Detection of OXA-48-Carbapenemase-Producing Enterobacteriaceae using ChromID OXA-48 in Critical Care Patients in Egypt

ABSTRACT:

Carbapenems are a class of beta-lactam antibiotics with broad spectrum of activity. They are often considered as a last resort in treatment of infections caused by multidrug resistant organisms. Carbapenemase-producing Enterobacteriaceae (CPE) have been reported worldwide. Class D OXA-48 carbapenemases is rapidly disseminating in Enterobacteriaceae leading to high mortality from resistant and invasive CPE infections. In the present study, we attempted to isolate OXA-48 carbapenemase-producing Enterobacteriaceae from different clinical specimens obtained from hospitalized patients at different ICU of kasr Kasr Al-Ainy hospital, Cairo University. Initial screening for carbapenemase-producing Enterobacteriaceae was done using ertapenem disc diffusion method and direct inoculation of the specimens into ChromID OXA-48. The phenotypic Modified Hodge Test (MHT) was used for confirmation of carbapenemse production among screened carbapenem resistant isolates.Out of 112 collected samples, 94 Enterobacteriaceae were isolated. Fifty five isolates (58.5%) were ertapenem disc resistant and 50 isolates (53%) showed positive growth on ChromID OXA-48. Fifty two (94.5%) out of 55 suspected carbapenemase-producing isolates by disc diffusion method and the 50 isolates (100%) grown on ChromID OXA-48 were MHT positive. Our study underlines the need to detect OXA-48 CPE as early as possible to minimize its spread in ICU and apply appropriate infection control measures.

KEYWORDS:

Enterobacteriaceae, class D OXA-48 carbapenemase, ChromID OXA-48, Modified Hodge Test.

INTRODUCTION

ICU patients are important potential reservoirs of CPE because of the severity of illness and the antibiotic selection pressure. The most important challenges for ICU practitioners are to identify admissions with the highest risk of communicable disease and to rapidly implement preventive measures in order to avoid nosocomial transmission (Memish et al., 2015).

Carbapenems are often used as last-line agents when patients with infections become gravely ill or are suspected of harboring resistant bacteria.  The rapid emergence and dissemination of Enterobacteriaceae that are resistant to carbapenems poses a considerable threat to clinical patient care and public health. Carbapenemase-producing strains are characterized by their resistance to virtually all beta-lactam antibiotics, including the cephalosporins and carbapenems, as well as to flouroquinolones, aminoglycosides and co-trimoxazole. Invasive infections with these strains are associated with high rates of morbidity and mortality (Gaibani et al., 2011; Lee et al., 2016).

Carbapenemase-producing Enterobacteriaceae (CPE) have been increasingly identified worldwide. Class A (KPC), Class B (VIM and IMP) and Class D (OXA-48) carbapenemase producing isolates have been involved in nosocomial outbreaks (Djahmi et al., 2014).This global spread of carbapenemase-producing Enterobactericeae has made the development of a simple test a desirable goal (Kim et al., 2015). A disc diffusion test using ertapenem (10μg) is the most sensitive screening test to identify CPE (CLSI, 2016).

Chromogenic media allow the presumptive identification of bacteria within 24 hours of incubation on the basis of clonal morphology and distinctive color patterns. These media have been shown to improve the culture based methodology by decreasing the turnaround time and by increasing the differentiation and detection of the target microbes of interest (Akteret al., 2014). Studies have highlighted the potential difficulty in isolating CPE, as such isolates often have low carbapenem MIC and maybe inhibited by some selective media that contain carbapenems(Yamamoto et al.,2017).

A chromogenic media (ChromID OXA-48) has been shown to identify class D CPE (Girlish et al., 2013). ChromID OXA-48 is the only selective media for identification of class D OXA-48 with very accurate sensitivity and specificity (Zarakolu et al., 2015).

Aim of the work

The purpose of the present study is to compare the results of the chromogenic medium ChromID OXA-48 used for rapid detection of carbapenemase-producing Enterobacteriaceae(CPE) from clinical samples with disc diffusion method results as a screening test and to compare both methods with the phenotypic confirmatory test (Modified Hodge Test).

