CAIM: coverage-based analysis for identification of microbiome.

Accurate taxonomic profiling of microbial taxa in a metagenomic sample is vital to gain insights into microbial ecology. Recent advancements in sequencing technologies have contributed tremendously toward understanding these microbes at species resolution through a whole shotgun metagenomic approach. In this study, we developed a new bioinformatics tool, coverage-based analysis for identification of microbiome (CAIM), for accurate taxonomic classification and quantification within both long- and short-read metagenomic samples using an alignment-based method. CAIM depends on two different containment techniques to identify species in metagenomic samples using their genome coverage information to filter out false positives rather than the traditional approach of relative abundance. In addition, we propose a nucleotide-count-based abundance estimation, which yield lesser root mean square error than the traditional read-count approach. We evaluated the performance of CAIM on 28 metagenomic mock communities and 2 synthetic datasets by comparing it with other top-performing tools. CAIM maintained a consistently good performance across datasets in identifying microbial taxa and in estimating relative abundances than other tools. CAIM was then applied to a real dataset sequenced on both Nanopore (with and without amplification) and Illumina sequencing platforms and found high similarity of taxonomic profiles between the sequencing platforms. Lastly, CAIM was applied to fecal shotgun metagenomic datasets of 232 colorectal cancer patients and 229 controls obtained from 4 different countries and 44 primary liver cancer patients and 76 controls. The predictive performance of models using the genome-coverage cutoff was better than those using the relative-abundance cutoffs in discriminating colorectal cancer and primary liver cancer patients from healthy controls with a highly confident species markers.

Integrating short- and full-length 16S rRNA gene sequencing to elucidate microbiome profiles in Pacific white shrimp (Litopenaeus vannamei) ponds.

Despite their immense economic value as a key aquaculture species, the production of Pacific white shrimp (Litopenaeus vannamei) faces significant challenges from intensive farming practices and disease outbreaks. Routine microbial profiling for disease surveillance could be a promising approach to anticipate and control disease outbreaks. To achieve this, accuracy in microbial profiling in shrimp ponds is crucial for enabling targeted action and prevention. Extensive documentation emphasizes that, beyond biological factors (related to the host, diet, or health status during the rearing period), technical elements, including sequencing techniques significantly influence bacterial community profiling. This study investigated the influence of short- and long-read sequencing of 16S rRNA genes on the microbial profiles in shrimp intestines, water, and sediments. The origin of the samples (intestine or environmental) in shrimp culture ponds primarily drove the observed differences in core microbial species. The ecological niches accounted for 56% of bacterial community variations in culture ponds. Both sequencing approaches showed consistent results in identifying higher-rank taxa and assessing alpha and beta diversity. However, at the species level, full-length 16S rRNA gene sequences provided better resolution than V3-V4 sequences. For routine microbial profiling in shrimp culture ponds, our study suggests that short-read sequences were sufficient for determining overall bacterial community.IMPORTANCEThis interdisciplinary study investigated the influence of sequencing techniques on bacterial communities profiling within Pacific white shrimp (Litopenaeus vannamei) ponds. By integrating aquaculture, microbiology, and environmental science, we revealed the role of ecological niches and factors like salinity and pH on microbiota diversity and composition in shrimp intestines, pond water, and sediment. Additionally, we compared the taxonomic resolution using partial versus full-length 16S rRNA gene sequences, highlighting the value of longer amplicons for precise identification of key taxa. These findings provide novel insights into microbial dynamics underlying environmental effects in shrimp aquaculture. Comprehensive characterization of the pond microbiome could lead to management strategies that promote shrimp health and productivity. Furthermore, the potential of a multi-omics approach for integrating complementary data streams to elucidate environment-microbiome-host interactions was highlighted.

Characterization of staphylococcal cassette chromosome mec in Staphylococcus haemolyticus ST3 and ST42 isolates from bovine mastitis milk: identification of novel variant in class C1 mec complex.

