MMIC blog post 2

Case 1

Patient: Khong Fay Seah, Female, 27, outpatient

Complaints: Fever, chills, dysuria

Diagnosis: Urinary Tract Infection (UTI)

Gram Stain	Oxidase	Lactose	TSI	Indole	Methyl-red	Voges-Proskauer	Citrate	Urease	Wet Mount	Anitbiotic Susceptibility Test
Escherichia Coli	-	-	+	slant & butt yellow (acidic), gas produced, H2S negative	+	+	-	-	-	Motile bacillus	Penicillin G (Ampicillin), Sulfonamides Cephalosporin, Aminoglycoside
Proteus mirabilis	-	-	-	slant red (alkaline) & butt yellow (acidic), gas & H2S produced	-	+	-	+	+	Highly motile bacillus	Trimethoprim-sulfamethoxazole, Ampicillin
Klebsiella pneumoniae	-	-	+	slant & butt yellow (acidic), gas produced, H2S negative	-	-	+	+	+	Non-motile bacillus	Penicillin, Cephalosporin, Aminoglycoside, Cephalosporin (e.g. Cefotaxime)
Enterobacter spp	-	-	+	slant & butt yellow (acidic), gas produced, H2S negative	-	-	+	+	-	Motile bacillus	Ciprofloxacin, Aminoglycoside (e.g. Gentamycin)
Pseudomonas aeriginosa	-	+	-	slant & butt red(alkaline), gas & H2S negative	+	-	-	+	-	Motile bacillus	Penicillin, Cephalosporin, Aminoglycoside, Tetramycin

Table 1.1 Biochemical,Microscopy and Antibiotic Susceptibility Tests Results of Gram-Negative Microbes

Fig1.1 Left indicates negative Fig 1.2 Left indicates negative Methyl-red
Indole result, right is positive result, right is positive

Taken from http://www.google.com/>images>search>indole tests)
Taken from http://www.google.com/>image>search>methyl-red test)

Fig1.3 Left indicates negative Voges- Fig1.4 Fig1.1 Left indicates positive citrate

Proskauer result, right is positive result, right is negative

Taken from http://www.google.com/>image>search>Voges-Proskauer test

Taken from http://www.google.com/>image>search>Citrate test

	Gram Stain	Catalase	Coagulase	Haemolysis on Blood Agar	Wet Mount	Antibiotic Susceptibility Test
Staphylococcus saprophytcus	+	+	-	Non-hemolytic	Non-motile cocci	Norfloxacin, Trimethoprim-sulfamethoxazole (e.g. Bactrim)
Staphylococcus aureus	+	+	+	Beta hemolytic colonies	Non-motile cocci	Vancomycin, Amoxicillin with clavulanic acid, Cloxacillin, Ampicillin, Cephalosporon
Enterococcus faecalis	+	-		Non-hemolytic colonies (24 hours) Alpha-hemolytic(48 hours)	Non-motile cocci	Ampicillin, Vancomycin, Aminoglycoside

Table 1.2 Biochemical,Microscopy and Antibiotic Susceptibility Tests Results of Gram-positive Microbes

Reference

1. http://www.cst.cmich.edu>Urinary Tract Infections and Sexually Transmitted Infections

2. http://merck.com>search>fungal urinary tract infections

3. Warren Levinson Review of Medical Mrcrobiology and Immunolgy Ninth Edition

Case 2 (Azhar)

Patient: Kwan Siew Yan, Female, 29, outpatient

Complaints: Diarrhea

Diagnosis: Enterocolitis

Salmonella spp.

Commonly used agars used for culturing:

Differential media:

• Eosin methylene blue (EMB) agar (growth of colourless colonies)


• MacConkey’s agar (growth of colourless colonies)


• Bismuth sulfite agar (growth of grey-brown or black colonies, they may or may not have a metallic sheen)

Selective media:

• Salmonella-Shigella (SS) agar (growth of black colonies)


• Hektoen enteric (HE) agar (growth of blue-green to blue colonies with black deposits in the middle of the colonies)


• Xylose-lisine-deoxycholate (XLD) agar (growth of red colonies, they may or may not have black centres)

Biochemical Tests:

• Indole negative (does not produce indole from tryptophan)


• Citrate positive (able to utilize citrate)


• Urease negative (does not produce urease)
  

Antimicrobial Sensitivity Testing:

• Ampicillin - Susceptible


• Ciprofloxacin - Susceptible


• Chloramphenicol - Susceptible


• Gentamycin - Susceptible

Microscopy:

• Wet mount (Motile rod)


• Gram stain (Gram-negative)

Shigella spp.

