2. 2
Arthur Dessi Roman MD MTM FPCP FPSMID
Internal Medicine – Infectious Diseases
Outline
• Basic Bacteriology
– Structure and Characteristics
– Bacterial Culture
• Host defenses
• Cases
– Gram positive Pathogens
– Gram negative Pathogens
– Bonus(es)
• Question and Answer Portion
• Sportswear Competition
3. 3
Highschool recall: Eukaryotes
• Eukaryote - organism whose cells contain complex
structures enclosed within membranes
– the defining membrane-bound structure is the nucleus,
which contains the genetic material
– also contain other membrane-bound organelles:
mitochondria, chloroplasts and the Golgi apparatus
– e.g. animals, plants and fungi
Highschool recall: Prokaryotes
• Prokaryote - NO cell nucleus , or any other
membrane-bound organelles.
– DNA and other sites of metabolic activity: openly
accessible, free-floating
– asexual reproduction, usually by binary fission or budding
– Genetic exchange and recombination still occur, but this is
a form of horizontal gene transfer and is
NOT a replicative process, simply
involving the transference of DNA
between two cells, as in
bacterial conjugation.
6. 6
Generalities
• Most cocci are gram positive
• Most bacilli are gram negative
Gram positive Bacilli/rods
• Bacillus sp.
– Normal flora of the skin
– B. anthracis, B. cereus
• Lactobacillus (shirota strain et al)
• Corynebacterium
• Clostridium sp.
• Listeria
• Erysipelothrix
11. 11
The Gram Stain
• Quick procedure that reveals bacterial cell
morphology (cocci vs. bacilli, G+ vs G-)
• Gram negative: red/pink
• Gram positive: violet/blue
• The color difference is based on the amount
of the peptidoglycan layer (G+=thick = more
dye uptake=blue/violet color)
• Results guide initial antibiotic therapy
The Prokaryotic genome has many
plasmids.
• encode genes that carry specialized functions
(eg. Resistance to bacteria)
• Also encode for genes that mediate their
transfer from one organism to another as well
as other genes associated with genetic
acquisition or rearrangement of DNA.
12. 12
Rise of the superbugs
Resistance of bacteria to antibiotics after their liberal
use in hospitals may be assimilated by plasmids of
other non-resistant bacteria
Gram-positive vs Gram-negative cell wall
13. 13
Gram-positive & Gram-negative cell wall
Gram + Gram -
Cell wall made of thick
peptidoglycan
Thin peptidoglycan
No outer membrane Outer membrane present
No endotoxin contains LPS/ Lipid A/ endotoxin
responsible for sepsis
More susceptible to penicillins
(lysozymes, detergents, etc.) due to
absence of outer layer
Outer membrane provides
barrier has porins for entry of
nutrients
NO periplasmic space Periplasmic space contains B-
lactamase/other enzymes that may
degrade antibiotics
Possess teichoic acids NO teichoic acids
14. 14
Bacterial cell wall peptidoglycan
PBPs
• Penicillin-binding proteins (PBPs) catalyze the
synthesis of the PG in the cell wall
• B-lactam antibiotics work by inhibiting PBPs
• Some bacteria produce enzymes that alter the
PBPs or inactivate the B lactam ring
resistance
17. 17
(Livermore and Woodford, Trends in Microbiol, 2006)
The Gram Negative Cell Wall
Porin
channels
PBPs
Efflux system
B-lactamases
Mechanism of Resistance to β-lactams
CELL WALL
PENICILLIN BINDING
PROTEIN (cell wall
synthesis)
S. aureus
19. 19
Anaerobic vs. aerobic bacteria
• Aerobic: need oxygen to grow
• Anaerobic: fail to grow in the presence of oxygen
• Obligate anaerobes: grow only in aerobic environment
• Facultative anaerobes: can grow with or without oxygen
• Microaerophilic bacteria: grow best in an environment with
reduced oxygen
• Aerotolerant: can grow in the presence of air, but do NOT use
oxygen
Important Bacterial Pathogens
Bacteria
Gram positive* Gram negative* Atypical
Cocci Bacilli Cocci Bacilli
*can be aerobic/anaerobic
20. 20
Spot Quiz
What are single-celled, prokaryotic
microorganisms that sometime produce disease
in humans and are usually susceptible to
antibiotics?
