Variations in colistin susceptibility among different species of the genus Acinetobacter

Sir, Bacteria of the genus Acinetobacter with Acinetobacter baumannii in particular have emerged as important nosocomial pathogens, especially for patients in intensive care.1A. baumannii has a remarkable capacity to develop resistance to all clinically relevant antimicrobial agents. Strains of this species resistant to most available agents are encountered in hospitals worldwide. The recent spread of carbapenem resistance has drastically narrowed options for treatment of A. baumannii infections and has led to the reintroduction of polymyxins with colistin in particular for the systemic treatment of infections caused by these bacteria.1 Even though A. baumannii is clinically and epidemiologically the most important species of the genus, other Acinetobacter species have also been implicated in human infections and strains of some of these species may also be multidrug resistant combined with the ability to spread among hospitalized patients.1 Currently, 22 species with valid names and at least 11 additional putative species are recognized within the genus with as many as 25 of them having been found in human specimens.1,2 However, the identification of Acinetobacter species in diagnostic laboratories is cumbersome owing to the lack of practical diagnostic systems providing reliable identification of these microorganisms.1 Consequently, acinetobacters identified by routine procedures as belonging to a particular species may actually represent another species, which can result in the incorrect association of an antimicrobial resistance phenotype or genotype with a given species. As only limited information was available in the literature on the susceptibility of non-A. baumannii strains to polymyxins, we decided to determine the in vitro colistin susceptibility of representatives of Acinetobacter species known to occur in human clinical specimens. A total of 154 strains of 21 species were studied (Table 1). The strains were selected from the collections of the National Institute of Public Health in Prague and Leiden University Medical Center. Each species included the Type or a reference strain and additional strains recovered from human clinical specimens. The vast majority (n = 126) of the strains were isolated in 1978–99, i.e. before the reintroduction of colistin for systemic treatment of Gram-negative infections, whereas only nine strains were collected from 2000 onwards, suggesting a small contribution of polymyxin selective pressure on the strains studied. The majority of the strains (n = 92) were selected from our previous study on Acinetobacter clinical isolates from the Czech Republic;3 the others originated mostly from other European countries. Based on data of origin in time and space, and typing, there was no indication of direct epidemiological relationships between the strains. All strains had been identified by methods enabling reliable identification at the species level, i.e. by the so-called consensus identification,3 which is based on the congruence of the results of amplified rRNA gene restriction analysis (ARDRA) and biochemical tests, and/or by AFLP fingerprinting.2 Colistin Etest (AB Biodisk, Solna, Sweden) was carried out using Mueller–Hinton II agar (BBLTM BD, USA) as suggested by the manufacturer. Colistin (MAST Group, Bootle, UK) susceptibility testing using agar dilution was performed with ∼104 cfu per inoculation spot according to the CLSI guidelines.4 MICs determined by Etest that fell between the 2-fold dilutions of agar dilution MICs were elevated to the next highest drug concentration so that they matched the 2-fold dilution scheme. Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were included in all experiments as quality control strains and values obtained were in agreement with published standards. Distribution of colistin MICs (mg/L) for 154 isolates of Acinetobacter spp. aTogether referred to as the A. calcoaceticus–A. baumannii complex. A. baumannii, genomic species 3 and genomic species 13TU represent the vast majority of hospital clinical isolates of Acinetobacter. Distribution of colistin MICs (mg/L) for 154 isolates of Acinetobacter spp. aTogether referred to as the A. calcoaceticus–A. baumannii complex. A. baumannii, genomic species 3 and genomic species 13TU represent the vast majority of hospital clinical isolates of Acinetobacter. The results of the susceptibility testing are shown in Table 1. Colistin MICs obtained by Etest were in good correlation with those of agar dilution in the concentration range 0.064–32 mg/L, with 88% and 100% agreement within two and three log2 dilutions, respectively. Using the CLSI (≤2 mg/L) susceptibility breakpoint, categorical agreement was achieved for all except two strains. Based on MICs, species could be classified into three groups. The first group included four non-glucose-acidifying and non-haemolytic species (Acinetobacter lwoffii, Acinetobacter johnsonii, Acinetobacter ursingii and Acinetobacter schindleri) associated with MICs of ≤0.125 mg/L while the second group encompassed 12 species with MICs between 0.064 and 1.0 mg/L. The last group included five species with at least some strains yielding colistin MICs of >2 mg/L, i.e. Acinetobacter junii, Acinetobacter parvus, Acinetobacter beijerinckii, genomic species (gen. sp.) 13BJ and gen. sp. 16. It is of practical significance that all strains with an MIC ≥4 mg/L could be easily differentiated from A. baumannii on the basis of their basic phenotypic features such as the presence of clear haemolysis on sheep blood agar plates (A. beijerinckii, gen. sp. 13BJ and gen. sp. 16) or the inability to acidify media with glucose (all except for gen. sp. 13BJ). The most noteworthy were the results for A. beijerinckii, A. junii and gen. sp. 13BJ. Whereas the former two species included both susceptible and resistant strains all eight strains of gen. sp. 13BJ yielded MICs ≥16 mg/L. It has recently been shown that therapy with colistin can be compromised by the selection and spread of colistin-resistant A. baumannii strains.5 Our data further indicate that resistance to colistin may be commonly present (A. junii, A. beijerinckii) in or even intrinsic (gen. sp. 13BJ) to some non-A. baumannii species. Although the colistin-resistant non-A. baumannii strains are rarely isolated from clinical specimens and these strains are usually susceptible to other antimicrobials (data on file), our findings emphasize the importance of precise species identification especially in reports on polymyxin resistance in Acinetobacter spp. Notably, resistance to colistin in A. baumannii has recently been associated with mutations in the PmrAB two-component system6 and it remains to be established whether this system also plays a role in the resistance of non-A. baumannii strains. This work was supported by the Grant Agency of the Czech Republic (grant number 310/08/1747). None to declare. We are grateful to Mrs Martina Maixnerová for technical assistance in susceptibility testing.