1. Introduction
At present, the genus Acinetobacter comprises 108 species (https://lpsn.dsmz.de/genus/acinetobacter). An increasing incidence during the 1970s of resistant members of the family Enterobacteriaceae involved in nosocomial infections was followed by the therapeutic introduction of newer broad-spectrum antibiotics in hospitals and a subsequent increase in the importance of strictly aerobic gram-negative bacilli, including Pseudomonas aeruginosa, Stenotrophomonas (Xanthomonas) maltophilia, and Acinetobacter species.
Of these microorganisms, it is currently recognized that Acinetobacter species play an important role in the nosocomial infection. The genus Acinetobacter is now defined as including gram-negative coccobacilli, with a DNA G + C content of 39 to 47 mol%, that are strictly aerobic, nonmotile, catalase positive, and oxidase negative. These organisms have been implicated in a variety of nosocomial infections, including bacteremia, urinary tract infection, and secondary meningitis, but their predominant role is as agents of nosocomial pneumonia, particularly ventilator-associated pneumonia in patients confined to hospital intensive care units (ICUs). Such infections are often extremely hard for the clinician to treat because of the widespread resistance of these bacteria to the principal groups of antibiotics. Various mechanisms of antibiotic resistance have been recognized in these organisms, and combination therapy is usually required for effective treatment of Acinetobacter nosocomial infections. These therapeutic difficulties are coupled with the fact that these organisms have a significant capacity for long-term survival in the hospital environment, with corresponding enhanced opportunities for transmission between patients, either via human reservoirs or via medical materials.
It is currently considered that most clinically significant Acinetobacter species are Acinetobacter baumannii, Acinetobacter nosocomialis, and Acinetobacter pittii. These species are not differentiated by phenotypic or biochemical methods and are often grouped together diagnostically as the A. baumannii complex, along with Acinetobacter calcoaceticus which is generally not considered pathogenic. Species-level identification based on genetic methods (e.g., rpoB sequencing, rRNA intergenic spacer sequencing) has enabled differentiation of the A. baumannii complex species for research purposes. It has been demonstrated that matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can be utilized as an alternative method to reliably identify A. baumannii complex species [1] [2] [3] [4] . Using these methods, the prevalence of A. baumannii, A. nosocomialis, and A. pittii within the A. baumannii complex has been shown to be highly variable among clinical isolates depending on the epidemiological settings [5] [6] [7] [8] .
The detection of A. baumannii complex species at the species level in clinical specimens is important, as it may affect the prognosis and patient management, but identification by conventional biochemical methods can be difficult. The accurate identification and enumeration of A. baumannii complex species are required to determine their role in various systemic diseases. These organisms can be identified by the sequence analysis of several target genes and MALDI-TOF MS. However, because these methods are laborious, expensive, and time-consuming for use in the detection or discrimination of the clinical isolates of A. baumannii complex species at the species level, epidemiological studies on the relationship between these organisms and various diseases are limited. Thus, a simple and more reliable assay for identifying A. baumannii complex species is required.
The purpose of the present study was to design species-specific primers in order to identify and detect A. baumanii complex species, i.e., A. baumannii, A. nosocomialis, A. pittii and A. calcoaceticus, and Acinetobacter lwoffii which is frequently detected from the human specimens, and to investigate the distribution of these organisms in the dental hospital using a multiplex PCR.
2. Materials and Methods
2.1. Bacterial Strains and Culture Conditions
Bacterial strains were obtained from Japan Collection of Microorganisms (JCM; Japan) and Center for Conservation of Microbial Genetic Resource, Gifu University (GTC; Japan). The following bacterial strains were used in the present study: A. baumannii JCM 6841, A. calcoaceticus JCM 6842, A. lwoffii JCM 6840, A. pittii GTC 00524, and A. nosocomialis GTC 03314. These strains were maintained by cultivating them on BactTM Brain Heart Infusion (BHI, Becton, Dickinson and Co., Sparks, MD, USA) and 1.5% agar (BHI agar). These organisms were cultured at 30˚C overnight under an aerobic condition.
2.2. Design of Species-Specific Primers for A. baumanii Complex Species and A. lwoffii
Design of species-specific primers for A. baumanii complex species, i.e., A. baumannii, A. nosocomialis, A. pittii and A. calcoaceticus, and A. lwoffii was performed as described previously [9] . Briefly, the 16S rRNA gene sequences of A. baumannii (accession no. AB594765), A. nosocomialis (HQ180192), A. pittii (MN307289), A. calcoaceticus (AB626122) and A. lwoffii (X81665), and the DNA gyrase subunit B (gyrB) gene sequences of A. baumannii (AB594765), A. nosocomialis (HQ180192), A. pittii (MN307289), A. calcoaceticus (AB626122) and A. lwoffii (X81665) were obtained from the DNA Data Bank of Japan (DDBJ; https://www.ddbj.nig.ac.jp/services.html, Mishima, Japan), and a multiple sequence alignment analysis was performed with the CLUSTAL W program; i.e., the 16S rRNA and gyrB gene sequences of four A. baumanii complex species and A. lwoffii were aligned and analyzed, respectively. Homology among the primers selected for each Acinetobacter species and their respective 16S rRNA and gyrB gene sequences was confirmed by a BLAST search.