Commonly used agars used for culturing:

Differential media:

• EMB agar (growth of colourless colonies)


• MacConkey’s agar (growth of colourless colonies)

Selective media:

• SS agar (growth of colourless colonies)


• Hektoen enteric (HE) agar (growth of blue-green to blue colonies)


• Xylose-lisine-deoxycholate (XLD) agar (growth of red colonies)

Biochemical Tests:

• Indole negative (does not produce indole from tryptophan)


• Citrate negative (unable to utilize citrate)


• Urease negative (does not produce urease)

Antimicrobial Sensitivity Testing:

• Cefuroxime - Susceptible


• Chloramphenicol - Susceptible


• Gentamycin - Susceptible

Microscopy:

• Wet mount (Non-motile rod)


• Gram stain (Gram-negative rod)

Campylobacter spp.

Commonly used agars used for culturing:

Differential media:

• MacConkey’s agar (no growth)

Selective media:

• Campylobacter Selective medium (grey to colourless colonies depending on species)

*incubation in microaerophilic condition (reduced oxygen [5%] and 10% carbon dioxide condition)

Biochemical Tests:

• Catalase positive (contains catalase which converts hydrogen peroxide to water and oxygen)


• Oxidase positive (contains cytochrome c oxidase)

Antimicrobial Sensitivity Testing:

• Chloramphenicol - Susceptible


• Gentamycin - Susceptible


• Erythromycin - Susceptible

Microscopy:

• Gram stain (Gram-negative spiral rods)


• Wet mount (motile spiral rods)

Clostridium difficile

Commonly used agars used for culturing:

Differential media:

• Blood agar plate (presence of haemolysis)

No biochemical tests available

Other tests:

• Immunoenzymatic assay to detect toxins (either toxin A or B)

Antimicrobial Sensitivity Testing:

• Ampicillin - Susceptible


• Ceftazidime - Susceptible

Microscopy:

• Gram stain (Gram-positive rods with a bulge on their terminal ends)

Vibrio spp

Commonly used agars used for culturing:

Differential media:

• Blood agar plate (non-haemolytic colonies with a greenish hue)

Selective media:

• Thiosulfate-citrate-bile-sucrose (TCBS) agar (growth of yellow colonies)

Biochemical Tests:

• Oxidase positive (contains cytochrome c oxidase)


• Lysine decarboxylase positive (able to decarboxylate lysine)


• Arginine dihydrolase positive (converts arginine to citrulline and finally citrulline to ornithine)

Antimicrobial Sensitivity Testing:

• Gentamycin - Susceptible


• Ampicillin – Susceptible


• Penicillin G - Resistant

Microscopy:

• Gram stain (Gram-negative rods)


• Wet mount (motile curved rods)
  

Escherichia coli

Commonly used agars used for culturing:

Differential media:

• EMB agar (growth of purple colonies with a characteristic green sheen)


• MacConkey’s agar (growth of red colonies)


• Cysteine Lactose Electrolyte Deficient (CLED) agar (growth of yellow colonies)

Biochemical Tests:

• Indole positive (produces indole from tryptophan)


• Lysine decarboxylase positive (able to decarboxylate lysine)

Antimicrobial Sensitivity Testing:

• Ciprofloxacin - Susceptible

Microscopy:

• Gram stain (Gram-negative rod)


• Wet mount (motile rods)

Yersinia spp.

Commonly used agars used for culturing:

Differential media:

• MacConkey’s agar (growth of small colourless colonies)

*poor growth

Differential Selective media:

• Cefsulodin-irgasan-novobiocin (CIN) agar (growth of colonies with a deep red centre that has a transparent margin)

Antimicrobial Sensitivity Testing:

• Ampicillin - Resistant


• Chloramphenicol - Susceptible


• Ciprofloxacin - Susceptible


• Gentamycin – Susceptible


• Imipenam - Susceptible

Biochemical Tests:

• Urease positive (produces urease)


• Oxidase negative (does not contain cytochrome c oxidase)

Microscopy:

• Gram stain (Gram-negative rod)


• Wet mount (non-motile rods at 37°C) *motile at 25°C

Parasites

Microscopy:

• Wet mount (presence of either ova [eggs] or parasite)


• Stool ova & cyst (presence of either ova [eggs] or parasite)

Case 3 (Ming Boon)

Patient: Maisy Hong, Female, 67, inpatient

Complaints: Fever, chills, bladder distension, on indwelling catheter

Diagnosis: UTI

From the list of common causative microorganisms in the previous post, these are the microorganisms that are the most probable causative agents for the UTI in this patient as they are the infections are nosocomial and associated with catheter. The following tests are required in order to differentiate between the organisms.

