|Year : 2015 | Volume
| Issue : 4 | Page : 223-227
Phenotypic determination of carbapenemase producing enterobacteriaceae isolates from clinical specimens at a tertiary hospital in Lagos, Nigeria
OO Oduyebo1, OM Falayi2, P Oshun1, AO Ettu1
1 From the Department of Medical Microbiology and Parasitology, Lagos University Teaching Hospital, Idi-Araba, Lagos, Nigeria
2 Department of Medical Microbiology and Parasitology, College of Medicine, University of Lagos, Idi-Araba, Lagos, Nigeria
|Date of Web Publication||14-Jan-2016|
O M Falayi
Department of Medical Microbiology and Parasitology, College of Medicine, University of Lagos, Idi-Araba, Lagos
Source of Support: None, Conflict of Interest: None
Aims and Objectives: Carbapenemase production among clinical isolates of Enterobacteriaceae has been widely reported with prevalence rates ranging from between 2.8% and 53.6%. The aim of this study was to assess the prevalence of carbapenemase production among clinical isolates of Enterobacteriaceae from a Tertiary Hospital in Lagos, Nigeria, and to characterize the type of carbapenemase produced.
Materials and Methods: Carbapenemase production was detected phenotypically using a commercially available combination disc test (Rosco Diagnostica carbapenemase detection Neo-Sensitab) containing inhibitors to the various carbapenemase classes. The Neo-Sensitabs were used for Enterobacteriaceae isolates that were resistant after the initial antibiotic susceptibility testing with meropenem (10 μg).
Results: A total of 177 Enterobacteriaceae isolates were investigated and 27 (15.2%) were carbapenem-resistant. From the 27 carbapenem non-susceptible isolates, 22 (12.4%) were carbapenemase producers while 5 (2.8%) exhibited carbapenem resistance due to extended spectrum beta-lactamase production. Of the 22 isolates that were positive for carbapenemase production, 15 (8.5%) were metallo beta-lactamase (MBL) producers, 6 (3.4%) produced oxacillinase-48 while 1 (0.5%) produced both MBL and Klebsiella pneumoniae carbapenemase. Hence, the overall prevalence of carbapenemase-producing Enterobacteriaceae in this study was 12.4%.
Conclusion: Carbapenemase-producing Enterobacteriaceae was indeed prevalent in our institution. The combination disc test was a cost effective and suitable method for the initial detection of carbapenemase-producing Enterobacteriaceae within the clinical setting especially when molecular detection methods are not available.
Keywords: Carbapenemase, carbapenems, Enterobacteriaceae, resistance
|How to cite this article:|
Oduyebo O O, Falayi O M, Oshun P, Ettu A O. Phenotypic determination of carbapenemase producing enterobacteriaceae isolates from clinical specimens at a tertiary hospital in Lagos, Nigeria. Niger Postgrad Med J 2015;22:223-7
|How to cite this URL:|
Oduyebo O O, Falayi O M, Oshun P, Ettu A O. Phenotypic determination of carbapenemase producing enterobacteriaceae isolates from clinical specimens at a tertiary hospital in Lagos, Nigeria. Niger Postgrad Med J [serial online] 2015 [cited 2019 May 22];22:223-7. Available from: http://www.npmj.org/text.asp?2015/22/4/223/173973
| Introduction|| |
The Enterobacteriaceae are a large family of Gram-negative rods found primarily in the colon of humans and other animals, many as part of the normal flora.  As an important cause of nosocomial and community-acquired bacterial infections,  the main-stay for treatment of infections caused by the Enterobacteriaceae was the beta-lactam group of antibiotics.  Excessive use of the beta-lactams in treating infections caused by the Enterobacteriaceae has selected strongly for the resistance displayed by this group of organisms in recent years, the major mechanism being beta-lactamase production. , Consequently, carbapenems became a preferred option among clinicians in the treatment of infections caused by multidrug-resistant Enterobacteriaceae isolates.  This was probably due to the fact that they are relatively resistant to inactivation by most beta-lactamases. 
