1. Amela Dedeić-Ljubović
Clinical Center University of Sarajevo
Institute of Clinical Microbiology
Bosnia and Herzegovina
Carbapenem resistant among Enterobacteriacea
Amela Dedeić-Ljubović

2. Carbapenem resistance
Great concern to health services worldwide
Significantly limits treatment options for life-threatening infections/high mortality rate
No new drugs for gram-negative bacilli
Emerging resistance mechanisms, carbapenemases are mobile.
Detection and implementation of infection control practices are necessary to limit spread
discovery void

3. Carbapenem Resistance: Mechanisms
Enterobacteriaceae
Cephalosporinase + porin loss
Carbapenemase
P. aeruginosa
Porin loss
Up-regulated efflux
Acinetobacter spp.

4. Carbapenemases Classification Enzyme Most Common Bacteria Class A
KPC, SME, IMI, NMC, GES
Enterobacteriaceae
(rare reports in P. aeruginosa)
Class B
(metallo-b-lactamse)
IMP, VIM, GIM, SPM
P. aeruginosa
Enterobacteriacea
Acinetobacter spp.
Class D
OXA

5. Class A Carbapenemases
Chromosome encoded (NmcA, Sme, IMI-1, SFC-1) .
Plasmid encoded (KPC, IMI-2, GES, derivatives).
All effectively hydrolyze carbapenems and are partially inhibited by
clavulanic acid.
Klebsiella Pneumoniae Carbapenemase (KPC ) are the most frequent
in this group.
First reported in 1996 in North Carolina, USA
Reported in other Enterobacteriaceae : K. oxytoca, Citrobacter freundii,
Enterobacter spp., Escherichia coli, Salmonella spp., Serratia spp.
Also reported in Pseudomonas aeruginosa

6. Klebsiella Pneumoniae Carbapenemase
blaKPC genes are flanked by a same transposon Tn4401 located on conjugative plasmids and are horizontally transferred .
Thirteen variants of KPC are known so far; KPC2 and KPC3 are the most frequent worldwide.
KPC-2 is clearly the most prevalent variant in Europe .
The mortality rate due to infection with a KPC producer ranged from 25% to 69%.

7. Susceptibility Profile of KPC-Producing K. pneumoniae
Antimicrobial
Interpretation
Amikacin I
Chloramphenicol R
Amox/clav
Ciprofloxacin
Ampicillin
Ertapenem
Aztreonam
Gentamicin
Cefazolin
Imipenem
Cefpodoxime
Meropenem
Cefotaxime
Pipercillin/Tazo
Cetotetan
Tobramycin
Cefoxitin
Trimeth/Sulfa
Ceftazidime
Polymyxin B
MIC >4mg/ml
Ceftriaxone
Colistin
Cefepime
Tigecycline S

8. KPC’s in Enterobacteriaceae
Species
Comments
Klebsiella spp.
K. pneumoniae-cause of outbreaks
K. oxytoca-sporadic occurrence
Enterobacter spp.
Sporadic occurrence
Escherichia coli
Salmonella spp.
Citrobacter freundii
Serratia spp.

9. Worldwide geographic distribution of Klebsiella pneumoniae carbapenemase (KPC) producers.

10. Class B carbapenemases (MBLs)
Italy was the first Mediterranean country to report MBLs:- VIM, IMP types, New Delhi
metallo-β-lactamases-1 (NDM-1) type.
MBLs can hydrolyze all β-lactams except monobactam (e.g. aztreonam).
Their activity is inhibited by EDTA but not by clavulanic acid.
Endemicity of VIM- and IMP-producing Klebsiella pneumoniae strains has
now been noted in Greece.
Most of the outbreaks indicated a link with the Indian subcontinent, Balkan countries
and the Middle East .
These enzymes are encoded on highly transmissible plasmids.
NDM are broadly resistant to many other drug classes
NDM-1 producers have been reported in the environment and in the
community: E. coli ST131, a well-known source of community infections
The death rates are high (18% to 67%).

