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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 29  |  Issue : 4  |  Page : 801-811

Detection of Klebsiella pneumoniae carbapenemases and metallo-β-lactamases among Klebsiella pneumoniae isolates from hospitalized patients at Menoufia University Hospitals, Egypt


1 Department of Microbiology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt

Date of Submission10-Nov-2014
Date of Acceptance18-Jan-2015
Date of Web Publication21-Mar-2017

Correspondence Address:
Asmaa M Elbrolosy
Department of Microbiology, Faculty of Medicine, Menoufia University, Marina Street, Elbagour, Menoufia Governorate, 32871
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.202524

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  Abstract 

Objectives
The aim of the study was to determine the incidence of carbapenem resistance among Klebsiella pneumoniae isolates in Menoufia University Hospitals and detect the production of Klebsiella pneumoniae carbapenemases (KPCs) and metallo-b-lactamases (MbLs) using phenotypic and molecular methods and correlate their presence with in-vitro susceptibility to carbapenems.
Background
Carbapenem-hydrolyzing b-lactamases are the most powerful b-lactamases. KPCs and MbLs are able to hydrolyze carbapenems, which cause resistance to multiple classes of antibiotics.
Materials and methods
The study was conducted on 128 K. pneumoniae isolates. Carbapenemase production was detected by antibiotic susceptibility testing against imipenem (IPM), meropenem, and ertapenem and by IPM minimal inhibitory concentration assays. Results were confirmed by the modified Hodge test, the phenylboronic acid combined disk (PBA-CD) test, and the IPM/EDTA combined disk (IPM/EDTA-CD) test. Detection of carbapenemase genes (blaKPC, blaVIM, and blaIMP) was performed by multiplex PCR.
Results
Eighty K. pneumoniae isolates were reported as IPM resistant. Class A carbapenemases (KPCs) were detected in 35% of IPM-resistant K. pneumoniae by the PBA-CD test. Class B carbapenemases (MbLs) were detected in 48.8% of IPM-resistant K. pneumoniae by the IPM/EDTA-CD test. Age, long hospital stay, invasive procedures, and history of drug intake (carbapenem) were high-risk factors for IPM-resistant K. pneumoniae. Among IPM-resistant isolates, 17.5% were positive for the blaKPC gene and 43.75% were positive for the blaVIM and blaIMP genes. In relation to PCR, the sensitivity and specificity of the PBA-CD test were 85.7 and 75.8%, respectively, whereas for the IPM/EDTA-CD test sensitivity and specificity were 94.3 and 86.7%, respectively.
Conclusion
Laboratory identification of carbapenemase-harboring clinical isolates is necessary for implementing contact precautions, for outbreak detection, and for proper choice of effective therapy. Phenotypic and molecular-based techniques identify carbapenemase producers with variable efficiencies.

Keywords: carbapenemase genes, Klebsiella pneumoniae carbapenemases, metallo-β-lactamases, phenylboronic acid


How to cite this article:
Melake NA, Mahmoud AB, Elraghy NA, Labib AZ, Hassan DM, Elbrolosy AM. Detection of Klebsiella pneumoniae carbapenemases and metallo-β-lactamases among Klebsiella pneumoniae isolates from hospitalized patients at Menoufia University Hospitals, Egypt. Menoufia Med J 2016;29:801-11

How to cite this URL:
Melake NA, Mahmoud AB, Elraghy NA, Labib AZ, Hassan DM, Elbrolosy AM. Detection of Klebsiella pneumoniae carbapenemases and metallo-β-lactamases among Klebsiella pneumoniae isolates from hospitalized patients at Menoufia University Hospitals, Egypt. Menoufia Med J [serial online] 2016 [cited 2020 Feb 27];29:801-11. Available from: http://www.mmj.eg.net/text.asp?2016/29/4/801/202524


  Introduction Top


The emergence and rapid dissemination of carbapenemase-producing Gram-negative bacilli worldwide is a cause for concern [1]. Klebsiella pneumoniae carbapenemases (KPCs) are an important mechanism of resistance for an increasingly wide range of Gram-negative bacteria and are no longer limited to Klebsiella pneumoniae [2]. K. pneumoniae has been listed as one of the most important emerging threats in this century [3].

