A total of 47 A. baumannii clinical isolates were derived from the blood, sputum or swab samples of patients from different departments at the First Affiliated Hospital of Chengdu Medical College, China from 2014 to 2015. Bacteria were aerobically grown at 37°C in Mueller-Hinton (MH) broth or agar (Oxoid, England) or in Luria-Bertani (LB) broth (Lin et al., 2009). These isolates were identified by ATB New (bioMérieux, France) (Chang et al., 2015) and further verified by PCR products of rpoB (Wang et al., 2014) and 16S rRNA gene (Chiang et al., 2011) with the primers included in Table S1. The PCR products were sequenced by Tsingke Biological Technology (Tsingke, Chengdu, China), followed by sequence alignment using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Antibiotics and biocides used in this study were purchased commercially. Chlorhexidine acetate and triclosan were purchased from Dalian Meilun Biological Technology (Dalian, China), benzalkonium bromide from Chengdu Dingdangchem Medical Technology (Chengdu, China), hydrogen peroxide, ethanol and sodium hypochlorite from Chengdu Kelong Chemical Reagent Factory (Chengdu, China). Biocide stock solutions of 10 mg/L of chlorhexidine acetate or benzalkonium bromide in deionized water and triclosan in dimethyl sulfoxide were prepared. Susceptibility testing for antibiotics and biocides was carried out using the broth microdilution method in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2014). The ranges of biocide concentrations evaluated were 0.25–256 μg/ml (chlorhexidine acetate), 0.25–256 μg/ml (benzalkonium bromide), 0.25–256 μg/ml (triclosan), 1.25–2,560 μg/ml (sodium hypochlorite), 2.9–6,021 mM (hydrogen peroxide), and 7.5–75% (vol/vol) (ethanol). The minimal inhibitory concentration (MIC) was defined as the lowest concentration of an antimicrobial agent at which no bacterial growth was observed visually after incubation at 37°C for 18 h (CLSI, 2015).

Eleven known drug resistance transporter genes abeD (Srinivasan et al., 2015), aceI (Hassan et al., 2013), adeB, adeG, adeJ (Coyne et al., 2011), adeT1, adeT2 (Srinivasan et al., 2011), abeM (Su et al., 2005), amvA (Rajamohan et al., 2010a), qacE, qacEΔ1 (Mahzounieh et al., 2014; Babaei et al., 2015), and the triclosan target gene, fabI (Rajamohan et al., 2010b; Fernando et al., 2014), were PCR amplified to determine their distribution in the collected clinical isolates. The primers used are listed in Table S1. PCR amplification of the target genes was performed in a 50 μl volume containing 0.2 mM of each deoxy nucleotide, 0.5 μM of each primer, 1.25 U of Taq polymerase, and 5 μl of 10 × buffer (Vazyme, China). The reaction was amplified using a program consisting of 94°C for 5 min followed by 30 cycles of 94°C for 30 or 60 s, annealing at 50–55°C for 30 or 50 s and extension at 72°C for 30–90 s, and a final extension at 72°C for 10 min. The PCR products were verified by DNA sequencing.

MLST was used to characterize the seven housekeeping genes: gltA (encoding citrate synthase), gyrB (DNA gyrase subunit B), gdhB (glucoside hydrogenase B), recA (homologous recombination factor), cpn60 (60-kDa chaperonin), gpi (glucose-6-phosphate isomerase) and rpoD (RNA polymerase sigma factor) (http://pubmlst.org/abaumannii/info/primers-Oxford.shtml). Amplification reactions were carried out as described previously (Woo et al., 2011). eBURST (Version3, http://eburst.mlst.net/) was used to compare the A. baumannii clinical isolate sequence types (ST) to the existing STs and to assign isolates to their clonal complexes. A clonal complex (CC) is defined as a set of similar ST(s) having the same alleles at ≥6 of 7 loci (Aanensen and Spratt, 2005).

