Persistence of Marine Bacterial Plasmid in the House Fly (Musca domestica): Marine-Derived Antimicrobial Resistance Genes Have a Chance of Invading the Human Environment.

The house fly is known to be a vector of antibiotic-resistant bacteria (ARB) in animal farms. It is also possible that the house fly contributes to the spread of ARB and antibiotic resistance genes (ARGs) among various environments. We hypothesized that ARB and ARGs present in marine fish and fishery food may gain access to humans via the house fly. We show herein that pAQU1, a marine bacterial ARG-bearing plasmid, persists in the house fly intestine for 5 days after fly ingestion of marine bacteria. In the case of Escherichia coli bearing the same plasmid, the persistence period exceeded 7 days. This interval is sufficient for transmission to human environments, meaning that the house fly is capable of serving as a vector of marine-derived ARGs. Time course monitoring of the house fly intestinal microflora showed that the initial microflora was occupied abundantly with Enterobacteriaceae. Experimentally ingested bacteria dominated the intestinal environment immediately following ingestion; however, after 72 h, the intestinal microflora recovered to resemble that observed at baseline, when diverse genera of Enterobacteriaceae were seen. Given that pAQU1 in marine bacteria and E. coli were detected in fly excrement (defined here as any combination of feces and regurgitated material) at 7 days post-bacterial ingestion, we hypothesize that the house fly may serve as a vector for transmission of ARGs from marine items and fish to humans via contamination with fly excrement.

Cellulolytic enzymes in Microbulbifer sp. Strain GL-2, a marine fish intestinal bacterium, with emphasis on endo-1,4-ß-glucanases Cel5A and Cel8.

Cellulose is an abundant biomass on the planet. Various cellulases from environmental microbes have been explored for industrial use of cellulose. Marine fish intestine is of interest as one source of new enzymes. Here, we report the discovery of genes encoding two ß-glucosidases (Bgl3A and Bgl3B) and four endo-1,4-ß-glucanases (Cel5A, Cel8, Cel5B, and Cel9) as part of the genome sequence of a cellulolytic marine bacterium, Microbulbifer sp. Strain GL-2. Five of these six enzymes (excepting Cel5B) are presumed to localize to the periplasm or outer membrane. Transcriptional analysis demonstrated that all six genes were highly expressed in stationary phase. The transcription was induced by cello-oligosaccharides rather than by glucose, suggesting that the cellulases are produced primarily for nutrient acquisition following initial growth, facilitating the secondary growth phase. We cloned the genes encoding two of the endo-1,4-ß-glucanases, Cel5A and Cel8, and purified the corresponding recombinant enzymes following expression in Escherichia coli. The activity of Cel5A was observed across a wide range of temperatures (10-40 ˚C) and pHs (6-8). This pattern differed from those of Cel8 and the commercial cellulase Enthiron, both of which exhibit decreased activities below 30 ˚C and at alkaline pHs. These characteristics suggest that Cel5A might find use in industrial applications. Overall, our results reinforce the hypothesis that marine bacteria remain a possible source of novel cellulolytic activities.

Marine bacteria harbor the sulfonamide resistance gene sul4 without mobile genetic elements.

Marine bacteria are possible reservoirs of antibiotic-resistance genes (ARGs) originating not only from clinical and terrestrial hot spots but also from the marine environment. We report here for the first time a higher rate of the sulfonamide-resistance gene sul4 in marine bacterial isolates compared with other sul genes. Among four sulfonamide-resistance genes (sul1, sul2, sul3, and sul4), sul4 was most abundant (45%) in 74 sulfonamide-resistant marine isolates by PCR screening. The order of abundance was sul4 (33 isolates) >sul2 (6 isolates) >sul3 (5 isolates) >sul1 (1 isolate). Whole-genome sequencing of 23 isolates of sul4-expressing α- and γ-proteobacteria and bacilli revealed that sul4 was not accompanied by known mobile genetic elements. This suggests that sul4 in these marine isolates is clonally transferred and not horizontally transferable. Folate metabolism genes formed a cluster with sul4, suggesting that the cluster area plays a role in folate metabolism, at which sul4 functions as a dihydropteroate synthase. Thus, sul4 might be expressed in marine species and function in folate synthesis, but it is not a transferable ARG.

