3- Staphylococci aureus considered susceptible to vancomycin if its MIC (minimum inhibitory concentration) is ≤ 2

µg/ml, intermediate if MIC 4-8 µg/ml which known as (VISA) and resist if MIC ≥ 16 µg/ml (VRSA). Staphylococci resistance

is due to bacteria cell wall changes and not due vancomycin resistance gene (van gene) which occur in Enterococci.

4- Tolerance; it is usually due to failure of the drug to induce the cell wall autolytic enzymes.

Staphylococci certain colonies sometimes differ from other population in shape, color, hemolysis, enzymes production,

and pathogenicity, in nafcillin resistance as 107 will be resist at 37 ºC but in 30 ºC incubation only 103 will be resist.

Staphylococci antigen

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Its cell wall peptidoglycan is important in production of endogenous pyrogen (interlukin-1) plus opsonic antibodies by

monocytes beside it act as chemotactant for the polymorphonuclear leucocytes (PMNs), also it has endotoxin like

activity to enhance the complements.

In addition the teichonic acid in the cell wall (composed of glycerol or ribitol phosphate polymers) which connects to

the peptidoglycan can enhance antibodies detected by gel diffusion in patients with Staph endocarditis.

Another antigen is the cell wall protein A act as antigen and adhesin of bacteria to the target cell wall and it attach to Fc

portion in the IgG molecules to enhance the agglutination process to specific antigen (coagglutination).

Some strains gain a capsule that inhibits bacteria phagocytosis by PMNs cells.

Coagulase (aggregation or clumping factor) also found in the cell wall, that connect enzymatically to fibrinogen

Staphylococci extracellular enzymes & toxins

The main pathogenesis is by invasion + multiplication + enzymes & toxins production

The bacterial enzymes are under plasmids or chromosomal control and they are:

1- Catalase: that convert H2O2 to H2O and O2 in Staph but not in Strep

2- Coagulase & clumping factor: it coagulate oxalated or citrated plasma; as it bind to prothrombin to be activated and

initiate fibrin polymerization, there after the clumping factor is responsible for bacteria adherence to the fibrinogen.

3- Hyalorunidase, staphylokinase that produce fibrinolysis, proteinase, lipase.

The toxins are:

1- α-toxins (hemolysin) act upon leucocytes cell wall

2- β-toxins act upon RBCs act upon leucocytes wall

3- γ-toxins (hemolysin) act upon leucocytes wall

4- δ-toxins act upon human epithelial cells

5- Exfoliative toxins (A+B) they are epidermolytic toxins, cause skin desquamation (scalded skin), type A is under

chromosomal control and it heat stable while type B is under plasmid control and it heat labile.

6- Toxic shock syndrome toxin (TSST-1) it bind to major histocompatability complex class II (MHC-II) molecules and this

will lead to T-lymphocytes stimulation.

7- Enterotoxins (emetic effect) and it types are (A-E, G-J, K-R, and U, V); it preformed as Staph grow in carbohydrate or

protein diets and the personnel ingest the already produced toxins not the microbe itself.

Pathogenesis

S epidermidis usually found normally onto skin, respiratory, GIT. 20-50% of people normally harboring S aureus

nasally (this is important in nursery), also found into fomites, clothing and lines.

Staphylococci infections

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Local infection or abscess with painful inflammatory reaction that goes to central suppuration and heals as soon the pus

is drained. The fibrin and immune cells around the lesion try to prevent infection spread, so must prevent its

manipulation or trauma rupture.

Staphylococci occur also directly through wound contamination (surgical or traumatic) as meningitis after skull fracture

or osteomyelitis after open fracture surgery. If the bacteria transmitted by blood stream we get bacteremia, pulmonary,

osteomyelitis, meningitis, arthritis, etc.

In food poisoning the incubation period is short (1-8 hours); with vomiting and diarrhea & NO FEVER

Staphylococci laboratory diagnosis

The specimens are pus swab, blood, tracheal or abscess aspirates, urine, CSF

1-Gram stained smears

2-Culture onto 5% blood agar plate (BAP), Mannitol salt plate as S aureus ferment it to gain yellow colonies, in

suspected mixed growth use CAN (Columbia agar plate) which contain colistin + nalidixic acid in order to suppress the

G- bacteria; in addition we can use chromogenic culture media especially for nasal carriers detection.

