Klebsiella pneumoniae species complex: From wastewater to the environment.

Klebsiella pneumoniae plays a significant role in nosocomial infections and spreading antibiotic resistance, and therefore forms a major threat to public health. In this study, we investigated the role of the wastewater pathway in the spread of pathogenic bacteria and more specifically, in the spread of antibiotic resistant Klebsiella pneumoniae subspecies. Whole-genome sequencing was performed of 185 K. pneumoniae isolates collected from hospital, nursing home, and community wastewater, the receiving wastewater treatment plant (WWTP), and clinical isolates from the investigated hospital. K. pneumoniae isolates from different sources were not genetically related, except for WWTP influent (46.5%) and effluent (62.5%), revealing survival of bacteria from wastewater treatment. The content of antibiotic resistance (ARGs), virulence, and plasmid replicon genes differed between K. pneumoniae subspecies and their origin. While chromosomal bla genes were specific for each K. pneumoniae subspecies, bla genes predicted in plasmid contigs were found in several K. pneumoniae subspecies, implying possible gene transfer between subspecies. Transferable ARGs were most abundant in patients and hospital isolates (70%), but the average number of plasmid replicon genes per isolate was similar across all sources, showing plasmid content being more relevant than plasmid quantity. Most patient (90%) and hospital wastewater (34%) isolates were K. pneumoniae subsp. pneumoniae, and the yersiniabactin cluster genes ybt, fyuA, and irp12 were only found in this subspecies, as were the IncFII(pECLA), IncHI2A, and IncHI2 plasmid replicon genes, suggesting the clinical origin of these type of plasmids.

Targeted phage hunting to specific Klebsiella pneumoniae clinical isolates is an efficient antibiotic resistance and infection control strategy.

Klebsiella pneumoniae is one of the most threatening multi-drug-resistant pathogens today, with phage therapy being a promising alternative for personalized treatments. However, the intrinsic capsule diversity in Klebsiella spp. poses a substantial barrier to the phage host range, complicating the development of broad-spectrum phage-based treatments. Here, we have isolated and genomically characterized phages capable of infecting each of the acquired 77 reference serotypes of Klebsiella spp., including capsular types widespread among high-risk K. pneumoniae clones causing nosocomial infections. We demonstrated the possibility of isolating phages for all capsular types in the collection, revealing high capsular specificity among taxonomically related phages, in contrast to a few phages that exhibited broad-spectrum infection capabilities. To decipher the determinants of the specificity of these phages, we focused on their receptor-binding proteins, with particular attention to depolymerases. We also explored the possibility of designing a broad-spectrum phage cocktail based on phages isolated in reference capsular-type strains and determining the ability to lyse relevant clinical isolates. A combination of 12 phages capable of infecting 55% of the reference Klebsiella spp. serotypes was tested on a panel of carbapenem-resistant K. pneumoniae clinical isolates. Thirty-one percent of isolates were susceptible to the phage cocktail. However, our results suggest that in a highly variable encapsulated bacterial host, phage hunting must be directed to the specific Klebsiella isolates. This work is a step forward in the understanding of the complexity of phage-host interactions and highlights the importance of implementing precise and phage-specific strategies to treat K. pneumoniae infections worldwide.IMPORTANCEThe emergence of resistant bacteria is a serious global health problem. In the absence of effective treatments, phages are a personalized and effective therapeutic alternative. However, little is still known about phage-host interactions, which are key to implementing effective strategies. Here, we focus on the study of Klebsiella pneumoniae, a highly pathogenic encapsulated bacterium. The complexity and variability of the capsule, where in most cases phage receptors are found, make it difficult for phage-based treatments. Here, we isolated a large collection of Klebsiella phages against all the reference strains and in a cohort of clinical isolates. Our results suggest that clinical isolates represent a challenge, especially high-risk clones. Thus, we propose targeted phage hunting as an effective strategy to implement phage-derived therapies. Our results are a step forward for new phage-based strategies to control K. pneumoniae infections, highlighting the importance of understanding phage-host interactions to design personalized treatments against Klebsiella spp.

Characterizing carbapenemase-producing Escherichia coli isolates from Spain: high genetic heterogeneity and wide geographical spread.

