Larisa Ivanova

Antimicrobial use in aquaculture re‐examined: its relevance to antimicrobial resistance and to animal and human health

The worldwide growth of aquaculture has been accompanied by a rapid increase in therapeutic and prophylactic usage of antimicrobials including those important in human therapeutics. Approximately 80% of antimicrobials used in aquaculture enter the environment with their activity intact where they select for bacteria whose resistance arises from mutations or more importantly, from mobile genetic elements containing multiple resistance determinants transmissible to other bacteria. Such selection alters biodiversity in aquatic environments and the normal flora of fish and shellfish. The commonality of the mobilome (the total of all mobile genetic elements in a genome) between aquatic and terrestrial bacteria together with the presence of residual antimicrobials, biofilms, and high concentrations of bacteriophages where the aquatic environment may also be contaminated with pathogens of human and animal origin can stimulate exchange of genetic information between aquatic and terrestrial bacteria. Several recently found genetic elements and resistance determinants for quinolones, tetracyclines, and β-lactamases are shared between aquatic bacteria, fish pathogens, and human pathogens, and appear to have originated in aquatic bacteria. Excessive use of antimicrobials in aquaculture can thus potentially negatively impact animal and human health as well as the aquatic environment and should be better assessed and regulated.

Salmon Aquaculture and Antimicrobial Resistance in the Marine Environment

Antimicrobials used in salmon aquaculture pass into the marine environment. This could have negative impacts on marine environmental biodiversity, and on terrestrial animal and human health as a result of selection for bacteria containing antimicrobial resistance genes. We therefore measured the numbers of culturable bacteria and antimicrobial-resistant bacteria in marine sediments in the Calbuco Archipelago, Chile, over 12-month period at a salmon aquaculture site approximately 20 m from a salmon farm and at a control site 8 km distant without observable aquaculture activities. Three antimicrobials extensively used in Chilean salmon aquaculture (oxytetracycline, oxolinic acid, and florfenicol) were studied. Although none of these antimicrobials was detected in sediments from either site, traces of flumequine, a fluoroquinolone antimicrobial also widely used in Chile, were present in sediments from both sites during this period. There were significant increases in bacterial numbers and antimicrobial-resistant fractions to oxytetracycline, oxolinic acid, and florfenicol in sediments from the aquaculture site compared to those from the control site. Interestingly, there were similar numbers of presumably plasmid-mediated resistance genes for oxytetracycline, oxolinic acid and florfenicol in unselected marine bacteria isolated from both aquaculture and control sites. These preliminary findings in one location may suggest that the current use of large amounts of antimicrobials in Chilean aquaculture has the potential to select for antimicrobial-resistant bacteria in marine sediments.

Borrelia chilensis, a new member of the Borrelia burgdorferi sensu lato complex that extends the range of this genospecies in the Southern Hemisphere

Borrelia burgdorferi sensu lato (s.l.), transmitted by Ixodes spp. ticks, is the causative agent of Lyme disease. Although Ixodes spp. ticks are distributed in both Northern and Southern Hemispheres, evidence for the presence of B. burgdorferi s.l. in South America apart from Uruguay is lacking. We now report the presence of culturable spirochetes with flat-wave morphology and borrelial DNA in endemic Ixodes stilesi ticks collected in Chile from environmental vegetation and long-tailed rice rats (Oligoryzomys longicaudatus). Cultured spirochetes and borrelial DNA in ticks were characterized by multilocus sequence typing and by sequencing five other loci (16S and 23S ribosomal genes, 5S-23S intergenic spacer, flaB, ospC). Phylogenetic analysis placed this spirochete as a new genospecies within the Lyme borreliosis group. Its plasmid profile determined by polymerase chain reaction and pulsed-field gel electrophoresis differed from that of B. burgdorferi B31A3. We propose naming this new South American member of the Lyme borreliosis group B. chilensis VA1 in honor of its country of origin.