Carbapenemases in Enterobacteriaceae are represented by three molecular (Ambler) classes of b-lactamases: A, B, and D.KPC is Ambler class A b-lactamase, which typically resides on transferable plasmids. It poses a serious clinical challenge, as KPC-producing K. pneumoniae isolates are resistant to all b-lactam antibiotics and are rapidly spreading worldwide [4].

K. pneumoniae is the most common organism associated with KPC resistance determinants; however, KPCs are being increasingly reported in other genera of the Enterobacteriaceae family and have been detected among nonfermentative bacilli, such as Pseudomonas aeruginosa and Acinetobacter spp. [5]. The blaKPC gene was first identified in 2001 from an isolate in North Carolina and was named blaKPC-1 [6]. Subsequent variants of the KPC gene were named in sequential numeric order from blaKPC-2 to blaKPC-11 [5].

Metallo-b-lactamase (MbL) is Ambler class B b-lactamase, which typically requires zinc for its catalytic activity. MbLs hydrolyze all b-lactam antibiotics, including penicillin, cephalosporin, and carbapenem, with the exception of aztreonam. MbLs encoding genes are usually encoded by mobile DNA elements with a high capacity for dissemination [7].

Transferable MbLs are notable for their constant and efficient carbapenemase activity. This is a most worrisome feature because carbapenems, which are stable against the vast majority of serine b-lactamases produced by resistant pathogens, are the antibiotics with the broadest spectrum of activity and are among the few backup agents for use against multidrug-resistant Gram-negative pathogens. Moreover, MbLs are not susceptible to therapeutic b-lactamase inhibitors (such as clavulanate) and no new inhibitor of these enzymes is yet in the pipeline [8]. Transferable MbLs are divided into six groups including IMP, VIM, GIM, SPM, NDM, and SIM types [9].

The most common mechanism of carbapenem resistance in Klebsiella spp. is the production of carbapenemases, especially KPCs and MbLs such as IMP and VIM types [10]. Carbapenemase detection among species of Enterobacteriaceae remains a challenging issue. Carbapenemase-producing K. pneumoniae with low carbapenem MICs in the susceptible range according to the Clinical and Laboratory Standard Institute (CLSI) or the European Committee of Antimicrobial Susceptibility Testing (EUCAST) has been described [11].

Treatment options for infection from these multidrug-resistant organisms are extremely limited, and uncontrolled spread may lead to treatment failure with increased morbidity and mortality [12]. Reliable detection of carbapenemases is necessary for implementing contact precautions and for outbreak detection [11].

Many techniques can be used for detecting the production of carbapenemases, from phenotypic to advanced molecular-based techniques [13]. Currently, the detection of putative carbapenemase production is based on an initial phenotypic screen for carbapenem resistance, followed by the modified Hodge test (MHT) as a confirmatory test [14]. Inhibitor-based tests using boronic acid compounds such as KPC inhibitors have recently been proposed in disk potentiation tests that efficiently differentiate KPC producers from those producing MbLs or other broad-spectrum b-lactamases [15].

The aim of this study was to determine the incidence of carbapenem resistance among clinical isolates of K. pneumoniae from hospitalized patients at Menoufia University Hospitals to detect the production of KPCsand MbLs by phenotypic and molecular methods and correlate their presence with in-vitro susceptibility of isolates to carbapenems.


  Materials and Methods Top


The study was conducted from January 2013 to June 2014. It involved 295 patients with various clinical diseases who were admitted to Menoufia University Hospitals. The patients' personal and clinical histories were taken, including age, sex, residence, associated comorbidities, history of drug administration, admission to ward and/or ICUs, duration of hospitalization and exposure to invasive procedures.

Bacterial strains

The clinical specimens (n = 295) included sputum, endotracheal aspirations, burns and wound swabs; urine, ascetic fluid, and blood samples were collected from each patient. The specimens were processed according to standard microbiological methods. A total of 128 isolates of K. pneumoniae were isolated and identified by conventional techniques [16] and by use of the VITEK 2-Compact System (bioMerieux, Marcy l'Etoile, France).