Patients and Methods

The present study was conducted on 105 patients admitted at general surgery, neonatal, obstetric and chest intensive care units (ICU) of Kasr El-Ainy hospitals, Cairo University during the period from February 2016 through August 2016. The study included 60 males and 45 females their ages range from few days to 87years. A total of 112 different samples were obtained including 38 urine samples, 27 wound swabs, 25 sputum samples, 15 endotracheal tube aspirates and 7 blood samples. The study was carried out in the Department of Microbiology and Immunology, Faculty of Medicine, Cairo University.This study have been approved by the institutional research ethics committee and have been performed in accordance with the ethical standards.

 

Sample collection:

Different clinical samples were collected from ICU hospitalized patients after taking their informed consent, under complete aseptic conditions using sterile containers, swabs, suction catheters, syringes as follows:

– Wound swabs:

Twenty seven wound swab samples were collected from surgical site infections using sterile swabs.

– Sputum samples:

Twenty five morning sputum samples were collected using screw capped universal containers.

– Endotracheal tube aspirates:

Fifteen endotracheal  aspirates  were  obtained  from  patients  by  mean  of sterile  suction  catheters.  A portion of the catheter containing significant amount of the aspirate was aseptically cut into a sterile container.

– Urine samples:

Thirty eight catheter specimens of urine (CSU) were collected after clamping the catheter for 10 minutes, then the wall of the catheter was disinfected above  the  level  of  clamping  and  CSU were  aspirated  with  syringe through the disinfected part.

– Blood samples:

Seven blood samples were collected using a sterile syringe; 5 ml of blood was withdrawn after disinfection of the skin.

All specimens were labeled with the date, patient’s name, patient’s number, time of collection, specimen type, and then transported immediately to the microbiology department.

CULTIVATION OF THE SAMPLES

            Different   clinical  samples other  than  the  blood  were   cultured  on MacConkey’s agar plates number 3 (CM 0115 ) (Oxoid Ltd.,  Basingstoke,  Hampshire, England)   and  the  chromogenic  media Chrom ID OXA-48  agar  plates (BioMérieux, France). The plates were incubated aerobically at 37 ͦ C for 24-48 hours.

Chrom ID OXA-48 agar is a selective chromogenic medium for the screening of OXA-48 type carbapenemase–producing Enterobacteriaceae (CPE). It consists of a rich nutritive base combiningdifferent peptones that containsa mixture of antibiotics which enable selective growth of CPE in addition to three chromogenic substrates which enable the identification of the most frequently isolated CPE.

Blood  samples , 5 ml each,  were inoculated into blood culture bottles and  mixed  with the medium (Oxoid Ltd.,  Basingstoke,  Hampshire, England), then  incubated  aerobically  at 35 ͦ C . The medium is designed to create pressure in the sealed bottle when the organisms are growing. The detection of positive pressure is by means of a growth indicator device which is connected to the bottle after the blood sample is added. A positive result is indicated when the blood/broth mixture rises above the green locking sleeve of the growth indicator device. Subcultures were done on MacConkey’s and chrom agar. The blood culture bottles were considered negative after 14 days if no growth of the subculture was detected.

IDENTIFICATION OF THE ISOLATES

Identification of the isolates was done according to the conventional microbiological standard tests (Gram stain, glucose fermentation test and oxidase test). Isolates on MacConkey’s agar identified as Gram negative bacilli, glucose fermenters and oxidase negative were considered Enterobactericeae (Brookset al, 2007). Identification of different isolates was done using biochemical reactions (TSI, urease test, citrate test, MIO, LIA and oxidase tests) the interpretation in discussed in Table (1). Colonies on chromogenic media were subjected to biochemical reactions for further confirmation.

IṾ- Carbapenemase screening tests:

Isolates   were    screened   for   carbapenemase  production  using ertapenem (ETP) disc  (Oxoid, UK)  and   the chromogenic  medium ChromID OXA-48.The results of ertapenem disc were interpreted according to the standard guidelines (CLSI., 2016).

