Incidence of OXA-23 and OXA-58 Carbapenemases Coexpressed in Clinical Isolates of Acinetobacter baumannii in Tunisia

Abstract

Acinetobacter baumannii is an important opportunistic and multidrug-resistant pathogen responsible for nosocomial infections in health facilities. The aim of this study was to characterize the molecular mechanisms of carbapenem resistance in A. baumannii isolates isolated from Mohamed Kassab Orthopedic Institute in Tunis, Tunisia. Twenty-five imipenem-resistant A. baumannii clinical isolates collected between 2013 and 2016 were identified using API 20NE and were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Carbapenemase activity was detected using microbiological tests and PCR. The epidemiological relatedness of the isolates was studied using multilocus sequence typing (MLST). The isolates were resistant to all antibiotics tested with increased minimum inhibitory concentration values (>32 mg/L). The microbiological tests showed that the 25 A. baumannii were positive for modified Hodge test and for the Carba NP test; however, β-lactamase activity was not inhibited by EDTA. All the isolates harbored the naturally occurring bla_OXA-51-like gene and the bla_OXA-23-like carbapenemase gene. Among these isolates, one isolate coexpressed the bla_OXA-58 gene. MLST revealed several sequence types (STs) with the predominance of ST2 imipenem-resistant A. baumannii (14/25; 56%). In this study we report the prevalence of ST2 imipenem resistance and for the first time the coexpression of bla_OXA-23 and bla_OXA-58 in clinical isolates of A. baumannii in a Tunisian hospital.

Introduction

Acinetobacter baumannii is recognized as one of the most problematic nosocomial pathogens. Its clinical impact is controversial because the issue of attribute mortality frequently remains unsettled.^1,2 Indeed, the bacteria colonize patients and frequently the infected patient shows a reduced prognosis and frequently presents secondary infective complications.³ Treatment is complicated by the ability of A. baumannii to acquire resistance to multiple classes of antimicrobials.⁴ A. baumannii is naturally resistant to cephalosporins and chloramphenicol. However, it has a great ability to develop resistance against many antibiotics, including aminoglycosides, fluoroquinolones, and carbapenems by acquisition of mobile genetic elements like plasmids, transposons, integrons, insertion sequence IS, or insertion sequence common region ISCR.⁵ Carbapenem resistance in A. baumannii is increasingly being observed worldwide, and the most important resistance mechanism is enzymatic hydrolysis mediated by carbapenem-hydrolyzing β-lactamases, belonging to class A, class B, and class D.⁶ The frequency of carbapenem-resistant A. baumannii incidence has risen in many parts of the world. Large and sustained outbreaks caused by this bacteria have been described in Europe,^7–9 South America,¹⁰ North America,¹¹ Australia,¹² Asia,¹³ and Africa,^14,15 including North Africa.^13,16,17 In Tunisia, there are only few reports describing the emergence of carbapenem resistance in A. baumannii.^18–20 The aim of our study was to identify molecular support of imipenem resistance in A. baumannii clinical isolates collected in Mohamed Kassab Orthopedic Institute of Tunis. This study shows for the first time coexpression of OXA-23 and OXA-58 in carbapenemase-producing A. baumannii in Tunisia. Moreover, our report is the first in this country that uses multilocus sequence typing (MLST) to study the epidemiological relatedness of an autochthonous MDR A. baumannii. This report, in addition to recent observations in neighboring countries,^14,21 confirms the emergence of this resistance mechanism in North Africa.

Materials and Methods

Bacterial isolates and antibiotic susceptibility testing

Twenty-five A. baumannii clinical isolates from bronchial aspirate, pus, blood, and urine were collected from February 2013 to March 2016 in Mohamed Kassab Orthopedic Institute of Tunis. Isolates were identified using an API 20NE system (bioMérieux, Marcy-l'Etoile, France) and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) using the Biotyper database and a Microflex spectrometer (Bruker Daltonics, Bremen, Germany). Correct identification at the species level was defined as a MALDI-TOF score >1.9 as previously described.²²

Antibiotic susceptibility testing was performed on Mueller-Hinton agar (bioMérieux, Craponne, France) by the standard disk-diffusion procedure according to the Antibiogram Committee of the French Society for Microbiology (CA-SFM) (www.sfm.microbiologie.org/). Seventeen antibiotics were tested: ticarcillin, ticarcillin/clavulanic acid, piperacillin/tazobactam, ceftazidime, cefotaxime, cefepime, aztreonam, amikacin, tobramycin, gentamicin, ciprofloxacin, rifampicin, trimethoprim/sulfamethoxazole, ertapenem, meropenem, imipenem, and colistin (Bio-Rad, France). Isolates were considered resistant to imipenem if the diameter of the inhibition zone was <17 mm. For isolates with inhibition diameter <17 mm, minimum inhibitory concentrations (MICs) for imipenem were determined by E-test (AB BIODISK, Solna, Sweden), and isolates were considered resistant when displaying an imipenem MIC >8 μg/mL (CA-SFM). MICs for colistin were also evaluated. Disk-diffusion susceptibility testing and the MICs of antibiotics were measured in accordance with CA-SFM guidelines.

