Development of two microbial source tracking markers for detection of wastewater-associated Escherichia coli isolates.

Escherichia coli has been used as an indicator of fecal pollution in environmental waters. However, its presence in environmental waters does not provide information on the source of water pollution. Identifying the source of water pollution is paramount to be able to effectively reduce contamination. The present study aimed to identify E. coli microbial source tracking (MST) markers that can be used to identify domestic wastewater contamination in environmental waters. We first analyzed wastewater E. coli genomes sequenced by us (n = 50) and RefSeq animal E. coli genomes of fecal origin (n = 82), and identified 144 candidate wastewater-associated marker genes. The sensitivity and specificity of the candidate marker genes were then assessed by screening the genes in 335 RefSeq wastewater E. coli genomes and 3318 RefSeq animal E. coli genomes. We finally identified two MST markers, namely W_nqrC and W_clsA_2, which could be used for detection of wastewater-associated E. coli isolates. These two markers showed higher performance than the previously developed human wastewater-associated E. coli markers H8 and H12. When used in combination, W_nqrC and W_clsA_2 showed specificity of 98.9 % and sensitivity of 25.7 %. PCR assays to detect W_nqrC and W_clsA_2 were also developed and validated. The developed PCR assays are potentially useful for detecting E. coli isolates of wastewater origin in environmental waters, though users should keep in mind that the sensitivity of these markers is not high. Further studies are needed to assess the applicability of the developed markers to a culture-independent approach.

Chromosomal integration of bla CTX-M genes in diverse Escherichia coli isolates recovered from river water in Japan.

Occurrence of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (ESBLEC) in environmental waters is of great concern. However, unlike clinical ESBLEC, their genetic characteristics, in particular the genetic contexts of ESBL genes, are not well understood. In this study, we sequenced and analyzed the genomes of CTX-M-producing E. coli isolates recovered from river water to fully characterize the genetic contexts of bla CTX-M genes. Among the 14 isolates with completed genomes, bla CTX-M genes were detected on the chromosome in nine isolates. All but one chromosomal bla CTX-M genes were associated with ISEcp1 and were carried on different transposition units ranging in size from 2,855 bp to 11,093 bp; the exception, bla CTX-M-2, was associated with ISCR1. The remaining five isolates carried bla CTX-M genes on epidemic IncI1 plasmids of different sequence types (STs) (ST3, ST16, ST113, and ST167) (n = 4) or on an IncB/O/K/Z plasmid (n = 1). This study revealed that environmental E. coli carry bla CTX-M genes in diverse genetic contexts. Apparent high prevalence of chromosomal bla CTX-M potentially indicates that some E. coli can stably maintain bla CTX-M genes in environmental waters, though further studies are needed to confirm this.

Genomic dissection of Klebsiella pneumoniae infections in hospital patients reveals insights into an opportunistic pathogen.

Klebsiella pneumoniae is a major cause of opportunistic healthcare-associated infections, which are increasingly complicated by the presence of extended-spectrum beta-lactamases (ESBLs) and carbapenem resistance. We conducted a year-long prospective surveillance study of K. pneumoniae clinical isolates in hospital patients. Whole-genome sequence (WGS) data reveals a diverse pathogen population, including other species within the K. pneumoniae species complex (18%). Several infections were caused by K. variicola/K. pneumoniae hybrids, one of which shows evidence of nosocomial transmission. A wide range of antimicrobial resistance (AMR) phenotypes are observed, and diverse genetic mechanisms identified (mainly plasmid-borne genes). ESBLs are correlated with presence of other acquired AMR genes (median n = 10). Bacterial genomic features associated with nosocomial onset are ESBLs (OR 2.34, p = 0.015) and rhamnose-positive capsules (OR 3.12, p < 0.001). Virulence plasmid-encoded features (aerobactin, hypermucoidy) are observed at low-prevalence (<3%), mostly in community-onset cases. WGS-confirmed nosocomial transmission is implicated in just 10% of cases, but strongly associated with ESBLs (OR 21, p < 1 × 10-11). We estimate 28% risk of onward nosocomial transmission for ESBL-positive strains vs 1.7% for ESBL-negative strains. These data indicate that K. pneumoniae infections in hospitalised patients are due largely to opportunistic infections with diverse strains, with an additional burden from nosocomially-transmitted AMR strains and community-acquired hypervirulent strains.

