- Research article
- Open Access
Co-occurrence of carbapenemase encoding genes in Acinetobacter baumannii, a dream or reality?
- Linda Hadjadj1,
- Sofiane Bakour1 and
- Jean-Marc Rolain1Email authorView ORCID ID profile
- Received: 11 December 2017
- Accepted: 28 August 2018
- Published: 5 September 2018
Abstract
Background
Acinetobacter baumannii is an important opportunistic pathogen that is rapidly evolving towards multidrug resistance and is responsible for life-threatening infections. Carbapenems are commonly used to treat A. baumannii infections but the emergence of carbapenemase encoding genes, such as blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaNDM has been reported. Moreover, several studies have reported the co-occurrence of two distinct carbapenemases in some isolates. The aim of the present study is to demonstrate whether the phenomenon of co-occurrence of two distinct carbapenemase encoding genes in a single isolate still exists.
Results
We studied six strains of A. baumannii including one harboring blaOXA-23-like and blaOXA-24-like genes and five with blaOXA-23-like and blaNDM genes. One colony of each strain was inoculated in sterile water and diluted ten-fold. Each dilution was cultivated on trypticase soy agar plates for 24 h at 37 °C and the isolated bacteria were analyzed. For two of the six tested strains, we identified two different populations of A. baumannii, each with a different carbapenemase, genes encoding aminoglycoside modifying enzymes, resistance phenotype, and clonal type. In addition, the two different populations had the same aspect on the agar plate.
Conclusions
Here, we demonstrate that A. baumannii infections could be linked to multiple clones harboring different carbapenemase encoding genes in the same sample. In addition, we describe an easy method of verifying the presence of co-occurrence of carbapenemase in one isolate.
Keywords
- Acinetobacter baumannii
- Carbapenemase
- Co-occurrence
Background
Acinetobacter baumannii is an important opportunistic Gram-negative bacteria pathogen that is rapidly evolving towards multidrug resistance. Worldwide, this bacterium is responsible for nosocomial infections and life-threatening infections [1]. The most common treatment is the use of carbapenems but extensive use of antimicrobial agents within hospitals has contributed to resistance against these antibiotics. The principal mechanism of resistance to carbapenems in A. baumannii is the production of OXA-type carbapenemases, such as OXA-23, OXA-24, and OXA-58 enzymes, and the new metallo-β-lactamase (MBL), New Delhi Metallo-β-lactamase 1 (NDM-1) [1].
The location of the carbapenemase genes is not fixed, since some studies have described blaOXA-23, blaOXA-24 and blaOXA-58 as chromosomal or plasmidic [2]. In epidemiologic studies, co-occurrences of carbapenemase genes, such as blaOXA-23-blaOXA-24, blaOXA-23-blaNDM, and blaOXA-58-blaNDM have been described [3–8]. In blaOXA-23 and blaNDM co-expressing Acinetobacter spp. strains, blaNDM can be acquired by plasmid [4, 9]. However, it remains unclear whether and how these co-existing carbapenemase genes are expressed and how they contribute to drug resistance. The aim of our study was to provide an alternative explanation for the coexistence of blaOXA and blaNDM genes in A. baumannii. For this purpose, we describe an easy method of determining whether the co-occurrence of carbapenemase genes in such bacteria was “real” or whether it is due to the existence of different bacterial clones harboring different genes in the same sample.
Results
Analysis of the original A. baumannii strains and their clones
Strain | Date of isolation (month/ year) | Sample/Ward | Carbapenemase genes | Aminoglycoside genes | ST | CTX | CAZ | TIM | TPZ | ATM | IPM | CN | TOB | AK | CIP | SXT | DO | CT |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
(μg/mL) | ||||||||||||||||||
519 (n = 10) | 03/2013 | NA/ Intensive Care Unit | blaOXA-23- blaNDM-1 | / | 25 | R (> 32) | R (> 256) | R (> 256) | R (> 256) | R (32) | R (> 32) | R (> 256) | R (> 256) | R (32) | R (> 32) | R (> 32) | R (32) | S (0.5) |
598 (n = 10) | 03/2013 | NA/ Burns | blaOXA-23- blaNDM-1 | / | 25 | R (> 32) | R (> 256) | R (> 256) | R (> 256) | R (32) | R (> 32) | R (> 256) | R (> 256) | R (32) | R (> 32) | R (> 32) | R (32) | S (0.5) |
624 (n = 10) | 03/2013 | NA/ Intensive Care Unit | blaOXA-23- blaNDM-1 | / | 25 | R (> 32) | R (> 256) | R (> 256) | R (> 256) | R (32) | R (> 32) | R (> 256) | R (> 256) | R (32) | R (> 32) | R (> 32) | R (32) | S (1) |
679 (n = 10) | 03/2013 | NA/ Pediatric | blaOXA-23- blaNDM-1 | / | 25 | R (> 32) | R (> 256) | R (> 256) | R (> 256) | R (32) | R (> 32) | R (> 256) | R (> 256) | R (32) | R (> 32) | R (> 32) | R (32) | S (0.5) |
924 (n = 10) | 05/2011 | Pus/ Pus unit | blaOXA-23- blaNDM-1 | aph(3′)-VI | 2 | R (> 32) | R (> 256) | R (> 256) | R (> 256) | R (128) | R (> 32) | R (> 256) | R (> 256) | R (> 256) | R (> 32) | R (> 32) | R (> 256) | S (1) |
924-A (n = 4) | bla OXA-23 | aph(3′)-VI | 2 | R (> 32) | R (> 256) | R (> 256) | R (> 256) | R (16) | R (> 32) | S (0.093) | S (2) | R (> 256) | R (> 32) | R (> 32) | R (> 256) | S (0.5) | ||
924-B (n = 6) | bla NDM-1 | / | 25 | R (> 32) | R (> 256) | R (> 256) | R (> 256) | R (128) | R (> 32) | R (> 256) | R (> 256) | R (16) | R (> 32) | R (> 32) | R (24) | S (1) | ||
AH35 (n = 10) | 05/2011 | Urine/ Intensive Care Unit | blaOXA-23- blaOXA-24 | aph(3′)-VI, aac(3)-Ia, ant(2″)-I | 2 | R (> 32) | R (> 256) | R (> 256) | R (> 256) | R (> 256) | R (> 32) | R (8) | R (> 256) | R (24) | R (> 32) | R (> 32) | R (> 256) | S (0.5) |