Answer: BACTERIA
21. 21
What is the rigid structure outside the cell
membrane which protects and maintains the
shape of bacteria?
Answer: CELL WALL
What is the main component of bacterial cell
walls?
Answer: PEPTIDOGLYCAN
22. 22
What are the proteins that facilitate formation
of peptidoglycans?
Answer: PENICILLIN-BINDING PROTEINS
(PBPs)
What is the procedure that can help visualize
bacteria under the microscope and differentiate
them into gram-positive and gram-negative
bacteria?
Answer: GRAM STAIN
23. 23
Differentiate the call walls of gram-positive and
gram-negative cell walls.
Answer: Gram-negative cell walls have an outer
membrane made up of a thinner layer
of peptidoglycan and an outer
membrane, which is in turn made up
of lipopolysacharide or endotoxin.
What is the enzyme that can degrade beta
lactam antibiotics found in the periplasmic
space of gram-negative bacteria?
Answer: BETA LACTAMASE
24. 24
The outer membrane of gram-negative bacteria
act as a barrier to some antibiotics, but
antibiotics and other nutrients can enter the
bacteria via “holes” called:
Answer: PORINS
Differentiate aerobes from anaerobes.
Answer: Aerobes are bacteria that need
oxygen to grow, while anaerobes
are bacteria that cannot grow in the
presence of oxygen.
26. 26
Bacterial Identification
• Subsequent bacterial identification is based
from initial Gram staining results from
bacteria from isolated colonies on agar plate
• Subsequent testing uses bacterial nutritional
requirement and metabolic capacities
• Most bacteria will grow in 24-48 hours except for
fastidious or anaerobes which may require more time.
• Culture/identification is performed so a physician can
come up with a diagnosis and prescribe the most
appropriate antibiotic.
28. 28
Bacterial growth curve
Pathogens
• Microorganisms causing disease
• Pathogenicity: ability to cause disease
(increased by the presence of bacterial
enzymes and bacterial toxins)
• Stages of infection: adherence, invasion,
multiplication, dissemination in the host
29. 29
Host Defenses
Types of Infection
Acute: Chronic:
- develop rapidly - develop more slowly
- usually associated with fever - milder symptoms
- generally of short duration - longer-lasting
Acute infections may become chronic, chronic infections may
become acute.
Local: Systemic:
- Limited to one site - throughout the body
Local infections may occasionally cause systemic symptoms or
may serve as nucleus for spread of infection to other body
areas (e.g. dental infection).
30. 30
Types of Infection
• Bacteremia: presence of bacteria in the blood.
• Septicemia: blood poisoning; systemic disease associated
with presence and persistence of pathogenic microorganisms
or their toxins in the blood.
Phases of Infection
1. Invasion: Bacteria gain entry into the host, or host tissues,
where it’s not part of normal flora.
2. Incubation: Period before any manifestation of illness. It
represents the time required for pathogens to multiply to a
critical population size. Patient can be infectious.
3. Prodrome: Period characterized by nonspecific symptoms
(e.g. headache, malaise).
4. Clinical illness: Period when specific symptoms (fever)
appear. Body damage may result due to toxin, death of host
cells, actions of host’s immune system.
5. Convalescence or Resolution: Period of recovery, even
without symptoms patient may still be infectious.
31. 31
Symptoms of Infection
• Provide subjective information on patient’s condition.
• Inflammation: Process that occurs in tissues as a
reaction to injury. It involves vasodilation, increased
permeability of capillaries to fluid and large molecules,
and attraction and accumulation of WBCs and other
immune system components in injured area.
- Results in pain, swelling, heat and throbbing.
• Fever: Not true in all infections. Microbes produce
pyrogens (fever-causing substances) that cause release
of cytokines that cause the fever.
• Symptoms depend on organs or systems affected and
are helpful to MDs in diagnosing specific conditions.
Signs of Infection
• Provide objective measures of disease; vary depending on nature
and severity of condition.
