Introduction

Infections caused by multi-drug resistant

members of the Enterobacteriaceae, especially

Klebsiella pneumoniae is a rapidly growing

problem worldwide. In the past decade

carbapenem resistant Enterobacteriaceae (CRE)

has emerged and rapidly caused outbreaks

worldwide with high morbidity and mortality.

Therapeutic option for treating CRE were

limited, in part to colistin. With the uncontrolled

prescription of antibiotics in Palestine, colistin

resistant CRE’s rapidly emerged. Here we report

the characterization of 24 Klebsiella pneumoniaeisolates, detected in patient samples that were

CRE and resistant to colistin.

Materials and Methods

Carbapenem and colistin resistant Klebsiellapneumoniae (N=24) were isolated from 7 referral

hospitals across Palestine from different patient

samples between 2016 and 2018. Antimicrobial

sensitivity testing by disk diffusion and minimal

inhibitory concentration (MIC) was performed

and interpreted according to the CLSI, 2018

standards. Carbapenem resistant was confirmed

phenotypically by the mCIM protocol and

genotypically with real time PCR for the blaOXA-

48, blaNDM1, blaKPC, and blaVIM-1. Colistin None

Wild Type (NWT) Epidemiological Cutoff Value

(ECV) was determined by microbroth dilution

for the 24 K. pneumonia isolate. In addition, the

colistin resistant mechanisms were investigated

by Polymerase Chain Reaction (PCR)

amplification and sequencing the mgrB and mcr-

1 and mcr-2 genes. PFGE was performed on 12

isolates to determine the clonal relationship

between the isolates.

Conclusion1. K. pneumoniae with NWT ECV phenotype

emerged in Palestine.

2. Mutations in teh mgrB gene was the major

mechanism for colistin NWT ECV

3. Other resistance mechanisms should be

investigated for some of the isolates with

wild type mgrB gene like the TCRS PhopQ

4. Laboratories must start using the BMD

when testing for colistin AST

5. Colistin dose must be monitored in order to

avoid emergence of Colistin with NWT

ECV

Raed Ghneim1, Dina Nasser2 , Adham Abu Taha3, Rabee Adwan4, Ghada Safi2, Ayman Daoud3, Rifqa Daifi3, Issa Shtaieh5, Ibrahim Sahori6, Mamoun Ibdieh7, Abir Mohamad8, Caroline Hajal2, Mahmoud Ramlawi2, Suzan

Dkeidek4, Ihab Helou2, Ali Sabateen2, Musa Hindiyeh1,2.

1Caritas Baby Hospital, Bethlehem, Palestine, 2Augusta Victoria Hospital, Jerusalem, Palestine, 3Najah Hospital, Nablus, Palestine, 4Makassed Hospital, Jerusalem, Palestine, 5Palestine Reference Laboratory, Ramallah,

Palestine, 6Beit Jala Governmental Hospital, Beit Jala, Palestine, 7Palestine medical complex, Ramallah, Palestine, 8Rafidia Hospital, Nablus, Palestine

Figure 1: AST by Kirby Bauer method showed that the majority of these isolateswere resistant to the available antibiotics, complete resistance to the β-Lactamantibiotics, co-trimoxazole and high resistant to aminoglycosides.

Figure 2: AST as determined by MIC on Vitek 2.0 (bioMerieux, France) showedcompete resistance to β-Lactam antibiotics, high resistance to co-trimoxazole,aminoglycosides and fluoroquinolones

Figure 3: mCIM and the Carbapenem Resistance Genes were investigated. Themajority of the isolates encoded blaNDM (11 Isolates) or blaOXA-48 (11 isolates). Oneisolate encoded the blaKPC gene. One isolate was positive for blaNDM and blaOXA-48.No isolate encoded the blaVIM-1 gene. Two isolates that were negative for the genesinvestigated were mCIM negative.

Number Sample CT-DDmm

CT-E testµg/mL

CT -Vitek MICµg/mL

BMD µg/mL

1 KPCR 1 6 >256 >16 256 ECV = NWT2 KPCR 2 6 48 >16 32 ECV = NWT3 KPCR 3 6 32 >16 64 ECV = NWT4 KPCR 4 6 32 2 64 ECV = NWT5 KPCR 5 10 12 >16 32 ECV = NWT6 KPCR 7 10 4 <0.5 8 ECV = NWT7 KPCR 8 6 96 >16 256 ECV = NWT8 KPCR 9 11 4 >16 256 ECV = NWT9 KPCR 10 11 4 >16 32 ECV = NWT