  1. Disc diffusion method (CLSI, 2016):
    • The diameters of the inhibition zones were measured in millimeters and compared to the reference table (Table 1) to differentiate the isolates into susceptible(S), intermediate(I) or resistant(R).
    • Carbapenemase –producing isolates of Enterobacteriaceae usually test intermediate or resistant to one or more carbapenems using the interpretive criteria according to (CLSI, 2016).
  2. Chromogenic media (ChromID OXA-48):

On the chromogenic media ChromID OXA-48, carbapenemase producing  E.coli produce pink to burgundy color, while carbapenemase producing KlebsiellaEnterobacterSerratiaand Citrobacter isolates produce bluish-green to bluish-grey color.

  1. V) Phenotypic confirmatory test for carbapenemase production

Isolates that were intermediate or resistant to ertapenem or yeilded positive growth on ChromID OXA-48 media were subjected to the confirmatory Modified Hodge Test (CLSI, 2016).

THE MODIFIED HODGE TEST (CLSI, 2016)

  • A 0.5 McFarland turbidity standard suspension of E.coli ATCC 25922 was diluted 1:10 in sterile physiological saline solution (NaCl 0.9%).
  • A Muller-Hinton agar plate was inoculated with this dilution as for disc diffusion test.
  • An Ertapenem disc (Oxoid, UK) was placed on the plate.
  • An inoculation loop or swab was used to pick up three to five colonies of test organism grown overnight on blood agar and were streaked from the edge of the ertapenrm disc. The streak was 20-25 mm long. Three to four isolates were examined in the same plate.
  • The plate was incubated overnight at 35 ± 2 ͦ C in an aerobic atmosphere.
  • Following incubation for 16-20 hours, the intersection of the edge of the inhibition zone and the streak of the test isolate was examined.
Reading and Interpretation:
  • Indentation of the inhibition zone(s) indicated that the test organism hydrolyzes carbapenems.
  • The test result is non –determinable if the growth of E.coli ATCC  25922 was inhibited by the test isolate (inhibition zone parallel to the streak of the test isolate)
  • The test is considered negative for carbapenemase production if there was no enhanced growth.
Statistical analysis:

Data were statistically described in terms of mean ± standard deviation (± SD), and range, or frequencies (number of cases) and percentages when appropriate. Comparison of numerical variables between the study groups was done using Chi-square (c2) test. Exact test was used instead when the expected frequency is less than 5. P values less than 0.05 was considered statistically significant. All statistical calculations were done using computer program IBM SPSS (Statistical Package for the Social Science; IBM Corp, Armonk, NY, USA) release 22 for Microsoft Windows.

Results

The current study was conducted on 105 patients of different intensive care units of Kasr Al-Ainy hospitals, Cairo University during the period from February 2016 through August 2016. A total of 134 isolates were recovered. The distribution of isolates among collected samples was as follows: 40 isolates (30%) were isolated from urine samples, 35 isolates (26%) from wound swabs, 30 isolates (22.4%) from sputum samples, 22 isolates (16.4%) from endotracheal tube aspirates and 7 isolates (5.2%) from blood samples. The isolates were identified using the standard biochemical reactions (TSI, urease test, citrate test, MIO, LIA and oxidase tests) as 94 (70%) Enterobacteriaceae species, 25 (17%) Acinetobacter spp. and 15 (11.2%) Pseudomonas spp.. The Enterobactericeae isolates were further identified to 35 (37.2%) E. coli, 57 (60.6%) Klebsiellaspp. and 2 (2%)Proteusspp. as shown in Table 2.

Table 1: Inhibition zone diameters interpretive standards for Enterobacteriaceae(CLSI, 2016)

Antimicrobial agent Disc content(µg) Zone diameter(mm)
S I R
Ertapenem 10 ≥ 22 19-21 ≤18

Ertapenem non susceptibility is the most sensitive indicator of carbapenemase production (CLSI, 2016).

Table 2: Distribution of Gram-negative bacilli isolates recovered from different clinical samples

Type and number of specimens E.coli KlebsiellaSpp. ProteusSpp. AcinetobacterSpp. PseudomonasSpp. Total number of isolates
Urine (38) 25 11 0 3 1 40
Wound swab(27) 7 15 0 8 5 35
Sputum (25) 1 13 0 11 5 30
Endotracheal tube aspirates(15) 2 11 2 3 4 22
Blood (7) 0 7 0 0 0 7
Total (112) 35 57 2 25 15 134

 

I- CARBAPENEMASE SCREENING TESTS

1-Disc diffusion method

Out of 94 Enterobacteriaceae isolates, 55 isolates (58.5 %) were resistant to ertapenem disc (49 Klebsiella and 6 E.coli isolates), while 39 isolates (41.5%) were sensitive (8 Klebsiella., 29 E.coli and 2 Proteus isolates) fig. 1.

