Batteri nonBatteri non--fermentanti fermentanti nonnon--PseudomonasPseudomonas
Stefania StefaniStefania StefaniDipartimento di ScienzeDipartimento di Scienze
MicrobiologicheMicrobiologicheEmail [email protected] [email protected]
Ottobre 2010Ottobre 2010
Characteristics of nonCharacteristics of non--fermentersfermenters
Oxidative gram-negative bacilli, including Pseudomonasspp., produce acids from glucose or other carbohydrates only in the presence of oxygen(nonfermenters).
Clinically Important nonfermentative Clinically Important nonfermentative GramGram--Negative BacilliNegative Bacilli
ACINETOBACTER EVOLUTIONACINETOBACTER EVOLUTION
Family: NeisseriaceaeNeisseriaceae to MoraxellaceaeMoraxellaceae
Gram-negative, coccobacillary rods
Genus: Acinetobacter31 genomics species
Only 17 valid species names
A.calcoaceticus-A.baumannii complex
Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter genomic species 3 e 13TU
ACINETOBACTER BAUMANNII
Gram-negative coccobacillus catalase(+),oxidase(-)
Ubiquitous, non motile Present in soil, fresh water and
fomites
ACINETOBACTER BAUMANNII
Despite the absence of data on the genetic competence of A.baumannii, other Acinetobacter spp, in particular A.baylyi, are highly
competent and recombinogenic 1, 2.
A.baumannii strain AYE: possesses an 86-kb resistance island (AbaR1); 82
out of the 88 ORF were predicted to have originated from other gram-
negative organisms, such as Pseudomonas, Salmonella, E.coli
Overall, 52 resistance genes were identified and 45 were localized into the
AbaR1 region3;
Other strains have a wide array of resistance markers but not in the same
hotspot already identified: enormous genetic flexibility!
1) Bacher JM. Et al J.Bacteriol 2006; 188:8534; 2) Vaneechoutte MDM et al AEM 2006; 72:932; 3) Fournier PE et al PLoS genetic 2006; 2: e7
ACINETOBACTER EPIDEMIOLOGYAchieving Fame and Lacking virulence
ColonizesHands,
Environment
Antibiotics Resistance Genes
Devices,Biofilm e VAP
Lower virulence
PathogenesisPathogenesis
Opportunistic pathogen
Survive under dry & iron-deficient conditions
1/3 polysaccharide capsule, prevents complement activation, phagocytosis
delayed
Fimbriae: adhesion to human bronchial epithelium
Pili: colonization of environmental surface to form biofilms
Hospital-Acquired Pneumonia
Community-Acquired Pneumonia
Bloodstream Infection Traumatic Battlefield
and Other Wounds UTI
Meningitis Other Manifestations
Acinetobacter baumanniiAcinetobacter baumannii
1. A. baumannii is often a source of nosocomial infections
2. Unfortunately it has the potential to often be multimulti--drug resistantdrug resistant
3. In recent years it has gotten more attention because of numerouscases being reported in soldiers from IraqIraq and AfghanistanAfghanistan
4. Interestingly, during the Vietnam war this was the most common gram negative bacillus to contaminate wounds
National Nosocomial Infection Surveillance (NNIS) System in the United States
reviewed that
Pneumonia isolates 6.9 %
Bloodstream infection isolates 2.4%
Surgical site infection isolates 2.1%
Urinary tract infection isolates 1.6%
PneumoniaPreviously colonized & Prolonged intubationMortality rate from 35 ~ 70%Bloodstream infectionRespiratory tract, intravenous catheters1/3 develop septic shockMortality rate range from 20 ~ 60%
EndocarditisRare, Acute onset & aggressive Higher mortality in native valves6 weeks antimicrobial agentMeningitisFever, meningeal signs, seizureMortality 20~30%,> 3 weeks antimicrobial agent
Urinary tract infectionPyuria + positive culture + symptoms10~14 days antimicrobial agent + catheter removal
MULTIDRUG-RESISTANTACINETOBACTER BAUMANNII
Multidrug resistance is resistance to more than two of theMultidrug resistance is resistance to more than two of thefollowing five drug classesfollowing five drug classes: cephalosporins
carbapenems, ampicillin, ampicillin--sulbactam, sulbactam, fluoroquinolonesand and aminoglycosides It needs to be acknowledgedIt needs to be acknowledged
that susceptibility testing of that susceptibility testing of --lactamlactamactamase inhibitoractamase inhibitorcombinations is highly problematic and that laboratories maycombinations is highly problematic and that laboratories may
not test piperacillinnot test piperacillin--tazobactam or ticarcillintazobactam or ticarcillin--clavulanateclavulanate
Pandrug resistance is often defined as resistance to all antimicPandrug resistance is often defined as resistance to all antimicrobials that undergo firstrobials that undergo first--line line susceptibility testing that have therapeuticsusceptibility testing that have therapeutic
This would include all This would include all --lactamslactamsincluding carbapenems and sulbactam (MICs of 4 g/including carbapenems and sulbactam (MICs of 4 g/ml]), fluoroquinolones, and aminoglycosides.ml]), fluoroquinolones, and aminoglycosides.
