Hospital and community wastewater as a source of multidrug-resistant ESBL-producing Escherichia coli.

Introduction: Hospitals and wastewater are recognized hot spots for the selection and dissemination of antibiotic-resistant bacteria to the environment, but the total participation of hospitals in the spread of nosocomial pathogens to municipal wastewater treatment plants (WWTPs) and adjacent rivers had not previously been revealed. Methods: We used a combination of culturing and whole-genome sequencing to explore the transmission routes of Escherichia coli from hospitalized patients suffering from urinary tract infections (UTI) via wastewater to the environment. Samples were collected in two periods in three locations (A, B, and C) and cultured on selective antibiotic-enhanced plates. Results: In total, 408 E. coli isolates were obtained from patients with UTI (n=81), raw hospital sewage (n=73), WWTPs inflow (n=96)/outflow (n=106), and river upstream (n=21)/downstream (n=31) of WWTPs. The majority of the isolates produced extended-spectrum beta-lactamase (ESBL), mainly CTX-M-15, and showed multidrug resistance (MDR) profiles. Seven carbapenemase-producing isolates with GES-5 or OXA-244 were obtained in two locations from wastewater and river samples. Isolates were assigned to 74 different sequence types (ST), with the predominance of ST131 (n=80) found in all sources including rivers. Extraintestinal pathogenic lineages frequently found in hospital sewage (ST10, ST38, and ST69) were also found in river water. Despite generally high genetic diversity, phylogenetic analysis of ST10, ST295, and ST744 showed highly related isolates (SNP 0-18) from different sources, providing the evidence for the transmission of resistant strains through WWTPs to surface waters. Discussion: Results of this study suggest that 1) UTI share a minor participation in hospitals wastewaters; 2) a high diversity of STs and phylogenetic groups in municipal wastewaters derive from the urban influence rather than hospitals; and 3) pathogenic lineages and bacteria with emerging resistance genotypes associated with hospitals spread into surface waters. Our study highlights the contribution of hospital and municipal wastewater to the transmission of ESBL- and carbapenemase-producing E. coli with MDR profiles to the environment.

Carp edema virus infection associated gill pathobiome: A case report.

Understanding disease aetiology and pathologic mechanisms is essential for fish health evaluation. Carp edema virus (CEV) is the causative agent of a disease (CEVD) responsible for high mortality rates in both wild and cultured common carp Cyprinus carpio. Inspection of two carp specimens from a pond with high fish mortality revealed CEV infection in both the host and its ectoparasite (Argulus foliaceus). In addition to flavobacteria, well known to be associated with gill lesions, we found that free-living eukaryotes (amoebae and ciliates) and a temporary parasite (Ichthyobodo spp.) colonizing the gills may also contribute to alterations in gill structure and/or function, either directly, through firm (Ichthyobodo) or weak (amoebae) attachment of trophozoites to the gill epithelium, or indirectly, through carriage of pathogenic bacteria. Bacterial assemblages rich in families and genera, with predominance of Cetobacterium spp. in low-intensity alteration of the gill tissue and of Flavobacterium spp. in gills with extensive necrotic lesions, were detected in gills and within the cytoplasm of associated amoebae using high-throughput sequencing. Quantitative PCR indicated F. swingsii as the prevailing flavobacterial species within amoebae from less affected gills and F. psychrophilum within amoebae from extensively affected gills. This case study suggests that eukaryotic organisms as part of the gill pathobiome may also contribute to irreversible gill lesions seen in CEVD. Emphasizing the complexity of mutual relationships between bacterial assemblages and eukaryotic co-pathogens, further studies regarding factors that trigger pathology and influence severity in the CEV-positive carp are needed.

Paraphocaeicola brunensis gen. nov., sp. nov., Carrying Two Variants of nimB Resistance Gene from Bacteroides fragilis, and Caecibacteroides pullorum gen. nov., sp. nov., Two Novel Genera Isolated from Chicken Caeca.