Escherichia coli

Gram stain: Gram negative bacillus

Wet mount: Motile

Culture
(Under anaerobic conditions)

Blood agar: Gamma hemolysis

Eosin Methylene Blue agar: Colonies with metallic green sheen

MacConkey agar: Pink colonies

Biochemical tests

Indole test: Positive
Methyl Red (MR) test: Positive
Voges-Proskauer (VP) test: Negative
Simmon’s citrate test: Negative
Oxidase test: Negative
Urease: Negative

Antimicrobial testing

- Susceptibility depends on the type of strains
- Beta-lactamase resistant strains are not sensitive to penicillin and cephalosporin
- Non-resistant strains are sensitive to ampicillin and trimethoprim-sulfamethoxazole

Pseudomonas aeruginosa

Gram stain: Gram negative bacillus

Wet mount: Motile

Culture
(Under aerobic conditions as it is a strict aerobe, able to grow at 42^oC)

Blood agar: Beta-hemolysis

MacConkey agar: Colourless colonies

Ordinary nutrient agar: Blue-green colonies

TSI agar: Growth with typical metallic sheen

Biochemical tests

Indole test: Negative
Methyl Red test: Negative
Voges-Prokauer test: Negative
Simmon’s citrate test: Positive
Catalase test: Positive
Oxidase test: Positive
- Able to distinguish between the Enterobacteriaceae family as all the bacteria in this family are oxidase negative

Antimicrobial testing

- Naturally resistant to a broad spectrum of antibiotics including sulfonamide, trimethoprim
- Susceptible to a combination of antipseudomonal penicillin (eg. ticarcillin, piperacillin) and aminoglycoside (eg. gentamicin, amikacin)

Proteus mirabilis

Gram stain: Gram negative bacillus

Wet mount: Highly motile

Culture
(Culture under anaerobic conditions)

Blood agar with phenylethyl alcohol: Colonies do not have swarming effect

Colonies of P. mirabilis have a swarming effect, making it difficult to obtain pure isolates of other species. Addition of phenylethyl alcohol inhibits the swarming.

Picture taken from http://www2.bakersfieldcollege.edu/bio16/Lab%20Manual/Lab%20WEb%20Exercises/lab_exercise3.htm

MacConkey agar: Colourless colonies

TSI agar: Black butt

Biochemical tests

Indole test: Negative
Methyl Red test: Positive
Voges-Prokauer test: Negative
Simmon’s citrate test: Positive
Catalase test: Positive
Urease test: Positive
- Differentiate Proteus species from other members of Enterobacteriaceae

Antimicrobial testing

- Less resistant to antibiotics compared to the indole positive species
- Sensitive to ampicillin, aminoglycosides and trimethoprim-sulfamethoxazole

Enterobacter cloacae

Gram stain: Gram negative bacillus

Wet mount: Motile

Culture
(Under anaerobic conditions)

MacConkey agar: Pink colonies

Biochemical tests

Indole test: Negative
Methyl Red test: Negative
Voges-Prokauer test: Positive
Simmon’s citrate test: Positive
Urease test: Positive

Antimicrobial testing

- Antibiotic resistance vary greatly
- Isolates from nosocomial infections are frequently resistant to multiple antibiotics
- Susceptible to aminoglycoside (eg. gentamicin) and cephalosporin (eg. cefotaxime)
- In severe infections, a combination of imipenem and gentamicin is required

Serratia marcescens

Gram stain: Gram negative bacillus

Wet mount: Motile

Culture
(Under anaerobic conditions)

MacConkey agar: Pink colonies (late lactose-fermenter so colonies might be colourless)

Ordinary nutrient agar: Red colonies

Picture taken from http://www.bact.wisc.edu/Microtextbook/index.php?module=Book&func=displayarticle&art_id=123

Biochemical tests

Indole test: Negative
Methyl Red test: Negative
Voges-Prokauer test: Positive
Simmon’s citrate test: Positive
Urease test: Negative

Antimicrobial testing

- Antibiotic resistance vary greatly
- Isolates from nosocomial infections are frequently resistant to multiple antibiotics
- Susceptible to aminoglycoside (eg. gentamicin) and cephalosporin (eg. cefotaxime)

