Molecular Markers of Ovarian Germ Cells of Banana Prawn (Fenneropenaeus merguiensis).

The banana prawn (Fenneropenaeus merguiensis) is a valuable prawn in the worldwide market. However, cultivation of this species is limited owing to the difficulty in culture management and limited knowledge of reproduction. Therefore, we studied the gene expression and molecular mechanisms involved in oogenesis for elucidating ovarian germ cell development in banana prawns. The tissue-specific distribution of certain genes identified from previous transcriptome data showed that FmCyclinB, FmNanos, and nuclear autoantigenic sperm protein (FmNASP) were only expressed in gonads. The in situ hybridization (ISH) of these three genes showed different expression patterns throughout oogenesis. FmCyclinB was highly expressed in pre-vitellogenic oocytes. FmNanos was expressed at almost the same level during oogenesis but showed the most expression in late pre-vitellogenic stages. Based on the highest expression of FmCyclinB and FmNanos in mid pre-vitellogenic and late pre-vitellogenic oocytes, respectively, we suggested that FmNanos may suppress FmCyclinB expression before initiation of vitellogenesis. Meanwhile, FmNASP expression was detected only in pre-vitellogenesis. Moreover, quantitative real-time polymerase chain reaction (qRT-PCR) analysis of FmNASP expression was supported by FmNASP ISH analysis based on high expression of FmNASP in sub-adult ovaries, which contain most of pre-vitellogenic oocytes. In this study, we found three reliable ovarian markers for banana prawns and also found a dynamic change of molecular mechanism during the sub-stage of pre-vitellogenesis. We determined the expression levels of these genes involved in oogenesis. Our findings provide information for further studies on banana prawn reproduction which may assist in their cultivation.

Antimicrobial Susceptibility and Molecular Features of Colonizing Isolates of Pseudomonas aeruginosa and the Report of a Novel Sequence Type (ST) 3910 from Thailand.

Pseudomonas aeruginosa is an important pathogen as it can cause hospital-acquired infections. Additionally, it can also colonize in patients and in other various environments. Hence, this study aimed to investigate the antimicrobial susceptibility, and to study the molecular features, of colonizing isolates of P. aeruginosa from Songklanagarind Hospital, Thailand. Genomic DNA extraction, whole-genome sequencing (WGS), and bioinformatics analysis were performed in all studied isolates. The findings demonstrated that the majority of isolates were non-susceptible to colistin and carbapenem. For in silico study, multilocus sequence typing (MLST) revealed one novel sequence type (ST) 3910 and multiple defined STs. The isolates carried several antimicrobial resistance genes (blaOXA-50, aph(3')-IIb, etc.) and virulence-associated genes (fleN, waaA, etc.). CRISPR-Cas sequences with different spacers and integrated bacteriophage sequences were also identified in these isolates. Very high SNPs were found in the alignments of the novel ST-3910 isolate with other isolates. A comparative genomic analysis exhibited phylogenetic clustering of our colonizing isolates with clinical isolates from many countries. Interestingly, ST-3981, ST-3982, ST-3983, ST-3984, ST-3985, ST-3986, ST-3986, ST-3986, ST-3987, and ST-3988, the new STs from published genomes, were assigned in this study. In conclusion, this WGS data might be useful for tracking the spread of P. aeruginosa colonizing isolates.

Whole-genome analysis of carbapenem-resistant Acinetobacter baumannii from clinical isolates in Southern Thailand.

The worldwide spread of carbapenem-resistant Acinetobacter baumannii (CRAB) has become a healthcare challenge for some decades. To understand its molecular epidemiology in Southern Thailand, we conducted whole-genome sequencing (WGS) of 221 CRAB clinical isolates. A comprehensive bioinformatics analysis was performed using several tools to assemble, annotate, and identify sequence types (STs), antimicrobial resistance (AMR) genes, mobile genetic elements (MGEs), and virulence genes. ST2 was the most prevalent ST in the CRAB isolates. For the detection of AMR genes, almost all CRAB isolates carried the bla OXA-23 gene, while certain isolates harbored the bla NDM-1 or bla IMP-14 genes. Also, various AMR genes were observed in these CRAB isolates, particularly aminoglycoside resistance genes (e.g., armA, aph(6)-Id, and aph(3″)-Ib), fosfomycin resistance gene (abaF), and tetracycline resistance genes (tet(B) and tet(39)). For plasmid replicon typing, RepAci1 and RepAci7 were the predominant replicons found in the CRAB isolates. Many genes encoding for virulence factors such as the ompA, adeF, pgaA, lpxA, and bfmR genes were also identified in all CRAB isolates. In conclusion, most CRAB isolates contained a mixture of AMR genes, MGEs, and virulence genes. This study provides significant information about the genetic determinants of CRAB clinical isolates that could assist the development of strategies for improved control and treatment of these infections.

