Elevated Antibiotic Resistance Research Articles

«One Health» approach: transfer of antimicrobial resistance from animals to humans

Antimicrobials are valuable therapeutics whose efficacy is seriously compromised by the emergence and spread of antimicrobial resistance. The current science provides overwhelming evidence that antibiotic use is a powerful selector of resistance that can appear not only at the point of origin but also nearly everywhere else. The latter phenomenon occurs because of the enormous ramifications of horizontal gene transfer. A mounting body of evidence shows that antimicrobial use in animals, including the nontherapeutic use of antimicrobials, leads to the propagation and shedding of substantial amounts of antimicrobial-resistant bacteria — both as pathogens, which can directly and indirectly infect humans, and as commensals, which may carry transferable resistance determinants across species borders and reach humans through multiple routes of transfer. These pathways include not only food but also water and sludge and manure applications to food crop soils. Continued nontherapeutic use of antimicrobials in food animals will increase the pool of resistance genes, as well as their density, as bacteria migrate into the environment at large. The lack of species barriers for gene transmission argues that the focus of research efforts should be directed toward the genetic infrastructure and that it is now imperative to take an ecological approach toward addressing the impacts of NTA use on human disease. The study of animal-to-human transmission of antibiotic resistance therefore requires a greater understanding of the genetic interaction and spread that occur in the larger arena of commensal and environmental bacteria. The provision of antibiotics to food animals encompasses a wide variety of nontherapeutic purposes that include growth promotion. The concern over resistance emergence and spread to people by nontherapeutic use of antimicrobials has led to conflicted practices and opinions. Considerable evidence supported the removal of nontherapeutic antimicrobials in Europe and North America, based on the «precautionary principle». Still, concrete scientific evidence of the favorable versus unfavorable consequences of nontherapeutic antimicrobials is not clear to all stakeholders. Substantial data show elevated antibiotic resistance in bacteria associated with animals fed nontherapeutic antimicrobials and their food products. This resistance spreads to other animals and humans-directly by contact and indirectly via the food chain, water, air, and manured and sludge-fertilized soils. Modern genetic techniques are making advances in deciphering the ecological impact of nontherapeutic antimicrobials, but modeling efforts are thwarted by deficits in key knowledge of microbial and antibiotic loads at each stage of the transmission chain. Still, the substantial and expanding volume of evidence reporting animal-to-human spread of resistant bacteria, including that arising from use of nontherapeutic antimicrobials, supports eliminating antibiotics use in order to reduce the growing environmental load of resistance genes.

Antibiotics and antibiotic resistance from animal manures to soil: a review

SummaryThe overuse of veterinary antibiotics in animal production and the subsequent land applications of manures contribute to the elevated antibiotic resistance in the soil environment. To minimize the risk of antibiotic resistance, it is important to understand the fate of antibiotics and the spread of antibiotic resistance genes (ARGs) from animal production systems to soil. In this paper, we review recent studies on veterinary antibiotic use, the concentrations of antibiotics and the abundance and diversity of AGRs in animal manures and in soil that receives manures or manure composts. The mechanisms of ARG dissemination in the environment are also discussed. Although we focus on China where around 3 billion tons of animal manures are produced and more than 84 000 tons of antibiotics are consumed annually in animal production industries, the problem is worldwide. Approximately 58% of the veterinary antibiotics consumed are excreted into the environment, more than half of which end up in the soil. The abundance of ARGs in manures can reach up to 10−1 of the 16S rRNA genes. Applications of manures or manure composts can enrich soil ARGs in at least three ways: (i) by the direct introduction of manure‐derived ARGs, (ii) by elevating the intrinsic soil ARGs and (iii) by imposing a selection of ARGs with the antibiotics in the manures. We also discuss the need for more stringent regulations on the use of veterinary antibiotics and future research directions on the mechanisms of antibiotic resistance and resistance management.Highlights Soil is a natural reservoir of antibiotics and antibiotic resistance genes (ARGs). Manure applications introduce antibiotics and enrich soil ARGs through different mechanisms. Horizontal gene transfer plays an important role in the spread of ARGs from manures. More stringent regulations are needed to reduce the spread of ARG from animal sources.

