Extended-spectrum ß-lactamase-producing bacteria and their resistance determinants in different wastewaters and rivers in Nepal.

Wastewaters serve as significant reservoirs of antibiotic resistant bacteria. Despite the evidence of antimicrobial resistance in wastewaters and river water in Kathmandu, direct linkage between them is not discussed yet. This study investigated the prevalence of extended-spectrum ß-lactamase (ESBL)-producing bacteria and associated resistance genes in wastewaters and river water. Out of 246 bacteria from wastewaters, 57.72% were ESBL producers and 77.64% of them were multidrug resistant (MDR). ESBL producing E. coli was dominant in municipal and hospital wastewaters (HWW) as well as in river water while K. pneumoniae was common in pharmaceutical wastewater. The blaSHV and blaTEM genes were prevalent and commonly co-occurred with aac(6')-Ib-cr in K. pneumoniae isolated pharmaceutical wastewater. blaCTX-M carrying E. coli from hospital co-harbored aac(6')-Ib-cr while that from municipal influent and river water co-harbored qnrS. Whole genome sequencing data revealed the presence of diverse ARGs in bacterial isolates against multiple antibiotics. In average, an E. coli and a K. pneumoniae isolate contained 55.75 ± 0.96 and 40.2 ± 5.36 ARGs, respectively. Multi-locus sequence typing showed the presence of globally high-risk clones with wider host range such as E. coli ST10, and K. pneumoniae ST15 and ST307 in HWW and river indicating frequent dissemination of antimicrobial resistance in wastewater of Kathmandu. Whole genome sequence data aligned with phenotypic antibiograms and resistance genes detected by PCR in selected isolates. The presence of significant plasmid replicons (IncF, IncY) and mobile genetic elements (IS903, IS26) indicate high frequency of spreading antibiotic resistance. These findings indicate burden and dissemination of antimicrobial resistance in the environment and highlight the need for effective strategies to mitigate the antibiotic resistance.

Application of Skimmed-Milk Flocculation Method for Wastewater Surveillance of COVID-19 in Kathmandu, Nepal.

Wastewater surveillance (WS) has been used globally as a complementary tool to monitor the spread of coronavirus disease 2019 (COVID-19) throughout the pandemic. However, a concern about the appropriateness of WS in low- and middle-income countries (LMICs) exists due to low sewer coverage and expensive viral concentration methods. In this study, influent wastewater samples (n = 63) collected from two wastewater treatment plants (WWTPs) of the Kathmandu Valley between March 2021 and February 2022 were concentrated using the economical skimmed-milk flocculation method (SMFM). The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was tested by qPCR using assays that target the nucleocapsid (N) and envelope (E) genes. Overall, 84% (53/63) of the total samples were positive for SARS-CoV-2 according to at least one of the tested assays, with concentrations ranging from 3.5 to 8.3 log10 gene copies/L, indicating the effectiveness of the SMFM. No correlation was observed between the total number of COVID-19 cases and SARS-CoV-2 RNA concentrations in wastewater collected from the two WWTPs (p > 0.05). This finding cautions the prediction of future COVID-19 waves and the estimation of the number of COVID-19 cases based on wastewater concentration in settings with low sewer coverage by WWTPs. Future studies on WS in LMICs are recommended to be conducted by downscaling to sewer drainage, targeting a limited number of houses. Overall, this study supports the notion that SMFM can be an excellent economical virus-concentrating method for WS of COVID-19 in LMICs.

Application of wastewater-based epidemiology for monitoring COVID-19 in hospital and housing wastewaters.

