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Carbapenem-Resistant Klebsiella pneumoniae Strains: Susceptibility to Novel Antibiotics and Molecular Detection of the Resistance Mechanisms - A Study from Two Greek Tertiary Teaching Hospitals

Chatzidimitriou M1*, Chatzivasileiou P2, Sakellariou G3, Kyriazidi MA2, Chatzidimitriou D2, Chatzopoulou F2, Rousis D2, Katsifa E4, Vagdatli E5 and Lialiaris TH3

1School of Biomedical Sciences, International Hellenic University, Greece

2Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece

3Medical School, Demokritus University of Thrace, Thessaloniki, Greece

4General Teaching Hospital “G. Papanikolaou”, Thessaloniki, Greece

5General Teaching Hospital “Ippokrateio”, Thessaloniki, Greece

*Corresponding Author:
Chatzidimitriou M
International Hellenic University School of Biomedical Sciences Thessaloniki, Greece
Tel: 306944208095
E-mail: [email protected]

Received Date: May 08, 2020; Accepted Date: May 26, 2020; Published Date: June 05, 2020

Citation: Chatzidimitriou M, Chatzivasileiou P, Sakellariou G, Kyriazidi MA, Chatzidimitriou D, et al. (2020) Carbapenem-Resistant Klebsiella pneumoniae Strains: Susceptibility to Novel Antibiotics and Molecular Detection of the Resistance Mechanisms; A Study from Two Greek Tertiary Teaching Hospitals. Arch Clin Microbiol Vol. 11 No. 3:108

Copyright: © 2020 Chatzidimitriou M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

 
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Abstract

Objectives: We evaluated the carbapenem resistance mechanisms of Klebsiella pneumoniae strains isolated in two Greek tertiary teaching hospitals and their susceptibility to currently used and novel antimicrobial agents.

Materials and methods: Forty-seven carbapenem resistant K. pneumoniae strains were collected in G. Papanikolaou and Ippokrateio hospital of Thessaloniki from 1/11/2016 to 5/1/2018 and 26/1/2017 to 19/4/2017 respectively. Strain identification and antimicrobial susceptibility was conducted by Vitek 2 system (Biomérieux France). Susceptibility against new antimicrobial agents was examined by disk diffusion method. Polymerase chain reaction (PCR) was used for the detection of blaKPC, blaVIM, blaNDM and blaOXA-48 genes.

Results: The EDTA-boronic acid disk synergy test performed on the 24 K. pneumoniae strains from G. Papanikolaou hospital demonstrated that 8 (33.3%) yielded positive for metallo-b-lactamases (MBL) and 16 (66.6%) for K. pneumoniae carbapenemases (KPC) production. Gentamycin demonstrated the highest in vitro activity (82.6%) among the 23 K. pneumoniae strains from Ippokrateio hospital followed by colistin (73.9%) and tigecycline (69.5%). All strains from G. Papanikolaou hospital were sensitive to colistin whereas the 70.8% of them displayed susceptibility to gentamycin. Ceftazidime/ avibactam showed the highest sensitivity (76.6%) in all strains followed by eravacyclin (66.6%). The blaKPC gene was present in 30 strains (63.8%), the blaNDM in 11 (23.4%) and the blaVIM in 6 (12.8%). The blaOXA-48 gene was not detected.

Conclusions: Well established antimicrobial agents such as colistin, gentamycin and tigecycline and novel antibiotics like ceftazidime/avibactam and eravacycline may be reliable options for the treatment of invasive infections caused by KPC-producing pathogens.

Keywords

K. pneumonia; Carbapenem-resistance; Carbapenemases; Carbapenems

Introduction

Carbapenems are widely considered the treatment of choice for severe infections caused by Enterobacteriaceae producing extended-spectrum b-lactamases (ESBL). The increasing incidence of Gram-negative bacteria resistant to carbapenems is becoming a serious global health problem since the therapeutic options are significantly restricted [1]. Carbapenemase production is a major -yet not the solecarbapenem- resistance mechanism of Enterobacteriaceae [2] since other mechanisms such as impermeability of the outer microbial membrane combined with overexpression of β- lactamases are also involved [3]. Carbapenemases are a large group of various enzymes capable of hydrolysing several betalactam antibiotics including carbapenems [1].