Three difficult-to-cultivate, strictly anaerobic strains, AN20T, AN421T, and AN502, were analyzed within a project studying possible probiotics for newly hatched chickens. Phylogenetic analyses showed that strains AN20T, AN421T, and AN502 formed two well-separated phylogenetic lineages in all phylogenetic and phylogenomic trees comprising members of the family Bacteroidaceae. Comparison to reference genomes of type species Bacteroides fragilis NCTC 9343T, Phocaeicola abscessus CCUG 55929T, and Capsularis zoogleoformans ATCC 33285T showed low relatedness based on the calculated genome-to-genome distance and orthologous average nucleotide identity. Analysis of fatty acid profiles showed iso-C15:0, anteiso-C15:0, C16:0, C18:1ω9c, and iso-C17:0 3OH as the major fatty acids for all three strains and additionally C16:0 3OH for AN421T and AN502. A specific combination of respiratory quinones different from related taxa was found in analyzed strains, MK-5 plus MK-11 in strain AN20T and MK-5 plus MK-10 in strains AN421T and AN502. Strains AN421T and AN502 harbor complete CRISPR loci with CRISPR array, type II-C, accompanied by a set of cas genes (cas9, cas1, and cas2) in close proximity. Interestingly, strain AN20T was found to harbor two copies of nimB gene with >95% similarity to nimB of B. fragilis, suggesting a horizontal gene transfer between these taxa. In summary, three isolates characterized in this study represent two novel species, which we proposed to be classified in two novel genera of the family Bacteroidaceae, for which the names Paraphocaeicola brunensis sp. nov. (AN20T = CCM 9041T = DSM 111154T) and Caecibacteroides pullorum sp. nov. (AN421T= CCM 9040T = DSM 111155T) are proposed. IMPORTANCE This study represents follow-up research on three difficult-to-cultivate anaerobic isolates originally isolated within a project focused on strains that are able to stably colonize newly hatched chickens, thus representing possible probiotics. This project is exceptional in that it successfully isolates several miscellaneous strains that required modified and richly supplemented anaerobic media, as information on many gut-colonizing bacteria is based predominantly on metagenomic studies. Superior colonization of newly hatched chickens by Bacteroides spp., Phocaeicola spp., or related taxa can be considered of importance for development of future probiotics. Although different experiments can also be performed with provisionally characterized isolates, precise taxonomical definition is necessary for subsequent broad communication. The aim of this study is therefore to thoroughly characterize these isolates that represent novel genera and precisely determine their taxonomic position among related taxa to facilitate further research and communication involving these strains.

Multi-Drug Resistant Plasmids with ESBL/AmpC and mcr-5.1 in Paraguayan Poultry Farms: The Linkage of Antibiotic Resistance and Hatcheries.

Poultry represents a common source of bacteria with resistance to antibiotics including the critically important ones. Selective cultivation using colistin, cefotaxime and meropenem was performed for 66 chicken samples coming from 12 farms in Paraguay while two breeding companies supplied the farms. A total of 62 Escherichia coli and 22 Klebsiella pneumoniae isolates were obtained and representative isolates were subjected to whole-genome sequencing. Relatively high prevalence of phylogenetic group D and F was observed in E. coli isolates and several zoonotic sequence types (STs) including ST457 (14 isolates), ST38 (5), ST10 (2), ST117 (2) or ST93 (4) were detected. Isolates from three farms, which purchased chicken from a Paraguayan hatchery showed higher prevalence of mcr-5.1 and blaCTX-M-8 compared to the other nine farms, which purchased chickens from a Brazilian hatchery. Moreover, none of the K. pneumoniae isolates were linked to the Paraguayan hatchery. ESBL/AmpC and mcr-5-carrying multi-drug resistant (MDR) plasmids were characterized, and complete sequences were obtained for eight plasmids. The study shed light on Paraguayan poultry farms as a reservoir of antibiotic resistance commonly conferred via MDR plasmids and showed linkage between resistance and origin of the chickens at the hatcheries level.

