Antimicrobial Susceptibility Testing
“A Historical Perspective”
Stephen G. Jenkins, Ph.D. Emeritus Professor of Pathology
and Laboratory Medicine Professor of Pathology in Medicine Weill Cornell Medical College New York, NY
At the conclusion of this presentation, participants will be able to:
1. modify antimicrobial susceptibility testing practices in the clinical microbiology laboratory that address changes that have occurred in antimicrobial resistance,
2. discuss recent trends in antimicrobial resistance among Enerobacterales (Enterobacteriaceae) that will necessitate in vitro testing of additional antibiotics,
3. prioritize implementation of new testing methods for assessment of antimicrobial resistance among bacterial pathogens, and
4. assess needs in clinical microbiology laboratories for accurate determination of antibiotic activity.
One of the most infamous quotes in the history of biomedicine: “It is time to close the book on infectious diseases and declare the war against pestilence won.”
Long attributed to the United States Surgeon General, Dr. William H. Stewart (1965-1969)
No proof that it was actually his quote, but many of his contemporary physicians believed such, and for years research and interest in the field stagnated
Sir Alexander Fleming
4
The prepared mind, luck or both?
Fleming was going through old plates with staphylococci growing on them that had been left out while he was on vacation for 4-5 weeks
The plates had been placed in detergent, but a few were not entirely covered
He observed mould growing on one of the plates and noted that colonies of staphylococci were not growing near the mold
At this point there are many biologic facts that do not equate, but the fact is that he launched an investigation into the inhibitory substance produced by the mould
6
The famous plate with Penicillium mould (preserved in the archives of St. Mary’s hospital)
Penicillin: 10 Years Later
Florey and Chain
Fleming was unable to concentrate the substance from the mould due to lack of “chemical assistance”
Howard Florey and Ernst Chain
first looked at lysozyme
renewed interest following the success of sulfonamides
decided to look at penicillin
Learned to extract, concentrate and stabilize penicillin
“miraculous” results obtained in a mouse model
Early Warning
Fleming was working with mutants of S. aureus that could be grown in the presence of increasing concentrations of penicillin
He was concerned that if patients did not take a full course of treatment, resistant strains would appear
Another concern: an oral form of penicillin was produced and was available without prescription
“the greatest possibility of evil in self-medication is the use of too small doses so that instead of clearing up infection, the microbes are educated to resist penicillin…”
31
Since the then experimental “miracle” drug penicillin was rushed to a Boston hospital to treat victims of the Coconut Grove nightclub fire in 1942, >100 new antibiotics have been developed and marketed
AND, we have required each and every one of them
BUT, they have proven to be insufficient
Estimated that the # of deaths due to AR infections quintupled between 2007 and 2015
CDC has reported that 2 million people in U.S. become infected each year by bacteria or fungi resistant to commonly prescribed antibiotics, and that 23,000 (probably an underestimate) die from such infections
Recently, treatment-resistant variants of the yeast Candida auris have reared their ugly buds in medical centers in New York City and Chicago, resulting in the death of half of all so infected patients
Studies suggest that cost to U.S. health care system for treatment of patients infected with these pathogens surpasses $3 billion per year, and the problem is only expected to exacerbate
WHO predicts that worldwide death rates from AR microbes will ↑ from the current 700,000/year to 10 million by 2050
At that point, they will have surpassed cancer, heart disease, and diabetes as the leading cause of death in humans
Especially challenging are MDR-GNB including members of the Enterobacterales (e.g., CRE), Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter baumannii
‘Post-antibiotics’ world is here”
April 2019 UN report to the Secretary General of the United Nations
Penicillin (1941)
moments to get first resistant strain
2003: 97% of isolates are resistant to penicillin
Methicillin (1959): 2 years to get first resistant strain (1961)
now 30-70% resistant
CA-MRSA
Vancomycin (1950’s)
1995 VISA in France, 1996 VISA in Japan
2002: first VRSA, Now 14
Linezolid (2000)
Resistance reported in 2001
Why Do Bacteria Become
Resistant to Antibiotics?
We are trying to kill them
They are trying to eat and reproduce
What would you do if someone was trying to
kill you while you were trying eat and/or
reproduce?