PFGE was carried out as previously described (Seifert et al., 2005). A. baumannii isolates were grown on MH plates overnight at 37°C. Strains were lysed with proteinase K (20 mg/ml stock solution). The plugs were thoroughly washed and then digested using ApaI restriction enzyme (Takara, China). Restricted DNA fragments were separated using CHEF Mapper XA system (Bio-Rad, Hercules, CA, USA). The gels were run at 14°C in 0.5 × TBE buffer for 18 h, with pulse time of 5–35 s. XbaI-digested Salmonella enterica ser. Braenderup H9812 was used as the molecular marker (Dhanoa et al., 2015). Data analysis was based on the Dice coefficient of similarity analyzed by the BioNumerics 3.0 software (Applied Maths, Belgium) using the Unweighted Pair Group Method with Arithmetic Averages (UPGMA) algorithm at 1.5% band position tolerance. Pulsotype designation was based on isolates showing ≥80% relatedness.

Overnight cultures of bacterial strains were subcultured in LB (1:100 dilution) and allowed to grow to an OD₆₀₀ of 0.6–0.8. A. baumannii cells were exposed to a subinhibitory concentration (1/2 MIC of triclosan or chlorhexidine; see results for the MIC values of chlorhexidine and triclosan) and grown in parallel with control (no biocide addition) for 2 h. One milliliter of the cell culture was centrifuged and cells were collected for total RNA extraction using TRIzol reagent (Invitrogen). To remove contaminating genomic DNA, the samples were treated with RNase free DNase I based on the manufacturer's instructions (Takara). qRT-PCR analysis was carried out for three RND pump-encoding genes (adeB, adeG, and adeJ), the MATE family pump gene abeM, the Acinetobacter chlorhexidine efflux protein gene aceI, and the triclosan target-encoding gene fabI. The primers used for qRT-PCR analysis are listed in Table S1. Reverse transcription was performed by using the M-MLV Reverse Transcriptase (Invitrogen). The expression of genes was determined by quantitative PCR using SsoFast EvaGreen Supermix (Bio-Rad) with the following cycle profile: 1 cycle at 95°C for 3 min, followed by 40 cycles at 95°C for 15 s, 55°C for 15 s. Relative expression was determined using the cycle threshold (ΔΔCt) method on the Bio-Rad CFX96 real-time system (Bio-Rad). All reactions were carried out in triplicate with at least two biological replicates. The relative expression levels of target genes were normalized to those of the 16S rRNA gene (Arroyo et al., 2011; Fernando and Kumar, 2012; Kuo et al., 2012; Lin et al., 2017a,b). Data analysis was carried out according to the manufacturer's instructions using the Bio-Rad CFX 3.0 software which is designed for Bio-Rad CFX real-time PCR detection system. Expression data is presented by the mean plus/minus one standard deviation.

A. baumannii clinical isolates (n = 47) were collected from three different locations including sputum (n = 42), throat swabs (n = 4) and blood (n = 1) from patients at the First Affiliated Hospital of Chengdu Medical College. Epidemiological analysis of the 47 patients (30 males and 17 females) revealed that 22 were ≥70 years old and 15 were between 60 and 70 years old. Overall, these two patient cohorts account for 78.81% (37/47) of the total isolates. Among the isolates, 22 were collected from intensive care units and 9 were collected from the respiratory department. Most of the clinical specimens were sputum, which is consistent with A. baumannii as a major pathogen associated with respiratory tract infection (Bergogne-Berezin and Towner, 1996; Babaei et al., 2015). The clinical characteristics of the 47 patients with A. baumannii infection are summarized in Table 1. Based upon previous antimicrobial resistance and treatment concerns for A. baumannii, we decided to investigate the susceptibility to antibiotics and biocides in A. baumannii and underlying resistance mechanisms.

The results of antibiotic and biocide susceptibility testing are shown in Table 2 and Tables S2, S3. Together, 15 antibiotics and 6 biocides were tested. Based on the interpretative categories from the CLSI (CLSI, 2015), the tested isolates were frequently multidrug resistant (64% [30/47]), including resistance to β-lactams, aminoglycosides, fluoroquinolones and tetracyclines (Table 2 and Table S2). In particular, the rates of resistance to carbapenems and third-generation cephalosporins, amikacin, and minocycline were, respectively, about 60, 40, and 34%.