Klebsiella pneumoniae

Gram stain: Gram negative bacillus, a large capsule can be observed

Wet mount: Non-motile

Culture
(Under anaerobic conditions)

MacConkey agar: Pink colonies with mucoid appearance

Biochemical tests

Indole test: Negative
Methyl Red test: Negative
Voges-Prokauer test: Positive
Simmon’s citrate test: Positive
Urease test: Positive

Antimicrobial testing

- Antibiotic resistance vary greatly
- Isolates from nosocomial infections are frequently resistant to multiple antibiotics
- Susceptible to aminoglycoside (eg. gentamicin) and cephalosporin (eg. cefotaxime)

Enterococcus faecalis

Gram stain: Gram positive cocci, in chains

Wet mount: Non-motile

Culture
(Under anaerobic conditions)

Blood agar: Gamma hemolysis

MacConkey agar: Pink colonies with mucoid appearance

Bile Esculin Agar: Ferric citrate indicator will turn black

Biochemical tests

Indole test: Negative
Voges-Prokauer test: Positive
Simmon’s citrate test: Positive
Catalase test: Negative

Antimicrobial testing

- Resistant to aminoglycoside, penicillin and vancomycin when given individually
- Requires a combination of aminoglycoside (eg. gentamicin) and penicillin or vancomycin
-Linezolid can be used against vancomycin-resistant enterococci

References:

1. Levinson W. 2006. Review of Medical Microbiology and Immunology, 9^th edition. McGraw Hill Companies
2. http://en.wikipedia.org>Pseudomonas
3. http://www.bact.wisc.edu>Microbiology Web Textbook
4. http://gsbs.utmb.edu>Current Students>Medical Microbiology>Chapter 27
5. http://en.wikipedia.org>Proteus mirabilis
6. http://www2.truman.edu/~kes251>Proteus mirabilis
7. http://gsbs.utmb.edu>Current Students>Medical Microbiology>Chapter 26

Case 4 (Michelle)
Name: Tong Wei Hong (Outpatient)
Sex: Male
Age: 68 years old
Complaints: Fever, chills, excessive phlegm, breathing problems
Diagnosis: Bronchitis

Streptococcus pneumoniae: The bacterium is a gram-positive coccus, lancet shaped, 0.5-1.2um in diameter that firms short chains or more commonly in pairs, hence by gram staining test, we are able to identify these colony characteristics for this bacterium under a light microscope.
This bacterium does not have the catalase enzyme thus a negative result is expected when a biochemical identification test to detect the presence of catalase is carried out.
Colonies of this bacterium are alpha-hemolytic on blood agar when grown aerobically and beta-hemolytic when grown anaerobiaclly. The colonies are bile-soluble in which they are lysed in the presence of bile and their growth is inhibited by optochin. Hence this bacterium is optochin sensitive and it will soloubilise in bile. Blood cultures are positive in 15-25% of pneumoncoccal infections.
Antibiotic susceptibility test using penicillin, erythromycin, cephalosporin, chloramphenicol, vancomycin can be conducted. Penicillin has been the drug of choice against this bacterium though penicillin-resistant strains have been developing,thus vancomycin is used for these increasingly penicillin-resistant strains.
Additional serology tests such as Quellung reaction can be used to confirm the presence of pneumoncocci in gram stains of sputum smears in which anticapsular antibodies will cause the capsule polysaccharide of this bacterium to swell when viewed under the light microscope.

Haemophilus influenza: The bacterium is a small gram-negative rod (coccobacillus) with a polysaccharide capsule. Serologic typing is based on the antigenicity of the capsular polysaccharide in which of the six serotypes, type b causes most of the severe, invasive diseases hence by gram staining test we are able to identify these colony characteristics for this bacterium under a light microscope.
This bacterium contains the enzyme oxidase thus a positive result is expected when a biochemical identification test to detect the presence of oxidase is carried out.
They are pleomorphic bacilli that require heme (factor X) and NAD+(factor V) for growth on heated-blood (“chocolate”) agar enriched with these growth factors. The blood is heated to inactivate non-specific inhibitors of the growth of this bacterium.
Antibiotic susceptibility test using ceftriaxone, trimethoprim-sulfamethoxazole, amoxicillin-clavulanate can be conducted. Penicillin is not effective against it.
Definitive identification can be made by serology testing by capsular swelling (Quellung) reaction in which the polysaccharide capsule of this bacterium will swell when viewed under the light microscope. Additional means of identifying encapsulated strains include fluorescent-antibody staining of the organism or latex agglutination test.

