of the antigen diversity of Gonococci and there is delay in the development of these antibodies, so it

is more important to detect gonococcal antigens using elisa (EIA) or using radioimmunoassay (RIA).

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 PCR (molecular method): Detection of gonococcal nucleic acid using DNA probe which can detect

nucleic acid of the microorganism, this is the most sensitive and specific method.

Antigenic and virulence structures

Gonococci are antigenically heterogeneous both in vivo and in vitro. These antigenic changes help the

microorganism to escape from the immune system and it includes:

a- Pilli which is responsible for attachment to mucosal surfaces, also inhibit phagocytic uptake, and

antigenic (immunogenic) variation as more than 1 million variants are found

b- Porin (por) protein (protein I) that extends through the gonococcal cell membrane, each strain of

gonococcus expresses only one type of por.

c- Opacity (Opa) proteins (protein II) responsible for attachment to mucosal surfaces

d- RMP (protein III) It is associated with (Opa) protein in the formation of pores

e- Lipooligosaccharide (LOS) it has endotoxin effect and it is responsible for toxicity of gonococci

f- IgA protease it split IgA making it in non-functioning form, so IgA protease is a virulent factor that

enhances colonization of bacteria.

Treatment and prevention

More than 20% of current isolates of N. gonorrhoea are resistant to penicillin, A single intramuscular dose of

ceftriaxone is the recommended therapy for uncomplicated gonococcal infections. Intramuscular

spectinomycin is indicated in patients who are allergic to cephalosporines. Many patients with gonorrhea have

coexisting Chlamydia infections; therefore Doxycycline is effective against Chlamydia.

Prevention of gonorrhea involves evaluation and management of sexual contacts, plus antibiotic

prophylactically in an exposed individual even in the absence of symptoms. The use of barrier methods is also

a preventive measure against gonorrhea as is the case for all sexually transmitted infections. No vaccine is

available for gonorrhea.

Neisseria meningitidis (meningococcus)

N. meningitidis is one of the most frequent causes of meningitis. Infection with N. meningitidis can also take the

form of a fulminant meningococcemia, with intravascular coagulation, circulatory collapse, and potentially fatal

shock, but without meningitis.

In each case, symptoms can occur with extremely rapid onset and great intensity. Outbreaks of meningitis, most

common in winter and early spring, are favored by close contact between individuals, such as occurs in schools,

institutions, and military barracks. N. meningitidis tends to strike young, previously well individuals and can

progress over a matter of hours to death.

It is gram-negative, kidney coffee bean-shaped diplococci with large antigenic capsule, grows on chocolate (not

blood) agar in 5% C02 atmosphere and ferments maltose in contrast to gonococci

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It reservoir is human nasopharynx (5-10% carriers) and its transmission is through respiratory droplets; as it is

an oropharyngeal colonizer, so it spreads to the meninges via the bloodstream and disease occurs in only small

percentage of colonized individuals.

Antigenic and virulence structures

a- Capsule: At least 13 serogroups of meningococci have been identified by immunologic specificity and

capsular polysaccharides the most important serogroups associated with disease in human are

A,B,C,Y, W135 ,they are associated with fulminant sepsis with or without meningitis, which is used

for the preparation of vaccines

b- Outer membrane protein (OMP): about 20 antigenic types, used for serotyping

c- Pilli and outer membrane proteins important in ability to colonize and invasion

d- IgA protease it cleaves IgA and allows oropharynx colonization

e- Opa protein for attachment

f- Lipooligopolysaccharide (Endotoxin) responsible for fever, septic shock in meningococcemia.

Epidemiology

Transmission occurs through inhalation of respiratory droplets from a carrier or a patient in the early stages of

the disease. In addition to contact with a carrier

Risk factors for disease include:

1- Recent viral or Mycoplasma upper respiratory tract infection

2- Active or passive smoking

3- Complement deficiency (C5-C8)

In susceptible persons, pathogenic strains may invade the bloodstream and cause systemic illness after an

incubation period of 2 to 10 days. An incidence peak among adolescents and young adults led the Centers for

Disease Control (CDC) to recently recommend vaccination of this at risk group. Humans are the only natural

host

Diseases and pathogenicity

The source of the infection is either the patient or the carriers, the route of entry is through the nasopharynx by

droplet, and meningococci will attach to the epithelial cell by pili.