Fig. 1: a- Ertapenem sensitive isolate (zone diameter >22mm)

           b- Ertapenem resistant isolate (zone diameter <18mm)

2- Chromogenic media (ChromID OXA-48)

            Out of 94 Enterobacteriaceae isolated from 112 clinical samples which were directly inoculated intoChromID OXA-48, 50 Enterobactericeae isolates were observed. Forty two Klebsiella isolates were observed as green colonies, while 8 E.coli isolates showed pink colonies on the chromogenic media. All the 50 isolates grown on ChromID OXA-48 were resistant to ertapenem disc. Number and percentage of Enterobacteriaceae grown on Chrom ID OXA-48 agar are illustrated in table 3.

Comparison between Chrom ID OXA-48 and ertapenem disc diffusion results

            Out of 55 ertapenem disc resistant isolates, 50 (91%) Enterobacteriaceaeisolates grew on the Chrom ID OXA-48 agar Table4.

 П- MODIFIED HODGE TEST AS PHENOTYPIC CONFIRMATORY TEST

  1. Ertapenem disc diffusion

Out of the 55 suspected carbapenemase-producing Enterobacteriaceae isolates (resistant to ertapenem), 52 isolates (94.5%) were found to be MHT positive as evidenced by the indentation of the inhibition zones of ertapenem discs, while 3 isolates (5.5%) were found to be MHT negative as there was no indentation of the inhibition zones Table 5& fig. 2.

Fig. 2: Modified Hodge Test

Isolates number 4, 12, 33 and 36 showed positive results. Isolates number 13 and 34 were negative.

Table 3: Number and percentage of Enterobacteriaceae isolated on Chrom ID OXA-48 agar

Growth on chrom agar No & percentage
E. coli 8 (8.5%)
Klebsiella spp. 42 (44.6%)
Total number of Enterobacteriaceae 94 (100%)

 

Table 4: Comparison between Chrom ID OXA-48 and ertapenem disc diffusion results for 94 Enterobacteriaceae isolates

Ertapenem disc resistant isolates Isolates grown on ChromID  OXA-48
55 (58.5%) 50 (53.2%)

 

Chrom ID OXA-48

Testing the 50 isolates by ChromID OXA-48 using MHT showed statistically significant agreement between both results (p value = 0.001), the 50 positive isolates by ChromID OXA-48 gave also positive results with MHT Table 6.

Ш-Distribution of carbapenemase-producing Enterobacteriaceae among different specimens

The results of the screening tests (ertapenem disc diffusion and ChromID OXA-48) and the confirmatory MHT for each specimen are illusterated in Tables 7 & 8.

The total prevalence of CPE among 94 Enterobactericeae isolates was 55% (52/94). The frequency of CPE spread among 112 samples collected from ICUs was 46.5% (52/112).

DISCUSSION

Carbapenemase producing Gram-negative pathogen are of great concern for physicians. The challenging aspects are the treatment options and infection control measures. Monitoring of the respective carbapenem resistance mechanism is necessary to prevent outbreaks. Carbapenemase production is the main mechanism of carbapenem resistance in Enterobacteriaceae. The most prevalent carbapenemases in Enterobacteriaceae are KPC, VIM, NDM and OXA-48 (Shahcheraghiet al., 2017).

The rapid emergence of oxacillinases (OXA-48 and OXA-48 like enzymes) is alarming. The frequency of OXA-48 is more than the classical carbapenemases (KPC, NDM, IMP, and VIM) across the world (Bakthavatchalam et al., 2016). Class D OXA-48 is commonly identified in E. coli and Klebsiellapneumoniae. The transferrable plasmid of    OXA-48 is associated with rapid spread and inter-species dissemination(Temkin et al., 2014).