An alternative and complementary diagnostic method of surveillance is provided by wastewater-based surveillance (WBS), particularly in low-income nations like Nepal with scant wastewater treatment facilities and clinical testing infrastructure. In this study, a total of 146 water samples collected from two hospitals (n = 63) and three housing wastewaters (n = 83) from the Kathmandu Valley over the period of March 2021-Febraury 2022 were investigated for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using quantitative reverse transcription TaqMan PCR assays targeting the N and E genes. Of the total, 67 % (98/146) samples were positive for SARS-CoV-2 RNA either by using N- or E-gene assay, with concentrations ranging from 3.6 to 9.1 log10 copies/L. There was a significant difference found between positive ratio (Chi-square test, p < 0.05) and concentration (t-test, p = 0.009) of SARS-CoV-2 RNA detected from hospital wastewater and housing waters. Wastewater data are correlated with COVID-19 active cases, indicating significance in specific areas like the Hospital (APFH) (p < 0.05). According to the application of a bivariate linear regression model (p < 0.05), the concentrations of N gene may be used to predict the COVID-19 cases in the APFH. Remarkably, SARS-CoV-2 RNA was detected prior to, during, and following clinical case surges, implying that wastewater surveillance could serve as an early warning system for public health decisions. The significance of WBS in tracking and managing pandemics is emphasized by this study, especially in resource-constrained settings.

Antibiotic resistance determinants among carbapenemase producing bacteria isolated from wastewaters of Kathmandu, Nepal.

The emergence of carbapenem resistant bacteria (CRB) possesses a remarkable threat to the health of humans. CRB and carbapenem resistance genes (CRGs) have frequently been reported in clinical isolates from hospitals, however, their occurrence and distribution in wastewaters from various sources and river water have not been emphasized in Nepal. So, this study aimed to detect carbapenem resistant bacterial isolates and their resistance determinants in river water and different types of wastewaters. River water and both untreated and treated wastewater samples from hospitals, pharmaceutical industries, and municipal sewage were collected in summer and winter seasons. From 68 grab wastewater samples, CRB were detected only in 16 samples, which included eight hospital wastewater, and four each from untreated municipal sewage and river water. A total of 25 CRB isolates were detected with dominance of E. coli (44.0%) and K. pneumoniae (24.0%). The majority of the isolates harbored blaNDM-1 (76.0%), followed by blaOXA (36.0%) and blaKPC (20.0%) genes. Hospital wastewater majorly contributed to the presence of blaNDM-1, blaKPC, and blaOXA along with intI1 genes compared to river water and untreated municipal sewage, especially during the winter season. However, CRB were not detected in treated effluents of hospitals and municipal sewage, and both influents and effluents from pharmaceutical industries. The combined presence of each blaNDM-1 & blaOXA and blaKPC & blaOXA occurred in 16.0% of the bacterial isolates. The increased minimum inhibitory concentration (MIC) of meropenem was significantly associated with the presence of CRGs. The results of this study highlight the significance of carbapenem resistance in bacteria isolated from wastewater and river water, and underscore the necessity for efficient monitoring and control strategies to prevent the dispersion of carbapenem resistance in the environment and its potential consequences on human health.

Distribution of MecA and Erm Genes among Methicillin-resistant Staphylococcus Aureus with Inducible Resistance to Clindamycin.

BACKGROUND: The emergence of Methicillin-resistant Staphylococcus aureus and its ability to confer cross-resistance to macrolide-lincosamide-streptogramin B has complicated the treatment against it. Gene-based studies among phenotypic methicillin-resistant isolates with inducible resistance to clindamycin are less available in Nepal. This work was undertaken to detect the mecA and erm genes among such phenotypes isolated from clinical samples. METHODS: S. aureus isolated from different clinical samples was identified by standard microbiological procedures (Gram-staining, colony morphology, and different biochemical tests). Methicillin-resistant and inducible resistant to clindamycin phenotypes were detected by using cefoxitin disc (30 µg) and a double disk diffusion test according to the Clinical and Laboratory Standards Institute guidelines and mecA and erm genes were detected by polymerase chain reaction. RESULTS: Among 120 S. aureus isolates, 51.67% (n=62) were MRSA, and the prevalence of inducibly-resistant, constitutively-resistant and Macrolide-Streptogramin phenotypes were 15.83% (n=19), 28.33% (n=34) and 15.83% (n=19) respectively. While 35.84% (n=43) of isolates showed sensitivity to both antibiotics, erythromycin and clindamycin. Out of 14 inducibly-resistant phenotypes, 57.14% (n=8) were found carrying ermC and 28.57% (n=4) phenotypes contained both ermA and ermC. All phenotypes were positive for the mecA gene. CONCLUSIONS: Macrolides-Lincosamide-Streptogramin B resistance was predominant among methicillin-resistant S. aureus. While all isolates with inducible clindamycin resistance harbored mecA gene, most of them also harbored ermC gene. The higher prevalence of inducible-resistant to clindamycin indicated the need for rational use of antimicrobial agents.

