Batteri nonBatteri non--fermentanti fermentanti nonnon--PseudomonasPseudomonas

Stefania StefaniStefania StefaniDipartimento di ScienzeDipartimento di Scienze

MicrobiologicheMicrobiologicheEmail stefanis@unict.itEmail stefanis@unict.it

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