Phenotypic detection of carbapenemase activity

Imipenem-resistant isolates were screened for carbapenemase production using the modified Hodge test, the modified Carba NP test, and the ethylenediaminetetraacetic acid (EDTA) test as described.^23–26

PCR amplification and sequencing

Real-time PCR was performed to screen the presence of bla_OXA-51, bla_OXA-23, bla_OXA-24, bla_OXA-58, and bla_OXA-48 genes. Conventional PCR was performed for bla_NDM, bla_KPC, bla_IMP, bla_VIM, bla_SIM, and bla_GIM genes as previously described.²⁷ Positive PCR products for any carbapenemase-encoding gene were sequenced using BigDye^® Terminator chemistry on an automated ABI 3130 sequencer (PE Applied Biosystems, Foster City, CA). The sequences of the obtained genes were analyzed using BlastN and BlastP available at the National Center for Biotechnology Information (NCBI) database (www.ncbi.nlm.nih.gov/blast) and using ARG-ANNOT (Antibiotic Resistance Gene-ANNOTation) for identification.²⁸

Multilocus sequence typing

Molecular typing of the isolates was determined by full MLST using the seven housekeeping genes (cpn60, fusA, gltA, pyrG, recA, rplB, and rpoB) as described on the Institut Pasteur MLST website (www.pasteur.fr/mlst).

Results

From February 2013 to March 2016, 25 clinical isolates, identified as A. baumannii by the API 20 NE identification system and confirmed by MALDI-TOF/MS, were recovered from Tunisian patients (16 male and 9 female) hospitalized in Mohamed Kassab Orthopedic Institute and isolated from different services, mostly Anesthesiology (40%).

Among the 25 isolates, 10 (40%) were recovered from urine, 5 (20%) from tracheas, 3 (12%) from bedsores, 2 (8%) from pus, 2 (8%) from catheters, 2 (8%) from blood, and 1 (4%) from expectoration (Table 1). The results of antibiotic susceptibility testing revealed that the isolates were resistant to almost all antibiotics, including β-lactams, aminoglycosides, and fluoroquinolones. All isolates showed high-level resistance to carbapenems with MICs for imipenem >32 mg/L. Colistin was the only antibiotic active against all the isolates. Using the modified Hodge test, the modified Carba NP test, and the EDTA-disk test, all imipenem-resistant A. baumannii were modified Hodge and Carba NP test positive, suggesting carbapenemase production. However, β-lactamase activity was not inhibited by EDTA, indicating that imipenem-resistant A. baumannii isolates do not produce metallo-β-lactamases.

Table 1.

Phenotypic and Genotypic Features of 25 Clinical Imipenem-Resistant Acinetobacter baumannii Tunisian Strains Producing Carbapenemases

Isolate	Date of isolation (da-mo-yr)	Gender	Ward	Type of samples	IMP MIC (μg/mL)	MHT	EDTA test	Carba test	Carbapenemase genes	ST
29	25/03/2013	M	Anesthesiology	Tracheal	>32	+	−	−	OXA-51, OXA-23	2
27	03/04/2013	M	PMAIR	Bedsore	>32	+	−	−	OXA-51, OXA-23	2
25	14/05/2013	F	Septic surgery	Sepsis	>32	+	−	−	OXA-51, OXA-23	602
23	23/05/2013	M	Anesthesiology	Catheter	>32	+	−	−	OXA-51, OXA-23, OXA-58	1
21	22/08/2013	M	Septic surgery	Sepsis	>32	+	−	−	OXA-51, OXA-23	2
20	16/09/2013	M	PMAIR	Urine	>32	+	−	−	OXA-51, OXA-23	164
19	17/09/2013	M	Anesthesiology	Blood culture	>32	+	−	−	OXA-51, OXA-23	2
18	21/09/2013	M	Septic surgery	Urine	>32	+	−	−	OXA-51, OXA-23	602
14	06/11/2013	F	PMAIR	Urine	>32	+	−	−	OXA-51, OXA-23	602
16	17/11/2013	F	PMAIR	Urine	>32	+	−	−	OXA-51, OXA-23	310
15	02/12/2013	F	PMAIR	Urine	>32	+	−	−	OXA-51, OXA-23	602
13	23/12/2013	M	Anesthesiology	Catheter	>32	+	−	−	OXA-51, OXA-23	2
10	25/03/2014	M	Anesthesiology	Tracheal	>32	+	−	−	OXA-51, OXA-23	623
9	02/04/2014	F	Anesthesiology	Blood culture	>32	+	−	−	OXA-51, OXA-23	2
8	13/06/2014	M	Septic surgery	Bedsore	>32	+	−	−	OXA-51, OXA-23	602
7	01/12/2014	F	Anesthesiology	Tracheal	>32	+	−	−	OXA-51, OXA-23	2
6	25/03/2015	F	Adult orthopedic surgery	Bedsore	>32	+	−	−	OXA-51, OXA-23	2
5	30/03/2015	M	Trauma	Urine	>32	+	−	−	OXA-51, OXA-23	2
4	27/05/2015	F	Adult orthopedic surgery	Urine	>32	+	−	−	OXA-51, OXA-23	2
3	08/06/2015	M	PMAIR	Urine	>32	+	−	−	OXA-51, OXA-23	2
1	07/09/2015	M	PMAIR	Urine	>32	+	−	−	OXA-51, OXA-23	2
2	07/09/2015	M	PMAIR	Urine	>32	+	−	−	OXA-51, OXA-23	2
24	15/03/2016	F	Anesthesiology	Expectoration	>32	+	−	−	OXA-51, OXA-23	636
31	18/03/2016	M	Anesthesiology	Tracheal	>32	+	−	−	OXA-51, OXA-23	570
32	22/03/2016	M	Anesthesiology	Tracheal	>32	+	−	−	OXA-51, OXA-23	2

