Screening of pathogenic microbiota harbouring antibiotic resistance genes from healthcare wastes in Malaysia : a high-throughput amplicon sequencing approach

The disposal of healthcare waste without prior elimination of pathogens and hazardous contaminants has negative effects on the environment and public health. In past research the microbiological assessment of healthcare wastes employed a culture approach that resulted in the identification of Bacillus sp. in a sample of treated solid healthcare wastes. The effectiveness of microwave in hazardous waste treatment studied based on the survival of tested microorganisms using the culture method may overlook the presence of other pathogens after treatment. Yet, there is scarce data reported on the complete microbial community in microwave-treated healthcare waste using next-generation sequencing technology. This study aimed to profile the complete microbial community, identify viable antibiotic-resistant bacteria in microwave-treated healthcare wastes collected from three different waste operators (FC, FV, and FA) in Peninsular Malaysia, and characterize pathogenic gene markers in isolated organisms. The samples were subjected to bacterial and fungal amplicon sequencing for microbial community characterization, by targeting the full-length 16S ribosomal RNA (rRNA) gene and partial 18S rRNA gene with full-length internal transcribed spacer (ITS) 1 and ITS 2 regions, respectively. Bacterial cultivation was performed to identify viable bacteria in healthcare wastes. The isolated antibiotic-resistant bacteria were subjected to species identification and whole genome sequencing for complete genome characterization. In addition, antibiotic susceptibility testing was performed on the confirmed isolates using the disk diffusion technique to determine the antibiotic resistance patterns. Based on the results of objective 1, the bacterial composition in FC samples was dominated by the Aerococcus, Comamonas, and Pseudomonas genera, while FV and FA were dominated by Bacillus, Paenibacillus, and unclassified Bacilli. All three sets of samples showed significant differences in bacterial diversity, as evidenced by the alpha- (p-value = 0.048) and beta-diversity (p-value < 0.006) analyses. The fungal composition differed significantly between three groups of samples, as evidenced by the alpha- (p-value = 0.045) and beta-diversity (p-value < 0.002). The deep bioinformatic analysis confirmed the presence of blaTEM-1 and penP, which are associated with the production of class A beta-lactamase and beta-lactam resistance pathways. Based on objective 2, the viable bacteria in VFC, VFV, and VFA samples were represented by Proteus, Stenotrophomonas, and Pseudomonas genera, respectively, with significant beta diversity (p-value = 0.003). Based on the BLASTN results, the primary antibiotic-resistant bacteria isolated from VFC, VFV, and VFA samples were Proteus mirabilis, Stenotrophomonas maltophilia, and Pseudomonas sp., respectively. As no specific Pseudomonas species were identified from the database, this bacterium is potentially present as a novel bacterium. For objective 3, P. mirabilis and S. maltophilia were discovered to contain genes associated with virulence function and transposable element expression. The antibiotic resistance genes blaOXA-10 and sul1 were identified in P. mirabilis and S. maltophilia, conferring resistance to beta-lactam and folate pathway antagonist antibiotics. The antibiotic susceptibility tests revealed that P. mirabilis and S. maltophilia were multidrug-resistant bacteria, exhibiting resistance to drugs from multiple classes, including carbapenem. In conclusion, microorganisms and contaminants, which serve as putative indicators in healthcare waste treatment evaluation, revealed the limitations of the microwave sterilization method in microbial inactivation. Our findings suggested that the occurrence of clinically relevant microorganisms, antibiotic contaminants, and associated antibiotic resistance genes represents environmental and human health hazards when released into landfills via horizontal gene transfer

Whole genome sequencing analysis of Komagataeibacter nataicola reveals its potential in food waste valorisation for cellulose production

Background: Komagataeibacter nataicola (K. nataicola) is a gram-negative acetic acid bacterium that produces natural bacterial cellulose (BC) as a fermentation product under acidic conditions. The goal of this work was to study the complete genome of K. nataicola and gain insight into the functional genes in K. nataicola that are responsible for BC synthesis in acidic environments. Methods and result; The pure culture of K. nataicola was obtained from yeast-glucose-calcium carbonate (YGC) agar, followed by genomic DNA extraction, and subjected to whole genome sequencing on a Nanopore flongle flow cell. The genome of K. nataicola consists of a 3,767,936 bp chromosome with six contigs and 4,557 protein coding sequences. The maximum likelihood phylogenetic tree and average nucleotide identity analysis confirmed that the bacterial isolate was K. nataicola. The gene annotation via RAST server discovered the presence of cellulose synthase, along with three genes associated with lactate utilization and eight genes involved in lactate fermentation that could potentially contribute to the increase in acid concentration during BC synthesis. Conclusion: A more comprehensive genome study of K. nataicola may shed light into biological pathway in BC productivity as well as benefit the analysis of metabolites generated and understanding of biological and chemical interactions in BC production later