K. pneumoniae carbapenemases (KPCs) are classified as Ambler class A β-lactamases (penicillinases) with an antibiotic spectrum activity like that of ESBLs and carbapenems. Ambler class B carbapenemases are metallo-β-lactamases (MBLs) which hydrolyse all β-lactams except aztreonam. Among them, VIM, IMP and NDM group are the most described. The Ambler class D carbapenemases or oxacillinases comprise several OXA-48 derivatives with low hydrolytic activity against carbapenems [3]. Since the first description of the KPC enzyme in a K. pneumoniae isolate from North Carolina in 2001 [4], carbapenemases have been widely spread [4] and although are predominantly carried by K. pneumoniae they have also been harboured by several other Enterobacteriaceae genera [2,4].

In recent years, the predominant carbapenem-resistance mechanism among K. pneumoniae strains in many geographical regions is either KPC or MBL production, unlike the past when it was mainly attributed to decreased membrane permeability and ESBL production [5]. The geographic reservoir of MBL producing K. pneumoniae strains was initially located in southern Europe and Far East while KPC producing strains were first detected in northeastern regions of the United States [5]. Until 2004 KPC producing K. pneumoniae strains were restricted in the United States [1] and were either endemic in several areas or caused sporadic outbreaks [2]. The first outbreak outside United States was reported in Israel in 2006 [1,2] and the fact that it was attributed to strains genetically identical to those previously described in the United States is indicative of a KPC-producer transfer between countries [1].

In the following years, KPC-producers have been spread worldwide [2] and not only Israel [2,3] but other European countries such as Greece [1-3] and Italy [3] are nowadays considered endemic areas for KPC-producing bacteria [1-3]. Additionally, the Oxa-48 producers which are occasionally responsible for outbreaks, are more prevalent in Turkey, France, and the United Kingdom [3]. KPCs are encoded by the blaKPC gene located within the Tn4401 transposon which is highly transferable into different plasmids of Gram-negative bacteria, hence favouring its inter-species and geographic dissemination [2], outbreak onset [3] and multi-drug resistance [2,3].

The phenotypic detection of KPC producers is important for restricting resistance and should be adjusted according to the prevalent resistant type in each region. In the laboratory practice, MBL production is ascertained by double-disk synergy tests in combination with EDTA and imipenem disks.

The modified Hodge test (MHT) and the susceptibility to ertapenem are the most indicated methods to reveal the production of these enzymes, especially in endemic areas [5]. The MHT is the only phenotypic screening method recommended by the Clinical and Laboratory Standard Institute (CLSI) [2,6,7] and is based on the inhibition zone formation around a carbapenemase disk when a KPC suspected organism is cultured on Mueller-Hinton agar [2]. The sensitivity of the test reaches almost the 100% [2] but diversities in specificity values [2,7] and false positivity of the results raise a concern [6,7].

Culture of the isolates on chromogenic agar have also been recommended for KPC detection, demonstrating 100% sensitivity and specificity comparable to those of polymerase chain reaction (PCR) [2]. Moreover, the use of boronic acid disk test in combination with several antibiotic substrates has been evaluated as sensitive and highly specific for the phenotypic detection of KPC producing K. pneumoniae clinical isolates [5]. Undoubtedly, however, molecular techniques are the most indicated methods to confidently confirm KPC production [2], especially the Oxa-type carbapenemases for which no specific phenotypic test has been established yet [6].

The aim of the present study was to define the susceptibility profile of KPC producing K. pneumoniae strains collected in two Geek tertiary teaching hospitals against the currently used and novel antimicrobial agents as well as the molecular detection of the underlying resistance mechanisms.

Material and Methods

Forty-seven clinical samples (including urine, bronchial secretions, sputa, venous blood, venous catheter tips, tissue fragments, rectal swabs, trauma materials, soft tissue collections, drainage and ascitic fluids) were obtained from equal number of patients either hospitalized or attended the outpatient clinic of G. Papanikolaou General hospital and Ippokrateio hospital, both sited in Thessaloniki, Greece (Figures 1 and 2).

acmicrob-clinical

Figure 1: Distribution per clinical department of the 24 clinical samples obtained from G. Papanikolaou hospital.

acmicrob-obtained

Figure 2: Distribution per clinical department of the 23 clinical samples obtained from Ippokrateio General hospital.