Antimicrobial susceptibility screening was performed on K. pneumoniae isolates by the disk diffusion method against different antimicrobial agents (Oxoid): piperacillin (100 µg), amoxicillin/clavulanic acid (20/10 µg), piperacillin/tazobactam (100/10 µg), cefazolin (30 µg), cefoxitin (30 µg), cefotaxime (30 µg), ceftriaxone (30 µg), cefepime (30 µg), aztreonam (30 µg), amikacin (30 µg), gentamicin (10 µg), cefoperazone (75 µg), ceftazidime (30 µg), levofloxacin (5 µg), colistin (10 µg), norfloxacin (10 µg), trimethoprim/sulfamethoxazole (1.25/23.75 µg), chloramphenicol (30 µg), and tigecycline (15 µg). The procedures were performed and results were interpreted according to the Clinical and Laboratory Standard Institute guidelines [13].

Carbapenems susceptibility testing was performed by the disk diffusion method, the agar dilution method (MIC), and the MHT. K. pneumoniae isolates were tested against imipenem (IPM), meropenem (MEM), and ertapenem (ETP) disks (10 µg for each) (Oxoid) by the disk diffusion method. The average diameters of zones of inhibition were measured and interpreted according to CLSI guidelines (2013) (for IPM and meropenem: S ≥ 23, R≤19; and for ertapenem: S ≥ 22, R ≤ 18). Evaluation of MIC was achieved by the agar dilution method using IPM powder (Sigma, USA). IPM powder (St. Louis, MO, USA) was incorporated into two-fold serial concentrations of MHA. The IPM concentration used in this experiment ranged from 0.25 to 16 µg/ml [13],[17]. A total of 80 IPM-resistant isolates were selected for further study according to resistance to one or more carbapenems by the disk diffusion method and IPM MIC.

The MHT was performed according to the CLSI guidelines (2013). The indicator organism (E. coli ATCC 25922) was inoculated on MHAplates. One disk of IPM (10 µg) was placed on the plate and three to five colonies of test and quality control organisms (QC; K. pneumoniae ATCC BAA-1705, MHT positive and K. pneumoniae ATCC BAA-1706, MHT negative; Microbiologics) were picked out and inoculated in a straight line from the edge of the disk. The streak was at least 20–25 mm in length. After incubation at 37° C for 18–24 h, the plate was examined for enhanced growth around the test or for QC organism streak at the intersection of the streak and the zone of inhibition (cloverleaf-like indentation), which is a positive indication for carbapenemase production, as shown in [Figure 1] [13].
Figure 1: Modified Hodge test. A represents negative control strain (K. pneumoniae ATCC BAA-1706). B represents positive control strain (K. pneumoniae ATCC BAA-1705). C and E represent negative isolated strains. D represents positive isolated strain. The presence of growth of the indicator strain toward an imipenem disk (an indentation) was interpreted as a positive result for carbapenem hydrolysis (carbapenemase pattern).

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Inhibitor-based methods

The phenylboronic acid combined disk (PBA-CD) test was used for detection of class A carbapenemases (KPCs), and the IPM/EDTA combined disk (IPM/EDTA-CD) test was used for detection of class B carbapenemases (MbLs).

Suspected KPCs were confirmed by the PBA-CD test. Two (10 µg) IPM disks were placed on the plate at a distance of 25 mm apart and 10 µl of a 30 mg/ml PBA solution (300 µg of PBA/disk) was added to one of the IPM disks and incubated aerobically at 37°C for 18–24 h. A filter disk containing the same amount of PBA only was placed on the PBA-CD plate and was assessed simultaneously to confirm the lack of an inhibitory effect of PBA by itself. An increase of 4 mm in the inhibition zone diameter of the IPM/PBA disk compared with the IPM disk alone revealed the presence of a KPC producer [18].

Suspected MbLs were confirmed by the IPM/EDTA-CD test. Two (10 µg) IPM disks were placed on the plate at a distance of 25 mm apart and 4 µl of sterile EDTA solution (930 µg EDTA) was added to one of the IPM disks and incubated aerobically at 37°C for 18–24 h. If the increased inhibition zone with IPM/EDTA disk was at least 7 mm compared with the IPM disk alone, it was considered MbL positive [19].

A molecular study was conducted by using multiplex PCR assay. Detectionof blaKPC, blaVIM, and blaIMP genes was carried out for all IPM-resistant isolates (n = 80) to confirm the carbapenemase status.