2.3. Development of a Multiplex PCR Method Using Designed Primers
Bacterial cells were cultured in BHI supplemented with 0.5% yeast extract for 24 h, and 1 ml of the samples were then collected in microcentrifuge tubes and resuspended at a density of 1.0 McFarland standard (approximately 107 colony-forming units (CFU)/ml) in 1 ml of sterile distilled water. A total of 3.6 μl of the suspension was then used as a PCR template. The detection limit of PCR was assessed by serially diluting known numbers of bacterial cells in sterile distilled water and then subjecting each suspension to PCR. The multiplex PCR mixture contained 0.2 μM of each primer, 10 μl of 2 × MightyAmp Buffer Ver.3 (Takara Bio Inc., Shiga, Japan), 0.4 μl of MightyAmp DNA Polymerase (Takara), and 5 μl of the template in a final volume of 20 μl. PCR reactions were performed in a DNA thermal cycler (Applied Biosystems 2720 Thermal Cycler; Applied Biosystems, CA, USA). PCR conditions included an initial denaturation step at 98˚C for 2 min, followed by 30 cycles consisting of 98˚C for 10 s, 62˚C for 15 s, and 68˚C for 1 min. PCR products were analyzed by 2.0% agarose gel electrophoresis before being visualized by electrophoresis in 1 × Tris-borate-EDTA on a 2% agarose gel stained with ethidium bromide. A 100-bp DNA ladder (Takara Biomed, Shiga, Japan) was used as a molecular size marker. All experiments were performed in triplicate.
2.4. Environmental Samples
Swab samples were collected from ten dental spittoon units in a dental hospital (Nihon University Hospital, School of Dentistry at Matsudo), and the distribution of A. baumanii complex species and A. lwoffii in the dental hospital environment was investigated using a multiplex PCR. Swab samples which wiped the sink drain traps of each dental spittoon unit were collected in a sterile microcentrifuge tube. All samples were dispersed by sonication for 30 s in an ice bath (50 W, 20 kHz, Astrason® System model XL 2020, NY, USA) and were used as the PCR templates.
3. Results
3.1. Primer Design
Ten specific primers covering the upstream regions of the 16S rRNA and gyrB gene sequences of four A. baumanii complex species and A. lwoffii were designed in the present study (Figure 1 and Figure 2). The specific forward primers were designated as ABF for A. baumannii, ACF for A. calcoaceticus, ANF for A. nosocomialis, APF for A. pittii, and ALF for A. lwoffii, whereas the specific reverse primers were designated as ABR for A. baumannii, ACR for A. calcoaceticus, ANR for A. nosocomialis, APR for A. pittii, and ALR for A. lwoffii. The amplicon sizes of A. baumannii, A. calcoaceticus, A. nosocomialis, A. pittii, and A. lwoffii were 564 bp, 397 bp, 731 bp, 105 bp, and 274 bp, respectively.
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Figure 1. Species-specific primers for 16S rRNA gene of A. baumannii and A. calcoaceticus. The nucleotide sequence of each primer has been underlined.
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Figure 2. Species-specific primers for the gyrB gene of A. pittii, A. lwoffii and A. nosocomialis. The nucleotide sequence of each primer has been underlined.
3.2. Multiplex PCR
Our multiplex PCR method for identifying and detecting five Acinetobacter species, i.e., A. baumannii, A. calcoaceticus, A. nosocomialis, A. pittii, and A. lwoffii successfully amplified DNA fragments of the expected size for each species (Figure 3). The detection limit was assessed in the presence of titrated bacterial cells, and the sensitivity of the PCR assay was between 5 × 1 and 5 × 10 CFU per PCR template (5.0 μl) for the F A. baumannii-specific primer set with strain JCM 6841, the A. calcoaceticus-specific primer set with strain JCM 6842, the A. nosocomialis-specific primer set with strain GTC 03314, the A. pittii-specific primer set with strain GTC 00524 and the A. lwoffii-specific primer set with strain JCM 6840 (data not shown).
3.3. Distribution of A. baumanii Complex Species and A. lwoffii in Dental Hospital Environment
Table 1 shows the detection frequency of five Acinetobacter species in ten dental units. A. baumanii and A. calcoaceticus were detected at 20.0% and 10.0% in ten swab samples, respectively. Among them, two Acinetobacter species were found from one unit. On the other hand, A. nosocomialis, A. lowffii, and A. pittii were detected from no sample.
