Distribution of Kudoa thyrsites (Cnidaria, Myxozoa) myoliquefactive stages in Northeast Atlantic mackerel (Scomber scombrus) inferred from qPCR and histology.

Kudoa thyrsites is a myxosporean parasite (Cnidaria, Myxozoa) that infects the skeletal and cardiac muscle of Northeast Atlantic (NEA) mackerel (Scomber scombrus). Heavy infections are associated with post-mortem myoliquefaction of the host skeletal muscle which reduces the quality of the fish product. The biological infection characteristics of the parasite in NEA mackerel are poorly known. This study examined the distribution of K. thyrsites in various organs of NEA mackerel from the northern North Sea, and elucidates the relationship between density of infection, developmental stage and parasite distribution in the musculature, and the extent of visible flesh myoliquefaction. Quantitative polymerase chain reaction (qPCR) data showed that K. thyrsites is unevenly distributed in the somatic musculature of the fish host, with highest density in the anterior ventral muscle sections-the belly flaps. A weak positive correlation was observed between the level of myoliquefaction and the parasite density in the fish host muscle. This relationship was also reflected by the amount and distribution of parasite developmental stages seen during histological examinations. Histological findings indicate an association between the dispersion of free myxospores and the level of myoliquefaction of the fish host muscle. Visceral organs were also found infected using qPCR, although at lower densities compared to the musculature.

Resistance profiles and diversity of ß-lactamases in Escherichia coli strains isolated from city-scale sewage surveillance in Bergen, Norway mimic clinical prevalence.

The aim of this study was to examine antibiotic resistance profiles and diversity of ß-lactamases in Escherichia coli present within the population and the potential spread of resistant E. coli into the receiving environment using city-scale sewage surveillance. In E. coli isolates from ECC plates without antibiotics from ten influent samples (n = 300), highest resistance was observed against ampicillin (16.6%), sulfamethoxazole (9.7%) and trimethoprim (9.0%), while in effluent samples (n = 262) it was against sulfamethoxazole (11.8%), ampicillin (11.5%) and tetracycline (8.8%). All isolates (n = 123) obtained on cefotaxime-containing plates were multidrug-resistant. Several clinically important antibiotic resistance genes (ARGs) were detected in 46 E. coli isolates subjected to whole-genome sequencing, including carbapenemases like NDM-6, VIM-1 and OXA-48-variant, as well as tigecycline resistance gene tet(X4). CTX-M-15 was the most prevalent (42.9%) extended-spectrum ß-lactamase among cefotaxime-resistant isolates, followed by CTX-M-27 (31.4%) and CTX-M-14 (17.1%), resembling clinical prevalence in Norway. Most of the sequenced isolates carried other clinically relevant ARGs, such as dfrA17, sul1, sul2, tet(A), aph(6)-Id, aph(3'')-Ib and aadA5. Sixteen different sequence types (STs) were identified, including ST131 (39.1%), ST38 (10.9%) and ST69 (8.7%). One E. coli isolate belonging to novel ST (ST11874) carried multiple virulence factors including genotoxin, salmochelin, aerobactin and yersiniabactin, suggesting that this isolate has potential to cause health concerns in future. Our study reveals presence of clinically relevant ARGs like blaNDM-6 and tet(X4) in pathogenic strains, which have so far not been reported from the clinics in Norway. Our study may thus, provide a framework for population-based surveillance of antibiotic resistance.

Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1.

BACKGROUND: MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to ß-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure-activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure. OBJECTIVES: To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1. METHODS: The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different ß-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1). RESULTS: Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity. CONCLUSIONS: These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.

Nanopore sequencing reveals genomic map of CTX-M-type extended-spectrum ß-lactamases carried by Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway.