Plasmid DNA extraction was performed as follows: the isolates were inoculated into 5 ml of trypticase soy broth and incubated for 24 h at 37°C. Cells from 1.5 ml of an overnight culture were harvested by centrifugation for 5 min at 10 000 rpm where the pellet was resuspended in 500 µl of distilled water. The cells were lysed by heating them at 95°C for 10 min and cellular debris was removed by centrifugation for 5 min. The supernatant was used as a source of template DNA for amplification [20]. Each PCR reaction mixture (50 µl) consisted of 25 µl Taq green PCR Master Mix, 1 µl forward primer, 1 µl reverse primer (Qiagen, Germany), 1 µl template DNA, and 22 µl of nuclease-free water ([Table 1]).
Table 1 Primers used in the study (Qiagen)

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The PCR program was performed in a thermal cycler (Biometra, Germany) and consisted of an initial denaturation step at 94°C for 5 min, followed by repeated 30 cycles of DNA denaturation at 94°C for 1 min, primer annealing for 1 min, and primer extension at 72°C for 1.5 min The optimum annealing temperature was determined for each primer pair in the optimization step. Finally, an extended 72°C step for 10 min was recorded to ensure that all of the products are full-length [20]. The amplified DNA products were detected on 1.5% agarose gels by ethidium bromide staining (Sigma). A DNA ladder (100–1000 bp) (Fermentas, Germany) was used to estimate allele sizes in base pairs (bp) for the gel.

Statistical analysis

Analyses were carried out using SPSS program, version 17. The results were expressed by applying ranges, means ± SD, the χ2-test, and P values. P values less than 0.05 were considered significant.


  Results Top


This study included 295 patients (200 male and 95 female patients). The mean age of the studied patients was 54.32 ± 19.12 years.

K. pneumoniae isolates represented 43.4% of all of the isolated organisms, followed by P. aeruginosa (26.1%) and others [Figure 2].
Figure 2: Percentages of K. pneumoniae versus other isolated organisms.

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Resistance to IPM, meropenem, and ertapenem was observed in 62.5, 56.2, and 60.2% of K. pneumoniae isolates, respectively, with no significant difference between the three members of carbapenems (P > 0.05) [Figure 3].
Figure 3: Susceptibility pattern of K. pneumoniae against carbapenems.

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[Figure 4] shows the results of IPM susceptibility testing of K. pneumoniae by carbapenems susceptibility screening (the disk diffusion method, MIC determination by agar dilution, and MHT) and by inhibitor-based methods (PBA-CD and IPM/EDTA-CD tests). IPM resistance was detected in 62.5, 55.6, and 72.5% by the disk diffusion method, agar dilution method, and MHT, respectively. Class A carbapenemases (KPCs) were detected in 35% of IPM-resistant K. pneumoniae by the PBA-CD test. Class B carbapenemases (MbLs) were detected in 48.8% of IPM-resistant K. pneumoniae by the IPM/EDTA-CD test.
Figure 4: Results of imipenem susceptibility testing by the disk diffusion method, MIC determination by agar dilution, MHT, PBA-CD, and IPM/EDTA tests.

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[Table 2] shows that the highest percentage of IPM-resistant K. pneumoniae was in ICUs (32.5%), followed by the chest unit (21.3%), urology department (16.3%), burn unit (13.8%), and surgery department (10%), with no statistically significant difference (P > 0.05). In the hepatology department the percentage of IPM resistance was 5%, with a statistically significant difference (P< 0.05), and in the neonatology unit the percentage was 1.2% with no statistically significant difference (P > 0.05).
Table 2 Distribution of different specimens and imipenem-resistant K. pneumoniae isolates among different hospital departments

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[Table 3] shows that isolation of K. pneumoniae was higher among adult patients, aged 20–65 years (63.2%), with high statistically significant difference (P< 0.001) between different age groups. K. pneumoniae was isolated from male patients (59.4%) more than from female patients, with no significant difference (P > 0.05). There was statistically significant difference with regard to the duration of hospital stay of more than 14 days (43.7%), with respect to patients with invasive procedures (58.6%), and with respect to history of drug intake (64.8%). No statistically significant difference was found between patients from rural areas and those coming from urban areas and between patients with a history of associated comorbidities (53.9%) compared with those with no history of associated comorbidities. The same demographic characteristics were observed among patients harboring IPM-resistant K. pneumoniae, with the exception to statistically significant difference being found for associated comorbidities (P < 0.001).
Table 3 Demographic characters and clinical data of studied patients regarding total K. pneumoniae (n = 128) and imipenem-resistant K. pneumoniae (n = 80) isolates

