Identification of novel Carnobacterium maltaromaticum strains in bone marrow samples of patients with acute myeloid leukemia using a metagenomic binning approach.

The aim of this study aimed to examine the existence of a bacterial metagenome in the bone marrow of patients with acute myeloid leukemia (AML). We re-examined whole-genome sequencing data from the bone marrow samples of seven patients with AML, four of whom were remitted after treatment, for metagenomic analysis. After the removal of human reads, unmapped reads were used to profile the species-level composition. We used the metagenomic binning approach to confirm whether the identified taxon was a complete genome of known or novel strains. We observed a unique and novel microbial signature in which Carnobacterium maltaromaticum was the most abundant species in five patients with AML or remission. The complete genome of C. maltaromaticum "BMAML_KR01," which was observed in all samples, was 100% complete with 8.5% contamination and closely clustered with C. maltaromaticum strains DSM20730 and SF668 based on single nucleotide polymorphism variations. We identified five unique proteins that could contribute to cancer progression and 104 virulent factor proteins in the BMAML_KR01 genome. To our knowledge, this is the first report of a new strain of C. maltaromaticum in patients with AML. The presence of C. maltaromaticum and its new strain in patients indicates an urgent need to validate the existence of this bacterium and evaluate its pathophysiological role.

Spatial and temporal effects of fish feed on antibiotic resistance in coastal aquaculture farms.

For the first time, both spatial and temporal effects of fish feed on changes in abundance of antibiotic resistance genes (ARGs) were investigated in South Korea via quantifying ARGs and analyzing physicochemical parameters in the influent (IN) and effluent before (BF) and 30 min after (AF) the fish feeding time of sixteen flow-through fish farms. The absolute abundance of ARGs in AF samples was 5 times higher than in BF and 12 times higher than in IN samples. Values of physicochemical parameters such as ammonia, total nitrogen, suspended solids and turbidity in the effluent significantly increased by 21.6, 4.2, 2.6 and 1.65 times, respectively, after fish feeding. Spatially, the fish farms on Jeju Island exhibited higher relative abundance (3.02 × 10-4 - 6.1 × 10-2) of ARGs compared to the farms in nearby Jeollanam-do (3.4 × 10-5 - 8.3 × 10-3). Seasonally, samples in summer and autumn showed a higher abundance of ARGs than in winter and spring. To assess risk to the food chain as well as public health, further studies are warranted to explore the pathogenic potential of these ARGs.

Functional outcome of surgery in patients with acetabular fractures.

BACKGROUND: High velocity injuries due to road traffic accidents can be managed conservatively or surgically depending whether the fracture is displaced or not. The objective of this study was to evaluate functional outcome of surgery in patients of acetabular fractures. METHODS: It was a case series study conducted in Department of Orthopaedic Surgery, Pakistan Institute of Medical Sciences from 1st october 2007 to 15th December 2008. Thirty patients were included in the study fulfilling the inclusion criteria. Patients were followed up in the OPD and were evaluated for functional outcome by using Harris Hip Scoring System. RESULTS: Total 30 patients were operated during the study period. Mean age of patients was 33.5 years. Males were 22 and females were 8. Twenty-three patients had road traffic accident as a cause of injury and 7 were due to fall. Ninety-three percent of patients were operated within 2 weeks. Most common type of fracture was both column fractures. In only one patient sciatic nerve injury was present postoperatively. Average hospital stay was 7 days. Out of 30 patients, 21 patients were classified to have good score, 4 patients were classified to have excellent score while 5 patients were having poor result. CONCLUSION: The surgical management of displaced acetabular fractures yields good results.

Variations in antibiotic resistance genes and microbial community in sludges passing through biological nutrient removal and anaerobic digestion processes in municipal wastewater treatment plants.

