Microbiology, Public Health and the Murals of Diego Rivera.

Diego Rivera, an acclaimed Mexican painter active during the first half of the twentieth century, painted multiple frescoes in Mexico and the United States. Some include depictions of bacteria, their interactions with human hosts, and processes related to microbiology and public health including the microbial origin of life, diagnosis of infection, vaccine production and immunization. Microbiological subjects in Rivera's murals at the Mexican Ministry of Health in Mexico City; the Detroit Institute of Art, Detroit; Rockefeller Center, New York/Palacio de Bellas Artes, Mexico City; Chapultepec Park, Mexico City; and the Institute of Social Security, Mexico City, span almost 25 years, from 1929 to 1953. Illustrating the successes of the application of microbiological discoveries and methods to public health and the prevention and treatment of infectious diseases, they benefited from Rivera's creativity in melding microbiology's unique technological and scientific aspects and public health elements with industrial and political components.

Freshwater salmon aquaculture in Chile and transferable antimicrobial resistance.

Large amounts of antimicrobials are used in salmonid aquaculture in Chile. Most are used in marine aquaculture, but appreciable amounts are also employed in freshwater aquaculture. Much research and many publications have examined transferable antimicrobial resistance in bacteria isolated from marine salmon farms, but much less attention has been paid to this area in freshwater salmon farming. A recent paper by Domínguez et al. (2019) has as least in part remedied this situation. We now comment on some of its interpretations and have attempted to point out its areas of strength and weakness in light of the published scientific literature. Seen in this setting, the important results presented by Domínguez et al. (2019) underline the need for increased awareness of the challenge to animal and human health posed by excessive use of antimicrobials in aquaculture.

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.

Sleeper cells: the stringent response and persistence in the Borreliella (Borrelia) burgdorferi enzootic cycle.

Infections with tick-transmitted Borreliella (Borrelia) burgdorferi, the cause of Lyme disease, represent an increasingly large public health problem in North America and Europe. The ability of these spirochetes to maintain themselves for extended periods of time in their tick vectors and vertebrate reservoirs is crucial for continuance of the enzootic cycle as well as for the increasing exposure of humans to them. The stringent response mediated by the alarmone (p)ppGpp has been determined to be a master regulator in B. burgdorferi. It modulates the expression of identified and unidentified open reading frames needed to deal with and overcome the many nutritional stresses and other challenges faced by the spirochete in ticks and animal reservoirs. The metabolic and morphologic changes resulting from activation of the stringent response in B. burgdorferi may also be involved in the recently described non-genetic phenotypic phenomenon of tolerance to otherwise lethal doses of antimicrobials and to other antimicrobial activities. It may thus constitute a linchpin in multiple aspects of infections with Lyme disease borrelia, providing a link between the micro-ecological challenges of its enzootic life-cycle and long-term residence in the tissues of its animal reservoirs, with the evolutionary side effect of potential persistence in incidental human hosts.

Even therapeutic antimicrobial use in animal husbandry may generate environmental hazards to human health.

The potential negative impact for human health of veterinary use of antimicrobials in prophylaxis, metaphylaxis and growth promotion in animal husbandry was first established in the 1960s and 1970s. Determination of the molecular structure of antimicrobial resistance plasmids at that time explained the ability of antimicrobial resistance genes to disseminate among bacterial populations and elucidated the reasons for the negative effects of antimicrobials used in food animals for human health. In this issue of Environmental Microbiology, Liu et al. (2016) show that even therapeutic use of antimicrobials in dairy calves has an appreciable environmental microbiological footprint. We discuss the negative implications of this footprint for human health and the possibility they may lead to calls for increased regulation of veterinary antimicrobial use in terrestrial and aquatic environments.

Antimicrobial resistance and antimicrobial resistance genes in marine bacteria from salmon aquaculture and non-aquaculture sites.

Antimicrobial resistance (AR) detected by disc diffusion and antimicrobial resistance genes detected by DNA hybridization and polymerase chain reaction with amplicon sequencing were studied in 124 marine bacterial isolates from a Chilean salmon aquaculture site and 76 from a site without aquaculture 8 km distant. Resistance to one or more antimicrobials was present in 81% of the isolates regardless of site. Resistance to tetracycline was most commonly encoded by tetA and tetG; to trimethoprim, by dfrA1, dfrA5 and dfrA12; to sulfamethizole, by sul1 and sul2; to amoxicillin, by blaTEM ; and to streptomycin, by strA-strB. Integron integrase intl1 was detected in 14 sul1-positive isolates, associated with aad9 gene cassettes in two from the aquaculture site. intl2 Integrase was only detected in three dfrA1-positive isolates from the aquaculture site and was not associated with gene cassettes in any. Of nine isolates tested for conjugation, two from the aquaculture site transferred AR determinants to Escherichia coli. High levels of AR in marine sediments from aquaculture and non-aquaculture sites suggest that dispersion of the large amounts of antimicrobials used in Chilean salmon aquaculture has created selective pressure in areas of the marine environment far removed from the initial site of use of these agents.