An Integrative and Conjugative Element (ICE) Found in Shewanella halifaxensis Isolated from Marine Fish Intestine May Connect Genetic Materials between Human and Marine Environments.

Integrative and conjugative elements (ICEs) play a role in the horizontal transfer of antibiotic resistance genes (ARGs). We herein report an ICE from Shewanella halifaxensis isolated from fish intestine with a similar structure to both a clinical bacterial ICE and marine bacterial plasmid. The ICE was designated ICEShaJpn1, a member of the SXT/R391 family of ICEs (SRIs). ICEShaJpn1 has a common core structure with SRIs of clinical and fish origins and an ARG cassette with the pAQU1 plasmid of Photobacterium damselae subsp. damselae, suggesting that the common core of SRIs is widely distributed and ARG cassettes are collected from regional bacteria.

Macrolide resistance genes and mobile genetic elements in waterways from pig farms to the sea in Taiwan.

OBJECTIVES: Macrolides have a long history of use in animals and humans. Dynamics of macrolide-antibiotic resistance genes (ARGs) in waterways from the origin to the sea has not been reported. METHODS: Resistant bacterial rate was measured by culture method, and copy numbers of macrolide-ARGs, mef(A), erm(B), mph(B), mef(C)-mph(G), and mobile genetic elements (MGEs) traI and IntI1 were quantitated in environmental DNA. Community composition in each site was investigated by 16S rRNA gene metagenomic sequencing. In Yilan area, antibiotics were quantitated. RESULTS: Surface water samples from pig farms to the sea in southern and northern areas in Taiwan were monitored. Macrolide-resistant bacteria accounted for 3%-28% of total colony-forming bacteria in aquaculture ponds and rivers, whereas in pig farm wastewater it was 26%-100%. Three common macrolide-ARGs mef(A), erm(B), and mph(B) and the relatively new mef(C)-mph(G) were frequently detected in pig farms, but not in aquaculture ponds and the sea. Rivers receiving pig wastewater showed ARG contamination similar to the pig farms. Among the MGEs, IntI1 was frequently distributed in all sites and was positively related to mef(A), erm(B), and mph(B) but not to mef(C)-mph(G). CONCLUSION: Pig farms are the origin of macrolide-ARGs, although macrolide contamination is low. Since lincomycin was detected in pig farms in the northern area, the increase of macrolide-ARGs is a future concern due to cross-resistance to lincomycin. ARGs abundance in aquaculture ponds was low, though MGEs were detected. Relation of IntI1 to ARG suggests convergence of ARGs to specific MGEs might be time/history dependent.

Adsorption of sulfonamides to marine diatoms and arthropods.

Sulfonamides are frequently detected in the environment, where these compounds adsorb to soil particles and are retained in the environment. However, adsorption of sulfonamides to planktonic particles in the sea is not known. Here we demonstrate that sulfonamides adsorb to a diatom (Chaetoceros) and an arthropod (Artemia), albeit at low levels, under laboratory conditions. In both plankton, sulfamethazine (SMT) was more readily adsorbed than was sulfamethoxazole (SMX). The adsorption occurred quickly and the concentration on the plankton was stable for at least 24 h (Chaetoceros) or 5 h (Artemia). These data suggest that marine plankton may retain sulfonamides, although the adsorbed concentration per cell or individual is not high.

Contamination of antibiotics and sul and tet(M) genes in veterinary wastewater, river, and coastal sea in Thailand.