+, positif test; −, negatif test; M, Male; F, Female; PMAIR, physical medicine and internal rehabilitation; IMP, imipenem; MHT, modified Hodge test; ST, sequence type.

Screening for carbapenemase-encoding genes by PCR showed that all the isolates produced the naturally occurring bla_OXA-51-like gene and the acquired OXA-carbapenemase bla_OXA-23-like genes (Table 1). In addition, among these isolates, one isolate coexpressed the bla_OXA-58 gene, which was isolated from a male hospitalized in the Anesthesiology service (Table 1). None of the isolates contained bla_OXA-24-like and bla_OXA-48-like or metallo-β-lactamase-encoding genes.

According to MLST analysis, several different STs were assigned to the clinical imipenem-resistant A. baumannii isolates, including ST1, ST2, ST164, ST310, ST570, ST602, ST623, and ST636. The most common ST was ST2 (14/25; 56%) (Fig. 1). This clone was found circulating in all services and was associated with the production of OXA-23 enzymes (Table 1). It is worth mentioning that the only isolate that harbored bla_OXA-23 and bla_OXA-58 belonged to ST1 and the patient treated during three days with imipenem and colistin died at the age of 20 after being hospitalized for a month in the ICU (Table 1).

FIG. 1.

Phylogenetic tree of the 25 imipenem-resistant Acinetobacter baumannii clinical isolates based on the MLST concatenated gene sequences of each isolate aligned with reference strains SDF and AYE. MLST, multilocus sequence typing.

Discussion

Antimicrobial resistance in A. baumannii is one of the most problematic nosocomial threats worldwide. A. baumannii has been associated with healthcare-associated infections, often isolated from urine and/or respiratory specimens obtained from hospitalized patients, as confirmed in this study, in which 40% of A. baumannii isolates were recovered from urine followed by 20% from tracheas.^29,30 In this study we report a high prevalence of imipenem resistance (100%) in A. baumannii clinical isolates recovered recently in Tunis over a 3-year period.

Interestingly in our study we identified one isolate, belonging to the ST1, that harbored both bla_OXA-58 and bla_OXA-23 genes and this is the first description of the coexpression of OXA-23 and OXA-58 in the same A. baumannii clinical isolate. Carbapenems are the most potent β-lactams against Gram-negative bacteria and the drugs of choice for serious A. baumannii infections.³¹ However, resistance to these antibiotics is increasing, owing to the production of carbapenem-hydrolyzing β-lactamases, including metallo-β-lactamases, and particularly OXA-type carbapenemases.³² Indeed, in this study, the bla_OXA-23 gene was found in all imipenem-resistant A. baumannii isolates. These data confirm previous studies performed recently in North Africa that concluded that the main oxacillinase produced by A. baumannii is OXA-23.^4,5,22,30

In fact, in Algeria, several studies have shown also the dissemination of OXA-23-producing carbapenem-resistant A. baumannii by the detection of bla_OXA-23-like gene in 29 imipenem-resistant A. baumannii isolated from Sétif, as well as in two imipenem-resistant isolates obtained from Tizi-Ouzou.³³ Other work published in the same year reported that 22 imipenem-resistant clinical isolates of A. baumannii from the University Hospital of Annaba also harbored bla_OXA-23 gene.³⁴ One year later, bla_OXA-23-like gene was identified among 50% (40/80) of Acinetobacter spp. clinical isolates recovered from three different hospitals in western Algeria.³⁵ Recently, bla_OXA-23 gene was again detected in carbapenem-resistant A. baumannii clinical isolates recovered from Algerian hospitals.³⁶

