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Aix-Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198, Inserm 1095, IHU Méditerranée Infection, Faculté de Médecine et de Pharmacie, 27 Bd Jean Moulin, 13385 Marseille Cedex 05, FranceLaboratoire Antibiotiques, Antifongiques: Physico-chimie, Synthèse et Activité Biologique (LAPSAB), Faculté des Sciences de la Nature et de la Vie et Sciences de la Terre et de l’Univers, Université Abou Bekr Belkaid-Tlemcen, Tlemcen, Algeria
Aix-Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198, Inserm 1095, IHU Méditerranée Infection, Faculté de Médecine et de Pharmacie, 27 Bd Jean Moulin, 13385 Marseille Cedex 05, FranceLaboratoire Antibiotiques, Antifongiques: Physico-chimie, Synthèse et Activité Biologique (LAPSAB), Faculté des Sciences de la Nature et de la Vie et Sciences de la Terre et de l’Univers, Université Abou Bekr Belkaid-Tlemcen, Tlemcen, Algeria
Laboratoire Antibiotiques, Antifongiques: Physico-chimie, Synthèse et Activité Biologique (LAPSAB), Faculté des Sciences de la Nature et de la Vie et Sciences de la Terre et de l’Univers, Université Abou Bekr Belkaid-Tlemcen, Tlemcen, Algeria
Aix-Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198, Inserm 1095, IHU Méditerranée Infection, Faculté de Médecine et de Pharmacie, 27 Bd Jean Moulin, 13385 Marseille Cedex 05, France
Nosocomial infections caused by carbapenem-resistant Acinetobacter spp are a global health problem. The aim of this study was to investigate the molecular epidemiology and the genetic support of carbapenem resistance in Acinetobacter spp clinical isolates recovered from three different hospitals in western Algeria from 2008 to 2012.
Methods
A total of 113 Acinetobacter spp isolates were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Antimicrobial susceptibility testing was carried out, and minimum inhibitory concentrations (MICs) were determined by the dilution method on Mueller–Hinton agar for β-lactams, aminoglycosides, fluoroquinolones, and colistin. The characterization of β-lactamases was investigated by phenotypic tests for the detection of metallo-β-lactamases and oxacillinases. Resistance genes were screened for by quantitative PCR and sequenced when positive.
Results
Among the 113 isolates, 80 (70.8%) were found to be resistant to imipenem with MICs ranging from 64 to 512 μg/ml. The blaOXA−23−like gene was detected in 50% (40/80) of the isolates and the blaOXA−24−like gene was detected in 21.2% (17/80) of the isolates. In addition, the metallo-β-lactamase blaNDM-1−like was detected in five isolates (6.2%).
Conclusions
This study represents the first description of autochthonous Acinetobacter spp producing metallo-β-lactamase blaNDM-1−like and oxacillinases blaOXA−23−like and blaOXA−24−like in western Algeria.
Acinetobacter spp are major nosocomial pathogens. The genus currently consists of more than 40 species, including validly published species and genomic species.
Of these, Acinetobacter baumannii is the most clinically relevant Acinetobacter species. It has emerged as a major cause of healthcare-associated infections including pneumonia, urinary tract infection, and septicemia.
Closely related species, Acinetobacter nosocomialis (formerly named Acinetobacter genomic species (gen. sp.) 13TU) and Acinetobacter pittii (formerly named Acinetobacter gen. sp. 3), have also been associated with nosocomial infections and outbreaks.
Genotypic and phenotypic characterization of the Acinetobacter calcoaceticus–Acinetobacter baumannii complex with the proposal of Acinetobacter pittii sp. nov. (formerly Acinetobacter genomic species 3) and Acinetobacter nosocomialis sp. nov. (formerly Acinetobacter genomic species 13TU).
These three clinically important species are phenotypically and genotypically difficult to differentiate, thus they are grouped together into the Acinetobacter calcoaceticus–Acinetobacter baumannii (ACB) complex.