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.

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 ß-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.

Misunderstandings and misinterpretations: Antimicrobial use and resistance in salmon aquaculture.

The exponential growth of aquaculture over the past 30 years has been accompanied by a parallel increase in the use of antimicrobials. This widespread use has had negative effects on animal, human and environmental health and affected the biodiversity of the environments where aquaculture takes place. Results showing these harmful effects have been resisted and made light of by the aquaculture industry and their scientific supporters through introduction of misunderstandings and misinterpretations of concepts developed in the evolution, genetics, and molecular epidemiology of antimicrobial resistance. We focus on a few of the most obvious scientific shortcomings and biases of two recent attempts to minimise the negative impacts of excessive antimicrobial use in Chilean salmon aquaculture on human and piscine health and on the environment. Such open debate is critical to timely implementation of effective regulation of antimicrobial usage in salmon aquaculture in Chile, if the negative local and worldwide impacts of this usage are to be avoided.

Borreliella burgdorferi Antimicrobial-Tolerant Persistence in Lyme Disease and Posttreatment Lyme Disease Syndromes.

The annual incidence of Lyme disease, caused by tick-transmitted Borreliella burgdorferi, is estimated to be at least 476,000 cases in the United States and many more worldwide. Ten to 20% of antimicrobial-treated Lyme disease patients display posttreatment Lyme disease syndrome (PTLDS), a clinical complication whose etiology and pathogenesis remain uncertain. Autoimmunity, cross-reactivity, molecular mimicry, coinfections, and borrelial tolerance to antimicrobials/persistence have been hypothesized and studied as potential causes of PTLDS. Studies of borrelial tolerance/persistence in vitro in response to antimicrobials and experimental studies in mice and nonhuman primates, taken together with clinical reports, have revealed that B. burgdorferi becomes tolerant to antimicrobials and may sometimes persist in animals and humans after the currently recommended antimicrobial treatment. Moreover, B. burgdorferi is pleomorphic and can generate viable-but-nonculturable bacteria, states also involved in antimicrobial tolerance. The multiple regulatory pathways and structural genes involved in mediating this tolerance to antimicrobials and environmental stressors by persistence might include the stringent (rel and dksA) and host adaptation (rpoS) responses, sugar metabolism (glpD), and polypeptide transporters (opp). Application of this recently reported knowledge to clinical studies can be expected to clarify the potential role of bacterial antibacterial tolerance/persistence in Lyme disease and PTLDS.

Patterns and regulation of ribosomal RNA transcription in Borrelia burgdorferi.

BACKGROUND: Borrelia burgdorferi contains one 16S and two tandem sets of 23S-5S ribosomal (r) RNA genes whose patterns of transcription and regulation are unknown but are likely to be critical for survival and persistence in its hosts. RESULTS: RT-PCR of B. burgdorferi N40 and B31 revealed three rRNA region transcripts: 16S rRNA-alanine transfer RNA (tRNA Ala); tRNA Ile; and both sets of 23S-5S rRNA. At 34°C, there were no differences in growth rate or in accumulation of total protein, DNA and RNA in B31 cultured in Barbour-Stoenner-Kelly (BSK)-H whether rabbit serum was present or not. At 23°C, B31 grew more slowly in serum-containing BSK-H than at 34°C. DNA per cell was higher in cells in exponential as compared to stationary phase at either temperature; protein per cell was similar at both temperatures in both phases. Similar amounts of rRNA were produced in exponential phase at both temperatures, and rRNA was down-regulated in stationary phase at either temperature. Interestingly, a rel Bbu deletion mutant unable to generate (p)ppGpp did not down-regulate rRNA at transition to stationary phase in serum-containing BSK-H at 34°C, similar to the relaxed phenotype of E. coli relA mutants. CONCLUSIONS: We conclude that rRNA transcription in B. burgdorferi is complex and regulated both by growth phase and by the stringent response but not by temperature-modulated growth rate.