English

Streptococcus pseudopneumoniae is a mischaracterized species of Streptococci that is usually overlooked during examination of sputum samples of patients with chest infections. The association of this organism with lower respiratory tract diseases is still unclear and its isolation and description is underestimated in our locality. To our knowledge, there are no published studies on the isolation of S. pseudopneumoniae from pathological specimens in Egypt. The aims of this study were to isolate S. pseudopneumoniae from sputum specimens of patients admitted to Chest Department of Mansoura University Hospitals (MUHs) and to differentiate it from Streptococcus pneumoniae and other viridans group Streptococci, also to determine its prevalence and associated risk factors. Sixteen isolates of S. pseudopneumoniae were diagnosed phenotypically by optochin susceptibility and bile solubility tests followed by genotypic characterization by multiplex polymerase chain reaction (PCR). All of the isolates were subjected to antibiotic susceptibility testing using the disk diffusion method. The prevalence of S. pseudopneumoniae among studied patients was 4.9% (16/329). All of the examined isolates were found to be positive for aliB-like ORF2 and negative for cpsA and lytA genes by multiplex PCR. Elevated resistance rate of the isolates was recorded for erythromycin, penicillin and co-trimoxazole. Infection by S. pseudopneumoniae was found to be significantly associated with Chronic Obstructive Pulmonary Diseases (COPD). Although, S. pseudopneumoniae recorded a low prevalence in this study, their elevated antibiotic resistance together with the association with COPD and their pure isolation from sputum samples underline the necessity of spending more effort for their detection and characterization in the microbiology laboratories. Key words: Chronic obstructive pulmonary diseases (COPD), optochin, respiratory diseases, Streptococcus pseudopneumoniae.

Elevated antibiotic resistance of Sudanese urinary tract infection bacteria.

This study determined the prevalence of urinary tract infections in the Sudanese state of Khartoum and antimicrobial susceptibility pattern of isolated bacterial species. 200 adult patient urine specimens were collected and cultivated to identify the growing bacteria and their susceptibility to antibiotics. 35 % of specimens had significant bacterial growth. The most frequent isolates in this study were E. coli, E. faecalis and S. aureus. Most of the isolates were resistant to many antibiotics; Gram-negative and Gram-positive isolates were resistant to 67 % and 44 % of the examined antibiotics, respectively. E. coli was the most frequent bacterium in the studied samples and it was highly resistant to first-line antibiotics. The most resistant bacteria isolated were Pseudomonas species and the lowest was for S. saprophyticus. The results highlighted the need for knowledge about antibiotic susceptibility profile of the bacteria causing UTI prior to antibiotic prescription in order to ensure optimal treatment.

Antimicrobial Chemicals Are Associated with Elevated Antibiotic Resistance Genes in the Indoor Dust Microbiome.

Antibiotic resistance is increasingly widespread, largely due to human influence. Here, we explore the relationship between antibiotic resistance genes and the antimicrobial chemicals triclosan, triclocarban, and methyl-, ethyl-, propyl-, and butylparaben in the dust microbiome. Dust samples from a mixed-use athletic and educational facility were subjected to microbial and chemical analyses using a combination of 16S rRNA amplicon sequencing, shotgun metagenome sequencing, and liquid chromatography tandem mass spectrometry. The dust resistome was characterized by identifying antibiotic resistance genes annotated in the Comprehensive Antibiotic Resistance Database (CARD) from the metagenomes of each sample using the Short, Better Representative Extract Data set (ShortBRED). The three most highly abundant antibiotic resistance genes were tet(W), blaSRT-1, and erm(B). The complete dust resistome was then compared against the measured concentrations of antimicrobial chemicals, which for triclosan ranged from 0.5 to 1970 ng/g dust. We observed six significant positive associations between the concentration of an antimicrobial chemical and the relative abundance of an antibiotic resistance gene, including one between the ubiquitous antimicrobial triclosan and erm(X), a 23S rRNA methyltransferase implicated in resistance to several antibiotics. This study is the first to look for an association between antibiotic resistance genes and antimicrobial chemicals in dust.

Antibiofilm peptides increase the susceptibility of carbapenemase-producing Klebsiella pneumoniae clinical isolates to β-lactam antibiotics.

Multidrug-resistant carbapenemase-producing Klebsiella pneumoniae (KpC) strains are becoming a common cause of infections in health care centers. Furthermore, Klebsiella can develop multicellular biofilms, which lead to elevated adaptive antibiotic resistance. Here, we describe the antimicrobial and antibiofilm activities of synthetic peptides DJK-5, DJK-6, and 1018 against five KpC isolates. Using static microplate assays, it was observed that the concentration required to prevent biofilm formation by these clinical isolates was below the MIC for planktonic cells. More-sophisticated flow cell experiments confirmed the antibiofilm activity of the peptides against 2-day-old biofilms of different KpC isolates, and in some cases, the peptides induced significant biofilm cell death. Clinically relevant combinations of DJK-6 and β-lactam antibiotics, including the carbapenem meropenem, also prevented planktonic growth and biofilm formation of KpC strain1825971. Interestingly, peptide DJK-6 was able to enhance, at least 16-fold, the ability of meropenem to eradicate preformed biofilms formed by this strain. Using peptide DJK-6 to potentiate the activity of β-lactams, including meropenem, represents a promising strategy to treat infections caused by KpC isolates.