More recently, high prevalence of imipenem-resistant A. baumannii in Algerian hospitals, mediated mainly by bla_OXA-23-like genes, was reported.³⁷ In Tunisia, only few studies described the dissemination of OXA-23-producing carbapenem-resistant A. baumannii. The first description of OXA-23-producing carbapenem-resistant A. baumannii was reported in thirteen isolates during 2008. All the OXA-23-positive isolates were clonally related, and the bla_OXA-23 gene was found to be chromosomally located and associated with an upstream-located insertion sequence ISAba.¹⁹ In 2011, a second work was published reporting the presence of bla_OXA-23 gene among imipenem-resistant A. baumannii recovered from different wards at Charles Nicolle Hospital.³⁸ More recently, another work reported the production of OXA-23 in A. baumannii clinical isolates recovered in a neonatology center in the center-east of Tunisia.²⁰

In Libya, only two recent publications described the emergence of OXA-23-producing carbapenem-resistant A. baumannii.^16,21 These findings may reflect the current spread of OXAs and especially OXA-23 in clinically relevant A. baumannii isolates throughout northern Africa and also show that the Tunisian isolates share the same genetic pool with A. baumannii species worldwide. Moreover, interestingly in our study we identified one isolate that harbored both bla_OXA-58 and bla_OXA-23 genes. In Tunisia, to the best of our knowledge, only one publication reported the bla_OXA-58-like gene in multidrug-resistant (MDR) A. baumannii isolates recovered from hospitalized patients at the Sahloul Hospital in Sousse in 2008.

Sequencing of the bla_OXA-58-positive amplicons obtained from all isolates identified a gene with a single base-pair substitution with respect to the bla_OXA-58 sequence. This substitution gave rise to OXA-97.¹⁸ However, the coexistence in an isolate of both bla_OXA-23 and bla_OXA-58 genes has never been reported in Tunisia. So in this study we present the first report showing the emergence of MDR A. baumannii with the coexistence of bla_OXA-58 and bla_OXA-23-like carbapenemase encoding genes in Tunisia. This may suggest that the epidemiology of carbapenemase-encoding genes has changed in Tunisia with the occurrence of the bla_OXA-23 gene and the incidence of new variants like bla_OXA-58; it can be explained by the intensive introduction of imipenem as a therapy in Tunisian hospitals.

The bla_OXA-58 gene has been reported worldwide, but in North African countries, such as Libya and Morocco, no studies on the emergence and spreading of OXA-58-producing MDR A. baumannii have been reported, in contrast with Algeria where two studies reported isolates harboring bla_OXA-58 collected from Annaba and Tlemcen.^34,39 All of the isolates were analyzed by MLST, and different sequence types (STs) were obtained; this analysis showed that the 25 isolates belonged to eight different STs. These results confirm the clonal diversity of A. baumannii clinical isolates in Tunisia. The most common ST in this study was ST2 (Table 1). This ST has been reported in several countries in the Mediterranean area.

The results of this work are consistent with a recent study which also reported that imipenem-resistant A. baumannii belonging to ST2 was the predominant clone among isolates recovered from some Algerian hospitals.³⁶ In addition, in 2015 another study found that the ST2 clonal group predominated (41.2%; 54/131) among other ST clonal groups in a large series of 150 A. baumannii clinical isolates collected in Egypt.¹⁴ This clone also corresponds to the most prevalent Mediterranean A. baumannii clone according to a study conducted in Spanish hospitals.⁴⁰

Conclusion

In conclusion, these results reemphasize the worldwide dissemination of OXA-23 carbapenemase gene carbapenem-resistant clinical isolates of A. baumannii and the emergence of carbapenem resistance due also to the bla_OXA-58 gene in Tunisia. Because of the extensive use of carbapenems within Tunisian hospitals, as the last resort for treating infections due to third-generation cephalosporin-resistant isolate, the emergence of MDR A. baumannii isolates has become alarming. Strengthening prevention measures is required to control further spread of carbapenemases in this country.

Footnotes

Acknowledgments

The authors thank Linda Hadjadj (Technician in URMITE [Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes]), for her assistance.

This work was partly funded by CNRS and IHU Méditerranéen Infection (France). This work was supported by the Tunisian Ministry of Higher Education, Scientific Research and Information and Communication Technologies, which offered a scholarship to NM to attend Aix-Marseille Université (Marseille, France).

Disclosure Statement

No competing financial interests exist.

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