BACKGROUND: Environmental surveillance of antibiotic resistance can contribute towards better understanding and management of human and environmental health. This study applied a combination of long-read Oxford Nanopore MinION and short-read Illumina MiSeq-based sequencing to obtain closed complete genome sequences of two CTX-M-producing multidrug-resistant Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway, in order to understand the potential for mobility of the detected antibiotic resistance genes (ARGs). RESULTS: The complete genome sequence of strain 631 (E. coli sequence type 38) was assembled into a circular chromosome of 5.19 Mb and five plasmids (between 98 kb and 5 kb). The majority of ARGs cluster in close proximity to each other on the chromosome within two separate multidrug-resistance determining regions (MDRs), each flanked by IS26 transposases. MDR-1 carries blaTEM-1, tmrB, aac(3)-IId, aadA5, mph(A), mrx, sul1, qacEΔ1 and dfrA17; while MDR-2 harbors aph(3″)-Ib, aph(6)-Id, blaTEM-1, catA1, tet(D) and sul2. Four identical chromosomal copies of blaCTX-M-14 are located outside these regions, flanked by ISEc9 transposases. Strain 1500 (E. coli sequence type 191) exhibited a circular chromosome of 4.73 Mb and two plasmids (91 kb and 4 kb). The 91 kb conjugative plasmid belonging to IncI1 group carries blaCTX-M-15 and blaTEM-1 genes. CONCLUSION: This study confirms the efficacy of combining Nanopore long-read and Illumina short-read sequencing for determining complete bacterial genome sequences, enabling detection and characterization of clinically important ARGs in the marine environment in Norway, with potential for further dissemination. It also highlights the need for environmental surveillance of antibiotic resistance in low prevalence settings like Norway.

Molecular profiling of mucosal tissue associated microbiota in patients manifesting acute exacerbations and remission stage of ulcerative colitis.

Dysbiosis of intestinal microflora has been postulated in ulcerative colitis (UC), which is characterized by imbalance of mucosal tissue associated bacterial communities. However, the specific changes in mucosal microflora during different stages of UC are still unknown. The aim of the current study was to investigate the changes in mucosal tissue associated microbiota during acute exacerbations and remission stages of UC. The mucosal microbiota associated with colon biopsy of 12 patients suffering from UC (exacerbated stage) and the follow-up samples from the same patients (remission stage) as well as non-IBD subjects was studied using 16S rRNA gene-based sequencing and quantitative PCR. The total bacterial count in patients suffering from exacerbated phase of UC was observed to be two fold lower compared to that of the non-IBD subjects (p = 0.0049, Wilcox on matched-pairs signed rank tests). Bacterial genera including Stenotrophomonas, Parabacteroides, Elizabethkingia, Pseudomonas, Micrococcus, Ochrobactrum and Achromobacter were significantly higher in abundance during exacerbated phase of UC as compared to remission phase. The alterations in bacterial diversity with an increase in the abnormal microbial communities signify the extent of dysbiosis in mucosal microbiota in patients suffering from UC. Our study helps in identifying the specific genera dominating the microbiota during the disease and thus lays a basis for further investigation of the possible role of these bacteria in pathogenesis of UC.

Computational discovery and functional validation of novel fluoroquinolone resistance genes in public metagenomic data sets.

BACKGROUND: Fluoroquinolones are broad-spectrum antibiotics used to prevent and treat a wide range of bacterial infections. Plasmid-mediated qnr genes provide resistance to fluoroquinolones in many bacterial species and are increasingly encountered in clinical settings. Over the last decade, several families of qnr genes have been discovered and characterized, but their true prevalence and diversity still remain unclear. In particular, environmental and host-associated bacterial communities have been hypothesized to maintain a large and unknown collection of qnr genes that could be mobilized into pathogens. RESULTS: In this study we used computational methods to screen genomes and metagenomes for novel qnr genes. In contrast to previous studies, we analyzed an almost 20-fold larger dataset comprising almost 13 terabases of sequence data. In total, 362,843 potential qnr gene fragments were identified, from which 611 putative qnr genes were reconstructed. These gene sequences included all previously described plasmid-mediated qnr gene families. Fifty-two of the 611 identified qnr genes were reconstructed from metagenomes, and 20 of these were previously undescribed. All of the novel qnr genes were assembled from metagenomes associated with aquatic environments. Nine of the novel genes were selected for validation, and six of the tested genes conferred consistently decreased susceptibility to ciprofloxacin when expressed in Escherichia coli. CONCLUSIONS: The results presented in this study provide additional evidence for the ubiquitous presence of qnr genes in environmental microbial communities, expand the number of known qnr gene variants and further elucidate the diversity of this class of resistance genes. This study also strengthens the hypothesis that environmental bacterial communities act as sources of previously uncharacterized qnr genes.

Draft Genome Sequences of Yersinia pestis Strains from the 1994 Plague Epidemic of Surat and 2002 Shimla Outbreak in India.