• Fever: Both a sign and symptom.
• Increased number of neutrophils in blood.
• Increased in proportion of immature WBC (shift to the left).
• Neutropenia or granulocytopenia: abnormally, low neutrophils in
overwhelming, severe infections. (Normal value: 1,500-7,800 /mL).
The incidence of infection rises as neutrophil count declines.
• Anemia: reduction below normal in number of RBCs per mm3,
hemoglobin, or volume of packed RBC per 100 ml of blood.
• Increased ESR (erythrocyte sedimentation rate) which measures the
rate at which RBCs settle in uncoagulated blood – a non-specific
measure of inflammation or disease activity.
32. 32
Signs of Infection
• Elevated blood levels of C-reactive protein.
• Increase in pulse rate, respiration rate, anxiety, confusion,
delirium, and other abnormalities in mental status.
• Hyperglycemia: elevated blood sugar levels, especially in
diabetics.
• Changes in kidney and liver function.
Non-specific host defenses
• Directed against any pathogen
• Mechanical
• Chemical
• Phagocytes and complement system
35. 35
Spot quiz(zes)
What do you call the microorganisms that
normally live in our bodies?
Answer: NORMAL FLORA
36. 36
What do you call a microorganism that cause
disease?
Answer: PATHOGEN
What do you have when bacteria invade body
tissues, multiply in them, and cause damage to
the host’s cells?
Answer: INFECTION
37. 37
What do you call the body’s response to
infection?
Answer: IMMUNITY
What is the importance of knowing the normal
flora in terms of dealing with infections?
Answer: It helps in determining
EMPIRIC THERAPY.
38. 38
Antibiotic Classes
32
Case 1
• 35/male who was admitted for severe
abdominal pain and fever.
• Took paracetamol but did not improveER
• PE: febrile, direct and rebound tenderness
RLQ
39. 39
32
Case 1
• A> Acute appendicitis underwent OR
• He was discharged improved.
• On ff-up with the surgeon after 5 days,
STAPHYLOCOCCUS
40. 40
Staphylococcus aureus
• frequently found in the human respiratory tract and on
the skin
• NOT always pathogenic
• Causes skin infections (e.g. boils), respiratory disease
(e.g. sinusitis), and food poisoning
• Producing potent toxins
• Common cause of device-related infections
• Carriage in healthy individuals (nostrils)
• The emergence of antibiotic-resistant forms of
pathogenic S. aureus (e.g. MRSA) is a worldwide
problem in clinical medicine.
Staphylococcus aureus
• Virulence factors
– Coagulase
– Staphylokinase
– Hyaluronidase
43. 43
Staphylococcus aureus
1. Local Stap. Infection – drainage
> antibiotics –semi synthetic PCN
Cloxacillin Erythromycin
Cehalosporin (1st gen.)
2. Systemic Infection: Parenteral
Vancomycin Oxacillin
Methiallin Cephalosporin
Question
What is the current MRSA rate in the Philippines
according to the ARSP 2014?
A. <5%
B. 35%
C. 60%
D. 90%
44. 44
Question
What is the current MRSA rate in the Philippines
according to the ARSP 2014?