Emergence of rare carbapenemases (FRI, GES-5, IMI, SFC and SFH-1) in Enterobacterales isolated from surface waters in Japan.

OBJECTIVES: Carbapenemase-producing Enterobacterales (CPE) pose serious threats to public health. Compared with clinical CPE, the genetic characteristics of environmental CPE are not well understood. This study aimed to characterize the genetic determinants of carbapenem resistance in CPE isolated from environmental waters in Japan. METHODS: Eighty-five water samples were collected from rivers and a lake in Japan. CPE were identified using selective media, and genome sequencing was performed for the obtained isolates (n = 21). RESULTS: Various rare/novel carbapenemases were identified: GES-5 in Raoultella planticola (n = 1), FRI-8 and FRI-11 in Enterobacter spp. (n = 8), IMI-22 and IMI-23 in Serratia ureilytica (n = 3), and SFC-1, SFC-2 and SFH-1 in Serratia fonticola (n = 9). Genomes of 11 isolates could be closed, allowing the elucidation of the genetic contexts of the carbapenemase genes. The blaGES-5 gene was located within a class 1 integron, In2071 (cassette array, blaGES-5-aacA3-aadA16), on a 33 kb IncP6 plasmid. The blaFRI-8 genes were carried on IncFII(Yp) plasmids ranging in size from 191 kb to 244 kb, and the blaFRI-11 genes were carried on 70 kb and 74 kb IncFII(pECLA)/IncR plasmids. The blaIMI-22 and blaIMI-23 genes were co-located on a 107 kb plasmid. The blaSFC and blaSFH-1 genes were found on putative genomic islands inserted at tRNA-Phe genes in chromosomes. CONCLUSIONS: This study revealed the presence of rare/novel carbapenemases among CPE in aquatic environments, suggesting that the environment may act as a potential reservoir of these minor carbapenemases.

Detection of plasmid contigs in draft genome assemblies using customized Kraken databases.

Plasmids play an important role in bacterial evolution and mediate horizontal transfer of genes including virulence and antimicrobial resistance genes. Although short-read sequencing technologies have enabled large-scale bacterial genomics, the resulting draft genome assemblies are often fragmented into hundreds of discrete contigs. Several tools and approaches have been developed to identify plasmid sequences in such assemblies, but require trade-off between sensitivity and specificity. Here we propose using the Kraken classifier, together with a custom Kraken database comprising known chromosomal and plasmid sequences of Klebsiella pneumoniae species complex (KpSC), to identify plasmid-derived contigs in draft assemblies. We assessed performance using Illumina-based draft genome assemblies for 82 KpSC isolates, for which complete genomes were available to supply ground truth. When benchmarked against five other classifiers (Centrifuge, RFPlasmid, mlplasmids, PlaScope and Platon), Kraken showed balanced performance in terms of overall sensitivity and specificity (90.8 and 99.4 %, respectively, for contig count; 96.5 and >99.9 %, respectively, for cumulative contig length), and the highest accuracy (96.8% vs 91.8-96.6% for contig count; 99.8% vs 99.0-99.7 % for cumulative contig length), and F1-score (94.5 % vs 84.5-94.1 %, for contig count; 98.0 % vs 88.9-96.7 % for cumulative contig length). Kraken also achieved consistent performance across our genome collection. Furthermore, we demonstrate that expanding the Kraken database with additional known chromosomal and plasmid sequences can further improve classification performance. Although we have focused here on the KpSC, this methodology could easily be applied to other species with a sufficient number of completed genomes.

Molecular Analysis of a blaIMP-1-Harboring Class 3 Integron in Multidrug-Resistant Pseudomonas fulva.