The 24 clinical samples were collected in G. Papanikolaou hospital between 1/11/2016 and 5/1/2018 and the 23 ones in Ippokrateio hospital between 26/1/2017 and 19/4/2017 and sent to the corresponding clinical laboratory. All samples were cultured according to the standard microbiological methods. Isolated K. pneumoniae strains were inoculated in nutrient broth containing 16% glycerol and stored at -70ᵒC until assessment.

Culture methods

The clinical samples were cultured according to their origin: urine samples were inoculated on 5% horse blood agar and MacConkey 2 agar and incubated for 24 h at 37ᵒC. Blood samples were distributed into dedicated bottles and placed in the automated blood culture monitoring system, BacT/Alert (BioMérieux, France).

Positive blood cultures were subcultured on 5% horse blood agar, MacConkey 2, Chapman and Sabouraud agar. The other samples were plated on 5% horse blood agar and MacConkey 2 agar and incubated at 37ᵒC for 24 h as well as on chocolate agar incubated accordingly.

All specimens excluding sputa, were enriched with nutrient broth, and incubated for another 24 h at 37ᵒC under aerobic conditions. K. pneumoniae isolates grown on cultures were stored at -70ᵒC into Brain Heart Infusion Broth (BHIB), containing 16% glycerol until being processed for the molecular detection of specific resistance genes.

Identification methods

K. pneumoniae isolates were identified to the species level with the automated system VITEK 2 Compact ((bioMérieux, Marc L’ Έtoile, France) using the GN ID test panel according to the manufacturer’s recommendations. The GN card is based on documented biochemical methods and implementation of innovative substrates which measure the use of carbon source, enzymatic activity, and resistance.

The result is available within 10 hours or less [8]. For both identification and susceptibility, the isolates were suspended into 3 ml normal saline and the turbidity of the inoculum was adjusted to 0.5-0.63 (±0.1) of the MacFarland (McF) scale using Densichek (bioMérieux).

Antimicrobial susceptibility testing

The susceptibility to antibiotic agents was also evaluated with VITEK 2 system using the AST card for Gram-negative bacilli. The AST card methodology is based on the minimum inhibitory concentration (MIC) technique and is basically a miniaturised and abbreviated version of the doubling dilution technique for MIC determined by the microdilution method. Each card contains a control well filled only with nutrient medium and multiple wells filled with selected antibiotics of increasing concentrations in the medium.

The system continuously monitors the organism growth in each well over time and at the completion of the incubation period the MIC values for each antibiotic are automatically reported [8]. The strains were defined as sensitive, intermediate, or resistant according to the CLSI interpretive criteria whereas the EUCAST (European Committee of Antimicrobial Susceptibility Testing) breakpoints were used to define sensitivity to colistin.

EDTA-boronic acid synergy test

All 24 K. pneumoniae strains from G. Papanikolaou hospital resistant to carbapenems were tested for MBL and KPC production by synergy test using disks containing meropenem- EDTA and meropenem-boronic acid respectively. The phenotypic differentiation of KPC-type was performed as follows: 20 μL of phenylboronic acid solution (containing 400 μg of boronic acid) and 10 μL 0.1 M EDTA were used as reagents and dispensed onto commercially available meropenem containing antibiotic disks.

A microbial suspension of 0.5 McF turbidity was inoculated on Mueller-Hinton plate onto which four meropenem disks - a meropenem-phenylboronic acid disk, a meropenem-EDTA disk, a meropenem-phenylboronic acid plus EDTA disk and a meropenem disk alone - were placed at suitable distance. After incubation at 37ᵒC for 18 hours inhibition zones >5 mm of diameter around disks were evaluated.

The test was considered positive for KPC or MBL production when the inhibition zone around the disk of meropenemboronic acid or meropenem-EDTA respectively was greater than the disk containing meropenem alone. Similarly, concurrent production of KPC and MBL was suspected when the inhibition zone was >5 mm only around the disk containing both reagents.

Susceptibility to new antimicrobial agents

The strains were tested for susceptibility to the following antibiotics: minocycline, tetracycline, ceftazidime/clavulanic acid, doripenem, ertapenem/cloxacillin, meropenem/ dipicolinic acid, eravacycline and ceftazidime/avibactam. The susceptibility testing was carried out by the disk diffusion method on Mueller-Hinton agar according to the CLSI guidelines and interpretive standards (Table 1).