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The antimicrobial susceptibility pattern of K. pneumoniae and IPM-resistant K. pneumoniae isolates is shown in [Table 4]. Hundred percent resistance from K. pneumoniae and IPM-resistant K. pneumoniae isolates was observed against piperacillin, amoxicillin/clavulanic acid, cefazolin, ceftriaxone, and cefotaxime. Also 100% resistance was detected against other studied cephalosporins (cefoxitin, cefoperazone and ceftazidime) from IPM-resistant K. pneumoniae isolates.
Table 4 Antimicrobial susceptibility patterns of K. pneumoniae and imipenem-resistant K. pneumoniae isolates

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[Table 5] shows that 10% of IPM-resistant isolates were positive for the blaKPC gene alone, 27.5% were positive for the blaVIM gene alone, and 1.25% was positive for the blaIMP gene alone. Both blaKPC and blaVIM were detected among 7.5% of isolates and blaIMP and blaVIM were detected among 3.75% of isolates. Class A (blaKPC) was detected among 17.5% and class B (blaVIM + blaIMP) was detected among 43.75%. The total number of PCR-positive isolates comprised 50% of IPM-resistant K. pneumoniae isolates.
Table 5 Results of multiplex PCR for detection of blaKPC, blaIMP, and blaVIM genes among imipenem-resistant K. pneumoniae isolates

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[Table 6] shows that all PCR-positive isolates (100%) were IPM resistant by the disk diffusion test, whereas 95% were IPM resistant by the MIC agar dilution method (sensitivity and specificity were 100% for the disk diffusion test and 95 and 17.5% for the agar dilution method, respectively). MHT detected 31/40 PCR-positive isolates (77.5%) (sensitivity was 77.5% and specificity was 32.5%). The PBA-CD test detected 12/14 blaKPC-positive isolates (85.7%) (sensitivity was 85.7% and specificity was 75.8%), whereas the IPM/EDTA-CD test detected 33/35 blaVIM-positive and blaIMP-positive isolates (94.3%) (sensitivity was 94.3% and specificity was 86.7%).
Table 6 Sensitivity and specificity of phenotypic methods in relation to genotypic method for detection of carbapenemases among K. pneumoniae isolates

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[Table 7] shows that 97.5% of cases harboring PCR-positive isolates had a history of b-lactam intake, of which 82.5% involved carbapenems, and 95%of cases harboring PCR-positive isolates had undergone invasive procedures (e.g. cannula insertion, mechanical ventilation, and urinary catheterization) with statistically significant difference (P < 0.05) for both as risk factors.
Table 7 History of drug administration and invasive procedures as risk factors among imipenem-resistant K. pneumoniae isolates in relation to PCR results

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  Discussion Top


In the present study, 128 K. pneumoniae isolates were obtained from hospitalized patients at Menoufia University Hospitals. Among these isolates, 80 K. pneumoniae isolates(62.5%) were IPM resistant by the disk diffusion method, which is much higher than what was reported by Marschall et al. [21], who detected only 2.9% of K. pneumoniae isolates having resistance to one or more carbapenems. However, in the USA, carbapenem resistance among clinical isolates of K. pneumoniae increased significantly from 0.6% in 2006 to 5.6% in 2009. In New York City Hospitals, the rate of carbapenem-resistant K. pneumoniae isolates ranged up to 24% [2].

Our results revealed that there was no significant difference between susceptibility patterns of K. pneumoniae isolates against the three carbapenems. Willems et al. [11] revealed thatertapenem is not an appropriate carbapenemase indicator as its sensitivity is too low for non-KPC class A carbapenemase detection. Further, ertapenem is not advised as an indicator because it has a lower specificity than IPM and meropenem, as isolates with AmpC/ESBL and decreased permeability have higher MICs for ertapenem than for IPM or meropenem. Also, Benenson et al. [15] documented that a disk diffusion test using IPM was superior to ertapenem and meropenem disks with sensitivity and specificity of 100 and 96%, respectively, in identifying carbapenemase-producing organisms.

