1. Introduction
At present, the genus Klebsiella comprises 27 species and 8 subspecies (http://lpsn.dsmz.de/genus/klebsiella). Klebsiella species is a ubiquitous Enterobacteriaceae responsible for various human infectious diseases in immunocompromised individuals [1] . Type species of the genus Klebsiella is Klebsiella pneumoniae. For the high similarity based on DNA-DNA hybridization, Klebsiella pneumoniae species is divided into three subspecies: K. pneumoniae subsp. pneumoniae, that is the most frequently implicated into human diseases, more specifically urinary tract infections and pneumonia, K. pneumoniae subsp. ozaenae (formerly Klebsiella ozaenae), that is recognized as the main cause of a rhinopharynx chronic inflammatory disease named ozena and as a cause of tracheo bronchopathia [2] , and K. pneumoniae subsp. rhinoscleromatis (formerly Klebsiella rhinoscleromatis), involved in a chronic granulomatous disease of upper airway respiratory tract named rhinoscleroma [3] . Because K. pneumoniae susp. pneumoniae is frequently responsible for nosocomial infection cases, many pathophysiological studies had been previously performed using murine models [4] .
K. pneumoniae subsp. pneumoniae plays an important role in hospital-acquired infections [5] . Moreover, they are a frequent cause of infections in immunocompromised patients [6] [7] and a potential hazard to patients with extensive burns [8] . As potential members of the commensal flora of the human gut, K. pneumoniae subsp. pneumoniae are most frequently found in human feces. Recently, it was reported that this organism is related to the onset of inflammatory bowel disease including the Crohn disease [9] . The appearance and spread of hyper-virulent K. pneumoniae strains have increased the number of people susceptible to infections; also, strains of this organism have become increasingly resistant to antibiotics, making antibiotic therapy more challenging. Several new antimicrobial-resistance genes were discovered in K. pneumoniae subsp. pneumoniae before spreading to other pathogens; blaKPC, blaOXA-48-like and blaNDM-1 are examples [10] . Molecular epidemiology analyses allow us to determine the global spread of high-risk clones, thus, providing the necessary data to develop strategies to limit the spread of clinically dangerous strains [11] .
A reliable method to isolate three K. pneumoniae subspecies from various clinical samples would be an important contribution to epidemiological studies concerning nosocomial infections. Conventional isolation techniques, however, have the disadvantage of being rather insensitive for K. pneumoniae, due to the abundance of other bacteria in the samples, which mask the presence of smaller numbers of this organism.
Regrettably, it currently remains unclear whether K. pneumoniae is part of the normal oral flora. In our previous pilot study, we tried to detect this organism from the oral samples using conventional selective media for K. pneumoniae [12] [13] . However, it was impossible to identify this organism accurately, because those media were not able to completely inhibit the growth of oral bacteria whose number was reported to exceed 600 species [14] and inhibited the growths of some K. pneumoniae strains. Thus, a suitable selective medium is needed to assess the prevalence of oral K. pneumoniae involved in the onset of inflammatory bowel disease including the Crohn disease. The monitoring of K. pneumoniae levels at subspecies level in the oral cavities might be useful for the diagnosis and prevention of K. pneumoniae infectious disease.
The detection of K. pneumoniae at subspecies 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 K. pneumoniae at subspecies level are required to determine their role in various systemic diseases. Although conventional biochemical assays are used to identify three K. pneumoniae subspecies, they are often imprecise due to the phenotypic variations displayed by these bacteria. Although sequence analysis of several target genes is the most reliable method, it is expensive, laborious, and time-consuming. Thus, a simple and more reliable assay for identifying three K. pneumoniae subspecies is required.
The purpose of the present study was to develop selective media for the isolations of three K. pneumoniae subspecies from the human oral samples, and a simple and more reliable assay for identifying them, and also to assess the prevalence of these organisms in the oral cavity.
2. Materials and Methods
2.1. Bacterial Strains and Culture Conditions
All bacterial strains used in the present study are listed in Table 1. K. pneumoniae strains used in the present study 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.
Strains other than Klebsiella species were maintained by cultivating them also on BHI agar. These organisms were cultured at 37˚C overnight in an atmosphere of 5% CO2 in a CO2 incubator (NAPCO® Model 5400; Precision Scientific, Chicago, IL, USA).
