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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 18  |  Issue : 3  |  Page : 302-309

Phenotypic and molecular detection of nosocomial carbapenem-resistant blaOXA-48 Klebsiella pneumoniae isolated from Al-Azhar Assiut University Hospital ICUs


1 Department of Microbiology and Immunology, Assiut Faculty of Medicine, Al-Azhar University, Assiut, Egypt
2 Department of Microbiology and Immunology, Cairo Faculty of Medicine, Al-Azhar University, Cairo, Egypt
3 Department of Anaesthesia and Intensive Care, Assiut Faculty of Medicine, Al-Azhar University, Assiut, Egypt

Date of Submission14-Mar-2020
Date of Decision21-Apr-2020
Date of Acceptance25-Jun-2020
Date of Web Publication30-Oct-2020

Correspondence Address:
Mostafa H.A Hammam
12 Sayed Khashaba Street, Assiut 71111
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/AZMJ.AZMJ_53_20

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  Abstract 


Purpose The aim were to detect the prevalence of blaOXA-48 carbapenem-resistant Klebsiella pneumoniae (CRKP) (K. pneumoniae) among the K. pneumoniae isolates isolated from the ICUs of Al-Azhar Assiut University Hospital by phenotypic and molecular methods and to investigate susceptibility of other alternative drugs for CRKP strains.
Patients and methods This was a hospital-based cross-sectional study that was conducted on 63 isolates of K. pneumoniae isolated from clinical samples of patients .All isolates were subjected to phenotypic and genotypic susceptibility for carbapenem resistance. For identification of K. pneumoniae, it was done using conventional methods. The test for antibiotic susceptibility was done using disc diffusion method. Strains were resistant for carbapenems, with antibiotic sensitivity testing being investigated for confirmation of carbapenem resistance. Phenotypic confirmation was done using modified Hodge test, whereas the molecular confirmation was done using PCR.
Results K. pneumoniae isolates were most frequently seen in sputum (38%) followed by blood (20%) and sputum (16%). Of 63 K. pneumoniae isolates, 24 (38%) isolates were carbapenem resistant by disc diffusion method. By using modified Hodge test, 22 (91.7%) isolates were positive by modified-Hodge test and two (8.3%) isolates were negative, which was confirmed by PCR for blaOXA-48, giving results that 22 (91.7%) isolates were positive and two (8.3%) were negative.
Conclusion OXA-48 gene is a common source of carbapenem resistance in CRKP in the hospital environment in our country. Antibiotic susceptibility testing by disc diffusion method and modified Hodge test are cost-effective and suitable methods for the initial detection of CRKP, especially when molecular detection methods are not available.

Keywords: blaOXA-48, carbapenem-resistant, Klebsiella pneumoniae, nosocomial


How to cite this article:
Hammam MH, Goda AA, Alshahat MS, Badr BM, Aly WM. Phenotypic and molecular detection of nosocomial carbapenem-resistant blaOXA-48 Klebsiella pneumoniae isolated from Al-Azhar Assiut University Hospital ICUs. Al-Azhar Assiut Med J 2020;18:302-9

How to cite this URL:
Hammam MH, Goda AA, Alshahat MS, Badr BM, Aly WM. Phenotypic and molecular detection of nosocomial carbapenem-resistant blaOXA-48 Klebsiella pneumoniae isolated from Al-Azhar Assiut University Hospital ICUs. Al-Azhar Assiut Med J [serial online] 2020 [cited 2020 Dec 4];18:302-9. Available from: http://www.azmj.eg.net/text.asp?2020/18/3/302/299572




  Introduction Top


Klebsiella pneumoniae is considered one of the most common causes of hospital-acquired infections. It has a high carrier rate in gastrointestinal tracts, nasopharnyx, and hands of patients. With increased rates of K. pneumoniae antibiotic resistance, there has been increased colonization efficiency of K. pneumoniae, allowing it to persist and spread rapidly in health care settings [1].