We report the first draft genome sequences of the strains of plague-causing bacteria, Yersinia pestis, from India. These include two strains from the Surat epidemic (1994), one strain from the Shimla outbreak (2002) and one strain from the plague surveillance activity in the Deccan plateau region (1998). Genome size for all four strains is ~4.49 million bp with 139-147 contigs. Average sequencing depth for all four genomes was 21x.

Tigecycline-resistant Klebsiella pneumoniae strains from sewage in Norway carry heavy-metal resistance genes encoding conjugative plasmids.

OBJECTIVES: Tigecycline is a last-resort antibiotic used for treatment of infections with carbapenem-resistant Klebsiella pneumoniae. The aim of the study was to understand the genetic mechanism of resistance and the genetic context of resistance genes in two tigecycline-resistant K. pneumoniae strains isolated from sewage in Bergen, Norway. METHODS: Complete genome sequencing of the two strains was accomplished using a combination of short-read Illumina MiSeq-based and long-read Oxford Nanopore MinION-based sequencing. Conjugation experiments were performed using filter mating and a green fluorescent protein (GFP)-tagged Escherichia coli strain. RESULTS: The complete genome sequences of strain K6-320.1 and strain K7-325 were assembled into two contigs for each strain, one contig representing the complete circular chromosomes of 5 223 440 bp (K6-320.1) and 5 263 092 bp (K7-325), respectively, and the other representing plasmids with sizes of 276 509 bp (pK6-320.1) and 246 731 bp (pK7-325). Strain K6-320.1 belonged to sequence type (ST)869, whereas strain K7-325 belonged to the pathogenic ST307. Both plasmids belonged to the IncFIB(K)/IncFII(K) group and carried several antibiotic resistance genes (ARGs), including tet(A) and blaCTX-M. Both plasmids (pK6-320.1 and pK7-325) were transferred to a GFP-tagged E. coli strain, leading to the acquisition of resistance against multiple classes of antibiotics. Several heavy-metal resistance genes (HMRGs) encoding resistance against silver (sil), copper (pco), and arsenic (ars) were also present on both plasmids. CONCLUSIONS: Our study demonstrates the presence of multidrug-resistant K. pneumoniae strains carrying conjugative plasmids encoding both ARGs and HMRGs that have potential for persistence in the environment and human microbiota.

Multidrug-resistant Enterococcus faecium strains enter the Norwegian marine environment through treated sewage.

This study aimed to understand the antibiotic resistance prevalence among Enterococcus spp. from raw and treated sewage in Bergen city, Norway. In total, 517 Enterococcus spp. isolates were obtained from raw and treated sewage from five sewage treatment plants (STPs) over three sampling occasions, with Enterococcus faecium as the most prevalent (n = 492) species. E. faecium strains (n = 307) obtained from the influent samples, showed the highest resistance against quinupristin/dalfopristin (67.8%). We observed reduced susceptibility to erythromycin (30.6%) and tetracycline (6.2%) in these strains. E. faecium strains (n = 185) obtained from the effluent samples showed highest resistance against quinupristin/dalfopristin (68.1%) and reduced susceptibility to erythromycin (24.9%) and tetracycline (8.6%). We did not detect resistance against last-resort antibiotics, such as linezolid, vancomycin, and tigecycline in any of the strains. Multidrug-resistant (MDR) E. faecium strains were detected in both influent (2.3%) and effluent (2.2%) samples. Whole genome sequencing of the Enterococcus spp. strains (n = 25) showed the presence of several antibiotic resistance genes, conferring resistance against aminoglycosides, tetracyclines, and macrolides, as well as several virulence genes and plasmid replicons. Two sequenced MDR strains from the effluents belonged to the hospital-associated clonal complex 17 and carried multiple virulence genes. Our study demonstrates that clinically relevant MDR Enterococcus spp. strains are entering the marine environment through treated sewage.

Escherichia coli novel sequence type 11873 harbours a new CTX-M-15-carrying multidrug resistance type 1/2 hybrid IncC plasmid.