Moraxella catarrhalis: This bacterium is a gram-negative rod (coccobacillus) hence by gram staining test we are able to identify these colony characteristics for this bacterium under a light microscope.
This bacterium contains the enzyme oxidase thus a positive result is expected when a biochemical identification test to detect the presence of oxidase is carried out.
Antibiotic susceptibility test using cephalosporins, erythromycin, tetracycline can be conducted.

Mycoplasma pneumoniae: It is a very small, free-living bacterium, in the class of Mollicutes. Many are as small as 0.3um in diameter and their most striking feature is the absence of a cell wall. They stain poorly with gram stain hence diagnosis is usually not by culturing sputum samples as it will take at least 1 week for colonies to appear on special media. Culture on regular media reveals only normal flora.
Serologic testing is the mainstay of diagnosis. A cold-agglutinin titer of 1:128 or higher is an indication of recent infection. However, the test is non-specific and false positive results occur in influenza virus and adenovirus infections.
The diagnosis of this bacterium can be confirmed by a 4-fold or greater in specific antibody titer in the complement fixation test.

Chlamydia pneumoniae: They are obligate intracellular bacteria. They lack the ability to produce sufficient energy to grow independently and therefore can grow only inside host cells. They are able to form cytoplasmic inclusions which can be seen by Giemsa stain or by immunofluorescence. Gram stain is not useful in this case.
They can be grown in cell cultures treated with cycloheximide which inhibits host cell but not chlamydial protein synthesis hence enhancing the bacterium replication. The bacterium form inclusions that do not contain glycogen in which glycogen-filled inclusions are visualised by staining with iodine.
Antibiotic susceptibility test using tetracycline such as doxycycline and macrolides such as erythromycin can be conducted.

Bordetella pertussis: It is a small, coccobacillary, encapsulated gram-negative rod, hence by gram staining test we are able to identify these colony characteristics for this bacterium under a light microscope.
This bacterium contains the enzyme oxidase thus a positive result is expected when a biochemical identification test to detect the presence of oxidase is carried out.
Additional identification test such as culturing the bacterium in Bordet-Gengou medium (potato-blood-glycerol) can be conducted. Bordetella pertussis produces small, domed, glistening colonies that resemble bisected pearls. The colonies are
usually surrounded by a zone of hemolysis; however, some strains of B. pertussis are not hemolytic.
Identification can be made also by agglutination with specific antiserum or direct fluorescent-antibody staining.

Case 5 (Yvonne)

Patient: Wong Fei Hong, Male, 37, outpatient

Complaints: Fever, swelling around the operation wound

Diagnosis: Wound infection

Introduction of SSI (Surgical Site Infection)
A wound is the result of physical disruption of the integument or the mucous membrane due to external trauma, or some mechanical agency. The establishment of infection is caused by the presence of bacterial pathogens within internal tissues. Wound infection can be classified under traumatic open-wound infection which is caused by trauma, and surgical-wound infection, acquired surgically or via intravenous medical devices. The latter category would be the investigation of interest for the case study as one of the symptoms shown was swelling around the operation wound.

There are conditions that are more effective than the choice of antimicrobial prophylaxis to minimize microorganism invasion during surgery, such as:

The skill of the surgeon and his ability to prevent the formation of fluid collections

- Limiting the extent of residual devitalized tissue

- Prompt removal of drains and devices that traverse and potentially injure the primary mucocutaneous barrier

Therefore, the risk of surgical wound infections is determined by technical problems with the operation, particularly bleeding, the amount of devitalized tissue created, and the need for drains within the wound, as well as such metabolic factors as obesity and diabetes. The environment plays an important role too. In the study carry out by Rubin¹, Staphylococcus aureus and Streptococcus pyogenes were found to be particularly virulent in surgical-acquired infection.

Definition of SSIs According to the National Nosocomial Surveillance Infections:

- Superficial Incisional SSI*

• Occurs within 30 days after the operation;
• Involves only the skin or subcutaneous tissue; and
• At least 1 of the following:
  
• Purulent drainage (culture documentation not required)
• Organisms isolated from fluid/tissue of superficial incision
• At least 1 sign of inflammation (eg, pain or tenderness, induration, erythema, local warmth of the wound)
• Wound is deliberately opened by the surgeon
• Surgeon or attending physician declares the wound infected.