2.2. Development of New Selective Medium
2.2.1. Evaluation of Base Medium
BHI agar supplemented with 1% yeast extract (BHI-Y), BHI-Y supplemented with 5% sheep blood (BHI-Y blood), and Nutrient agar (NA) were examined as the base medium in the selective medium. Ten-fold dilutions of cultures were
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Table 1. Recovery of K. pneumoniae and other bacteria on BHI agar and AISM.
aAve ± SD.
made in 0.9 ml of Tris-HCl buffer (0.05 M, pH 7.2) and aliquots of 0.1 ml were spread onto the test media. The plates on which bacteria, except Klebsiella species, were inoculated were cultured at 37˚C for 48 h in an atmosphere of 5% CO2 in a CO2 incubator, and the plates on which Klebsiella species were inoculated were cultured at 30˚C for 48 h under an aerobic condition. After cultivation, the number of colony-forming units (CFU)/ml was counted.
2.2.2. Susceptibility Tests
Preliminary studies of antibiotic selection were also performed using disk susceptibility tests (Sensi-Disk, Becton Dickinson Co., MD, USA). The microbroth dilution method was used for susceptibility testing [15] .
2.3. Recovery of K. pneumoniae and Other Representative Oral Bacteria
The recoveries of the K. pneumoniae reference strains, Klebsiella isolates, and other representative oral bacteria were calculated as CFU/ml on selective medium and compared with those on BHI agar for total cultivable bacteria. All bacterial strains used in the present study are listed in Table 1.
All bacterial strains, except K. pneumoniae, were pre-incubated in BHI broth at 37˚C overnight in an atmosphere of 5% CO2 in a CO2 incubator. K. pneumoniae were pre-incubated in BHI broth at 30˚C overnight in an atmosphere of 5% CO2 in a CO2 incubator. Ten-fold dilutions of cultures were made in 0.9 ml of Tris-HCl buffer (0.05 M, pH 7.2) and aliquots of 0.1 ml were spread onto the test media. The plates on which bacteria, except K. pneumoniae, were inoculated were cultured at 37˚C for 72 h in an atmosphere of 5% CO2 in a CO2 incubator, and those on which K. pneumoniae were inoculated were cultured at 30˚C for 24 h under an aerobic condition. After cultivation, the number of CFU/ml was counted.
2.4. Clinical Samples
Thirty volunteers (13 men, 17 women; mean age 43 years, range 18 - 65 years) participated in the present study. They had no systemic disease and received no antibiotic therapy for at least 3 months. All participants were asked not to brush, rinse, or smoke immediately prior to the assessment and not to eat or drink for at least 2 h beforehand.
Paraffin-stimulated whole saliva samples 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 0.1 ml of each was diluted and inoculated on BHI-Y and selective medium plates. BHI-Y plates for total cultivable bacteria were cultured at 37˚C for 2 days in an atmosphere of 5% CO2 in a CO2 incubator, and selective medium plates for K. pneumoniae were cultured at 30˚C for 24 h under an aerobic condition. After cultivation, CFU/ml in each sample was calculated. The present study was conducted in accordance with the principles of the Declaration of Helsinki, and was approved by the Ethics Committee of Nihon University School of Dentistry at Matsudo, Japan (EC20-017).
2.5. Identification of K. pneumoniae Isolated from Clinical Samples
Twenty-four of the approximately 50 colonies that grew on the selective medium plate per subject were randomly isolated and subcultured, and their identity was then confirmed by a PCR analysis. After K. pneumoniae was isolated from the K. pneumoniae-positive subjects, the isolations of this organism from the same subjects were performed six months later again.
2.6. Design of Species-Specific Primers for Three K. pneumoniae Subspecies
The design of species-specific primers for three K. pneumoniae subspecies was performed as follows. The 16S rRNA sequences of K. pneumoniae subsp. pneumoniae (accession no. X87276), K. pneumoniae subsp. ozaenae (AF130982), and K. pneumoniae subsp. rhinoscleromatis (Y17657), and the wzc gene sequences of K. pneumoniae subsp. pneumoniae (AB719996), K. pneumoniae subsp. ozaenae (AB719988), and K. pneumoniae subsp. rhinoscleromatis (AB719987) were obtained from the DNA Data Bank of Japan (DDBJ; Mishima, Japan), and multiple sequence alignment analyses were performed using the CLUSTAL W program; i.e., the 16S rRNA sequences of four species were aligned and analyzed. Homologies among the primers selected for three K. pneumoniae subspecies were confirmed by a BLAST search.