Owing to increased rates of multidrug resistance (MDR) among K. pneumoniae nosocomial isolates, it has urged medical doctors to use carbapenems as a ‘last resort’ against nosocomial MDR K. pneumoniae. However, the frequent use of carbapenems was a leading cause of increased numbers and mechanisms of carbapenem resistance in MDR bacteria [2].

Centers for Disease Control defines carbapenem-resistant Enterobacteriaceae as Enterobacteriaceae isolates that are nonsusceptible to doripenem, imipenem, and/or meropenem, together with resistance to ceftriaxone, cefotaxime, and ceftazidime [3].

Bacterial resistance to carbapenems may be associated with a mixture of reduced permeability of the bacterial outer membrane, excess production of extended-spectrum B-Lactamases (ESBLs) and Amp C B-lactamases, and expression of B-lactamases like carbapenemases [4].

The carbapenemases genes can be classified into classes A, B and C. KPC is a member of class A, NDM and VIM are members of class Bm, and OXA-48 is a member of class D. It is thought to be involved in rapid spread of carbapenem-resistant K. pneumoniae (CRKP) [5].

It was noted that CRKP strains are involved in hospital outbreaks and cause fatal infections in ICUs. The most common site of infection was the respiratory system, followed by catheter-related infection, surgical site, and urinary infection [6].

Good diagnosis of carbapenem-resistant organisms is an urgent issue, for choosing appropriate treatment plans and infection control measures. However, diagnosis has a number of problems, because it cannot simply rely on resistance patterns, and its methodology has not been standardized in such a way enough to date [7].

Diagnosis of production of carbapenemase enzymes is not so easy owing to some carbapenemase-producing Enterobacteriaceae demonstrating elevated but susceptible carbapenem minimal inhibitory concentrations (MICs). Centers for Disease Control has published guidelines for detection of isolates producing carbapenemases. For isolates that test susceptible to a carbapenem but demonstrate reduced susceptibility either by disk diffusion or MIC testing, performing a phenotypic test for carbapenemase activity, the modified-Hodge test (MHT), is recommended [3].


  Aim Top


The aim was to detect the prevalence of blaOXA-48 CRKP among the K. pneumoniae isolates isolated from Al-Azhar Assiut University Hospital ICUs by phenotypic and molecular methods and to investigate susceptibility of other alternative drugs for CRKP strains.


  Patients and methods Top


Patients

The present study is a hospital-based cross-sectional study that was conducted on 63 isolates of K. pneumoniae isolated from clinical samples of patients who had been admitted to the intensive care units of Al Azhar Assiut University Hospital between September 2016 and June 2018. Ethical approval to perform the study was obtained from the Ethics Committee in the Faculty of Medicine, Al Azhar University. All the included patients consented to the collection of specimens.

Materials

Sample collection

Isolates of K. pneumoniae were recovered from blood, tips of endotracheal tube, sputum, urine, and wound swabs as follows:
  1. Blood cultures were collected:
    1. In adults, 8–10 ml of blood was collected by venipuncture, under complete aseptic condition, and was inoculated immediately into blood culture bottles (Oxoid, Basingstoke, UK).
    2. In neonates, 2 ml of blood was collected by venipuncture, under complete aseptic condition, and was inoculated immediately into blood culture bottles (Oxoid).
  2. Tips of endotracheal tubes were aseptically cut using sterile scalpels and were immediately transferred to sterile cups containing trypticase soya broth as transport media.
  3. Morning sputum samples were collected using screw-capped universal containers.
  4. Urine samples were gathered after the catheter has been clamped for 10 min, and then urine samples were aspirated with syringe through the disinfected part after catheter wall was disinfected above the level of clamping.
  5. Samples from wounds were collected by sterile cotton swabs.