Introduction: Carbapenemase-Producing Escherichia coli (CP-Eco) isolates, though less prevalent than other CP-Enterobacterales, have the capacity to rapidly disseminate antibiotic resistance genes (ARGs) and cause serious difficult-to-treat infections. The aim of this study is phenotypically and genotypically characterizing CP-Eco isolates collected from Spain to better understand their resistance mechanisms and population structure. Methods: Ninety representative isolates received from 2015 to 2020 from 25 provinces and 59 hospitals Spanish hospitals were included. Antibiotic susceptibility was determined according to EUCAST guidelines and whole-genome sequencing was performed. Antibiotic resistance and virulence-associated genes, phylogeny and population structure, and carbapenemase genes-carrying plasmids were analyzed. Results and discussion: The 90 CP-Eco isolates were highly polyclonal, where the most prevalent was ST131, detected in 14 (15.6%) of the isolates. The carbapenemase genes detected were bla OXA-48 (45.6%), bla VIM-1 (23.3%), bla NDM-1 (7.8%), bla KPC-3 (6.7%), and bla NDM-5 (6.7%). Forty (44.4%) were resistant to 6 or more antibiotic groups and the most active antibiotics were colistin (98.9%), plazomicin (92.2%) and cefiderocol (92.2%). Four of the seven cefiderocol-resistant isolates belonged to ST167 and six harbored bla NDM. Five of the plazomicin-resistant isolates harbored rmt. IncL plasmids were the most frequent (45.7%) and eight of these harbored bla VIM-1. bla OXA-48 was found in IncF plasmids in eight isolates. Metallo-ß-lactamases were more frequent in isolates with resistance to six or more antibiotic groups, with their genes often present on the same plasmid/integron. ST131 isolates were associated with sat and pap virulence genes. This study highlights the genetic versatility of CP-Eco and its potential to disseminate ARGs and cause community and nosocomial infections.

Corrigendum: Phenotypic and molecular characterization of IMP-producing Enterobacterales in Spain: predominance of IMP-8 in Klebsiella pneumoniae and IMP-22 in Enterobacter roggenkampii.

[This corrects the article DOI: 10.3389/fmicb.2022.1000787.].

An increase in erythromycin resistance in methicillin-susceptible Staphylococcus aureus from blood correlates with the use of macrolide/lincosamide/streptogramin antibiotics. EARS-Net Spain (2004-2020).

Objectives: To describe and analyse erythromycin resistance trends in blood isolates of Staphylococcus aureus (EARS-Net Spain, 2004-2020) and the association of these trends with the consumption of macrolide, lincosamide, and streptogramin B (MLSB) antibiotics. To assess molecular changes that could be involved in erythromycin resistance trends by whole genome analysis of representative isolates. Materials and methods: We collected antibiotic susceptibility data for all first-blood S. aureus isolates in patients from 47 Spanish hospitals according to EARS-Net criteria. MLSB antibiotic consumption was obtained from the Spanish Agency for Medicines and Medical Devices (2008-2020). We sequenced 137 representative isolates for core genome multilocus sequence typing, resistome and virulome analysis. Results: For the 36,612 invasive S. aureus isolates, methicillin resistance decreased from 26.4% in 2004 to 22.4% in 2020. Erythromycin resistance in methicillin-susceptible S. aureus (MSSA) increased from 13.6% in 2004 to 28.9% in 2020 (p < 0.001); however, it decreased from 68.7 to 61.8% (p < 0.0001) in methicillin-resistant S. aureus (MRSA). Total consumption of MLSB antibiotics increased from 2.72 defined daily doses per 1,000 inhabitants per day (DID) in 2014 to 3.24 DID in 2016. By WGS, the macrolide resistance genes detected were erm (59.8%), msrA (46%), and mphC (45.2%). The erm genes were more prevalent in MSSA (44/57, 77.2%) than in MRSA (38/80, 47.5%). Most of the erm genes identified in MSSA after 2013 differed from the predominant ermC gene (17/22, 77.3%), largely because ermT was significantly associated with MSSA after 2013 (11/29, 37.9%). All 13 ermT isolates in this study, except one, belonged to ST398 and came from 10 hospitals and six Spanish provinces. Conclusion: The significant increase in erythromycin resistance in blood MSSA correlated with the consumption of the MLSB antibiotics in Spain. These preliminary data seem support the hypothesis that the human ST398 MSSA clade with ermT-mediated resistance to erythromycin may be involved in this trend.

Widespread Detection of Yersiniabactin Gene Cluster and Its Encoding Integrative Conjugative Elements (ICEKp) among Nonoutbreak OXA-48-Producing Klebsiella pneumoniae Clinical Isolates from Spain and the Netherlands.