The rate of isolation of IPM-resistant K. pneumoniae was highest in ICUs (32.5%) followed by the chest department (21.3%), the urology department (16.3%), and finally the neonatology department (1.2%). These results were in accordance with those of Benenson et al. [15], who reported that the prevalence of carbapenem-resistant K. pneumoniae was highest in ICUs (18.5%), followed by chest (16.4%) and urology (11.8%) departments. The high prevalence of carbapenem-resistant isolates in ICUs can be explained by the immunocompromised state of most ICU patients, heavy pressure of antibiotic use, especially carbapenems, and excessive exposure to invasive procedures, all of which increase the probability of infections.

In the current study, the 20–65-year-old group was the most infected by K. pneumoniae (63.2%). Seventy percent of IPM-resistant K. pneumoniae strains were isolated from the same age group. A similar result was obtained by Viren et al. [22], who found that 61.6% of carbapenem-resistant K. pneumoniae strains were isolated from the age group of 20–65 years at a tertiary care hospital, Gujarat, in 2008.

About 54% of our patients had underlying health impairment and associated comorbidities, with no significant difference, whereas 67.5% of patients infected with IPM-resistant K. pneumoniae had at least one underlying medical condition with significant difference. This was in agreement with a study by Ho et al. [23].

Regarding the duration of hospital stay, the current study showed that the longer the duration of hospital stay, the higher the rate of IPM-resistant K. pneumoniae isolation (55% at > 14 days, 25% at 7–14 days, and 20% at 7 days). These observations were matched by those of Graffunder et al. [24] in the USA who found that the rate of carbapenemase-producing Klebsiella spp. was 29.4% in patients undergoing 7 days of ventilation, 67.1% in patients undergoing 7–27 days of ventilation, and 100% in patients undergoing ventilation exceeding 27 days. A possible explanation for that observation is that prolonged hospital stay favors the transmission of nosocomial infections.

In this study, patients who were exposed to invasive procedures (mechanical ventilation, urinary catheterization, central venous line, etc.), especially those admitted to ICUs for a prolonged time, suffered from K. pneumoniae infections, especially because of IPM-resistant strains. This observation was in accordance with that of Helal et al. [25] at Cairo University Hospitals where they observed that 100% of KPC-positive infected cases had been exposed to invasive procedures.

In the present study, about 85% of patients infected with IPM-resistant K. pneumoniae had a history of b-lactams intake, of which 73.5% involved carbapenems. This observation was in agreement with that of Freitas et al. [26], who reported that the use of carbapenems, mainly IPM, has been indicated as one of the major risk factors for carbapenemase acquisition.

Susceptibility of the infecting isolate is one of the key factors in deciding the most suitable antimicrobial chemotherapy. In-vitro susceptibility data from numerous studies throughout the world indicate that tigecycline is one of the most effective antibacterial agents against Gram-negative bacteria producing either KPCs or MbLs [27].

The current study evaluated in-vitro susceptibility to tigecycline as a therapeutic option for IPM-resistant K. pneumoniae isolates. It revealed that 75% of K. pneumoniae isolates were sensitive to tigecycline and that 57.5% of IPM-resistant K. pneumoniae were susceptible to tigecycline. These results were in accordance with those of Livermoreet al. [28], who found that tigecycline was active against 60.5% of carbapenem-resistant K. pneumoniae isolates. In contrast, Souha et al. [29] reported that isolates may show in-vitro susceptibility to tigecycline, but clinical experience with carbapenem-resistant strains is limited. Despite tigecycline being one of the first-line agents for use in the setting of carbapenemase-producing isolates, clinical failures have been reported in the literature, as exemplified by a brief report by Anthonyet al. [30] in which some patients with multidrug-resistant Gram-negative pathogens, including ESBL-producing and KPC-producing isolates, had a negative clinical and/or microbiological outcome with tigecycline.

In another study on carbapenemases in K. pneumoniae and other Enterobacteriaceae, Tzouvelekis et al. [27] performed a systematic search to estimate the efficacies of different antimicrobials in relation to their MICs for the infecting organisms. A total of 301 patients were identified, including 161 infected with KPC-producing K. pneumoniae and 140 infected with MbL-producing K. pneumoniae. They found that the best results were obtained in patients whoreceived combination therapies including a carbapenem and their study raised concerns about the use of tigecycline as a single agent in the treatment of serious carbapenemase-producing K. pneumoniae infections and support the notion of administering drug combinations preferentially, including a carbapenem, when susceptibility data allow.

