1. Introduction
At present, the genus Pseudomonas comprises 604 species (https://lpsn.dsmz.de/genus/pseudomonas). This organism can be ubiquitously found in humans, animals, soil, and plants [1] [2] . Pseudomonas species were extensively studied for their beneficial or deleterious associations with plants but also for their roles in soil bioremediation due to specific biodegradation properties [3] [4] [5] . Among the genus Pseudomonas, some species are detected from humans and animals, and Pseudomonas aeruginosa is the most frequently reported pathogen.
The genus Pseudomonas is a ubiquitous microorganism frequently detected from immunocompromised patients. The inherent resistance to numerous antimicrobial agents contributes to the opportunistic character of this pathogen. The emergence of healthcare-associated infection is considered a challenge to public health [6] . The problem increases when AmpC-producing Pseudomonas species are isolated from the healthcare professionals. The enzyme confers resistance to cephalosporin and penicillin. The blaAmpC genes are found in the chromosome of the genera Pseudomonas, Citrobacter, Enterobacter, Serratia and Providencia. Bacteria can eventually present sensitivity to cefoxitin, whereas mutant strains express resistance to it due to the loss of repressor genes [7] [8] . Asymptomatic carriers play a fundamental role in the epidemiology of healthcare-associated infection, since they act as disseminators through droplets of saliva expelled during hospital assistance. The asymptomatic colonization may progress to lower respiratory tract infection (pneumonia) and result in health problems to workers and users attended. It is important to establish control strategies to prevent the spread of nosocomial infections.
The detection of the genus Pseudomonas 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 Pseudomonas 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 Pseudomonas 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 Pseudomonas species is required.
Pseudomonas species, especially Pseudomonas putida, P. aeruginosa, Pseudomonas stutzeri and Pseudomonas fluorescens are frequently detected from the human oral cavities [9] [10] ; however, it remains unclear whether those are an oral resident microorganism or not, and where the source of infection is. The purpose of the present study was to design species-specific primers in order to identify and detect four Pseudomonas species, i.e., P. putida, P. aeruginosa, P. stutzeri and P. fluorescens, and to investigate the distribution of these organisms in the living environment 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). The following bacterial strains were used in the present study: P. putida JCM 13063 and JCM 6156, P. aeruginosa JCM 5962 and JCM 2776, P. fluorescens JCM 5963 and JCM 13057, and P. stutzeri JCM 5965 and JCM 20778. 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 Four Pseudomonas Species
Design of species-specific primers for four Pseudomonas species, i.e., P. putida, P. aeruginosa, P. stutzeri and P. fluorescens was performed as described previously [11] . Briefly, the RNA polymerase sigma factor (rpoD) gene sequences of P. putida (accession no. AB 039581), P. aeruginosa (AB 719996), P. stutzeri (KR 780035) and P. fluorescens (D 86033) 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 rpoD gene sequences of four Pseudomonas species were aligned and analyzed, respectively. Homology among the primers selected for each Pseudomonas species and their respective rpoD 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 5.6 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 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. PCR Analysis for Genus Level Detection of Pseudomonas Species
A PCR method for detecting Pseudomonas species at the genus level was performed using the previous reported primers [12] . The multiplex PCR mixture contained 0.5 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 5.6 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 and 68˚C for 1 min.
2.5. Environmental Samples
Fifty living environmental samples were collected by wiping the basin and drain of a washstand with a sterile cotton swab, and the distribution of Pseudomonas species was investigated using a conventional PCR at genus level and a multiplex PCR at species level. 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 1 rpoD gene sequences of four Pseudomonas species were designed in the present study (Figure 1). The specific forward primers were designated as PPF for P. putida, PAF for P. aeruginosa, PSF for P. stutzeri and PFF for A. P. fluorescens, whereas the specific reverse primers were designated as PPR for P. putida, PAR for P. aeruginosa, PSR for P. stutzeri and PFR for A. P. fluorescens. The amplicon sizes of P. putida, P. stutzeri, P. fluorescens and P. aeruginosa were 160 bp, 270 bp, 403 bp and 518 bp, respectively.
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Figure 1. Species-specific primers for rpoD gene of four Pseudomonas species. The nucleotide sequence of each primer has been underlined.
3.2. Multiplex PCR
Our multiplex PCR method for identifying and detecting four Pseudomonas species, i.e., P. putida, P. aeruginosa, P. stutzeri and P. fluorescens successfully amplified DNA fragments of the expected size for each species (Figure 2). 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 P. putida-specific primer set with strain JCM 6152, the P. aeruginosa-specific primer set with strain JCM 5962, the P. stutzeri-specific primer set with strain JCM 5965 and the P. fluorescens-specific primer set with strain JCM 5963 (data not shown).
3.3. Distribution of Four Pseudomonas Species in Living Environment
Table 1 shows the detection frequency of four Pseudomonas species in fifty washstand samples. The genus Pseudomonas was detected from all samples. P. putida, P. aeruginosa, P. stutzeri and P. fluorescens were detected at 44.8%, 8.0%, 4.0% and 2.0% in fifty swab samples, respectively.