In this study, we determined the presence of virulence factors in nonoutbreak, high-risk clones and other isolates belonging to less common sequence types associated with the spread of OXA-48-producing Klebsiella pneumoniae clinical isolates from The Netherlands (n = 61) and Spain (n = 53). Most isolates shared a chromosomally encoded core of virulence factors, including the enterobactin gene cluster, fimbrial fim and mrk gene clusters, and urea metabolism genes (ureAD). We observed a high diversity of K-Locus and K/O loci combinations, KL17 and KL24 (both 16%), and the O1/O2v1 locus (51%) being the most prevalent in our study. The most prevalent accessory virulence factor was the yersiniabactin gene cluster (66.7%). We found seven yersiniabactin lineages-ybt 9, ybt 10, ybt 13, ybt 14, ybt 16, ybt 17, and ybt 27-which were chromosomally embedded in seven integrative conjugative elements (ICEKp): ICEKp3, ICEKp4, ICEKp2, ICEKp5, ICEKp12, ICEKp10, and ICEKp22, respectively. Multidrug-resistant lineages-ST11, ST101, and ST405-were associated with ybt 10/ICEKp4, ybt 9/ICEKp3, and ybt 27/ICEKp22, respectively. The fimbrial adhesin kpi operon (kpiABCDEFG) was predominant among ST14, ST15, and ST405 isolates, as well as the ferric uptake system kfuABC, which was also predominant among ST101 isolates. No convergence of hypervirulence and resistance was observed in this collection of OXA-48-producing K. pneumoniae clinical isolates. Nevertheless, two isolates, ST133 and ST792, were positive for the genotoxin colibactin gene cluster (ICEKp10). In this study, the integrative conjugative element, ICEKp, was the major vehicle for yersiniabactin and colibactin gene clusters spreading. IMPORTANCE Convergence of multidrug resistance and hypervirulence in Klebsiella pneumoniae isolates has been reported mostly related to sporadic cases or small outbreaks. Nevertheless, little is known about the real prevalence of carbapenem-resistant hypervirulent K. pneumoniae since these two phenomena are often separately studied. In this study, we gathered information on the virulent content of nonoutbreak, high-risk clones (i.e., ST11, ST15, and ST405) and other less common STs associated with the spread of OXA-48-producing K. pneumoniae clinical isolates. The study of virulence content in nonoutbreak isolates can help us to expand information on the genomic landscape of virulence factors in K. pneumoniae population by identifying virulence markers and their mechanisms of spread. Surveillance should focus not only on antimicrobial resistance but also on virulence characteristics to avoid the spread of multidrug and (hyper)virulent K. pneumoniae that may cause untreatable and more severe infections.

Carbapenemase-Producing Klebsiella pneumoniae in COVID-19 Intensive Care Patients: Identification of IncL-VIM-1 Plasmid in Previously Non-Predominant Sequence Types.

During the COVID-19 pandemic, intensive care units (ICUs) operated at or above capacity, and the number of ICU patients coinfected by nosocomial microorganisms increased. Here, we characterize the population structure and resistance mechanisms of carbapenemase-producing Klebsiella pneumoniae (CP-Kpn) from COVID-19 ICU patients and compare them to pre-pandemic populations of CP-Kpn. We analyzed 84 CP-Kpn isolates obtained during the pandemic and 74 CP-Kpn isolates obtained during the pre-pandemic period (2019) by whole genome sequencing, core genome multilocus sequence typing, plasmid reconstruction, and antibiotic susceptibility tests. More CP-Kpn COVID-19 isolates produced OXA-48 (60/84, 71.4%) and VIM-1 (18/84, 21.4%) than KPC (8/84, 9.5%). Fewer pre-pandemic CP-Kpn isolates produced VIM-1 (7/74, 9.5%). Cefiderocol (97.3-100%) and plazomicin (97.5-100%) had the highest antibiotic activity against pandemic and pre-pandemic isolates. Sequence type 307 (ST307) was the most widely distributed ST in both groups. VIM-1-producing isolates belonging to ST307, ST17, ST321 and ST485, (STs infrequently associated to VIM-1) were detected during the COVID-19 period. Class 1 integron Int1-blaVIM-1-aac(6')-1b-dfrB1-aadAI-catB2-qacEΔ1/sul1, found on an IncL plasmid of approximately 70,000 bp, carried blaVIM-1 in ST307, ST17, ST485, and ST321 isolates. Thus, CP-Kpn populations from pandemic and pre-pandemic periods have similarities. However, VIM-1 isolates associated with atypical STs increased during the pandemic, which warrants additional monitoring and surveillance.