A multidrug-resistant (MDR) Pseudomonas fulva strain was isolated in 2006 from a urine sample. The isolate harbored the blaIMP-1 gene, which was located in a chromosomal Tn402-like class 3 integron as a gene cassette array of aacA31-fosE-blaIMP-1 Two mutations in gyrA and one mutation in parC were detected in quinolone-resistance-determining regions (QRDRs). We report a full-length, novel, blaIMP-1-carrying class 3 integron. This integron, together with mutations in QRDRs, could have influenced the MDR phenotype.

Characteristics of Carbapenemase-Producing Enterobacteriaceae in Wastewater Revealed by Genomic Analysis.

Wastewater is considered a major source of antibiotic-resistant bacteria released into the environment. Here, we characterized carbapenemase-producing Enterobacteriaceae (CPE) in wastewater by whole-genome analysis. Wastewater samples (n = 40) were collected from municipal wastewater treatment plants and hospital wastewater in Japan and Taiwan. Samples were screened for CPE using selective media, and the obtained isolates were sequenced using an Illumina MiSeq. The isolates (n = 45) included the following microorganisms: Klebsiella quasipneumoniae (n = 12), Escherichia coli (n = 10), Enterobacter cloacae complex (n = 10), Klebsiella pneumoniae (n = 8), Klebsiella variicola (n = 2), Raoultella ornithinolytica (n = 1), Citrobacter freundii (n = 1), and Citrobacter amalonaticus (n = 1). Among the 45 isolates, 38 harbored at least one carbapenemase-encoding gene. Of these, the blaGES (blaGES-5, blaGES-6, and blaGES-24) genes were found in 29 isolates. The genes were situated in novel class 1 integrons, but the integron structures were different between the Japanese (In1439 with blaGES-24 and In1440 with blaGES-5) and Taiwanese (In1441 with blaGES-5 and In1442 with blaGES-6) isolates. Other carbapenemase-encoding genes (blaVIM-1, blaNDM-5, blaIMP-8, blaIMP-19, and blaKPC-2) were found in one to three isolates. Notably, class 1 integrons previously reported among clinical isolates obtained in the same regions as the present study, namely, In477 with blaIMP-19 and In73 with blaIMP-8, were found among the Japanese and Taiwanese isolates, respectively. The results indicate that CPE with various carbapenemase-encoding genes in different genetic contexts were present in biologically treated wastewater, highlighting the need to monitor for antibiotic resistance in wastewater.

Rapid Identification of Different Escherichia coli Sequence Type 131 Clades.

Escherichia coli sequence type 131 (ST131) is a pandemic clonal lineage that is responsible for the global increase in fluoroquinolone resistance and extended-spectrum-ß-lactamase (ESBL) producers. The members of ST131 clade C, especially subclades C2 and C1-M27, are associated with ESBLs. We developed a multiplex conventional PCR assay with the ability to detect all ST131 clades (A, B, and C), as well as C subclades (C1-M27, C1-nM27 [C1-non-M27], and C2). To validate the assay, we used 80 ST131 global isolates that had been fully sequenced. We then used the assay to define the prevalence of each clade in two Japanese collections consisting of 460 ESBL-producing E. coli ST131 (2001-12) and 329 E. coli isolates from extraintestinal sites (ExPEC) (2014). The assay correctly identified the different clades in all 80 global isolates: clades A (n = 12), B (n = 12), and C, including subclades C1-M27 (n = 16), C1-nM27 (n = 20), C2 (n = 17), and other C (n = 3). The assay also detected all 565 ST131 isolates in both collections without any false positives. Isolates from clades A (n = 54), B (n = 23), and C (n = 483) corresponded to the O serotypes and the fimH types of O16-H41, O25b-H22, and O25b-H30, respectively. Of the 483 clade C isolates, C1-M27 was the most common subclade (36%), followed by C1-nM27 (32%) and C2 (15%). The C1-M27 subclade with blaCTX-M-27 became especially prominent after 2009. Our novel multiplex PCR assay revealed the predominance of the C1-M27 subclade in recent Japanese ESBL-producing E. coli isolates and is a promising tool for epidemiological studies of ST131.