Extended-spectrum ß-lactamases producing Enterobacteriaceae (ESBL-PE) prevalence in Nepal: A systematic review and meta-analysis.

An alarming increase in the occurrence of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE) has threatened the treatment and management of bacterial infections. This systematic review and meta-analysis aimed to provide a quantitative estimate of the prevalence of ESBL among the members of the Enterobacteriaceae family by analyzing the community-based and clinical studies published between 2011 and 2021 from Nepal and determine if ESBL-PE correlates with multidrug resistance (MDR). The Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines were followed for systematic review and meta-analysis and the articles' quality was assessed using the Newcastle-Ottawa scale. Of the 2529 articles screened, 65 articles were systematically reviewed, data extracted, and included in in-depth meta-analysis. The overall pooled prevalence of ESBL-producers in Enterobacteriaceae was 29 % (95 % CI: 26-32 %) with high heterogeneity (I2 = 96 %, p < 0.001). Escherichia coli was the predominant ESBL-producing member of the Enterobacteriaceae family, followed by Citrobacter spp. and Klebsiella spp. The prevalence of ESBL-PE increased from 18.7 % in 2011 to 29.5 % in 2021. A strong positive correlation (r = 0.98) was observed between ESBL production and MDR in Enterobacteriaceae. ESBL-PE isolates showed high resistance to ampicillin, cephalosporins, and amoxicillin-clavulanic acid, and blaCTX-M type was the most reported gene variant among ESBL-PE. In conclusion, this study demonstrated an increased prevalence of ESBL-PE in Nepal over the last decade, and such isolates showed a high level of MDR against the ß-lactams and non-ß-lactam antibiotics. Tackling the rising antibiotic resistance (AR) and MDR in ESBL-PE would require concerted efforts from all stakeholders to institute effective infection control programs in the community and clinical settings.

High efficient removal of 4-aminophenylarsonic acid from aqueous solution via enhanced FeOOH using Mn(VII).

Efficient removal of 4-aminophenylarsonic acid from contaminated water sources is essential to mitigate arsenic pollution. We proposed a competent technique to achieve 4-aminophenylarsonic acid removal via adsorption on enhanced α-FeOOH using various concentrations of Mn(VII). The elimination rate of 4-aminophenylarsonic acid applying FeOOH with Mn(VII) was dependent on acidic conditions. More than 99.9% of 4-aminophenylarsonic acid was eliminated in a 6-min reaction time under acidic conditions. The reaction of 4-aminophenylarsonic acid was fast at 4.0 and 5.0 pH, with its complete oxidation into arsenate and the liberation of manganese Mn(II) in the initial stage of the reaction. Similarly, the reaction rate constant (kobs) decreased from 0.7048 ± 0.02 to 0.00155 ± 0.00007 as the pH increased from 4.0 to 9.0. Oxidation capacity was considerably enhanced via the removal of electrons from 4-aminophenylarsonic acid to Mn(VII) after the creation of its radical intermediate and further change in Mn(III) to Mn(II) in the solution. The results showed that Mn(VII) played a crucial role in 4-aminophenylarsonic acid degradation at a low pH (e.g., 4.0), and the oxidation process proceeded in different manners, namely, electron transfer, hydroxylation, and ring-opening. These results illustrated that Mn(VII) is an effective, economic purification process to mitigate 4-aminophenylarsonic acid generated from poultry waste.

