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.

Phylogeny, resistome and mobile genetic elements of emergent OXA-48 and OXA-245 Klebsiella pneumoniae clones circulating in Spain.

OBJECTIVES: The global emergence of OXA-48-producing Klebsiella pneumoniae clones is a significant threat to public health. We used WGS and phylogenetic analysis of Spanish isolates to investigate the population structure of blaOXA-48-like-expressing K. pneumoniae ST11 and ST405 and to determine the distribution of resistance genes and plasmids encoding blaOXA-48-like carbapenemases. METHODS: SNPs identified in whole-genome sequences were used to reconstruct phylogenetic trees, identify resistance determinants and de novo assemble the genomes of 105 blaOXA-48-like-expressing K. pneumoniae isolates. RESULTS: Genome variation was generally lower in outbreak-associated isolates compared with those associated with sporadic infections. The relatively limited variation observed within the outbreak-associated isolates was on average 7-10 SNPs per outbreak. Of 24 isolates from suspected sporadic infections, 7 were very closely related to isolates causing hospital outbreaks and 17 were more diverse and therefore probably true sporadic cases. On average, 14 resistance genes were identified per isolate. The 17 ST405 isolates from sporadic cases of infection had four distinct resistance gene profiles, while the resistance gene profile differed in all ST11 isolates from sporadic cases. Sequence analysis of 94 IncL/M plasmids carrying blaOXA-48-like genes revealed an average of two SNP differences, indicating a conserved plasmid clade. CONCLUSIONS: Whole-genome sequence analysis enabled the discrimination of outbreak and sporadic isolates. Significant inter-regional spread within Spain of highly related isolates was evident for both ST11 and ST405 K. pneumoniae. IncL/M plasmids carrying blaOXA-48-like carbapenemase genes were highly conserved geographically and across the outbreaks, sporadic cases and clones.

Isolates of ß-lactamase-negative ampicillin-resistant Haemophilus influenzae causing invasive infections in Spain remain susceptible to cefotaxime and imipenem.

OBJECTIVES: The epidemiology of invasive Haemophilus influenzae has changed in recent years. ß-Lactamase-negative ampicillin-resistant (BLNAR) invasive isolates have recently been described in Europe but their clinical significance is unclear. Our main goal was to determine whether invasive H. influenzae remains susceptible to ß-lactam antibiotics indicated in the treatment of invasive infections. METHODS: The antibiotic susceptibility of 307 invasive H. influenzae isolates to seven ß-lactam antibiotics was determined by microdilution and interpreted by EUCAST and CLSI breakpoints. We also identified the bla genes, the amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3), the molecular epidemiology of invasive BLNAR isolates by PFGE and MLST, and the time-kill curves of two isolates with PBP3 mutations conferring reduced susceptibility to aminopenicillins and cephalosporins. RESULTS: Of the invasive isolates, 86.6% were non-typeable and 62% were isolated from adults. Decreased susceptibility to ß-lactams was due to the BLNAR genotype (gBLNAR; 19.2%) and to ß-lactamase production (16.9%). Susceptibility rates to amoxicillin/clavulanic acid, cefotaxime, cefixime and imipenem were greater than 98%. Of 18 gBLNAR non-typeable isolates studied by MLST, 15 different STs were obtained. Amoxicillin and cefotaxime were bactericidal after 2 and 4 h of incubation, respectively. CONCLUSIONS: Invasive H. influenzae disease was mainly due to non-typeable isolates infecting adults, and the most common mechanism of ß-lactam resistance was mutations in the transpeptidase domain of PBP3. The gBLNAR non-typeable isolates were genetically diverse. The majority of invasive H. influenzae remained susceptible to third-generation cephalosporins; amoxicillin and cefotaxime were bactericidal in two gBLNAR isolates.

Frequent carriage of resistance mechanisms to ß-lactams and biofilm formation in Haemophilus influenzae causing treatment failure and recurrent otitis media in young children.