Occurrence of Clinically Important Lineages, Including the Sequence Type 131 C1-M27 Subclone, among Extended-Spectrum-ß-Lactamase-Producing Escherichia coli in Wastewater.

Contamination of environmental waters by extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli (ESBLEC) is of great concern. Wastewater treatment plants (WWTPs) and hospitals release large amounts of ESBLEC into the environment. In the present study, we isolated ESBLEC strains from wastewater collected from a WWTP and a hospital in Japan and performed whole-genome sequencing to characterize these strains. Genomic analysis of 54 strains (32 from the WWTP and 22 from hospital wastewater) revealed the occurrence of clinically important clonal groups with extraintestinal pathogenic E. coli status in the WWTP and hospital wastewater. Fine-scale phylogenetic analysis was performed to further characterize 15 sequence type 131 (ST131) complex strains (11 from the WWTP and 4 from hospital wastewater). These ST131 complex strains were comprised of the following different subgroups: clade A (n = 2), C1-M27 (n = 8), and C1 (non-C1-M27) (n = 1) for strains from the WWTP and clade A (n = 2), C1-M27 (n = 1), and C1 (non-C1-M27) (n = 1) for strains from hospital wastewater. The results indicate that ESBLEC strains belonging to clinically important lineages, including the C1-M27 clade, may disseminate into the environment through wastewater, highlighting the need to monitor for antibiotic resistance in wastewater.

Whole-Genome Analysis of Antimicrobial-Resistant and Extraintestinal Pathogenic Escherichia coli in River Water.

Contamination of surface waters by antimicrobial-resistant bacteria and pathogenic bacteria is a great concern. In this study, 531 Escherichia coli isolates obtained from the Yamato River in Japan were evaluated phenotypically for resistance to 25 antimicrobials. Seventy-six isolates (14.3%) were multidrug resistant (MDR), 66 (12.4%) were nonsusceptible to one or two classes of agents, and 389 (73.3%) were susceptible. We performed whole-genome sequencing of selected strains by using Illumina technology. In total, the genome sequences of 155 strains were analyzed for antibiotic resistance determinants and phylogenetic characteristics. More than 50 different resistance determinants, including acquired resistance genes and chromosomal resistance mutations, were detected. Among the sequenced MDR strains (n = 66), sequence type 155 (ST155) complex (n = 9), ST10 complex (n = 9), and ST69 complex (n = 7) were prevalent. Among extraintestinal pathogenic E. coli (ExPEC) strains (n = 58), clinically important clonal groups, namely, ST95 complex (n = 18), ST127 complex (n = 8), ST12 complex (n = 6), ST14 complex (n = 6), and ST131 complex (n = 6), were prevalent, demonstrating the clonal distribution of environmental ExPEC strains. Typing of the fimH (type 1 fimbrial adhesin) gene revealed that ST131 complex strains carried fimH22 or fimH41, and no strains belonging to the fimH30 subgroup were detected. Fine-scale phylogenetic analysis and virulence gene content analysis of strains belonging to the ST95 complex (one of the major clonal ExPEC groups causing community-onset infections) revealed no significant differences between environmental and clinical strains. The results indicate contamination of surface waters by E. coli strains belonging to clinically important clonal groups.IMPORTANCE The prevalence of antimicrobial-resistant and pathogenic E. coli strains in surface waters is a concern because surface waters are used as sources for drinking water, irrigation, and recreational purposes. In this study, MDR and ExPEC strains in river water were characterized by genomic sequencing and analysis. We detected more than 50 resistance determinants and identified clonal groups specific to MDR and ExPEC strains. This study showed contamination of surface waters by E. coli strains belonging to clinically important clonal groups. Overall, this study advances our understanding of environmental MDR and ExPEC strains.

Global Escherichia coli Sequence Type 131 Clade with blaCTX-M-27 Gene.