*A wound is not considered a superficial site infection if a stitch abscess is present, the infection is at an episiotomy or circumcision site or a burn wound, or the SSI extends into the fascia or muscle.

- Deep Incisional SSIs

• Occurs within 30 days of operation or within 1 year if an implant is present;
• Involves deep soft tissues (eg, fascia and/or muscle) of the incision; and
• At least 1 of the following:
  
• Purulent drainage from the deep incision but without organ/space involvement
• Fascial dehiscence or fascia is deliberately separated by the surgeon due to signs of inflammation
• Deep abscess is identified by direct examination or during reoperation, by histopathology, or by radiologic examination
• Surgeon or attending physician declares that deep incisional infection is present.

- Organ/Space SSI

• Occurs within 30 days of operation or within 1 year if an implant is present;
• Involves anatomic structures not opened or manipulated during the operation; and
• At least 1 of the following:
  
• Purulent drainage from a drain placed by a stab wound into the organ/space
• Organisms isolated from organ/space by aseptic culturing technique
• Identification of abscess in the organ/space by direct examination, during reoperation, or by histopathologic or radiologic examination
• Diagnosis of organ/space SSI by surgeon or attending physician.

Source: National Nosocomial Infections Surveillance (NNIS) System Report, data summary from January 1992 to June 2002, issued August 2002.

Centers for Disease Control, Atlanta, Georgia.
http://www.cdc.gov/ncidod/hip/NNIS/2002NNISReport_AJIC.PDF

As the site and the degree of infection were not stated in the case given, we are unable to determine the severity and the possible bacteria strains.

Pathogenesis of SSI

All surgical wounds involved bacteria invasion while only a minority demonstrates clinical infection. In most patients, infection does not develop because innate host defenses are quite efficient in the elimination of contaminants at the surgical site.

However for the case of Wong Fei Hong, fever and swelling of the operation wound was manifested. This indicates that inflammation have taken place.

Determinants of Infection

4 important determinants that leads to SSI: (1) inoculum of bacteria, (2) virulence of bacteria, (3) adjuvant effects of microenvironment, and (4) innate and acquired host defenses.

These are the list of suspected microorganisms involved in SSI:

1. Enterobacter spp. (gram-negative rod)

Reasons for selection:

- opportunistic pathogens (large intestine) that causes nosocomial infections

- related to hospitalization, especially to invasive procedure such as intravenous catheterization

Biochemical tests:

- MacConkey’s or EMB agar: lactose-fermenting colonies (colored)

- Triple sugar iron (TSI) agar: Slant – acid; Butt – acid; Gas – (+); H₂S – (-)

Treatment (antibiotic susceptibility):

- Imipenem: 95%

- Netilmicin: 93%

- Ciprofloxacin: 90%

In severe Enterobacter infection, a combination of imipenem and gentamicin is often used.

2. Escherichia coli (gram-negative rods)

Reasons for selection:

- most abundant facultative anaerobe in the colon

- Endotoxin released causes fever

- Most frequent cause of nosocomial infections due to indwelling urinary catheters

Biochemical tests:

- Triple sugar iron (TSI) agar: Slant – acid; Butt – acid; Gas – (+); H₂S – (-)

- Blood agar

- EMB agar: green sheen colonies

Other tests:

- Presence of indole from tryptophan

- Microscopy: motile

Treatments (antibiotic susceptibility):

span >- Ceftriaxone: 100%

- Imipenem: 100%

- Ampicillin: 66%

3. Klebsiella spp.(gram-negative rods)

Reasons for selection:

- Opportunistic pathogens that cause nosocomial infections, especially pneumonia

Biochemical tests:

- Triple sugar iron (TSI) agar: Slant – acid; Butt – acid; Gas – (+); H₂S – (-)

- MacConkey’s or EMB agar: lactose-fermenting colonies (colored)

Treatment (antibiotic susceptibility):

- Ceftriaxone: 100%

- Imipenem: 100%

- Netilmicin: 100%

4. Proteus spp. (gram-negative rods)

Reasons for selection:

- Hospital-acquired infection, causing urinary tract infection

- Present in human colon

Biochemical tests:

- Urease test

- Blood agar: “swarming” overgrowth

- MacConkey’s or EMB agar: non-lactose fermenting (colorless)

- Triple sugar iron (TSI) agar: results varies in different species

Other test:

- Presence of enzyme phenylalanine deaminase

Treatment (antibiotic susceptibility):

