Characterization of MultidrugResistant serogroup 19 Streptococcus pneumoniae isolated from healthy children below 5 years of age in Indonesia.

We investigated the resistance genes, pilus islets, biofilm formation ability and sequence types of multidrug-resistant Streptococcus pneumoniae (MDRSP) isolated from healthy children below 5 years of age in Indonesia. In all, 104 archived MDRSP isolates from previous carriage studies in Indonesia in 2016-2019 were screened for the presence of antibiotic resistance genes and the rrgC (pilus islet 1) and pitB (pilus islet 2) genes. Multilocus sequence typing and biofilm formation were determined by PCR sequencing and the ability of cells to adhere to the walls, respectively. Results have shown that the mefA, ermB and tetM genes were found in 93, 52 and 100 % of MDRSP isolates, respectively. Insertions of arginine, proline and Ile-100-Leu were the most common mutations in the folA and folP genes. Pilus islets 1 and 2 were discovered in 93 and 82 % of MDRSP isolates, respectively. The MDRSP isolates showed no biofilm formation ability (64 %), and 5 out of 10 strains of MDRSP strains were ST1464. This finding can be used to provide further considerations in implementing and monitoring pneumococcal vaccination in Indonesia.

Nasopharyngeal carriage of Streptococcus pneumoniae among children <5 years of age in Indonesia prior to pneumococcal conjugate vaccine introduction.

Pneumococcal conjugate vaccines (PCVs) prevent nasopharyngeal colonization with vaccine serotypes of Streptococcus pneumoniae, leading to reduced transmission of pneumococci and stronger population-level impact of PCVs. In 2017 we conducted a cross-sectional pneumococcal carriage study in Indonesia among children aged <5 years before 13-valent PCV (PCV13) introduction. Nasopharyngeal swabs were collected during visits to community integrated health service posts at one peri-urban and one rural study site. Specimens were analyzed by culture, and isolates were serotyped using sequential multiplex polymerase chain and Quellung reaction. Antibiotic susceptibility was performed by broth microdilution method. We enrolled 1,007 children in Gunungkidul District, Yogyakarta (peri-urban) and 815 in Southwest Sumba, East Nusa Tenggara (rural). Pneumococcal carriage prevalence was 30.9% in Gunungkidul and 87.6% in Southwest Sumba (combined: 56.3%). PCV13 serotypes (VT) carriage was 15.0% in Gunungkidul and 52.6% in Southwest Sumba (combined: 31.8%). Among pneumococcal isolates identified, the most common VT were 6B (16.4%), 19F (15.8%), and 3 (4.6%) in Gunungkidul (N = 323) and 6B (17.6%), 19F (11.0%), and 23F (9.3%) in Southwest Sumba (N = 784). Factors associated with pneumococcal carriage were age (1-2 years adjusted odds ratio (aOR) 1.9, 95% CI 1.4-2.5; 3-4 years aOR 1.5, 95% CI 1.1-2.1; reference <1 year), other children <5 years old in the household (aOR 1.5, 95% CI 1.1-2.0), and presence of ≥1 respiratory illness symptom (aOR 1.8, 95% CI 1.4-2.2). Overall, 61.5% of the pneumococcal isolates were non-susceptible to ≥1 antibiotic class and 13.2% were multi-drug non-susceptible (MDNS) (non-susceptible to ≥3 classes of antibiotics). Among 602 VT isolates, 73.9% were non-susceptible and 19.9% were MDNS. These findings are critical to establish a pre-PCV13 carriage prevalence and demonstrate the complexity in evaluating the impact of PCV13 introduction in Indonesia given the wide variability in the carriage prevalence as shown by the two study sites.

Epidemiology of Haemophilus influenzae in children on Lombok Island, Indonesia.

The Haemophilus influenzae serotype b (Hib) conjugate vaccine routine immunization programme has been implemented for almost a decade; however, there is limited surveillance of H. influenzae carriage rates in the Indonesian population. H. influenzae was isolated from nasopharyngeal (NP) swab specimens of healthy children on Lombok Island, West Nusa Tenggara Province, Indonesia from 2018 to 2019. Serotyping was performed using quantitative polymerase chain reaction. We identified H. influenzae in 40 of the 96 (41.6 %) NP swab specimens. We identified 39 non-typeable H. influenzae (NTHi) isolates and 1 Hib isolate.

Antibacterial activity of medicinal plants in Indonesia on Streptococcus pneumoniae.