Antimicrobial resistance (AMR) represents a relentless, silent pandemic. Contributing to this are wastewater treatment plants (WWTPs), a potential source of antibiotic resistance genes' (ARGs) transmission to the environment, threatening public health. The presence of ARGs in pathogenic bacteria and their release into the environment by WWTPs threatens the public health. The current study investigated changes in ARGs' abundance in biological nutrient removal (BNR) processes and anaerobic digestion (AD) reactors of two WWTPs. Also, microbial community structure, which is known to shape the distribution and abundance of ARGs, was also analyzed. The relative abundance of eight ARGs (tetX, tetA, tetM, TEM, sul1, sul2, ermB and qnrD) was quantified as ARGs' copies/16 S rRNA gene copies using quantitative polymerase chain reaction (qPCR). Microbial community composition was assessed by 16 S rRNA microbiome sequencing analysis. TetX was prevalent among the eight ARGs, followed by TEM and sul1. However, its abundance was decreased in the AD sludges compared to BNR sludges. Proteobacteria was the major bacterial phylum found in all the sludge samples, while Arcobacter, 12up and Acidovorax were the predominant genera. Acinetobacter and Flavobacterium were significantly more abundant in the BNR sludges, while 12up and Aeromonas were predominant in AD sludges. Principal component analysis (PCA) revealed a clear difference in dominant ARGs and bacteria between the sludges in the processes of BNR and AD of the two WWTPs. Clinically relevant bacterial genera, Klebsiella and Enterococcus, found in both the BNR and AD sludges, were significantly correlated with the tetX gene. Throughout this study, the relationship between microbial communities and specific ARGs was revealed, illustrating that the composition of the microbial community could play a vital role in the abundance of ARGs. These results will better inform future studies aimed at controlling the spread of ARGs and their potential hosts from WWTPs.

Mitigation of N2O emissions via enhanced denitrification in a biological landfill leachate treatment using external carbon from fermented sludge.

The effects of an external carbon source (C-source) on the mitigation of N2O gas (N2O(g)) emissions from landfill leachate were investigated via enhanced denitrification using anaerobically fermented sewage sludge. Anaerobic fermentation of sewage sludge was conducted under thermophilic conditions with progressively increasing organic loading rates (OLR). Optimal conditions for fermentation were determined based on the efficiency of hydrolysis and the concentrations of sCOD and volatile fatty acids (VFAs) as follows: at an OLR of 40.48 ± 0.77 g COD/L·d with 1.5 days of solid retention time (SRT), 14.68 ± 0.59% of efficiency of hydrolysis, 14.42 ± 0.30 g sCOD/L and 7.85 ± 0.18 g COD/L of VFAs. Analysis of the microbial community in the anaerobic fermentation reactor revealed that degradation of sewage sludge might be potentially affected by proteolytic microorganisms producing VFAs from proteinaceous materials. Sludge-fermentate (SF) retrieved from the anaerobic fermentation reactor was used as the external C-source for denitrification testing. The specific nitrate removal rate (KNR) of the SF-added condition was 7.54 mg NO3-N/g VSS·hr, which was 5.42 and 2.43 times higher than that of raw landfill leachate (LL) and a methanol-added condition, respectively. In the N2O(g) emission test, the liquid phase N2O (N2O-N(l)) of 20.15 mg N/L was emitted as N2O(g) of 19.64 ppmv under only LL-added condition. On the other hand, SF led to the specific N2O(l) reduction rate (KN2O) of 6.70 mg N/g VSS hr, resulting in mitigation of 1.72 times the N2O(g) emission compared to under the only-LL-added condition. The present study revealed that N2O(g) emissions from biological landfill leachate treatment plants can be attenuated by simultaneous reduction of NO3-N and N2O(l) during enhanced denitrification via a stable supply of an external C-source retrieved from anaerobically fermented organic waste.

Association between gut microbiota and anxiety symptoms: A large population-based study examining sex differences.