2.7. Development of a Multiplex PCR Method Using Designed Primers
A multiplex PCR method for identifying three K. pneumoniae subspecies using the designed primers was developed as follows. Bacterial cells were cultured in a BHI broth overnight, and 1 ml of the sample was then collected in a microcentrifuge tube and resuspended at a density of 1.0 McFarland standard (approximately 107 CFU 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 for PCR was assessed by serially diluting known numbers of bacterial cells in sterile distilled water and then subjecting each suspension to PCR. The PCR mixture contained 2 μM of each primer, 10 μl of 2 × MightyAmp Buffer Ver.2 (Takara Bio Inc., Shiga, Japan), 0.4 μl of MightyAmp DNA Polymerase (Takara), and 3.6 μl of the template in a final volume of 20 μl. PCR was performed in a DNA thermal cycler (Applied Biosystems 2720 Thermal Cycler; Applied Biosystems, Carlsbad, CA). 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.
2.8. Genotyping by AP-PCR Analysis
Genotyping by AP-PCR Analysis AP-PCR analysis for genotyping of K. pneumoniae isolates was performed as follows. Subcultured K. pneumoniae isolates were suspended in 1.0 McFarland standard in 100 μl of distilled water, and 7.6 μl of the suspension was used as a template for AP-PCR. AP-PCR was performed as described previously [16] . Briefly, the PCR mixture contained 0.2 μM of OPA-7 primer (5’-GAAACGGGTG-3’), 10 μl of 2 × MightyAmp Buffer Ver.3 (Takara Bio Inc., Shiga, Japan), 0.4 μl of MightyAmp DNA Polymerase (Takara), and 7.6 μl of the template in a final volume of 20 μl. AP-PCR was carried out in a DNA thermal cycler (Applied Biosystems 2720 Thermal Cycler). AP-PCR conditions included an initial denaturation step at 98˚C for 2 min, 50˚C for 2 min, and 35˚C for 1 min, followed by 30 cycles consisting of 72˚C for 2.5 min, 92˚C for 1.5 min, and 35˚C for 1 min and final extension period of 72˚C for 5 min. PCR products were analyzed by 2.0% agarose gel electrophoresis and visualized by gel staining with ethidium bromide. A 100-bp DNA ladder was used as a molecular size marker (Takara Biomed).
3. Results
3.1. Development of Selective Medium
3.1.1. Selection of Base Medium
The selection of a base medium for the growth of K. pneumoniae was performed. Three K. pneumoniae subspecies grew well on NA as same as BHI-Y and BHI-Y blood (data not shown). Because of the low cost, NA was ultimately selected as the base medium.
3.1.2. Susceptibility to Antibiotics
Three K. pneumoniae subspecies, i.e., K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, and K. pneumoniae subsp. rhinoscleromatis was more resistant to bacitracin than oral Gram-positive cocci and rods, such as genera Streptococcus, Actinomyces, Corynebacterium, and Rothia. The minimal inhibitory concentration (MIC) of bacitracin for K. pneumoniae was more than 1000 μg/ml. K. pneumoniae was more resistant to lincomycin than oral Gram-negative rods, such as genera Aggregatibacter, Leptotrichia, and Eikenella. The MIC of lincomycin for K. pneumoniae was 10 μg/ml. Oral Gram-negative rods were sensitive to 2 μg/ml of lincomycin. K. pneumoniae was more resistant to penicillin than oral Gram-negative cocci, such as genus Neisseria. The MIC of penicillin for K. pneumoniae was 10 μg/ml. Oral Gram-negative cocci were sensitive to 1 μg/ml of penicillin.
3.1.3. Composition of New Selective Medium
The new selective medium, designated oral K. pneumoniae selective medium (OKPSM), was composed of the following (per liter): 35 g of nutrient agar, 10 g of lactose, 20 mg of bromocresol purple, 300 mg of bacitracin, 2 mg of lincomycin, 1 mg of penicillin, and 7.5 mg of amphotericin B. Antibiotics, i.e., bacitracin, lincomycin, penicillin, and amphotericin B were added after the base medium had been sterilized and cooled to 50˚C.
3.2. Multiplex PCR Method for Identifying K. pneumoniae
3.2.1. Primer Design
Six specific primers covering the upstream region of the 16S rRNA and wzc gene sequence of three K. pneumoniae subspecies was designed in the present study (Figure 1 and Figure 2). The specific forward primers were designated as KPF for K. pneumoniae subsp. pneumoniae, KOF3 for K. pneumoniae subsp. ozaenae, and KRF for K. pneumoniae subsp. rhinoscleromatis, whereas the specific reverse primers were designated as KPR for K. pneumoniae subsp. pneumoniae, KOR3 for K. pneumoniae subsp. ozaenae, and KRR for K. pneumoniae subsp. rhinoscleromatis. The amplicon sizes of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, and K. pneumoniae subsp. rhinoscleromatis were 549 bp, 152 bp, and 674 bp, respectively.