Extended-spectrum beta-lactamase-producing Escherichia coli and antimicrobial resistance in municipal and hospital wastewaters in Czech Republic: Culture-based and metagenomic approaches.

Wastewaters serve as important hot spots for antimicrobial resistance and monitoring can be used to analyse the abundance and diversity of antimicrobial resistance genes at the level of large bacterial and human populations. In this study, whole genome sequencing of beta-lactamase-producing Escherichia coli and metagenomic analysis of whole-community DNA were used to characterize the occurrence of antimicrobial resistance in hospital, municipal and river waters in the city of Brno (Czech Republic). Cefotaxime-resistant E. coli were mainly extended-spectrum beta-lactamase (ESBL) producers (95.6%, n = 158), of which the majority carried blaCTX-M (98.7%; n = 151) and were detected in all water samples except the outflow from hospital wastewater treatment plant. A wide phylogenetic diversity was observed among the sequenced E. coli (n = 78) based on the detection of 40 sequence types and single nucleotide polymorphisms (average number 34,666 ± 15,710) between strains. The metagenomic analysis revealed a high occurrence of bacterial genera with potentially pathogenic members, including Pseudomonas, Escherichia, Klebsiella, Aeromonas, Enterobacter and Arcobacter (relative abundance >50%) in untreated hospital and municipal wastewaters and predominance of environmental bacteria in treated and river waters. Genes encoding resistance to aminoglycosides, beta-lactams, quinolones and macrolides were frequently detected, however blaCTX-M was not found in this dataset which may be affected by insufficient sequencing depth of the samples. The study pointed out municipal treated wastewater as a possible source of multi-drug resistant E. coli and antimicrobial resistance genes for surface waters. Moreover, the combination of two different approaches provided a more holistic view on antimicrobial resistance in water environments. The culture-based approach facilitated insight into the dynamics of ESBL-producing E. coli and the metagenomics shows abundance and diversity of bacteria and antimicrobial resistance genes vary across water sites.

Carbapenemase-Producing Gram-Negative Bacteria from American Crows in the United States.

Wild corvids were examined for the presence of carbapenemase-producing Gram-negative bacteria in the United States. A total of 13 isolates were detected among 590 fecal samples of American crow; 11 Providencia rettgeri isolates harboring blaIMP-27 on the chromosome as a class 2 integron gene cassette within the Tn7 transposon, 1 Klebsiella pneumoniae ST258 isolate carrying blaKPC-2 on a pKpQIL-like plasmid as a part of Tn4401a, and 1 Enterobacter bugandensis isolate with blaIMI-1 located within EcloIMEX-2.

Effects of host genetics and environmental conditions on fecal microbiota composition of pigs.

Since microbiota may influence the physiology of its host including body weight increase, growth rate or feed intake, in this study we determined the microbiota composition in high or low residual feed intake (HRFI and LRFI) pig lines, of different age and/or subjected to sanitary stress by sequencing the V3/V4 variable region of 16S rRNA genes. Allisonella, Megasphaera, Mitsuokella, Acidaminococcus (all belonging to Firmicutes/class Negativicutes), Lactobacillus, Faecalibacterium, Catenibacterium, Butyrivibrio, Erysipelotrichaceae, Holdemania, Olsenella and Collinsella were more abundant in HRFI pigs. On the other hand, 26 genera including Bacteroides, Clostridium sensu stricto, Oscillibacter, Paludibacter, Elusimicrobium, Bilophila, Pyramidobacter and TM7 genera, and Clostridium XI and Clostridium XIVa clusters were more abundant in LRFI than HRFI pigs. Adaptation of microbiota to new diet after weaning was slower in LRFI than in HRFI pigs. Sanitary stress was of relatively minor influence on pig microbiota composition in both tested lines although abundance of Helicobacter increased in LRFI pigs subjected to stress. Selection for residual feed intake thus resulted in a selection of fecal microbiota of different composition. However, we cannot conclude whether residual feed intake was directly affected by different microbiota composition or whether the residual feed intake and microbiota composition are two independent consequences of yet unknown genetic traits differentially selected in the pigs of the two lines.