OBJECTIVE: The aim of the current study was to determine the genomic map of the resistance genes of two CTX-M-15-carrying Escherichia coli strains belonging to novel sequence type (ST) 11873. Complete, closed genome sequences of the E. coli strains were obtained by applying a combination of short-read Illumina and long-read Oxford Nanopore-based sequencing. METHODS: Isolation of E. coli was performed using ECC CHROMagar and antibiotic sensitivity patterns were determined using Sensititre EUVSEC plates. Whole-genome sequencing was performed for two E. coli strains (3-338 and 5-325) using Illumina MiSeq- and Oxford Nanopore MinION-based sequencing. RESULTS: The complete genome of strain 3-338 (GenBank accession no. CP130007-17) was assembled into a circular chromosome of 4.65 Mb and 10 plasmids (between 2 and 148 kb). Strain 5-325 (CP130018-27) exhibited a circular chromosome of 4.7 Mb and 9 plasmids (between 2 and 148 kb). Both strains carried an identical type 1/2 hybrid IncC plasmid (∼148 kb) harbouring multiple antibiotic resistance genes (ARGs), including blaCTX-M-15, blaOXA-1, blaTEM-1, qnrS1, sul2, aphA1, aacC2, mph(A) and floR. This plasmid also carried heavy metal resistance genes, such as chrA and arsR. Strain 5-325 carried an additional IncFIB plasmid (∼78 kb) harbouring additional ARGs, including blaTEM-1, qnrS1, tet(A), dfrA14, sul2, strA and strB. CONCLUSIONS: Our study shows the emergence of a CTX-M-15-carrying type 1/2 hybrid IncC plasmid in novel E. coli ST11873. These findings emphasise the need for population-based sewage surveillance for understanding the prevalence of antibiotic resistance in pathogens in order to mitigate the further spread of such resistance factors.

In vitro antibacterial activity of Tabernaemontana alternifolia (Roxb) stem bark aqueous extracts against clinical isolates of methicillin resistant Staphylococcus aureus.

BACKGROUND: The rise of antibiotic resistance among methicillin resistant Staphylococcus aureus (MRSA), have caused concerns for the treatment of MRSA infections. Hence, search for an alternative therapy for these infections is inevitable. Folk Indian medicine refers to the use of leaf and stem bark powder of Tabernaemontana alternifolia (Roxb) in treatment of skin infections, but no scientific report establishes its antibacterial activity. METHODS: Direct aqueous extracts and sequential aqueous extracts of the stem bark of T. alternifolia (using petroleum ether and ethyl acetate as other solvents) were prepared by soxhlet extraction. The antibiotic sensitivity profiles of the clinical isolates were determined against 18 antibiotics using disc diffusion method. The isolates were identified by 16S rRNA gene sequencing. The methicillin resistance among S. aureus (MRSA) was confirmed by PCR amplification of mecA gene. The disc diffusion method was used to determine the antibacterial activity of the extracts. The micro-dilution method was used to determine the minimum inhibitory concentration (MIC) of the extract against the test organism. To further evaluate the therapeutic potential of the extract, cell cytotoxicity was checked on Vero cells by MTT assay. Chemical profiling of the extract was done by HPTLC method. RESULTS: The aqueous extracts of T. alternifolia stem bark exhibited antibacterial activity against Gram-positive microorganisms, particularly against clinical isolates of MRSA and vancomycin resistant S. aureus (VRSA). The minimum inhibitory concentration (MIC) of extract against the isolates ranged from 600-800 µg/ml. The extract did not exhibit cytotoxic activity against Vero cells even at the concentration of 4 mg/ml. The chemical profiling revealed presence of alkaloids, flavonoids, coumarins, saponins and steroids. Petroleum ether and ethyl acetate extracts did not exhibit antibacterial activity. CONCLUSION: Our results offer a scientific basis for the traditional use of T. alternifolia in the treatment of skin infections, showing that the plant extract has an enormous potential as a prospective alternative therapy against MRSA skin infections. The present study lays the basis for future studies, to validate the possible use of T. alternifolia as a candidate in the treatment of MRSA infections.

Genome sequence of Vibrio anguillarum isolates carrying a novel class A ß-lactamase VAN-1: do migratory fish transport novel resistance factors?