Staphylococcus aureus populations from the gut and the blood are not distinguished by virulence traits-a critical role of host barrier integrity.

BACKGROUND: The opportunistic pathogen Staphylococcus aureus is an asymptomatically carried member of the microbiome of about one third of the human population at any given point in time. Body sites known to harbor S. aureus are the skin, nasopharynx, and gut. In particular, the mechanisms allowing S. aureus to pass the gut epithelial barrier and to invade the bloodstream were so far poorly understood. Therefore, the objective of our present study was to investigate the extent to which genetic differences between enteric S. aureus isolates and isolates that caused serious bloodstream infections contribute to the likelihood of invasive disease. RESULTS: Here, we present genome-wide association studies (GWAS) that compare the genome sequences of 69 S. aureus isolates from enteric carriage by healthy volunteers and 95 isolates from bloodstream infections. We complement our GWAS results with a detailed characterization of the cellular and extracellular proteomes of the representative gut and bloodstream isolates, and by assaying the virulence of these isolates with infection models based on human gut epithelial cells, human blood cells, and a small animal infection model. Intriguingly, our results show that enteric and bloodstream isolates with the same sequence type (ST1 or ST5) are very similar to each other at the genomic and proteomic levels. Nonetheless, bloodstream isolates are not necessarily associated with an invasive profile. Furthermore, we show that the main decisive factor preventing infection of gut epithelial cells in vitro is the presence of a tight barrier. CONCLUSIONS: Our data show that virulence is a highly variable trait, even within a single clone. Importantly, however, there is no evidence that blood stream isolates possess a higher virulence potential than those from the enteric carriage. In fact, some gut isolates from healthy carriers were more virulent than bloodstream isolates. Based on our present observations, we propose that the integrity of the gut epithelial layer, rather than the pathogenic potential of the investigated enteric S. aureus isolates, determines whether staphylococci from the gut microbiome will become invasive pathogens. Video Abstract.

Phenotypic and molecular characterization of IMP-producing Enterobacterales in Spain: Predominance of IMP-8 in Klebsiella pneumoniae and IMP-22 in Enterobacter roggenkampii.

Objectives: Little is known about IMP-producing Enterobacterales (IMP-Ent) in Europe. We analyzed at genomic and phenotypic level IMP-Ent isolates circulating in Spain in a 9-year period. Materials and methods: IMP-Ent isolates submitted to our reference laboratory were included. Antibiotic susceptibility was performed using microdilution method (EUCAST), and IMP-carbapenemase activity was measured with carbapenemase inhibitors, the ß-CARBA method, the modified Hodge test (MHT), and the modified carbapenemase inhibition method (mCIM). All isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis. Results: Fifty IMP-Ent isolates, collected from 19 hospitals in 13 Spanish provinces, were detected: Klebsiella pneumoniae (IMP-Kpn) (24; 48%), Enterobacter roggenkampii (13; 26%), Enterobacter hormaechei (8, 16%), Klebsiella oxytoca (two; 4%), Enterobacter asburiae (one, 2%), Serratia marcescens (one; 2%) and Escherichia coli (one; 2%). All isolates were positive by the MHT and ß-CARBA tests; 48 (96%) were mCIM positive; 12 (24%) and 26 (52%) displayed positive inhibition with dipicolinic (meropenem) and EDTA (ertapenem), respectively. Five IMP-carbapenemase types were identified: IMP-8 (22; 44%), IMP-22 (17; 34%), IMP-13 (7; 14%), IMP-28 (two; 4%), and IMP-15 (two; 4%), predominating IMP-8 in K. pneumoniae and IMP-22 in E. roggenkampii. IMP-28 was exclusively identified in K. oxytoca and IMP-15 in E. hormaechei. Predominant STs were ST405 (29.2%), ST15 (25%) and ST464 (20.8%) in IMP-Kpn; ST96 (100%) in E. roggenkampii and ST182 (62.5%) in E. hormachei. Colistin and amikacin were the most active non-carbapenem antibiotics against IMP-Ent. Conclusion: IMP-Ent isolates remain infrequent in Spain, although in recent years have been circulating causing nosocomial outbreaks, being IMP-8-producing K. pneumoniae and IMP-22-producing E. roggenkampii the most frequently detected in this study. Inhibition with EDTA or dipicolinic acid presented false negative results in some IMP-producing strains. Active microbiological and molecular surveillance is essential for a better comprehension and control of IMP-Ent dissemination.

