Co-occurrence of carbapenemase encoding genes in Acinetobacter baumannii, a dream or reality?

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 bla_OXA-23, bla_OXA-24 and bla_OXA-58 as chromosomal or plasmidic [2]. In epidemiologic studies, co-occurrences of carbapenemase genes, such as bla_OXA-23-bla_OXA-24, bla_OXA-23-bla_NDM, and bla_OXA-58-bla_NDM have been described [3–8]. In bla_OXA-23 and bla_NDM co-expressing Acinetobacter spp. strains, bla_NDM 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 bla_OXA and bla_NDM 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.

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].

Bla_OXA-23, bla_OXA-24 and bla_NDM 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.

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.

Six A. baumannii strains (924, 519, 598, 624, and 679) isolated from two Algerian hospitals were studied; five A. baumannii co-expressed bla_OXA-23 and bla_NDM genes recovered from a hospital in Algiers, and one A. baumannii (AH35) co-expressed bla_OXA-23 and bla_NDM 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].

For each strain, one colony was inoculated in sterile water and diluted ten-fold to isolate a single clone. Each dilution was cultivated on Trypticase Soy Agar (TSA) plates for 24 h at 37 °C and isolated bacterial were analyzed. Antibiotic susceptibility was determined by minimum inhibitory concentrations (MICs) using broth microdilution method (Biocentric) for colistin and using the E-test method (bioMérieux) for others antibiotics tested in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Real time PCR was performed to verify the presence of the bla_OXA-23, bla_OXA-24 and bla_NDM genes [16, 17]. Standard PCR amplification was carried out using primers for amplification of the aph(3′)-VI, ant(2″)-I, aac(3)-Ia, aac(6′)-Ib and armA genes [18]. Clonal types of the isolates were determined using multilocus sequence typing (MLST) in line with the Pasteur schemes (https://pubmlst.org/abaumannii/). Each stage of the materials and methods section is summarized in Fig. 2.