Several studies have confirmed the presence of carbapenem-resistant Enterobacteriaceae, especially within the hospital setting. ,,, The more common mechanism of carbapenem resistance among the Enterobacteriaceae was the production of carbapenemases which are specific beta-lactamases with the ability to hydrolyse the carbapenems.  They belong to three classes of beta-lactamases based on the Ambler classification scheme namely: Class A family consisting of the Klebsiella pneumoniae carbapenemase (KPC), IMI, SME and GES enzymes, the Class B family which may also be referred to as metallo beta-lactamases (MBL) and include the IMP, VIM, NDM, and SPM enzymes and the Class D family which consists of the oxacillinases (OXAs) most notable of which was the OXA-48 enzyme. 
Carbapenemase production among Enterobacteriaceae has been reported in several studies. In New York City, 39% of patients had faecal colonisation with KPC-producing Klebsiella pneumonia.  In Morocco, the prevalence of carbapenemase production among clinical isolates of Enterobacteriaceae was determined to be 2.8%.  In Kano, Northern Nigeria prevalence of carbapenemase production among Enterobacteriaceae isolates from patients at Aminu Kano Teaching Hospital was 11.8%. 
The effects of carbapenemase production among Enterobacteriaceae were far reaching. It causes increased morbidity and mortality among patients with infections caused by these carbapenemase-producing bacteria.  There was also the issue of increased healthcare costs due to the extended hospital stay which patients with such infections may encounter. ,
Carbapenemase production in Enterobacteriaceae may be detected phenotypically or by molecular methods.  Though the molecular technique was considered the gold standard for optimal identification of carbapenemase-producing Enterobacteriaceae,  phenotypic tests such as the combination disc test can also be used. 
The combination disc test consists of meropenem discs to which various carbapenemase inhibitors have been added. Usually, boronic acid inhibits Class A carbapenemase (KPC), dipicolinic acid/ethylenediaminetetraacetic acid inhibits Class B carbapenemase (MBL) while cloxacillin was used to differentiate between AmpC hyperproduction and carbapenemase production.  The Class D carbapenemase presently has no inhibitor but high-level temocillin resistance usually in form minimum inhibitory concentration >32 mg/L , or tentative zone diameter ≤10 mm with temocillin 30 μg disc was a phenotypic indicator of OXA-48 production.  The combination disc test employed in this study was produced by Rosco Diagnostica and has been well validated in several studies. ,,
Based on this background, the aim of this study was to assess the prevalence of carbapenemase production among clinical isolates of carbapenem-resistant Enterobacteriaceae in a tertiary hospital in Lagos and to characterise the type of carbapenemase produced using a combination disc test (Rosco Diagnostica Carbapenemase detection test).
| Materials and Methods|| |
One hundred and seventy-seven bacterial isolates belonging to the family Enterobacteriaceae were isolated from various clinical specimens submitted to the Department of Medical Microbiology, Lagos University Teaching Hospital, a 750 bed hospital. The bacteria isolates were collected between April 2013 and July 2013. Two hundred and seventeen hospital specimens were cultured to obtain the bacterial isolates. The specimens include 66 urine (30.4%), 61 blood (28.1%), 18 sputum (8.3%), 46 wound (21.2%) and 26 pus (11.9%). The aforementioned hospital specimens were cultured on blood agar (Oxoid) and MacConkey agar (Oxoid) and incubated at 35-37°C for 24 h. Thereafter, the isolates were Gram-stained and the Gram-negative rods were identified using the MICROBACT 12A and 12B identification kit (Oxoid).
Antimicrobial susceptibility testing
The Modified Kirby-Bauer Susceptibility testing technique as described by Cheesebrough  was carried out. The Enterobacteriaceae isolates were tested with meropenem (10 μg) (Cypress Diagnostics) on Mueller-Hinton (Oxoid) agar plates. Incubation was performed at 35°C for 18-24 h. After incubation isolates with zone diameters <25 mm to meropenem (10 μg) as stated in the EUCAST guidelines for the detection of resistance  were classified as carbapenem-resistant and were tested for carbapenemase production.