OBJECTIVES: The aim of the study was to understand the genetic basis of resistance of five ß-lactam resistant Vibrio anguillarum isolates obtained from the gut content of Atlantic mackerel (Scomber scomberus), using whole genome sequencing and to characterize a novel ß-lactamase (VAN-1) from these isolates. METHOD: Antibiotic sensitivity pattern was determined using Sensititre™ plates and whole genome sequencing was carried out using Illumina MiSeq-based sequencing. The blaVAN-1 gene was synthesized and expressed in Escherichia coli Top10 cells. RESULTS: Five isolates obtained (out of 73) from the gut content of Atlantic mackerel were identified as Vibrio anguillarum. Whole genome assemblies ranged from 3.894 to 3.906 million bases in length with an average of 50 contigs. A novel ß-lactamase blaVAN-1, sharing 77.7% nucleotide identity with a known mobile ß-lactamase from Vibrio species was detected. The blaVAN-1 gene in these isolates is flanked by a truncated IS5 family transposase on one end and a hypothetical protein and outer membrane protein followed by another IS5 family transposase on the other end, suggesting its potential for mobility. The blaVAN-1 gene was absent in V. anguillarum type strain (ATCC 14181) and V. anguillarum isolates from bivalves and sea water in Norway. VAN-1 conferred ampicillin resistance when expressed in E. coli, thus confirming the functionality of this gene. CONCLUSIONS: Our study highlights the importance of the marine environment as a reservoir of new antibiotic resistance genes. Our results suggest that migratory fish may transport novel antibiotic resistance determinants over long distances.

Co-localization of clinically relevant antibiotic- and heavy metal resistance genes on plasmids in Klebsiella pneumoniae from marine bivalves.

Klebsiella pneumoniae is an opportunistic pathogen frequently associated with antibiotic resistance and present in a wide range of environments, including marine habitats. However, little is known about the development, persistence, and spread of antibiotic resistance in such environments. This study aimed to obtain the complete genome sequences of antibiotic-resistant K. pneumoniae isolated from marine bivalves in order to determine the genetic context of antibiotic- and heavy metal resistance genes in these isolates. Five antibiotic-resistant K. pneumoniae isolates, of which four also carried heavy metal resistance genes, were selected for complete genome sequencing using the Illumina MiSeq platform and the Oxford Nanopore Technologies GridION device. Conjugation experiments were conducted to examine the transfer potential of selected plasmids. The average length of the complete genomes was 5.48 Mbp with a mean chromosome size of 5.27 Mbp. Seven plasmids were detected in the antibiotic-resistant isolates. Three IncFIB, one IncFIB/IncFII, and one IncFIB/IncHIB plasmid, respectively, carried antibiotic resistance genes such as qnrS1, aph(6)-Id and aph(3')-Ia, aadA1, and aadA2. Four of these plasmids also carried genes encoding resistance to copper (pco), silver (sil), and arsenic (ars). One plasmid carrying tet(D) and blaSHV-1 as well as pco, sil, and ars genes was transferred to Escherichia coli by conjugation. We show the co-occurrence of antibiotic- and heavy metal resistance genes on a conjugative IncFIB plasmid from K. pneumoniae from marine bivalves. Our study highlights the importance of the marine environment and seafood as a possible dissemination route for antimicrobial resistance and provides insights into the potential for co-selection of antibiotic resistance genes by heavy metals.

Complete sequence of a new conjugative multidrug-resistance encoding IncFII/IncFIA/IncFIB plasmid carrying NDM-6 metallo-ß-lactamase from pathogenic Escherichia coli sequence type 167 isolated from sewage in Norway.

OBJECTIVES: The aim of the current study was to determine the genomic map of resistance genes and to understand the potential for mobility of a new NDM-6-carrying plasmid from a pathogenic Escherichia coli strain. A complete and closed genome sequence of the E. coli strain was obtained by applying a combination of short-read Illumina and long-read Nanopore-based sequencing. METHODS: Isolation of E. coli was performed, using ECC CHROMagar™, and antibiotic sensitivity patterns were determined, using Sensititre™ EUVSEC3 plates. Whole-genome sequencing was performed, using Illumina MiSeq- and Oxford Nanopore MinION-based sequencing. Conjugation experiments were performed, using filter-mating and a green fluorescent protein (GFP)-tagged E. coli strain. RESULTS: Two carbapenem-resistant E. coli strains were isolated from sewage. These strains (2-331 and 2-333) belonged to sequence type (ST) 167 and carried an NDM-6 carbapenemase. The complete genome of strain 2-331 (GenBank accession no.: CP110117-22.1) was assembled into six contigs, representing a complete circular chromosome of 4 947 178 bp and five plasmids, ranging from 143 596 bp to 1549 bp. Plasmid p2-331_1 (∼144 kb), belonging to the IncFII/IncFIA/IncFIB group, carried multiple antibiotic resistance genes, including mph(A), mrx(A), blaTEM-1, rmtB1, blaNDM-6, ble, sul1, qacEΔ1, aadAΔ, dfrA12, and tet(A). Plasmid p2-331_1 was transferred from strain 2-331, via conjugation, conferring resistance against eight different classes of antibiotics to a GFP-tagged E. coli strain. CONCLUSIONS: Our study showed the emergence of a new conjugative plasmid-carrying NDM-6 carbapenemase from pathogenic E. coli ST167 for the first time in Norway. The importance of population-based sewage-surveillance for understanding the antimicrobial resistance situation within the community is highlighted.