Streptococcus pneumoniae is a human pathogenic bacterium able to cause invasive pneumococcal diseases. Some studies have reported medicinal plants having antibacterial activity against pathogenic bacteria. However, antibacterial studies of medicinal plants against S. pneumoniae remains limited. Therefore, this study aims to describe the antibacterial activity of medicinal plants in Indonesia against S. pneumoniae. Medicinal plants were extracted by maceration with n-hexane, ethanol, ethyl acetate and water. Antibacterial activity was defined by inhibition zone and minimum inhibitory concentration (MIC). Bactericidal activity was measured by culture and time-killing measurement. Methods used to describe the mechanism of action of the strongest extract were done by absorbance at 595 nm, broth culture combined with 1% crystal violet, qRT-PCR targeting lytA, peZT and peZA, and transmission electron microscope to measure bacterial lysis, antibiofilm, LytA and peZAT gene expression, and ultrastructure changes respectively. Among 13 medicinal plants, L. inermis Linn. ethyl acetate extract showed the strongest antibacterial activity against S. pneumoniae with an MIC value of 0,16 mg/ml. Bactericidal activity was observed at 0,16 mg/ml for 1 hour incubation. Lawsonia inermis extract showed some mechanism of actions including bacterial lysis, antibiofilm, and ultrastructure changes such as cell wall disruption, decreasing cell membrane integrity and morphological disorder. Increasing of lytA and decreasing of peZA and peZT expression were also observed after incubation with the extract. In addition, liquid chromatography mass spectrophotometer showed phenolic compounds as the commonest compound in L. inermis ethyl acetate extract. This study describes the strong antibacterial activity of L. inermis with various mechanism of action including ultrastructure changes.

Serotype Distribution and Antimicrobial Resistance Profile of Haemophilus influenzae Isolated from School Children with Acute Otitis Media.

Haemophilus influenzae is a Gram-negative opportunistic bacterial pathogen of the human respiratory tract. This study describes the prevalence, serotype distribution, and susceptibility profiles of H. influenzae strains isolated from the nasopharynx of school children with acute otitis media (AOM) in Banyumas Regency, Central Java, Indonesia. H. influenzae was isolated from nasopharyngeal swab specimens using chocolate agar plates supplemented with IsoVitaleX and bacitracin. Serotyping was performed using quantitative polymerase chain reaction. Antimicrobial susceptibility profiles were determined using a microdilution broth assay. H. influenzae was present in 69.7% of samples (85/122). Nontypeable H. influenzae (NHTi) was the most common serotype (95.3%), followed by H. influenzae type b (3.5%) and H. influenzae type f (1.2%). All the H. influenzae isolates were susceptible to levofloxacin, ceftriaxone, imipenem, meropenem, cefuroxime, and cefixime. Most isolates were susceptible to sparfloxacin (99%), cefepime (99%), amoxicillin/clavulanic acid 2 : 1 (99%), ampicillin/sulbactam 2 : 1 (96%), chloramphenicol (94%), tetracycline (93%), ampicillin (87%), and clarithromycin (82%). Nineteen percent of the isolates were resistant to cotrimoxazole, and 11% of the isolates were resistant to ampicillin. This study showed that H. influenzae carriage among samples was dominated by NTHi and less susceptible to cotrimoxazole.

Nasopharyngeal carriage rate, serotype distribution, and antimicrobial susceptibility profile of Streptococcus pneumoniae isolated from children under five years old in Kotabaru, South Kalimantan, Indonesia.

BACKGROUND: Streptococcus pneumoniae is a bacterial pathogen that colonizes the human nasopharynx. Colonization is frequently reported to be high in young children. In this study, we investigated the nasopharyngeal (NP) carriage rate, serotype distribution, and antibiotic susceptibility of S. pneumoniae in children under five years of age in Kotabaru, South Kalimantan, Indonesia. METHODS: NP swab specimens were collected from 399 young children (mean age: 30 months) who participated in the Rampa Village Community Health Center, with 74% of the participants being Bajau children. S. pneumoniae was identified using optochin susceptibility and bile solubility tests. Serotyping was performed by sequential multiplex PCR, and antimicrobial susceptibility profiling was performed by disk diffusion and microdilution methods. RESULTS: The NP carriage rate of S. pneumoniae was 45% (180/399). The most commonly serotypes were 6A/6B (18%), followed by 15B/15C (17%), 19F (16%), 34 (8%), and 23F (5%); 46% of them were identified as strains of the PCV13 vaccine type. Additionally, almost half of the pneumococcal isolates were non-susceptible to penicillin (40%), whereas non-susceptibility to tetracycline (36.8%), trimethoprim/sulfamethoxazole (29.7%), erythromycin (16.8%), chloramphenicol (9.7%), and clindamycin (8.6%) was also found. We identified 18% (n = 34) of S. pneumoniae isolates as multidrug-resistant (MDR) strains, and serotype 19F was the most common (74%) among them. CONCLUSIONS: MDR S. pneumoniae vaccine type strains were dominated by serotype 19F. The implementation of a pneumococcal conjugate vaccine program in Indonesia might reduce MDR strains circulating in the community in the future.

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.

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.

Identification and antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus-associated subclinical mastitis isolated from dairy cows in Bogor, Indonesia.

