Sudeshna Ghosh

The effects of subtherapeutic antibiotic use in farm animals on the proliferation and persistence of antibiotic resistance among soil bacteria

The use of antibiotics at subtherapeutic concentrations for agricultural applications is believed to be an important factor in the proliferation of antibiotic-resistant bacteria. The goal of this study was to determine if the application of manure onto agricultural land would result in the proliferation of antibiotic resistance among soil bacteria. Chlortetracycline-resistant bacteria were enumerated and characterized from soils exposed to the manure of animals fed subtherapeutic concentrations of antibiotics and compared to the chlortetracycline-resistant bacteria from soils at farms with restricted antibiotic use (dairy farms) and from non-agricultural soils. No significant differences were observed at nine different study sites with respect to the numbers and types of cultivated chlortetracycline-resistant bacteria. Genes encoding for tetracycline resistance were rarely detected in the resistant bacteria from these sites. In contrast, soils collected from a tenth farm, which allowed manure to indiscriminately accumulate outside the animal pen, had significantly higher chlortetracycline-resistance levels. These resistant bacteria frequently harbored one of 14 different genes encoding for tetracycline resistance, many of which (especially tet(A) and tet(L)) were detected in numerous different bacterial species. Subsequent bacterial enumerations at this site, following the cessation of farming activity, suggested that this farm remained a hotspot for antibiotic resistance. In conclusion, we speculate that excessive application of animal manure leads to the spread of resistance to soil bacteria (potentially by lateral gene transfer), which then serve as persistent reservoir of antibiotic resistance.

The role of anaerobic digestion in controlling the release of tetracycline resistance genes and class 1 integrons from municipal wastewater treatment plants

In this study, the abilities of two anaerobic digestion processes used for sewage sludge stabilization were compared for their ability to reduce the quantities of three genes that encode resistance to tetracycline (tet(A), tet(O), and tet(X)) and one gene involved with integrons (intI1). A two-stage, thermophilic/mesophilic digestion process always resulted in significant decreases in the quantities of tet(X) and intI1, less frequently in decreases of tet(O), and no net decrease in tet(A). The thermophilic stage was primarily responsible for reducing the quantities of these genes, while the subsequent mesophilic stage sometimes caused a rebound in their quantities. In contrast, a conventional anaerobic digestion process rarely caused a significant decrease in the quantities of any of these genes, with significant increases occurring more frequently. Our results demonstrate that anaerobic thermophilic treatment was more efficient in reducing quantities of genes associated with the spread of antibiotic resistance compared to mesophilic digestion.

Sphingobacterium sp. strain PM2-P1-29 harbours a functional tet(X) gene encoding for the degradation of tetracycline

Aims:  The tet(X) gene has previously been found in obligate anaerobic Bacteroides spp., which is curious because tet(X) encodes for a NADP‐dependent monooxygenase that requires oxygen to degrade tetracycline. In this study, we characterized a tetracycline resistant, aerobic, Gram‐negative Sphingobacterium sp. strain PM2‐P1‐29 that harbours a tet(X) gene.

Triclosan Promotes Staphylococcus aureus Nasal Colonization

ABSTRACT The biocide triclosan is used in many personal care products, including toothpastes, soaps, clothing, and medical equipment. Consequently, it is present as a contaminant in the environment and has been detected in some human fluids, including serum, urine, and milk. Staphylococcus aureus is an opportunistic pathogen that colonizes the noses and throats of approximately 30% of the population. Colonization with S. aureus is known to be a risk factor for several types of infection. Here we demonstrate that triclosan is commonly found in the nasal secretions of healthy adults and the presence of triclosan trends positively with nasal colonization by S. aureus. We demonstrate that triclosan can promote the binding of S. aureus to host proteins such as collagen, fibronectin, and keratin, as well as inanimate surfaces such as plastic and glass. Lastly, triclosan-exposed rats are more susceptible to nasal colonization with S. aureus. These data reveal a novel factor that influences the ability of S. aureus to bind surfaces and alters S. aureus nasal colonization. IMPORTANCE Triclosan has been used as a biocide for over 40 years, but the broader effects that it has on the human microbiome have not been investigated. We demonstrate that triclosan is present in nasal secretions of a large portion of a test population and its presence correlates with Staphylococcus aureus nasal colonization. Triclosan also promotes the binding of S. aureus to human proteins and increases the susceptibility of rats to nasal colonization by S. aureus. These findings are significant because S. aureus colonization is a known risk factor for the development of several types of infections. Our data demonstrate the unintended consequences of unregulated triclosan use and contribute to the growing body of research demonstrating inadvertent effects of triclosan on the environment and human health. Triclosan has been used as a biocide for over 40 years, but the broader effects that it has on the human microbiome have not been investigated. We demonstrate that triclosan is present in nasal secretions of a large portion of a test population and its presence correlates with Staphylococcus aureus nasal colonization. Triclosan also promotes the binding of S. aureus to human proteins and increases the susceptibility of rats to nasal colonization by S. aureus. These findings are significant because S. aureus colonization is a known risk factor for the development of several types of infections. Our data demonstrate the unintended consequences of unregulated triclosan use and contribute to the growing body of research demonstrating inadvertent effects of triclosan on the environment and human health.