3.2.2. Detection Limit
Our multiplex PCR method for identifying and detecting three K. pneumoniae subspecies, i.e., K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp.
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Figure 1. Species-specific primers for 16S rRNA gene of K. pneumoniae subsp. pneumoniae. The nucleotide sequence of each primer has been underlined.
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Figure 2. Species-specific primers for the wzc gene of K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis. The nucleotide sequence of each primer has been underlined.
ozaenae and K. pneumoniae subsp. rhinoscleromatis 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 K. pneumoniae subsp. pneumoniae-specific primer set with strain JCM 1662, the K. pneumoniae subsp. ozaenae-specific primer set with strain JCM 1663, and the K. pneumoniae subsp. rhinoscleromatis-specific primer set with strain JCM 1664.
3.3. Recovery of K. pneumoniae and Inhibition of Other Representative Oral Bacteria on Selective Medium
Table 1 shows the recovery of some K. pneumoniae reference strains on OKPSM relative to BHI-Y. The growth recoveries of the K. pneumoniae reference strains on OKPSM were between 97.5% and 99.5% (average 98.1%) that on BHI-Y.
Table 1 also shows the inhibition of other representative oral bacteria on OKPSM relative to BHI-Y. The growth of other representative oral bacteria was markedly inhibited on the selective medium.
3.4. Clinical Examination
The detection frequencies of K. pneumoniae in saliva samples from thirty healthy subjects are shown in Table 2. K. pneumoniae was detected from only three subjects (10.0%). Subspecies of them were K. pneumoniae subsp. pneumoniae. On the other hand, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis were detected from no one. The mean numbers of this organism in the positive subjects were 7.4 × 102 CFU/ml.
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Figure 3. Multiplex PCR for Klebsiella pneumoniae subspecies level. Lane: 1: K. pneumoniae subsp. ozaenae JCM 1663; 2: K. pneumoniae subsp. pneumoniae JCM 1662; 3: K. pneumoniae subsp. rhinoscleromatis JCM 1664; 4: Mixture of K. pneumoniae subsp. ozaenae JCM 1663, K. pneumoniae subsp. pneumoniae JCM 1662 and K. pneumoniae subsp. rhinoscleromatis JCM 1664. M, molecular size marker (100 bp DNA ladder).
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Table 2. Detection frequencies of K. pneumoniae in saliva samples.
In the first isolation, K. pneumoniae colonies on OKPSM commonly had a smooth appearance such as a teardrop.
The colony colors of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis on OKPSM were light yellow, white and light purple, respectively. Therefore, OKPSM could distinguish three K. pneumoniae subspecies by each colony color using differences in acid production from galactose which was added to the medium. The average colony sizes of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis on OKPSM were 4.6 mm, 2.2 mm and 4.2 mm in diameter, respectively (Figure 4).
3.5. Comparison of Genotype of Each K. pneumoniae Isolate at 0 Month and after 6 Months
Figure 5 shows the results of genotyping by AP-PCR using OPA-7 primer of each K. pneumoniae subsp. pneumoniae isolates from subject A, B and C at 0 month and after 6 months. The AP-PCR patterns of K. pneumoniae subsp.
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Figure 4. Colony appearances of three Klebsiella subspecies reference strains on OPKSM.
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Figure 5. Genetic differences among K. pneumoniae subsp. pneumoniae isolates from each subject at 0 month and after 6 months with AP-PCR. Lane: 0: K. pneumoniae subsp. pneumoniae isolates from each subject at 0 month; Lane 6: K. pneumoniae subsp. pneumoniae isolates after 6 months; Lane M, molecular size maker (100 bp DNA ladder).
pneumoniae that were isolated from each subject at 0 month and after 6 months showed identical genotypes among each individual.
4. Discussion
A reliable selective medium to isolate K. pneumoniae from various clinical samples would be an important contribution to epidemiological studies concerning nosocomial infections and antimicrobial resistant strains. Conventional isolation techniques, however, have the disadvantage of being rather insensitive for Klebsiella species, due to the abundance of other bacteria in the samples, which mask the presence of smaller numbers of this organism.