Acknowledgement: S. Brecher
21
MRSA - History
In the 1950s, years before MRSA reported, three independent developments led to an appreciation of potential future healthcare implications with S. aureus
1. Slow, but persistent rise in penicillin resistance (noted soon after its clinical trials) – by end of 1940s, 50% resistance in U.S.
2. Development of an effective typing system based on strain-specific lysis by a set of bacteriophages
3. Emergence of an especially invasive, transmissible, and penicillin-resistant clone of S. aureus
First in Australia
Rapidly appeared in different continents with a “speed and virulence reminiscent of an influenza pandemic” (Fleming)
Staphylococcus aureus
• Readily acquires resistance by one of two distinct mechanisms
– Mutation of an existing gene
– Horizontal transfer of a resistance gene from another bacterium
• Several mobile genetic elements carrying exogenous antibiotic resistance genes can mediate acquisition of resistance
• Most important of these – methicillin resistance because a single genetic element (mecA) confers resistance to all currently available β-lactam antibiotics (penicillins, cephalosporins, monobactams and carbapenems) except ceftaroline
MRSA History
• By 1957 this strain (termed 80/81 or the “hospital Staphylococcus”) responsible for nearly all epidemics in maternity units in US and 50% of all hospital outbreaks in UK
• 1/3 of hospitalized patients who were nasal carriers of this strain went on to develop septicemia as compared to only 2-5% colonized with other strains
• Concern not only because of high death rate, but because outbreaks were not just confined to patients, but often caused invasive skin infections in health care workers
MRSA History
• 80/81 strain began to decline in 1960s when methicillin (1st semisynthetic, penicillinase stable penicillin) was released
• 6 months after methicillin marketed (October 1960), 3 MRSA were reported
• All 3 had same phenotype and were from same hospital in southern England
• Because MRSA were still rare and only occurred under lab conditions very different than those at sites of infection (low temperatures and high salt concentrations), medical community lulled into a false sense of security
MRSA History
• By 1967, multidrug resistant MRSA reported from numerous nations
• Most documented MRSA infections were acquired nosocomially
• Community acquired infections were restricted to persons with frequent contact with health care facilities and IV drug users
• 1993 – Novel MRSA strains reported from Western Australia among indigenous patients not previously exposed to healthcare systems
MRSA History
• Heralded WW recognition of evolution of true CA-MRSA differing from HA-MRSA in several ways:
– More susceptible to antibiotic classes other than β-lactams
– Genotypically different than HA strains
– Harbored different methicillin-resistance cassettes
– More likely to produce a putative virulence factor (Panton-Valentine leukocidin; PVL)
mecB
Phenotypically categorized MRSA - negative for mecA and mecC (Germany)
Isolate carried mecB gene only previously described in Macrococcus caseolyticus
Part of an MDR plasmid encoding resistance to aminoglycosides, macrolides, and tetracyclines
Potential of spread because of potential for plasmid transfer of concern
Becker K et al. EID Vol. 24. February, 2018
Additional genetic determinant encoding for oxacillin resistance (most frequently in veterinarian medicine)
Not detected by chromogenic screening agars or oxacillin disk diffusion testing
Frequently missed by oxacillin MIC testing
Detected quite well by cefoxitin MIC testing and almost uniformly by cefoxitin disk testing
Kriegeskorte A, Idelevich EA, Schlattmann A, Layer F, Strommenger B, Denis O, Paterson GK, Holmes MA, Werner G, Becker K.. J Clin Microbiol. 2017 Dec 26;56(1). Print: January, 2018
mecD
Described in clinical Macrococcus caseolyticus strains from canine and bovine sources
Conferred resistance to all β-lactams
including anti-MRSA cephalosporins (e.g.,
ceftaroline)
Concern for spread to Staphylococcus aureus and Bacillus spp. because of relevant integrases
Until the availability of penicillin, the most common bacterial cause of otitis media in children was:
1. Streptococcus (Diplococcus) pneumoniae
2. Haemophilus influenzae
3. Moraxella (Neisseria; Branhamella) catarrhalis
4. Streptococcus pyogenes
Streptococcus pyogenes
(Group A ß-hemolytic Streptococci)
Personal communication : Jerome Klein, MD (pediatric ID physician;
1995 Pediatric ID Society Distinguished Physician Award Recipient)
Frequently serious infection resulting in mastoiditis, brain abscess, and occasionally death
Treatment of choice: penicillin
Alternative: macrolide
A Shocking Case
• A 28-year old previously healthy female presented to the ED of a local hospital with cellulitis of the lower leg, high fever, and significant hypotension
• Her two children and husband had been diagnosed the previous week with “strep throat”
• Blood cultures were collected
• She was admitted, treated to control her hypotension, and started empirically on IV clindamycin
Shocking Case
• The following day her cultures yielded
gram-positive cocci in chains
• Penicillin was added to her antibiotic
regimen but despite all interventional
efforts the patient expired on the second
hospital day
• Streptococcus pyogenes was recovered from
her admission blood cultures
Streptococcus pyogenes
• Susceptibility testing indicated that the
isolate was susceptible to penicillin,
clindamycin, and vancomycin but resistant
to erythromycin
• What, if any, additional antimicrobial
susceptibility testing should have been
considered?
