The emergence of a multidrug-resistant Candida species, C. auris and C. haemulonii, has been reported worldwide. In Thailand, information on them is limited. We collected clinical isolates from Thai patients with invasive candidiasis. Both species were compared with a laboratory C. albicans strain. In vitro antifungal susceptibility and thermotolerance, and pathogenesis in the zebrafish model of infection were investigated. Both species demonstrated high minimal inhibitory concentrations to fluconazole and amphotericin B. Only C. auris tolerated high temperatures, like C. albicans. In a zebrafish swim-bladder-inoculation model, the C. auris-infected group had the highest mortality rate and infectivity, suggesting the highest virulence. The case fatality rates of C. auris, C. haemulonii, and C. albicans were 100%, 83.33%, and 51.52%, respectively. Further immunological studies revealed that both emerging Candida species stimulated genes involved in the proinflammatory cytokine group. Interestingly, the genes relating to leukocyte recruitment were downregulated only for C. auris infections. Almost all immune response genes to C. auris had a peak response at an early infection time, which contrasted with C. haemulonii. In conclusion, both emerging species were virulent in a zebrafish model of infection and could activate the inflammatory pathway. This study serves as a stepping stone for further pathogenesis studies of these important emerging species.

Candida albicans, an opportunistic pathogen, has the ability to form biofilms in the host or within medical devices in the body. Biofilms have been associated with disseminated/invasive disease with increased severity of infection by disrupting the host immune response and prolonging antifungal treatment. In this study, the in vivo virulence of three strains with different biofilm formation strengths, that is, non-, weak-, and strong biofilm formers, was evaluated using the zebrafish model. The survival assay and fungal tissue burden were measured. Biofilm-related gene expressions were also investigated. The survival of zebrafish, inoculated with strong biofilms forming C. albicans,, was significantly shorter than strains without biofilms forming C. albicans. However, there were no statistical differences in the burden of viable colonogenic cell number between the groups of the three strains tested. We observed that the stronger the biofilm formation, the higher up-regulation of biofilm-associated genes. The biofilm-forming strain (140 and 57), injected into zebrafish larvae, possessed a higher level of expression of genes associated with adhesion, attachment, filamentation, and cell proliferation, including eap1, als3, hwp1, bcr1, and mkc1 at 8 h. The results suggested that, despite the difference in genetic background, biofilm formation is an important virulence factor for the pathogenesis of C. albicans. However, the association between biofilm formation strength and in vivo virulence is controversial and needs to be further studied.

Candida albicans is one of the most common human fungal pathogens. Candidemia has significant mortality globally. No epidemiological study of C. albicans based on multilocus sequence typing (MLST) has been conducted in Thailand. Therefore, MLST was used to study the molecular epidemiology of C. albicans blood strains in a large Thai teaching hospital. In vitro virulence phenotypes and antifungal susceptibility testing by broth microdilution were also conducted. Forty-six C. albicans blood strains from 37 patients were collected from the Department of Microbiology, Siriraj Hospital, in 2016 and 2017. Most patients (71.8%) were more than 60 years old, and the case fatality rate was 54.8%. The male-to-female ratio was 5:3. Thirty-four diploid sequence types (DSTs), including six new DSTs, were identified, with DST2514 (8.7%) and DST2876 (8.7%) as the most common DSTs. Strains were clustered into nine clades. Unlike other studies of C. albicans blood strains in Asia, clade 17 was the most common (13 strains, 28.3%). Sequential allelic changes were evident in sequential strains from one patient. All strains produced phospholipase and hemolysin, while none produced proteinase. The ability to form biofilm was found in 82.6% of the strains. Clade 17 strains showed significantly stronger hemolytic activity than non–clade 17 strains (69.2% versus 27.3%; p = 0.022). However, no significant association existed between clades and patient mortalities. All were susceptible or wild type to anidulafungin (MIC range = 0.015–0.12 and GM = 0.030), micafungin (MIC range = ≤ 0.008–0.015 and GM = 0.008), caspofungin (MIC range = 0.008–0.12 and GM = 0.036), and amphotericin B (MIC range = 0.25–0.5 and GM = 0.381). Only one strain was resistant to voriconazole (MIC range = ≤ 0.008 to ≥ 8 and GM = 0.010) and fluconazole (MIC range = 0.12–16 and GM = 0.398). In conclusion, a high prevalence of clade 17 C. albicans blood strains was found in Thailand, in contrast to other Asian countries. This unique finding might be explained by the strong hemolytic activity that is required for bloodstream infection of C. albicans.