Table 1 Susceptibility breakpoints of the new antibiotics tested by disk diffusion method.

Antimicrobial agent Inhibition zone (mm)
S I R
*Minocycline (MN) 30 μg ≥ 16 13-15 ≤ 12
*Tetracycline (TE) 30 μg ≥ 15 14-Dec ≤ 11
**Ceftazidime/Clavulanic acid (CAL) 40(30/10) μg ≥ 26 23-25 ≤ 22
*Doripenem (DOR) 10 μg ≥ 23 20-22 ≤ 19
**Ertapenem/Cloxacillin (ET/CL) ≥ 22 19-21 ≤ 18
**Meropenem/Dipicolinic acid (MR/DP) ≥ 23 20-22 ≤ 19
**Eravacycline (ERV) 20 μg ≥ 15 14-Dec ≤ 11
**Ceftazidime/Avibactam (CZA) 50(30/20) μg ≥ 21 - ≤ 20

Molecular detection of resistance genes

For the microbial DNA extraction 2-3 colonies of each strain were diluted into 100 μl distilled water. After incubation at 95ᵒC for 10 minutes into water bath and centrifugation for 2 minutes at 14500 rpm the supernatant containing the microbial DNA (template DNA) was transferred into sterile tubes and either used for amplification or stored at -20oC for future assessment.

For each K. pneumoniae strain four different PCRs were conducted to detect the carbapenemase genes blaKPC, blaVIM, blaNDM and blaOXA-48 using four different primer couples (Table 2).

Table 2 Primer couples used for the detection of carbapenemase genes.

Carbapenemase genes DNA primer couples PCR product
blaKPC F: 5΄-TGTCACTGTATCGCCGTC-3΄ 798 bp
R: 5΄-CTCAGTGCTCTACAGAAAACC-3΄
blaVIM F: 5΄-GTTTGGTCGCATATCGCAAC-3΄ 390 bp
R: 5΄-AATGCGCAGCACCAGGATAG-3΄
blaNDM F: 5΄-GCAGCTTGTCGGCCATGCGGGC-3΄ 621 bp
R: 5΄-GGTCGCGAAGCTGAGCACCGCAT-3΄
blaOXA-48 F: 5΄-GCGTGGTTAAGGATGAACAC-3΄ 438 bp
R: 5΄-CATCAAGTTCAACCCAACCG-3΄

Two μl of the template DNA and 48 μl of reagent mixture (50 μl final volume) were used for each amplification. The reagent mixture contained deoxynucleotide triphosphate mix (dNTPs) (Invitrogen 55083, 55082, 55084, 55085), MgCl2, Taq DNA polymerase (AmpliTaq Gold Biosystems 5 Units/μl 4486226), the primer couples (Invitrogen) and the reaction buffer at a content as shown in Table 3.

Table 3 PCR reagent mixture.

Substance Volume
10cc X PCR buffer 5 μL
MgCl2 1.5 mM 3 μL
dNTPS 0.125 mM 0.25 μL
Forward primer 0.3 pmol/μL 1.5 μL
Reverse primer 0.3 pmol/μL 1.5 μL
Taq DNA Polymerase 2U/μL 0.4 μL
Distilled water 36.35 μL
Template DNA 2 μL
Final volume 50 μL

The DNA amplification was carried out using the 2720 Thermal Cycler (Applied Biosystems) according to the following protocol: one PCR cycle for the initial DNA denaturation at 94ᵒC for 10 min and 36 PCR cycles including a) denaturation of the DNA at 94ᵒC for 30 sec, b) annealing at 52ᵒC for 40 sec, allowing primers to bind at target sites on the template and c) primer extension at 72ᵒC for 50 sec. Subsequently, the final product was extended at 72ᵒC for 6 min. The PCR products were electrophorised on 2% (w/v) agarose gel for 30 min using a horizontal electrophoresis device of 100V constant voltage. The electrophoresis buffer used was 1x TAE solution (40 mmol/L Tris-HCl [pH 8,3], 2 mmol/L acetate, 1 mmol/L EDTA). The electrophoresis gel was stained with ethidium bromide 0.5 μg/ml and read under UV light.

Results

EDTA-boronic acid disk tests

The EDTA-boronic acid disk synergy test for the 24 K. pneumoniae isolates from G. Papanikolaou hospital demonstrated that 8 strains were positive for MBL and 16 for KPC production.