12. Class D carbapenemases
Also named OXAs for oxacillinases include 232 enzymes.
OXA-48 represents the main enzyme isolated around the world.
Its high level of resistance to temocillin is interesting to detect this enzyme
OXA-48 was initially identified in K. pneumoniae isolate from Turkey in
2001.
OXA-48 producing strains have been extensively reported as sources of
nosocomial outbreaks in many part of the world notably in Mediterranean
countries.
Found in different Enterobacteriaceae, such as Citrobacter freundii, Providencia
rettgeri, and Enterobacter cloacae and even in E. coli
The death rates associated with MBL producers are unknown.

13. Geographic distribution of oxacillinase-48 (OXA-48) type

14. Geographic distribution of NewDelhi metallo-β-lactamase-1 producers, July 15, Red stars indicate infections traced back to India,Pakistan, or Bangladesh; green stars indicate infections traced back to the Balkan states or the Middle East; and black stars indicate unknown origin.

15. WHO Global report 2014- K. pneumoniae resistance to carbapenems
Countries
Resistance %
No. tested isolates/data source
Period for data colection
Austria
0.2
National data
2011
Belgium
0.3
National data
Bulgaria
National data
Croatia
National data
2012
Cyprus
15.7
National data
Czech Republic
0.1
National data
Denmark
National data
Estonia
National data
Finland
National data
France
National data
Georgia 2
Publication
2003/04
Germany
National data
Greece
68,2
National data
Ireland
National data
Hungary
1.9
National data
Israel 7
Publication
2007/08
Italy
26,7
National data

16. WHO Global report 2014- K. pneumoniae resistance to carbapenems
Countries
Resistance %
No. tested isolates/data source
Period for data colection
Malta
3.8
National data
2011
Netherlands
0.3
National data
Norway
National data
Poland
0.5
National data
Portugal
National data
Romania
National data
Russia
3.1, 5.2, 18.5
National data
2011/12
Serbia
11,2
National data
2012
Slovakia
0.7
National data
Slovenia
National data
Spain
National data
Sweden
National data
Switzerland 1
National data
FYR Macedonia
National data
Turkey
Publication
2013
United Kingdom
0.4
National data

17. Occurrence of CPE according to ECDS
39 national experts (NEs) from Europe rated the occurrence of CPE in 2013:
37 were fully aware of the current epidemiology of CPE
(Iceland, Montenegro and the former Yugoslav Republic of Macedonia) reported no case of CPE.
21 NEs reported sporadic cases, single or sporadic hospital outbreaks.
11 countries, regional or national spread was reported.
Greece, Italy and Malta reported that CPE are regularly isolated from patients in most hospitals, corresponding to an endemic situation.
33 NEs indicated that Klebsiella pneumoniae was the most frequent Enterobacteriaceae species harbouring carbapenemases and IMP, KPC, NDM, OXA-48 and VIM are the five most common.
Bosnia and Herzegovina, Estonia, Montenegro, Serbia and the former Yugoslav Republic of Macedonia these data were not available

18. Occurrence of carbapenemase-producing Enterobacteriaceae in 38 European countries based on self-assessment by the national experts (European Centre for Disease Prevention and Control. Carbapenemase-producing bacteria inEurope: interim results from the European Survey on carbapenemase-producing Enterobacteriaceae (EuSCAPE) project. Stockholm: ECDC; 2013.)

19. Strategies for CPE detection
Accurate detection of colonized patients at an early stage of hospitalization or on admission/discharge either to the hospital or to a specific unit.
The accurate and rapid laboratory identification of carbapenem-resistant isolates.
Screening should include as a minimum ‘at-risk’ patients.
Stools and rectal swabs are the most suitable specimens for performing such screening.
Screened patients should be kept in strict isolation before obtaining results of the screening (at least 24–48 hours).