A. <5%
B. 35%
C. 60%
D. 90%
Percent resistance of S. aureus,
(n=3,529), ARSP, 2014
ANTIMICROBIAL RESISTANCE SURVEILLANCE PROGRAM 2014 ANNUAL REPORT
FIGURE 32. Yearly penicillin, oxacillin and vancomycin resistance rates of S. aureus,
ARSP, 2005-2014
ANTIMICROBIAL RESISTANCE SURVEILLANCE PROGRAM (ARSP) 2014 ANNUAL REPORT,
Research Institute for Tropical Medicine, Department of Health Philippines
45. 45
Percent resistance of S. aureus,
(n=3,529), ARSP, 2014
ANTIMICROBIAL RESISTANCE SURVEILLANCE PROGRAM (ARSP) 2014 ANNUAL REPORT,
Research Institute for Tropical Medicine, Department of Health Philippines
Percent resistance of S. aureus,
ARSP, 2014
ANTIMICROBIAL RESISTANCE SURVEILLANCE PROGRAM 2014 ANNUAL REPORT
Antimicrobial Resistance
The 2014 resistance rate of S. aureus against the β-lactam oxacillin is at 60.3%
(n=3,323; 95% CI: 58.6-62). The 2014 rate of methicillin-resistant S. aureus (MRSA) rates
increased significantly from 53.2% in 2013 to the 60.3% in 2014 (p value 0.0001). Resistance
rates against the antibiotics tested against S. aureus for 2014 and the past decade are see n
FIGURES 40-43. The 2014 resistance rates to the rest of the antibiotics tested did not differ
significantly from the reports from 2013 except for the significant increase in co-trimoxazole
resistance from 14% in 2013 to 22% for 2014 (p value 0.0001). Although there were 17 isolates
reported as vancomycin resistant, none of these S. aureus isolates were sent for confirmatory
testing at the reference laboratory.
FIGURE 30. Percent resistance against penicillins, vancomycin, clindamycin and
erythromycin of S. aureus, ARSP, 2014
ANTIMICROBIAL RESISTANCE SURVEILLANCE PROGRAM (ARSP) 2014 ANNUAL REPORT,
Research Institute for Tropical Medicine, Department of Health Philippines
• Overall cumulative MRSA rate for 2014 was at 60.3%
(from 53.2%, p=0.0001)
• 85% were isolated from specimens taken from patients in
the OPD, emergency room and admission within their 1st
2 hospital days
• 60% MRSA rate from all blood isolates (n=570)
46. 46
Enterococci
• Gram-positive cocci in chain
44
Enterococci
• Gram-positive cocci in chain
• Commensals of the GI tract of humans & animals
• Diseases :
• Consist of 16 species :- E. faecalis, E. faecium
- Urinary tract infection (UTI)
- Bacteremia, endocarditis
44
47. 47
Vancomycin-Resistant
Enterococci (VRE)
46
• An emerging pathogen but none yet in PH
• Acquired via plasmids
• Cephalosporin use is a risk factor
• Can be carried by healthy people
32
Case 2
• 65/male DM, HPN admitted for sudden left
sided weaknessER
• PE: no motor movement left UE and LE,
shallow right NSLF, slurred speech
48. 48
32
Case 2
• Treated as stroke and underwent rehab after 5
days doing well
• On Day 6 cough and difficulty of breathing,
high fever, auscultation revealed rales on right
lung field
• Eventually intubated due to respiratory
distress
Case 2
• Started empirically on PipTazo
• ETA GS revealed many PMNs and many Gram
_____________.
49. 49
Pseudomonas aeruginosa
•Aerobic gram-negative bacilli, non-
spore forming
•Produces cyanin pigment
•Produces grape-like odor
•Inhabitant of the GI tract
•Saprophytic (natural habitat: soil
and water)
•An opportunistic pathogen
Pseudomonas aeruginosa
•
50. 50
• MOST important hospital-acquired pathogen
• Other pseudomonads: Burkholderia mallei,
Burkholderia cepacia
•High tendency to develop antibiotic resistance
Pseudomonas aeruginosa
21
Resistance Pattern of P. aeruginosa, ARSP 2011
51. 51
23
• Causes nosocomial infections- extensive burns, trauma
to the skin or conjunctiva, urinary tract manipulations
• cystic fibrosis.