D-testing • CLSI SAST recommends that D-testing be
performed on ß-hemolytic streptococci for which the erythromycin and clindamycin antimicrobial susceptibility testing results are discordant
• Evolving evidence that strains harboring the genome for inducible MLSB resistance may not respond optimally to clindamycin
• S. pyogenes: three types of macrolide resistance
– mef(A) (efflux)
– erm(A) subclass erm(TR): inducible ribosomal methylation
– erm(B): constitutive ribosomal methylation
Susceptibility Testing CLSI Recommendations
• Susceptibility and resistance to azithromycin,
clarithromycin, and roxithromycin can be
predicted by testing erythromycin
• Susceptibility testing of penicillins and other
β-lactams approved by the FDA for treatment
of group A streptococci is not necessary for
clinical purposes and need not be done
routinely since, as with vancomycin, resistant
strains have not been recognized
Group A Streptococci - Penicillin
• BUT in patients with pharyngo-tonsillitis, the
presence of -lactamase producing bacteria is
associated with failure to eradicate Group A -
hemolytic streptococci
Group A Streptococci - Penicillin
• Amoxicillin failure – 21% (48/228 children)1
• Prior to treatment, -lactamase-producing bacteria recovered from throats of 54% of children who failed therapy versus 16% of those who did not fail therapy (P<0.001)
• 163 -lactamase-producing bacteria recovered including Haemophilus influenzae (34), Moraxella catarrhalis (31), Staphylococcus aureus (28), Prevotella spp. (39), and Fusobacterium spp. (31)
1Brook I, Gober AE. 2007. Acta Paediatrica. 97: 193-195
Group A Streptococcal Resistance:
Host Issues • In acute pharyngo-tonsillitis, epithelial cells are the
initial sites of host invasion by Group A streptococci
• Some strains of this bacterium can internalize into epithelial cells
• Organisms that express fibronectin-binding protein F1 (promotes adherence to and entry into human cells) have proven to be responsible for antibiotic treatment failures1
• Streptococcal internalization might contribute to eradication failure and persistent throat carriage
1Passali D et al. 2007. Acta Otorhinolaryngol Ital. 27:27-32
Case Presentation
31-year old male computer programmer
returned home to India for a two-week visit
Upon returning to the U.S., he went directly
to an area hospital ED because of fever,
headache, chills, abdominal cramping, and
myalgias
Blood cultures were collected
He was diagnosed with a “viral syndrome”
and sent home on oral ciprofloxacin
Case Presentation
The following morning he returned to the ED with a temperature of 103.40F and was admitted for IV therapy
The patient denied diarrhea or dysuria
A urine specimen was submitted for culture
Blood cultures were reported positive with gram-negative bacilli
Possible diagnoses?
