Persistence and evidence for accelerated biodegradation of streptomycin in agricultural soils.

Some antibiotics are used for the treatment of various bacterial crop diseases, and there is a concern that this practice may represent a selection pressure that increases the reservoir of antibiotic resistance carried by bacteria in crop production systems. Since the 1950s the aminoglycoside antibiotic streptomycin has been widely used for the treatment of some bacterial crop diseases such as fire blight in apples and pears. Following application, the time that bacteria will be exposed to the antibiotic, and therefore the pressure for selection of resistance, will vary according to the environmental persistence of the antibiotic. In the present study, the dissipation of streptomycin was examined in soils supplemented with 5 mg streptomycin/kg soil and incubated for 21 days under laboratory conditions. The impact of two key rate-controlling variables, soil texture (sandy loam, loam, clay loam) and temperature (4, 20, 30 °C) on streptomycin persistence were explored. -Robust methods for streptomycin extraction and analysis by LC-MS/MS were developed. Streptomycin dissipation followed first order kinetics, with the time to dissipate 50 % of the parent compound (DT50) in soils of varying texture incubated at 20 °C ranging from about seven to 15 days. In contrast, the DT50 of streptomycin in autoclaved loam soil incubated at 20 °C was about 111 days. At 4 °C the DT50 ranged from 49 to 137 days. Under no incubation conditions were any extractable transformation products obtained. Streptomycin was dissipated significantly more rapidly in field soil that had a prior history of exposure to the antibiotic than in soil that did not. Taken together, these results indicate that streptomycin is amenable to biodegradation in agricultural soils with DT50s of several days when temperature is permissive.

Complete genome sequence of Bradyrhizobium ottawaense strain MIAE 01942 isolated from soybean nodules grown in antibiotic-amended soil.

Bradyrhizobium ottawaense MIAE 01942 is a symbiotic nitrogen-fixing bacterium isolated from the root nodules of soybeans grown in agricultural soils amended with veterinary antibiotics. The genome consists of a single 8.45 Mb circular chromosome that harbors genes involved in nitrogen fixation, denitrification, and antibiotic and metal resistance.

Dynamic effects of black soldier fly larvae meal on the cecal bacterial microbiota and prevalence of selected antimicrobial resistant determinants in broiler chickens.

BACKGROUND: We had earlier described the growth-promoting and -depressive effects of replacing soybean meal (SBM) with low (12.5% and 25%) and high (50% and 100%) inclusion levels of black soldier fly larvae meal (BSFLM), respectively, in Ross x Ross 708 broiler chicken diets. Herein, using 16S rRNA gene amplicon sequencing, we investigated the effects of replacing SBM with increasing inclusion levels (0-100%) of BSFLM in broiler diets on the cecal bacterial community composition at each growth phase compared to broilers fed a basal corn-SBM diet with or without the in-feed antibiotic, bacitracin methylene disalicylate (BMD). We also evaluated the impact of low (12.5% and 25%) inclusion levels of BSFLM (LIL-BSFLM) on the prevalence of selected antimicrobial resistance genes (ARGs) in litter and cecal samples from 35-day-old birds. RESULTS: Compared to a conventional SBM-based broiler chicken diet, high (50 to100%) inclusion levels of BSFLM (HIL-BSFLM) significantly altered the cecal bacterial composition and structure, whereas LIL-BSFLM had a minimal effect. Differential abundance analysis further revealed that the ceca of birds fed 100% BSFLM consistently harbored a ~ 3 log-fold higher abundance of Romboutsia and a ~ 2 log-fold lower abundance of Shuttleworthia relative to those fed a BMD-supplemented control diet at all growth phases. Transient changes in the abundance of several potentially significant bacterial genera, primarily belonging to the class Clostridia, were also observed for birds fed HIL-BSFLM. At the finisher phase, Enterococci bacteria were enriched in the ceca of chickens raised without antibiotic, regardless of the level of dietary BSFLM. Additionally, bacitracin (bcrR) and macrolide (ermB) resistance genes were found to be less abundant in the ceca of chickens fed antibiotic-free diets, including either a corn-SBM or LIL-BSFLM diet. CONCLUSIONS: Chickens fed a HIL-BSFLM presented with an imbalanced gut bacterial microbiota profile, which may be linked to the previously reported growth-depressing effects of a BSFLM diet. In contrast, LIL-BSFLM had a minimal effect on the composition of the cecal bacterial microbiota and did not enrich for selected ARGs. Thus, substitution of SBM with low levels of BSFLM in broiler diets could be a promising alternative to the antibiotic growth promoter, BMD, with the added-value of not enriching for bacitracin- and macrolide-associated ARGs.