3-Catalase test to detect cytochrome oxidase enzyme as using 3% hydrogen peroxide solution

4-Hemolysis in BAP

5-Coagulase test which is carried out in 2 ways (slide); we use particles covered with fibrinogen and IgG antibodies

that bind coagulase to gain clumping within 20 seconds if the IgG is present in low concentration we turn onto tube

method as we mix equal volumes from bacterial broth and citrated plasma then incubate it for 1-4 hours at 37 ºC to gain

the clot in positive reaction (Coagulase positive Staph) (CPS); S intermedius usually give positive test although it is not

regarded as human pathogen.

((Most of the CPS is pathogenic to human))

1-Susceptibility testing done by broth microdilution or disk diffusion and as known 90% of S aureus is producing β –

lactamase.

Resistance to nafcillin, oxacillin, and methicillin occur in 65% of S aureus and in 75% of S epidermidis isolates. As

commercially methicillin disk production is ceased, so can be replaced with oxacillin disk; if the bacteria resist it that

mean it resist methicillin i.e., it is MRSA.

2-Detection of mec gene through PCR, most laboratories use Mueller-Hinton agar (MHA) contain 4% NaCl + 6 µg/ml

oxacillin in order to inoculate S aureus on it, if the bacteria grow that’s mean it resist nafcillin and gain mec gene.

3-Rapid staph detection through quantitative PCR (qPCR) also called real-time (RT-PCR) as in the ordinary culture we

need few days to gain the bacteria while by PCR few copies of bacterial DNA enough to detect its presence within few

hours; in it we duplicate DNA using thermus aquaticus (Taq polymerase) and to start duplication need free nucleotides

(primers).

4-Serology by agglutination to the isolates of little values nowadays.

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As most persons harboring Staph on skin or nose and throat and they are eliminated only from the skin in eczema cases

but later they will return as usual, the bacteria is transmitted in-between persons from fingers or clothes and fomites, so

local antiseptics are of value in infection control.

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Lecture Five

Genus Streptococcus

It is gram positive cocci (spheres or ovoid) found in pairs or chains which divided in a perpendicular manner so

sometimes this will give like a diplococci appearance. The bacteria elaborates wide range of substances and

enzymes, its classification depend on colony morphology and blood agar plate hemolysis type; cell wall groupspecific substances (Lancefield classification) and capsular antigens especially to S.pneumoniae

(Pneumococcus; over 90 serotypes are present) and for S.agalactiae (group B), biochemical reactions (through

sugar fermentation or for detection of enzymes or susceptibility and resistance for certain chemicals), molecular

study of species.

Many Streptococci able to hemolysed RBCs invitro, if it’s a complete it called (β-hemolysis), partial

destruction with green pigment formation called (α-hemolysis), or they non-hemolytic which called (γhemolysis).

Group-specific substance is carbohydrate in the cell wall; it classified to A-H and K-U, and it determined by its

amino-sugars e.g., for group A it is rhamnose-N-acetylglucosamine; for group B it is rhamnose-glucosamine

polysaccharide; for group C it is rhamnose-N-acetylgalactosamine; for group D it is glycerol teichoic acid; for

group E it is glucopyranosyl-N-acetylgalactosamine.

This group specific antigen is extract by colony centrifugation with:

1- Hot HCl treatment

2- Enzymatic extraction with trypsin or pepsin

3- Through bacterial broth autoclaving

Most streptococcal human infections are caused by groups A, B, C, F, and G.

Group A β–hemolytic Streptococcus pyogenes

It cause local and systemic infections plus post-streptococcal immunological disorders, usually it produce large

hemolysis zone (1 cm) in diameter and colony size equal to 0.5 mm, it is susceptible to bacitracin, PYR test

(+ve) (hydrolyze 1-Pyrrolidonyl-2-naphthalamide); its main habitat at pharynx and skin, and its main diseases

are pharyngitis, impetigo, rheumatic fever, glomerulonephritis, toxic shock.

Most of group A produce a capsule composed of hyaluronic acid, it inhibit phagocytosis and it play role in

bacterial virulence as it bind to hyaluronic acid binding protein (CD44) which found onto human epithelial

cells, so disrupt the epithelium to allow the microbes to be inside; other capsule belonging S pneumoniae or S

agalactiae is different.

Also its cell wall contain proteins (M, T, R antigens), M protein compose mainly the capsule projections (pili)

which covered with lipoteichoic acid and it important for bacteria attachments.

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Its growth onto solid media is enhanced by CO2; they are facultative anaerobes grow well at 37 ºC; most of

streptococci are grow under aerobic and anaerobic conditions except Peptostreptococcus which is strict

anaerobic.