Antibiotic Resistance, Biofilm Formation and Sub-Inhibitory Hydrogen Peroxide Stimulation in Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the most prevalent cause of urinary tract infections (UTIs). Biofilm formation and antibiotic resistance could be high among the causative agent. The purpose of this study was to determine antibiotic resistance, biofilm production, and biofilm-associated genes, bcsA and csgD, and sub-inhibitory hydrogen peroxide (H2O2) stimulation in UPEC for biofilm formation. A total of 71 UPEC were collected from a tertiary care hospital in Kathmandu and subjected to identify antibiotic susceptibility using Kirby-Bauer disk diffusion. The biofilm formation was assessed using microtiter culture plate method while pellicle formation was tested by a tube method. In representative 15 isolates based on biofilm-forming ability, bcsA and csgD were screened by conventional polymerase chain reaction, and treated with sub-lethal H2O2. The UPEC were found the most susceptible to meropenem (90.2%), and the least to ampicillin (11.3%) in vitro and 90.1% of them were multi-drug resistant (MDR). Most UPEC harbored biofilm-producing ability (97.2%), and could form pellicle at 37°C. Among representative 15 isolates, csgD was detected only among 10 isolates (66.67%) while bcsA gene was present in 13 isolates (86.67%). This study revealed that level of biofilm production elevated after sub-lethal H2O2 treatment (P = .041). These findings suggested that the pathogens are emerging as MDR. The biofilm production is high and the majority of selected strains contained bcsA and csgD genes. Pellicle formation test was suggestive to be an alternative qualitative method to screen biofilm production in UPEC. The sub-inhibitory concentration of H2O2 may contribute in increasing biofilm formation in UPEC.

Seasonal taxonomic composition of microbial communal shaping the bioaerosols milieu of the urban city of Lanzhou.

Here, the taxonomical composition and seasonal dynamics of airborne microbial communities were described in the urban city of Lanzhou, Northwest China. Year-long samples were studied in two filter membranes (Quartz and PTFE). Higher microbial loads were reported in the PTFE than in the quartz filter. Onefold decrease was reported in bacterial loads in spring and summer than winter and autumn for both filters. The fungal loadings were lowest during winter and highest during autumn, followed by summer. The microbial communities included Actinobacteria and Proteobacteria, Ascomycota, and Basidiomycota as major components. Maximum abundance of the members from Gammaproteobacteria, Coriobacteria and Clostridia were studied in all seasons on PTFE membrane, followed by, Erysipelotrichia, Negativicutes and Fusobacteria. Members of Actinobacteria and Bacilli showed higher abundance in spring and winter, with a small proportion during autumn. Members of Clostridia, Gammaproteobacteria, Bacilli, and Actinobacteria showed maximum abundance on the quartz filter in all the seasons. Similarly, on the PTFE, fungi including Dothideomycetes and Agaricomycetes were dominant, followed by Saccharomycetes during summer and winter. The result showed that PM2.5, SO42-, NO2-, Na+, EC, and OC are important environmental parameters influencing the seasonal microbial community. However, the relation of the microbiome with the environment cannot be confidently defined because the environmental factors are changeable and yet interrelated.

Detection of SARS-CoV-2 RNA in wastewater, river water, and hospital wastewater of Nepal.