Case Presentation
The urine culture failed to yield a pathogen
GNR identified as Salmonella typhi
Antimicrobial susceptibility studies were performed with the following results:
– TMP-SXT: 2/38 g/mL (S)
– Ampicillin: 2 g/mL (S)
– Ciprofloxacin: 1 g/mL
– Ceftriaxone: 2 g/mL (S)
2qq \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\iu ``````````````````````````````````````````````````````` Additional Considerations
Test ciprofloxacin at lower concentrations and report isolates for which the MIC is 0.125 g/mL as resistant to the fluoroquinolones based upon revised CLSI/FDA breakpoints
Isolates with a single mutation in gyr(A) are fully resistant to nalidixic acid and have elevated MICs for the newer fluoroquinolones (0.125 – 2 g/mL)
FLUOROQUINOLONE USE AND RESISTANCE RATES
0
5
10
15
20
25
30
35
1990–1993 1994 1995 1996 1997 1998 1999 2000
Year
0
50
100
150
200
250
Pseudomonas aeruginosa
Gram-negative bacilli
Fluoroquinolone use
1. Neuhauser MM et al. JAMA. 2003;289:885–888.
The 1990–1993 data points represent composite susceptibility and
fluoroquinolone use for those 4 years.
Qu
ino
lon
e U
se
(kg
x 1
03)
Str
ain
s R
es
ista
nt
to C
ipro
flo
xa
cin
(%
)
43 states; 35,790 isolates
Mount Sinai Levofloxacin Data (% Resistant)
2001 2002 2003 2004 2005 2006 2007
E. coli 10 17 28 32 39 32 41
Klebsiella pneumoniae 20 18 31 41 54 51 48
Enterobacter cloacae 11 13 13 11 21 32 22
Proteus mirabilis 4 7 14 8 21 23 21
Acinetobacter baumannii 59 78 89 77 73 78 70
Pseudomonas aeruginosa 33 35 38 34 38 37 37
CSF Gram Stain - 1975
Haemophilus influenzae
What was the drug of choice for treating infections caused by H. influenzae in 1976?
Fanny Allen Hospital
No AST on H. influenzae
Letter: Penicillinase production by Haemophilus influenzae. Williams JD, Kattan S, Cavanagh P. Lancet. 1974 Jul 13;2(7872):103.
Beta-lactamase shown to be a TEM
Now approximately 25% are Beta-lactamase positive and approximately 4% are BLNAR
Which of the following is expected to be the most difficult clinical problem to address over the next two decades?
MRSA
ESBLs
MDR-TB
MDR-GC
30
Penicillin once drug of choice
First suggestions of possible penicillin resistance as early as 1945
Studies on the action of penicillin; development of penicillin resistance by gonococcus. MILLER CP, BOHNHOFF M. Proc Soc Exp Biol Med. 1945 Dec;60:354-6.
Both modified PBPs (not detected by beta-lactamase testing) and penicillinase production are mechanisms
Ceftriaxone resistance first confirmed in Japan in 2015; now spreading globally
Genetic relatedness of ceftriaxone-resistant and high-level azithromycin resistant Neisseria gonorrhoeae cases, United Kingdom and Australia, February to April 2018. Jennison AV et al. Euro Surveill. 2019 Feb;24(8).
Few labs culture.
As a result resistance (no AST testing being performed) “snuck up on us”
Now resistance being seen to the tetracyclines, macrolides, ceftriaxone, and spectinomycin
New antibiotic in final phase 3 clinical trials for gonorrhea and UTIs
Gepotidacin: first of a new class of drugs
Works by selectively interacting with two bacterial enzymes that aid in bacterial replication, making its mechanism of action different from other approved antibiotics
CLSI Rationale for Development of
AR Detection, Characterization &
Reporting Guidelines
• Clinical microbiology laboratories are charged with the testing of infectious agents for susceptibility to currently available antimicrobial agents
• Such testing, depending on the organism and the AST method employed, can take a protracted period of time to complete
The ticking time bomb for
sepsis 7.6% reduced survival
for each hour after the
onset of hypotension
before initiation of
effective
antimicrobials
inappropriate
antimicrobials within
first 6 hours of septic
shock = 5-fold higher
mortality (52 vs.