They are so much alike that they cannot be differentiated using routine commercial systems. Genotypic methodologies can be used to differentiate them, such as the determination of specific gene sequences, including the 16sRNA, recA, rpoB, and gyrB genes, in combination with the technology of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS).
The efficacy of carbapenems against multidrug-resistant Acinetobacter spp has been undermined by the emergence of Ambler class B and class D carbapenemase-hydrolyzing β-lactamases.
The class D carbapenemase (oxacillinase) found in A. baumannii can be clustered into four distinct groups: OXA–23-like (OXA–23, OXA–27 and OXA–49), OXA–24-like (OXA–24/40, OXA–25, OXA–26 and OXA–72), OXA–58-like (OXA–58 and OXA–96), and OXA–51-like enzymes.
The high-level carbapenem resistance due to the expression of genes encoding the class D carbapenemases, requires a strong promoter such as that provided by the mobile insertion sequence ISAba1.
This is characteristic of A. baumannii, and most outbreaks of carbapenem-resistant A. baumannii associated with blaOXA−23 have been identified using primers based on ISAba1.
The new metallo-β-lactamase (MBL), New Delhi metallo-β-lactamase 1 (NDM-1), was initially reported in Klebsiella pneumoniae clinical isolates from a Swedish patient who had previously been hospitalized in India.
Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India.
Recently, cases of NDM-producing A. baumannii have been described in several countries worldwide, including Canada, USA, Sweden, UK, Austria, Belgium, France, Netherlands, Germany, Japan, Africa, Oman, and Australia.
At present, the worldwide caseload is probably being driven by people infected or colonized in the Indian subcontinent before traveling elsewhere. However, there is already evidence of others reservoirs of infected patients in the Balkan states, the Middle East, and Israel, suggesting that the gene may become endemic worldwide, similar to the blaKPC gene, which is now endemic in Greece and Israel.
In the present study, we evaluated the prevalence of antibiotic resistance and the genetic background of carbapenem resistance in a series of 113 A. baumannii strains isolated in western Algeria between October 2008 and April 2012. We report five blaNDM-1-positive A. baumannii strains recovered from autochthonous cases in the same area.
2. Materials and methods
Bacterial isolates of Acinetobacter spp were recovered from three different hospitals situated in north-western Algeria (Tlemcen, Oran, and Sidi Bel Abbes). All of them were isolated from the hospital environment and patients admitted to the intensive care unit (ICU) and hematology, surgery, and neurosurgery wards, during the study period of October 2008 to April 2012. They were identified using MALDI-TOF MS, which was performed with a Bruker Daltonics Microflex (Bremen, Germany) using 96-spot polished-steel targets.
Antimicrobial susceptibility was determined by disk diffusion and agar dilution methods, in accordance with the Comité de l’Antibiogramme de la Société Française de Microbiologie (CA-SFM) 2008 guidelines. Antibiotic disks were purchased from Bio-Rad (Marnes-la-Coquette, France). The minimum inhibitory concentrations (MICs) were determined by agar dilution method in Mueller–Hinton medium (Fluka BioChemika, Spain) and E-test strips for carbapenems (imipenem, meropenem) (bioMérieux, Marcy l’Etoile, France). Isolates with MICs of imipenem >8 μg/ml and inhibition zone diameter <17 mm were investigated in this study. The double-disk synergy test (DDST) was used to detect MBL.