Functional analysis of Borrelia burgdorferi uvrA in DNA damage protection

Bacterial pathogens face constant challenges from DNA-damaging agents generated by host phagocytes. Although Borrelia burgdorferi appears to have much fewer DNA repair enzymes than pathogens with larger genomes, it does contain homologues of uvrA and uvrB (subunits A and B of excinuclease ABC). As a first step to exploring the physiologic function of uvrA(Bbu) and its possible role in survival in the host in the face of DNA-damaging agents, a partially deleted uvrA mutant was isolated by targeted inactivation. While growth of this mutant was markedly inhibited by UV irradiation, mitomycin C (MMC) and hydrogen peroxide at doses that lacked effect on wild-type B. burgdorferi, its response to pH 6.0-6.8 and reactive nitrogen intermediates was similar to that of the wild-type parental strain. The sensitivity of the inactivation mutant to UV irradiation, MMC and peroxide was complemented by an extrachromosomal copy of uvrA(Bbu). We conclude that uvrA(Bbu) is functional in B. burgdorferi.

Plasmid-Related Quinolone Resistance Determinants in Epidemic Vibrio parahaemolyticus, Uropathogenic Escherichia coli, and Marine Bacteria from an Aquaculture Area in Chile

Marine bacteria from aquaculture areas with industrial use of quinolones have the potential to pass quinolone resistance genes to animal and human pathogens. The VPA0095 gene, related to the quinolone resistance determinant qnrA, from clinical isolates of epidemic Vibrio parahaemolyticus conferred reduced susceptibility to quinolone after cloning into Escherichia coli K-12 either when acting alone or synergistically with DNA gyrase mutations. In addition, a plasmid-mediated quinolone resistance gene from marine bacteria, aac(6′)-Ib-cr, was identical to aac(6′)-Ib-cr from urinary tract isolates of E. coli, suggesting a recent flow of this gene between these bacteria isolated from different environments. aac(6′)-Ib-cr from E. coli also conferred reduced susceptibility to quinolone and kanamycin when cloned into E. coli K-12.

Plasmid-Mediated Quinolone Resistance (PMQR) Genes and Class 1 Integrons in Quinolone-Resistant Marine Bacteria and Clinical Isolates of "Escherichia coli" from an Aquacultural Area

Antimicrobial usage in aquaculture selects for antimicrobial-resistant microorganisms in the marine environment. The relevance of this selection to terrestrial animal and human health is unclear. Quinolone-resistance genes qnrA, qnrB, and qnrS were chromosomally located in four randomly chosen quinolone-resistant marine bacteria isolated from an aquacultural area with heavy quinolone usage. In quinolone-resistant uropathogenic clinical isolates of Escherichia coli from a coastal area bordering the same aquacultural region, qnrA was chromosomally located in two E. coli isolates, while qnrB and qnrS were located in small molecular weight plasmids in two other E. coli isolates. Three quinolone-resistant marine bacteria and three quinolone-resistant E. coli contained class 1 integrons but without physical association with PMQR genes. In both marine bacteria and uropathogenic E. coli, class 1 integrons had similar co-linear structures, identical gene cassettes, and similarities in their flanking regions. In a Marinobacter sp. marine isolate and in one E. coli clinical isolate, sequences immediately upstream of the qnrS gene were homologous to comparable sequences of numerous plasmid-located qnrS genes while downstream sequences were different. The observed commonality of quinolone resistance genes and integrons suggests that aquacultural use of antimicrobials might facilitate horizontal gene transfer between bacteria in diverse ecological locations.

Genome Sequence of Borrelia chilensis VA1, a South American Member of the Lyme Borreliosis Group

ABSTRACT Borrelia chilensis strain VA1 is a recently described South American member of the Borrelia burgdorferi sensu lato complex from Chile. Whole-genome sequencing analysis determined its linear chromosome and plasmids lp54 and cp26, confirmed its membership in the Lyme borreliosis group, and will open new research avenues regarding its pathogenic potential.

Efficacy of Human Placental‐Derived Stem Cells in Collagen VII Knockout (Recessive Dystrophic Epidermolysis Bullosa) Animal Model

Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating inherited skin blistering disease caused by mutations in the COL7A1 gene that encodes type VII collagen (C7), a major structural component of anchoring fibrils at the dermal‐epidermal junction (DEJ). We recently demonstrated that human cord blood‐derived unrestricted somatic stem cells promote wound healing and ameliorate the blistering phenotype in a RDEB (col7a1−/−) mouse model. Here, we demonstrate significant therapeutic effect of a further novel stem cell product in RDEB, that is, human placental‐derived stem cells (HPDSCs), currently being used as human leukocyte antigen‐independent donor cells with allogeneic umbilical cord blood stem cell transplantation in patients with malignant and nonmalignant diseases. HPDSCs are isolated from full‐term placentas following saline perfusion, red blood cell depletion, and volume reduction. HPDSCs contain significantly higher level of both hematopoietic and nonhematopoietic stem and progenitor cells than cord blood and are low in T cell content. A single intrahepatic administration of HPDSCs significantly elongated the median life span of the col7a1−/− mice from 2 to 7 days and an additional intrahepatic administration significantly extended the median life span to 18 days. We further demonstrated that after intrahepatic administration, HPDSCs engrafted short‐term in the organs affected by RDEB, that is, skin and gastrointestinal tract of col7a1−/− mice, increased adhesion at the DEJ and deposited C7 even at 4 months after administration of HPDSCs, without inducing anti‐C7 antibodies. This study warrants future clinical investigation to determine the safety and efficacy of HPDSCs in patients with severe RDEB. Stem Cells Translational Medicine 2018;7:530–542

Cord Blood-Derived Stem Cells Suppress Fibrosis and May Prevent Malignant Progression in Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by mutations in the Col7a1 gene. Patients with RDEB suffer from recurrent erosions in skin and mucous membranes and have a high risk for developing cutaneous squamous cell carcinoma (cSCCs). TGFβ signaling has been associated with fibrosis and malignancy in RDEB. In this study, the activation of TGFβ signaling was demonstrated in col7a1−/− mice as early as a week after birth starting in the interdigital folds of the paws, accompanied by increased deposition of collagen fibrils and elevated dermal expression of matrix metalloproteinase (MMP)‐9 and MMP‐13. Furthermore, human cord blood‐derived unrestricted somatic stem cells (USSCs) that we previously demonstrated to significantly improve wound healing and prolong the survival of col7a1−/− mice showed the ability to suppress TGFβ signaling and MMP‐9 and MMP‐13 expression meanwhile upregulating anti‐fibrotic TGFβ3 and decorin. In parallel, we cocultured USSCs in a transwell with RDEB patient‐derived fibroblasts, keratinocytes, and cSCC, respectively. The patient‐derived cells were constitutively active for STAT, but not TGFβ signaling. Moreover, the levels of MMP‐9 and MMP‐13 were significantly elevated in the patient derived‐keratinocytes and cSCCs. Although USSC coculture did not inhibit STAT signaling, it significantly suppressed the secretion of MMP‐9 and MMP‐13, and interferon (IFN)‐γ from RDEB patient‐derived cells. Since epithelial expression of these MMPs is a biomarker of malignant transformation and correlates with the degree of tumor invasion, these results suggest a potential role for USSCs in mitigating epithelial malignancy, in addition to their anti‐inflammatory and anti‐fibrotic functions. Stem Cells 2018;36:1839–12

Resistencia a los antimicrobianos en Chile y el paradigma de Una Salud: manejando los riesgos para la salud pública humana y animal resultante del uso de antimicrobianos en la acuicultura del salmón y en medicina

The emergence and dissemination of antimicrobial-resistant bacteria (ARB) is currently seen as one of the major threats to human and animal public health. Veterinary use of antimicrobials in both developing and developed countries is many-fold greater than their use in human medicine and is an important determinant in selection of ARB. In light of the recently outlined National Plan Against Antimicrobial Resistance in Chile, our findings on antimicrobial use in salmon aquaculture and their impact on the environment and human health are highly relevant. Ninety-five percent of tetracyclines, phenicols and quinolones imported into Chile between 1998 and 2015 were for veterinary use, mostly in salmon aquaculture. Excessive use of antimicrobials at aquaculture sites was associated with antimicrobial residues in marine sediments 8 km distant and the presence of resistant marine bacteria harboring easily transmissible resistance genes, in mobile genetic elements, to these same antimicrobials. Moreover, quinolone and integron resistance genes in human pathogens isolated from patients in coastal regions adjacent to aquaculture sites were identical to genes isolated from regional marine bacteria, consistent with genetic communication between bacteria in these different environments. Passage of antimicrobials into the marine environment can potentially diminish environmental diversity, contaminate wild fish for human consumption, and facilitate the appearance of harmful algal blooms and resistant zoonotic and human pathogens. Our findings suggest that changes in aquaculture in Chile that prevent fish infections and decrease antimicrobial usage will prove a determining factor in preventing human and animal infections with multiply-resistant ARB in accord with the modern paradigm of One Health.