• Ear infections – swimmer’s ear
• Bacteremia in debilitated patients, accounts for 10% of
all Gram(-) associated with a 50% mortality rate
Pseudomonas aeruginosa
21
52. 52
Treatment
• Aminoglycosides
• Third and fourth generation cephalosporins
(ceftazidime, cefepime)
• Carbapenems (imipenem, meropenem)
• Pip-tazo
• Colistin
Pseudomonas aeruginosa
Clinical manifestation of P. aeruginosa
infections
53. 53
32
Case 3
• 15/male with cerebral palsy recurrently
admitted for pneumonia
• He already has a tracheostomy tube in place
• Developed progressive cough and respiratory
distressER
32
Case 3
• PE: drowsy, highly febrile, rales all over R>L
• Hooked to MV and started on Pip-Tazo
54. 54
Case 3
• After 3 days of antibiotics, he was still febrile
• Developed hypotension and further
respiratory distress
Case 4
• EC = few PMN=moderate
• Gram negative diplococci-few
• Gram negative coccobacilli-many
55. 55
Acinetobacter
• Aerobic gram-negative coccobacilli,
non-spore forming
• > 25 species
• A common cause of ventilator-
associated pneumonia (VAP)
• Opportunistic pathogen
28
• Local isolates are very highly
resistant to antibiotics
A. baumannii
Clinical manifestation of A. baumannii
infection1.Hospital-acquired pneumonia
especially VAP
2.Community-acquired pneumonia
:The source of infection may be throat
carriage, which occurs in 10% of
community residents with excessive
alcohol consumption
3.Bloodstream infection & endocarditis
4.Skin/soft tissue infection
5.UTI
6.Meningitis
29
56. 56
A. baumannii
Persistence in the hospital
environment : 3 days to 5 months
1. Resistance to major antimicrobial
drugs
2. Resistance to desiccation
3. Resistance to disinfectant
Three factors contributing to the
persistence
31
A. baumannii
31
57. 57
A. baumannii
Treatment:
• Meropenem
• Colistin
• Tigecycline
• Ampicillin-Sulbactam
31
Acinetobacter
• Aerobic gram-negative coccobacilli,
non-spore forming
• > 25 species
• A common cause of ventilator-
associated pneumonia (VAP)
• Opportunistic pathogen
28
• Local isolates are very highly
resistant to antibiotics
58. 58
A. baumannii
Clinical manifestation of A. baumannii
infection1.Hospital-acquired pneumonia
especially VAP
2.Community-acquired pneumonia
:The source of infection may be throat
carriage, which occurs in 10% of
community residents with excessive
alcohol consumption
3.Bloodstream infection & endocarditis
4.Skin/soft tissue infection
5.UTI
6.Meningitis
29
A. baumannii
Persistence in the hospital
environment : 3 days to 5 months
1. Resistance to major antimicrobial
drugs
2. Resistance to desiccation
3. Resistance to disinfectant
Three factors contributing to the
persistence
31
60. 60
Enterobactereaceae
• Gram-negative, nonspore-forming rods
• Facultative anaerobic
• Grow in simple media (MacConkey agar)
• Ferment glucose (LF)
• Motile with peritrichous flagella except Shigella
• Some possess capsule
Impt. Human pathogens
• Escherichia - E. coli
• Enterobacter (E. aerogenes, E. cloacae)
• Citrobacter - C. freundii
• Serratia - S. marcescens
• Hafnia - H. alvei
• Klebsiella
61. 61
*Proteus Group
– P. mirabilis
– P. vulgaris
– Morganella – M. morganii
– Providencia – P. rettgeri
*Edwardsiella – E. hoshinae
Escherichia coli
• Escherichia coli and certain related bacteria (e.g.,
Klebsiella, Enterobacter, Citrobacter) form the
group known as the coliforms
• presence in drinking water is considered evidence
of fecal contamination
• used as an index of fecal pollution.
62. 62
Epidemiology
• Fecal-oral transmission transmission among infants in
hospital nurseries.
• Food-borne transmission of E. coli 0157:H7 -
contaminated meat, from cattle; this serotype behaves as
a zoonotic agent.
• UTI –perineum; In women, UTI is associated with sexual
activity, hence the term "honeymoon cystitis".
Diseases associated with
Escherichia coli:
• Diarrhea
• Meningitis – impt. cause of neonatal
meningitis in the neonates
• Sepsis
• Urinary Tract Infection -most
common cause
63. 63
Gram’s Stain and Culture
Klebsiellae
• Encapsulated
• The species of Klebsiella include
- Klebsiella pneumoniae- most infections
-Klebsiella oxytoca
-Klebsiella ozaenae
-Klebsiella rhinoscleromatis
64. 64
• Mucoid colony
• มีแคปซูลหนา
Klebsiella
18
Diseases:
• Community acquired and nosocomial
pneumonia w/ underlying medical problems
such as alcoholism, COPD or diabetes mellitus
• Lung abscess may also develop.