The current study applied the commercial multiplex PCR technique for detection of blaKPC, blaVIM, and blaIMP genes among IPM-resistant K. pneumoniae. The same technique was also applied by Kasse et al. [31], who documented that the sensitivity for the detection of KPC-encoding, VIM-encoding, and IMP-encoding genes by this technique was 100%.

This study showed that total class A-positive isolates comprised 17.5% and total class B-positive isolates comprised 43.75% of IPM-resistant K. pneumoniae. The total PCR-positive isolates were represented by 50%. These results were in accordance with those of Helal et al. [25] at Cairo University Hospitals who detected 22% of carbapenem-resistant K. pneumoniae strains to harbor blaKPC genes. Kasse et al. [31] observed that 35.3% of IPM-resistant K. pneumoniae isolates were KPC producers, 12.3% were blaVIM positive, and only two isolates were blaIMP-positive enzymes.

In the current study, the multiplex PCR assay revealed that97.5% of cases infected with PCR-positive IPM-resistant K. pneumoniae isolates had a history of b-lactam intake, of which 82.5% involved carbapenems. These results were in accordance with those of Helalet al. [25], who found that history of b-lactam antibiotic intake was 73.3% in cases infected with blaKPC-positive isolates, whereas history of carbapenem antibiotic intake was 46.7% in these cases. According to Kwak et al. [32], it was postulated that previous use of carbapenems was identified as an independent risk factor for the acquisition of carbapenem-resistant K. pneumoniae. The present study highlights the extensive prescription of broad-spectrum antimicrobial agents in Menoufia University Hospitals, coupled with high resistance rates among the Gram-negative bacilli surveyed. Carbapenem prescription has increased mostly as a consequence of increasing IPM prescription.

Reliable detection of carbapenemases is necessary for implementing contact precautions and for outbreak detection. However, carbapenemase detection is challenging as carbapenemase-producing K. pneumoniae isolates (harboring carbapenemase genes) with low carbapenem MICs in the susceptible range have been described [11].

In the present study, screening K. pneumoniae isolates for carbapenemases (class A and class B) was achieved by the initial disk diffusion test, followed by the IPM MIC agar dilution method and MHT. MHT cannot discriminate between KPCs and other carbapenemases. Therefore, inhibitor-based tests (PBA-CD and IPM/EDTA) have recently been proposed to efficiently differentiate KPC producers from those producing MbLs or other broad-spectrum b-lactamases [14].

With regard to CLSI guidelines [13], in our study, 38/40 PCR-positive isolates (95%) were IPM resistant by MIC and two PCR-positive isolates (5%) were IPM susceptible by MIC. However, all PCR-positive isolates were IPM resistant by the disk diffusion method (100%). These data were observed by Thomson [33], who stated that low-level resistance and even susceptibility to carbapenems have been observed for carbapenemase producers of any type. In a study by Rapp and Urban [34], most carbapenemase-producing Enterobacteriaceae had a carbapenem MIC of at least 2 µg/ml, but some had been reported to be susceptible to carbapenems. The reason for this discrepancy is that full resistance to carbapenems usually requires the presence of a second mechanism of resistance such as a defect in the permeability of outer membrane proteins.

In another study presented by Benenson et al. [15], in which they used CLSI guidelines, all strains of K. pneumoniae that were sensitive to IPM by the disk diffusion test were found to be carbapenemase negative by PCR (100%) and strains that were resistant to IPM were all found to be carbapenemase producers by PCR (100%). However, this was not detected in our study in which only 50% of phenotypic IPM-resistant K. pneumoniae isolates were carbapenemase positive by PCR. Absence of phenotypic resistance to carbapenem could be due to a lack of gene expression and a pronounced inoculum effect on MIC determinations for IPM with some KPC-producing Klebsiella spp.

The sensitivity and specificity of MHT were 77.5 and 32.5%, respectively, in relation to PCR results. Among the 58 MHT-positive isolates, 31 were PCR positive and among 22 MHT-negative isolates nine isolates were PCR positive, as explained by CLSI guidelines [13], which stated that not all carbapenemase-producing isolates of Enterobacteriaceae are MHT positive and that MHT-positive results may be encountered in isolates with carbapenem resistance mechanisms other than carbapenemase production and that some isolates showed a slight indentation but did not produce carbapenemase. MHT can give false-positive results with extended-spectrum b-lactamase-positive or AmpC-producing Enterobacteriaceae.