Sewage-based surveillance shows presence of Klebsiella pneumoniae resistant against last resort antibiotics in the population in Bergen, Norway.

The aim of this study was to understand the prevalence of antibiotic resistance in Klebsiella pneumoniae present in the population in Bergen city, Norway using city-scale sewage-based surveillance, as well as the potential spread of K. pneumoniae into the marine environment through treated sewage. From a total of 30 sewage samples collected from five different sewage treatment plants (STPs), 563 presumptive K. pneumoniae isolates were obtained on Simmons Citrate Agar with myo-Inositol (SCAI) plates, and 44 presumptive K. pneumoniae isolates on SCAI plates with cefotaxime. Colistin resistance was observed in 35 isolates, while cefotaxime resistance and tigecycline resistance was observed in only five isolates each, out of 563 presumptive K. pneumoniae isolates. All 44 isolates obtained on cefotaxime-containing plates were multidrug-resistant, with 25% (n = 11) showing resistance against tigecycline. Clinically important acquired antibiotic resistance genes (ARGs), like blaCTX-M-14, blaCTX-M-15, qnrS1, aac(3)-IIe, tet(A), and sul1, were detected in several sequenced Klebsiella spp. isolates (n = 53). All sequenced colistin-resistant isolates (n = 13) had a mutation in the mgrB gene with nucleotide substitution at position C88T creating a premature stop codon. All sequenced tigecycline-resistant isolates (n = 4) harbored a Tet(A) variant with 22 amino acid (aa) substitutions compared to the reference protein. The sequenced K. pneumoniae isolates (n = 44) belonged to 22 different sequence types (STs) with ST730 (29.5%) as most prevalent, followed by pathogenic ST307 (11.4%). Virulence factors, including aerobactin (iutA), enterobactin (entABCDEFS and fepABCDG), salmochelin (iro), and yersiniabactin (ybt) were detected in several sequenced K. pneumoniae isolates, suggesting pathogenicity potential. Heavy metal resistance genes were common in sequenced K. pneumoniae isolates (n = 44) with silver (silABCEFPRS) and copper (pcoABDRS) resistance genes present in 79.5% of the isolates. Sewage-based surveillance can be a useful tool for understanding antibiotic resistance in pathogens present within a population and to provide up-to date information on the current resistance situation. Our study presents a framework for population-based surveillance of resistance in K. pneumoniae.

Knockout of Targeted Plasmid-Borne ß-Lactamase Genes in an Extended-Spectrum-ß-Lactamase-Producing Escherichia coli Strain: Impact on Resistance and Proteomic Profile.

Resistance to ß-lactams is known to be multifactorial, although the underlying mechanisms are not well established. The aim of our study was to develop a system for assessing the phenotypic and proteomic responses of bacteria to antibiotic stress as a result of the loss of selected antimicrobial resistance genes. We applied homologous recombination to knock out plasmid-borne ß-lactamase genes (blaOXA-1, blaTEM-1, and blaCTX-M15) in Escherichia coli CCUG 73778, generating knockout clone variants lacking the respective deleted ß-lactamases. Quantitative proteomic analyses were performed on the knockout variants and the wild-type strain, using bottom-up liquid chromatography tandem mass spectrometry (LC-MS/MS), after exposure to different concentrations of cefadroxil. Loss of the blaCTX-M-15 gene had the greatest impact on the resulting protein expression dynamics, while losses of blaOXA-1 and blaTEM-1 affected fewer proteins' expression levels. Proteins involved in antibiotic resistance, cell membrane integrity, stress, and gene expression and unknown function proteins exhibited differential expression. The present study provides a framework for studying protein expression in response to antibiotic exposure and identifying the genomic, proteomic, and phenotypic impacts of resistance gene loss. IMPORTANCE The critical situation regarding antibiotic resistance requires a more in-depth effort for understanding underlying mechanisms involved in antibiotic resistance, beyond just detecting resistance genes. The methodology presented in this work provides a framework for knocking out selected resistance factors, to study the adjustments of the bacterium in response to a particular antibiotic stress, elucidating the genetic response and proteins that are mobilized. The protocol uses MS-based determination of the proteins that are expressed in response to an antibiotic, enabling the selection of strong candidates representing putative resistance factors or mechanisms and providing a basis for future studies to understand their implications in antibiotic resistance. This allows us to better understand how the cell responds to the presence of the antibiotic when a specific gene is lost and, consequently, identify alternative targets for possible future treatment development.