Effects of Clinical Wastewater on the Bacterial Community Structure from Sewage to the Environment.

This study pertains to measure differences in bacterial communities along the wastewater pathway, from sewage sources through the environment. Our main focus was on taxa which include pathogenic genera, and genera harboring antibiotic resistance (henceforth referred to as "target taxa"). Our objective was to measure the relative abundance of these taxa in clinical wastewaters compared to non-clinical wastewaters, and to investigate what changes can be detected along the wastewater pathway. The study entailed a monthly sampling campaign along a wastewater pathway, and taxa identification through 16S rRNA amplicon sequencing. Results indicated that clinical and non-clinical wastewaters differed in their overall bacterial composition, but that target taxa were not enriched in clinical wastewater. This suggests that treatment of clinical wastewater before release into the wastewater system would only remove a minor part of the potential total pathogen load in wastewater treatment plants. Additional findings were that the relative abundance of most target taxa was decreased after wastewater treatment, yet all investigated taxa were detected in 68% of the treated effluent samples-meaning that these bacteria are continuously released into the receiving surface water. Temporal variation was only observed for specific taxa in surface water, but not in wastewater samples.

Predominance of CTX-M-15-producing ST131 strains among ESBL-producing Escherichia coli isolated from asylum seekers in the Netherlands.

OBJECTIVES: Numerous studies show increased prevalence of MDR bacteria amongst asylum seekers, but data on the molecular profiles of such strains are limited. We aimed to evaluate the molecular profiles of ESBL-producing Escherichia coli (ESBL-E. coli) strains isolated from asylum seekers and investigate their phylogenetic relatedness. METHODS: WGS data of ESBL-E. coli isolates from asylum seekers, retrieved from 1 January to 31 December 2016, were analysed to assess MLST STs, fim types, phylogroups and resistance genes. Fifty-two ESBL-E. coli isolates from the Dutch-German border region were used for genome comparison purposes as a control group. RESULTS: Among 112 ESBL-E. coli isolates from asylum seekers, originating mostly from Syria (n = 40) and Iraq (n = 15), the majority belonged to ST131 (21.4%) and ST10 (17.0%). The predominant gene for ß-lactam resistance was blaCTX-M-15 (67.9%), followed by the often co-detected blaTEM-1B (39.3%). No mcr or carbapenemase genes were detected. The majority of the strains belonged to phylogroups B2 (38.4%) and A (32.1%), carrying fimH27 (25%) and fimH30 (19.6%). A core genome MLST minimum spanning tree did not reveal clusters containing strains from the asylum seekers and the control group. Five clusters were formed within the asylum seeker group, by strains isolated from people originating from different countries. CONCLUSIONS: The most frequently isolated clones in this study were isolated on a regular basis within the Dutch population before the increase in the asylum seeker population. No mcr- or carbapenemase-producing clones were detected among the asylum seeker population. Minor clustering was observed amongst the asylum seeker strains.

Farm dust resistomes and bacterial microbiomes in European poultry and pig farms.

BACKGROUND: Livestock farms are a reservoir of antimicrobial resistant bacteria from feces. Airborne dust-bound bacteria can spread across the barn and to the outdoor environment. Therefore, exposure to farm dust may be of concern for animals, farmers and neighboring residents. Although dust is a potential route of transmission, little is known about the resistome and bacterial microbiome of farm dust. OBJECTIVES: We describe the resistome and bacterial microbiome of pig and poultry farm dust and their relation with animal feces resistomes and bacterial microbiomes, and on-farm antimicrobial usage (AMU). In addition, the relation between dust and farmers' stool resistomes was explored. METHODS: In the EFFORT-study, resistomes and bacterial microbiomes of indoor farm dust collected on Electrostatic Dust fall Collectors (EDCs), and animal feces of 35 conventional broiler and 44 farrow-to-finish pig farms from nine European countries were determined by shotgun metagenomic analysis. The analysis also included 79 stool samples from farmers working or living at 12 broiler and 19 pig farms and 46 human controls. Relative abundance of and variation in resistome and bacterial composition of farm dust was described and compared to animal feces and farmers' stool. RESULTS: The farm dust resistome contained a large variety of antimicrobial resistance genes (ARGs); more than the animal fecal resistome. For both poultry and pigs, composition of dust resistomes finds (partly) its origin in animal feces as dust resistomes correlated significantly with fecal resistomes. The dust bacterial microbiome also correlated significantly with the dust resistome composition. A positive association between AMU in animals on the farm and the total abundance of the dust resistome was found. Occupational exposure to pig farm dust or animal feces may contribute to farmers' resistomes, however no major shifts in farmers resistome towards feces or dust resistomes were found in this study. CONCLUSION: Poultry and pig farm dust resistomes are rich and abundant and associated with the fecal resistome of the animals and the dust bacterial microbiome.

