Emerging pathogens 2008
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Peter H. Gilligan PhD
Clinical Microbiology-Immunology Labs
UNC Hospitals
How I became a clinical microbiologist
• Obtained doctoral degree in microbiology at the University
of Kansas
• Did post-doctoral training (2 years) in medical and public
health microbiology at UNC Hospitals
• Director of Microbiology Labs at St Christopher’s Hospital
for Children (Philadelphia) for 4 years
• Past 20+ years, Associate Director then Director of the
Clinical Microbiology-Immunology Labs at UNC Hospitals
• Have served on medical school admission committee for
approximately 15 years and the MD/PhD advisory
(admissions) committee for the past 10 years
What do clinical microbiologists do?
• We serve:
» our patients
» our health care-providing colleagues, physicians,
nurses, physician assistants, pharmacy colleagues
» hospital administrators
• We make money for the institution
» general public by insuring the public health
• Involved in studying outbreaks of several emerging
infectious diseases
How do we serve?
• central role in the diagnosis and management of
infectious diseases
• central role in infection control and antimicrobial use
• recognize emerging disease threats and outbreaks
including bioterrorism events
• we educate & train health care providers
• we create new knowledge (research) to deal with
practical problems
Best things about my job
• Direct impact on patient care and public health of the
community
• Intellectually challenging job requiring a broad fund of
knowledge-need to know a little about a lot of things –I am
never bored!!!!!!!
• Work with highly motivated and intelligent individuals
• Get to be at the cutting edge of infectious disease
diagnosis
Worst things about my job
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Incredible amounts of governmental oversight
Increasing emphasis on financial aspects of the job
Declining talent pool of technologists
Need to be responsible for an organization that run
24/7/365-we never close. Personally have worked through
ice storms, blizzards, and hurricanes.
How you can become a clinical
microbiologist
• CLS programs available here, ECU, WCU, WSSU, Wake
Forest, UNC-CH
» Education is also available on line
• 2 more years of school to get a BS in CLS
» There is no unemployment in this group
• Take ASCP certification exam to become certified as a MT.
» Starting salary is 38,000 and up
» Career options are amazingly diverse; many former UNC
students work in leadership positions in the pharmaceutical
and biotech industries
Emerging/Re-emerging Infectious Diseases in the
past 25 years
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HIV*
Avian influenza
SARS*
Cryptosporidium*
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E. coli O157:H7*
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Nipah virus
nv Creutzfeldt-Jakob disease
Sin Nombre Virus
West Nile Virus
Clostridium difficle*
Bacillus anthracis (BT agent)
Cyclospora
CA-MRSA*
Rapidly growing mycobacterium*
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Rotavirus*
BK virus*
Chlamydia pneumoniae
Pencillinum marneffei
Legionella*
MDR- TB and pneumococcus*
Burkholderia cepacia complex*
VRE/VRSA*
Helicobacter pylori*
Invasive Group A streptococcal
disease*
HHV-6*
HPV*
HCV*
How do new pathogens emerge
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Organisms that jump species barriers
Changing ecosystems
Changes in food production techniques
Evolution of medical devices and care
» Long term survival of immunosuppressed
• Pathogens that are detected because of new
technology
• Misuse of micro-organisms
» Biocrime/bioterrorism
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Organism evolution as a result of human intervention
» Antibiotic pressure
How do microbes change?
• Bacteria, because they evolve very quickly, can readily
adapt to hostile environments
» Assume a generation time for a bacteria of 50 minutes
» 30 generations/day; or 220,000 bacterial generations for
each human generation (assume generation is 20 years)
» Bacteria have a huge evolutionary advantage over humans
How emerging pathogens develop?
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Mutation drives evolution
» constantly occurring
» usually silent or lethal
» environmental pressure such as antibiotics may select
“resistance” mutation
• Key feature of success of antibiotic resistant strains is their
genetic fitness I.e. their ability to compete in a complex microbial
environment
» Recognition that certain bacteria may be hypermutators
because of mutation in DNA repair genes
• These strains may not be as “fit” as wild-types but may
predominant in certain chronic infections such as
P.
aeruginosa causing chronic pulmonary infections in CF patients
How do emerging pathogens develop?
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Recombination
» Resistance genes from antibiotic producing
organisms
» genetic exchange of resistant genes can occur
among organisms which are genetically
diverse
• Think Cholera toxin genes to E. coli
» transfer of resistance/virulence genes can be
mediated by plasmids/phage/transposons/
integrons
Organisms that jump species barriers
• HIV, SARS, Avian flu
» HIV likely jumped from primates to humans
» SARS from pigs(?)
» Avian flu-hasn’t yet made the jump from birds to humans
because human to human spread is rare, if it occurs at all.
However mutation may result in that occurring.