Sample processing

  1. Bottles of blood culture and endotracheal tubes tips were moved to microbiological laboratory for immediate aerobic incubation at 37°C.
  2. Subcultures from blood cultures and endotracheal tubes tips cultures were done on blood agar, chocolate agar, MacConkey’s agar, and Sabouraud’s dextrose agar plates and were incubated aerobically at 37°C for a whole day (24 h).
  3. Subcultures from blood cultures were done day after day, and after 14 days of no growth, they were considered negative for growth.
  4. Sputum samples, wound swabs, and urine samples were immediately transferred to a microbiological laboratory and were subjected to culture on routine laboratory media including blood agar plate, chocolate agar, MacConkey’s agar, and Sabouraud’s dextrose agar plates.


Methodology

Isolation and identification of K. pneumoniae isolates from clinical specimens

Macroscopic examination was done for colony morphology of the cultures, and the suspected colonies were examined by Gram stain. All gram-negative bacilli isolates were subcultured on MacConkey agar media for purity and further identification tests. Different standard biochemical reactions, including sugar fermentations, oxidase test, triple sugar iron agar, urease production, citrate utilization, and indole production, were performed for their identification (all the aforementioned media were obtained from Oxoid) [8].

The isolates were stored in brain heart infusion broth, to which 15% sterile glycerol was added, at −20C.

Antimicrobial-susceptibility testing by disc diffusion method according to Clinical and Laboratory Standard Institute Guidelines [9] for detection of carbapenem-resistant K. pneumoniae

Antibiotics used were as follows: amoxicillin/clavulanic acid, ampicillin/sulbactam, cefazolin, gentamicin, ceftriaxone, ciprofloxacin and trimethoprim-sulfamethoxazole, imipenem, meropenem, colistin, and tigecycline.

Bacterial colonies were suspended in sterile normal saline; the suspension was prepared with a turbidity matched to 0.5 McFarland turbidity. Muller-Hinton agar was inoculated with the prepared suspension by a sterile swab and allowed to dry for 3–5 min. The antibiotic discs were placed on the surface of the inoculated plates using a sterile forceps and were gently pressed down onto the agar to ensure complete contact with the surface. The spatial arrangement of the discs was done as they were at 15 mm from edge of the plate and 25 mm apart. The plates were incubated at 35°C for 18–24 h. Results were interpreted according to Clinical and Laboratory Standard Institute Guidelines [9].

Phenotypic detection of carbapenemases

Phenotypic detection of carbapenemases production by modified-Hodge test [9]

To uncover carbapenemase secretion in CRKP isolates, it is recommended to use MHT; this is accomplished by inoculating the CRKP isolate together with a carbapenem-sensitive indicator strain and detection of indicator strain’s inhibition zone deformation owing to carbapenemase secretion by CRKP. Regardless of its helpfulness, this test presents a burden as it recognizes the presence of carbapenemases only, without having the option to discriminate between various carbapenemase types.

Procedure

Suspension of 0.5 McFarland turbidity of the indicator organism (Escherichia coli ATCC 25922) was prepared in a sterile normal saline. Overall, 300 µl of 0.5 McFarland suspension of E. coli ATCC 25922 was added to 2700 µl sterile normal saline, to prepare a 1 : 10 dilution. Muller-Hinton agar was inoculated with the prepared (E. coli ATCC 25922) dilution by a sterile swab and allowed to dry for 3–5 min. Then, 10 µg meropenem disk was placed in the center of the agar plate. Three to four colonies of the test strain was streaked on the plate from meropenem disc toward the periphery. Positive control K. pneumoniae and negative control K. pneumoniae were streaked on the same plate. The plate was examined after 24 h of incubation.

Interpretation

  1. Modified-Hodge test positive test: carbapenemases enzyme secreting CRKP was detected by when the test isolate secrets the enzyme and allows the growth of the carbapenem-susceptible E. coli ATCC 25922 strain toward the circle prompting clover leaf-like space of the E. coli 25922 developing along the CRKP streak within the disk diffusion zone.
  2. Modified-Hodge test negative test: there is no E. coli 25922 growth along the CRKP growth streak within the disc diffusion zone demonstrating no carbapenemase enzyme secretion.


Molecular detection of carbapenemases

Polymerase chain reaction-based identification of carbapenemase genes was done.