Phenotypic testing for carbapenemase production
An inhibitor combination disc test called the KPC, MBL and OXA-48 detection discs for Enterobacteriaceae (Rosco Diagnostica) was used to confirm carbapenemase production among the carbapenem-resistant Enterobacteriaceae isolates. This combination disc test was made up of 5 discs namely: Disc A containing meropenem (10 μg); disc B containing an MBL inhibitor (dipicolinic acid); disc C containing a KPC inhibitor (aminophenyl boronic acid) and disc D containing a AmpC/extended spectrum beta-lactamase (ESBL) inhibitor (cloxacillin) and disc E consisting of temocillin (30 μg). The discs were tested on Mueller Hinton Agar. Quality control was carried out to ensure the accuracy of the results obtained. K. pneumoniae ATCC 1705 (KPC positive) and K. pneumoniae ATCC BAA-2146 (MBL positive) were used as positive control strains while K. pneumoniae ATCC 700603 was used as a negative control strain. The interpretation of the test was as follows: The zone of inhibition of disc A was compared to the zones of inhibition of each of the carbapenem-plus-inhibitor disc (B, C, and D). If tablet B showed a zone difference of ≥5 mm from disc A, the organism was recorded as demonstrating MBL activity. If disc C showed a zone difference ≥4 mm from tablet A, the organism was recorded as demonstrating KPC activity. If disc D showed a zone difference of ≥5 mm from tablet A, then resistance to meropenem was due to AmpC production. However if disc D showed a zone difference of ≥3 mm from tablet A, then meropenem resistance was due to ESBL production. No zone of inhibition around the temocillin (30 μg) disc was a presumptive indication of OXA-48 production.
| Results|| |
A total of 177 Enterobacteriaceae isolates were tested for carbapenem resistance and carbapenemase production. Twenty-seven (15.2%) of the total isolates tested were carbapenem-resistant [Table 1] while carbapenemase production was detected among 22 (12.4%) of the carbapenem resistant isolates [Table 2]. Of the 27 carbapenem-resistant isolates, 15 (55.5%) produced MBL, 6 (22.2%) produced OXA-48, 1 (3.7%) co-produced both KPC and MBL enzymes while 5 (18.5%) exhibited carbapenem resistance due to ESBL production [Table 2].
|Table 1: Organisms tested and proportion of which were carbapenem-resistant|
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|Table 2: Various resistance mechanisms displayed by the carbapenem-resistant isolates|
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| Discussion|| |
In this study, the prevalence of carbapenem resistance among clinical isolates of Enterobacteriaceae in our institution was 15.2%. This was quite high for a drug which was normally reserved for last resort use. A previous study carried out in our institution showed the prevalence of carbapenem resistance among K. pneumoniae isolates to be 5.2%.  Comparing the results of the two studies indicates that the prevalence of carbapenem resistance among Enterobacteriaceae isolates in our institution has increased.
In contrast, other studies report a lower prevalence of carbapenem-resistant Enterobacteriaceae when compared with our study. For instance, in a study carried out in Enugu, Nigeria the prevalence of carbapenem-resistant Enterobacteriaceae was 2.5%.  A similar study performed in China, determined the prevalence of carbapenem resistance to be 1.2%.  Higher levels of carbapenem use, especially within the hospital setting, may select for its resistance leading to the higher prevalence of carbapenem resistance among Enterobacteriaceae isolates.
The screening cut-off used in this study (25 mm zone diameter to meropenem [10 μg]) comprises both the intermediate and resistant isolates, and this was done to ensure that every carbapenemase-producing isolate was identified. Besides an intermediate reading to the carbapenems may be indicative of low-level resistance which if left undetected may escalate to high-level resistance. Meropenem was used to determine carbapenem non-susceptibility because it offers the best balance between sensitivity and specificity in terms of detecting carbapenemase producers. ,
We also detected that carbapenem resistance in our study was due to two major mechanisms namely (i) carbapenemase production (12.4%) (ii) ESBL production (2.8%). The implication of this finding was that both carbapenemases and ESBL production are plasmid mediated resistance mechanisms. Hence, there was a high tendency that these resistance plasmids could spread, thus rendering previously sensitive bacterial populations resistant to carbapenems. That supports the fact that preserving the clinical efficacy of carbapenems as drugs of last resort in treating infections caused by multidrug resistant Enterobacteriaceae was of utmost importance.