- AMX-CLV (amoxicillin-clavulanate): 100%

- Ceftriaxone: 100%

- Imipenem: 100%

5. Serratia spp. (gram-negative rods)

Reasons for selection:

- Opportunistic pathogens that cause nosocomial infection, pneumonia

- Due to invasive procedures such as intravenous catheterization and respiratory intubation

Biochemical tests:

- Triple sugar iron (TSI) agar: Slant – alkaline; Butt – acid; Gas – (-); H₂S – (-)

- MacConkey’s or EMB agar: late lactose fermenters (S. marcescens produces red-pigmented colonies)

Treatment (antibiotic susceptibility):

- Imipenem: 96%

- Ceftriaxone: 92%

- Netilmicin: 94%

6. Pseudomonas aeruginosa (gram-negative rods)

Reasons for selection:

- Able to withstand disinfectants; hospital-acquired infection

- Found growing in hexachlorophene-containing soap solutions, in antiseptics.

- Colonizes the upper respiratory tract of hospitalized patients

- Contaminates anesthesia equipment, intravenous fluids

- Opportunistic pathogens, most common cause of gram-negative nosocomial pneumonia

Biochemical tests:

- MacConkey’s or EMB agar: non-lactose fermenting (colorless)

- Oxidase test: positive

- Triple sugar iron (TSI) agar: Slant – alkaline; Butt – alkaline; Gas – (-); H₂S – (-)

- Ordinary nutrient agar: blue-green pigment

Treatment (antibiotic susceptibility):

- Imipenem: 89%

- Ceftazidime: 87%

- Netilmicin: 72%

7. Acinetobacter spp. (gram-negative coccobacillary rods) – minor bacterial pathogens

Reasons for selection:

- Opportunistic pathogens that readily colonize patients with compromised immune system

- Causes disease in a hospital setting, associated with respiratory therapy equipment and indwelling catheters

Biochemical test:

- Gram stain

Treatment (antibiotic susceptibility):

- Imipenem: 95.2%

- Ciprofloxacin: 92.8%

- Netilmicin: 92.8%

8. Staphylococcus spp. / S. aureus (gram-positive cocci)

Reasons for selection:

- Most common microorganism present in surgical-wound infection

- Often found in the nose and skin in hospital staff and patients

- Affects individuals with compromised immune system

- A major cause of hospital acquired (nosocomial) infection of surgical wounds and infections associated with indwelling medical devices

- Localized host response to staphylococcal infection is inflammation, characterized by fever and swelling

Biochemical tests (S. aureus):

- Coagulase test: positive

- Mannitol-salt agar: golden-yellow colonies, beta-hemolytic

Biochemical tests (Staphylococcus spp. other than S. aureus)

- Coagulase test: negative

- White colonies, non-hemolytic

Other tests:

- Smears reveal gram-positive cocci in grapelike clusters

Treatment (antibiotic susceptibility):

• MS (methicillin-susceptible) Staphylococcus aureus

- AMX-CLV (amoxicillin-clavulanate): 100%

- Teicoplanin: 100%

- Vancomycin: 100%

• MR (methicillin-resistant) Staphylococcus aureus

- Teicoplanin: 100%

- Vancomycin: 99.1%

- Netilmicin: 90.4%

• MS (methicillin-susceptible) coagulase-negative staphylococci

- Imipenem: 100%

- Teicoplanin: 100%

- Vancomycin: 100%

• MR (methicillin-resistant) coagulase-negative staphylococci

- Teicoplanin: 98.6%

- Vancomycin: 98.6%

- Netilmicin: 91.5%

9. Enterococcus faecalis (gram-positive cocci)

Reasons for selection:

- Important cause of hospital-acquired urinary tract infections and endocarditis

- Indwelling urinary catheters and urinary tract instrumentation are frequent predisposing factors

- Cause intra-abdominal and pelvic infections, in combination with anaerobes

Biochemical tests:

- Catalase test: negative

- Blood agar: alpha-, beta-, or non-hemolytic

Other tests:

- Grows in 6.5% NaCl

- Hydrolyzes esculin in the presence of 40% bile

Treatment (antibiotic susceptibility):

- Imipenem: 100%

- Teicoplanin: 97.9%

- AMX-CLV (amoxicillin-clavulanate): 72.9%

VRE (vancomycin-resistant enterococci) are important causes of nosocomial infections. Linezolid would be the drug of choice for the treatment of VRE.