Hidden in plain sight: Borrelia burgdorferi and the extracellular matrix.

Borrelia burgdorferi, the tick-transmitted etiologic agent of Lyme borreliosis, can colonize and persist in multiple tissue sites despite vigorous host immune responses. The extracellular matrix appears to provide a protective niche for the spirochete. Recent studies in mice suggest that B. burgdorferi interacts in various ways with collagen and its associated molecules, exploiting molecular and structural features to establish microcolonial refugia. Better knowledge of the genetic and structural bases for these interactions of B. burgdorferi with the extracellular matrix will be required before an understanding of the persistence of B. burgdorferi in the tissues and development of chronic infections can be achieved.

Antimicrobial resistance in Chile and The One Health paradigm: Dealing with threats to human and veterinary health resulting from antimicrobial use in salmon aquaculture and the clinic. / 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.

Aquaculture as yet another environmental gateway to the development and globalisation of antimicrobial resistance.

Aquaculture uses hundreds of tonnes of antimicrobials annually to prevent and treat bacterial infection. The passage of these antimicrobials into the aquatic environment selects for resistant bacteria and resistance genes and stimulates bacterial mutation, recombination, and horizontal gene transfer. The potential bridging of aquatic and human pathogen resistomes leads to emergence of new antimicrobial-resistant bacteria and global dissemination of them and their antimicrobial resistance genes into animal and human populations. Efforts to prevent antimicrobial overuse in aquaculture must include education of all stakeholders about its detrimental effects on the health of fish, human beings, and the aquatic ecosystem (the notion of One Health), and encouragement of environmentally friendly measures of disease prevention, including vaccines, probiotics, and bacteriophages. Adoption of these measures is a crucial supplement to efforts dealing with antimicrobial resistance by developing new therapeutic agents, if headway is to be made against the increasing problem of antimicrobial resistance in human and veterinary medicine.

Genetic and physiological characterization of 23S rRNA and ftsJ mutants of Borrelia burgdorferi isolated by mariner transposition.

Borrelia burgdorferi contains one 16S rRNA gene and two tandem sets of 23S and 5S rRNA genes located in a single chromosomal region. This unusual rRNA gene organization has been speculated to be involved in the slow growth of this organism. Because we were repeatedly unable to isolate a 23S ribosomal mutant in B. burgdorferi by allelic exchange, we developed a transposition mutagenesis system for this bacterium. To this end, Himar1 transposase is expressed in B. burgdorferi from a resident plasmid containing an erythromycin resistance marker, and this strain is then electroporated with suicide plasmids containing mariner transposons and kanamycin resistance genes expressible in B. burgdorferi. This system permitted us to generate hundreds of erythromycin/kanamycin-resistant B. burgdorferi clones with each of three suicide plasmids. DNA sequencing of several kanamycin-resistant clones generated with one of the suicide plasmids showed stable and random insertion of the transposon into the B. burgdorferi chromosomal and plasmid genome. One mutant was inactivated in rrlA (23S rRNA), another in ftsJ (rrmJ). rrlA disruption had no effect on growth rate under a wide range of culture conditions, but disruption of ftsJ interfered significantly with growth rate and bacterial morphology. These data show it is possible to isolate random and stable B. burgdorferi transposition mutants for physiological analysis of this pathogenic spirochete.

Antimicrobial resistance genes in marine bacteria and human uropathogenic Escherichia coli from a region of intensive aquaculture.

Antimicrobials are heavily used in Chilean salmon aquaculture. We previously found significant differences in antimicrobial-resistant bacteria between sediments from an aquaculture and a non-aquaculture site. We now show that levels of antimicrobial resistance genes (ARG) are significantly higher in antimicrobial-selected marine bacteria than in unselected bacteria from these sites. While ARG in tetracycline- and florfenicol-selected bacteria from aquaculture and non-aquaculture sites were equally frequent, there were significantly more plasmid-mediated quinolone resistance genes per bacterium and significantly higher numbers of qnrB genes in quinolone-selected bacteria from the aquaculture site. Quinolone-resistant urinary Escherichia coli from patients in the Chilean aquacultural region were significantly enriched for qnrB (including a novel qnrB gene), qnrS, qnrA and aac(6')-1b, compared with isolates from New York City. Sequences of qnrA1, qnrB1 and qnrS1 in quinolone-resistant Chilean E. coli and Chilean marine bacteria were identical, suggesting horizontal gene transfer between antimicrobial-resistant marine bacteria and human pathogens.

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

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.