Genomic and functional characterization of carbapenem-resistant Klebsiella pneumoniae from hospital wastewater.

BACKGROUND: The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) attracted extensive attention. Information on CRKP from hospital wastewater (HWW) is limited. The aims of this study were to investigate the genomic characteristics and to evaluate the survivability characteristics of 11 CRKP from HWW in a Chinese teaching hospital in Fujian province. RESULTS: A total of 11 CRKP from HWW were recovered in this study. All CRKP from HWW were resistant to most antibiotics. Comparative genetic analysis demonstrated that all CRKP isolates were clustered into the three distinct phylogenetic clades and clade 2 and clade 3 were mixtures of samples collected from both HWW and clinical settings. Varieties of resistance genes, virulence genes and plasmid replicon types were detected in CRKP from HWW. In vitro transfer of blaKPC-2 was successful for 3 blaKPC-2-positive CRKP from HWW with high conjugation frequency. Our study demonstrated that the genetic environments of blaKPC-2 shared core structure with ISKpn27-blaKPC-2-ISKpn6. Group analysis showed that CRKP from HWW had a lower survivability in serum compared to clinical CRKP (p < 005); and CRKP from HWW had no significant difference in survivability in HWW compared to clinical CRKP (p > 005). CONCLUSIONS: We analyzed the genomic and survivability characteristics of CRKP from HWW in a Chinese teaching hospital. These genomes represent a significant addition of genomic data from the genus and could serve as a valuable resource for future genomic studies about CRKP from HWW.

Profile and actual transmissibility of Carbapenem resistance genes: Intracellular and extracellular DNA in hospital wastewater.

The current worldwide spread of carbapenem resistance genes (CRGs) has posed a major public health threat, which continues to grow in severity. Hospital wastewaters (HWWs) are major reservoirs for antibiotic resistance genes, while resistomes in HWWs are still poorly characterized when it comes to CRGs. We comprehensively characterized the profile and actual transmissibility of extracellular CRGs (eCRGs) and intracellular CRGs (iCRGs) in HWWs for the first time. In this study, CRGs showed similar relative abundance in treated and untreated HWWs. Meanwhile, HWWs treatments led to the enrichment of blaIMP-8, probably attributed to the promotion of Novosphingobium and Prosthecobacter after treatment. To evaluate the transmission potential of CRGs, extracellular and intracellular carbapenem-resistant plasmids were captured from HWWs by transformation and conjugation, respectively. We found an interesting phenomenon regarding the transmission characteristics of CRGs: blaKPC-carrying plasmids could only be captured by transformation, while blaNDM-carrying plasmids were captured by conjugation. Further experiments showed that HWW treatments increased the conjugation ability of blaNDM. In conclusion, our study demonstrated that HWWs are significant reservoirs of CRGs and various CRGs exhibit different modes of transmission in HWWs. CRGs cannot be removed by membrane bioreactor and chlorine disinfection. An urgent need is to develop more efficient wastewater treatments to limit CRG dissemination.

YouTube™ as a source of information for Candida auris infection: a systematic review.

BACKGROUND: Candida auris is a novel Candida species, and has emerged globally as a multidrug-resistant health care-associated fungal pathogen. YouTube™ (http://www.youtube.com) as the largest free video-sharing website is increasingly used to search health information. Thus, the aim of this study was to evaluate the content, reliability and quality of YouTube™ videos regarding Candida auris infection, and to identify whether it is a useful resource for people. METHODS: The YouTube™ was used to search systematically for videos using the keywords: "Candida auris infection" and "Candida auris". Strict inclusion and exclusion criteria were used to select the videos. The videos were reviewed and scored by two independent reviewers and recorded the "title", "length", "views", "comments", "dislike", "like", "posted days" and "category of videos". The videos were categorized as "poor", "good" and "excellent" by the score. The DISCERN tool was used to assess the reliability of the YouTube™ videos. RESULTS: Seventy-six videos were included in final analysis in our study. Most videos (59.2%, 55/76) had better quality. There were no statistically significant differences between groups in respect of the number of likes, dislikes, views, comments, percentage positivity, likebility, view rate and viewers' interaction. Length and posted days were significantly associated with the classification. The videos were categorized as "educational video", "new report", "personal experience and blog entertainment" and "interview". Significant differences were found in the source of videos and the characteristics of the individuals appearing in a video between the groups. CONCLUSION: YouTube™ has striking potential to be an effective user-friendly learning interface for people to obtain information of Candida auris infection.