BACKGROUND: This study aimed to examine the association between anxiety symptoms and gut microbial composition and to infer their functional pathways. METHODS: In total, 605 participants were included in this study. Participants were categorized into anxious and non-anxious groups according to their Beck Anxiety Inventory scores, and their fecal microbiota was profiled using 16S ribosomal RNA gene sequencing. The microbial diversity and taxonomic profiles of the participants with anxiety symptoms were analyzed using generalized linear models. The function of the gut microbiota was inferred by comparing 16S rRNA data between the anxious and non-anxious groups. RESULTS: The gut microbiome of the anxious group showed lower alpha diversity than that of the non-anxious group, and there were prominent differences in the community structure of the gut microbiota between the two groups. Male participants with anxiety had lower relative abundances of the family Oscillospiraceae, fibrolytic bacteria including those of the family Monoglobaceae, and short-chain fatty acid-producing bacteria such as those of the genus Lachnospiraceae_NK4A136 than those without anxiety symptoms. Female participants with anxiety symptoms had a lower relative abundance of the genus Prevotella than those without anxiety symptoms. LIMITATION: The direction of causality between anxiety symptoms and the gut microbiota was unclear owing to the cross-sectional design of the study. CONCLUSION: Our results elucidate the association between anxiety symptoms and gut microbiota and provide insights for developing interventions to treat anxiety symptoms.

Higher abundance of core antimicrobial resistant genes in effluent from wastewater treatment plants.

Wastewater treatment plants (WWTPs) receive sewage water from a variety of sources, including livestock farms, hospitals, industries, and households, that contain antimicrobial resistant bacteria (ARB) and antimicrobial resistant genes (ARGs). Current treatment technologies are unable to completely remove ARB and ARGs, which are eventually released into the aquatic environment. This study focused on the core resistome of urban WWTPs that are persistent through wastewater treatment processes. We adopted the Hiseq-based metagenomic sequencing approach to identify the core resistome, their genetic context, and pathogenic potential of core ARGs in the influent (IN) and effluent (EF) samples of 12 urban WWTPs in South Korea. In this study, the abundance of ARGs ranged from 0.32 to 3.5 copies of ARGs per copy of the 16S rRNA gene, where the IN samples were relatively higher than the EF samples, especially for the macrolide-lincosamide-streptogramin (MLS)- and tetracycline- resistant genes. On the other hand, there were 43 core ARGs sharing up to 90% of the total, among which the relative abundance of sul1, APH(3'')-lb, and RbpA was higher in EF than in IN (p < 0.05). Moreover, tetracycline and sulfonamide-related core ARGs in both EF and IN were significantly more abundant on plasmids than on chromosomes (p < 0.05). We also found that the majority of core ARGs were carried by opportunistic pathogens such as Acinetobacter baumannii, Enterobacter cloacae, and Pseudomonas aeruginosa in both IN and EF. In addition, phages were the only mobile elements whose abundance correlated with that of core ARGs in EF, suggesting that transduction may play a major role in disseminating ARGs in the receiving water environment of the urban WWTP. The persistent release of core ARGs with pathogenic potential into environmental water is of immediate concern. The mobility of ARGs and ARBs in the environment is a major public health concern. These results should be taken into consideration when developing policy to mitigate environmental dissemination of ARG by WWTPs.

Dynamics of Genotypic and Phenotypic Antibiotic Resistance in a Conventional Wastewater Treatment Plant in 2 Years.

Wastewater treatment plants (WWTPs) are considered a sink and a source of antibiotic resistance. In this study, we applied both culture-dependent and SmartChip-based culture-independent approaches for the investigation of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) at Jungnang (JN), located in the Han River, Seoul, South Korea, for 2 years, i.e., 2017 and 2018. The JN WWTP reduced the diversity and abundance of ARB and ARGs but was not sufficient for removing them all. Interestingly, through the treatment process in the JN WWTP, the composition of diverse multidrug-resistant (MDR) bacteria was concentrated mainly into some genera of the Gammaproteobacteria class (Citrobacter, Escherichia-Shigella, and Stenotrophomonas), which could be key carriages to spread ARGs into the environments. In addition, SmartChip analyses showed that the relative abundance and the number of ARGs were significantly decreased from the influents to the effluents in both 2017 and 2018. SmartChip analyses for 2 years also allowed to notify the core ARGs in the influents and the effluents with the presence of clinically relevant core ARGs, such as vanC, bla OXA , and bla NDM , which persisted in the treatment process. Considering diverse bacterial mechanisms for exchanging and transferring ARGs, the occurrence of MDR bacteria and core ARGs could be a source for the blooming of the antibiotic resistome in the WWTP and nearby environments.