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Figure 3. Specificity of the multiplex PCR assays for A. baumanii complex species and A. lwoffii. The primer mixture contained ABF, ABR, ACF, ACR, ANF, ANR, APF, APR, ALF and ALR. Lanes: 1: A. pittii GTC 00524; 2: A. lwoffii JCM 6840; 3: A. calcoaceticus JCM 6842; 4: A. baumannii JCM 6841; 5: A. nosocomialis GTC 03314; 6: Mixture of A. pittii, A. lwoffii, A. calcoaceticus, A. baumannii and A. nosocomialis. M, molecular size marker (100-bp DNA ladder).
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Table 1. Detection frequency of five Acinetobacter species in ten dental units.
4. Discussion
To develop a PCR-based technique more applicable for clinical use than conventional PCR, we established a multiplex PCR system for identifying and detecting simultaneously five medically important Acinetobacter species, using only one PCR tube per sample. A multiplex-PCR method is a rapid tool that allows for the simultaneous amplification of more than one sequence of target DNA in a single reaction, thereby saving time and reagents [10] . The most significant problem with regard to this method is the possibility of hybridization among the different sequences of primers. Higgins et al. reported a PCR strategy allowing the identification of four A. baumanii complex species as same as the present study [11] . However, the findings of our pilot study showed that the size of each PCR fragment by this method was similar; therefore, it was difficult to accurately identify each A. baumanii complex species. Moreover, including DNA extraction, it took more than 3 hours to finish the identification. Therefore, a reliable identification method is needed to accurately assess the prevalence of A. baumanii complex species.
Identification of Acinetobacter isolates to the species level is often difficult, especially in routine diagnostic laboratories [12] . The clinically relevant species A. baumannii, A. nosocomialis (formerly genomic species 13TU) and A. pittii (formerly genomic species 3) are often grouped together alongside the environmental A. calcoaceticus species as A. baumannii complex because they are genetically closely related and phenotypically very difficult to differentiate from each other [13] . However, there are considerable epidemiological and clinically relevant differences among these species. A. calcoaceticus is an environmental organism that, to our knowledge, has never been involved in serious human disease, and therefore it should not be misidentified as A. baumannii. The natural habitats of A. baumannii and A. nosocomialis are unknown, as are the differences in their epidemic behaviors, resistance mechanisms, and pathogenicities. A. pittii can be found regularly on human skin, as well as in aquatic environments [14] . A. pittii has also been implicated in nosocomial infections, but its tendency for epidemic spread and resistance development is far less pronounced than that of A. baumannii [14] [15] . For epidemiological and clinical purposes, it is therefore highly desirable to differentiate among these species correctly.
In the present study, we designed species-specific primers with the already mentioned means, for the identification of medically important A. baumanii complex species, i.e., A. baumannii, A. calcoaceticus, A. nosocomialis, A. pittii, and A. lwoffii with a PCR method. These primers were able to distinguish each Acinetobacter species and did not display cross-reactivity with each other. Moreover, we developed a one-step multiplex PCR method with the ability to identify and differentiate four A. baumanii complex species and A. lwoffii using only each one PCR tubes per sample. Species-specific primers for five medically important Acinetobacter species were designed based on the sequences of 16S rRNA and gyrB genes. The protein-coding genes that have been tested for the assessment of microbial diversity include the genes for DNA gyrase subunit B (gyrB) [16] , RNA polymerase subunit B (rpoB) [17] , the TU elongation factor (tuf) [18] , the 60 kDa chaperonin protein (cpn60) [19] . The gyrB gene has a number of potential advantages. Previous reports have shown these markers to be suitable for phylogenetic analyses, as it provides a better resolution at species level than the 16S rRNA gene [20] .
In the present study, Acinetobacter species were detected from only two of ten dental spittoon units in a dental hospital. Numerous studies have documented the presence of Acinetobacter species in the hospital environment, but rates of positive cultures may vary widely, depending on the epidemiological setting. In the previous study, Acinetobacter species have been found in 27% of hospital sink traps and 20% of hospital floor swab cultures [21] . The result of the present study was similar to that of the previous study.
Our multiplex PCR method is easy because the use of MightyAmp DNA Polymerase Ver.3 (Takara) means that DNA extraction may be avoided, and the subspecies identification and detection using this method only takes approximately 2 hours. Thus, the method described herein will allow the prevalence of A. baumannii, A. calcoaceticus, A. nosocomialis, A. pittii and A. lwoffii and their involvement in the various infections, to be fully clarified in future studies.
5. Conclusion
Our developed multiplex PCR method enables the reliable identification of the clinically most relevant Acinetobacter species. Its simplicity means that it can be employed readily in most laboratories, where it might contribute to a better understanding of the epidemiology and clinical significance of the most important Acinetobacter species, i.e., A. baumanii complex species.
Authors’ Contributions
Fukatsu A, Tsuzukibashi O, Yamamoto H, Takahashi Y, Usuda K, Fuchigami M, Uchibori S, Komine C, Umezawa K, Hayashi S and Asano T corrected the data. Fukatsu A, Tsuzukibashi O, Wakami M, Murakami H, Kobayashi T and Fukumoto M drafted and wrote the manuscript. The concept of this manuscript was devised by Fukatsu A. All authors read and approved the final manuscript.