10 KPCR 11 15 2 2 8 ECV = NWT11 KPCR 17 12 4 >16 64 ECV = NWT12 KPCR 18 13 2 4 8 ECV = NWT13 KPCR 20 13 4 8 16 ECV = NWT14 KPCR 23 11 8 >16 16 ECV = NWT15 KPCR 24 6 48 >16 32 ECV = NWT16 KPCR 25 11 6 >16 8 ECV = NWT17 KPCR 26 12 8 >16 32 ECV = NWT18 KPCR 27 8 32 >16 32 ECV = NWT19 KPCR 28 13 2 ND 64 ECV = NWT20 KPCR 29 12 4 ND 8 ECV = NWT21 KPCR 30 9 16 ND 256 ECV = NWT22 KPCR 31 10 8 ND 64 ECV = NWT23 KPCR 32 10 256 ND 64 ECV = NWT24 KPCR 33 13 2 ND 64 ECV = NWT

Figure 5: Analysis of the mgrB gene with its promoter regionrevealed that the majority of the isolates had the 253bpamplicon. Sequence analysis showed that some isolates had astop codon at the beginning and at the middle of the gene, somehad insertion elements and some had deletions. The mcr-1and 2were not detected by PCR in our isolates.

22%

44%

11%6%

11%6%

0%

10%

20%

30%

40%

50%

WT Stop codon88N

Stop codon7N

Del-136N InsertionSequences

mgrb Del

Distribution of mgrB mutation in the isolates

Figure 6: Summary of the distribution of mgrB mutations that led to colistin NWT ECV’s in the isolates. (Insertion sequence: IS5 at 90N and in the promoter region)

Figure 4: Comparison between colistin reading by Disk Diffusion, E-Test, Vitek and Broth Microdilution

ResultsPart One

Antibiotic Susceptibility Testing (AST) & Carbapenem resistance

mechanism were investigated

Part Two

.

Part Three

Alteration of the mgrB gene and the acquisition of the mcr-1 and mcr-2 was evaluated in all 24 K.

pneumoniae with the Colistin NWT ECV’s

Figure 7: PFGE shows that there is no clonal spread of the resiatntisolates. On the contrary the emergence could be due antibioticmissuses.

91.7%

8.3% 4.2%

45.8% 45.8%

0.0%8.3%

0%

20%

40%

60%

80%

100%

mCIM- p

ositive

mCIM-negativ

e

blaKPC

blaNDM

blaOXA-48

blaVIM-1

Negative

References1. Cannatelli, A., et al., MgrB inactivation is a common mechanism of colistin resistance in

KPC-producing Klebsiella pneumoniae of clinical origin. Antimicrobial Agents

Chemotherapy, 2014. 58(10): p. 5696-703.

2. Poirel, L., A. Jayol, and P. Nordmann, Polymyxins: Antibacterial Activity, Susceptibility

Testing, and Resistance Mechanisms Encoded by Plasmids or Chromosomes. Clinical

Microbiology Reviews, 2017. 30(2): p. 557-596.

3. Clinical and Laboratory Standards Institute. 2018. Performance standards for

antimicrobial susceptibility testing; 27th informational supplement.M100-S28. Clinical

and Laboratory Standards Institute, Wayne, PA.

4. ISO 20776-1:2006 - Clinical laboratory testing and in vitro diagnostic test systems --

Susceptibility testing of infectious agents and evaluation of performance of antimicrobial

susceptibility test devices - Part 1: Reference method for testing the in vitro activity of

antimicrobial agents against rapidly growing aerobic bacteria involved in infectious

diseases.

0%

20%

40%

60%

80%

100%

Amikacin

Gentamicin

Nitrofurantoin

Amoxicillin

-clavulanate

Ampicillin

Cefotaxim

e

Ceftazid

ime

Ceftria

xone

Cefuroxim

e

Cefoxitin

Meropenem

Ertapenem

Imipenem

co-trim

oxazole

Ciprofloxacin

Resistant Susceptible Intermediate

Perc

ent

Part Four

N=18 N=18 N=24 N=24 N=24 N=18 N=24 N=24N=24

N=24 N=18N=24 N=18

N=18

N=24

0%

20%

40%

60%

80%

100%

Ticarcillin

Pipercillin

Piperacillin/Tazobactam

CeftazidimeCefepime

AztreonamImipenem

MeropenemAmikacin

Gentamycin

Topramycin

Ciprofloxacin

Pefloxacin

Minocycline

co-trimoxazole

Perc

ent

Resistant Susceptible Intermediate

P-1408