BACKGROUND: Methicillin-resistant Staphylococcus haemolyticus (MRSH) is an important pathogenic agent of bovine mastitis. Among the prominent clone lineages in dairy cows are MRSH sequence types ST3 and ST42. Little information is available on the complete characterization of SCCmec elements in MRSH. OBJECTIVE: In this study, two clinical isolates of MRSH ST3 and ST42 from bovine mastitis milk were selected, and their nontypable SCCmec structures were compared. METHODS: Two MRSH strains, MRSH-ST3 strain M62.3 and MRSH-ST42 strain M81.1, were identified from bovine mastitis milk in Thailand in 2022. Minimum inhibitory concentration was used to screen for antimicrobial resistance susceptibility. Oxford Nanopore Technologies and Illumina sequencing were performed in combination to complete the genome. Their gene organization and structure of SCCmec types were analysed and compared with the whole sequences of other strains in the same sequence types. RESULTS: Both MRSH-ST3 strain M62.3 and MRSH-ST42 strain M81.1 possessed the class C1 mec complex but lacked the ccr gene complex. Notably, MRSH-ST42 strain M81.1 contained a novel variant of C1 mec complex, which consisted of IS431-mecA-ISSha1-paaZ-upgQ-IS431, with IS431 organized in the same orientation. Apart from class C1 mec and the heavy metal-resistant cluster, the gene composition and order of the SCCmec element varied. In ST3, variations in the SCCmec type, gene content and organization were observed. CONCLUSIONS: The distinct evolution of the MRSH lineage was indicated by the various SCCmec elements. The insertion of ISSha1 resulted in a unique variant of class C1 mec complex that demonstrated the important role of the insertion sequence in SCCmec diversification.

Genomic analysis of carbapenem- and colistin-resistant Klebsiella pneumoniae complex harbouring mcr-8 and mcr-9 from individuals in Thailand.

The surge in mobile colistin-resistant genes (mcr) has become an increasing public health concern, especially in carbapenem-resistant Enterobacterales (CRE). Prospective surveillance was conducted to explore the genomic characteristics of clinical CRE isolates harbouring mcr in 2015-2020. In this study, we aimed to examine the genomic characteristics and phonotypes of mcr-8 and mcr-9 harbouring carbapenem-resistant K. pneumoniae complex (CRKpnC). Polymerase chain reaction test and genome analysis identified CRKpnC strain AMR20201034 as K. pneumoniae (CRKP) ST147 and strain AMR20200784 as K. quasipneumoniae (CRKQ) ST476, harbouring mcr-8 and mcr-9, respectively. CRKQ exhibited substitutions in chromosomal-mediated colistin resistance genes (pmrB, pmrC, ramA, and lpxM), while CRKP showed two substitutions in crrB, pmrB, pmrC, lpxM and lapB. Both species showed resistance to colistin, with minimal inhibitory concentrations of 8 µg/ml for mcr-8-carrying CRKP isolate and 32 µg/ml for mcr-9-carrying CRKQ isolate. In addition, CRKP harbouring mcr-8 carried blaNDM, while CRKQ harbouring mcr-9 carried blaIMP, conferring carbapenem resistance. Analysis of plasmid replicon types carrying mcr-8 and mcr-9 showed FIA-FII (96,575 bp) and FIB-HI1B (287,118 bp), respectively. In contrast with the plasmid carrying the carbapenemase genes, the CRKQ carried blaIMP-14 on an IncC plasmid, while the CRKP harboured blaNDM-1 on an FIB plasmid. This finding provides a comprehensive insight into another mcr-carrying CRE from patients in Thailand. The other antimicrobial-resistant genes in the CRKP were blaCTX-M-15, blaSHV-11, blaOXA-1, aac(6')-Ib-cr, aph(3')-VI, ARR-3, qnrS1, oqxA, oqxB, sul1, catB3, fosA, and qacE, while those detected in CRKQ were blaOKP-B-15, qnrA1, oqxA, oqxB, sul1, fosA, and qacE. This observation highlights the importance of strengthening official active surveillance efforts to detect, control, and prevent mcr-harbouring CRE and the need for rational drug use in all sectors.

Complete genome sequences of paired isogenic Burkholderia pseudomallei isolated from a Thai pediatric patient with a urinary tract infection.

Burkholderia pseudomallei is the causative agent of melioidosis, the disease endemic in Southeast Asia and northern Australia. We report complete genome sequences of paired isogenic B. pseudomallei isolated from a 12-year-old Thai male presenting with acute urinary tract infection before (SCBP001) and after (SCBP007) a decrease in susceptibility to ceftazidime.

Genomic characterization of carbapenem and colistin-resistant Klebsiella pneumoniae isolates from humans and dogs.