Exposure of juvenile Leghorn chickens to lead acetate enhances antibiotic resistance in enteric bacterial flora

Heavy metals have been implicated for their ability to increase antibiotic resistance in bacteria collected from polluted waters, independent of antibiotic exposure. Specific-pathogen-free Leghorn chickens were therefore given Pb acetate in the drinking water to expose the enteric bacteria to Pb and to determine if antibiotic resistance changed in these bacteria. Concentrations of Pb used were 0.0, 0.01, 0.1, 1.0, or 10.0 mM; birds given the highest 2 concentrations showed signs of moribundity and dehydration and were removed from the study. Vent culture samples were collected for bacterial cultures on d 0 before Pb exposure, d 7 and 14, and then birds were euthanized by CO2 gas for necropsy on d 14, at which time intestinal contents were also collected for bacterial cultures. Fecal swabs but not intestinal samples from Pb-exposed birds contained isolates that had significantly elevated antibiotic resistance. Some of the isolates contained bacteria that were resistant to up to 20 antibiotics. These results suggest the need for repeated studies in chickens infected with zoonotic pathogens.

Food animals and antimicrobials: impacts on human health.

Antimicrobials are valuable therapeutics whose efficacy is seriously compromised by the emergence and spread of antimicrobial resistance. The provision of antibiotics to food animals encompasses a wide variety of nontherapeutic purposes that include growth promotion. The concern over resistance emergence and spread to people by nontherapeutic use of antimicrobials has led to conflicted practices and opinions. Considerable evidence supported the removal of nontherapeutic antimicrobials (NTAs) in Europe, based on the "precautionary principle." Still, concrete scientific evidence of the favorable versus unfavorable consequences of NTAs is not clear to all stakeholders. Substantial data show elevated antibiotic resistance in bacteria associated with animals fed NTAs and their food products. This resistance spreads to other animals and humans-directly by contact and indirectly via the food chain, water, air, and manured and sludge-fertilized soils. Modern genetic techniques are making advances in deciphering the ecological impact of NTAs, but modeling efforts are thwarted by deficits in key knowledge of microbial and antibiotic loads at each stage of the transmission chain. Still, the substantial and expanding volume of evidence reporting animal-to-human spread of resistant bacteria, including that arising from use of NTAs, supports eliminating NTA use in order to reduce the growing environmental load of resistance genes.

Influence of Pseudomonas aeruginosa pvdQ Gene on Altering Antibiotic Susceptibility Under Swarming Conditions

In Pseudomonas aeruginosa PAO1, the pvdQ gene has been shown to have at least two functions. It encodes the acylase enzyme and hydrolyzes 3-oxo-C12-HSL, the key signaling molecule of quorum sensing system. In addition, pvdQ is involved in swarming motility. It is required for up-regulated during swarming motility, which is triggered by high cell densities. As high-density bacterial populations also display elevated antibiotic resistance, studies have demonstrated that swarm-cell differentiation in P. aeruginosa promotes increased resistance to various antibiotics. PvdQ acts as a signal during swarm-cell differentiation, and thus may play a role in P. aeruginosa antibiotic resistance. The aim of this study is to examine whether pvdQ was involved in modifying antibiotic susceptibility during swarming conditions, and to investigate the mechanism by which this occurred. We constructed the PAO1pMEpvdQ strain, which overproduced PvdQ. PAO1pMEpvdQ promotes swarming motility, while PAO1ΔpvdQ abolishes swarming motility. In addition, both PAO1 and PAO1pMEpvdQ acquired resistance to ceftazidime, ciprofloxacin, meropenem, polymyxin B, and gentamicin, though PAO1pMEpvdQ exhibited a two to eightfold increase in antibiotic resistance compared to PAO1. These results indicate that pvdQ plays an important role in elevating antibiotic resistance via swarm-cell differentiation and possibly other mechanisms as well. We analyzed outer membrane permeability. Our data also suggest that pvdQ decreases P. aeruginosa outer membrane permeability, thereby elevating antibiotic resistance under swarming conditions. Our results suggest new approaches for reducing P. aeruginosa resistance.