Materials and equipment

  1. DNA thermal cycler (Biometra, Analytik Jena AG, Germany).
  2. Primers: as has been discussed later.
  3. Agarose gel (Acros Organics, Scotia court, Whitby, Ontario, Canada).
  4. Gel electrophoresis instrument (Biometra).
  5. Ethidium bromide.
  6. Base pair ladder (100 bp).
  7. Phosphate buffer saline.
  8. Tris-borate EDTA buffer (10×).
  9. Ultraviolet transilluminator.
  10. Readymade master mix (My Taq Red mix; Bioline Reagents Limited, Unit 16, The Edge Business Centre, Humber Road, London, UK).


Primers [10]

OXA-48 gene: amplicon size:300 bp.

Forward: 5′-TGTTTTTGGTGGCATCGAT-3′

Reverse: 5′-GATCGCGATTCCAAGTGG-3′

DNA extraction

DNA was extracted from all K. pneumoniae isolates manually by heat shock. From overnight cultures, two colonies of the target strain of K. pneumoniae were transferred into microcentrifuge tubes, each containing 200 µl of sterile phosphate buffer saline (PBS 1×). The cell suspension was mixed thoroughly and heated for 15 min at 100°C and then transferred into an ice tray for 2 min. Finally, the suspension was centrifuged for 10 min at 13 000 g to create supernatant containing bacterial DNA. Two microliters of the supernatant was used for PCR reaction.

PCR conditions

For each primer, the following was carried out for all isolates according to previously described methods in Zarakolu et al. [10].

A total reaction mixture of 25 µl, containing 12.5 µl of Taq PCR master mix, 8.5 µl sterile RNase-free water, 2 µl of the primer, and 2 µl of DNA template, underwent PCR amplification. Positive and negative controls were included. PCR amplification was carried out with the use of a DNA thermal cycler (Biometra), with an initial incubation at 95°C for 5 min, followed by 36 cycles at 94°C for 25 s, 53°C for 25 s, and 72°C for 25 sec, and final extension was at 72°C for 7 min.

Agarose gel electrophoresis

The resulting amplicons were separated using a 1.5% agarose gel containing 0.5 µg/µl ethidium bromide. The gel was poured in 1x TBE buffer at 100 V for 45 min in an electrophoresis system. DNA marker 100–1500 bp ladder was used as a molecular size marker.

Statistical analysis

Information was input into the computer and analyzed using IBM SPSS software package version 22.0. Information analysis was done by count percentage. The sensitivity and specificity of the phenotypic carbapenemases identification strategies were assessed by PCR as the best quality level:



where a= true positives, b=false positives, c=false negatives, and d=true negatives [11].


  Results Top


This study was conducted on a clinical sample of 385 patient admitted to the intensive care units of Al Azhar Assiut University Hospital in the period from September 2016 to June 2018. Of them, 241 (62.6%) were males and 144 (37.4%) were females. Of 385 clinical samples, 77 samples showed no growth. One microbial isolate growth was recovered in 253 samples and 55 samples recovered double microbial isolates growth. A total of 363 microbial isolates were recovered from 385 clinical samples as follows: sputum samples, 137 (37.7%); blood, 73 (20.1%); endotracheal tube samples (ETT), 58 (16%); urine samples, 33 (9.1%); and wound swabs, 62 (17.1%). Gram-negative bacilli were the most prevalent isolates (n= 154) (42.4%), followed by gram-positive cocci (n= 133, 36.6%), Candida spp. (n=42, 11.5%), and gram-negative coccobacilli (n=34, 9.4%).

Of the 154 isolate of gram negative bacilli, 63 isolates of K. pneumoniae were the subject of this study. K. pneumoniae were recovered as follows: from sputum samples, 24 (38%), from blood, 13 (20%); from endotracheal tube samples (ETT), 10 (16%); from urine samples, 5 (9%), and from wound swabs, n=11 (17%) ([Figure 1] and [Table 1]).
Figure 1 Klebsiella pneumoniae isolates on MacConkey agar.