Strains showing non-susceptibility to carbapenems were screened for the production of acquired carbapenem-hydrolyzing class D β-lactamase: blaOXA−23, blaOXA−24, blaOXA−58, intrinsic β-lactamase blaOXA−51, and MBL blaNDM-1. Quantitative real-time PCR (CFX96, C1000 Thermal Cycler, Bio-Rad) and standard PCR were carried out to detect the encoding genes. MasterMix was prepared in accordance with the manufacturer's instructions and positive controls carrying each gene were used to determine the efficacy of the real-time PCR assay. The experimental run protocol used was as follows: denaturation program (95 °C for 15 min), amplification and quantification programs repeated 35 times (95 °C for 30 s, 60 °C for 1 min). Samples were considered positive if a threshold cycle was reached during the 35 cycles or less. Standard PCR analysis was performed for blaVIM−like, blaGIM−like, blaIMP−like, blaKPC−like, blaNDM-1−like, blaCTX-M−like, blaSHV−like, blaTEM−like, blaPER−like, and blaGES−like. PCR screening was also performed for aminoglycoside-modifying enzyme and fluoroquinolone resistance genes (aac(3)-Ia, aac(6′)-Ib, aadA, ant(2′′)-I, aph(3′)-VI, armA, rmtA, rmtF, arr-2, qnrA, and qnrB). Oligonucleotide primers and probes used are listed in the Supplementary Material (Table S1). Purified PCR products were sequenced using BigDye terminator chemistry on an automated ABI 3730 sequencer (PE Applied Biosystems, Foster City, CA, USA) based on Sanger's sequencing method. Data collection and analysis were performed using CodonCode Aligner 3.7.1.1 sequencing analysis software.
3. Results
In total, 100 human isolates and 13 hospital environment isolates were collected from Tlemcen Hospital, Oran Hospital, and Sidi Bel Abbes Hospital (51, 45, and 17, respectively). Overall, 106 strains were identified as A. baumannii, one strain as A. radioresistens (from Sidi Bel Abbes Hospital), two strains as A. nosocomialis (from Oran Hospital), and four strains as A. pittii (from Tlemcen, Oran, and Sidi Bel Abbes hospitals). All the isolates were identified to the species level with a log score >2.0; the mass spectrometry-based identification scheme yielded identical results compared against the default Bruker database. A mean spectra projection (MSP) dendrogram was generated on the basis of consensus spectra obtained from each bacterium (Figure 1).
Figure 1Mean spectra projection (MSP) dendrogram generated by BIOTYPER software (version 2; Bruker Daltonics) of Acinetobacter spp strains.
The overall susceptibility of all the strains according to the French CA-SFM breakpoints showed that most of the isolates were characterized by resistance to β-lactams (piperacillin 92.2%, piperacillin–tazobactam 88%, ticarcillin 95.9%, ticarcillin–clavulanic acid 96.2%, ceftazidime 98.6%), to fluoroquinolones (ciprofloxacin 85%, with MICs ranging from 0.125 to 0.25 μg/ml), and to aminoglycosides (amikacin 79.1%, gentamicin 56.1%, and tobramycin 38.9%, with MICs ranging from 1 to 512 μg/ml), whilst they differed in their susceptibility to imipenem (70.8%) and showed different levels of resistance with MICs ranging from 0.5 to 512 μg/ml. However, all isolates were susceptible to colistin (MIC 0.125–0.25 μg/ml) (Table 1).
Table 1Resistance rates for Acinetobacter spp isolates in this study
Eighty imipenem-resistant strains (with MIC ranging from 64 to 512 μg/ml), including 42 (82%) imipenem-resistant A. baumannii from Tlemcen Hospital, 31 (69%) imipenem-resistant Acinetobacter spp from Oran Hospital (30 A. baumannii and one A. nosocomialis), and seven (41%) imipenem-resistant Acinetobacter spp from Sidi Bel Abbes Hospital (six A. baumannii and one A. radioresistens) were screened for the presence of carbapenemase-encoding genes (Table 2). Real-time PCR results showed that 40 out of 80 imipenem-resistant isolates were positive for the blaOXA−23 gene (31 A. baumannii from Tlemcen Hospital, seven A. baumannii and one A. nosocomialis from Oran Hospital, and one A. baumannii from Sidi Bel Abbes Hospital) and 17 isolates harbored the blaOXA−24 gene (four A. baumannii from Tlemcen Hospital, 11 A. baumannii from Oran Hospital, and one A. baumannii and one A. radioresistens from Sidi Bel Abbes Hospital), of which five coexisted with the OXA–23 gene (Table 3).