Candidemia, a bloodstream infection caused by genus Candida, has a high mortality rate. Candida albicans was previously reported to be the most common causative species among candidemia patients. However, during the past 10 years in Thailand, Candida tropicalis has been recovered from blood more frequently than C. albicans. The cause of this shift in the prevalence of Candida spp. remains unexplored. We conducted in vitro virulence studies and antifungal susceptibility profiles of 48 C. tropicalis blood isolates collected during 2015–2017. To compare to global isolates of C. tropicalis, multilocus sequence typing (MLST), a minimum spanning tree, and an eBURST analysis were also conducted. C. tropicalis and C. albicans were the most (47–48.7%) and second-most (21.5–33.9%) common species to be isolated from candidemia patients, respectively. Of the C. tropicalis blood isolates, 29.2, 0, 100, and 93.8% exhibited proteinase activity, phospholipase activity, hemolytic activity, and biofilm formation, respectively. Moreover, 20.8% (10/48) of the isolates were resistant to voriconazole and fluconazole, and also showed high minimum inhibitory concentrations (MICs) to posaconazole and itraconazole. In contrast, most of the isolates were susceptible to anidulafungin (97.9%), micafungin (97.9%), and caspofungin (97.9%), and showed low MICs to amphotericin B (100%) and 5-flucytosine (100%). The MLST identified 22 diploid sequence types. Based on the eBURST analysis and minimum spanning tree, 9 out of 13 members (69.2%) of an eBURST group 3 were resistant to voriconazole and fluconazole, and also showed high MICs to posaconazole and itraconazole. Association analysis revealed the eBURST group 3 was significantly associated with the four triazole resistance (p < 0.001). In conclusion, the eBURST group 3 was associated with the triazole resistance and resistance to many antifungal drugs might be collectively responsible for the prevalence shift.

Group B streptococcus (GBS) or Streptococcus agalactiae is an opportunistic pathogen that causes serious illness in newborns, pregnant women, and adults. However, insufficient detection methods and disease prevention programs have contributed to an increase in the incidence and fatality rates associated with this pathogen in non-neonatal patients. This study aimed to investigate factors of the observed increased incidence by investigation of serotype distribution, virulence factors, and antimicrobial susceptibility patterns from invasive GBS disease among non-neonatal patients in Thailand. During 2017–2018, a total of 109 S. agalactiae isolates were collected from non-pregnant patients. There were 62 males and 47 females, with an average age of 63.5 years (range: 20 – 96). Serotypes were determined by latex agglutination assay and multiplex polymerase chain reaction (PCR)-based assay. Among those isolates, seven virulence genes (rib, bca, pavA, lmb, scpB, cylE, and cfb) were detected by PCR amplification, and were determined for their susceptibility to 20 antimicrobial agents using a Sensititre^TM Streptococcus species STP6F AST plate. Among the study isolates, serotype III was predominant (52.3%), followed by serotype V and serotype VI (13.8% for each), serotype Ib (11.9%), and other serotypes (8.2%). Of the seven virulence genes, pavA was found in 67.0%. Except for one, there were no significant differences in virulence genes between serotype III and non-serotype III. Study isolates showed an overall rate of non-susceptibility to penicillin, the first-line antibiotic, of only 0.9%, whereas the resistance rates measured in tetracycline, clindamycin, azithromycin, and erythromycin were 41.3, 22.0, 22.0, and 22.0%, respectively. Strains that were resistant to all four of those drugs were significantly associated with non-serotype III (p < 0.001). Using multi-locus sequence typing (MLST), 40.0% of the four-drug-resistant isolates belonged to serotype VI/ST1, followed by serotype Ib/ST1 (35.0%). Cluster analysis with global GBS isolates suggested that the multiple drug-resistant isolates to be strongly associated with the clonal complex (CC) 1 (p < 0.001). Compared to the 2014 study of 210 invasive GBS isolates conducted in 12 tertiary hospitals in Thailand, the proportion of serotype III has dramatically dropped from nearly 90% to about 50%. This suggests that resistances to the second-line antibiotics for GBS might be the selective pressure causing the high prevalence of non-serotype III isolates.