The tested isolates also showed a variety of susceptibility to biocides. The isolates had MICs ranging from 2 to 256 μg/mL for triclosan, 8 to 128 μg/mL for chlorhexidine, 4–32 μg/mL for benzalkonium bromide, 47–376 mM (1.6–13 mg/mL) for hydrogen peroxide, and 7.5–22.5% (vol/vol) (60–180 mg/mL) for ethanol (Table 2 and Table S3; Figure 1). The MIC values of antibiotics and biocides for the individual isolates are included in Tables S2, S3. Because of the absence of the clinically-relevant interpretative criteria for biocide susceptibility, we could not categorize the isolates as either susceptible or resistant (Table 2). However, the biocide MIC distribution (Figure 1) still allowed for the determination of susceptibility trends of the tested isolates toward biocides. In particular, the bimodal distribution of triclosan and chlorhexidine MIC values clearly indicates the presence of the two subgroups for each of the two biocides (Figure 1). These two subgroups likely represent the wild-type MIC and resistant (or non-susceptible) MIC. In this regard, identification of the wild-type MIC population for bacterial isolates such as the establishment of the microbiologically-based epidemiological cutoff values is critical for resistance surveillance (Turnidge et al., 2006; Martínez et al., 2015). Epidemiological cutoff values would allow for the discrimination of wild-type strains from strains with acquired resistance. Based on the biocide susceptibility results (Figure 1 and Table S3), it is feasible to consider the value of ≤16 μg/ml and ≥64 μg/ml, respectively, as the wild-type MIC and reduced susceptibility (non-susceptible/resistance) cutoff for both triclosan (53 vs. 34%) and chlorhexidine (36 vs. 47%) (Table 2 and Table S3; Figure 1). However, even though the four other biocides tested (benzalkonium, hypochlorite, hydrogen peroxide and ethanol) displayed variations in biocide susceptibility, only one major population can be identified for the isolates for their susceptibility toward the individual agents. Furthermore, these populations likely reflect the intrinsic susceptibility or overlapping wild-type/resistant MICs of A. baumannii (Figure 1). Additionally, comparing the absolute MIC values of antibiotics and biocides (Tables S2, S3), biocide agents are less active than antibiotics against susceptible isolates, suggesting a higher intrinsic resistance level of A. baumannii isolates to biocides.

The tested isolates are mostly multiple antibiotic resistant (64%; Table S2). However, this resistance phenotype does not clearly link to biocide susceptibility (Tables S2, S3). Moreover, a clear correlation cannot be established for the susceptibility to the six tested biocides (Table S3). In this regard, cross-resistance (reduced susceptibility) to triclosan and chlorhexidine is generally not observed as 13 isolates with reduced triclosan susceptibility (MICs of ≥64 μg/ml) are relatively susceptible to chlorhexidine (MICs of ≤32 μg/ml) while 18 isolates with decreased chlorhexidine susceptibility (MICs of ≥64 μg/ml) are still susceptible to triclosan (MICs of ≤32 μg/ml). It is noted that there were four isolates that had triclosan and chlorhexidine MIC values of ≥64 μg/ml (Table S3).

Multidrug resistance including resistance to structurally-unrelated antibiotics and biocides can be attributable to broad-specific efflux transporters. Consequently, we assessed the distribution of 11 known drug efflux pump genes, which encode the characterized drug efflux pumps of five different families (i.e., AdeB, AdeG, AdeJ, AdeT1, and AdeT2 of the RND family, AbeD and Amv of the MFS family, AbeM of the MATE family, QacE and QacEΔ1 of the SMR, and AceI of PACE family). Based upon literature information including published genome sequence data, most of these target genes (if not all) are expected to be located on the chromosomes (Li et al., 2015). Indeed, many of the genes were present in the majority of isolates (e.g., 95–100% for aceI, adeJ, adeT2, abeD, abeM, and amvA). The adeB and adeG genes were both positive, in 85 and 89% isolates, respectively. Only three genes, adeT1, qacE, and qacEΔ1, were detected at lower prevalence at 75, 70, and 68%, respectively (Table 3).

Triclosan target gene, fabI, was present in all tested isolates. As resistance to triclosan is known to be attributable to specific mutations in fabI, DNA sequencing was carried out for PCR-amplified complete fabI gene products from 13 highly triclosan resistant isolates (triclosan MICs of ≥128 μg/ml) and 1 triclosan-susceptible isolates (triclosan MICs of 16 μg/ml) (Table S3). Unexpectedly, only one high-level triclosan-resistant isolate (i.e., AB30 [Table S3]) was found to carry a point mutation at amino acid position 95 of FabI, Gly95Ser. Although altered base pairs were observed in fabI gene of various isolates, these changes did not alter the amino acid residues of FabI (data not shown).