Antimicrobial susceptibility

Susceptibility testing results of all K. pneumoniae strains conducted with the automated system Vitek 2 are illustrated in Tables 4 and 5.

Table 4 Antibiotic susceptibility (MIC values) of the 23 isolates from Ippokrateio hospital.

Antimicrobial agent Number of isolates
S I R
Ampicilin/Sulbactam     23
Piperacillin/Tazobactam     23
Cefoxitin     23
Ceftazidime     23
Ceftriaxone     23
Cefepime     23
Aztreonam     23
Imipenem     23
Meropenem     23
Amikacin 10   13
Gentamicin 19   4
Ciprofloxacin 2   21
Levofloxacin 2   21
Tigecycline 16 5 2
Fosfomycin 11   12
Colistin 17   6
Trimethoprim/Sulfamethoxazole 11   12

The isolates from Ippokrateio hospital were resistant to carbapenems, all demonstrating MIC values ≥ 4 μg/mL for imipenem and meropenem. The highest in vitro activity was shown for gentamycin (82.6% sensitivity) followed by colistin (73.9%) and tigecycline (69.5%) (Table 4). Similarly, all 24 isolates from G. Papanikolaou hospital were resistant to carbapenems while the highest in vitro activity was displayed for colistin (100 % sensitivity), followed by sulfamethoxazole/ trimethoprim and gentamycin (75% and 70.8% respectively) (Table 5).

Table 5 Antibiotic susceptibility (MIC values) of the 24 isolates from G. Papanikolaou hospital.

Antimicrobial agent Number of isolates
S I R
Ampicilin/Sulbactam     24
Piperacillin/Tazobactam     24
Cefoxitin     24
Ceftazidime     24
Ceftriaxone     24
Cefepime     24
Aztreonam     24
Imipenem     24
Meropenem     24
Amikacin 15   9
Gentamicin 17 1 6
Ciprofloxacin   1 23
Levofloxacin     24
Tigecycline 8 4 12
Fosfomycin 6   18
Colistin 24    
Trimethoprim/Sulfamethoxazole 18   6
Amoxicillin/Clavulanic acid     24
Ticarcillin     24
Cefotaxime     24
Ertapenem     24

Susceptibility to novel antibiotics

The susceptibility profile of all KPC strains to novel antibiotics was examined by disk diffusion method and results are illustrated in Table 6. The combination of ceftazidime/ avibactam had the highest activity (76.6% sensitivity) followed by eravacycline (66%). The 55.3% of strains demonstrated susceptibility to minocycline, followed by doripenem (27.7%) and tetracycline (25.5%). Susceptibility to meropenem/ dipicolinic acid and ceftazidime/clavulanic acid was reported in 8.5% of strains whereas only 2.1% were sensitive to ertapenem/cloxacillin. It is noteworthy that all K. pneumoniae strains carrying the blaKPC gene were susceptible to ceftazidime/avibactam.

Table 6 Susceptibility of all isolates to new antibiotics by disk diffusion method.

Antimicrobial agent Number of isolates (%)
S I R
Minocycline 26 (55.3) 2 (4.3) 19 (40.4)
Tertacycline 12 (25.5) 3 (6.4) 32 (68.1)
Ceftazidime/Clavulanic acid 4 (8.5) 3 (6.4) 40 (85.1)
Doripenem 13 (27.7) 6 (12.7) 28 (59.6)
Ertapenem/Cloxacillin 1 (2.1) 2 (4.3) 44 (93.6)
Meropenem/Dipicolinic acid 4 (8.5) 3 (6.4) 40 (85.1)
Eravacycine 31 (66.6) 6 (12.7) 10 (21.3)
Ceftazidime/Avibactam 36 (76.6) - 11 (23.4)
 

Molecular screening for carbapenemase production

According to PCR results the blaKPC gene was present in 30 isolates (63.8%), the blaNDM in 11 (23.4%) while 6 (12.8%) were carriers of the blaVIM gene. In none of the examined strains the blaOXA-48 gene was detected (Figure 3).

acmicrob-electrophoresis

Figure 3: PCR product electrophoresis of the 39 out of 47 K. pneumoniae strains. The bands across the first three lanes at each raw are positive controls for blaKPC, blaVIM and blaNDM gene.