Description and determinants of the faecal resistome and microbiome of farmers and slaughterhouse workers: A metagenome-wide cross-sectional study.

BACKGROUND: By studying the entire human faecal resistome and associated microbiome, the diversity and abundance of faecal antimicrobial resistance genes (ARGs) can be comprehensively characterized. Prior culture-based studies have shown associations between occupational exposure to livestock and carriage of specific antimicrobial resistant bacteria. Using shotgun metagenomics, the present study investigated 194 faecal resistomes and bacteriomes from humans occupationally exposed to ARGs in livestock (i.e. pig and poultry farmers, employees and family members and pig slaughterhouse workers) and a control population (Lifelines cohort) in the Netherlands. In addition, we sought to identify determinants for the human resistome and bacteriome composition by applying a combination of multivariate (NMDS, PERMANOVA, SIMPER and DESeq2 analysis) and multivariable regression analysis techniques. RESULTS: Pig slaughterhouse workers and pig farmers carried higher total ARG abundances in their stools compared to broiler farmers and control subjects. Tetracycline, ß-lactam and macrolide resistance gene clusters dominated the resistome of all studied groups. No significant resistome alpha diversity differences were found among the four populations. However, the resistome beta diversity showed a separation of the mean resistome composition of pig and pork exposed workers from broiler farmers and controls, independent of their antimicrobial use. We demonstrated differences in resistome composition between slaughter line positions, pig versus poultry exposed workers, as well as differences between farmers and employees versus family members. In addition, we found a significant correlation between the bacteriome and resistome, and significant differences in the bacteriome composition between and within the studied subpopulations. Finally, an in-depth analysis of pig and poultry farms - of which also farm livestock resistomes were analysed - showed positive associations between the number of on-farm working hours and human faecal AMR loads. CONCLUSION: We found that the total normalized faecal ARG carriage was larger in persons working in the Dutch pork production chain compared to poultry farmers and controls. Additionally, we showed significant differences in resistome and bacteriome composition of pig and pork exposed workers compared to a control group, as well as within-population (farms, slaughterhouse) compositional differences. The number of on-farm working hours and the farm type (pig or broiler) that persons live or work on are determinants for the human faecal resistome. Overall, our results may suggest direct or indirect livestock contact as a determinant for human ARG carriage. Future studies should further focus on the connection between the human and livestock resistome (i.e. transmission routes) to substantiate the evidence for livestock-associated resistome acquisition.

Abundance and Antimicrobial Resistance of Three Bacterial Species along a Complete Wastewater Pathway.

After consumption, antibiotic residues and exposed bacteria end up via the feces in wastewater, and therefore wastewater is believed to play an important role in the spread of antimicrobial resistance (AMR). We investigated the abundance and AMR profiles of three different species over a complete wastewater pathway during a one-year sampling campaign, as well as including antimicrobial consumption and antimicrobial concentrations analysis. A total of 2886 isolates (997 Escherichia coli, 863 Klebsiella spp., and 1026 Aeromonas spp.) were cultured from the 211 samples collected. The bacterial AMR profiles mirrored the antimicrobial consumption in the respective locations, which were highest in the hospital. However, the contribution of hospital wastewater to AMR found in the wastewater treatment plant (WWTP) was below 10% for all antimicrobials tested. We found high concentrations (7-8 logs CFU/L) of the three bacterial species in all wastewaters, and they survived the wastewater treatment (effluent concentrations were around 5 log CFU/L), showing an increase of E. coli in the receiving river after the WWTP discharge. Although the WWTP had no effect on the proportion of AMR, bacterial species and antimicrobial residues were still measured in the effluent, showing the role of wastewater contamination in the environmental surface water.