Table 2Isolates of Acinetobacter spp in relation to the presence of carbapenemase enzymes
In addition, among all the isolates, five from Oran were positive for the MBL NDM-1. All five isolates showed positivity by DDST. The gene was sequenced and revealed 99% identity to the sequence reported in the GenBank database under accession number . The five NDM-1-positive isolates were from autochthonous cases in five patients admitted to the ICU and hematology wards of Oran Hospital. All five blaNDM-1-positive isolates were identified as A. baumannii. The earliest positive isolate was collected in April 2011 from a 38-year-old man hospitalized on the hematology ward who was then transferred to the ICU of Oran Hospital for a severe cranial trauma subsequent to a stair fall. He had no relevant travel history and neither did his family. Antibiotics used were ceftazidime, amikacin, and colistin, then imipenem and colistin. The patient died in July 2011. The four other patients were all men aged up to 38 years who were admitted to the same ICU as the first patient during the period April to August 2011. Unfortunately no additional clinical records were available for these patients.
No blaVIM−like, blaGIM−like, blaIMP−like, blaCTX-M−like, blaSHV−like, blaTEM−like, blaPER−likeblaGES−like, blaKPC−like, or blaOXA−58 were detected in the collected strains. Resistance to aminoglycosides (gentamicin, tobramycin, and amikacin) observed in almost all the isolates was due to the expression of aac(3)-Ia (77 isolates), aadA (57 isolates), ant(2′′)-I (60 isolates), aph(3′) (70 isolates), and aac(6′)-Ib (one isolate) genes. No isolates were positive for rmtA, rmtF, armA, arr-2, or the qnr genes.
4. Discussion
Acinetobacter spp has recently emerged as one of the most important opportunistic nosocomial pathogens. Although A. baumannii is the most important species in clinical settings, the other Acinetobacter spp, such as A. pittii and A. nosocomialis, are also frequently isolated in hospitals and have been involved in a number of outbreaks in ICUs.
The analysis of the dendrogram generated by BIOTYPER software showed that the protein signatures formed five separate clusters related to each one of the species, excluding A. baumannii strains that form two separates clusters. This is consistent with the findings of Espinal et al., who showed that MALDI-TOF MS is able to identify and class Acinetobacter strains in separate clusters.
In our study, we investigated the high prevalence of carbapenemase-encoding genes (OXA-type carbapenemase and blaNDM-1) in Acinetobacter spp. OXA-type carbapenemase-producing A. baumannii are increasingly reported from Europe, South America, Asia Oceania, and Africa.
Emergence of bla(OXA–23) and bla(OXA–58) carbapenemase-encoding genes in multidrug-resistant Acinetobacter baumannii isolates from University Hospital of Annaba, Algeria.
Emergence of bla(OXA–23) and bla(OXA–58) carbapenemase-encoding genes in multidrug-resistant Acinetobacter baumannii isolates from University Hospital of Annaba, Algeria.
Rapid detection of carbapenem resistance in Acinetobacter baumannii using matrix-assisted laser desorption ionization-time of flight mass spectrometry.
In our series of isolates, the main molecular support explaining the resistance to carbapenems is the presence of blaOXA−23 carbapenemase-encoding genes, along with the coexistence of blaOXA−24. Consequently, the isolates demonstrated high rates of co-resistance to all other classes of antimicrobial agents tested. A limited number of antimicrobial agents maintain reliable levels of activity against OXA–23-producing A. baumannii.
Neither of the non-baumannii Acinetobacter showed the coexistence of blaOXA−23 with blaOXA−24, in contrast to A. baumannii isolates, of which five harbored both genes at the same time.
OXA–23 (formerly ARI-1) was originally reported in an A. baumannii detected in Scotland in 1985.