Metagenome meta-analysis reveals an increase in the abundance of some multidrug efflux pumps and mobile genetic elements in chemically polluted environments.

Many human activities contaminate terrestrial and aquatic environments with numerous chemical pollutants that not only directly alter the environment but also affect microbial communities in ways that are potentially concerning to human health, such as selecting for the spread of antibiotic-resistance genes (ARGs) through horizontal gene transfer. In the present study, metagenomes available in the public domain from polluted (with antibiotics, with petroleum, with metal mining, or with coal-mining effluents) and unpolluted terrestrial and aquatic environments were compared to examine whether pollution has influenced the abundance and composition of ARGs and mobile elements, with specific focus on IS26 and class 1 integrons (intI1). When aggregated together, polluted environments had a greater relative abundance of ARGs than unpolluted environments and a greater relative abundance of IS26 and intI1. In general, chemical pollution, notably with petroleum, was associated with an increase in the prevalence of ARGs linked to multidrug efflux pumps. Included in the suite of efflux pumps were mexK, mexB, mexF, and mexW that are polyspecific and whose substrate ranges include multiple classes of critically important antibiotics. Also, in some instances, ß-lactam resistance (TEM181 and OXA-541) genes increased, and genes associated with rifampicin resistance (RNA polymerases subunits rpoB and rpoB2) decreased in relative abundance. This meta-analysis suggests that different types of chemical pollution can enrich populations that carry efflux pump systems associated with resistance to multiple classes of medically critical antibiotics.IMPORTANCEThe United Nations has identified chemical pollution as being one of the three greatest threats to environmental health, through which the evolution of antimicrobial resistance, a seminally important public health challenge, may be favored. While this is a very plausible outcome of continued chemical pollution, there is little evidence or research evaluating this risk. The objective of the present study was to examine existing metagenomes from chemically polluted environments and evaluate whether there is evidence that pollution increases the relative abundance of genes and mobile genetic elements that are associated with antibiotic resistance. The key finding is that for some types of pollution, particularly in environments exposed to petroleum, efflux pumps are enriched, and these efflux pumps can confer resistance to multiple classes of medically important antibiotics that are typically associated with Pseudomonas spp. or other Gram-negative bacteria. This finding makes clear the need for more investigation on the impact of chemical pollution on the environmental reservoir of ARGs and their association with mobile genetic elements that can contribute to horizontal gene transfer events.

Molecular mechanism of plasmid-borne resistance to sulfonamide antibiotics.

The sulfonamides (sulfas) are the oldest class of antibacterial drugs and inhibit the bacterial dihydropteroate synthase (DHPS, encoded by folP), through chemical mimicry of its co-substrate p-aminobenzoic acid (pABA). Resistance to sulfa drugs is mediated either by mutations in folP or acquisition of sul genes, which code for sulfa-insensitive, divergent DHPS enzymes. While the molecular basis of resistance through folP mutations is well understood, the mechanisms mediating sul-based resistance have not been investigated in detail. Here, we determine crystal structures of the most common Sul enzyme types (Sul1, Sul2 and Sul3) in multiple ligand-bound states, revealing a substantial reorganization of their pABA-interaction region relative to the corresponding region of DHPS. We use biochemical and biophysical assays, mutational analysis, and in trans complementation of E. coli ΔfolP to show that a Phe-Gly sequence enables the Sul enzymes to discriminate against sulfas while retaining pABA binding and is necessary for broad resistance to sulfonamides. Experimental evolution of E. coli results in a strain harboring a sulfa-resistant DHPS variant that carries a Phe-Gly insertion in its active site, recapitulating this molecular mechanism. We also show that Sul enzymes possess increased active site conformational dynamics relative to DHPS, which could contribute to substrate discrimination. Our results reveal the molecular foundation for Sul-mediated drug resistance and facilitate the potential development of new sulfas less prone to resistance.