Molecular Epidemiological Characteristics of Carbapenem Resistant Aeromonas from Hospital Wastewater.

Background: Hospital wastewater (HWW) promotes the spread of carbapenem resistance genes (CRGs). Aeromonas carry a large number of CRGs in HWW, they may play a role as a suitable reservoir for CRGs, while resistomes in HWW are still poorly characterized regarding carbapenem resistant Aeromonas. Thus, the aim of the study was to evaluate the molecular epidemiological characteristics of carbapenem resistant Aeromonas in HWW. Methods: A total of 33 carbapenem resistant Aeromonas were isolated from HWW. Antimicrobial susceptibility testing and polymerase chain reaction (PCR) were used to assess the antimicrobial resistance profiles. Molecular typing was performed using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and multilocus sequence typing (MLST). The horizontal transmission mode of bla KPC was explored through conjugation and transformation experiments. The stability of bla KPC-IncP-6 plasmids was assessed through plasmid stability and in vitro competition test. The PCR mapping method was used to investigate the structural diversity of bla KPC. Results: The detection rates of bla KPC and cphA in Aeromonas were 97.0% and 39.4% respectively. Aeromonas caviae were grouped into 13 clusters by ERIC-PCR and 12 STs by MLST. Aeromonas veronii were grouped into 11 clusters by ERIC-PCR and 4 STs by MLST. 56.3% bla KPC were located on mobilizable IncP-6 plasmids. bla KPC-IncP-6 plasmid showed high stability and low cost fitness. Conclusion: Carbapenem resistant Aeromonas from HWW mainly carried bla KPC, which exhibited great structural diversity. Aeromonas might serve as reservoirs for bla KPC and bla KPC might spread mainly through transformation in HWW.

Epidemic Potential of Escherichia coli O16:H41-ST131: Compared with Pandemic O25b:H30-ST131 Lineage.

BACKGROUND: O16:H41 is an important subclone among Escherichia coli (E. coli) sequence type (ST) 131, which has risen dramatically in recent years. However, reasons for the rapid increase of E. coli O16:H41-ST131 remain unclear. The aim of this study was to compare the pathogenicity and survivability features of E. coli O16:H41-ST131 with global epidemic O25b:H30-ST131 lineage. METHODS: Sixteen E. coli ST131 were divided into two groups: group O16:H41-ST131 (n=6) and group O25b:H30-ST131 (n=10). Adhesion and invasion activity of different isolates were measured using human T24 cells. Biofilm production was quantified by crystal violet staining. Fifty percent human serum was used to detect serum sensitivity. Resistance to hydrogen peroxide was detected by broth microdilution method, and anti-phagocytic function was determined by phagocytosis experiments. RESULTS: E. coli O16:H41-ST131 and O25b:H30-ST131 lineage showed similar biofilm formation, adhesion and invasion abilities. In terms of survivability, resistance to serum and hydrogen peroxide of E. coli O16:H41-ST131 was similar as that of E. coli O25b:H30-ST131. But anti-phagocytic function of E. coli O16:H41-ST131 was significantly weaker than that of E. coli O25b:H30-ST131. CONCLUSION: The pathogenicity and survivability of E. coli O16:H41-ST131 were similar to those of E. coli O25b:H30-ST131, which may be important reasons for its increasing prevalence. Our study may contribute to a better understanding of the prevalence of E. coli O16:H41-ST131.