In 1970, Thom [12] developed the selective medium for Klebsiella species from the human feces, the MacConkey-inositol-carbenicillin (MIC) agar. This medium was based on the MacConkey agar in which lactose was replaced by 1% inositol, with the addition of 100 mg of carbenicillin per liter. This medium owes its elective capacity to the fact that about 97% to 99% of Klebsiella species and only 0% to 1% of E. coli strains are capable of fermenting inositol and hence appear as red colonies. The selectivity of the medium is due to the presence of carbenicillin to which most E. coli strains are susceptible. Resistant E. coli strains will appear as pale colonies. Since about 10% to 15% of Klebsiella strains are also susceptible to this concentration of carbenicillin, these strains will be missed when this medium is used. Therefore, some investigators reduced the carbenicillin concentration to 10 mg/liter [13] [17] .
Cooke et al. [13] used MIC agar simultaneously with Simmons citrate agar (SCA), the latter medium allowing the growth of only those types of bacteria that are capable of utilizing citrate as the only carbon source. On SCA, ca. 97% to 99% of Klebsiella strains appear as small blue colonies, whereas E. coli is unable to grow on this medium. However, several other types of bacteria are capable of growing on citrate, thus impairing the detection of Klebsiella species. So far, however, this method combined with two enrichment media yielded the highest isolation rates of Klebsiella species.
Regrettably, it currently remains unclear whether K. pneumoniae is part of the normal oral flora. In our previous pilot study, we tried to detect this organism from the oral samples using MIC agar and SCA. However, it was impossible to identify this organism accurately, because those media were not able to completely inhibit the growth of oral bacteria whose number was reported to exceed 600 species [14] and inhibited the growths of some K. pneumoniae strains. Thus, a suitable selective medium is needed to assess the prevalence of oral K. pneumoniae involved in the onset of inflammatory bowel disease including the Crohn disease.
A useful selective medium for isolating K. pneumoniae may contribute to the correct and rapid diagnosis of infectious diseases caused by this organism. However, a selective medium that is useful for the isolation of K. pneumoniae from various clinical samples has not ever been developed. In the present study, K. pneumoniae strains were more resistant to bacitracin, lincomycin, and penicillin than other representative oral bacteria. The growth of oral bacteria detected in the oral cavity was inhibited by the addition of 300 mg/L bacitracin, 2 mg/L lincomycin, and 1 mg/L penicillin to NA. All of the K. pneumoniae reference strains grew well on the new selective medium, designated as OKPSM, while the growth of other bacteria was markedly inhibited (Table 1). Moreover, OKPSM allowed for the identification of K. pneumoniae by its characteristic colony morphology.
In the present study, we designed species-specific primers with the already mentioned means, for the identification at the subspecies level of K. pneumoniae with a PCR method. These primers were able to distinguish K. pneumoniae at the subspecies level and did not display cross-reactivity with each other. Moreover, we developed a multiplex PCR method with the ability to identify and differentiate K. pneumoniae at the subspecies level using only each one PCR tubes per sample. Species-specific primers for three subspecies were designed based on the sequences of 16S rRNA and wzc gene. Moreover, the PCR method in the present study directly uses bacterial cells with MightyAmp DNA Polymerase Ver.3 (Takara) and is completed within approximately 2 hours.
In this study, K. pneumoniae isolates from the same subject at 0 month and after 6 months showed the identical genotype on AP-PCR using OPA-07, respectively. Also, K. pneumoniae subsp. pneumoniae was detected at only 10.0% in 30 saliva samples. K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis were not detected. The mean numbers of K. pneumoniae subsp. pneumoniae in those samples were 7.4 × 102 CFU/ml. These results indicated that oral cavity may be only transitory reservoir as K. pneumoniae subsp. pneumoniae is coming here due to regurgitation or vomiting.
5. Conclusion
We developed a selective medium, designated OKPSM, to isolate K. pneumoniae in the oral cavity of humans. Since OKPSM is highly selective for K. pneumoniae, it will be useful for assessing the distribution and role of this organism at various locations in humans. The selective medium (OKPSM) and our multiplex PCR method as isolation and identification methods, respectively, for K. pneumoniae may contribute to making clear the role of this organism in the aetiology of inflammatory bowel disease including Crohn disease.
Authors’ Contributions
Tsuzukibashi O, Fukatsu A, Yamamoto H, Takahashi Y, Usuda K, Fuchigami M, Uchibori S, Komine C, Umezawa K, Hayashi S and Asano T corrected the data. Tsuzukibashi O, Wakami M, Murakami H, Kobayashi T and Fukumoto M drafted and wrote the manuscript. The concept of this manuscript was devised by Tsuzukibashi O. All authors read and approved the final manuscript.