Effects of partial or complete replacement of soybean meal with commercial black soldier fly larvae (Hermetia illucens) meal on growth performance, cecal short chain fatty acids, and excreta metabolome of broiler chickens.

Black soldier fly larvae meal (BSFLM) is receiving great attention as a rich source of protein and antimicrobials for poultry. Therefore, we evaluated the effects of partially or completely replacing soybean meal (SBM) with commercial BSFLM on growth performance, tibia traits, cecal short chain fatty acid (SCFA) concentrations, and excreta metabolomes in broiler chickens (Gallus gallus domesticus). A total of 480 day-old male Ross × Ross 708 chicks were assigned to 6 diets (8 replicates/diet): a basal corn-SBM diet with in-feed bacitracin methylene disalicylate (BMD), a corn-SBM diet without BMD (0% BSFLM), and four diets in which the SBM was substituted with 12.5, 25, 50, and 100% BSFLM. Body weight (BW), feed intake (FI) and cumulative feed conversion ratio (cFCR) were monitored on days 14, 28, and 35. Cecal SCFA levels were determined on days 14, 28, and 35. Tibia traits and excreta metabolomes were determined on day (d) 35. On d14, birds fed 12.5 and 25% BSFLM had a similar BW, FI, and cFCR as birds fed BMD (P > 0.05). On d 35, birds fed 12.5% BSFLM had a similar BW, FI and cFCR as birds fed BMD or 0% BSFLM (P > 0.05). For each phase, birds fed 100% BSFLM had a lower BW, FI and higher cFCR than birds fed BMD or 0% BSFLM (P < 0.05). On d 35, BW decreased linearly, quadratically, and cubically with increasing levels of BSFLM (P < 0.01). Overall (d 0-35), BSFLM linearly, quadratically, and cubically decreased FI and quadratically and cubically increased cFCR (P < 0.01). Quadratic responses were observed for tibia fresh weight (P = 0.049) and ash content (P = 0.022). BSFLM did not impact cecal SCFAs levels. The excreta metabolome of birds fed 100% BSFLM clustered independently from all other groups and exhibited greater levels of putatively identified methionine, lysine, valine, glutamine, histidine and lower levels of arginine as compared to all diets. Taken together, substitution of SBM with ≤25% of BSFLM in the starter phase may be used as an alternative to BMD.

The aminoglycoside resistance-promoting AmgRS envelope stress-responsive two-component system in Pseudomonas aeruginosa is zinc-activated and protects cells from zinc-promoted membrane damage.

Exposure of wild-type (WT) Pseudomonas aeruginosa PAO1 to ZnCl2 (Zn) yielded a concentration-dependent increase in depolarization of the cytoplasmic membrane (CM), an indication that this metal is membrane-damaging. Consistent with this, Zn activated the AmgRS envelope stress-responsive two-component system (TCS) that was previously shown to be activated by and to protect P. aeruginosa from the membrane-damaging effects of aminoglycoside (AG) antibiotics. A mutant lacking amgR showed enhanced Zn-promoted CM perturbation and was Zn-sensitive, an indication that the TCS protected cells from the CM-damaging effects of this metal. In agreement with this, a mutant carrying an AmgRS-activating amgS mutation was less susceptible to Zn-promoted CM perturbation and more tolerant of elevated levels of Zn than WT. AG activation of AmgRS is known to drive expression of the AG resistance-promoting mexXY multidrug efflux operon, and while Zn similarly induced mexXY expression this was independent of AmgRS and reliant on a second TCS implicated in mexXY regulation, ParRS. MexXY did not, however, contribute to Zn resistance or protection from Zn-promoted CM damage. Despite its activation of AmgRS and induction of mexXY, Zn had a minimal impact on the AG resistance of WT P. aeruginosa although, given that Zn-tolerant AmgRS-activated amgS mutant strains are AG resistant, there is still the prospect of this metal promoting AG resistance development in this organism.

Aminoglycoside-inducible expression of the mexAB-oprM multidrug efflux operon in Pseudomonas aeruginosa: Involvement of the envelope stress-responsive AmgRS two-component system.