20. Laboratory Detection of KPC-Producers
Problems:
1) Some isolates demonstrate low-level carbapenem resistance
2) Some automated systems fail to detect low-level resistance

21. MIC range of carbapenems CPE
MIC, mg/L
Carbapenemase Imipenem Meropenem Ertapenem
KPC –> –> –>64
Metalloβ-lactamases –> –> –>64
OXA-48 type –> –> –>64
Table 2. Breakpoint values (MIC, mg/L) for carbapenems according to guidelines in Europe (EUCAST) and the United States (CLSI)
EUCAST CLSI
CARBAPENEMS S R S R
Ertapenem < > < >1
Imipenem < > < >4
Meropenem < > < >4

22. European Committee on Antimicrobial Susceptibility Testing
European Committee on Antimicrobial Susceptibility Testing Breakpoint tables for interpretation of MICs and zone diameters Version 4.0, valid from
Carbapenems
S ≤
MIC breakpoint (mg/L)
R > S≤
DISC (10μg)
R<
DISC
(10μg)
Ertapenem
0.5 1 25 22
Imipenem 2 8 16
Meropenem
The carbapenem breakpoints for Enterobacteriaceae will detect all clinically important resistance mechanisms (including the majority of carbapenemases). Some isolates that produce carbapenemase are categorised as susceptible with these breakpoints and should be reported as tested, i.e. the presence or absence of a carbapenemase does not in itself influence the categorisation of susceptibility. In many areas, carbapenemase detection and characterisation is recommended or mandatory for infection control purposes.

23. Proposition for testing carbapenemases
Detection of carbapenemase activity should be performed on enterobacterial isolates exhibiting MIC values of ertapenem ≥0.5 mg/L or of imipenem or meropenem ≥1 mg/L.
Any enterobacterial isolate that exhibits even a slight decrease in susceptibility to carbapenems compared with a wild-type phenotype.
This detection will be useful for treating patients and for preventing nosocomial outbreaks of carbapenemase producers (and therefore multidrug-resistant isolates), whatever the carbapenem resistance level is.

24. Susceptibility of KPC-Producers to Imipenem
*12% of isolates test susceptible to imipenem

25. Susceptibility of KPC-Producers to Meropenem
*9% of isolates test susceptible to meropenem

26. Susceptibility of KPC-Producers to Ertapenem
None of the isolates test susceptible to ertapenem

27. Susceptibility testing- reading disk diffusion & Etest

28. Non-molecular tests for carbapenemase production
Modified Hodge Test
Lawn of E. coli ATCC 25922
1:10 dilution of a
0.5 McFarland suspension
Test isolates
Imipenem disk
Described by Lee et al. CMI, 7,

29. Modified Hodge Test
Preliminary results suggest that any of the three carbapenem disks work in the Modified Hodge Test

30. Etest MBL
Imipenem alone (IP) and imipenem plus EDTA (IPI). A reduction in the MIC of imipenem of ≥3 dilutions in the presence of EDTA is interpreted as a positive test.

31. Discs combination test
AmpC - Cloxacillin inhibits AmpC. A difference of >/= 5mm between meropenem (MRP) and meropenem + cloxacillin (MRPCX) indicates AmpC. MBL - Dipicolinic acid inhibits MBL. A difference of >/= 5mm between meropenem (MRP) and meropenem + dipicolonic acid (MRPDP) indicates MBL. KPC - Boronic acid inhibits KPC and AmpC. A difference of >/= 4 mm between meropenem (MRP) and meropenem + Boronic acid (MRPBO) but no zone difference with MRPCX indicates KPC. OXA 48 - For enterobactericeae no zone to Temocillin suggests OXA48

32. UV spectrophotometer MALDI-TOF
It is based on several steps, including:
(i) an 18 h culture (which can be shortened in some cases to 8 h);
(ii) a protein extraction step;
(iii) measurement of imipenem hydrolysis using a UV spectrophotometer.
this spectrophotometry-based technique has 100% sensitivity and 98.5% specificity for detecting any kind of carbapenemase activity.
Recently, the use of mass spectrometry for detection of carbapenemase activity has been proposed, based on the analysis of the degradation of a carbapenem molecule
MALDI-TOF