Microbial source tracking using metagenomics and other new technologies.

The environment is under siege from a variety of pollution sources. Fecal pollution is especially harmful as it disperses pathogenic bacteria into waterways. Unraveling origins of mixed sources of fecal bacteria is difficult and microbial source tracking (MST) in complex environments is still a daunting task. Despite the challenges, the need for answers far outweighs the difficulties experienced. Advancements in qPCR and next generation sequencing (NGS) technologies have shifted the traditional culture-based MST approaches towards culture independent technologies, where community-based MST is becoming a method of choice. Metagenomic tools may be useful to overcome some of the limitations of community-based MST methods as they can give deep insight into identifying host specific fecal markers and their association with different environments. Adoption of machine learning (ML) algorithms, along with the metagenomic based MST approaches, will also provide a statistically robust and automated platform. To compliment that, ML-based approaches provide accurate optimization of resources. With the successful application of ML based models in disease prediction, outbreak investigation and medicine prescription, it would be possible that these methods would serve as a better surrogate of traditional MST approaches in future.

Fish farm effluents as a source of antibiotic resistance gene dissemination on Jeju Island, South Korea.

The abuse or misuse of antibiotics is directly linked to the emergence of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs) in the environment. Most fish farms located on Jeju Island operate a flow-through system that pumps in seawater for fish farming and discharges it back to the ocean. To investigate the amount of ARGs that these fish farm effluents discharge into the marine environment, we conducted a metagenomic-based resistome analysis. We observed higher levels of ARGs in fish farm effluents than in seawater at beach and residential areas. A greater proportion of ARGs was found on plasmid rather than on chromosomal DNA, especially for sulfonamide and phenicol classes. The distribution of ARGs did not differ between summer and winter, but the microbial community did. In addition, fish farm samples contained significantly more opportunistic pathogens (i.e., Vibrio, Photobacterium, Aliivibrio, and Tenacibaculum) and virulence factors than non-fish farm samples. Vibrio was the most frequently identified host of ARGs and virulence factors. The presence of Vibrio in the coastal area has been increasing owing to the recent rise in the temperature of seawater. This study suggests the need for actions to treat or monitor ARGs in the coastal areas where fish farms operating a flow-through system are located.

A hybrid DNA sequencing approach is needed to properly link genotype to phenotype in multi-drug resistant bacteria.

Antibiotic resistance genes (ARGs) are now viewed as emerging contaminants posing a potential worldwide human health risk. The degree to which ARGs are transferred to other bacteria via mobile genetic elements (MGEs), including insertion sequences (ISs), plasmids, and phages, has a strong association with their likelihood to function as resistance transfer determinants. Consequently, understanding the structure and function of MGEs is paramount to assessing future health risks associated with ARGs in an environment subjected to strong antibiotic pressure. In this study we used whole genome sequencing, done using MinION and HiSeq platforms, to examine antibiotic resistance determinants among four multidrug resistant bacteria isolated from fish farm effluent in Jeju, South Korea. The combined data was used to ascertain the association between ARGs and MGEs. Hybrid assembly using HiSeq and MinION reads revealed the presence of IncFIB(K) and pVPH2 plasmids, whose sizes were verified using pulsed field gel electrophoresis. Twenty four ARGs and 95 MGEs were identified among the 955 coding sequences annotated on these plasmids. More importantly, 22 of 24 ARGs conferring resistance to various antibiotics were found to be located near MGEs, whereas about a half of the ARGs (11 out of 21) were so in chromosomes. Our results also suggest that the total phenotypic resistance exhibited by the isolates was mainly contributed by these putatively mobilizable ARGs. The study gives genomic insights into the origins of putatively mobilizable ARGs in bacteria subjected to selection pressure.

Metagenomic exploration of antibiotic resistome in treated wastewater effluents and their receiving water.