From the nasopharynx the meningococci will spread through the blood stream to the target organ causing

bacteremia, so the squeal is either meningitis or suffers from fulminating meningococcemia.

Meningitis: mainly in adult ages 11 to 55 years, it is usually begun suddenly with headache, vomiting and stiff

neck, this will progress to coma within few hours. Fulminating meningococcemia is more sever with high fever

and hemorrhagic rash; there may be disseminated intravascular coagulation and circulatory collapse (waterhouse- friderichsen syndrome).

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Laboratory diagnosis

a- Specimens: Blood, CSF, Nasopharyngeal swab are taken for carrier surveys

b- Smears as gram stained smear of CSF show typical Neisseria within polymorphonuclear leukocytes

c- Culture onto chocolate agar and Thayer - Martin media, incubated at 37 ºC

d- Serology as rapid detection test to measure antigens or antibodies to meningococcal polysaccharides

through latex agglutination test, to identify N. meningitidis capsular antigens in CSF, or by

Immunoelectrophoresis

e- PCR which is the most sensitive and specific method

Treatment

Bacterial meningitis is a medical emergency. Accordingly, antibiotic treatment cannot await a definitive

bacteriologic diagnosis.

Penicillin G is the drug of choice for treating meningococcal disease. In patient allergic to penicillin

chloramphenicol and cefotaxime (or ceftriaxone) can be used.

Prevention

a- Irradiation of the carrier states (major source)

b- Isolation of the patient

c- Chemoprophylaxis for contact people

d- Vaccination with a conjugate meningococcal vaccine which was approved in the United States in 2005

for use in adolescents and adults ages 11 to 55 years, and has replaced the Unconjugated

polysaccharide vaccine; this is a tetravalent vaccine that contains capsular polysaccharides from

serogroups A, C, W-135, and Y conjugated to diphtheria toxoid

Moraxella catarrhalis

Previously they are called Neisseria catarrhalis, the name changed to Branhamella and now they are a

separated genus “Moraxella”. They are normally found in upper respiratory tract especially among school

children as (50% of school children carry this microorganism), it may cause pneumonia, otitis media, sinusitis

and other infections.

It is nonmotile, gram-negative coccobacilli that are generally found in pairs. Moraxella are aerobic, oxidase

positive, non-fastidious organisms, can grow on nutrient agar, and does not ferment carbohydrates.

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Lecture Nine

Legionella

Legionella was first recovered from the blood of a soldier more than 50 years ago, but its importance as a

human pathogen was not recognized until 1976, when a mysterious epidemic of pneumonia struck members of

the Pennsylvania American Legion.

The disease was named Legionnaire's disease by the press. Within 6 months a bacterium, subsequently named

Legionella pneumophila, had been isolated and definitively established as the agent. A general term for disease

produced by Legionella species is legionellosis.

The most common presentation of Legionella pneumophila is acute pneumonia (legionellosis); potentially any

species of Legionella may cause the disease.

Extra pulmonary disease (e.g., pericarditis and endocarditis) is rare. Less often, disease presents as a

nonpneumonic epidemic, influenza like illness called Pontiac fever.

Structure, Classification, and Antigenic Types

Legionella species are Gram-negative bacilli. There are currently 39 species and 60 distinct antigenic types of

Legionella.

Legionella cells are thin, somewhat pleomorphic Gram-negative bacilli that measure 2 to 20 μm. Long,

filamentous forms may develop, particularly after growth on the surface of agar. Ultra structurally, Legionella

has the inner and outer membranes typical of Gram-negative bacteria. It possesses pili (fimbriae), and most

species are motile by means of a single polar flagellum.

Pathogenesis

Legionella bacilli reside in surface and drinking water and are usually transmitted to humans in aerosols. The

bacteria multiply intracellularly in alveolar macrophages.

Recruited neutrophils and monocytes, as well as bacterial enzymes, produce destructive alveolar inflammation.

Direct inoculation of surgical wounds by contaminated tap water has been described.

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Figure shows smear from the lung of a patient fatally infected with L pneumophila serogroup1,

demonstrating many thin, Gram-negative bacilli (arrows). These bacteria stain less intensely with safranin

than do enteric bacilli

Host Defenses

Nonspecific physical and inflammatory pulmonary defenses are important, but cell-mediated immunity is