Isolation and Identification of Optochin-Resistant Viridans Group Streptococci from the Sputum Samples of Adult Patients in Jakarta, Indonesia

Aim To investigate optochin-resistant viridans group streptococci (VGS) strains isolated from the sputum sample of adult patients with different clinical symptoms. Materials and Methods Optochin-resistant VGS isolates were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). recA sequencing was used to confirm identified isolates at the genus level by MALDI-TOF MS. Finding. We identified 79% of tested isolates (148/187) at the species-level identification using the MALDI-TOF MS tool. We identified that the most common species isolated from sputum specimens were S. oralis (44.9%) followed by S. mitis (25.7%), S. infantis (9.1%), S. parasanguinis (7.5%), S. peroris (3.7%), S. anginosus (2.7%), and S. sanguinis (2.1%). Discussion. The S. oralis strains were majority of optochin-resistant VGS isolates obtained from sputum of adult patients in Jakarta, Indonesia. MALDI-TOF MS showed potential for the rapid identification tool to identify optochin-resistant VGS isolates. Although there were discrepancies in identifying isolates at the genus/species level, the performance could be improved by expanding its database.


Introduction
e high-level similarities between Streptococcus pneumoniae, a human pathogen, and viridans group Streptococci (VGS), particularly within the nonpneumococcal mitis group including Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae, often cause difficulties in species discrimination [1,2]. In clinical laboratory testing, conventional tests such as optochin sensitivity and bile solubility are still applied as key identifications for S. pneumoniae isolates [3]. However, some S. pneumoniae isolates were reported as optochin resistant in different geographical regions [4]. strongly correlates with dental caries development, and S. mitis, S. oralis, and S. sanguinis are taking roles in infective endocarditis [8].
Specific and accurate species-level identification of VGS is one of the important factors in patient clinical management and is also important for understanding their pathogenicity and virulence [1,9]. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) has become an indispensable tool for clinical microbiology laboratories and shown to be a potential alternative for organism identification with a rapid and cost-saving method for VGS identification [2,5,9]. Previously, we reported that thirteen S. pneumoniae (pneumococcus) strains were susceptible to the optochin test and one hundred and eighty-nine of alpha haemolytic nonpneumococcus strains were resistant to the optochin test from the sputum of adult patients with nonspecific clinical symptoms in Jakarta, Indonesia [10]. In this study, we investigate further nonpneumococcus strains from adult patients for optochin-resistant VGS identification by the MALDI-TOF MS.

Sample Preparation.
All isolates were subcultured on a tryptone soya agar plate with 5% sheep blood and then incubated overnight at 37°C with 5% CO 2 [10]. A single colony of overnight pure growth bacteria was spotted to the MSP 96 ground plate (Bruker Daltonik, Germany) using a sterile toothpick and air dried in room temperature for approximately 5 minutes as a direct method sample preparation. e dried spots were then mixed with 1 μL matrix (saturated solution of α-cyano-4-hydroxycinnamic acid/ HCCA in 50% acetonitrile and 2.5% trifluoroacetic acid (TFA)). e solution was air dried in room temperature for approximately 10 mins. Standard protein extraction method was used to confirm the isolates with MALDI-TOF identification score <2.000 [11]. A 2.0 McFarland of bacterial suspension was made in 300 μL of water and then mixed with 900 μL of ethanol. e suspension was homogenised and centrifuged at 20000 × g for 2 minutes. e supernatant was removed, and the pellet was dried at 55 o C for 30 minutes. e dried pellet was resuspended in 50 μL of acetonitrile followed by centrifugation at 20000 × g for 2 minutes. A 1 μL supernatant was spotted to the ground plate and air dried for 10 mins in room temperature. en, 1 μL of matrix was added to the same spot as in the direct colony method as described above.