33. Carba NP test
The Carba NP test was performed with a noncarbapenemase producer (Escherichia coli producing the extended-spectrum β-lactamase CTX-M-15, upper panel) and with a carbapenemase producer (Klebsiella pneumoniae–producing New Delhi metallo-β-lactamase-1, lower panel) in a reaction medium without (left panel) and with (right panel) imipenem. Uninoculated wells are shown as controls. Photographs were taken after a 1.5-hour incubation. Rapid Detection of Carbapenemase-producing Enterobacteriaceae Patrice Nordmann , Laurent Poirel, and Laurent Dortet

34. Molecular tests for carbapenemase genes
Remain the gold standard for the precise identification of carbapenemase genes.
Most of these techniques are based on PCR and may be followed by a sequencing step (e.g. VIM type, KPC type, NDM type or OXA-48 type).
They are either single or multiplex PCR techniques.
PCR technique performed directly on colonies can give results
within 4–6 h (or less when using real-time PCR technology)
The main disadvantages of the molecular-based technologies are their cost, the requirement for trained microbiologists and the inability to
detect novel unidentified genes.
Sequencing of the genes is interesting mostly for research and epidemiological purposes.

35. Screening of colonized patients
Screening should include:
‘at-risk’patients,: intensive care units, transplant recipients immunocompromised, and
those transferred from any foreign hospital (unknown prevalence of carbapenemase
producer carriage) or from non-foreign hospitals but known to face a high risk of carriage
of carbapenemase producers.
Stools and rectal swabs are the most suitable specimens for performing such screening.Three screening media are currently known:
CHROMagar KPC medium, which contains a carbapenem (CHROMagar, Paris,
France). its lack of sensitivity since it does not detect carbapenemase producers exhibiting
a low level of carbapenem resistance, as observed for several MBL or OXA-48 producers.
CRE Brilliance, Thermo Fisher Scientific, UK).14 It detects KPC andMBL producers well, and most but not all OXA-48 producers.
SUPERCARBA contains cloxacillin, zinc and ertapenem. It shows excellent sensitivity and specificity for detection of any kind of carbapenemase

36. Proposed algorithm for carbapenemase detection
(i) Infecting strains: Carba NP test on isolated colonies with decreased susceptibility to carbapenems. If positive, molecular identification of the genes, mainly for epidemiological reasons.
(ii) Screening of carriers: screening of carbapenem-resistant isolates using e.g. SUPERCARBA medium.
NP test to be performed on selected colonies. If the latter test is positive, molecular identification of the genes, mainly for epidemiological reasons.

37. Detection algorithms for CPE
Rapid Detection of Carbapenemaseproducing Enterobacteriaceae Patrice Nordmann, Laurent Poirel,
and Laurent Dortet Emerging Infectious Diseases • • Vol. 18, No. 9, September 2012

38. KPC – Questions
If I have detect KPC-production, should I change susceptible carbapenem results to resistant?
Not enough data to make a clear recommendation
Clinical outcomes data will be necessary

39. Testing Other Drugs Tigecycline:
Test by Etest if possible – disk diffusion tends to overcall resistance
No CLSI breakpoint, but there are FDA breakpoint
Susceptible ≤ 2 mg/ml
Intermediate = 4 mg/ml
Resistant ≥ 8 mg/ml

40. Testing Other Drugs Polymixin B or Colistin
Could test either, but colistin used clinically
Disk diffusion test does not work – don’t use!
Etest – works well
Broth microdilution – reference labs
Breakpoints - none
MIC ≤ 2 mg/ml, normal MIC range
MIC ≥ 4 mg/ml indicates increased resistance

41. THANK YOU