Acquisition of a Stable and Transferable blaNDM-5-Positive Plasmid With Low Fitness Cost Leading to Ceftazidime/Avibactam Resistance in KPC-2-Producing Klebsiella pneumoniae During Treatment.

The emergence and prevalence of carbapenem-resistant Enterobacteriaceae (CRE) have drawn worldwide attention. Ceftazidime/avibactam (CAZ/AVI) gives us a valuable alternative strategy to treat CRE infections. Unfortunately, CAZ/AVI resistance could occur during CAZ/AVI treatment. The CAZ/AVI-resistant Carbapenem-resistant Klebsiella pneumoniae (CR-KP) (KP137060) and earlier CAZ/AVI-susceptible isolate (KP135194) from the same hospitalized patient were collected at Fujian Medical University Union Hospital between October and November 2019. In this study, CAZ/AVI MICs of CAZ/AVI-susceptible and -resistant isolates (KP135194 and KP137060) were 4 mg/L and 128 mg/L, respectively; and the two isolates had the same antibiotic resistance pattern to other carbapenems. Two strains were then submitted for whole-genome sequencing and bioinformatic analysis. ompK36 was not detected in two isolates. No mutation was observed in blaKPC-2, ompK35 and ompK37 in this study and there was no significant difference of the expression in blaKPC-2, ompK35 and ompK37 between the two isolates (p>0.05). Two isolates were sequence type 11 and harbored blaKPC-2, blaSHV-182 and blaTEM-1B. Compared with KP135194, KP137060 harbored an additional blaNDM-5 positive plasmid. blaNDM-5 gene could be successfully transferred into E. coli J53 at a conjugation frequency of 1.14×10-4. Plasmid stability testing showed that blaKPC-2- and blaNDM-5-harboring plasmids were still stably maintained in the hosts. Growth assay and growth competition experiments showed there was no significant difference in fitness cost between two CR-KP isolates. Our study described the acquisition of a blaNDM-5-harboring plasmid leading to resistance to ceftazidime/avibactam in KPC-2-producing Klebsiella pneumoniae during treatment. This phenomenon deserves further exploration.

Phylogenetic Analysis Reveals Distinct Evolutionary Trajectories of the Fluoroquinolones-Resistant Escherichia coli ST1193 From Fuzhou, China.

Escherichia coli (E. coli) ST1193 is an emerging fluoroquinolones-resistant and virulent lineage. Large gaps remain in our understanding of the evolutionary processes and differences of this lineage. Therefore, we used 76 E. coli ST1193 genomes to detect strain-level genetic diversity and phylogeny of this lineage globally. All E. coli ST1193 possessed fimH64, filCH5, and fumC14. There was 94.7% of isolates classified as O-type O75. There was 9.33% of E. coli ST1193 that possessed K5 capsular, while 90.67% of isolates possessed K1 capsular. The core genome analysis revealed that all isolates were divided into two phylogenetic clades (clade A and B). Clade A included 25 non-Chinese E. coli ST1193, and clade B contained all isolates collected from Fuzhou, China, respectively. The results of comparative genomics indicated Indels were identified in 150 clade-specific genes, which were enriched into the biological process and molecular function. Accessory genome phylogenetic tree showed a high degree of correlation between accessory genome clusters and core genome clades. There was significant difference in antibiotic resistance genes (ARGs) [bla CTX-M-55 , bla TEM-1 , sul2, tet(B), tet(R), APH(6)-Id, and AAC(3)-IId], virulence factors (cia, neuC, gad, and traT), and plasmid replicon types (IncQ1, Col156, and IncB/O/K/Z) between clade A (non-Chinese isolates) and clade B (Chinese isolates) (p < 0.05). Further analysis of the genetic environments of bla CTX-M-55 demonstrated that the flanking contexts of bla CTX-M-55 were diverse. In conclusion, our results reveal the distinct evolutionary trajectories of the spread of E. coli ST1193 in Fuzhou, China and non-China regions. This supports both global transmission and localized lineage expansion of this lineage following specific introductions into a geographic locality.

