Phenotypic Detection of Enterobacterales Strains Susceptible of Producing OXA-48 Carbapenemase ()
1. Introduction
Bacterial resistance to antibiotics is steadily increasing and constitutes a global public health problem. According to estimates by World Health Organization (WHO), human and economic impacts of antimicrobial resistance between now and 2050 will be considerable [1] . Over last ten years, emergence and spread of Carbapenem-Resistant Enterobacterales (CRE) and, in particular, Klebsiella pneumoniae, have become worldwide [2] .
Carbapenems are antibiotics of choice for serious infections caused by multi-resistant gram-negative bacteria [3] . CRE can cause treatment failures and constitute a major public health problem [4] . Resistance due to production of a carbapenemase is transferred horizontally among Enterobacteriaceae, and there are many types of carbapenemases: clavulanic acid-inhibited ß-lactamases (Class A: KPC, NMC, IMI, SME and GES), metallo-betalactamases (Class B: IMP, VIM, NDM, GIM, SPM and SIM) and extended-spectrum oxacillinases (Class D: OXA-48-like) [5] . With exception of OXA-48, these enzymes confer high resistance to most beta-lactam compounds such as penicillins and cephalosporins, but affect carbapenems to a variable extent [6] . OXA-type carbapenemases are considered to be penicillinases capable of hydrolyzing oxacillin and cloxacillin and are weakly inhibited by clavulanic acid and EDTA. OXA-48 is one of the few members of this family to have significant carbapenem hydrolysis activity [7] .
OXA-48 carbapenemases have been frequently reported in North African and other Mediterranean countries [6] [7] . In Morocco, an increased spread of OXA-48-producing Enterobacteriaceae isolates linked to healthcare-associated infections has been reported [8] [9] .
In Senegal, emergence of plasmid-encoded blaOXA-48 gene has been reported in multi-resistant Enterobacteriaceae of community and hospital origin in Dakar [10] . Detection of OXA-48 carbapenemases remains difficult due to multitude of phenotypes and resistance mechanisms [11] . A phenotypic test for detection of OXA-48 CPE has been proposed with temocillin disc. Temocillin is a derivative of ticarcillin (6-α-methoxy ticarcillin) which has a narrow spectrum for enterobacteria and is not hydrolyzed by β-lactamases (ESBL, AmpC) and cephalosporinases. Hafnia alvei has natural resistance to temocillin [12] .
The aim of this study was to use a phenotypic identification method to detect OXA-48 CPE in strains of Enterobacteriaceae isolated in Dakar.
2. Material and Methods
2.1. Bacterial Strains
This retrospective study was carried out at Medical Biology Laboratory of Pasteur Institute in Dakar on enterobacteria strains isolated from 2015 to 2017. Strains resistant to ertapenem (10 µg) with an inhibition diameter of less than 25 mm according to EUCAST 2020 recommendations were included in the study. Phenotypic detection of OXA-48 CPE was performed using temocillin disc (30 µg).
2.2. Phenotypic Detection of Resistance to Temocillin
Antibiotic susceptibility profiling was performed using Mueller-Hinton agar diffusion method (Becton Dickinson, Heidelberg, Germany) with antibiotic disc temocillin (30 µg) according to guidelines of the European Committee of Antimicrobial Susceptibility Testing (EUCAST, 2020).
2.3. Molecular Confirmation
Presence of OXA-48 CPE was confirmed by PCR using following specific primers.
O48-F CCA AGC ATT TTT ACC CGC ATC KAC C
O48-R GYT TGA CCA TAC GCT GRC TGC G
PCR and sequencing were performed searching of blaOXA-48 gene as described previously [13] .
Control strain NCTC-13442 was used as a reference.
2.4. Data Analysis
Data were entered and analysed using Excel 2018 and logiciel R. We estimated the sensitivity, specificity and kappa coefficient of the phenotypic test in reference to PCR.
3. Results
3.1. Bacterial Strains
Forty-one strains of enterobacteria isolated from various pathological samples were analysed for this study. These strains were mainly composed of K. pneumoniae (n = 16), E. coli (n = 11) and E. cloacae (n = 9) (Table 1).
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Table 1. Distribution of enterobacteria strains tested.
3.2. Resistance to Ertapenem
Antibiotic susceptibility profiling by diffusion method showed that all strains of enterobacteria were resistant to ertapenem. Inhibition diameters range from 8 to 21 mm depending on the species (Table 2).
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Table 2. Enterobacteria strains resistant to ertapenem.
3.3. Resistance to Temocillin
Of the 41 strains resistant to ertapenem, 34 isolates were resistant to temocillin (Table 1), and therefore suspected of being EPC probably type OXA-48.