Revealing the Virulence Potential of Clinical and Environmental Aspergillus fumigatus Isolates Using Whole-Genome Sequencing.

Aspergillus fumigatus is considered a common causative agent of human fungal infections. A restricted number of virulence factors have been described, and none of them lead to a differentiation in the virulence level among different strains. Variations in the virulence phenotype depending on the isolate origin, measured as survival percentage in animal infection models, have been previously reported. In this study, we analyzed the whole-genome sequence of A. fumigatus isolates from clinical and environmental origins to determine their virulence genetic content. The sample included four isolates sequenced at the University Medical Center Groningen (UMCG), three clinical (two of them isolated from the same patient) and the experimental strain B5233, and the draft genomes of one reference strain, two environmental and two clinical isolates obtained from a public database. The fungal genomes were screened for the presence of virulence-related genes (VRGs) using an in-house database of 244 genes related to thermotolerance, resistance to immune responses, cell wall formation, nutrient uptake, signaling and regulation, and production of toxins and secondary metabolites and allergens. In addition, we performed a variant calling analysis to compare the isolates sequenced at the UMCG and investigated their genetic relatedness using the TRESP (Tandem Repeats located within Exons of Surface Protein coding genes) genotyping method. We neither observed a difference in the virulence genetic content between the clinical isolates causing an invasive infection and a colonizing clinical isolate nor between isolates from the clinical and environmental origin. The four novel A. fumigatus sequences had a different TRESP genotype and a total number of genetic variants ranging from 48,590 to 68,352. In addition, a comparative genomics analysis showed the presence of single nucleotide polymorphisms in VRGs and repetitive genetic elements located next to VRG groups, which could influence the regulation of these genes. In conclusion, our genomic analysis revealed a high genetic diversity between environmental and clinical A. fumigatus isolates, as well as between clinical isolates from the same patient, indicating an infection with a mixed-population in the latter case. However, all isolates had a similar virulence genetic content, demonstrating their pathogenic potential at least at the genomic level.

Decreasing prevalence of contamination with extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) in retail chicken meat in the Netherlands.

Retail chicken meat is a potential source of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E). In the past decade, vast national efforts were undertaken to decrease the antibiotic use in the veterinary sector, resulting in a 58% decrease in antibiotic sales in the sector between 2009 and 2014. This decrease in antibiotic use was followed by a decrease in ESBL-E prevalence in broilers. The current study investigates the prevalence of contamination with ESBL-E in retail chicken meat purchased in the Netherlands between December 2013 and August 2015. It looks at associations between the prevalence of contamination with ESBL-E and sample characteristics such as method of farming (free-range or conventional), supermarket chain of purchase and year of purchase. In the current study, 352 chicken meat samples were investigated for the presence of ESBL-E using selective culture methods. Six samples were excluded due to missing isolates or problems obtaining a good quality sequence leaving 346 samples for further analyses. Of these 346 samples, 188 (54.3%) were positive for ESBL-E, yielding 216 ESBL-E isolates (Escherichia coli (n = 204), Klebsiella pneumoniae (n = 11) and Escherichia fergusonii (n = 1)). All ESBL-E isolates were analysed using whole-genome sequencing. The prevalence of contamination with ESBL-E in retail chicken meat decreased from 68.3% in 2014 to 44.6% in 2015, absolute risk difference 23.7% (95% confidence interval (CI): 12.6% - 34.1%). The ESBL-E prevalence was lower in free-range chicken meat (36.4%) compared with conventional chicken meat (61.5%), absolute risk difference 25.2% (95% CI: 12.9% - 36.5%). The prevalence of contamination with ESBL-E varied between supermarket chains, the highest prevalence of contamination was found in supermarket chain 4 (76.5%) and the lowest in supermarket chain 1 (37.8%). Pairwise isolate comparisons using whole-genome multilocus sequence typing (wgMLST) showed that clustering of isolates occurs more frequently within supermarket chains than between supermarket chains. In conclusion, the prevalence of contamination with ESBL-E in retail chicken in the Netherlands decreased over time; nevertheless, it remains substantial and as such a potential source for ESBL-E in humans.

Extended-spectrum beta-lactamase producing Enterobacteriaceae (ESBL-E) isolated from bean sprouts in the Netherlands.