The applicability of wastewater-based epidemiology (WBE) has been extensively studied throughout the world with remarkable findings. This study reports the presence and reduction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at two wastewater treatment plants (WWTPs) of Nepal, along with river water, hospital wastewater (HWW), and wastewater from sewer lines collected between July 2020 and February 2021. SARS-CoV-2 RNA was detected in 50%, 54%, 100%, and 100% of water samples from WWTPs, river hospitals, and sewer lines, respectively, by at least one of four quantitative PCR assays tested (CDC-N1, CDC-N2, NIID_2019-nCOV_N, and N_Sarbeco). The CDC-N2 assay detected SARS-CoV-2 RNA in the highest number of raw influent samples of both WWTPs. The highest concentration was observed for an influent sample of WWTP A (5.5 ± 1.0 log10 genome copies/L) by the N_Sarbeco assay. SARS-CoV-2 was detected in 47% (16/34) of the total treated effluents of WWTPs, indicating that biological treatments installed at the tested WWTPs are not enough to eliminate SARS-CoV-2 RNA. One influent sample was positive for N501Y mutation using the mutation-specific qPCR, highlighting a need for further typing of water samples to detect Variants of Concern. Furthermore, crAssphage-normalized SARS-CoV-2 RNA concentrations in raw wastewater did not show any significant association with the number of new coronavirus disease 2019 (COVID-19) cases in the whole district where the WWTPs were located, suggesting a need for further studies focusing on suitability of viral as well as biochemical markers as a population normalizing factor. Detection of SARS-CoV-2 RNA before, after, and during the peaking in number of COVID-19 cases suggests that WBE is a useful tool for COVID-19 case estimation in developing countries.

Methicillin-resistant Staphylococcus aureus nasal colonization in people living with HIV and healthy people in Kathmandu, Nepal.

AIM: This study aimed to compare methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization in people living with HIV (PLHIV) and healthy people from Kathmandu. METHODS: MRSA isolated from 400 nasal swabs was screened using a cefoxitin disc and confirmed by the presence of the mecA gene. RESULTS: MRSA nasal carriers among the PLHIV and control cohorts were 3.5% (7 out of 200) and 5.0% (10 out of 200), respectively. All the MRSA from PLHIV and most of MRSA from healthy controls were PVL positive. Longer duration of antiretroviral therapy significantly reduces the risk of MRSA nasal colonization in PLHIV. CONCLUSION: There is no significant difference in MRSA nasal colonization in PLHIV and healthy controls in this study region.

Detection of SARS-CoV-2 and Other Viruses in Wastewater: Optimization and Automation of an Aluminum Hydroxide Adsorption-Precipitation Method for Virus Concentration.

This study aimed to provide a low-cost technique for virus detection in wastewater by improving an aluminum hydroxide adsorption-precipitation method. The releasing efficiency of viruses trapped by the aluminum hydroxide precipitates was improved by adding ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) to dissolve the precipitates at a Na2EDTA·2H2O:AlCl3 molar ratio of 1.8-3.6. The recovery rates of the improved method for seven viruses, including SARS-CoV-2-abEN pseudovirus and six animal viruses, were 5.9-22.3% in tap water and 4.9-35.1% in wastewater. Rotavirus A (9.0-4.5 × 103 copies/mL), porcine circovirus type 2 (5.8-6.4 × 105 copies/mL), and porcine parvovirus (5.6-2.7 × 104 copies/mL) were detected in China's pig farm wastewater, while rotavirus A (2.0 × 103 copies/mL) was detected in hospital wastewater. SARS-CoV-2 was detected in hospital wastewater (8.4 × 102 to 1.4 × 104 copies/mL), sewage (6.4 × 10 to 2.3 × 103 copies/mL), and river water (6.6 × 10 to 9.3 × 10 copies/mL) in Nepal. The method was automized, with a rate of recovery of 4.8 ± 1.4% at a virus concentration of 102 copies/mL. Thus, the established method could be used for wastewater-based epidemiology with sufficient sensitivity in coping with the COVID-19 epidemic and other virus epidemics.

Detection of Plasmid-Mediated Colistin Resistant mcr-1 Gene in Escherichia coli Isolated from Infected Chicken Livers in Nepal.