The Escherichia coli sequence type (ST) 131 C2/H30Rx clade with the blaCTX-M-15 gene had been most responsible for the global dissemination of extended-spectrum ß-lactamase (ESBL)-producing E. coli. ST131 C1/H30R with blaCTX-M-27 emerged among ESBL-producing E. coli in Japan during the late 2000s. To investigate the possible expansion of a single clade, we performed whole-genome sequencing for 43 Japan and 10 global ST131 isolates with blaCTX-M-27 (n = 16), blaCTX-M-14 (n = 16), blaCTX-M-15 (n = 13), and others (n = 8). We also included 8 ST131 genomes available in public databases. Core genome-based analysis of 61 isolates showed that ST131 with blaCTX-M-27 from 5 countries formed a distinct cluster within the C1/H30R clade, named C1-M27 clade. Accessory genome analysis identified a unique prophage-like region, supporting C1-M27 as a distinct clade. Our findings indicate that the increase of ESBL-producing E. coli in Japan is due mainly to emergence of the C1-M27 clade.

Interspecies Dissemination of a Mobilizable Plasmid Harboring blaIMP-19 and the Possibility of Horizontal Gene Transfer in a Single Patient.

Carbapenemase-producing Gram-negative bacilli have been a global concern over the past 2 decades because these organisms can cause severe infections with high mortality rates. Carbapenemase genes are often carried by mobile genetic elements, and resistance plasmids can be transferred through conjugation. We conducted whole-genome sequencing (WGS) to demonstrate that the same plasmid harboring a metallo-ß-lactamase gene was detected in two different species isolated from a single patient. Metallo-ß-lactamase-producing Achromobacter xylosoxidans (KUN4507), non-metallo-ß-lactamase-producing Klebsiella pneumoniae (KUN4843), and metallo-ß-lactamase-producing K. pneumoniae (KUN5033) were sequentially isolated from a single patient and then analyzed in this study. Antimicrobial susceptibility testing, molecular typing (pulsed-field gel electrophoresis and multilocus sequence typing), and conjugation analyses were performed by conventional methods. Phylogenetic and molecular clock analysis of K. pneumoniae isolates were performed with WGS, and the nucleotide sequences of plasmids detected from these isolates were determined using WGS. Conventional molecular typing revealed that KUN4843 and KUN5033 were identical, whereas the phylogenetic tree analysis revealed a slight difference. These two isolates were separated from the most recent common ancestor 0.74 years before they were isolated. The same resistance plasmid harboring blaIMP-19 was detected in metallo-ß-lactamase-producing A. xylosoxidans and K. pneumoniae Although this plasmid was not self-transferable, the conjugation of this plasmid from A. xylosoxidans to non-metallo-ß-lactamase-producing K. pneumoniae was successfully performed. The susceptibility patterns for metallo-ß-lactamase-producing K. pneumoniae and the transconjugant were similar. These findings supported the possibility of the horizontal transfer of plasmid-borne blaIMP-19 from A. xylosoxidans to K. pneumoniae in a single patient.

Assessment of Genetic Markers for Tracking the Sources of Human Wastewater Associated Escherichia coli in Environmental Waters.

In this study, we have evaluated the performance characteristics (host-specificity and -sensitivity) of four human wastewater-associated Escherichia coli (E. coli) genetic markers (H8, H12, H14, and H24) in 10 target (human) and nontarget (cat, cattle, deer, dog, emu, goat, horse, kangaroo, and possum) host groups in Southeast Queensland, Australia. The overall host-sensitivity values of the tested markers in human wastewater samples were 1.0 (all human wastewater samples contained the E. coli genetic markers). The overall host-specificity values of these markers to differentiate between human and animal host groups were 0.94, 0.85, 0.72, and 0.57 for H8, H12, H24, and H14, respectively. Based on the higher host-specificity values, H8 and H12 markers were chosen for a validation environmental study. The prevalence of the H8 and H12 markers was determined among human wastewater E. coli isolates collected from a wastewater treatment plant (WWTP). Among the 97 isolates tested, 44 (45%) and 14 (14%) were positive for the H8 and H12 markers, respectively. A total of 307 E. coli isolates were tested from environmental water samples collected in Brisbane, of which 7% and 20% were also positive for the H8 and H12 markers, respectively. Based on our results, we recommend that these markers could be useful when it is important to identify the source(s) of E. coli (whether they originated from human wastewater or not) in environmental waters.