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Table 1 Distribution of Klebsiella pneumoniae in different clinical samples

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The K. pneumoniae isolates were tested for carbapenem resistance by testing their susceptibility to meropenem. Of the 63 clinical isolates of K. pneumoniae, 24 isolates (38%) were CRKP ([Figure 2] and [Table 2]).
Figure 2 Detection of carbapenem-resistant Klebsiella pneumoniae. (a) Meropenem-sensitive Klebsiella pneumoniae isolate.

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Table 2 Frequency of carbapenem-resistant Klebsiella pneumoniae isolates

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Highest resistance was detected against amoxicillin/clavulanic acid, ampicillin/sulbactam, cefazolin, and ceftriaxone, as all CRKP isolates were resistant to them. Resistance to gentamicin, ciprofloxacin, trimethoprim-sulfamethoxazole was 87.5, 79.2, and 91.7%, respectively. The least resistance was to tigecycline and colistin, as only 33.3 and 29.2%, respectively. ([Table 3]).
Table 3 Antibiotic resistance pattern of carbapenem-resistant Klebsiella pneumoniae isolates

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CRKP isolates were tested for carbapenemase production by MHT. Among the 24 CRKP isolates, 22 (91.7%) isolates were positive by MHT and two (8.3%) isolates were negative ([Figure 3] and [Table 4]).
Figure 3 Detection of carbapenemases production by carbapenem-resistant K. pneumoniae isolates by MHT. (17 and 18) Positive MHT (leaf-like indentation of the Escherichia coli ATCC 25922 growing along the test organism growth streak within the disk diffusion zone). *(19) Negative MHT (no indentation of the E. coli 25922 along the test organism growth streak within the disc diffusion zone). MHT, modified-Hodge test.

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Table 4 Detection of carbapenemases production by carbapenem-resistant Klebsiella. pneumoniae isolates by modified Hodge test

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In this study, CRKP isolates were tested for the most common carbapenemase gene (blaOXA-48) by PCR. Among the 24 carbapenemase-producing isolates, 22 (91.6%) were blaOXA-48 producers ([Figure 4] and [Table 5]).
Figure 4 Agarose gel electrophoresis analysis of the PCR amplification products of OXA-48.

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Table 5 Detection of blaOXA-48 by PCR in carbapenem-resistant K. pneumoniae isolates

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  Discussion Top


CRKP isolates are becoming a significant emergent health challenge. CRKP infection has few treatment options, and its associated mortality rates are high. According to the data from the European Antimicrobial Resistance Surveillance Network, The resistant isolate percentages ranged from 0% (Bulgaria, Finland, Iceland, Latvia, Lithuania, and Sweden) to 59.4% (Greece) [3].

In Egypt, US Naval Medical Research Unit No 3 noted that of 594 K. pneumoniae isolates, 5.6% were carbapenem resistant in a study conducted in Egypt as part of the National surveillance 2002–2010 [12].

Prevalence detected within the present study was higher (38%). This might be explained by the continual use of carbapenems in treatment because of high prevalence of resistant strains and similar finding was reported by Metwally et al. [13] who reported high prevalence of K. pneumoniae carbapenemase-mediated resistance in K. pneumoniae isolates from Egypt (37.8%). In Tehran, a higher prevalence of CRKP was recorded by Azimi et al. [14] (64%).

In the present study, antibiotic susceptibly pattern of CRKP showed the highest resistance to amoxicillin/clavulanic acid, ampicillin/sulbactam, cefazolin, and ceftriaxone, as all CRKP isolates were resistant to them. These results are in accordance with Shawky et al. and El-Ghazzawy et al. [15],[16], who reported that 100% of CRPK isolates were resistant to ampicillin, ampicillin/sulbactam, cefazolin, ceftriaxone, imipenem, and meropenem. Overall, 87.5, 78.8, and 91% were resistant to aminoglycosides, ciprofloxacin, and trimethoprime-sulfamethoxazole, respectively. A less resistance pattern was recorded by Baran et al. [17] who reported resistance pattern to aminoglycosides and trimethoprime-sulfamethoxazole of 63.8 and 55%, respectively.