Introduction: Carbapenem and colistin-resistant Enterobacteriaceae, including Klebsiella pneumoniae, have become a growing global concern, posing a significant threat to public health. Currently, there is limited information about the genetic background of carbapenem and colistin-resistant K. pneumoniae isolates infecting humans and dogs in Thailand. This study aimed to characterize carbapenem and colistin-resistant genes in six resistant K. pneumoniae clinical isolates (three from humans and three from dogs) which differed in their pulse field gel electrophoresis profiles. Methods: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), antimicrobial susceptibility testing, and whole-genome sequencing were employed to identify and analyze the isolates. Results and discussion: All six isolates were carbapenemase-producing K. pneumoniae isolates with chromosomally carried blaSHV, fosA, oqxA and oqxB genes, as well as nine to 21 virulence genes. The isolates belonged to five multilocus sequence types (STs): one isolate from a human and one from a dog belonged to ST16, with the other two human isolates being from ST340 and ST1269 and the other two dog isolates were ST147 and ST15. One human isolate and two dog isolates harbored the same blaOXA-232 gene on the ColKP3 plasmid, and one dog isolate carried the blaOXA-48 gene on the IncFII plasmid. Notably, one human isolate exhibited resistance to colistin mediated by the mcr-3.5 gene carried on the IncFII plasmid, which co-existed with resistance determinants to other antibiotics, including aminoglycosides and quinolones. In conclusion, this study provides a comprehensive characterization of both chromosome- and plasmid-mediated carbapenem and colistin resistance in a set of K. pneumoniae clinical isolates from unrelated humans and dogs in Thailand. The similarities and differences found contribute to our understanding of the potential widescale dissemination of these important resistance genes among clinical isolates from humans and animals, which in turn may contribute to outbreaks of emerging resistant clones in hospital settings.

Metagenomic insights into isolable bacterial communities and antimicrobial resistance in airborne dust from pig farms.

This study aims to investigate bacterial communities and antimicrobial resistance (AMR) in airborne dust from pig farms. Airborne dust, pig feces and feed were collected from nine pig farms in Thailand. Airborne dust samples were collected from upwind and downwind (25 meters from pig house), and inside (in the middle of the pig house) of the selected pig house. Pig feces and feed samples were individually collected from the pen floor and feed trough from the same pig house where airborne dust was collected. A direct total bacteria count on each sampling plate was conducted and averaged. The ESKAPE pathogens together with Escherichia coli, Salmonella, and Streptococcus were examined. A total of 163 bacterial isolates were collected and tested for MICs. Pooled bacteria from the inside airborne dust samples were analyzed using Metagenomic Sequencing. The highest bacterial concentration (1.9-11.2 × 103 CFU/m3) was found inside pig houses. Staphylococcus (n = 37) and Enterococcus (n = 36) were most frequent bacterial species. Salmonella (n = 3) were exclusively isolated from feed and feces. Target bacteria showed a variety of resistance phenotypes, and the same bacterial species with the same resistance phenotype were found in airborne dust, feed and fecal from each farm. Metagenomic Sequencing analysis revealed 1,652 bacterial species across all pig farms, of which the predominant bacterial phylum was Bacillota. One hundred fifty-nine AMR genes of 12 different antibiotic classes were identified, with aminoglycoside resistance genes (24%) being the most prevalent. A total of 251 different plasmids were discovered, and the same plasmid was detected in multiple farms. In conclusion, the phenotypic and metagenomic results demonstrated that airborne dust from pig farms contained a diverse array of bacterial species and genes encoding resistance to a range of clinically important antimicrobial agents, indicating the significant role in the spread of AMR bacterial pathogens with potential hazards to human health. Policy measurements to address AMR in airborne dust from livestock farms are mandatory.

Isolation and Genomic Characterization of Schaalia turicensis from a Patient with Gonococcal Urethritis, Thailand.

Schaalia turicensis is facultative anaerobic Gram-positive bacillus that commonly inhabits the oropharynx, gastrointestinal, and genitourinary tract of healthy individuals. This organism has been co-isolated with Neisseria gonorrhoeae from 15-year-old Thai male patient with gonococcal urethritis in Bangkok, Thailand. In this study, we characterized the class 1 integron in S. turicensis isolate using whole-genome sequencing and bioinformatics analysis. Sequencing analysis confirmed the presence of an imperfect class 1 integron located on chromosome and a novel 24.5-kb-long composite transposon, named Tn7083. The transposon Tn7083 carried genes encoding chloramphenicol resistance (cmx), sulfonamide resistance (sul1), and aminoglycoside resistance [aph(6)-Id (strB), aph(3'')-Ib (strA), aph(3')-Ia].

GLIMMERS: glioma molecular markers exploration using long-read sequencing.

Summary: The revised WHO guidelines for classifying and grading brain tumors include several copy number variation (CNV) markers. The turnaround time for detecting CNVs and alterations throughout the entire genome is drastically reduced with the customized read incremental approach on the nanopore platform. However, this approach is challenging for non-bioinformaticians due to the need to use multiple software tools, extract CNV markers and interpret results, which creates barriers due to the time and specialized resources that are necessary. To address this problem and help clinicians classify and grade brain tumors, we developed GLIMMERS: glioma molecular markers exploration using long-read sequencing, an open-access tool that automatically analyzes nanopore-based CNV data and generates simplified reports. Availability and implementation: GLIMMERS is available at https://gitlab.com/silol_public/glimmers under the terms of the MIT license.