OBJECTIVES: Non-typeable Haemophilus influenzae are a major cause of acute otitis media (AOM), including chronic and recurrent otitis in young children. The objective of this study was to determine whether non-typeable H. influenzae isolates causing these infections produce biofilms and carry resistance mechanisms to ß-lactams. METHODS: A collection of 48 H. influenzae isolates was obtained by tympanocentesis or from otorrhoea samples from individual patients <3 years of age and diagnosed with recurrent or treatment failure AOM. Each isolate was surveyed for the presence of blaTEM genes, amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3) and biofilm formation in microtitre plates. RESULTS: In 43 of the 48 isolates (89.6%), at least one of the three tested conditions was identified: biofilm formation (83.3%) and resistance mechanisms to ß-lactams (33.3%), modifications in the transpeptidase domain of PBP3 being the most prevalent (22.9%), followed by ß-lactamase production (10.4%). Additionally, 13 (27.1%) isolates had two or more of these three traits. In relation to biofilm formation, those isolates with an amoxicillin MIC ≤ 0.5 mg/L had higher optical density values than isolates with an amoxicillin MIC ≥ 1 mg/L (Mann-Whitney U-test, P=0.048). CONCLUSIONS: These findings suggest that the successful treatment of non-typeable H. influenzae causing chronic and recurrent AOM in young children may be compromised by the high biofilm-forming capacity of the isolates and the presence of ß-lactam resistance mechanisms, particularly PBP3 mutations.

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.

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.

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.

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.

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.

Significant ecological impact on the progression of fluoroquinolone resistance in Escherichia coli with increased community use of moxifloxacin, levofloxacin and amoxicillin/clavulanic acid.

OBJECTIVES: To determine trends in ciprofloxacin resistance and co-resistance to other antibiotic classes in blood isolates of Escherichia coli, and to investigate if there is an ecological relationship to the community use of fluoroquinolones and other antibiotics. METHODS: Forty-two Spanish hospitals of the European Antimicrobial Resistance Surveillance Network collected ciprofloxacin and other antibiotic susceptibility data for non-duplicate consecutive E. coli isolates from patients with bacteraemia between 2001 and 2009. The nationwide ambulatory use of antibiotics between 1997 and 2008 was determined by WHO methods, and the co-evolution of both parameters was further analysed. RESULTS: Of the 28 307 E. coli blood isolates, 27.9% were ciprofloxacin non-susceptible (CIPNS), increasing from 17.6% in 2001 to 32.7% in 2009. A continuous increase was observed between CIPNS and other resistances, including cephalosporin resistance due to the production of extended-spectrum ß-lactamases (ESBLs) and non-susceptibility to both amoxicillin/clavulanic acid and tobramycin. Although the total use of antibiotics did not increase, community use of levofloxacin, moxifloxacin and amoxicillin/clavulanic acid increased by 307.2%, 62.6% and 70.1%, respectively. Yearly rates of CIPNS E. coli strongly correlated with the use of levofloxacin, moxifloxacin and amoxicillin/clavulanic acid (r(2 )> 0.80; P < 0.005 in all cases). CONCLUSIONS: The rapid increase in CIPNS E. coli causing bacteraemia was closely related to the increase in resistance to amoxicillin/clavulanic acid, production of ESBLs and resistance to aminoglycosides. Community use of fluoroquinolones (mainly moxifloxacin and levofloxacin) and of amoxicillin/clavulanic acid represents a significant driver in the progression of fluoroquinolone resistance in bacteraemic E. coli.

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.

Haemophilus influenzae clinical isolates with plasmid pB1000 bearing blaROB-1: fitness cost and interspecies dissemination.

Plasmid pB1000 is a mobilizable replicon bearing the bla(ROB-1) beta-lactamase gene that we have recently described in Haemophilus parasuis and Pasteurella multocida animal isolates. Here we report the presence of pB1000 and a derivative plasmid, pB1000', in four Haemophilus influenzae clinical isolates of human origin. Pulsed-field gel electrophoresis showed unrelated patterns in all strains, indicating that the existence of pB1000 in H. influenzae isolates is not the consequence of clonal dissemination. The replicon can be transferred both by transformation and by conjugation into H. influenzae, giving rise to recipients resistant to ampicillin and cefaclor (MICs, > or =64 microg/ml). Stability experiments showed that pB1000 is stable in H. influenzae without antimicrobial pressure for at least 60 generations. Competition experiments between isogenic H. influenzae strains with and without pB1000 revealed a competitive disadvantage of 9% per 10 generations for the transformant versus the recipient. The complete nucleotide sequences of nine pB1000 plasmids from human and animal isolates, as well as the epidemiological data, suggest that animal isolates belonging to the Pasteurellaceae act as an antimicrobial resistance reservoir for H. influenzae. Further, since P. multocida is the only member of this family that can colonize both humans and animals, we propose that P. multocida is the vehicle for the transport of pB1000 between animal- and human-adapted members of the Pasteurellaceae.