Table 5: Confirmation of disc diffusion test results by Modified Hodge Test

MHT No & percentage
Positive 52 (94.5%)
Negative 3 (5.5%)
Total ertapenem disc resistant isolates 55 (100%)

 

Table 6: Confirmation of ChromID OXA-48 results by Modified Hodge Test

MHT No & percentage
Positive 50
Negative
Total number of isolates grown on ChromID OXA-48 50 (100%)

 

(p value < 0 .001is considered statistically significant)

             Identification of carbapenem resistance among Enterobacteriaceae is of primary importance since carbapenemase producers are resistant not only to most (if not all) β-lactams but also to other main classes of antibioticsincluding quinolones, aminoglycosides and trimethoprim–sulfamethoxazole (Hara et al., 2013).

            Reliable detection of carbapenemases is necessary to implement contact precautions to prevent hospital outbreaks (Cuzon et al., 2011 and Melake et al., 2016). The CLSI guidelines for the phenotypic detection of carbapenemase-producing Enterobacteriaceae is based on an initial screening test for carbapenem resistance by the disc diffusion method or MIC determination, followed by the MHT for confirmation (CLSI., 2017)

In the present study, we attempted to determine the presence of class D OXA-48 carbapenemases among 112 clinical samples by disc diffusion method and the chromogenic media (ChromID OXA-48), to compare both methods with Modified Hodge Test as a confirmatory phenotypic test and to estimate the frequency of CPE in critical care patients of Kasr Al-Ainy hospital.

In the current study, out of 112 clinical samples, 94 Enterobacteriaceae were isolated on MacConkey’s agar plates and identified by the conventional biochemical reactions. The antimicrobial susceptibility test was done for the 94 Enterobacteriaceae isolates using ertapenem discs as an initial screening method. Fifty five isolates (58.5%) showed resistance to ETP disc, while 39 isolates (41.5%) were sensitive. The high prevalence of carbapenem non-susceptibility in the current study could be explained by the fact that our samples were collected from ICU. Patients within the ICU underwent invasive procedures, treatment with antibiotic combinations and were exposed to other patients with multi-drug resistant pathogens. (Cisneros et al., 2005;Parveen et al., 2010 and Morrill et al., 2015).

However, lower rates were reported by study done by Mcgettigan et al., (2009) who stated that 3% of Enterobacteriaceae isolates were ETP resistant. Another study performed at Suez Canal university hospital by(Metwally et al., 2013) stated that 44.4% of Enterobacteriaceae isolates were ertapenem resistant.

Table 7: Comparison between screening tests results for each sample

Type of samples / number of EnterobacteriaceaeIsolates Growth on ChromID  OXA-48 ETP disc diffusion
Positive Negative Resistant Sensitive
Urine samples (36) 8 28 10 26
Wound swabs (22) 13 9 16 6
Endotracheal tube aspirates (15) 10 5 11 4
Sputum samples (14) 14 0 12 2
Blood samples (7) 5 2 6 1
Total  (94) 50 44 55 39

 

Table 8: Modified Hodge Test results in comparison to screening tests.

MHT
Positive Negative
ETP resistant isolates (55) 52 (94.5%) 3 (5.5%)
ChromID OXA-48 positive growth isolates (50) 50 (100%)

 

On the other hand, a higher prevalence of ETP resistance was reported in a study conducted by Birgy et al, (2012) in a teaching hospital in Paris, where the disc diffusion method revealed that 91% of Enterobacteriaceae isolates were non susceptible to ETP. Those variations in the prevalence of carpabenemase-producing Enterobacteriaceae could be explained by different sample size as well as different geographical distribution.

In the present study, 55 isolates (49.1%) fulfilled the CLSI criterion for performing carbapenemase detection by the MHT (they were non- susceptible to ETP). These isolates were tested for the presence of carbapenemases by the MHT using substrates recommended by the CLSI (2016). Out of 55 isolates, 52 (94.5%) proved to be carbapenem resistant.

This result was in line with previous study done by Deshpande et al., (2010) showed that 22 (91.6%) out of 24 carbapenem resistant Enterobacteriaceae were MHT positive. Birgy et al., (2012) stated that 95% (21/22) of tested isolates produced positive results with MHT. Mosca et al., (2013) showedthat32 isolates out of 38 (84%) gave positive results by MHT.