With With the increased use of the the increased use of the polymyxinspolymyxins and possibly and possibly tigecycline,tigecycline,this definition will likely have to encompass these other agentsthis definition will likely have to encompass these other agents..
Inherent: chromosomally encoded
AmpC cephalosporinases
Enzymatic: ESBLs and OXA
I ceppi di A.baumannii producono naturalmente una beta-lattamasi cromosomiale Amp-C che conferisce resistenza a ureidopenicilline, cefalosporine a spettro ristretto e cefamicine.
Al contrario di quanto avviene negli altri Gram-negativi, non inducibile ( se non per ISAbaI)
Limpermeabilit porinica spesso invocata nelle resistenze verso diverse classi di antibiotici inclusi i carbapenemici
Pompe ad efflusso: (gene adeABC: RND proteins) coinvolgimento nella resistenza agli aminoglucosidi, ma in grado di utilizzare altri substrati come fluoroquinoloni, tetracicline, cloramfenicolo, trimethoprim, eritromicina. Gene abeM: multi-drug efflux pump (FQs e AGs)
- Lactamas
TEM
SHV
VEB
- Lactamases PER
CTX-M
OXA
IMP
VIM
SIM
OMPS CarO (29 kDa)
OmpW
Efflux AdeABC
Assessment of ESBLs is difficult, especially in the presence of an AmpCAssessment of ESBLs is difficult, especially in the presence of an AmpC
OXA-24 Cluster
Distribution:Spain,Belgium,France, Portugal
EncodedPlasmid ( OXA-40) or chromosomal
Associated IS Elements:
None
OXA-58 Cluster
Distribution:Spain,Belgium,France, Portugal
EncodedPlasmid or chromosomal
Associated IS Elements:
ISAba1,ISAba2,ISAba3,IS18
48%48%
47%47%
56%56%
OXAOXA--23 Cluster23 ClusterDistribution:Distribution:
Europe,Australia,China.Korea,Sngapore Europe,Australia,China.Korea,Sngapore Vietnam LibyaVietnam Libya
EncodedEncodedPlasmid or chromosomalPlasmid or chromosomal
Associated IS Associated IS ElementsElements::ISAba1,ISAbaISAba1,ISAba
OXA-51 ClusterDistribution:
Naturally occuring in A.baumanni therefore global distribution.
Encoded:Cromosomal
Associated IS Elements:ISAba1
59%59%63%63%
CarbapenemCarbapenem--hydrolyzing hydrolyzing OXAs are most common in OXAs are most common in
MDR MDR AA. . baumanniibaumannii
Peleg, Seifert,PatersonCMI 2008
60 %60 %
Enzymes Phenotypic tests Limits
OXA type Not described Only PCR for specific genes
MBL Disk approsimation test: -
Imipenem or meropenem
+EDTA1
-2 mercaptoproprionic acid 2
- Etest MBL strips3
False positive results for
strains producing OXA-23
but lacking genes encoding
IMP and VIM4
Strips cannot be used in
strains with MIC < 4 mg/L5
1) Franklin C et al JCM 2006; 44: 3139; 2) Arakawa Y et al JCM 2000; 38:40; 3) Lee K et al JCM 2005; 43: 942; 4) Segal H. et al JAC 2005; 56: 598; 5) Yan JJ et al DMID 2004; 49: 5
CLINICAL LABORATORY DETECTION OF CARBAPENEMS
Phenotypic tests for evaluating the presenceof serine carbapenemases (OXA type) have
not yet been described.