Seawater from Bergen harbor is a reservoir of conjugative multidrug-resistance plasmids carrying genes for virulence.

Aquatic environments play important roles in the dissemination of clinically-relevant antibiotic resistance genes (ARGs) and pathogens. Limited knowledge exists about the prevalence of clinically-relevant acquired resistance genes in the marine environment, especially in Norway. The aim of the current study was to investigate the presence of and characterize self-transmissible resistance plasmids from Bergen harbor seawater, with exogenous-plasmid capture, using a green fluorescent protein (GFP)-tagged Escherichia coli strain as a recipient. We obtained transconjugants resistant against ampicillin and cefotaxime from four of the 13 samples processed. Nine transconjugants, selected on the basis of antibiotic sensitivity patterns, were sequenced, using Illumina MiSeq and Oxford Nanopore MinION platforms. Ten different plasmids (ranging from 35 kb to 136 kb) belonging to incompatibility groups IncFII/IncFIB/Col156, IncFII, IncI1 and IncB/O/K/Z were detected among these transconjugants. Plasmid p1A1 (IncFII/IncFIB/Col156, 135.7 kb) carried resistance genes blaTEM-1, dfrA17, sul1, sul2, tet(A), mph(A), aadA5, aph(3″)-Ib and aph(6)-Id, conferring resistance against six different classes of antibiotics. Plasmid p1A4 carried blaCTX-M-55, lnu(F), aadA17 and aac(3)-IId. Cephalosporinase blaCMY-2 was detected on plasmids captured from an area impacted by wastewater from a local marine aquarium. Along with ARGs, some plasmids also carried virulence factors, such as enterotoxins, adhesion factors and siderophores. Our study demonstrates the presence of clinically-important multidrug-resistance conjugative plasmids in seawater from Bergen harbor, which have the potential to be transferred to human microbiota. The results highlight the need for surveillance of antibiotic resistance in the environment, as suggested by the World Health Organization, especially in low prevalence settings like Norway.

Novel Plasmid Carrying Mobile Colistin Resistance Gene mcr-4.3 and Mercury Resistance Genes in Shewanella baltica: Insights into Mobilization of mcr-4.3 in Shewanella Species.

Shewanella species have been identified as progenitors of several clinically important antibiotic resistance genes. The aim of our study was to analyze Shewanella baltica strains isolated from the gut contents of wild Atlantic mackerel (Scomber scombrus) for the presence of both known and novel variants of antibiotic resistance genes (ARGs), using Illumina-based whole-genome sequencing (WGS). Thirty-three S. baltica strains were isolated from Atlantic mackerel collected in the northern North Sea. WGS revealed the presence of several new variants of class C and class D beta-lactamases. Nearly 42% (14/33) of the strains carried the mobile colistin resistance gene mcr-4.3. To understand the genetic context of mcr-4.3, we determined the complete genome sequence of strain 11FHM2, using a combination of Oxford Nanopore- and Illumina-based sequencing. The complete genome sequence is 5,406,724 bp long, with one contig representing a chromosome of 5,068,880 bp and three contigs representing novel plasmids (pSBP1, 194,145 bp; pSBP2_mcr4, 86,727 bp; and pSBP3, 56,972 bp). Plasmid pSBP2_mcr4 contains the mobile colistin resistance gene mcr-4.3, as well as the mercury resistance operon merRPAT. Plasmid pSBP1 carries genes encoding resistance against copper, zinc, chromium, and arsenic. Plasmid pSBP3 does not carry any antibiotic or heavy metal resistance genes. Analysis of the flanking region of mcr-4.3 suggests that a phage integrase may be involved in the mobilization of mcr-4.3 in Shewanella spp. Our results provide insights into the mobile mcr-4.3 present in Shewanella spp. and highlight the importance of the marine environment in the emergence and dissemination of clinically important resistance genes. IMPORTANCE We identified two new plasmids in Shewanella baltica isolated from wild Atlantic mackerel (Scomber scombrus) collected from the northern North Sea, one plasmid carrying the mcr-4.3 gene for colistin resistance and the operon merRPAT for mercury resistance and the other carrying multiple heavy metal resistance genes. The marine environment has been recognized as a source of new resistance genes that are found in human pathogens. Selection pressure from heavy metals is seen in the marine environment, especially associated with human activities, such as waste discharge, mining, and in aquaculture settings. This would help maintain and disseminate these plasmids in the environment. Our study provides insights into the mobilization of colistin resistance genes in Shewanella spp. and highlights the importance of the marine environment in the emergence and dissemination of clinically important antibiotic resistance genes.