The prevalence of carbapenemase-producing Enterobacteriaceae in this study was 12.4%. Our result was slightly higher than that obtained from a study carried out in Kano  where the prevalence of carbapenemase production was determined to be 10.2%. Studies carried out in Morocco (2.8%) and Taiwan (8.6%), respectively , revealed lower prevalence of carbapenemase-producing Enterobacteriaceae when compared
with that of our study.
However, our result was lower than that obtained in Tanzania where the prevalence of carbapenemase production was 35.24%  It should be noted that carbapenemase production, in this case, was detected among bacterial isolates resistant to at least three classes of antimicrobials (multi-drug resistant isolates). This was probably the reason the prevalence of carbapenemase production in the Tanzanian study was quite high. Thus, the aforementioned studies showed that higher or lower prevalence levels of carbapenemase production were dependent on the rate of consumption of the carbapenems. Our prevalence rate in this study may be slightly higher because there are no strict antibiotic policies guiding the sale of carbapenems in our region. Anecdotal reports indicate that in Lagos, Nigeria, carbapenems are being sold as over the counter drugs which could lead to indiscriminate use and development of resistance to the carbapenems.
In this study, 22 (12.4%) of the 177 Gram-negative bacteria isolates tested were positive for carbapenemase production. Of this 22 (12.4%) carbapenemase-producing Enterobacteriaceae, 15 (8.5%) produced MBL. Thus, we can say that the MBL enzyme was the most prevalent carbapenemase in our institution. The Kano and Enugu studies , reported that 85.5% and 100% of carbapenemase-producing Enterobacteriaceae produced MBLs. In Tanzania, 33.7% of the total isolates tested were MBL producers.  OXA-48 enzyme was also prevalent in our institution as 6 (3.4%) out of the 22 (12.4%) carbapenemase-producing Enterobacteriaceae isolates in our study were OXA-48 producers. However, OXA-48 carbapenemase was not peculiar to our institution alone; in a Tanzanian study, 4.8% of all the multidrug-resistant Enterobacteriaceae isolates tested were OXA-48 producers.  Similarly in Morocco, 2.2% of the total isolates tested produced OXA-48.  Co-production of MBL and KPC enzymes is found in 1 carbapenem-resistant isolate in our study. A similar result was obtained in India where a K. pneumoniae isolate produced both NDM and KPC enzyme.  It is pertinent to note that co-production of two carbapenemase enzymes by one bacteria isolate could enhance inactivation of the carbapenems by the isolate. This could, in turn, lead to high-level resistance to the carbapenems.
We also observed that 5 (2.8%) of the total isolates tested displayed carbapenem resistance due to ESBL production. The bacteria isolate that exhibited carbapenem resistance due to ESBL production belonged to the Klebsiella species. This result was lower than that obtained from a similar study carried out in China where the prevalence of carbapenem resistance due to ESBL production is 34.7%.  Our results show that the ESBL production by Enterobacteriaceae does not only cause resistance to the third-generation cephalosporins but can also lead to carbapenem resistance. This confirms the claim made by some authors , that the Class C beta-lactamases (ESBL and AmpC) are not robust carbapenemases but can lead to carbapenem resistance when combined with other resistance mechanisms such as porin loss.
Since carbapenems are the drugs of last resort in the treatment of infections caused by multidrug-resistant Enterobacteriaceae, the treatment options are severely limited as there are few drugs left for the treatment of infections caused by this group of Enterobacteriaceae that exhibit resistance to most classes of antibiotics including carbapenems. This greatly highlights the problem of antibiotic resistance within the healthcare setting and makes a strong case for the strict implementation of antibiotic policy within the healthcare setting.
| Conclusion|| |
Having established the presence of carbapenemase-producing Enterobacteriaceae in our institution, efforts should be geared towards limiting their spread. Preventive efforts such as infection control procedures, hand hygiene among healthcare workers, antibiotic stewardship as well as careful use of carbapenems and third generation cephalosporins should be aggressively pursued within the health care setting. This was because the treatment options for infections caused by carbapenemase-producing bacteria are limited.
Accurate detection of the carbapenemase-producing Enterobacteriaceae was also very important. Where molecular tests are not available, phenotypic tests such as the carbapenemase detection discs used in this study may be employed. They are easy to perform, interpret and introduce into the workflow of the clinical laboratory.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]
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