These isolates included K. pneumoniae (n = 14), E. cloacae (n = 8), E. coli (n = 7), C. freundii (n = 3), C. sakazakii (n = 1) and M. morganii (n = 1).
3.4. Resistance of PCR and Sequencing of blaOXA-48
All strains both resistant to ertapenem and temocillin harbored OXA-48 gene (Table 2). Sequencing confirmed presence of OXA-48 gene in all 34 temocillin-resistant isolates (Table 3): GenBank accession numbers NG049762.1 (n = 31) and NG 049482.1 (n = 3).
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Table 3. Temocillin-resistant strains of enterobacteria harboring the OXA-48 gene.
R: resistant, I: intermediate, S: sensitive.
3.5. Comparison Temocillin Resistance versus PCR
This study showed a sensitivity of 100% (95% CI: 0.90 - 1.0), a specificity of 100% (95% CI: 0.54 - 1.0), and a kappa coefficient equal to 1 for the phenotypic test compared with PCR (Table 4).
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Table 4. Comparison of phenotypic detection with temocillin disk versus.
4. Discussion
The aim of this study was to evaluate phenotypic detection of OXA-48 CPE strains isolated from 2015 to 2017 with a temocillin disc (30 µg). Confirmation was made by PCR for reschearch of OXA-48 gene.
Forty-one enterobacteria strains resistant to ertapenem (inhibition diameter < 22 mm, EUCAST 2020) were analyzed. Temocillin resistance was found in 83% of isolates (n = 34), suggesting a probable CPE type OXA-48. Molecular analysis of these strains confirmed the presence of OXA-48 gene in all temocillin-resistant isolates. For strains resistant to ertapenem, other carbapenem resistance genes (KPC, IMP, VIM, NDM, IMI, GES, etc.) should also be checked.
In Senegal, the circulation of enterobacteria strains harboring blaOXA-48 gene isolated in community and hospital settings has been reported since 2011 [10] . These CPE type OXA-48 are endemic in several African countries, including North Africa, Türkiye, Arabian Peninsula and India [14] .
In France in 2020, OXA-48 carbapenemases accounted for 63.3% of EPCs in circulation according to data from National Reference Center [15] .
In this study, 14 strains of K. pneumoniae (41%) produced carbapenemase type OXA-48. The spread of K. pneumoniae strains harboring genes encoding resistance to carbapenems has been described throughout the world [16] .
The increasing use of carbapenems in clinical practice is being followed by emergence of CPE, which are increasingly being detected throughout the world, and this trend has been observed in Senegal. Emergence of carbapenemases acquired from enterobacteria appeared early in Senegal (2008) after introduction of imipenem, and their frequency is probably underestimated, partly because of limited number of bacteriology laboratories equipped with qualified staff [17] . Multiple antibiotic resistance is prevalent among enterobacteria strains isolated in rural communities [18] , but especially in hospitals in Dakar where emergence of CPE harboring the blaOXA-48 gene is a major concern.
Sensitivity and specificity of phenotypic test using temocillin disk were 100% in reference to PCR detection of the blaOXA-48 gene. The good sensitivity and specificity of phenotypic test with temocillin may be a means of rapidly detecting ertapenem-resistant enterobacteria strains likely to be OXA-48 carbapenemase producers, and of managing patients infected with these strains. In fact, this test does not require any investment on the part of the laboratory, compared to the molecular biology technique, which is not accessible to most laboratories in developing countries, and also to rapid diagnostic test, which despite its lower cost is not yet within the reach of these laboratories. All strains of enterobacteria resistant to ertapenem and temocillin require confirmation of OXA-48 carbapenemase production by rapid diagnostic method (immunoassay) or molecular technique.
This study has some limitations, notably the fact that it only detects OXA-48 type carbapenemases and therefore cannot identify other classes of carbapenemases.
However, some bacterial species are naturally resistant to temocillin (e.g. Hafnia alvei), and other (fairly rare) mechanisms can induce resistance to this antibiotic.
5. Conclusion
The phenotypic test with temocillin disk has shown good sensitivity and specificity for detecting ertapenem-resistant enterobacteria strains likely to produce an OXA-48 carbapenemase. However, identification of carbapenemase type must be carried out by a rapid diagnostic method (immunoassay) or by PCR.
Authors’ Contributions
AS designed the study and revised manuscript version. AD and BN participated susceptibility testing and molecular test. CT carried out statistical analyses. All authors have read and approved final version of manuscript.
Acknowledgements
Medical Biology Laboratory of Pasteur Institute of Dakar (IPD).