In disagreement with the current study, Lari et al., (2013) reported that 19 out of 35 (54%) gave positive results with MHT. The later result showed partial agreement with studies done by                         Castanheira et al., (2011) andAmbretti et al., (2013) who reported that 26/39 (66.7%) and 71/108 (65.7%) Enterobacteriaceae isolates were positive by MHT, respectively. Meanwhile, lower rates than that of the current study was reported by Kiratisin and Henprasert, (2010) who found that out of 35 isolates suspected of carbapenemase production, only 6 (17%) isolates yielded positive results with MHT. Saharman and Lestari, (2013) also stated that 8 out of 29 CRE (27.59%) were MHT positive.Another study done in Bayero University, Kano, Nigeria by Yusuf et al., (2012) found that out of 135 Enterobacteriaceae, 19 isolates (14%) gave positive results by MHT. Moreover, Balan et al., (2012) tested 200 Enterobacteriaceae for carbapenemases production by MHT and reported that 45 isolates (22.5%) found to be CPE.

In the current study, results of Chrom ID OXA-48 differed from disc diffusion results, 50 Enterobactericaea isolates (53%) were observed as growth of colored colonies. All those fifty isolates were MHT positive (100% agreement).Klebsiellapneumoniea was the most dominant species and was isolated from 42 (44.6%) patients.

A study in Christian Medical College, India done by Bakthavatchalam et al., (2016)reported that out of 117 CPE screened isolates, 106 isolates (90.5%) were positive by Chrom ID OXA-48 media. Chrom ID OXA-48 is the only available chromogenic medium for selective identification of OXA-48. This result showed agreement with study conducted by Girlich et al., (2013) who showed 54 (94.5%) out of 57 class D OXA-48 isolates were recovered using ChromID OXA-48 medium.

A lower rate was reported by Zarakolu et al., (2015) at a university hospital in Turkeywho showed that 25 out of 33 CPE (75.8%) were found to be colonized by class D OXA-48 CPE. The failure to isolate CPE on ChromID OXA-48 was probably attributable to either relatively low amount of CPE in the sample or over growth by other bacteria. Another explanation of this find may due to presence of other classes of carbapenemases.

In our study the most prevalent CPE was Klebsiella (44.6%) and   E.coli (8.5%). Similarly, Ling et al., (2012) found that CPE were commonly seen in Klebsiellapneumoniae (42.2 %), E.coli (24.3 %) in 268 isolates.Another study done by Gijónet al., (2012) showed that the most common carbapenemase-producing Enterobacteriaceae identified from fecal samples were Klebsiellapneumoniae (72.7%) and                   E.coli (9.1%).

CONCLUSION

Antimicrobial resistance due to continuous selective pressure from the widespread use of antibiotics is a growing public health problem. OXA-48 enzymes are emerging as an important mechanism of resistance in Enterobacteriaceae. The prevalence of carbapenemase in Enterobacteriaceae by disc diffusion method and ChromID OXA-48 is 58.5 % (55/94) and 53% (50/94) respectively. Screening by disc diffusion method showed that 55 (58.5%) out of 94 Enterobacteriaceae isolates were ertapenem disc resistant. Screening by ChromID OXA-48 showed that out of 94 Enterobacteriaceae isolates collected from 112 samples which were directly inoculated into ChromID OXA-48, 50 Enterobacteriaceae isolates (53%) gave the expected color pattern on the chromogenic agar. The phenotypic confirmatory test (MHT) was used to confirm the results of both screening methods (disc diffusion and chromogenic media) as it is recommended by CLSI. Out of 55 ertapenem resistant isolates, 52 isolates (94.5%) were MHT positive, while the 50 chrom agar positive isolates were all positive by MHT (100%).

ACKNOWLEDGMENTS

This study was funded from Cairo University Faculty of Medicine Egypt.

Author contributions

All authors contribute equally in conception, design, practical work, supervision, manuscript writing, editorial help, collection of data and data analysis through the whole study.

Compliance with ethical standards

Conflict of interest: The authors declare that they have no competing interests.

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