Background: Plasmid-mediated resistance to the colistin in poultry is considered as an emerging problem worldwide. While poultry constitutes the major industry in Nepal, there is a paucity of evidence on colistin resistance in Escherichia coli isolates causing natural infections in poultry. This study aimed to explore the prevalence of plasmid-mediated colistin resistance gene, mcr-1 in E. coli isolated from liver samples of dead poultry suspected of E. coli infections. Methods: A total of two hundred and seventy liver samples (227 broilers and 43 layers) from dead poultry suspected of colibacillosis were collected from post-mortem in the Central Veterinary Laboratory (CVL), Kathmandu, between 1 February and 31 July 2019. The specimens were processed to isolate and identify E. coli; an antimicrobial susceptibility test (AST) using disk diffusion method was performed with 12 different antibiotics: Amikacin (30 µg), ampicillin (10 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), cefoxitin (30 µg), ceftazidime (30 µg), ceftriaxone (30 µg), cotrimoxazole (25 µg), gentamicin (10 µg), imipenem (10 µg), levofloxacin (5 µg) and tetracycline (30 µg). Colistin resistance was determined by agar dilution method and colistin-resistant strains were further screened for plasmid-mediated mcr-1 gene, using conventional polymerase chain reaction (PCR). Results: Out of 270 liver samples, 53.3% (144/270) showed growth of E. coli. The highest number (54%; 109/202) of E. coli isolates was obtained in the liver samples from poultry birds (of both types) aged less than forty days. In AST, 95.1% (137/144) and 82.6% (119/144) of E. coli isolates were resistant against tetracycline and ciprofloxacin, respectively, while 13.2% (19/144) and 25.7% (37/144) isolates were resistant to cefoxitin and imipenem, respectively. In the same assay, 76.4% (110/144) E. coli isolates were multi-drug resistant (MDR). The phenotypic prevalence of colistin resistance was 28.5% (41/144). In the PCR assay, 43.9% (18/41) of colistin-resistant isolates were screened positive for plasmid-mediated mcr-1. Conclusion: The high prevalence of mcr-1 in colistin-resistant E. coli isolates in our study is a cause of concern for the probable coming emergence of colistin resistance in human pathogens, due to horizontal transfer of resistant genes from poultry to human isolates.

Transcriptional and metabolic response against hydroxyethane-(1,1-bisphosphonic acid) on bacterial denitrification by a halophilic Pannonibacter sp. strain DN.

Biological denitrification is an environmentally sound pathway for the elimination of nitrogen pollution in wastewater treatment. Extreme environmental conditions, such as the co-existence of toxic organic pollutants, can affect biological denitrification. However, the potential underlying mechanism remains largely unexplored. Herein, the effect of a model pollutant, hydroxyethane-(1,1-bisphosphonic acid) (HEDP), a widely applied and consumed bisphosphonate, on microbial denitrification was investigated by exploring the metabolic and transcriptional responses of an isolated denitrifier, Pannonibacter sp. strain DN. Results showed that nitrate removal efficiency decreased from 85% to 50% with an increase in HEDP concentration from 0 to 3.5 mM, leading to nitrite accumulation of 204 mg L-1 in 3.5 mM HEDP. This result was due to the lower bacterial population count and reduction in the live cell percentage. Further investigation revealed that HEDP caused a decrease in membrane potential from 0.080 ±â€¯0.005 to 0.020 ±â€¯0.002 with the increase in HEDP from 0 to 3.5 mM. This hindered electron transfer, which is required for nitrate transformation into nitrogen gas. Moreover, transcriptional profiling indicated that HEDP enhanced the genes involved in ROS (O2-) scavenging, thus protecting cells against oxidative stress damage. However, the suppression of genes responsible for the production of NADH/FADH2 in tricarboxylic acid cycle (TCA), NADH catalyzation (NADH dehydrogenase) in (electron transport chain) ETC system and denitrifying genes, especially nor and nir, in response to 2.5 mM HEDP were identified as the key factor inhibiting transfer of electron from TCA cycle to denitrifying enzymes through ETC system.

Asymptomatic SARS-CoV-2 Carriers: A Systematic Review and Meta-Analysis.