Community-acquired carriage and infections due to extended-spectrum beta-lactamase producing Enterobacteriaceae (ESBL-E) are increasing worldwide, resulting in increased morbidity, mortality and healthcare costs. The origins of community-acquired ESBL-E carriage and infections remain unclear. Bean sprouts are a potential source of Enterobacteriaceae for the community, as illustrated by outbreaks of pathogenic Enterobacteriaceae in the past. The current study focuses on contamination of retail bean sprouts with ESBL-E in the Netherlands. Of 131 bean sprout samples purchased between 2013 and 2016, 25 (19%) were contaminated with ESBL-E. The detected isolates were almost exclusively Klebsiella spp. and co-resistance to other antibiotics was observed frequently. Over time there was substantial genetic diversity between isolates. On the other hand, isolates from samples closely matched in time were frequently clonally related, indicative of batch contamination. Remarkably, no Escherichia coli was found. In conclusion, bean sprouts frequently harbor ESBL-E, which is a potential source for consumers.

Detection of Legionella Anisa in Water from Hospital Dental Chair Units and Molecular Characterization by Whole-Genome Sequencing.

This study aims to assess contamination with Legionella spp. in water from dental chair units (DCUs) of a hospital dental ward and to perform its molecular characterization by whole-genome sequencing (WGS). We collect eight water samples (250 mL) from four DCUs (sink and water-syringe). Samples are tested for the presence of Legionella spp. (CFUs/mL) by culturing according to the Nederland Norm (NEN) 6265. Three DCUs are found positive for Legionella anisa, and four isolates are cultured (sink n = 2, water-syringe n = 1; two isolates from the same chair) with 1 × 10² CFU/mL. Whole-genome multi-locus sequence typing (wgMLST) results indicate that all strains belong to the same cluster with two to four allele differences. Classical culture combined with WGS allows the identification of a unique clone of L. anisa in several DCUs in the same hospital dental ward. This may indicate a common contamination source in the dental unit waterlines, which was fixed by replacing the chairs and main pipeline of the unit. Our results reveal tap water contamination in direct contact with patients and the usefulness of WGS to investigate bacterial molecular epidemiology.

Epidemiology of Extended-Spectrum ß-Lactamase-Producing E. coli and Vancomycin-Resistant Enterococci in the Northern Dutch-German Cross-Border Region.

Objectives: To reveal the prevalence and epidemiology of extended-spectrum ß-lactamase (ESBL)- and/or plasmid AmpC (pAmpC)- and carbapenemase (CP) producing Enterobacteriaceae and vancomycin-resistant enterococci (VRE) across the Northern Dutch-German border region. Methods: A point-prevalence study on ESBL/pAmpC/CP producing Enterobacteriaceae and VRE was carried out in hospitalized patients in the Northern Netherlands (n = 445, 2012-2013) and Germany (n = 242, 2012). Healthy individuals from the Dutch community (n = 400, 2010-2012) were also screened. In addition, a genome-wide gene-by-gene approach was applied to study the epidemiology of ESBL-Escherichia coli and VRE. Results: A total of 34 isolates from 27 patients (6.1%) admitted to Dutch hospitals were ESBL/pAmpC positive and 29 ESBL-E. coli, three pAmpC-E. coli, one ESBL-Enterobacter cloacae, and one pAmpC-Proteus mirabilis were found. In the German hospital, 18 isolates (16 E. coli and 2 Klebsiella pneumoniae) from 17 patients (7.7%) were ESBL positive. In isolates from the hospitalized patients CTX-M-15 was the most frequently detected ESBL-gene. In the Dutch community, 11 individuals (2.75%) were ESBL/pAmpC positive: 10 ESBL-E. coli (CTX-M-1 being the most prevalent gene) and one pAmpC E. coli. Six Dutch (1.3%) and four German (3.9%) hospitalized patients were colonized with VRE. Genetic relatedness by core genome multi-locus sequence typing (cgMLST) was found between two ESBL-E. coli isolates from Dutch and German cross-border hospitals and between VRE isolates from different hospitals within the same region. Conclusion: The prevalence of ESBL/pAmpC-Enterobacteriaceae was similar in hospitalized patients across the Dutch-German border region, whereas VRE prevalence was slightly higher on the German side. The overall prevalence of the studied pathogens was lower in the community than in hospitals in the Northern Netherlands. Cross-border transmission of ESBL-E. coli and VRE seems unlikely based on cgMLST analysis, however continuous monitoring is necessary to control their spread and stay informed about their epidemiology.