Environmental dissemination of antimicrobial resistance is a global health problem. Antimicrobial-resistant bacteria and antibiotic-resistant genes (ARGs) are constantly released into the environment through effluents (EFs) from wastewater treatment plants (WWTPs). Thus, requiring a better understanding of the selection and fate of ARGs in wastewater treatment processes. Therefore, we investigated the impacts of urban WWTP EFs on receiving water in the context of their resistomes and mobilomes. We used a HiSeq-based short read metagenomic approach to address the dynamics and diversity of ARGs in WWTP EF as well as the upstream (UP) and downstream (DN) river waters, followed by an investigation of plasmid-mediated ARGs. The abundance of ARGs at each site varied from 7.2 × 10-2 to 7.4 × 10-1 ARG copies per 16S rRNA gene copy, and EF samples showed the highest abundance, followed by DN and UP water samples. ARG diversity ranged from 121 to 686 types per site, and EF had the most diverse ARGs. Commonly identified ARGs in the EF and DN samples were clinically important and were absent in UP samples. The abundance of ARGs, mobile genetic elements (MGEs), and plasmid contigs found only in EF and DN were positively correlated with each other, indicating the importance of mobilomes in the dissemination of ARGs in the environment. Moreover, the proportions of plasmid-mediated ARGs was highest in the EF samples, followed by the DN and UP samples. These findings suggest that WWTP EF may act as a driving factor shaping the resistomes and mobilomes of receiving waters. In particular, a higher abundance of plasmid-mediated ARGs in WWTP EF suggests higher transmissibility in the DN environment.

Comparison of de-novo assembly tools for plasmid metagenome analysis.

BACKGROUND: With the advent of next-generation sequencing techniques, culture-independent metagenome approaches have now made it possible to predict possible presence of genes in the environmental bacteria most of which may be non-cultivable. Short reads obtained from the deep sequencing can be assembled into long contigs some of which include plasmids. Plasmids are the circular double stranded DNA in bacteria and known as one of the major carriers of antibiotic resistance genes. OBJECTIVE: Metagenomic analyses, especially focused on plasmids, could help us predict dissemination mechanisms of antibiotic resistance genes in the environment. However, with the availability of a myriad of metagenomic assemblers, the selection of the most appropriate metagenome assembler for the plasmid metagenome study might be challenging. Therefore, in this study, we compared five open source assemblers to suggest most effective way of plasmid metagenome analysis. METHODS: IDBA-UD, MEGAHIT, SPAdes, SOAPdenovo2, and Velvet are compared for conducting plasmid metagenome analyses using two water samples. RESULTS: Our results clearly showed that abundance and types of antibiotic resistance genes on plasmids varied depending on the selection of assembly tools. IDBA-UD and MEGAHIT demonstrated the overall best assembly statistics with high N50 values with higher portion of longer contigs. CONCLUSION: These two assemblers also detected more diverse plasmids. Among the two, MEGAHIT showed more memory efficient assembly, therefore we suggest that the use of MEGAHIT for plasmid metagenome analysis may offer more diverse plasmids with less computer resource required. Here, we also summarized a fundamental plasmid metagenome work flow, especially for antibiotic resistance gene investigation.

Prevalence and Genetic Relatedness of Extended Spectrum-ß-Lactamase-Producing Escherichia coli Among Humans, Cattle, and Poultry in Pakistan.

Objective: To determine the prevalence and genetic relatedness of blaCTX-M-type extended spectrum-ß-lactamase (ESBL)-producing Escherichia coli at the human-animal interface in Pakistan. Materials and Methods: A total of 150 human, cattle, and poultry fecal samples (50 each) were screened for ESBL-producing E. coli using ESBL CHROMagar®. Bacterial species confirmation as well as determination of minimum inhibitory concentrations (µg/mL) to different antibiotics was performed using the automated VITEK®-2 compact system. Phenotypic confirmation of ESBL production was performed according to the Clinical Laboratory Standards Institute (CLSI) guidelines. Genetic analysis of blaCTX-M was carried out by PCR and DNA sequencing. Plasmids and clonal similarity of the E. coli strains were determined by PCR-based replicon typing and pulsed-field gel electrophoresis (PFGE), respectively. Results: Of 150 samples, 29 (19.3%) ESBL-producing E. coli were recovered, and majority of them originated from human (n = 16; 55%), followed by cattle (n = 9; 31%) and poultry (n = 4; 13.7%). blaCTX-M-15 was predominant ESBL genotype (n = 25; 86.2%), mainly identified from human (n = 15) and cattle (n = 9). This is also the first report of the occurrence of CTX-M-15 and CTX-M-55 in cattle and poultry E. coli isolates of Pakistan, respectively. The majority of the ESBL-producing E. coli (96.5%) showed a multidrug resistance phenotype. All isolates carried IncFII or IncFIA plasmids, and the phylogroup B1 was dominant (44.8%) followed by phylogroups A (31%), D (17.2%), and B2 (6.8%). PFGE revealed that isolates from different hosts were genetically unrelated. Conclusion: Presence of CTX-M-15-type ESBL-producing E. coli in different reservoirs is alarming and has the potential to impact both veterinary and human therapeutic treatment options.