» Technology allows us to quickly develop diagnostics for new
pathogens
• Took years to develop HIV diagnostics
• Took weeks to develop SARS diagnostics
Changing ecosystems
• Lyme disease
» A perfect storm
• Farmland in New England returned to forest
• Natural predators for deer were eliminated
• Deer populations and the ticks they carried increased
because of ecosystem changes
• People built homes and spent increasing amounts of time
in the woods
• This resulted in increased exposure to deer ticks that
carried Borrelia
» Ticks were pencil point in size and often difficult to see
Changes in food production techniques
• Increased use of factory farming
• Feedlots bring together large numbers of animals who
produce large amounts of waste
» Waste can lead to run-off of EHEC that can contaminant
adjacent fields as was seen in recent spinach outbreaks
• Large meat packing operations can result in 50 ton lots of
ground meat containing 100s of animals
» Meat can be distributed throughout the US
» Contaminated lots can then lead to large scale outbreaks
Changes in medical care
• Immunosuppression either as a result of HIV or medically
therapy (ex. transplants) results in emerging infections
» Pneumocystis, MAC, toxoplasma and CMV in HIV patients
» CMV, adenovirus and HHV-6 in transplant patients
• The use of indwelling artificial materials such as catheters,
shunts, artificial joints present new ecosystems and new
organisms
» Examples-coagulase negative staphylococci growing as a
biofilm on artificial joints/catheters/shunts
» Rapidly growing mycobacteria causing keratitis following
LASIK surgery
Pathogens detected with new technology
• Prime example is HCV
» Viral genome elucidated using molecular cloning techniques
• Broad range 16S RNA primers are used to detect noncultivable bacteria
• Next big thing- application of molecular tools to
understand how mixed microbial populations cause
disease
» Likely diseases caused by mixed microbial populations are
bacterial vaginosis, peridontal disease, inflammatory bowel
disease, CF lung disease
How does bacterial resistance
develop?
• Bacterial resistance develops in response to antimicrobial
pressure
» It is estimated that 3 million lbs of antimicrobials are used
each year in the US
• Much of it is used in children to treat viral respiratory
illness
• Estimated that 3/4 of children in US younger than two
receive antimicrobials
• Children then may serve as a key role for the emergence of
antimicrobial resistance
» 10x that amount are used in animals
» End result- tremendous selective pressure that results in the
emergence of bacterial resistance
Antibiotic associated adverse effects
• Antimicrobial toxicity and allergic reactions
» Anti-parasitic>anti-fungal>anti-viral>antibacterials
» 20% of ER visits for drug adverse events are due to
antimicrobials
• Alteration in the microbial flora
» Candida vaginitis and thursh
» C. difficile infection
» Salmonellosis
• Emergence of resistance
» Few organisms where resistance is not clinically important
Case
• Patient is an 80 yr female with primary pulmonary
hypertension who present with SOB, DOE and chest
radiograph consistent with lobar pneumonia
• Treated for 14 days with levofloxacin and discharged to a
skilled nursing facility
• 3 weeks later she has has the onset of loose watery stools
up to 10 episodes a day
Case
• She develops generalized weakness, abdominal pain, and
is unable to walk
• She develops vomiting and nausea and can’t eat
• She is re-admitted to the hospital after three weeks of
diarrhea; she presents with fever, chill, malaise, myalgias,
and dizziness
• PE is significant for a pulse rate of 120, RR of 24, and BP
of 82/45
Case
• Her abdominal examination is significant for decreased
bowel sounds, tenderness and mild distension.
• She had a white blood cell count of 29,000/ul
• Imaging studies were consistent with pseudomembranous
colitis
• Over a four day period her white blood cell count rose
from 29,000 to 127,000 ul; she had expanding abdominal
girth and decreasing bowel sounds.
• She died on the 4th hospital day
Clostridium difficile
• General characteristics
» Gram positive rod
» Spore former
» Anaerobic
» Can be part of human
microflora
» Pathogenicity due to the
production of two protein
exotoxins A and B
Chance favors only the prepared mind
Louis Pasteur
Key events in the discovery of C. difficile
• Larson and colleagues describe a toxin in the feces of a child with
pseudomembranous colitis (1977)
• Bartlett and colleagues show that C difficile can induce colitis in
hamsters given clindamycin and then a variety of antibiotics and then
proves that the organism can cause the same disease in humans
(1978)
» Serendipity is important- showed that C. sordelli antitoxin could
neutralize toxins produced by C. difficile in a tissue culture
cyotoxicity assay.
Key events in the discovery of C. difficile
• Among others, Gilligan and colleagues show that C.
difficile is the most common bacterial agent in a general
population (1980)
• Lylerly and colleagues purify two toxins, A and B, from C.
difficile and also produce an important anti-toxin against
these organisms (1982)
What makes C difficile an important
pathogen in the industrialized world?