Genomic insights into blaNDM-carrying carbapenem-resistant Klebsiella pneumoniae clinical isolates from a university hospital in Thailand.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates is a serious threat to global health. Here, we elucidate the genetic features of blaNDM-carrying CRKP clinical isolates from a university hospital in Thailand. The entire genomes of 19 CRKP isolates were extracted and then sequenced using the MGISEQ200 platform. Using various bioinformatics tools, we analyzed the antimicrobial resistance (AMR), virulence factors, gene transfer, bacterial defense mechanisms, and genomic diversity of the CRKP isolates. The sequence type (ST) 16 was found in most of the isolates, along with carriages of the blaNDM-1, blaOXA-232, and blaCTX-M-15 genes. The IncFIB(pQil), Col440II, and ColKP3 plasmids were identified with high frequency. The CRKP isolates harbored genes encoding for virulence factors such as adherence, biofilm formation, immune evasion, and iron uptake. The CRISPR-Cas region in the CRKP9 isolate consisted of 28 distinct spacer sequences. The genomes of the CRKP isolates presented restriction-modification (R-M) sites (M.Kpn34618Dcm and M.Kpn928I) and integrated bacteriophage genomes (Klebsiella phage ST16-OXA48phi5.4 and Enterobacteria phage mEp390). Bottromycin and sactipeptides were also identified. The isolates could be separated into three clades according to STs and pairwise single nucleotide polymorphism (SNP) distance. Pairwise average nucleotide identity (ANI) values revealed intra-species. These findings support the importance of whole-genome sequencing (WGS) to the rapid and accurate genomic analysis of clinical isolates of CRKP.

In vitro Synergistic Activities of Fosfomycin in Combination with Other Antimicrobial Agents Against Carbapenem-Resistant Escherichia coli Harboring bla NDM-1 on the IncN2 Plasmid and a Study of the Genomic Characteristics of These Pathogens.

Purpose: The spread of New Delhi metallo-ß-lactamase (NDM) encoded by the bla NDM gene has been a global health crisis for many years. Most of bla NDM-harboring bacteria commonly carry various antimicrobial resistance (AMR) genes on their chromosomes or plasmids, leading to limited treatment options. Thus, we aimed to evaluate the synergistic effects of fosfomycin in combination with other antimicrobial agents against bla NDM-harboring carbapenem-resistant Escherichia coli (CREC) and to characterize the whole-genome and plasmid sequences of these pathogens. Methods: Thirty-eight CREC isolates were collected from patients in the Medicine Ward, Songklanagarind Hospital, Thailand. The activity of fosfomycin in combination with other antimicrobial agents against CREC isolates harboring bla NDM on the plasmid was evaluated using the checkerboard method. In this method, the serial dilutions of two antibiotics were mixed with the cultured CREC, the mixtures were incubated, and FICI was calculated to interpret the synergistic activity of the combination. The whole-genome and particular plasmids of these pathogens were sequenced using next-generation sequencing. Sequence analysis, especially on antimicrobial resistance (AMR) genes, mobile-genetic elements (MGEs), and virulence genes was performed using many bioinformatics tools. Results: Of the E. coli 38 isolates, only 3 isolates contained the bla NDM-1 gene, which is located on the IncN2 plasmid. The combinations of fosfomycin with aminoglycosides, colistin, tigecycline, sitafloxacin, and ciprofloxacin were synergies against bla NDM-1-harboring CREC isolates. Genomic analysis revealed that these isolates harbored many ß-lactam resistance genes and other AMR genes that may confer resistance to aminoglycoside, fluoroquinolone, rifampicin, trimethoprim, sulfonamide, tetracycline, and macrolide. Also, various MGEs, especially the bla NDM-1-bearing IncN2 plasmid, were present in these isolates. Conclusion: Our study demonstrated some synergistic effects of antimicrobial combination against CREC isolates harboring bla NDM-1 on the IncN2 plasmid. Also, our data on the whole-genome and plasmid sequences might be beneficial in the control of the spread of bla NDM-1-harboring CREC isolates. The linkages between bla NDM-1-carrying plasmid, patient information, and time of collection will be elucidated to track the horizontal gene transfer in the future.