Asymptomatic cases of SARS-CoV-2 can be unknown carriers magnifying the transmission of COVID-19. This study appraised the frequency of asymptomatic individuals and estimated occurrence by age group and gender by reviewing the existing published data on asymptomatic people with COVID-19. Three electronic databases, PubMed, Embase, and Web of Science (WoS), were used to search the literature following the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). The study population for this review included asymptomatic individuals infected with SARS-CoV-2 reported in original articles published up to 30 April 2020. A random effects model was applied to analyze pooled data on the prevalence of asymptomatic cases among all COVID-19 patients and also by age and gender. From the meta-analysis of 16 studies, comprising 2,788 SARS-CoV-2 infected patients, the pooled prevalence according to the random effect size of asymptomatic cases was 48.2% (95% CI, 30-67%). Of the asymptomatic cases, 55.5% (95% CI, 43.6-66.8%) were female and 49.6% (95% CI, 20.5-79.1%) were children. Children and females were more likely to present as asymptomatic COVID-19 cases and could act as unknown carriers of SARS-CoV-2. Symptom-based screening might fail to identify all SARS-CoV-2 infections escalating the threat of global spread and impeding containment. Therefore, a mass surveillance system to track asymptomatic cases is critical, with special attention to females and children.

Characteristics of microbial community functional structure of a biological coking wastewater treatment system.

Nitrogenous heterocyclic compounds are key pollutants in coking wastewater; however, the functional potential of microbial communities for biodegradation of such contaminants during biological treatment is still elusive. Herein, a high throughput functional gene array (GeoChip 5.0) in combination with Illumina HiSeq2500 sequencing was used to compare and characterize the microbial community functional structure in a long run (500days) bench scale bioreactor treating coking wastewater, with a control system treating synthetic wastewater. Despite the inhibitory toxic pollutants, GeoChip 5.0 detected almost all key functional gene (average 61,940 genes) categories in the coking wastewater sludge. With higher abundance, aromatic ring cleavage dioxygenase genes including multi ring1,2diox; one ring2,3diox; catechol represented significant functional potential for degradation of aromatic pollutants which was further confirmed by Illumina HiSeq2500 analysis results. Response ratio analysis revealed that three nitrogenous compound degrading genes- nbzA (nitro-aromatics), tdnB (aniline), and scnABC (thiocyanate) were unique for coking wastewater treatment, which might be strong cause to increase ammonia level during the aerobic process. Additionally, HiSeq2500 elucidated carbozole and isoquinoline degradation genes in the system. These findings expanded our understanding on functional potential of microbial communities to remove organic nitrogenous pollutants; hence it will be useful in optimization strategies for biological treatment of coking wastewater.

Biotransformation of nitrogen- and sulfur-containing pollutants during coking wastewater treatment: Correspondence of performance to microbial community functional structure.

Although coking wastewater is generally considered to contain high concentration of nitrogen- and sulfur-containing pollutants, the biotransformation processes of these compounds have not been well understood. Herein, a high throughput functional gene array (GeoChip 5.0) in combination with Illumina MiSeq sequencing of the 16S rRNA gene were used to identify microbial functional traits and their role in biotransformation of nitrogen- and sulfur-containing compounds in a bench-scale aerobic coking wastewater treatment system operated for 488 days. Biotransformation of nitrogen and sulfur-containing pollutants deteriorated when pH of the bioreactor was increased to >8.0, and the microbial community functional structure was significantly associated with pH (Mantels test, P < 0.05). The release of ammonia nitrogen and sulfate was correlated with both the taxonomic and functional microbial community structure (P < 0.05). Considering the abundance and correlation with the release of ammonia nitrogen and sulfate, aromatic dioxygenases (e.g. xylXY, nagG), nitrilases (e.g. nhh, nitrilase), dibenzothiophene oxidase (DbtAc), and thiocyanate hydrolase (scnABC) were important functional genes for biotransformation of nitrogen- and sulfur-containing pollutants. Functional characterization of taxa and network analysis suggested that Burkholderiales, Actinomycetales, Rhizobiales, Pseudomonadales, and Hydrogenophiliales (Thiobacillus) were key functional taxa. Variance partitioning analysis showed that pH and influent ammonia nitrogen jointly explained 25.9% and 35.5% of variation in organic pollutant degrading genes and microbial community structure, respectively. This study revealed a linkage between microbial community functional structure and the likely biotransformation of nitrogen- and sulfur-containing pollutants, along with a suitable range of pH (7.0-7.5) for stability of the biological system treating coking wastewater.