S pyogenes also show colony variation as some matt (mostly virulent strains) due to excess M protein and other

are glossy (mostly a virulent strains)

S pyogenes antigens

1- M protein: it appear as hair like projections through the cell wall, especially in virulent strains also it

resist phagocytosis by polymorphonuclear leucocytes (PMNs); immunity to it infection related to

absence of antibodies against it, and as it has more than 150 types of M protein so the person could

gain repeated streptococcal infections. M protein is found in 2 functional structures, (class I, II), M

protein has a role in rheumatic fever as it enhance antibodies towards cardiac sacrolemma

2- T substance its role in pathogenesis still unclear

3- Nucleoproteins

Streptococcal pyogenes toxins and enzymes

1- Streptokinase (fibrinolysin) it convert human plasminogen to plasmin which digest the fibrin clot,

clinically streptokinase is given intravenously in cases of coronary or pulmonary or venous thrombosis

2- Streptodornase (deoxyribonuclease) it depolymerase DNA

3- Hyaluronidase it split hyaluronic acid which is important component of the connective tissue ground

substance, it assist M.O. to spread in infected area (spreading factor)

4- Pyrogenic (erythrogenic exotoxin) they are 3 types (A, B, and C); it associated with toxic shock

syndrome and scarlet fever; as it activate the T-lymphocytes through contact to the MHC class II

which found onto T-cell surface so the cell will release cytokines that mediate the shock; this

mechanism it seems to be the same as with staph enterotoxins.

5- Diphosphopyridine nucleotidase it related to killing of leucocytes

6- Proteinase and amylase produced by certain strains

7- Hemolysins it elaborate 2 hemolysin (streptolysin O) which rapidly inactivated by O2 so it

responsible for hemolysis in the deep area of BAP; it is responsible for antibodies elaborate after strep

infections known as antistreptolysin O (ASO) that its normal range from 160-200 units and any

elevation is due to recent infection or reactivation in hyperimmune personnel, the other streptolysin is

(S) which is O2 stable cause hemolysis at the BAP surface, elaboration occur as serum is found hence

get this name.

Streptococcal pyogenes infections

I-Infections due to bacterial invasion

Mostly they introduced through skin abrasion by burns or wound or surgical incision e.g. in:

1- Erysipelas

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2- Cellulitis

3- Necrotizing fasciitis (streptococcal gangrene) sometimes they called the microbe “flesh eating

bacteria”

4- Puerperal fever (endometritis) a sit enter the uterus after delivery

5- Bacteremia / sepsis it usually follow cellulitis and rarely due to pharyngitis

II-Local infection plus bacterial by-products

1- Sore throat or pharyngitis as the bacteria attach to pharynx epithelial cells receptors (glycoprotein

fibronectin) through its lipoteichoic acid which cover pili and hyaluronic acid in capsulated strains, in

children the infection occur as subacute nasopharyngitis with serous discharge and mild fever plus

tendency to extend to the mastoid, middle ear, and enlarged cervical lymph nodes.

In older children and adults get acute intense nasopharyngitis, tonsillitis with intense redness and purulent

exudate over the mucus membrane, high fever plus tender lymphadenitis. Similar picture occur due to

gonococcal infection or infectious mononucleosis or diphtheria or adenovirus infections.

2- Streptococcal pyoderma a skin local infection especially in children known as (impetigo) which

similar to S aureus impetigo, composed of vesicles which rupture to crusted area, it highly

communicable and prone to be occurred in eczematous or wounded or burned skin. Groups of M

proteins 49+57 and 59-61 are prone to yield skin infections rather than glomerulonephritis.

III-Invasive infections (TSS, scarlet fever)

It characterized with bacteremia and respiratory failure plus multi-organs involvement, its rash will appear on

the trunk and spread to extremities.

IV-post streptococcal diseases (rheumatic fever and glomerulonephritis)

After a latent period of Strep infection (1-4 weeks) and this period represent to the time needed to be hyper

immunized to the bacterial antigens; nephritis more prone after skin infections and rheumatic fever more prone

after respiratory infections.

The nephritogenic strains with M proteins 2, 42, 49, 56, 57, 60; nephritis initiated with Ag-Ab complex deposit

onto the glomerular basement membrane, which lead to RBCs and albumin in urine (edema) and elevated

blood pressure, low serum complements, its sequel the majority will recovered and the minority get renal

failure.