Near-complete whole-genome sequence of Paenibacillus sp. nov. Strain J5C2022, a sucretolerant and endospore-forming bacterium isolated from highly concentrated sugar brine

Here, we present a 7.62-Mbp genome sequence of Paenibacillus sp. nov. strain J5C2022, a Gram-positive facultatively anaerobic bacterium that was isolated from 4-month-old fruit pickle brine and sequenced using the Illumina platform

ITS1 amplicon sequencing of feline gut mycobiome of Malaysian local breeds using Nanopore Flongle

The gut mycobiome exhibits major influence on the gastrointestinal health and disease but received less attention due to low abundance. This study characterizes the fungal community and compares the microbial diversity between indoor and outdoor cats. Genomic DNA was extracted and sequenced by targeting the Internal Transcribed Spacer 1 (ITS1) region using Flongle flow cell on MinION™ sequencing platform. Results show the phylum Ascomycota and genus Peniophorella were numerous in indoor cats, whereas the Basidiomycota and Pichia were abundant in outdoor cats. Peniophorella formed the core mycobiome in both feline populations. Furthermore, alpha (p value = 0.0207) and beta diversities (p value = 0.009) results showed significant differences between the two groups. Overall, indoor cats have greater amounts of Peniophorella, whereas outdoor cats have higher Trichosporon and unclassified Sordariaceae. The study also suggests that keeping a cat indoors or left as a stray will affect their respective gut mycobiome

Evaluation of pre-treated healthcare wastes during COVID-19 pandemic reveals pathogenic microbiota, antibiotics residues, and antibiotic resistance genes against beta-lactams

The disposal of healthcare waste without prior elimination of pathogens and hazardous contaminants has negative effects on the environment and public health. This study aimed to profile the complete microbial community and correlate it with the antibiotic compounds identified in microwave pre-treated healthcare wastes collected from three different waste operators in Peninsular Malaysia. The bacterial and fungal compositions were determined via amplicon sequencing by targeting the full-length 16S rRNA gene and partial 18S with full-length ITS1–ITS2 regions, respectively. The antibiotic compounds were characterized using high-throughput spectrometry. There was significant variation in bacterial and fungal composition in three groups of samples, with alpha- (p-value = 0.04) and beta-diversity (p-values <0.006 and < 0.002), respectively. FC samples were found to acquire more pathogenic microorganisms than FA and FV samples. Paenibacillus and unclassified Bacilli genera were shared among three groups of samples, meanwhile, antibiotic-resistant bacteria Proteus mirabilis, Enterococcus faecium, and Enterococcus faecalis were found in modest quantities. A total of 19 antibiotic compounds were discovered and linked with the microbial abundance detected in the healthcare waste samples. The principal component analysis demonstrated a positive antibiotic-bacteria correlation for genera Pseudomonas, Aerococcus, Comamonas, and Vagococcus, while the other bacteria were negatively linked with antibiotics. Nevertheless, deep bioinformatic analysis confirmed the presence of blaTEM-1 and penP which are associated with the production of class A beta-lactamase and beta-lactam resistance pathways. Microorganisms and contaminants, which serve as putative indicators in healthcare waste treatment evaluation revealed the ineffectiveness of microbial inactivation using the microwave sterilization method. Our findings suggested that the occurrence of clinically relevant microorganisms, antibiotic contaminants, and associated antibiotic resistance genes (ARGs) represent environmental and human health hazards when released into landfills via ARGs transmission

Method for detection of pathogenic bacteria from indoor air microbiome samples using high-throughput amplicon sequencing

The exposure of the air microbiome in indoor air posed a detrimental health effect to the building occupants compared to the outdoor air. Indoor air in hospitals has been identified as a reservoir for various pathogenic microbes. The conventional culture-dependent method has been widely used to access the microbial community in the air. However, it has limited capability in enumerating the complex air microbiome communities, as some of the air microbiomes are uncultivable, slow-growers, and require specific media for cultivation. Here, we utilized a culture-independent method via amplicon sequencing to target the V3 region of 16S rRNA from the pool of total genomic DNA extracted from the dust samples taken from hospital interiors. This method will help occupational health practitioners, researchers, and health authorities to efficiently and comprehensively monitor the presence of harmful air microbiome thus take appropriate action in controlling and minimizing the health risks to the hospital occupants. Key features; • Culture-independent methods offer fast, comprehensive, and unbias profiles of pathogenic and non-pathogenic bacteria from the air microbiomes. • Unlike the culture-dependent method, amplicon sequencing allows bacteria identification to the lowest taxonomy levels

Whole-genome sequence of a stenotrophomonas maltophilia isolate from tap water in an intensive care unit