Discussion

The early detection of the resistance mechanisms as well as the reliability of the sensitivity tests are the cornerstone for limiting KPC strain dispersion and the treatment of caused diseases. In line with this, the present study was designed to investigate the resistance mechanisms to carbapenems of 47 KPC-producing K. pneumoniae strains reported from two Greek tertiary teaching hospitals as well as their susceptibility to both well-established and novel antimicrobial agents. The strains were originated from various clinical samples such as urine, bronchial secretions, sputa, venous blood, venous catheter tips, tissue fragments, rectal swabs, trauma materials, soft tissue collections, drainage and ascitic fluids. Strain identification to species level and detection of carbapenemase resistance were carried out by Vitek 2 system. All isolates demonstrated MIC values within the resistance range for imipenem, ertapenem and meropenem. The 24 isolates from G. Papanikolaou hospital were tested for carbapenemase production with EDTA-boronic acid disk synergy test which demonstrated KPC and MBL production in 16 and 8 strains, respectively. The EDTA-boronic acid disk synergy test was not performed on the 23 isolates from Ippokrateio hospital given that all 47 strains were processed for the detection of the carbapenemases genes blaKPC, blaNDM, blaVIM and blaOXA-48 by PCR. The blaKPC was the most prevalent variant (30%) followed by blaNDM (23.4%) and blaVIM (12.8%). None of the examined isolates was carrier of blaOXA-48 gene nor harboured KPC and MBL-producing genes concomitantly. KPC production is a common resistance mechanism of Enterobacteriaceae against carbapenems and is highly prevalent in Mediterranean countries, especially Italy and Greece. These countries have been declared endemic regions in regard to KPC in 2014–2015 [9], while in Greece, according to a 10-year single-center study, an alarming increase in the number of KPC-producing K. pneumoniae cases was recorded [10]. As reported by EARS-Net, Greece displayed the highest prevalence of carbapenem-resistant K. pneumoniae isolates in 2014 in Europe but with a decreasing trend from 68.2% to 62.3% between 2011 and 2014 [11]. Similarly, in the European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE) project conducted from 1/11/2013 to 30/04/2014, Grundmann et al. confirmed that KPC-producing K. pneumoniae strains have been detected in large proportions in several countries including Greece, Italy, Israel and Portugal [12]. Contrariwise, VIM was the sole carbapenemase detected in Greece until 2006 ([3]. In the early 2000s, the first nationwide epidemic of VIM-producing K. pneumoniae was reported [11,12] but the epidemiological status progressively changed with KPC having been the predominant carbapenem resistance mechanism since 2007 [12]. Therefore, a surveillance study among 119 Greek hospitals conducted by Maltezou et al. revealed that KPC production was the most prevalent resistance mechanism among K. pneumoniae strains (82.6%) followed by VIM production (9.7%) and their concomitant existence (7.7%) [13,14]. Currently, NDM is the second ranking carbapenemase in Greece [12] whereas the frequency of VIM is gradually decreased and restricted to 8.4% in 2016 [13]. In accordance to our results, a nationwide multicenter study conducted between 2014-2016 by Galani et al. demonstrated that the most prevalent carbapenemase detected among K. pneumoniae strains was KPC, followed by NDM and VIM [13]. In respect to other European countries, NDM producers are more commonly detected in Romania, Poland, and Denmark where an inter-regional spread has been documented [9,11]. Similarly, VIM is reported to be the predominant MBL in Spain, Italy, and Hungary where it was classified in epidemiological stage 4 in 2014-2015 [9,11]. In our study, none of the examined K. pneumoniae strains were positive for OXA-48 nor for a double carbapenemase production, possibly due to the relatively small number of our collection. OXA-48 carbapenemase is very commonly detected in Turkey in both K. pneumoniae and E. coli strains and is also frequently encountered in other European countries such as Romania, Spain, Belgium, Germany, and France [12]. In Greece, however, the prevalence of OXA-48 producing Enterobacteriaceae remains relatively low [11].