The most frequently used methods for detecting MBLs have been disk approximation methods comprising
imipenem and imipenem plus EDTA
Apparently, false-positive results were seen for isolates producing OXA-23 but lacking genes encoding IMP and VIM.Segal H., JAC 2005
5-mm increase of inhibition zone MBL
EDTA
Imipenem + EDTA
Imipenem
A reduction in the MIC of imipenem of 3 dilution in the presence of EDTA is interpreted as positive
PCR and sequencing
Other resistances
Aminoglycosides
AminoglycosideAminoglycoside--modifying enzymesmodifying enzymes
Ribosomal 16S rRNA methylation Ribosomal 16S rRNA methylation
EffluxEfflux
Quinolones
Modification to target binding siteModification to target binding site
EffluxEfflux
Tetracyclines and glycylcyclines
TetracyclineTetracycline--specific effluxspecific efflux
Ribosomal protection Ribosomal protection
Multidrug effluxMultidrug efflux
Comparison between EUCAST and CLSI
breakpointsAntibiotics EUCAST April 2010
(susceptibility/ resistance
breakpoints)
CLSI 2010
(susceptibility/resistance
breakpoints
Imipenem, Meropenem
Doripenem
2/8
1/4
4/16
-
Ciprofloxacin 1/1 1/4
Levofloxacin 1/ 2 2/8
Amikacin 8/16 16/64
Gentamicin/tobramycin 4/4 4/16
Netilmicin 4/4 8/32
Amp/sulbactam IE 8/32
Piperacillin/tazobactam IE 16/128
Cefazidime, cefepime - 8/32
Polymixin B/Colistin 2/2 2/4
Trimethprim/sulfamethoxazole 2/4 2/4
Doxycycline, minocycline,
tetracycline
IE 4/16
Tigecycline IE -
CRA EPIDEMIOLOGY
Countries that have reported an outbreak of carbapenem-resistant Acinetobacter baumannii. Red signifies outbreaks reported before 2008, and yellow signifies outbreaks reported since 2008.
Van Looveren, H. Goossens, CMI 2004
THERAPEUTIC STRATEGIES FORTHERAPEUTIC STRATEGIES FORACINETOBACTER BAUMANNII ACINETOBACTER BAUMANNII INFECTIONINFECTION
SulbactamSulbactam is a -lactamase inhibitors.It has clinically relevant intrinsic antimicrobial activity mediated by its binding to penicillin-binding protein 2. Its commercially available in a combined formulation with either ampicillin or cefoperazone and also as a single agent in France, Germany, and Spain.
SulbactamSulbactam
CarbapenemsCarbapenems
PolimixinsPolimixins
TigecyclineTigecycline
Rates of resistance to the polymyxins have recently been reported to
be as high as 3.2% for multidrug-resistant A. baumannii strains, with higher rates reported in Korea.
Tigecycline, a 9-t-butylglycylamido semisynthetic derivative of minocycline, that inhibits the 30S ribosomal subunit, but its unique feature is its ability to evade the major determinants of tetracycline resistance, i.e., the tet(A) to tet(E) and tet(K) efflux pumps and the tet(M) and tet(O) determinants that provide ribosomal protection
Semiautomated methods, such as those for the Vitek 2, Microscan,Semiautomated methods, such as those for the Vitek 2, Microscan,and BD Phoenix systems, are commonly used for antimicrobial and BD Phoenix systems, are commonly used for antimicrobial susceptibility testing by clinical microbiology laboratories.susceptibility testing by clinical microbiology laboratories.
Unfortunately, there is limited information about the performancUnfortunately, there is limited information about the performance e of these methods against of these methods against A. baumanniiA. baumannii. Studies from the 1990s . Studies from the 1990s with an early Vitek system showed that numerous isolates were with an early Vitek system showed that numerous isolates were reported as resistant to imipenem by Vitek but typically were reported as resistant to imipenem by Vitek but typically were susceptible to imipenem when tested by broth and agar dilutionsusceptible to imipenem when tested by broth and agar dilution
TIGECYCLINE
27 Rimini - AMCLI 2009
Discrepancies in in vitro evaluation of
tigecycline In a recent surveillance study where the susceptibility of
tigecycline against target pathogens was determined locally by Etest, the activity profile observed for tigecycline varied fromthat observed in a recent centralized surveillance study wich utilized broth microdilution (BMD)1.