The phenotypic detection of KPC-producing and MbL-producing organisms was originally based on reduced susceptibility to carbapenems, which has to be confirmed by MHT. However, MHT cannot discriminate between KPCs and other carbapenemases, and obviously its positive predictive value for KPC detection is low in regions where other carbapenem-hydrolyzing enzymes, like MbLs, also prevail. In that regard, inhibitor-based tests have recently been proposed to be inhibitors that can be used in disk potentiating tests and efficiently differentiate KPC producers from those producing MbLs or other broad-spectrum b-lactamases [14]. There has been increasing interest in the use of boronic acid compounds as KPC inhibitors for the phenotypic detection of KPCs [14].

The present study evaluated the performance of the PBA-CD test by using 300 µg PBA and IPM as a substrate for inhibition of KPC production among IPM-resistant K. pneumoniae. Out of 80 IPM-resistant K. pneumoniae isolates, 28 (35%) were BA-CD test positive and 52 (65%) isolates were negative. The test was able to detect 12/14 blaKPC-positive isolates (85.7%). Thus, the sensitivity of the IPM/boronic acid synergy test was 85.7%, specificity was 75.8%, and diagnostic accuracy was 77.5% with respect to PCR results. The false-positive results were explained by Pournaras et al. [35], who stated that isolates that hyperproduced a plasmid-mediated AmpC also showed a weak synergy when synergy test using IPM and PBA was applied.

During their comparative study on boronic acid compounds for the detection of KPC-producing Enterobacteriaceae, Tsakris et al. [14] chose 127 genotypically confirmed KPC-negative Enterobacteriaceae isolates that were nonsusceptible to at least one carbapenem for testing. When they tried disks containing IPM with and without 300 µg amino-phenylboronic acid (APBA), 600 µg APBA, and 400 µg PBA, 72, 92, and 112 of the KPC producers, respectively, gave positive results (sensitivities, 64.3, 82.1, and 100%, respectively). In contrast, when they used disks containing meropenem with and without 300 µg APBA, 600 µg APBA, and 400 µg PBA, 87, 108, and 112 of the KPC producers, respectively, gave positive results (sensitivities, 77.7, 96.4, and 100%, respectively). Among KPC producers, the disk potentiating tests using meropenem and PBA demonstrated the largest differences in inhibition zones. This comparative study showed that PBA is the most effective inhibitor of KPC enzymes, and its use in CD tests with IPM or meropenem may give the most easily interpreted results [14].

The sensitivity and specificity of the IPM/EDTA-CD test were 94.3 and 86.7%, respectively, in relation to PCR results. Out of 35 MbLs-PCR-positive isolates (31 blaVIM and four blaIMP), 33 (94.3%) were IPM/EDTA test positive. The same observations were obtained by Valenza et al. [36], who stated that the IPM/EDTA test could be a valid alternative to the molecular investigation of MbL genes. They reported that an initial screening by combined susceptibility testing of IPM followed by a confirmatory EDTA test represents a valid and less expensive alternative to the molecular investigation of MbL genes. This aspect is particularly important, as it makes MbL detection possible not only in reference laboratories but also in routine diagnostic microbiology laboratories [Figure 5].
Figure 5: Agarose gel electrophoresis for the multiplex PCR-amplified products of K. pneumoniae blaKPC, blaVIM, and blaIMP genes: lane M: DNA molecular size marker (1000 bp); lane 1: positive blaVIM (382 bp) and positive blaIMP (120 bp) genes; lane 6: positive blaKPC (785 bp) and positive blaVIM (382 bp) genes; lane 7: positive blaVIM gene (382 bp); and lanes 2, 3, 4, 5, 8, 9, 10, 11, and 12: negative samples.

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  Conclusion Top


Reliable detection of carbapenemases is necessary for implementing contact precautions, for outbreak detection, and for proper choice of effective therapy. However, carbapenemase detection in Enterobacteriaceae is challenging as carbapenemase-producing K. pneumoniae with low carbapenem MICs in the susceptible range has been described. Therefore, molecular detection of carbapenemase genes by PCR has been proposed as the gold standard for detection of carbapenemase-bearing organisms.


  Acknowledgements Top


Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

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