Performance and microbial community composition in a long-term sequential anaerobic-aerobic bioreactor operation treating coking wastewater.

The combined anaerobic-aerobic biosystem is assumed to consume less energy for the treatment of high strength industrial wastewater. In this study, pollutant removal performance and microbial diversity were assessed in a long-term (over 300 days) bench-scale sequential anaerobic-aerobic bioreactor treating coking wastewater. Anaerobic treatment removed one third of the chemical oxygen demand (COD) and more than half of the phenols with hydraulic retention time (HRT) of 42 h, while the combined system with total HRT of 114 h removed 81.8, 85.6, 99.9, 98.2, and 85.4 % of COD, total organic carbon (TOC), total phenols, thiocyanate, and cyanide, respectively. Two-dimensional gas chromatography with time-of-flight mass spectrometry showed complete removal of phenol derivatives and nitrogenous heterocyclic compounds (NHCs) via the combined system, with the anaerobic process alone contributing 58.4 and 58.6 % removal on average, respectively. Microbial activity in the bioreactors was examined by 454 pyrosequencing of the bacterial, archaeal, and fungal communities. Proteobacteria (61.2-93.4 %), particularly Betaproteobacteria (34.4-70.1 %), was the dominant bacterial group. Ottowia (14.1-46.7 %), Soehngenia (3.0-8.2 %), and Corynebacterium (0.9-12.0 %), which are comprised of phenol-degrading and hydrolytic bacteria, were the most abundant genera in the anaerobic sludge, whereas Thiobacillus (6.6-43.6 %), Diaphorobacter (5.1-13.0 %), and Comamonas (0.2-11.1 %) were the major degraders of phenol, thiocyanate, and NHCs in the aerobic sludge. Despite the low density of fungi, phenol degrading oleaginous yeast Trichosporon was abundant in the aerobic sludge. This study demonstrated the feasibility and optimization of less energy intensive treatment and the potential association between abundant bacterial groups and biodegradation of key pollutants in coking wastewater.

ß -Lactamase-Producing Multidrug-Resistant Bacterial Pathogens from Tracheal Aspirates of Intensive Care Unit Patients at National Institute of Neurological and Allied Sciences, Nepal.

The widespread use of tracheal intubation and mechanical ventilation to support the critically ill patients increases the risk of development of tracheobronchitis and bronchopneumonia. This cross-sectional study was conducted with an aim to isolate and identify bacterial pathogens from tracheal aspirates producing extended-spectrum ß -lactamase (ESBL), AmpC ß -lactamase, and metallo- ß -lactamase (MBL) from August 2011 to April 2012 at National Institute of Neurological and Allied Sciences (NINAS), Kathmandu, Nepal. ESBL was detected by combined disk assay using cefotaxime and cefotaxime with clavulanate, AmpC ß -lactamase by inhibitor-based method using cefoxitin and phenylboronic acid, and MBL by Imipenem-EDTA combined disk method. 167 bacterial strains were isolated from 187 samples and majority of them were Acinetobacter spp. followed by Klebsiella pneumoniae with 32.9% and 25.1%, respectively. 68.8% of isolates were multidrug resistant (MDR) and Acinetobacter spp. constituted 85.4%. ESBL, AmpC ß -lactamase, and MBL were detected in 35 (25%), 51 (37.2%), and 11 (36.7%) isolates, respectively. Pseudomonas spp. (42.8%) were the predominant ESBL producer while Acinetobacter spp. were the major AmpC ß -lactamase producer (43.1%) and MBL producer (54.5%).