In brief, the MIC90 for Acinetobacter spp, and Serratia marcescens, was fourfold higher by distributed Etest relative to that observed in the centralized BMD testing. To less extent, a similar problem was observed with S.pyogenes
1. Draghi D et al Abstr 46ICAAC D701 p.155
28 Rimini - AMCLI 2009
A.baumannii
S.pyogenes
S.pneumoniae
S.marcescens
The FDA, CLSI, and EUCAST have established no breakpoints for interpretation of antibiotic susceptibility testing of tigecycline versus A. baumannii. This has resulted in immense confusion as to appropriate methods for performing and interpreting antibiotic susceptibility testing for this drug-organism combination.
Suggestions have been made to utilize an inhibition zone diameter of 16 mm or 13 mm as an indicator of A. baumannii susceptibility to tigecycline. The British Society for Antimicrobial Chemotherapy (BSAC) has established tentative tigecycline breakpoints for Acinetobacter spp., as follows: MICs of 1 mg/l, susceptible; MIC of 2 mg/l, intermediate; and MICs of 2 mg/l, resistant.
EUCASTEUCAST notes that there is insufficient evidence that the species in question is a good target for therapy with the drug and CLSI breakpoints are pending,
ANTIBIOTIC SUSCEPTIBILITY ANTIBIOTIC SUSCEPTIBILITY TESTING FOR THE CLINICAL TESTING FOR THE CLINICAL MICROBIOLOGY LABORATORYMICROBIOLOGY LABORATORY
New antibiotics for MDR New antibiotics for MDR A.baumanniA.baumanni??
Doripenem : Doripenem, a1-methlycarbapenem, is a broad spectrum parental carbapenem recently approved in the United States and European Union for the treatment of complicated urinary tract and complicated intra-abdominal infections.
- More effective than other carbapenems- But susceptible to metallo--lactamase.
Doripenem in our experience
poster presented at the Congress of Acinetobacter Rome 2010
Strains and Antibiotics Range (mg/L) MIC90 (mg/L)
All strains (n.82)
Doripenem 0.25-64 16
Imipenem 4->256 128
Meropenem 8->256 256
OXA-58 positive (n.64)
Doripenem 0.25-64 4
Imipenem 4->256 128
Meropenem 8->256 256
OXA-58+23 positive (n.18)
Doripenem 4-64 16
Imipenem 16->256 128
Meropenem 32->256 256
Stenotrophomonas maltophiliaStenotrophomonas maltophilia
GENERAL OVERVIEW
Formerly Pseudomonas maltophilia and then Xanthomonas maltophilia
Nosocomial infections
Normal flora can infect wounds, urinary tract, &
blood
CLINICAL SYNDROMES
Opportunistic Nosocomial Infections
Bacteremia
Pneumonia
Meningitis
Wound Infections
EPIDEMIOLOGY
Hospital Epidemics from Contaminated Moist Reservoirs:
Disinfectant solutions
Respiratory equipment
Ice machines
Flower vases
Risk Factors
Hospitalization
Impaired host defense mechanisms (e.g., highly
immunocompromised)
Long-term broad-spectrum antibiotics (e.g., bone
marrow transplant patients)
TREATMENT, PREVENTION, AND CONTROL
Resistance to Multiple Antibiotics (e.g., Beta-
lactams; Aminoglycosides)
Susceptible to:
Trimethoprim-sulfamethoxazole
Chloramphenicol; Tigecycline
Ceftazidime
(CLSI BP
Antimicrobial resistance High-level intrinsic resistance to beta-lactams, quinolones,
aminoglycosides, tetracycline, disinfectant and heavy
methals.
Sequencing demonstrated: genes for multi-drug efflux pumps,
beta-lactamses, amiglycoside modifying enzymes..together
with low impermeability.
Resistence to cotrimoxazole has been linked to class I
integrons carrying sul1 and insertion sequence common
region (ISCR) eleemnts carrying sul2.
Resistance to FQ: over expression of efflux pumps
Difficult to test because results are affected by temperature of incubation and media content
All aminoglycosides, polymixins and carbapenems should be reported resistant
without testing
For beta-lactams and quinolones susceptibility testing is unreliable, but combinations of ciprofloxacin and a beta-
lactam or aztreonam and co-amoxyclav have had a favourable clinical response.