• Important ideas
» Organism can survive in the environment for months as spores;
spores are refractory to disinfectants especially alcohol and all
antimicrobials
» Alternation in the gut flora is important in predisposing patient’s to
disease with this organism- antibiotics mediate this change
» Most common diarrheal disease etiology in the industrialized world
requiring specific antimicrobial interventions
Changing C. difficile epidemiology
Changing C. difficile epidemiology
• Beginning in 2002, a highly virulent strain of C. difficile (NAP1/027)
emerged in the Quebec Province in Canada, several US states,
Britain, and just recently in Netherlands
» The disease is seen primarily in those over 65 years of age
» The incidence in one Canadian region in patients over 65 went
from:
• 102/100,000 in 1991-2
• 210/100,000 in 2002
• 866/100,000 in 2003
What factors has resulted in the re-emergence of
Clostridium difficile??
• Better case ascertainment
» Improvement in lab diagnosis
• Aging population
» Decline in Bifidobacterium with age, an organism important in
colonization resistance, in gut flora may create more permissive
environment for C. difficile
• Increased use of antimicrobials especially fluoroquinolones with antianaerobic activity to which C. difficile is resistant
» This is being debated in the infectious disease community
» 90% of C. difficile isolates are fluorquinolone resistant
What factors has resulted in the re-emergence of
Clostridium difficile?
• Increased contamination of health care setting with C.
difficile spores making infections more likely
» Cleanliness of British Public Health Service hospitals has
become a major political issue there
• Shared rooms and bathroom facilities
» Particular issue in Canada and Britain
Pathogenesis of C. difficile
• Key steps in pathogenesis
» Anaerobic gut flora confers “colonization resistance” to the host
from infection with C. difficile
» Alteration of this gut flora by antimicrobial therapy creates a
permissive environment for the vegetation of C. difficile spores
» Spores are either present in gut at time of alteration of gut flora or
are obtained from the hospital environment during the period of gut
flora alteration
• May take as long as six weeks for gut flora to return to normal
• Antimicrobials most impacting gut flora include clindamycin,
cephalosporins, and perhaps newer fluoroquinolones
Pathogenesis of C. difficile
• Organism grows and begins to produce both toxin A & B
» Toxins have high degree of sequence similarity
• Toxins bind to specific receptors on surface of the cell and
enter cell via receptor mediated endocytosis
• Toxins acts as a glucosyltransferase inactivating small
GTPase
Pathogenesis of C. difficile
• GTPase control a variety of cell functions resulting in:
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actin condensation which leads to cell rounding, membrane
blebbing, apoptosis, and cell death
increased permeability of the colonic epithelium
Chemokine expression which increases inflammatory
response
Increases neutrophil infiltration
Loss of tight junctions resulting in neutrophil migration into
the intestine
C. difficile: Spectrum of disease
Asymptomatic carriage
Mild diarrhea
Profuse diarrhea with non-specific colitis
Pseudomembranous colitis
Toxic megacolon
frequency
Clostridium difficile PMC and toxic
megacolon
Is there an emerging pandemic of a
highly virulent C. difficile strain?
• Recent outbreak in Canada, the regions of the US,
Britain, and now the Netherlands have recognized
the emergence of a highly virulent strain of C. difficile
• Characteristics of this new strain include:
» Mutation in tcdC gene which down-regulates toxin
production during logarithmic phase
» Mutation results in approximately 20-fold increase in
toxin production
Is there an emerging pandemic of a
highly virulent C. difficile strain?
» Organism also produce binary toxin, a 2nd virulence
factor typically produced by a small number of isolates
» Poor response to metronidazole has also been
reported
» Higher morbidity and mortality particular among those
over >75.
Treatment of C. difficile disease
• Initial studies showed that metronidazole and vancomycin
had similar initial response to therapy (90%) and similar
disease recurrence rates (5 to 12%)
• Metronidazole became the drug of choice because it was
much cheaper and because of concerns of vancomycin
use resulting in increased rates of VRE and concerns
about the emergence of VRSA
Treatment of C. difficile disease
• Two recent studies (published 6/05) have shown much higher rates of
treatment failures/recurrences than previously reported with
metronidazole
» One study (CID 40:1586, 2005) only 50% of patients were cured,
22% had symptoms continuous for > 10 days and 28% had
recurrences
» In a Canadian survey (Pepin et al. CID 40:1591-7), recurrence
rates increased from 21% in 1991-2002 to 48% in 2003-2004; in
those over 65 y.o., that rate was close to 60% in 2003-4
Treatment of C. difficile disease
» Whether resistance to metronidazole is the reason is not
reported in these studies; most recent report puts
metronidazole resistance at 6% ( Pelaez et al. AAC 46:
1647, 2002)
• Isolates were recovered from 1993-2000 before emergence of
the NAP 1/027 strain in 2002
As Brian the scientist would say, “Any
Questions?”