High Prevalence of CTX-M-15-Type ESBL-Producing E. coli from Migratory Avian Species in Pakistan.

The increased presence of clinically relevant multidrug resistant bacteria in natural environments is an emerging challenge for global health care. Little is known regarding the occurrence of extended-spectrum beta-lactamase producing Escherichia coli (ESBL-E. coli) from environmental sentinels in Pakistan. The goal of the current study was to gain insights into the prevalence and phylogenetic relationships of ESBL-E. coli recovered from wild birds in Pakistan during winter migration. After initial screening of fecal samples on selective chromogenic agar, ESBL-E.coli were analyzed phenotypically using the Vitek-2 automated system. Genotypic characterization was performed using whole genome sequencing (WGS) followed by an in-depth in silico analysis. Of 150 birds screened, 26 (17.3%) were fecal carriers of ESBL-E. coli. Of these, 88.4% isolates exhibited multidrug resistance (MDR) phenotypes. Resistance to cefotaxime, ceftazidime, ampicillin, doxycycline, tetracycline and sulfamethoxazole/trimethoprim (CTX-CAZ-AM-DC-TE-SXT) represented the most common pattern of MDR (76.9%). WGS data analysis found blaCTX-M-15 as the predominant ESBL genotype (92.3%). Other genes encoding resistance to sulfonamides (sul1/sul2/sul3), aminoglycosides (strA, strB, aadA1, aadA2, aadA5, aac(3)-IId-like, aac(3)-IVa-like and aph(4)-Ia), trimethoprim (dfrA14 or dfrA17), tetracyclines [tet(A)/tet(B)], and fluoroquinolones (qnrS1) were detected commonly, often encoded on IncF-type plasmids (76.9%). ESBL-E. coli were assigned to 17 different sequence types (STs) of which ST10 and ST7097 (4 isolates each) were the most abundant followed by ST4720, ST93, and ST1139 (2 isolates each). Core-genome phylogeny of the isolates found low numbers (0-29) of single nucleotide polymorphisms (SNPs) in isolates belonged to ST7097 originated from two different locations (Chashma barrage and Rasul barrage). Similar trends were found among isolates belong to ST1139. In addition, WGS-based plasmid typing and S1-digestion found plasmids of the same pMLST type (IncF[F-:A-:B53]) and similar sizes in different bacterial and avian hosts suggesting horizontal gene transfer as another possibility for the spread of ESBL-E. coli in avian wildlife in Pakistan.

First Report of bla CTX-M-15-Type ESBL-Producing Klebsiella pneumoniae in Wild Migratory Birds in Pakistan.

We investigated wild migratory birds faecal swabs for extended-spectrum ß-lactamases-producing Klebsiella pneumoniae (ESBL-K. pneumoniae) from wetland habitats in Pakistan. ESBL-K. pneumoniae were analysed for MDR phenotype, ESBL genotype and genetic diversity. A total of 13 (8.6%) ESBL-K. pneumoniae were recovered. Of these, 8 (61%) isolates were MDR. DNA sequencing confirmed bla CTX-M-15 as the dominant ESBL genotype. BOX-PCR fingerprints showed most of the isolates are unrelated. This study is the first to report the wildlife contamination of CTX-M-15-producing K. pneumoniae in Pakistan. Due to long-range migration, these birds could be responsible for trans-boundary spread of multidrug-resistant bacteria.