In rheumatic fever it a serious sequel as damage the heart muscle and valves; as some Strep strain gain cell wall

antigens cross react with cardiac muscles antigens, it major clinical picture as fever, malaise, non-suppurative

polyarthritis, signs of inflammation in all heart parts i.e. (endocardium which lead to thick deformed valves,

myocardium, which lead to perivascular granuloma “Aschoff bodies” and pericardium).

This carditis has tendency to be reactivated with recurrent Strep infections, whereas in nephritis this will not

happen; so the chemoprophylaxis is valuable.

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Streptococcus A laboratory diagnosis

1- Specimen throat swab which is less valuable as always viridians strep is founded or pus or blood

2- Smears shows cocci or pairs rather than chain, if the gram stained film is positive and the culture is

negative so anaerobes must be suspected

3- Culture on BA with the assist of bacitracin diagnostic disk that inhibit its growth and anaerobic culture

if suspected, incubate at 5% CO2 to enhance hemolysin elaboration, Strep in blood culture either grow

within hours or need days to be positive as it grow slowly especially with Enterococcus or viridians

streptococci

4- Antigen rapid detection with commercial kits, it based on enzymatic or chemical extraction of these

Ag, then use elisa or agglutination kit to identify it.

5- Serological test:

a- ASOT in pharyngitis case

b- Anti DNase

c- Antihyaluronidase

d- Antistreptokinase

e- Anti specific M protein

Although human carry S pyogenes asymptomatically in nasopharynx or perineal, its detection in culture should

be considered as significant finding, they spread the microbes by skin contact or respiratory droplets, many

other Strep as viridians or enterococci normally inhabitant the respiratory or intestinal or urinary flora and only

produce diseases if they reach sites which not belong to them especially after dental or surgical intervention; so

chemoprophylaxis is significant prior to it.

S agalactiae

It group B in Lancefield grouping, it classified to types depending on its capsular polysaccharides. It is βhemolytic and produce 1-2 mm in diameter hemolysis area around the colony, give +ve CAMP test (Christie

Atkins Munch Peterson) as using BA which inoculate with suspected S agalactiae and in the agar center

inoculate a streak of S aureus, so the hemolysis will be exaggerated to form arrowhead character also it

hydrolyze sodium hippurate.

S agalactiae is part of normal flora in 5-25% of women in the genital tract plus lower GIT (asymptomatic

carriers), and not all bacteria will cause babies infection as it depend upon membrane rupture before 37

gestational week, or rupture more than 18 hours prior delivery.

S agalactiae cause invasive infections in adults like bacteremia, UTIs, bone and joints infections, skin and soft

tissue, pneumonia, genitourinary.

Group C and G Streptococci

It habitat the nasopharynx and cause pharyngitis or sinusitis, bacteremia or endocarditis; it react like S pyogenes

as produce β-hemolysin or α - type or none; and got the same M protein.

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Group D Enterococcus (Streptococci faecalis and bovis)

S bovis group is the most valuable member in this group which cause human infections, it hemolysis is α - type

or none, it habitat colon and biliary tree, and growth in bile (deoxycholate) presence, hydrolyze esculin, S

faecalis grow in 6.5% NaCl and S bovis does not grow. They cause abdominal abscesses, UTIs, endocarditis,

biliary infections and bovis commonly seen in colonic cancer.

Streptococcus anginosus group (anginosus, intermedius, milleri, and constellatus)

Consist of groups F (A, C, G) and untypeable groups, its hemolysis β or α-type or none, habitat the throat,

colon, female genital tract; resist bacitracin, it cause Pyrogenic infections including brain abscesses. They are

PYR –ve and Vogas-Proskauer +ve.

Group N Streptococcus

Rarely isolated from human and they are normally contributed in milk coagulation (souring).

Groups E, F, H, and K-U

Rarely isolated usually they are an animal’s pathogens

Viridans Streptococci

It include S mitis, S intermedius, S sanguis, S slivarius, they hemolysis α –type or none, its growth not inhibited

by optochin and their colonies not soluble in bile (deoxycholate), it is the most usual flora in the upper

respiratory tract and they are important for the healthy state of the mucus membrane.

It reach blood stream through teeth manipulation to cause endocarditis, S mutans produce large polysaccharides

like dextrans or levans from sucrose which contribute to the formation of dental caries.

Enterococci or pneumococci or viridans Streptococci as they reach the blood stream (bacteremia) it will settle

on normal or already deformed heart valves.