In a report by Opazo et al., the blaOXA−23 gene is reported to have originated in the chromosome of A. radioresistens, which might be the natural reservoir of these enzymes
Emergence of bla(OXA–23) and bla(OXA–58) carbapenemase-encoding genes in multidrug-resistant Acinetobacter baumannii isolates from University Hospital of Annaba, Algeria.
none of the isolates in this series were positive for this gene. The blaOXA−51-like gene, originally intrinsic to A. baumannii, was detected in all the isolates except one A. pittii and one A. nosocomialis. These blaOXA−51-like genes, all preceded by ISAba1, may confer a high level of carbapenem resistance. They were probably located on plasmids that might have emerged between different clones of non-baumannii Acinetobacter species and also between A. baumannii clones. The plasmid-borne ISAba1–blaOXA−51-like in non-baumannii Acinetobacter species not only contributes to a high level of carbapenem resistance, but also affects the accuracy of using blaOXA−51-like detection as a tool for differentiating A. baumannii from other Acinetobacter species.
In the present study, we found five strains producing the blaNDM-1 gene in autochthonous cases in the ICU of Oran Hospital between April and August 2011. No bacterial isolates harboring the blaNDM-1 gene were detected in Algeria from the beginning of the study (2008) until this period. The global distribution of the blaNDM-1 gene has been extensively described.
Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India.
The potential presence of this gene in non-baumannii Acinetobacter should receive proper attention. All five of the blaNDM-1-positive isolates were identified as A. baumannii, suggesting that this species, which has a robust survival capability, can easily acquire foreign resistance genes.
hospitals. Patient histories were confirmed as lacking any foreign travel, suggesting that NDM-producing A. baumannii isolates may have already spread in North Africa.
A recent study has suggested that the putative original source of the blaNDM-1 gene could be from the chromosome of plant pathogens, such as Pseudoxanthomonas and related bacteria widespread in the environment.
The spread of strains carrying the blaNDM-1 gene will enhance the likelihood of variants emerging. Interestingly, we have evidence that NDM-encoding genes may be widespread in A. baumannii, and further molecular surveys will be necessary to evaluate their distribution in that species. Many studies have constituted reports on carbapenem-resistant A. baumannii whose carbapenem resistance is mediated mainly by OXA-type carbapenemases. Despite being less commonly identified in A. baumannii than oxacillinase, NDM-1 is currently spreading worldwide and could be reported with a high frequency as a mediator of carbapenem resistance. It is thus critical to survey the presence of this gene in multidrug-resistant (MDR) A. baumannii isolates worldwide.
Although polymyxins such as colistin (polymyxin E) have not typically been included in regimens to treat Acinetobacter infections because of their neurotoxicity and nephrotoxicity, they are now considered as one of the last resorts against MDR Acinetobacter infections. Owing to the increasing use of colistin against Gram-negative pathogens and the high recombination rate of Acinetobacter spp, it is of concern that colistin resistance in Acinetobacter spp isolates may increase rapidly.
In conclusion, the spread of NDM-1-positive A. baumannii isolates in the hospital setting reemphasizes the need for strict adherence to surveillance programs in order to prevent the colonization, the infection, and the dissemination of this gene in Algeria.
Acknowledgements
We thank Linda Hadjadj for technical assistance.
Funding: This work was partly funded by CNRS and IHU Méditerranée Infection in Marseille, France, and LAPSAB Tlemcen Laboratory, Algeria.
Genotypic and phenotypic characterization of the Acinetobacter calcoaceticus–Acinetobacter baumannii complex with the proposal of Acinetobacter pittii sp. nov. (formerly Acinetobacter genomic species 3) and Acinetobacter nosocomialis sp. nov. (formerly Acinetobacter genomic species 13TU).
Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India.
Emergence of bla(OXA–23) and bla(OXA–58) carbapenemase-encoding genes in multidrug-resistant Acinetobacter baumannii isolates from University Hospital of Annaba, Algeria.
Rapid detection of carbapenem resistance in Acinetobacter baumannii using matrix-assisted laser desorption ionization-time of flight mass spectrometry.
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