10.3% survival)
Kumar et al 2006 Crit Care Med 34:1589
Kumar et al 2009 Chest 136:1237
Rationale
• CLSI standards have been developed to guide laboratorians as to how to best conduct AST and, to expedite communication of actionable results to clinicians, rapidly detect and/or characterize a significant number of (AR) mechanisms
• CLSI standards additionally provide guidance as to how to best interpret and report results for many such AR detection assays
These recommendations are ever-evolving to address changing landscape of AR mechanisms being encountered
Detection Methods for AR
• Approaches for detection of AR mechanisms include both phenotypic and molecular methods
• As gene-based testing may not be universally available, phenotypic assays can provide useful information to prescribers in a relatively rapid manner for guidance of antimicrobial choices
• Additionally, phenotypic methods may, in some cases, better detect gene expression than molecular-based assays
Select AR Detection Methods
• AR among GNRs is especially challenging from a therapeutic perspective
• For example, resistance to a class of agents such as the carbapenems may be due to a host of variables including, but not restricted to, carbapenemases with differing spectra of activity and susceptibilities to inhibitors
• To address this issue, several assays have now been included in CLSI document M100 that allow microbiologists not only to detect such enzymes, but to characterize them as to type
GN AR Detection Methods
• Institutional infection control procedures or epidemiologic investigations may necessitate ID of carbapenemase-producing Enterobacterales and P. aeruginosa
• For such purposes and in some cases for provision of therapeutic direction, labs should consider performing: • the mCIM assay with or without eCIM; Blood mCIM
• the CarbaNP test,
• a molecular assay, and/or
• a phenotypic gene expression test for relevant enzymes
(e.g., CARBA-5)
Carbapenemase-producing isolates of Enterobacterales usually
Test intermediate or resistant 1 or more carbapenems using current CLSI breakpoints (NOTE: Ertapenem non-susceptibility is most sensitive (but least specific) indicator of carbapenemase production), and
Usually test resistant to 1 or more agents in cephalosporin subclass III (e.g., cefotaxime, ceftazidime, and ceftriaxone)
BUT…some isolates that produce carbapenemases such as IMI or SME often test susceptible to these cephalosporins
Tests Used for Epidemiologic or Infection Control
Related Testing CarbaNP
(Table 3B)
mCIM
(Table 3C)
mCIM with eCIM
(Table 3C) Other (e.g., molecular
assays)
Organisms Enterobacteriales and P.
aeruginosa, that are not
susceptible to one or
more carbapenems
Enterobacteriales
and P. aeruginosa
that are not
susceptible to one
or more
carbapenems
Enterobacteriales that are
positive by mCIM
Enterobacteriales and P.
aeruginosa that are not
susceptible to one or more
carbapenems to determine the
presence of a carbapenemase, or
to determine carbapenemase
type in isolates positive by
Carba NP or mCIM.
Strengths Rapid
No special
reagents or media
necessary
No special reagents or media
necessary
Determines type of
carbapenemase in addition to
absence or presence of the
enzyme
Limitations
Special reagents are
needed, some of which
necessitate in-house
preparation (and have a
short shelf life).
Invalid results occur with
some isolates.
Certain carbapenemase
types (e.g., OXA-type,
chromosomally encoded)
are not consistently
detected.
Requires
overnight
incubation
Requires overnight incubation Special reagents and equipment
are needed.
Specific to targeted genes; false-
negative result if specific
carbapenemase gene present is
not targeted.
mCIM separates carbapenem resistant organisms into those that are carbapenemase producers versus those that are non-carbapenemase producers, and
eCIM assay differentiates class A (serine) carbapenemase-producing Enterobacterales from those producing class B (metallo-) carbapenemases
This may be important in antibiotic decision-making in that class A enzymes are largely inhibited by avibactam, relebactam, and vaborbactam whereas class B enzymes are not
Class D (OXA-type) enzymes may not be effectively characterized by such methods
GN AR Detection Methods
The Modified Hodge Test, included in several earlier versions of M100, no longer recommended in latest iterations of the document due to poorer sensitivity and specificity than currently recommended approaches
“Dodge the Hodge”
Antimicrobial resistance is here to stay and will continue to evolve
Clinical microbiologists must stay on top of such changes and trends such that appropriate alternative antimicrobial agents can be tested and reported
New and more rapid tests need to be developed to accurately detect and characterize AR mechanisms
“Bacterial IQ”
Questions??