Water systems in Southeast Asia accumulate antibiotics and antibiotic resistance genes (ARGs) from multiple origins, notably including human clinics and animal farms. To ascertain the fate of antibiotics and ARGs in natural water environments, we monitored the concentrations of these items in Thailand. Here, we show high concentrations of tetracyclines (72,156.9 ng/L) and lincomycin (23,968.0 ng/L) in pig farms, followed by nalidixic acid in city canals. The city canals and rivers contained diverse distributions of antibiotics and ARGs. Assessments of targeted ARGs, including sul1, sul2, sul3, and tet(M), showed that freshwater (pig farm wastewater, rivers, and canals) consistently contained these ARGs, but these genes were less abundant in seawater. Although sulfonamides were low concentrations (<170 ng/mL), sul1 and sul2 genes were abundant in freshwater (minimum 4.4 × 10-3-maximum 1.0 × 100 copies/16S), suggesting that sul genes have disseminated over a long period, despite cessation of use of this class of antibiotics. Ubiquitous distribution of sul genes in freshwater appeared to be independent of selection pressure. In contrast, water of the coastal sea in the monitored area was not contaminated by these antibiotics or ARGs. The density of Enterobacteriales was lower in seawater than in freshwater, suggesting that the number of ARG-possessing Enterobacteriales falls after entering seawater. From the pig farms, through rivers/canals, to the coastal sea, the occurrence of tetracyclines and tet(M) exhibited some correlation, although not a strong one. However, no correlations were found between concentrations of total antibiotics and ARGs, nor between sulfonamides and sul genes. This is the first comprehensive study showing Thai features of antibiotics and ARGs contaminations. The pig farm is hot spot of antibiotics and ARGs, and sul genes ubiquitously distribute in freshwater environments, which become less abundant in seawater.

Tetracycline Resistance Gene Profiles in Red Seabream (Pagrus major) Intestine and Rearing Water After Oxytetracycline Administration.

Marine aquaculture fish and the environment are possible hot spots for the maintenance and spread of antibiotic resistance genes (ARGs). We here show the time courses of changes of six tetracycline resistance genes (tet) in fish rearing seawater and fish intestine in tank experiments. Experimental tanks were prepared as oxytetracycline (OTC) administration tanks and those without OTC. It was found that tet(B), tet(M), and tet(W) were dominant in seawater among the six tet genes. tet(B) and tet(M) abundances increased immediately after OTC administration, indicating that OTC served as a selective pressure to increase the proportion of tet-possessing bacteria. In contrast, the abundance of tet genes in the fish intestine did not differ between the with- and without-OTC administration groups, and clearly was not altered by OTC administration. Profile changing of tet in seawater and fish intestine did not synchronize. These observations suggested that the dynamics of intestinal tet-possessing bacteria do not directly reflect the environment, but reflect selection within the intestine.

Tetracycline resistance gene tet(M) of a marine bacterial strain is not accumulated in bivalves from seawater in clam tank experiment and mussel monitoring.

Antibiotic resistance genes (ARGs) are found in marine as well as terrestrial bacteria. Bivalves are known to accumulate chemical pollutants and pathogenic microbes, however, the fate of ARGs in bivalves after the intake of ARG-possessing bacteria is not known. Here we show that the copy number of oxytetracycline resistance gene tet(M) increased rapidly in the clam digestive tract by filtering water, then remained constant over 96h in a tank experiment even with the addition of tet(M)-possessing bacteria every 24h. >99.9% of the added tet(M) was decomposed, reaching a balanced state. Environmental sampling of mussel digestive tract and seawater supported the hypothesis that tet(M) was decomposed in bivalves as tet(M) was present in seawater from April to October at a concentration of 10-5 to 10-6 copies/16S, whereas tet(M) in mussels was mostly below the detection limit. Two (April) and three (July and October) individual mussels were positive for tet(M) with a concentration equivalent to that of seawater. We therefore conclude that bivalves do not accumulate tet(M) from seawater.