Exposure of P. aeruginosa to the aminoglycoside (AG) paromomycin (PAR) induced expression of the PA3720-armR locus and the mexAB-oprM multidrug efflux operon that AmgR controls, although PAR induction of mexAB-oprM was independent of armR. Multiple AGs promoted mexAB-oprM expression and this was lost in the absence of the amgRS locus encoding an aminoglycoside-activated envelope stress-responsive 2-component system (TCS). Purified AmgR bound to the mexAB-oprM promoter region consistent with this response regulator directly regulating expression of the efflux operon. The thiol-active reagent, diamide, which, like AGs, promotes protein aggregation and cytoplasmic membrane damage also promoted AmgRS-dependent mexAB-oprM expression, a clear indication that the MexAB-OprM efflux system is recruited in response to membrane perturbation and/or circumstances that lead to this. Despite the AG and diamide induction of mexAB-oprM, however, MexAB-OprM does not appear to contribute to resistance to these agents.

EsrC, an envelope stress-regulated repressor of the mexCD-oprJ multidrug efflux operon in Pseudomonas aeruginosa.

mexCD-oprJ is an envelope stress-inducible multidrug efflux operon of Pseudomonas aeruginosa. A gene encoding a homologue of the NfxB repressor of this operon, PA4596, occurs downstream of oprJ and was proposed as a second repressor of this efflux operon. Inactivation of this gene had no impact on mexCD-oprJ expression in cells not exposed to envelope stress although its loss under envelope stress conditions yielded a > 10-fold increase in mexCD-oprJ expression. Consistent with PA4596 functioning as a mexCD-oprJ repressor, the purified protein was able to bind to a DNA fragment carrying the mexCD-oprJ promoter region. Expression of PA4596 was induced under conditions of envelope stress dependent on the AlgU envelope stress sigma factor, consistent with PA4596 operating under envelope stress conditions where it possibly serves to moderate envelope stress-inducible mexCD-oprJ expression. nfxB mutants showed elevated PA4596 expression and purified NfxB bound to DNA encompassing the PA4596 upstream region, an indication that NfxB functions as a repressor of PA4596 expression. Elimination of PA4596 in P. aeruginosa lacking nfxB and hyperexpressing mexCD-oprJ had no additional impact on mexCD-oprJ expression, regardless of the presence of envelope stress, suggesting that PA4596 repressor activity may be dependent on NfxB. This envelope stress-regulated repressor of mexCD-oprJ has been renamed esrC.

Pentachlorophenol induction of the Pseudomonas aeruginosa mexAB-oprM efflux operon: involvement of repressors NalC and MexR and the antirepressor ArmR.

Pentachlorophenol (PCP) induced expression of the NalC repressor-regulated PA3720-armR operon and the MexR repressor-controlled mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa. PCP's induction of PA3720-armR resulted from its direct modulation of NalC, the repressor's binding to PA3720-armR promoter-containing DNA as seen in electromobility shift assays (EMSAs) being obviated in the presence of this agent. The NalC binding site was localized to an inverted repeat (IR) sequence upstream of PA3720-armR and overlapping a promoter region whose transcription start site was mapped. While modulation of MexR by the ArmR anti-repressor explains the upregulation of mexAB-oprM in nalC mutants hyperexpressing PA3720-armR, the induction of mexAB-oprM expression by PCP is not wholly explainable by PCP induction of PA3720-armR and subsequent ArmR modulation of MexR, inasmuch as armR deletion mutants still showed PCP-inducible mexAB-oprM expression. PCP failed, however, to induce mexAB-oprM in a mexR deletion strain, indicating that MexR was required for this, although PCP did not modulate MexR binding to mexAB-oprM promoter-containing DNA in vitro. One possibility is that MexR responds to PCP-generated in vivo effector molecules in controlling mexAB-oprM expression in response to PCP. PCP is an unlikely effector and substrate for NalC and MexAB-OprM--its impact on NalC binding to the PA3720-armR promoter DNA occurred only at high µM levels--suggesting that it mimics an intended phenolic effector/substrate(s). In this regard, plants are an abundant source of phenolic antimicrobial compounds and, so, MexAB-OprM may function to protect P. aeruginosa from plant antimicrobials that it encounters in nature.