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Enterobacteriaceae

Genera and species to be considered

Opportunistic Pathogens:

Citrobacter freundii

Citrobacter (diversus) koseri

Citrobacter braakii

Cronobacter sakazakii (previously Enterobacter sakazakii)

Edwardsiella tarda

Enterobacter aerogenes

Enterobacter cloacae

Enterobacter gergoviae

Enterobacter amnigenus

Enterobacter (cancerogenous) taylorae

Escherichia coli (including extraintestinal)

Ewingella americana

Hafnia alvei

Klebsiella pneumoniae

Klebsiella oxytoca

Morganella morganii subsp. morganii

Morganella psychrotolerans

Pantoea agglomerans (previously Enterobacter agglomerans)

Proteus mirabilis

Proteus vulgaris

Proteus penneri

Providencia alcalifaciens

Providencia heimbachae

Providencia rettgeri

Providencia stuartii

Serratia marcescens

Serratia liquefaciens group

Serratia odorifera

Pathogenic Organisms:

Primary Intestinal Pathogens

E. coli (diarrheagenic)

Plesiomonas shigelloides

Salmonella, all serotypes

Shigella dysenteriae (group A)

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Shigella flexneri (group B)

Shigella boydii (group C)

Shigella sonnei (group D)

Pathogenic Yersinia spp.

Yersinia pestis

Yersinia enterocolitica subsp. enterocolitica

Yersinia frederiksenii

Because of the large number and diversity of genera included in the Enterobacteriaceae, it is helpful to consider

the bacteria of this family as belonging to one of two major groups. The first group comprises species that

either commonly colonize the human gastrointestinal tract or are most notably associated with human

infections. Although many Enterobacteriaceae that cause human infections are part of our normal

gastrointestinal flora, there are exceptions, such as Yersinia pestis. The second group consists of genera capable

of colonizing humans but rarely associated with human infection and commonly recognized as environmental

inhabitants or colonizers of other animals. For this reason, the discovery of these species in clinical specimens

should alert laboratorians to possible identification errors; careful confirmation of both the laboratory results

and the clinical significance of such isolates is warranted.

General Characters

 Molecular analysis has not proven effective for definitively characterizing all the organisms and genera

included within the Enterobacteriaceae family. Therefore, species names and reclassification of organisms

continually evolve. In general, the Enterobacteriaceae consist of a diverse group of gram negative bacilli or

coccobacilli; they are non–spore forming, facultative anaerobes capable of fermenting glucose; they are oxidase

negative

(except for Plesiomonas sp.); and, with rare exception (Photorhabdus and Xenorhabdus spp.), they reduce

nitrates to nitrites. Furthermore, except for Shigella dysenteriae type 1, all commonly isolated

Enterobacteriaceae are catalase positive.

Epidemiology

Enterobacteriaceae inhabit a wide variety of niches, including the human gastrointestinal tract, the

gastrointestinal tract of other animals, and various environmental sites. Some are agents of zoonoses, causing

infections in animal populations (Table 1). Just as the reservoirs for these organisms vary, so do their modes of

transmission to humans. For species capable of colonizing humans, infection may result when a patient’s own

bacterial strains (i.e., endogenous strains) establish infection in a normally sterile body site. These organisms

can also be passed from one patient to another. Such infections often depend on the debilitated state of a

hospitalized patient and are acquired during the patient’s hospitalization (nosocomial). However, this is not

always the case. For example, although E. coli is the most common cause of nosocomial infections, it is also the

leading cause of community-acquired urinary tract infections. Other species, such as Salmonella spp., Shigella

spp., and Yersinia enterocolitica, inhabit the bowel during infection and are acquired by ingestion of

contaminated foodor water. This is also the mode of transmission for the various types of E. coli known to

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cause gastrointestinal infections. In contrast, Yersinia pestis is unique among the Enterobacteriaceae that infect

humans. This is the only species transmitted from animals by an insect vector (i.e., flea bite).

Table (1-1) Epidemiology of Clinically Relevant Enterobacteriaceae

Organism Habitat (Reservoir) Mode of Transmission

Varies with the type of infection. For

nongastrointestinal

infections, organisms may be endogenous or spread

person to person, especially in the hospital setting.

For gastrointestinal infections, the transmission mode

varies with the strain of E. coli (see Table 20-2); it

may involve fecal-oral spread between humans in

contaminated food or water or consumption of

undercooked beef or unpasteurized milk from

colonized cattle

Normal bowel flora of humans

and

other animals; may also inhabit

female genital tract

Escherichia coli