Genetic characterization of co-trimoxazole non-susceptible Streptococcus pneumoniae isolates from Indonesia

We investigated the genetic variation of folA and folP genes encoding dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) enzymes amongst trimethoprim/sulfamethoxazole (co-trimoxazole) non-susceptible Streptococcus pneumoniae isolated from the Indonesian population. Archived S. pneumoniae isolates were screened for the presence and analysis of folA and folP genes using the polymerase chain reaction sequencing method. We found that 80 % of co-trimoxazole non-susceptible isolates (n=30/39) showed a 6 bp insertion in the sulphonamide-binding site of DHPS. The Asp-92-Ala and Ile-100-Leu substitutions were more common on DHFR (42 %; 22/53) followed by the Asp-92–Ala, Glu-94–Asp and Ile-100–Leu substitutions (32 %; 17/53). The combination of the Ile-100–Leu substitution at the DHFR region and the 6 bp insertion was the most dominant combination among isolates having both folA and folP genes.


INTRODUCTION
Trimethoprim/sulfamethoxazole (co-trimoxazole) is an inexpensive and broad-spectrum antimicrobial drug that is still widely used in developing countries [1]. It is commonly administered as a prophylactic to protect against opportunistic infections for HIV-infected individuals and as a priority intervention for HIV-infected pregnant women [2]. It has been suggested that co-trimoxazole increased the risk of carrying pneumococci slightly among HIV-infected children in Zambia [3]. It has been used as a treatment option for a range of pneumococcal diseases, particularly in children [4]. In Indonesia, this drug was reported to be the second most common antibiotic used after the penicillin class to treat gastrointestinal disorders, respiratory system disorders, unspecified pyrexia, metabolism and nutrition disorders [5][6][7].
Resistance to co-trimoxazole among pneumococcal diseases has increased worldwide [4]. In Indonesia, the percentage of co-trimoxazole non-susceptible Streptococcus pneumoniae has increased over time. In 1997, it was reported that 12 % of S. pneumoniae isolated from 484 healthy children (0-25 months of age) in Lombok, Indonesia were non-susceptible to sulfamethoxazole [8]. Furthermore, a pneumococcal carriage study conducted in Lombok also reported that the percentage of co-trimoxazole non-susceptible S. pneumoniae increased to 62 % in 2012 [9]. Resistance to co-trimoxazole is associated with mutation on the folP and folA genes encoding dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) enzymes, respectively. Genetic variation of these genes plays an important role in co-trimoxazole resistance [10]. The polymorphisms of these genes have also been reported to have an association with the level of resistance [11] In this study, we aimed to analyse and characterize the genetic variations of folA and folP genes OPEN ACCESS correlated with co-trimoxazole resistance among S. pneumoniae isolates in Indonesia.
The polymerase chain reaction (PCR) and DNA sequencing targeting genes for folA and folP genes were performed as previously described [10]. The isolates were subcultured onto a 5 % sheep blood agar plate and incubated at 37 °C with 5 % CO 2 for 20 h. Fresh culture was harvested into 300 µl TE buffer in a 1.5 ml microcentrifuge tube and vortexed. The bacterial suspension was heated at 100 °C for 5 min and then immediately placed in −20 °C conditions for 5 min before being centrifuged at 13 000 g for 10 min [12,13]. The PCR reaction mixture comprised GoTaq Green Master Mix (Promega, Madison, WI, USA), the primers for the folP gene: folP forward 5′-GTCAA-GTAAAGCCAATCATG-3′ and folP reverse 5′-AATTTTC-CGCTTCATCAGC −3′ and the primers for the folA gene: folA forward 5′-TGTAAGCTATTCCAAACCAG-3′ and folA reverse 5′-CTACGTTCCATTAGACTTCC-3′ at 10 µM concentration, 1.0 µL of DNA template and nuclease-free water to a final volume of 50 µl. The PCR condition for folP gene was set as follows: 95 °C for 5 min followed by 35 cycles of 94 °C for 60 s, 51 °C for 60 s and 72 °C for 90 s, with a final extension at 72 °C for 7 min. The PCR condition for folA gene was set as follows: 95 °C for 5 min followed by 35 cycles of 94 °C for 60 s, 53 °C for 60 s and 72 °C for 30 s, with a final extension at 72 °C for 7 min. The amplicons for folP and folA genes were visualized with gel electrophoresis for approximately 600 and 900 bp, respectively. The amplicons were sequenced using the BigDye Terminator v3.1 labelled cycle sequencing kit (Applied Biosystems, USA) according to the manufacturer's instructions. DNA sequences of folP (n=32) and folA (n=51) genes were submitted to the GenBank database with accession numbers MW816655-6685, MW816687-6694, MW816696-6707, MW835933-5961 and MW835963-5965.
In this study, the combination of the Ile-100-Leu substitution at DHFR region and the 6 bp insertion was the most dominant combination (69 %; 25/36) among isolates having both folA and folP genes sequences, especially the combination of Asp-92-Ala, Glu-94-Asp, Ile-100-Leu and STRPRPGSSYVEIE and the combination of Asp-92-Ala, Ile-100-Leu and STRPRPGSSYVEIE (Table 3). Moreover, we also discovered that among isolates showing higher resistance (MIC ranged from 16/304-≥32/608 µg ml −1 ) to co-trimoxazole, the Ile-100-Leu substitution at the DHFR region and the 6 bp insertion were the most prevalent combination patterns. Moreover, we also discovered that among isolates showing higher resistance (MIC ranged from 16/304-≥32/608 µg ml −1 ) to co-trimoxazole, the Ile-100-Leu substitution at the DHFR region and the 6 bp insertion were the most prevalent combination patterns.