• Urinary tract infection
• Wound infection
• Epidemic diarrhea - newborns
• Septicemia
66. 66
Enterobacteriaceae: Treatment
• Empiric therapy with aminoglycoside and
third cephalosporin
Case 4
• 35/female who just delivered a baby boy via
NSD 1 week ago rushed back to the ER for
severe hypogastric pain
• PE: highly febrile, direct tenderness at the
hypogastric area, purulent vaginal discharge
• Diagnosis?
• Started on meropenem and clindamycin
(MRSA)
67. 67
Case 4
• She underwent EL on Day 3
• Doing well
• Day 10 of meropenem and clinda, developed
high fever
• No cough. No dysuria but she was having LBM
5-7x/day
• Diagnosis?
Clostridium Difficile
• Gm (+) bacillus, obligate anaerobe
• Spore forming contact precautions
• Causes pseudomembranous colitis and
antibiotic-associated diarrhea
68. 68
Laboratory Diagnosis
• Culture of stool specimens
• Cytotoxin Assay
– Tissue culture assay
– immunoassays
• Colonoscopy or Signoidoscopy with Biopsy
Endoscopic findings – pseudomembranes and
hyperemic rectal mucosa
Treatment
• Antimicrobial therapy should be
discontinued
• Maintenance of fluid & electrolyte balance
• Avoid drugs which intestinal motility
69. 69
• Drug of choice
– Metronidazole (30 mg/day in 4 divided
doses) – drug of choice in the initial
treatment of most patients with colitis
– Oral Vancomycin (40 mg/kg/day in 4 divided
doses) – indicated only for seriously ill
patients or those who do not respond to
vancomycin
– Bacitracin – ssecond alternative therapeutic
choice
Case 5
• 70/female post stroke and chronically bed
ridden was admitted for decreased sensorium
• Because of urinary incontinence, has been on
foley catheter and PEG for episodes of
aspiration
• PE: drowsy, T 37 C BP 70/50 HR 100
• Clear breath sounds, no nuchal rigidity
70. 70
Case 5
• Labs done: WBC 20 Neut 90%
• Chest xray: normal
• Urinalysis: WBC 50 RBC 0-3 EC few yeast cells
few
• Started on Ceftriaxone 1g IV q24h
• FC was changed
• Urine GS CS requested
Case 5
• Urine GS: PMN many yeast cells few G+cocci
in clusters many
71. 71
Candida spp.
• normal flora of the skin, mucous membranes, and
gastrointestinal tract
• colonize the mucosal surfaces of all humans during or soon
after birth
• Candida albicans – the most common pathogen
• Others: C albicans, C tropicalis, C parapsilosis, C glabrata, C
guilliermondii, and C dubliniensis)
• The widespread use of fluconazole has precipitated the
emergence of more azole-resistant species, such as C krusei
and C lusitaniae.
Cutaneous & Mucosal Candidiasis
• The risk factors: AIDS, pregnancy, diabetes, young or old age,
birth control pills, and trauma (burns, maceration of the skin),
corticosteroids or antibiotics, hyerglycemia, and cellular
immunodeficiency
• Cutaneous candidiasis- when the skin is weakened by trauma,
burns, or maceration.
Vulvovaginitis - irritation, pruritus, and vaginal discharge;
often preceded by factors such as diabetes, pregnancy, or
antibacterial drugs that alter the microbial flora, local acidity,
or secretions.
72. 72
Systemic Candidiasis (Candidemia)
• Causes:
– indwelling catheters, surgery, intravenous drug abuse,
aspiration, or damage to the skin or gastrointestinal tract
• most often associated with:
– chronic administration of corticosteroids or other
immunosuppressive agents; with hematologic diseases
such as leukemia, lymphoma, and aplastic anemia; or with
chronic granulomatous disease
Candidiasis: Treatment
• Systemic candidiasis: amphotericin B with/without oral
flucytosine, fluconazole, or caspofungin