Colistin, gentamycin and tigecycline are the last line treatment against carbapemen-resistant K. pneumoniae infections. Non-susceptibility rates against these agents have been steadily increasing [10] and are mainly attributed to their previous administration [15-18] as well as to the lack of preventive measures in health-care units [18,19]. In our study, resistance to colistin, gentamycin and tigecycline displayed the 26.1%, 17.4% and 30.5% respectively of the 23 carbapenemresistant K. pneumoniae strains from Ippokrateio hospital. By contrast, none of the 24 strains from G. Papanikolaou hospital was resistant to colistin but only 33.3% were sensitive to tigecycline. Among the examined strains 29.2% of them displayed resistance to gentamycin. An important finding of the present study was the relatively high susceptibility prevalence to trimethoprim/sulfamethoxazole demonstrated by the 12 out of 17 K. pneumoniae strains of our collection recovered from urine samples. Considering that trimethoprim/ sulfamethoxazole is mainly administered for urinary tract infections, this observation should raise concerns regarding the unwarranted use of new, powerful but costly antibiotics instead of earlier, less expensive and until today, effective drugs.

The 47 K. pneumoniae strains of our study were also examined for their susceptibility to other antibiotics such as ceftazidime/avibactam and eravacycline. Avibactam is a novel β-lactamase inhibitor efficient against class A and class D carbapenemases and less active against MBL-producing K. pneumoniae strains [20,21]. As an inhibitor, it allows the antimicrobial activity of ceftazidime against KPC-producing K. pneumoniae [20]. Eravacycline is a novel synthetic fluorocycline antimicrobial agent of the tetracycline antibiotic class with a potential mechanism-based inhibitory effect on the bacterial ribosome [22]. As it is not affected by tetracycline resistance mechanisms (efflux pumps, ribosomal proteins) [22] it is active against most Gram-negative species [23] including those producing KPC and NDM carbapenemases [21]. Clinical data suggest that ceftazidime/avibactam is active against most multi-drug resistant K. pneumoniae isolates and could be a valuable treatment option either alone or in combination with other drugs, such as aztreonam, in patients with KPCproducing K. pneumoniae infections [20]. An in vitro assessment of ceftazidime/avibactam activity against multidrug resistant K. pneumoniae strains confirmed that 96.6 % of them were susceptible, exhibiting MIC values of ≤ 8 μg/ml. Moreover, the combination was the most effective together with tigecycline and colistin [24]. In a study conducted. the activity of ceftazidime/avibactam in a large collection of clinical isolates was retained against the 99.3% of the examined Enterobacteriaceae harbouring blaKPC gene and the 99.2% of blaKPC K. pneumoniae carriers [25]. In line with this are the results of our study in which all 30 K. pneumoniae strains carrying blaKPC were susceptible to ceftazidime/ avibactam. As reported by Abdallah et al. the in vitro activity of eravacycline against a large Gram-negative pathogen collection was preserved against multidrug resistant isolates, including those producing OXA and KPC carbapenemases. For K. pneumoniae strains MIC values were 1 dilution lower than that of tigecycline [26]. Accordingly, Sutcliffe et al. demonstrated that eravacycline was ≥ 2-fold active in vitro compared to tigecycline for many Enterobacteriaceae including the 46% of the examined K. pneumoniae isolates, suggesting its potential therapeutic superiority against infections caused by multidrugresistant pathogens [22].

In our study, non-susceptibility to ceftazidime/avibactam and eravacycline was displayed by the 23.4% and 34% of the examined stains, respectively. Resistance to ceftazidime/ avibactam has been sporadically reported in the United States and Europe and has been associated to KPC gene or CTX-M-14 mutations. Mutations in blaKPC gene and subsequent resistance to ceftazidime/avibactam are generally resulted from its previous administration [27]. Several mechanisms are claimed to be involved in resistance and heteroresistance of K. pneumoniae to eravacycline such as overexpression of OqxAB and MacAB efflux pumps and the transcriptional regulator RamA [28,29].

Conclusion

In conclusion, the in vitro susceptibility of the 47 K. pneumoniae strains collected in both hospitals demonstrated that colistin, gentamycin and tigecycline may be beneficial alternatives for the treatment of invasive infections caused by KPC-producing pathogens. New antibiotic agents such as ceftazidime/avibactam and eravacycline possess key advantages in the treatment of those infections but careful monitoring of their susceptibility profile coupled with the identification of the carbapenemase produced are important for defining the optimal therapeutic regimen. Additionally, given the high morbidity and mortality of KPC-producing K. pneumoniae infections and the limited therapeutic choices, preventive strategies, such as strict adherence to basic hygiene practices in healthcare facilities and antimicrobial stewardship should be urgently implemented for restricting the spread of those infections.

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