MALDI-TOF-MS-Based Identification.
e isolates were identified using Microflex MALDI-TOF (Bruker Daltonik, Germany) and flexControl version 3.4 software as previously described [2,12]. Isolate identification was performed from spectrum acquisition was conducted in the positive linear mode with laser frequency at 60 Hz. Mass range started at 2.000-20.000 Da. Each voltage from ion source 1 and ion source 2 was set at 20 kV and 18.5 kV. Bacterial test standard protein was included in every test as instrument calibration. Automatic identification started after the spectra result was moved to Biotyper RTC software. e identification criteria were based on the similarity level, shown by the logarithmic score of isolates and database spectra prior to instructions by the manufacturer as follows: score <1.700 indicated isolates were not reliably identified; isolates with score 1.700-1.999 indicated identification accuracy up to the genus level; and isolates with score ≥2.000-3.000 indicated isolates accurately identified up to the species level. Mass spectra analysis was conducted using flexAnalysis software. All obtained spectra were saved in flexControl before undergoing the calibration, smoothing, and baseline subtraction process on flexAnalysis MBT-Standard, prior to the manufacturer's recommendation.

recA Sequencing Identification.
e recA sequencing tool was used to confirm all identified isolates at the genus level by MALDI-TOF MS [13]. We performed recA gene amplification and sequencing using forward primer [5′-GCCTTYATC-GATGCBCARCA-3′] and reverse primer [5′-GTTTC CGGRTTDCCRAACAT-3′] with the GoTaq Green Mastermix [13]. e obtained sequences were compared to the recA gene sequences database in NCBI GenBank and analysed using BLAST alignment (http://www.ncbi.nlm.nih.gov/blast) and MEGA-6 software. e obtained sequences with similarity ≥96% on published sequences in GenBank were assigned as cutoff for species identification.
We identified 11 optochin-resistant VGS isolates (30.6%) at the genus level by the MALDI-TOF MS tool matched with the results from recA sequencing confirmation. S. oralis isolates were the most common matched isolates between MALDI-TOF MS and recA sequencing tools (six strains), followed by S. mitis and S. peroris (two strains each) and S. infantis (one strain). In this study, we observed that only one optochin-resistant VGS isolate was identified at the genus level by the MALDI-TOF MS tool (Table 1). After recA sequencing confirmation, 17 optochin-resistant VGS isolated at the genus level by the MALDI-TOF MS tool were identified as S. infantis ( Table 2).

Discussion
In this study, we found that S. oralis and S. mitis were the major common optochin-resistant VGS isolates (70.6%) obtained from the sputum samples. e prevalence of S. mitis and S. oralis in this study was higher compared to other previous studies. Maeda et al. reported that the prevalence of S. mitis and S. oralis isolates from the sputum samples of adult patients with cystic fibrosis was 19% and 11%, respectively [14]. Meanwhile, the S. anginosus group (38.8%) and S. mitis (22.8%) group were the most common VGS species isolated from bloodstream infection detected by MALDI-TOF MS identification [15]. From oncologic patients, almost half of the VGS isolated from the blood culture was S. mitis isolates (46.5%) followed by S. anginosus (32.6%) and S. sanguinis (16.3%) by MALDI-TOF MS [6]. Oral streptococci isolates were reported as the most detected isolates from bronchoalveolar lavage fluid specimens obtained from pneumonia patients [16]. e oral streptococci isolates were all members of the S. mutans and S. mitis groups, the S. salivarius group, and the S. anginosus group except for S. pneumoniae [16]. S. mitis and S. oralis were significantly remaining species to be isolated from bloodstream isolates from neutropenic patients using the sodA gene detection [17].
In this study, we identified one isolate as S. infantis (2.6%) at the genus level. However, more S. infantis (9.1%) were identified from all optochin-resistant VGS isolates at the genus level by the MALDI-TOF MS tool after recA sequencing confirmation. Zbinden A et al. reported that the A 313-bp part of recA was selected on the basis of variability within the S. mitis group, showing <95.8% interspecies homology [13]. We found a mismatched pair of S. pneumoniae and S. infantis identified at the genus level by both MALDI-TOF MS and recA sequencing tools. is discrepancy was possibly due to high similarities in the molecular and proteomic profile of the mitis group including S. mitis and S. oralis, thus presenting a challenge to correctly identify species using DNA-or protein-based identification methods [2,18]. e peak analysis and most updated Bruker database may improve the correct species identification [19]. In conclusion, the S. oralis and S. mitis were the predominant VGS isolates obtained from sputum of adult patients in Jakarta, Indonesia. MALDI-TOF MS showed potential for rapid identification to identify non-Streptococcus pneumoniae isolates. Although there were discrepancies in identifying isolates at the genus/species level, the performance could be improved by expanding its database.
Data Availability e MALDI-TOF and recA sequencing data used to support the findings of this study are available from the corresponding author upon request. Disclosure e contents are solely the responsibility of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention or the Department of Health and Human Services. e funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflicts of Interest
e authors declare no conflicts of interest.