CLSI and EUCAST
Antibiotics CLSI EUCAST not species
related
Tycarcillin/clavulanate 16/2 128/2 8/16
Ceftazidime 8/32 4/8
Minocycline 4/16 IE
Levofloxacin 2/8
Trimethoprim/sulfa 2/38 4/76
Chloramphenicol 8/32
Tigecycline 0.25/0.5
Prevalence of resistance
Antibiotic Resistance in % Country or clinical setting
Cotrimoxazole 1997-203 4.7 worldwide
5-8-10 Asia-Pacific regions Europe
25 Taiwan
27 Spain
76-84 CF patients
7-25 Cancer patients
15 ICU
Ticacillin/clavulanate 1997-
2003
40 Worldwide
Cirpofloxacin 1997-2003 40 Worldwide
Looney WJ et al Lancet 2009; 9: 312
Single agent or combinations?
Cotrimoxazole alone or in combination is
considered the treatment of choice; in
patients in whom cotrimoxazole cannot be
used, ticarcillin/clavulanate has been
recommended as second terapeutic option.
New FQ, tigecycline.
Combinations are now current practice,
despite the lack of clinical trials!
Conclusion: what they have in
common Acinetobacter and Stenotrophomonas emerged as
important opportunistic, often MDR, pathogens;
Management of infection hampered by high-level
intrinsic resistance to many antibiotic classes;
recently, acquired resistance;
New therapeutics are necessary; Prevention of
acquisition and infections relies on the cornerstone
of modern infection control with higher emphasis
on control of antimicrobial use and consideration of
environmental reservoirs.
OXA CarbapenemasesOXA Carbapenemases
Hydrolysis spectrum: penicillins and early cephalosporins
No aztreonam hydrolysis
Variable hydrolysis of extended spectrum cephalosporins
Confer only reduced susceptibility to the carbapenems
MetalloMetallo----Lactamases (MBL)Lactamases (MBL)
Do not hydrolyze aztreonamThe most common MBL families are: (IMP 1988)
(VIM 1996) (SIM 2004 Korea)
Requires Zn2+ for activity
Inhibited by EDTA but not by CA Chromosomally or plasmid mediated
Broad substrate spectrum including penicillins, cephalosporins, and
carbapenemases
Most common in Europe
Italy, Greece, France, Germany, Spain
Also spread in other countries
Korea, Brazil, Argentina
Spread to USA
Necessary revival of the polypeptide antibiotics known as the polymyxins commercially available (colistin or polymyxin E and polymyxin B) discovered over 50 years ago.
Both forms are available for nebulization.
Colistin methanesulfonate (CMS; also known as colistimethate) is used inintravenous formulations of colistin, the human formulation should not be used for susceptibility testing because CMS is an inactive prodrug of colistin. Thus, dilution-based testing should always be done with colistin sulfate.
Agreement between agar dilution and Vitek 2 testing for colistinsusceptibility.
Disk diffusion testing is not recommended because the polymyxins are large polypeptides and diffuse poorly in agar, resulting in small zones of inhibition.
Use of higher concentrations of the polymyxin in the disks does not appear to improve the accuracy of test results.
Polymyxins
ANTIBIOTICSANTIBIOTICS
EUCAST 2009 CLSI 2009
MIC breakpoints MIC breakpoints ( mg/L)( mg/L)
MIC breakpoints MIC breakpoints ( mg/L)( mg/L)
ImipenemImipenemS
2
R
8
S
4
R
16
MeropenemMeropenem 2 8 4 16
Ciprofloxacin Ciprofloxacin 1 1 1 4
Levofloxacin Levofloxacin 8 16 2 8
Amikacin Amikacin 4 4 16 64
Gentamicin,Gentamicin, 4 4 4 16
AmpicillinAmpicillin--sulbactam sulbactam i.e i.e 8 32
PipPip--TazTaz i.e i.e 16 128
TicarcillinTicarcillin--clavulanateclavulanate i.e i.e 16 128
CeftazidimeCeftazidime i.e i.e 8 32
CeftriaxoneCeftriaxone i.e i.e 8 64
Polymyxin B, colistin Polymyxin B, colistin i.e i.e 2 4
TigecyclineTigecycline i.e i.e i.e i.e