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.

CTX-M-15-producing urinary Escherichia coli O25b-ST131-phylogroup B2 has acquired resistance to fosfomycin.

OBJECTIVES: To describe trends in fosfomycin resistance in urinary isolates of Escherichia coli producing extended-spectrum beta-lactamases (ESBLs) in relation to fosfomycin consumption and to characterize representative fosfomycin-resistant isolates. METHODS: In 2007-08, an unexpected increase in fosfomycin resistance in ESBL-producing urinary E. coli was observed. Laboratory records were reviewed and a prospective surveillance study was initiated on all urinary tract infections caused by ESBL-producing, fosfomycin-resistant E. coli. bla(ESBL) types, phylogroups, genetic environment and afa/dra operon were determined by PCR and sequencing. Molecular epidemiology was analysed by PFGE and multilocus sequence typing. To elucidate possible mechanisms of fosfomycin resistance, uhpT, glpT, uhpA, ptsI, cyaA and murA genes were analysed. Fosfomycin consumption was determined as recommended by WHO. RESULTS: From 2004 to 2008, fosfomycin consumption increased by 50%, while fosfomycin resistance in ESBL producers increased from 2.2% to 21.7%. Of 26 isolates studied, 24 produced CTX-M-15 and belonged to the O25b-ST131-phylogroup B2 clonal strain. PFGE revealed two clusters. Cluster I included 18 isolates, 16 of them indistinguishable from strains producing CTX-M-15 previously described in Madrid. The five isolates of Cluster II had the IS26 linked to bla(CTX-M-15) and the afa/dra operon. In Cluster I isolates, no mutations in glpT, uhpT, uhpA, ptsI, cyaA and murA were detected. Cluster II isolates showed a 15 bp deletion (A(169)-C(183)) in uhpA. CONCLUSIONS: Fosfomycin resistance in urinary E. coli has increased due to the acquisition of this resistance by a previously circulating CTX-M-15-producing E. coli O25b-ST131-phylogroup B2 strain. This happened during a period when the use of fosfomycin increased by 50%.

Emergence of CTX-M-15-producing Klebsiella pneumoniae of multilocus sequence types 1, 11, 14, 17, 20, 35 and 36 as pathogens and colonizers in newborns and adults.

OBJECTIVES: To characterize the population structure and resistance mechanisms of Klebsiella pneumoniae isolates that are highly resistant to third-generation cephalosporins, collected from five Spanish hospitals. METHODS: A total of 162 K. pneumoniae isolates from five hospitals located in three geographical areas of Spain were characterized. The number of isolates from each hospital ranged from 3 to 82. The genetic relationship between isolates was established by PFGE and multilocus sequence typing (MLST). bla(ESBL) types and other antibiotic resistance genes were analysed by PCR and sequencing. Plasmids were classified according to their incompatibility group by a PCR-based replicon-typing scheme. RESULTS: All 162 isolates carried the bla(CTX-15) gene. Fifty-eight isolates (35.8%) caused clinical infections and 104 (64.2%) were colonizers. Sixty-nine (42.6%) isolates were collected from newborns and 93 (57.4%) from adults. Using PGFE, the 162 isolates were grouped into seven clusters that were further identified as members of the MLST types 1, 11, 14, 17, 20, 35 and 36. Two hospitals each had two different clones and the remaining three hospitals had a single CTX-M-15-producing K. pneumoniae clone. All clones carried different antibiotic resistance genes, including bla(OXA-1), aac(3)-IIa, aac(6')-Ib-cr, qnrS1 and qnrB. In four of the seven (57.1%) clones the bla(CTX-M-15) gene was transferred by conjugation; in all cases plasmids of the incompatibility group IncF were identified by PCR. CONCLUSIONS: This study shows that multiresistant K. pneumoniae producing CTX-M-15 of MLST types 1, 11, 14, 17, 20, 35 and 36 are spreading as pathogens and colonizers among newborns and adult patients in Spain.

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.