Here, we present a 4,508,936-bp complete genome sequence of Stenotrophomonas maltophilia strain HW002Y, which was isolated from the tap water in an intensive care unit at Sultan Ahmad Shah Medical Centre at the International Islamic University of Malaysia (Kuantan, Pahang, Malaysia). Sequencing was performed using a Nanopore Flongle flow cell

Complete whole genome sequencing of Vibrio parahaemolyticus strain UMP001VA, isolated from sea cucumber Holothuria leucospilota from Malaysia harbouring blaCARB-33 gene

Pathogenic strain of Vibrio parahaemolyticus found in marine and coastal ecosystems can cause infections in marine animals. Increasing antibiotic resistance reported in Vibrio species may be linked to environmental contamination. Here, we present a 4.99-Mbp complete genome sequence of V. parahaemolyticus strain UMP001VA, a Gram-negative bacterium that was isolated from the gut content of a sea cucumber Holothuria leucospilota collected from Pulau Rhu, Terengganu, Malaysia. The whole genome sequencing was performed using hybrid de novo assembly using Illumina and the Oxford Nanopore Technology platforms. The genome analysis reported a total length of 4,991,208 bp of V. parahaemolyticus genome sequences with 45.3 % GC content. The functional annotation of the genome showcased that 56 genes of V. parahaemolyticus were associated with virulence, disease, and defence function. Virulence factors found in H. leucospilota were related to T3SS1, together with the presence of blaCARB-33 antibiotic resistance gene conferring ampicillin, piperacillin, and amoxicillin drug resistance were reported. The presence of virulence factors and antibiotic resistance genes reveals the considerable pathogenic potential of V. parahaemolyticus in sea cucumbers. Therefore, the whole genome sequencing presented here provides a fundamental genome insight for future assessment of V. parahaemolyticus in marine organisms

Characterization of bacterial communities in prebiotics and probiotics treated shrimp farms from Kuantan

Aims: Prebiotics and probiotics profoundly enhance water quality and shrimp development to tackle infectious disease in shrimp farming. This study evaluated the impact of prebiotics and probiotics treatments in water by assessing the physicochemical properties and bacterial communities in local shrimp ponds. Methodology and results: Water was collected from shrimp pond 1 (SP1), treated with prebiotics and probiotics, and shrimp pond 2 (SP2), treated with only prebiotics. The physicochemical parameters of water from two shrimp ponds were measured, including pH, dissolved oxygen (DO), ammonia concentration and temperature. The total environmental DNA (eDNA) was extracted from the water samples and sequenced using amplicon sequencing targeting the full length of the 16S rRNA gene region via the Oxford Nanopore Technology Flongle. The water quality analysis indicated that SP1 had better water quality than SP2 for shrimp aquaculture. The dominant phyla in both shrimp ponds were Proteobacteria and Bacteroidota. SP1 samples had unique microbiota at the phylum level, including Bdellovibrionota, Firmicutes A, Patescibacteria and unclassified Rhizobiales, Saprospiraceae, Vulcanococcus and HIMB114 at the genus level. The alpha- and beta-diversity showed insignificant differences in microbiota composition between SP1 and SP2 (p-value>0.05). Conclusion, significance and impact of study: Research findings demonstrated that the probiotic-treated shrimp pond (SP1) had better water quality and more diverse microbial communities than the shrimp pond that was not treated with probiotics (SP2)

Characterization of bacterial communities in prebiotics and probiotics treated shrimp farms from Kuantan

Aims: Prebiotics and probiotics profoundly enhance water quality and shrimp development to tackle infectious disease in shrimp farming. This study evaluated the impact of prebiotics and probiotics treatments in water by assessing the physicochemical properties and bacterial communities in local shrimp ponds. Methodology and results: Water was collected from shrimp pond 1 (SP1), treated with prebiotics and probiotics, and shrimp pond 2 (SP2), treated with only prebiotics. The physicochemical parameters of water from two shrimp ponds were measured, including pH, dissolved oxygen (DO), ammonia concentration and temperature. The total environmental DNA (eDNA) was extracted from the water samples and sequenced using amplicon sequencing targeting the full length of the 16S rRNA gene region via the Oxford Nanopore Technology Flongle. The water quality analysis indicated that SP1 had better water quality than SP2 for shrimp aquaculture. The dominant phyla in both shrimp ponds were Proteobacteria and Bacteroidota. SP1 samples had unique microbiota at the phylum level, including Bdellovibrionota, Firmicutes A, Patescibacteria and unclassified Rhizobiales, Saprospiraceae, Vulcanococcus and HIMB114 at the genus level. The alpha- and beta-diversity showed insignificant differences in microbiota composition between SP1 and SP2 (p-value>0.05). Conclusion, significance and impact of study: Research findings demonstrated that the probiotic-treated shrimp pond (SP1) had better water quality and more diverse microbial communities than the shrimp pond that was not treated with probiotics (SP2)