Characterization of the Complete Nucleotide Sequences of IMP-4-Encoding Plasmids, Belonging to Diverse Inc Families, Recovered from Enterobacteriaceae Isolates of Wildlife Origin.

The complete nucleotide sequences of six IMP-4-encoding plasmids recovered from Enterobacteriaceae isolates of wildlife origin were characterized. Sequencing data showed that plasmids of different incompatibility groups (IncM, IncI1, IncF, and nontypeable [including an IncX5_2 and two pPrY2001-like]) carried the blaIMP-4-carrying integrons In809 or In1460. Most of the plasmids carried an mph(A) region, and chrA-like, aac(3)-IId, and blaTEM-1b genes. Finally, plasmid analysis revealed the involvement of two different IS26- and Tn1696-associated mechanisms in the mobilization of IMP-4-encoding integrons.

Housing Systems Influence Gut Microbiota Composition of Sows but Not of Their Piglets.

Different housing systems can be used in pig production and little is known about their effect on gut microbiota composition. In this study we characterized fecal microbiota by sequencing the rRNA genes in sows kept during gestation in conventional pens with a slatted floor and in enriched pens with a floor covered with deep straw. After farrowing, microbiota of 1- and 4-day-old piglets were also monitored. Microbiota of sows from the enriched system contained significantly more Prevotella, Parabacteroides, CF231, Phascolarctobacterium, Fibrobacter, Anaerovibrio and YRC22 and significantly less Lactobacillus, Bulleidia, Lachnospira, Dorea, Ruminococcus and Oscillospira than microbiota of sows from the conventional system. The Firmicutes to Bacteroidetes ratio was 0.96 in the microbiota of sows kept in the enriched pens and this increased to 1.66 in the microbiota of sows kept in the conventional system. The production system therefore influenced microbiota composition, most likely due the ingestion of the straw. The microbiota of 1- and 4-day-old piglets differed from the microbiota of sows and sows therefore did not represent the most important source for their colonization in early days of life.

Composition of Gut Microbiota Influences Resistance of Newly Hatched Chickens to Salmonella Enteritidis Infection.

Since poultry is a very common source of non-typhoid Salmonella for humans, different interventions aimed at decreasing the prevalence of Salmonella in chickens are understood as an effective measure for decreasing the incidence of human salmonellosis. One such intervention is the use of probiotic or competitive exclusion products. In this study we tested whether microbiota from donor hens of different age will equally protect chickens against Salmonella Enteritidis infection. Newly hatched chickens were therefore orally inoculated with cecal extracts from 1-, 3-, 16-, 28-, and 42-week-old donors and 7 days later, the chickens were infected with S. Enteritidis. The experiment was terminated 4 days later. In the second experiment, groups of newly hatched chickens were inoculated with cecal extracts of 35-week-old hens either on day 1 of life followed by S. Enteritidis infection on day 2 or were infected with S. Enteritidis infection on day 1 followed by therapeutic administration of the cecal extract on day 2 or were inoculated on day 1 of life with a mixture of the cecal extract and S. Enteritidis. This experiment was terminated when the chickens were 5 days old. Both Salmonella culture and chicken gene expression confirmed that inoculation of newly hatched chickens with microbiota from 3-week-old or older chickens protected them against S. Enteritidis challenge. On the other hand, microbiota from 1-week-old donors failed to protect chickens against S. Enteritidis challenge. Microbiota from 35-week-old hens protected chickens even 24 h after administration. However, simultaneous or therapeutic microbiota administration failed to protect chickens against S. Enteritidis infection. Gut microbiota can be used as a preventive measure against S. Enteritidis infection but its composition and early administration is critical for its efficacy.