Hospital Wastewater as a Reservoir for Antibiotic Resistance Genes: A Meta-Analysis.

Background: The emergence and dissemination of antibiotic resistance genes (ARGs) in the environment poses a huge global health hazard. Hospital wastewater (HWW), in which a high density of antibiotic residues and antibiotic-resistant bacteria are present, may be a reservoir of ARGs dissemination into the environment. Our meta-analysis comprehensively analyzes the prevalence of ARGs in HWW, as well as the influencing factors in ARGs distribution. Methods: Online databases were used to search for literature using the subject terms: "Drug Resistance" AND "Genes" AND "Hospitals" AND "Wastewater." Two reviewers independently applied predefined criteria to assess the literature and extract data including "relative abundance of ARGs," "title," "authors," "country," "location," "sampling year," and "sampling seasons." The median values and 95% confidence intervals of ARGs abundance were calculated by Wilcox.test function in R. Temporal trends, spatial differences, seasonal variations and removal efficiency of ARGs were analyzed by Pearson correlation analysis and Kruskal-Wallis H test. Results: Resistance genes to carbapenems, sulfonamides, tetracyclines and mobile genetic elements were found at high relative abundance (>10-4 gene copies/16S rRNA gene copies) in HWW. The abundance of resistance genes to extended-spectrum ß-lactams, carbapenems, sulfonamides and glycopeptide significantly decreased, while tetracycline resistance genes abundance increased from 2014 to 2018. The abundance of ARGs was significantly different by country but not by season. ARGs could not be completely removed by on-site HWW treatments and the removal efficiency varies for different ARGs. Conclusions: HWW presents more types of ARGs, and their abundance is higher than those in most wastewater systems. HWW may be a reservoir of ARGs and play an important role in the dissemination of ARGs.

A Comparative Study of Fluoroquinolone-Resistant Escherichia coli Lineages Portrays Indistinguishable Pathogenicity- and Survivability-Associated Phenotypic Characteristics Between ST1193 and ST131.

BACKGROUND: Sequence type 1193 is a new such lineage among fluoroquinolone-resistant Escherichia coli, which has risen dramatically within the last several years. However, reasons for rapid emergence and successful spread of E. coli ST1193 remain unclear. The aim of this study was to compare the pathogenicity and survivability features of E. coli ST1193 with global epidemic lineage, ST131. METHODS: A total of 30 E. coli were used in this study. Isolates were divided into two groups, ST1193 (n=15) and ST131 (n=15). Adhesion and invasion to T24 cells and resistance to serum were quantified and compared among two groups. Biofilm formation capacity was assessed by crystal violet assay. Macrocolony formation was assessed on macrocolony formation plates. Resistance to hydrogen peroxide was performed by broth microdilution. RAW264.7 cells were used to assess the anti-phagocytic function of different isolates. RESULTS: Adhesion and invasion assays revealed that E. coli ST1193 could adhere and invade T24 cells (p <0.05). 93.3% of E. coli ST1193 could form biofilms. The majority of E. coli ST1193 (66.7%) possessed no curli/no cellulose on macrocolony formation plates. E. coli ST1193 showed significant growth in serum and hydrogen peroxide and illustrated higher anti-phagocytic function to RAW264.7 cells (p <0.05). Group analysis showed that E. coli ST1193 was similar to ST131 in pathogenicity- and survivability-associated phenotypic characteristics (p >0.05). CONCLUSION: Our study provided more insights into pathogenicity and survivability features of E. coli ST1193, which was similar to ST131. Our study could be of great importance in understanding the emergence of global spread E. coli ST1193. Strategic and continued surveillance should be carried out to prevent the infections caused by E. coli ST1193.