BACKGROUND AND AIM: Subclinical mastitis is an udder infection devoid of clinical symptoms, and Staphylococcus aureus is one of the bacteria causing this disease. This study aimed to identify and determine the prevalence and antibiotic susceptibility of methicillin-resistant S. aureus (MRSA)-associated subclinical mastitis isolated from dairy cows in Bogor, Indonesia. MATERIALS AND METHODS: S. aureus was isolated from subclinical mastitis milk specimens. All strains were confirmed by polymerase chain reaction-based detection of staphylococcal nuc, and MRSA was confirmed by the presence of mecA. Antibiotic susceptibility was determined using the disk diffusion method. RESULTS: From 86 milk samples, 49 isolates (57%) were confirmed as S. aureus. All S. aureus isolates were susceptible to tetracycline, gentamicin, chloramphenicol, erythromycin, and trimethoprim/sulfamethoxazole. Nine isolates were identified as MRSA (10.5%). CONCLUSION: In this study, we reported MRSA-associated subclinical mastitis in Bogor, Indonesia.

The prevalence of pilus islets in Streptococcus pneumoniae isolates from healthy children in Indonesia.

Streptococcus pneumoniae produces pili that function as adherence factors to bind to epithelial cells in the human upper respiratory tract. In this study, we investigated the prevalence of pilus islets (PIs) in S. pneumoniae strains carried by healthy children below 5 years of age prior to pneumococcal vaccination in 2012 in Lombok Island, Indonesia. In all, 347 archived S. pneumoniae isolates were screened using polymerase chain reactions for the presence of rrgC and pitB genes representing pilus islet 1 (PI-1) and pilus islet 2 (PI-2), respectively. We found that 40 isolates (11.5 %) contained the PI genes: 5.2% carried both PI-1 and PI-2, and 3.5 and 2.9% carried PI-1 and PI-2, respectively. Furthermore, we found that most of the strains carrying either of the PIs belonged to the vaccine serotypes 19F and 19A and were less susceptible to chloramphenicol and tetracycline.

Streptococcus pneumoniae carriage and antibiotic susceptibility among Indonesian pilgrims during the Hajj pilgrimage in 2015.

The Hajj is an annual pilgrimage to Mecca and one of the largest gathering of people in the world. Most Indonesian pilgrims are senior adults and elderly adults, who are more prone to acquire infections during the Hajj ritual. The aims of this study are to investigate the dynamics of Streptococcus pneumoniae colonization and to investigate antibiotic susceptibility of pneumococcal strains in Indonesian pilgrims. This was a prospective multi-site longitudinal study in Indonesian hajj pilgrims aged >18 years old in the year 2015. Nasopharyngeal swabs were collected from the same subject before departure and upon arrival at the airport. S. pneumoniae was identified using conventional and molecular approach, while antibiotic susceptibility was determined using a disk diffusion method. Among 813 Hajj pilgrims who were enrolled from five sites in this study, the prevalence of S. pneumoniae carriage rates before- and after-the Hajj were 8.6% (95% CI 6.7-10.5%) and 8.2% (95% CI 6.4-10.1%), (p value: 0.844) respectively. Serotype 16F, 6A/6B, 3, 18, and 23F were the five most prevalent serotypes before Hajj, whereas serotypes 3, 34, 13, 4, and 23F were the most prevalent serotypes after Hajj. Serotype 3 was identified as most acquired serotype during Hajj in Indonesian pilgrim. There was an increase in the percentage of isolates susceptible to co-trimoxazole after Hajj (42.9% versus 57.4%). The study provided an overview of the change of dynamics of S. pneumoniae serotype acquisition in Indonesian Hajj Pilgrims. Along with data of vaccination serotypes coverage and antimicrobial susceptibility, these findings may contribute to recommendation of vaccination and treatment policies in the future.

Prevalence, serotype distribution, and antimicrobial susceptibility profile of Streptococcus pneumoniae among patients with acute respiratory tract infection.

We investigated the nasopharyngeal (NP) carriage and antimicrobial susceptibility profile of Streptococcus pneumoniae among adults and children with acute respiratory tract infection (ARTI). NP swabs were collected from ARTI patients in Tabanan, Bali, Indonesia, in 2017. Serotyping of S. pneumoniae and antibiotic susceptibility profile were performed by multiplex sequential PCR and the disk diffusion method, respectively. Out of 200 ARTI patients, S. pneumoniae strains were carried by 22.6% and 2.4% of children (36/159) and adults (1/41), respectively. Serotype 6A/6B was the most common serotype among cultured strains (21%) followed by 19F (18%) and 14 (8%). Most isolates were susceptible to chloramphenicol (87%), followed by clindamycin (74%), erythromycin (72%), sulfamethoxazole-trimethoprim (59%), and tetracycline (44%). This finding provides baseline information on the serotype distribution of S. pneumoniae carriage among ARTI patients in Indonesia.