Characterization of Pathogenic Escherichia coli in River Water by Simultaneous Detection and Sequencing of 14 Virulence Genes.

The occurrence of pathogenic Escherichia coli in environmental waters increases the risk of waterborne disease. In this study, 14 virulence genes in 669 E. coli isolates (549 isolates from the Yamato River in Japan, and 30 isolates from each of the following hosts: humans, cows, pigs, and chickens) were simultaneously quantified by multiplex PCR and dual index sequencing to determine the prevalence of potentially pathogenic E. coli. Among the 549 environmental isolates, 64 (12%) were classified as extraintestinal pathogenic E. coli (ExPEC) while eight (1.5%) were classified as intestinal pathogenic E. coli (InPEC). Only ExPEC-associated genes were detected in human isolates and pig isolates, and 11 (37%) and five (17%) isolates were classified as ExPEC, respectively. A high proportion (63%) of cow isolates possessed Shiga-toxin genes (stx1 or stx2) and they were classified as Shiga toxin-producing E. coli (STEC) or enterohemorrhagic E. coli (EHEC). Among the chicken isolates, 14 (47%) possessed iutA, which is an ExPEC-associated gene. This method can determine the sequences as well as the presence/absence of virulence genes. By comparing the sequences of virulence genes, we determined that sequences of iutA were different among sources and may be useful for discriminating isolates, although further studies including larger numbers of isolates are needed. Results indicate that humans are a likely source of ExPEC strains in the river.

Fecal source tracking in water by next-generation sequencing technologies using host-specific Escherichia coli genetic markers.

High levels of fecal bacteria are a concern for the aquatic environment, and identifying sources of those bacteria is important for mitigating fecal pollution and preventing waterborne disease. Escherichia coli has been used as an indicator of fecal pollution, however less success has been achieved using this organism for library-independent microbial source tracking. In this study, using next-generation sequencing technology we sequenced the whole genomes of 22 E. coli isolates from known sources (9 from humans, 2 from cows, 6 from pigs, and 5 from chickens) and identified candidate host-specific genomic regions. Specificity testing on the candidate regions was performed using 30 E. coli isolates from each source. Finally, we identified 4 human-, 2 cow-, 3 pig-, and 4 chicken-specific genetic markers useful for source tracking. We also found that a combination of multiplex PCR and dual index sequencing is effective for detecting multiple genetic markers in multiple isolates at one time. This technique was applied to investigating identified genetic markers in 549 E. coli isolates obtained from the Yamato River, Japan. Results indicate that humans constitute a major source of water contamination in the river. However, further work must include isolates obtained from geographically diverse animal hosts to make this method more reliable.

Effect of dietary phytol on the expression of α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase, a key enzyme of tryptophan-niacin metabolism, in rats.

α-Amino-ß-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) plays a key role in the regulation of NAD biosynthesis or the production of quinolinate from tryptophan (Trp). We investigated in this study the effect of phytol, a phytochemical known as a peroxisome proliferator-activated receptor α (PPARα) ligand, on NAD synthesis and ACMSD expression in rats. Male Sprague-Dawley rats were fed a diet containing 0.5%, 1%, or 2% phytol for 7 d. Phytol decreased the ACMSD activity and its mRNA expression in a dose-dependent manner in the liver. Phytol similarly and significantly suppressed ACMSD mRNA expression in primary rat hepatocytes. However, the mRNA expression of ACO (a known PPARα target gene) was higher in the low-phytol groups than in the high-phytol group in vivo and in vitro. Phytol increased the blood NAD level by suppressing ACMSD mRNA expression in the liver of the rats. It is possible that this mechanism occurred by the activation of PPARα and also of other transcriptional factors.