AH35-A (n = 6) | bla OXA-23 | aph(3′)-VI, ant(2″)-I | 2 | R (> 32) | R (> 256) | R (> 256) | R (> 256) | R (> 256) | R (> 32) | S (0.064) | S (1) | R (16) | R (> 32) | R (> 32) | R (> 256) | S (0.5) | ||
AH35-B (n = 4) | bla OXA-24 | aac(3)-Ia | 2 | R (> 32) | R (> 256) | R (> 256) | R (> 256) | R (128) | R (> 32) | R (8) | R (> 256) | R (24) | R (> 32) | R (> 32) | R (> 256) | S (0.5) | ||
a Isolation of original strain AH35 blaOXA-23 and blaOXA-24 on TSA medium and isolation of clones. AH35A; blaOXA-23 on the left, AH35B; blaOXA-24 on the right. b Isolation of original strain 924 blaNDM and blaOXA-23 on TSA medium and isolation of clones 924A; blaOXA-23 on the left, 924B and blaNDM on the right
Discussion
Since A. baumannii is able to remodel its genome, antibiotic use and the host environment might impose selective forces that drive its rapid adaptive evolution. Microscale genome modification has been revealed through the analysis of single nucleotide polymorphisms (SNPs) between A. baumannii strains isolated from the same patient. This modification can lead the emergence of resistance and to different sequence typing by modifying a single allele [10]. However, this explanation cannot be applied to our case, because ST2 and ST25 have only two common alleles. Mutations in five alleles would be a substantial evolution. Moreover, this cannot be an evolution of a clone because the presence of different carbapenemase-encoding genes was also observed.
Another phenomenon, referred to as small colony variants (SCVs), mostly described in Staphylococcus aureus, was also identified from one sample with different susceptibility to antibiotics from the parent strain [11]. SCVs can cause latent and recurrent infections and have been observed in many genera of bacteria, including Gram-negative bacteria such as Pseudomonas aeruginosa [12]. In our case, the populations are morphologically identical and do not have the aspect of small colony variants.
The presence of different populations of a same species in the same sample had been already described in S. aureus [13]. Similarly to our study, different clones with different resistance phenotypes, such as methicillin-susceptible and methicillin-resistant S. aureus strains could be isolated from the same sample by increasing the number of colonies tested for each sample [14].
BlaOXA-23, blaOXA-24 and blaNDM can be located either on the chromosome or plasmid. For this reason, we tend to believe that co-occurrence with other resistance genes is due to the presence of different genes in the same strain. However, another explanation could be the presence of a polyclonal population of A. baumannii from the same sample. These populations are morphologically undetectable with the naked eye. Although the number of strains studied was limited, our work represents the proof of concept that co-occurrence of carbapenemases in A. baumannii could be due to multiple clones.
Conclusion
In this work, we demonstrated that from one sample and from an original “chimera” strain with two carbapenemase genes which were highly resistant to antibiotics, we were able to isolate two less resistant strains with only one gene encoding for carbapenems resistance. The presence of different clonal types and different genes encoding aminoglycoside modifying enzymes in each clone is also evidence of polyclonal coexistence in a single sample.
Another option, in similar case to this current study, is the coexistence in a single sample of different clones harboring one carbapenemase encoding gene as well as different resistance genes. In addition, this study has strong implications for clinical practice. The use of only one colony from one sample to conduct AST may omit another population which may be more resistant.
Methods
Six A. baumannii strains (924, 519, 598, 624, and 679) isolated from two Algerian hospitals were studied; five A. baumannii co-expressed blaOXA-23 and blaNDM genes recovered from a hospital in Algiers, and one A. baumannii (AH35) co-expressed blaOXA-23 and blaNDM gene isolated from a urine sample in an intensive care unit in a hospital in Setif (Table 1). These clinical isolates were taken for the purpose of this study. Identification was confirmed using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF) (Microflex, Brüker Daltonics, Bremen, Germany) [15].
Example of the study of the strain 924
Abbreviations
- AME:
-
Aminoglycoside-modifying enzyme
- AST:
-
Antibiotic susceptibility testing
- EUCAST:
-
European Committee on Antimicrobial Susceptibility Testing
- MALDI-TOF:
-
Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry
- MBL:
-
Metallo-β-lactamase
- MIC:
-
Minimum inhibitory concentration
- MLST:
-
Multilocus sequence typing
- SCV:
-
Small colony variants
- SNP:
-
Single nucleotide polymorphism
- TSA:
-
Trypticase soy agar
Declarations
Acknowledgments
We thank TradOnline for English corrections.
Funding
This work was supported by the French Government under the « Investissements d’avenir » program managed by the Agence Nationale de la Recherche, (reference: Méditerranée Infection 10-IAHU-03). This work was supported by Région Provence Alpes Côte d’Azur and European funding FEDER PRIMI.
Availability of data and materials
All data generated or analysed during this study are included in this published article.
Authors’ contributions
LH wrote the manuscript, performed experiments, and analyzed the data. SB provided the strains and helped draft the manuscript. JMR conceived the study, participated in its design and coordination, and helped draft the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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Authors’ Affiliations
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