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Figure 2. Specificity of the multiplex PCR assays for four Pseudomonas species. The primer mixture contained PPF, PPR, PSF, PSR, PFF, PFR, PAF and PAR. Lanes: 1, Pseudomonas putida JCM 6156; 2, Pseudomonas putida JCM 13063; 3, Pseudomonas stutzeri JCM 5965; 4, Pseudomonas stutzeri JCM 20778; 5, Pseudomonas fluorescens JCM 5963; 6, Pseudomonas fluorescens JCM 5965; 7, Pseudomonas aeruginosa JCM 5962; 8, Pseudomonas aeruginosa JCM 2776; 9, Pseudomonas straminea JCM 2783; 10, Pseudomonas oleovorans JCM 20560; 11, Mixture of ; Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens, Pseudomonas aeruginosa ;M, molecular size marker (100-bp DNA ladder).
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Table 1. Detection frequency of four Pseudomonas species in fifty washstand samples.
4. Discussion
P. aeruginosa is a ubiquitous bacterium normally found in water and soil, and also an opportunistic pathogen of humans, animals, and plants [13] . In humans, P. aeruginosa causes severe infections in patients with underlying conditions by nosocomial infection. Immunosuppressed or intubated-ventilated patients presenting compromised host defenses are particularly vulnerable to this pathogen. The simultaneous detection of Pseudomonas species other than P. aeruginosa, such as P. stutzeri, P. fluorescens, and P. putida, can be important since those organisms have sometimes been reported to be involved in opportunistic infections [14] . 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 four medically important Pseudomonas 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 [15] . The most significant problem with regard to this method is the possibility of hybridization among the different sequences of primers. A multiplex PCR for the detection of four medically important Pseudomonas species has not ever been developed. Therefore, a reliable identification method is needed to accurately assess the prevalence of four medically important Pseudomonas species, i.e., P. putida, P. aeruginosa, P. stutzeri and P. fluorescens.
The molecular detection and identification of microorganisms is widely used in microbiology and epidemiology. However, only limited information is now available on the molecular detection of microorganisms, and the development of appropriate strategies for their rapid identification and monitoring is needed. Some molecular approaches, such as ribotyping, PCR amplification of the 16S-23S rRNA gene spacer region, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, have been exploited for the analysis of the diversity of Pseudomonas isolates [16] [17] [18] [19] . The molecular identification of Pseudomonas species is often difficult and controversial. The sequence analysis of the 16S rRNA gene is widely employed for the identification of bacteria; however, this region is not satisfactorily discriminating between Pseudomonas species. Phylogenetic studies have highlighted that inferred phylogenies based on the 16S rRNA gene lack resolution at the intrageneric level because of its low rate of evolution [20] . Recent studies on the microbiota reported that it was difficult to achieve an unequivocal identification of Pseudomonas at the species level, even though variable regions of the 16S rRNA gene were analyzed [21] . Several authors have evaluated the use of alternative sequences for the identification and phylogenetic studies of Pseudomonas spp. For this purpose, the sequences of the carA, recA, gyrB, fliC, and rpoD genes of Pseudomonas species have been determined [22] [23] [24] . In the present study, the rpoD gene sequence was used as a target in order to design species-specific primers to selectively and simultaneously detect four medically important Pseudomonas species.
In the present study, we designed species-specific primers with the already mentioned means, for the identification of four medically important Pseudomonas species, i.e., P. putida, P. aeruginosa, P. stutzeri and P. fluorescens with a PCR method. These primers were able to distinguish each Pseudomonas 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 medically important Pseudomonas species using only each one PCR tubes per sample.
In the present study, the genus Pseudomonas was detected from all samples (100%), whereas P. putida, P, aeruginosa, P. stutzeri and P. fluorescens were detected at 44%, 8%, 4% and 2% in fifty swab samples, respectively. These results indicated that washstands were the uninhabitable environment for P. putida, P. aeruginosa, P. stutzeri and P. fluorescens and also these organisms detected from the human oral cavities were derived from the living environment other than the washstands.
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, using this method, the oral distribution of P. putida, P. aeruginosa, P. stutzeri, and P. fluorescens, which were not analyzed in this study, and their involvement in various infections, will be fully clarified in future studies.
5. Conclusion
Our developed multiplex PCR method enables the reliable identification of four clinically important Pseudomonas 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 Pseudomonas species, i.e., P. putida, P, aeruginosa, P. stutzeri and P. fluorescens.
Authors’ Contributions
Hayashi S, Umezawa K, Tsuzukibashi O, Fukatsu A, Fuchigami M, Komine C, Yamamoto H, Hagiwara-Hamano M, Iizuka Y and Uchibori S corrected the data. Hayashi S, Umezawa K, Tsuzukibashi O, Fukatsu A, Wakami M, Murakami H, Kobayashi T, Fukumoto M and Nomoto T drafted and wrote the manuscript. The concept of this manuscript was devised by Fukatsu A. All authors read and approved the final manuscript.