Chlorinated phenols control the expression of the multidrug resistance efflux pump MexAB–OprM in Pseudomonas aeruginosa by interacting with NalC

NalC is a TetR type regulator that represses the multidrug efflux pump MexAB–OprM in Pseudomonas aeruginosa. Here we explain the mechanism of NalC‐mediated regulation of MexAB–OprM. We show that NalC non‐covalently binds chlorinated phenols and chemicals containing chlorophenol side‐chains such as triclosan. NalC‐chlorinated phenol binding results in its dissociation from promoter DNA and upregulation of NalCs downstream targets, including the MexR antirepressor ArmR. ArmR upregulation and MexR–ArmR complex formation have previously been shown to upregulate MexAB–OprM. In vivo mexB and armR expression analyses were used to corroborate in vitro NalC‐chlorinated phenol binding. We also show that the interaction between chlorinated phenols and NalC is reversible, such that removal of these chemicals restored NalC promoter DNA binding. Thus, the NalC‐chlorinated phenol interaction is likely a pertinent physiological mechanism that P. aeruginosa uses to control expression of the MexAB–OprM efflux pump.

Application of rbcL based molecular diversity analysis to algae in wastewater treatment plants

The molecular diversity of algae in the final clarifier or denitrification filter outfall from three wastewater treatment plants (WWTPs) with activated sludge based treatment was analyzed using the rbcL gene as a phylogenetic marker. The rbcL gene encodes the large subunit of the CO(2) fixing enzyme RuBisCO. Among algae identified at the WWTPs were diatoms, green algae, cyanobacteria, Eustigmatophyceae, and unknown heterokonts. A high level of diversity was observed within WWTPs with 19-24 unique rbcL sequences detected at each plant. Algae composition also varied between treatment plants. Our results show that the rbcL gene can be used as a phylogenetic marker for algae diversity analysis in a wastewater treatment context.

Phenotypic and genotypic analysis of bacteria isolated from three municipal wastewater treatment plants on tetracycline-amended and ciprofloxacin-amended growth media

Aims:  The goal of this study was to determine the antimicrobial susceptibility of bacteria isolated from three municipal wastewater treatment plants.

Transformation of tetracycline by TetX and its subsequent degradation in a heterologous host

TetX is a flavin-dependent monooxygenase. It has been reported to inactivate all tested tetracyclines. In this study, Escherichia coli overexpressing TetX was added to soil bacterial enrichment cultures along with varying levels of tetracycline and was found to affect community-wide tetracycline resistance levels. Soil microbial communities developed lower levels of tetracycline resistance upon exposure to 25 μg/mL of tetracycline when an E. coli expressing TetX was present (6% of cultivable bacteria were resistant to 40 μg/mL tetracycline). In contrast, in the absence of TetX activity, a similar tetracycline exposure selected for greater levels of resistant bacteria in the soil microbial community (90% of cultivable bacteria were resistant to 40 μg/mL tetracycline). We also describe new metabolites formed after tetracycline transformation by TetX and report the transient generation of redox-active metabolite(s). The results presented here are particularly pertinent in the light of the recent emergence of tet(X) in different bacterial species, including clinical isolates.

Chlorinated phenol-induced physiological antibiotic resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen with the ability to rapidly develop multidrug resistance under selective pressure. Previous work demonstrated that upon exposure to the environmental contaminant pentachlorophenol (PCP), P. aeruginosa PAO1 increases expression of multiple multidrug efflux pumps, including the MexAB-OprM pump. The current study describes increases in the antibiotic resistance of PAO1 upon exposure to PCP and other chlorinated organics, including triclosan. Only exposure to chlorinated phenols induced the mexAB-oprM-mediated antibiotic-resistant phenotype. Thus, chlorinated phenols have the potential to contribute to transient phenotypic increases of antibiotic resistance that are relevant when both compounds are present in the environment.

The microbial colonization of activated carbon block point-of-use (PoU) filters with and without chlorinated phenol disinfection by-products

Activated carbon block (ACB)-based point-of-use (PoU) drinking water filters are widely used to remove residual disinfectant, heavy metals, and disinfection by-products (DBPs) from household tap water. The filters are not designed to remove bacteria, which can grow in the filter. Based on previous research, we hypothesized that more biofilm would form in PoU filters due to the presence of chlorinated phenolic DBPs, resulting in higher bacterial concentrations in filtered water. Therefore, studies were conducted by operating a manifold system with three sets of ACB PoU filters that received chloraminated tap water spiked with (50 and 100 ng L−1 added) and without (0 ng L−1 added) pentachlorophenol (PCP) as a surrogate chlorinated phenolic DBP. After processing 85% of the manufacturers recommended water volume, effluent heterotrophic plate counts plateaued at levels up to 100 times greater than influent counts. The upstream filter fabric developed a significant biofilm that lightened with increasing PCP concentration; however, the overall biofilm mass was not different between treatments. Influent, effluent, and fabric samples had distinct bacterial community structures, and the structure of effluent communities was more similar to that of fabric communities than that of influent communities. The bacterial community structure changed across the filter primarily due to the filter environment itself, while PCP effects were subtle yet significant. The relative abundance of Mycobacterium increased within fabric biofilms when PCP was added, and the absolute abundance was estimated to increase in the effluent relative to the influent. In conclusion, ACB PoU filters significantly alter the bacterial abundance and composition of drinking water.