Responses of carbapenemase-producing and non-producing carbapenem-resistant Pseudomonas aeruginosa strains to meropenem revealed by quantitative tandem mass spectrometry proteomics.

Pseudomonas aeruginosa is an opportunistic pathogen with increasing incidence of multidrug-resistant strains, including resistance to last-resort antibiotics, such as carbapenems. Resistances are often due to complex interplays of natural and acquired resistance mechanisms that are enhanced by its large regulatory network. This study describes the proteomic responses of two carbapenem-resistant P. aeruginosa strains of high-risk clones ST235 and ST395 to subminimal inhibitory concentrations (sub-MICs) of meropenem by identifying differentially regulated proteins and pathways. Strain CCUG 51971 carries a VIM-4 metallo-ß-lactamase or 'classical' carbapenemase; strain CCUG 70744 carries no known acquired carbapenem-resistance genes and exhibits 'non-classical' carbapenem-resistance. Strains were cultivated with different sub-MICs of meropenem and analyzed, using quantitative shotgun proteomics based on tandem mass tag (TMT) isobaric labeling, nano-liquid chromatography tandem-mass spectrometry and complete genome sequences. Exposure of strains to sub-MICs of meropenem resulted in hundreds of differentially regulated proteins, including ß-lactamases, proteins associated with transport, peptidoglycan metabolism, cell wall organization, and regulatory proteins. Strain CCUG 51971 showed upregulation of intrinsic ß-lactamases and VIM-4 carbapenemase, while CCUG 70744 exhibited a combination of upregulated intrinsic ß-lactamases, efflux pumps, penicillin-binding proteins and downregulation of porins. All components of the H1 type VI secretion system were upregulated in strain CCUG 51971. Multiple metabolic pathways were affected in both strains. Sub-MICs of meropenem cause marked changes in the proteomes of carbapenem-resistant strains of P. aeruginosa exhibiting different resistance mechanisms, involving a wide range of proteins, many uncharacterized, which might play a role in the susceptibility of P. aeruginosa to meropenem.

Insights into the genetic diversity, antibiotic resistance and pathogenic potential of Klebsiella pneumoniae from the Norwegian marine environment using whole-genome analysis.

Klebsiella pneumoniae (Kp) can cause hospital- and community acquired infections. Although, Kp is widespread in the environment, very little is known about the genetic diversity and pathogenicity of Kp from the marine environment. The aim of our study was to understand the genetic diversity, resistome and pathogenic potential of 87 Kp isolates from the Norwegian marine environment, using whole-genome sequencing. We identified 50 sequence types, including globally disseminated sequence types associated with multidrug resistance or hypervirulence. Ten isolates carried the yersiniabactin loci. Acquired antibiotic resistance genes were identified in six Kp isolates. Heavy metal resistance genes were widespread among the isolates, with 71% carrying genes encoding resistance to copper, silver, arsenic, nickel and/or mercury. Co-occurrence of antibiotic resistance genes and heavy metal resistance genes was seen in five Kp isolates. Phylogenetic analysis revealed a close genetic relationship between Kp 2016-1200 ST25 isolated from blue mussels (Mytilus edulis) and a clinical isolate reported in Germany. To the best of our knowledge, this study provides the first comprehensive account of genetic diversity among Kp from the marine environment. Our study reveals high diversity of Kp in the Norwegian marine environment and seafood, including globally disseminated pathogenic sequence types carrying clinically relevant antibiotic resistance genes and virulence factors, as well as several heavy metal resistance genes.