DISCUSSION
In this study, we observed an insertion mutation occurring in folP encoding dihydropteroate synthase (DHPS) and a substitution in folA encoding dihydrofolate reductase (DHFR), the enzymes involved in the folate biosynthesis pathway [14]. Insertions of 3 and 6 bp occurred in folP, with the 6 bp insertion showing more resistance compared to the 3 bp insertion. Furthermore, the 6 bp insertion was found to be the most prevalent insertion Table 2. Substitution variation of the folA gene encoding the DHFR enzyme in S. pneumoniae isolates from the Indonesian population   among co-trimoxazole non-susceptible isolates. This finding was consistent with previous studies that reported that these 3 and 6 bp insertions were also observed in the folP gene of co-trimoxazole non-susceptible isolates with the 6 bp insertion causing RP amino acid (STRPRPGSSYVEIE) as a common mutation [10,11]. In addition, we observed that the 6 bp insertion showed more resistance to co-trimoxazole compared to the 3 bp insertion and the wild-type. This finding was also reported in a study conducted in Tanzania, which showed that most isolates that were non-susceptible to co-trimoxazole carried multiple mutations in DHFR [15].
A substitution occurring in folA is also a factor causing resistance to co-trimoxazole. In this study, we discovered that some mutations occurred in the folA gene of co-trimoxazole non-susceptible isolates, with Asp-92-Ala and Ile-100-Leu substitutions being the most prevalent mutations ( Table 2). This finding is consistent with a previous study which mentioned that substitutions in amino acid 92 (Asp-92-Ala) and 100 (Ile-100-Leu) were the most prevalent substitutions in co-trimoxazole non-susceptible isolates [10,11,15,16]. Meanwhile, a study in Tanzania mentioned that Asp-92-Ala (53,1 %) and Ile-100-Leu (100 %) were also the dominant substitutions in the DHFR of trimethoprim/sulfamethoxazole non-susceptible isolates [15]. Further, we also detected the substitution at 94 (Glu-94-Asp) as the most common substitution after Asp-92-Ala and Ile-100-Leu, which is concordant with previously reported work [10,15]. We also discovered some substitution combinations in DHFR (Table 2), but combination with Ile-100-Leu resulted in higher resistance against co-trimoxazole. The Ile-100-Leu was reported as an essential amino acid change for resistance development, while other mutations on DHFR did not show significant influence on resistance development. Other mutations without combination with Ile-100-Leu will not develop high resistance to co-trimoxazole [10,11], but the substitution Asp-92-Ala without the substitution Ile-100--Leu resulted in trimethoprim resistance [15]. We discovered that the Ile-100-Leu substitution in DHFR combined with the 6 bp insertion in DHPS shows higher resistance against co-trimoxazole (MIC ranged from 16/304-32/608 µg ml −1 ). The presence of these mutations reduced the affinity of trimethoprim in binding DHFR and sulfamethoxazole in binding DHPS, causing resistance to co-trimoxazole [10].
In summary, insertion in DHPS and substitution in DHFR lead to resistance against co-trimoxazole. The insertion of the 6 bp sequence into DHPS and the amino acid substitution Ile-100-Leu in DHFR seem to correlate with the development of resistance against co-trimoxazole. In addition, the combination of these mutations is associated with higher resistance to co-trimoxazole.
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