The widespread prevalence of antimicrobial resistance among K. pneumoniae signifies a noteworthy clinical issue because of constrained treatment alternatives, as notwithstanding being resistant in vitro to all B-lactams and carbapenems, isolates are additionally being nonsusceptible to quinolones and aminoglycosides, leaving the helpful alternatives restricted to tigecycline or colistin [18].

However, reports of colistin-resistant and tigecycline-resistant isolates have surfaced; these isolates are generally known as pan-resistant, according to their resistance to all routine antibiotics. Panresistant K. pneumoniae spread in ICUs could prompt critical morbidity and mortality [19].

The highest sensitivity of CRKP isolates was to tigecycline and colistin at 66.7% and 70.8%, respectively. Similar findings were detected by Shawky et al. [15], as they found that tigecycline and colistin were active against 82.7 and 86% of CRKP, respectively.

CRKP isolates were tested for carbapenemase production by MHT. Among the 24 CRKP isolates, 22 (91.7%) isolates were positive by MHT and two (8.3%) isolates were negative. Similar result was obtained by Celikbilek et al. [20], as they detected 97.8% of CRKP isolates were positive with MHT.

In contrast to these results, in another study, MHT positivity was found only in 1 of 7 isolates (14.2%) with carbapenemase genes [21], and it was concluded that MHT might be false negative, especially in the presence of ESBL or AmpC with reduced porine activity.

In this study, CRKP isolates were tested for the most common carbapenemase genes (blaOXA48) by PCR. Among the 24 carbapenemase-producing isolates, 22 (91.6%) were blaOXA-48 producers. Rapid emergence of OXA-48 is disturbing. Frequency of bla OXA-48 is more than the other carbapenemases (KPC, NDM, IMP, and VIM) globally [22]. Identification of class D OXA-48 is usually in E. coli and K. pneumoniae. Transferrable plasmid of OXA-48 is related to its spread generally and inter-species dissemination [23].K. pneumoniae isolates producing OXA-48 carbapenemase were first identified in the Middle East (in Turkey) and has rapidly spread globally [24]. OXA-48 is considered the most common carbapenemase in the Middle-Eastern countries. Moreover, OXA 48-positive and NDM-positive K. pneumoniae have also been isolated from Saudi hospitals as well as many countries in the Arabian Peninsula, and both enzymes have been previously described as major carbapenemases of Enterobacteriaceae in countries in the Arabian Peninsula [25]. Similar findings were reported by Zarakolu et al. [10] as nearly all CRKP isolates in their study were producers of OXA-48-like enzymes. These results are consistent with those of Shibl et al. and Al-Zahrani et al. [25],[26], who detected OXA-48 in 78 and 81.5% of K. pneumoniae isolates, respectively.

By comparing modified Hodge test with OXA-48 genes for detection of carbapenemase production in the present study, of 22 OXA 48-producing clinical isolates, 21 (95.5%) were positive using the MHT, and one of the 22 OXA 48-producing isolates yielded negative results with the MHT (95.5% sensitivity).

It is known that MHT is reliable for detection of OXA-48-like enzymes [27]. A similar result was detected in a study of Doyle et al. [28] who found MHT had a sensitivity of 93% for OXA-48-like producers and 98% for detection of KPC-producers. In contrast to this result, Baran et al. [17] recorded that the sensitivity of MHT for blaOXA-48-positive isolates was found 70.9%.


  Conclusion Top


CRKP isolates represent a major cause of morbidity and mortality in hospitals and especially in intensive care units. However, the diagnosis and detection of suitable antibiotic for these patients is an issue. The phenotypic methods (disc diffusion antibiotic susceptibility test, modified Hodge test, and combined Meropenem disc test) and the genotypic method (PCR) were done for diagnosis of CRKP. The most common source of carbapenem resistance in CRKP in the hospital environment was OXA-48 gene in our country. Modified Hodge test was a cost-effective and suitable method for the initial detection of CRKP, especially when molecular detection methods are not available.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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