10. Bacteroides fragilis (gram-negative rods)

Reasons for selection:

- Caused a variety of infectio

- Usually arise from a break in a mucosal surface

- Predisposing factor such as surgery play an important role in pathogenesis

- Associated with intra-abdominal infections

Biochemical tests:

- Sugar fermentation test

- Blood agar

- Organic acid tests

Treatment:

- Metronidazole

- mnioglycosides: treatment of facultative gram-negative rods in mixed infections

11. Clostridium perfrigens (gram-positive rods)

Reasons for selection:

- Common open-wound infection

- Edema occur in the wound area

- A small inoculum to cause an especially severe necrotizing infection at the surgical site

Biochemical tests:

- Sugar fermentation test

- Organic acid tests

- Blood agar: double zone of hemolysis

- Egg yolk agar: presence of lecithinase

Treatment:

- Penicillin G is the antibiotic of choice

Other causative agents that cause wound infection, such as Pasturella multocida which are transmitted by animals and Wuchereria bancrofti, transmitted by mosquitoes primarily in Africa, were ruled out in this investigation.

From the various study, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterocuccus faecalis are some of the pathogens commonly observed in surgical wound infection.

Case 6 (Shahirah)

Patient's name: Ong Fei Fei

The following agents are the most likely causative agent in this patient.

Proteus mirabilis

Gram stain: gram-negative bacillus
Wet mount: motile (highly)
Biochemical Tests:
Indole test: negative
Methyl Red test: positive
Voges-Prokauer tests: negative
Simmon’s citrate test: positive
Urease test: positive
Catalase test: positive
Culture: (under anaerobic conditions)
Blood agar: non-hemolytic
MacConkey agar: Colourless colonies
TSI agar: Black butt
Antimicrobial susceptibility: Ampicilin, aminoglycoside

Pseudomonas aerugionsa
Gram stain: gram-negative rods
Wet mount: Non-motile
Biochemical Tests:
Indole test: negative
Methyl Red test: negative
Voges-Prokauer test: negative
Simmon’s citrate test: positive
Oxidase test: positive
Catalase test: positive
Culture: (aerobic condition)
Blood agar: B-hemolytic
MacConkey agar: Colourless colonies
TSI agar: Negative
Antimicrobial susceptibility: gentamicin, cefepime, ciprofloxacin

Trichomonas vaginalis
Gram stain: Not useful as microorganism is a protozoa
Wet mount: Motile (jerky motion, corkscrew motility)
Culture: Best cultured at 35-37oC, anaerobically , pH 5.5-6.0
Antimicrobial susceptibility: metronidazole

Staphylococcus aureus
Gram stain: gram- positive cocci, cluster (similar to grapes)
Wet mount: Non-motile
Biochemical tests:
Catalase test: positive
Coagulase test: positive
Culture: (aerobic conditions)
Mannitol salt agar: Agar turns yellow due to the acid produced
Blood agar: B-hemolytic
Antimicrobial susceptibility: vancomycin, nafcillin

Staphylococcus saprophyticus
Gram stain: gram-positive cocci
Wet mount: non-motile
Biochemical Tests:
Catalase test: positive
Coagulase: negative
Urease: positive

Culture:
· Blood agar: gamma-hemolytic
Antimicrobial susceptibility: Quinolones

Eschericia coli
Gram stain: gram-negative rods
Wet mount: Highly motile
Biochemical Tests:
Indole test: Positive
Methyl Red test: Positive
Voges-Prokauer test: Negative
Simmon’s citrate test: Negative
Oxidase test: negative
MUG test: positive
Culture:
Blood agar (hemolysis)
MacConkey agar: Pink/red colonies
Antimicrobial susceptibility: amoxicillin, nitrofurantoin, trimethoprim-sulfamethoxazole

Gardnerella vaginalis
Gram stain: gram-variable rods
Wet mount: clue cells
Biochemical Tests:
Catalase test: negative
Culture:
Starch agar with bromcresol blue
Chocolate agar
Antimicrobial susceptibility: metronidazole

References: http://www.asmpress.org/asmpress/files/ccLibraryFiles/Filename/000000000713/CMPH2_2007_update_Section_3_for_e-store.pdf

http://www.emedicine.com/med/topic841.htm
http://www.textbookofbacteriology.net/staph.html
Brooks, Geo F., Janet Butel, and Stephen Morse. Jawetz, Melnick, and Adelberg's Medical Microbiology, 23rd edition. 2004.
Levinson, Warren. Review of Medical Microbiology and Immunology, Ninth edition. 2006.