To investigate the role of efflux pumps in biocide resistance, we targeted the isolates with reduced susceptibility to triclosan and chlorhexidine to analyze gene expression of adeB, adeJ, adeG, abeM, aceI, and fabI. These genes were targeted based on their known or putative role in mediating antibiotic and/or biocide resistance. The relative expression of adeB, adeG, adeJ, abeM, aceI, and fabI in comparison with the expression of the housekeeping gene of 16S rRNA is shown in Table 4. Overall, there was generally detectable, variable expression of these genes which was isolate-specific (i.e., dependent on the gene and isolate). No clear relationship could be established for antibiotic or biocide susceptibility and efflux gene expression levels in individual isolates. However, based on the susceptibility to triclosan, chlorhexidine or antibiotics, the tested isolates (n = 24) were divided into three sets of pairs for comparison, i.e., wild-type (WT) (triclosan or chlorhexidine MIC ≤16 μg/ml, or antibiotic susceptible) and non-susceptible (NS) (triclosan or chlorhexidine MIC ≥64 μg/ml, or antibiotic resistant) groups as presented in Table 4. We noted the following gene expression patterns: (i) the relative expression of the adeB and adeJ genes was doubled or elevated in triclosan non-susceptible isolates compared to the wild-type isolates whereas no significant difference was observed for other four genes (adeG, abeM, aceI, and fabI); (ii) the relative expression of the abeM and fabI genes was 2- to 3-fold higher in chlorhexidine non-susceptible isolates compared to the wild-type isolates, while the expression of adeG decreased in chlorhexidine non-susceptible isolates; (iii) aceI expression in chlorhexidine non-susceptible isolates is 38% higher than that in wild-type isolates; (iv) the relative expression of the adeJ and abeM genes was 3- to 4-fold higher in antibiotic-resistant isolates than antibiotic-susceptible isolates and there was more than a 2-fold decrease in the aceI expression in antibiotic-resistant isolates compared to the susceptible isolates (Table 4).

Investigation was also performed to assess the induction of the above mentioned genes by subinhibitory levels of triclosan or chlorhexidine. Expression of adeB, adeG, adeJ, and fabI increased about 3-fold upon induction by triclosan while only adeB expression was elevated by 4-fold after the induction with chlorhexidine (Table 5).

To investigate the linkage of biocide-resistant A. baumannii, the molecular characteristics of 13 isolates with high-level resistance to triclosan (MICs of ≥128 μg/mL; Table S3) were typed by MLST and PFGE. One isolate with triclosan MICs of 16 μg/mL (i.e., isolate AB01) were included as triclosan-susceptible strain for comparison. The MLST analysis revealed a total of 8 different STs (Figures 2, 3), including 4 existing STs (ST92, ST184, ST195, and ST618) and 4 novel STs (ST1277, 1-34-80-28-1-22-3; ST1278, 1-3-3-28-2-7-45; ST1279, 51-12-49-13-48-103-4; ST1280, 1-3-67-6-2-163-5). ST92, accounting for 46.2% (6/13), was the major clone followed by two isolates as ST195 (2/13). The remaining 5 isolates were ST184, ST618, ST1278, ST1279, and ST1280. The triclosan-susceptible isolate belongs to ST1277. Clonal relation analysis showed that ST92 and ST195 belonged to the CC92 lineage, with only one locus (gpi) different between the 2 STs, accounting for 61.5% (8/13) of 13 triclosan-resistant isolates. This result suggests that CC92 represents the most widely distributed A. baumannii clone complex identified in the hospital for strains with reduced susceptibility to triclosan.

PFGE was analyzed by BioNumerics and >80% homology was used as a standard to distinguish the types. The PFGE profiles contained 14 strains (Figure 3), including pulsotype A (2/13, 15.4%) and pulsotype B (3/13, 23.1%), and the other 7 strains showed different genetic variability pulsotypes. In addition, the MLST results of the clinical isolates showed that CC92 corresponded to the pulsotype B obtained by PFGE.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.