Cysteine biosynthesis, oxidative stress and antibiotic resistance in Salmonella typhimurium

The efficacy of antibiotics varies under different growth conditions due to the induction of specific or more general defense pathways, but the mechanisms are not completely understood. Actively swarming Salmonella show elevated resistance to many types of antibiotics. Previously, we had shown that cysteine biosynthesis was important for the induced antibiotic resistance phenotype of swarm cells. Here we examine the connection of cysteine to oxidative stress and demonstrate that the antioxidant properties of cysteine or cysteine-derived metabolites contribute to the antibiotic resistance in both vegetative and swarm cell populations. We observed that cys auxotrophs were oxidatively stressed, and in wild-type cells expression of the cys regulon was induced during periods of oxidative stress. In swarm cells, we found a 6-fold increase in reduced glutathione compared to swim cells and a corresponding increased resistance to oxidants. Wild-type and cys auxotrophs exhibited the same sensitivities to gentamicin, polymyxin and ciprofloxacin when grown anaerobically, suggesting that induced oxidative stress defense was contributing to elevated antibiotic resistance in swarm cells aerobically. Induction of the CysB regulon by addition of exogenous inducer resulted in elevated antibiotic resistance independently of swarming.

L-Cysteine is required for induced antibiotic resistance in actively swarming Salmonella enterica serovar Typhimurium

Swarm-cell differentiation in Salmonella enterica serovar Typhimurium (S. typhimurium) results in a biosynthetic mode of growth, despite growing on a rich medium, and cells that have elevated antibiotic resistance. These phenotypes are not a prerequisite for swarm motility. By blocking the switch to anabolic growth using amino acid auxotrophs and screening for the presence of elevated antibiotic resistance in the swarm state, we found that cysteine biosynthesis is crucial for complete swarm-cell differentiation. Mutants were made in each cys biosynthetic operon and all had decreased antibiotic resistance in the swarm state, while swim-cell resistance remained the same as that of wild-type cells. This swarm-state-specific decreased resistance in Deltacys strains could be restored to wild-type levels by the addition of cysteine to swarm medium. Two regulatory mutants, DeltacysB and DeltacysE, failed to swarm unless cysteine was supplemented to the medium. We show that all CysB-responsive operons involved in cysteine biosynthesis are upregulated in the swarm state, even though swarm cells are cultivated on a medium that represses cysteine biosynthesis in the swim state. While swarm medium has sufficient cysteine for growth of S. typhimurium, it does not contain enough for swarm-cell differentiation. We hypothesize that in these cells, the additional cysteine requirement is for use in pathways not directly related to cell growth.

Frequency of transferable multiple antibiotic resistance amongst coliform bacteria isolated from a treated sewage effluent in Antofagasta, Chile

Antibiotic-resistant coliform bacteria from raw and treated water from activated sludge of a sewage treatment plant were isolated on eosin methylene blue agar (EMB) plates in the separate presence of ampicillin, tetracycline, chloramphenicol and streptomycin. Antibiotic resistant coliforms obtained from inflow and effluent waters were studied to determine influences of treatment on the species composition, resistance patterns and transferable resistance in these bacteria. The bacterial counts obtained showed a high proportion of antibiotic resistant coliforms in the treated effluent. The resistant coliform species were eliminated at different rates by the treatment. Escherichia coli decreased, whereas the proportion of Klebsiella sp increased. The percentage of multiple-resistant isolates increased at the end of the treatment process. The high frequency of resistance was to ampicillin, followed by tetracycline and sulpha-trimethoprim. Moderate incidence of resistance was to streptomycin, chloramphenicol and cephradine. The lowest rate of resistance was to kanamicin and nalidixic acid. Mating experiments of multiple-resistant coliforms with an E. coli K-12 donor confirmed the capability of transferring its resistance. The results of this study suggested that wastewater treatment could reduce the total number of enteric bacteria in sewage, but may increase the proportion of antibiotic resistant coliforms in effluent water. Environmental release and agricultural use of treated sewage effluent containing bacteria having elevated antibiotic resistance levels presents a potential public health risk, as well as the risk of transferring the antibiotic resistance to bacterial populations in the environment.

Exposure of Salmonella Enteritidis to chlorine or food preservatives increases susceptibility to antibiotics

Mutants of Salmonella Enteritidis selected following exposure to the sanitizer chlorine or to the preservatives sodium nitrite, sodium benzoate or acetic acid show resistance to multiple antibiotics (tetracycline, chloramphenicol, nalidixic acid, and ciprofloxacin). Complementation experiments with a functional marR restored antibiotic susceptibility of selected mutants to levels similar to wild-type strains, suggesting that mar mutation was responsible for resistance. The multiple antibiotic resistance ( mar) operon is a global regulator controlling intrinsic resistance towards structurally and functionally unrelated antibiotics and other noxious agents. Mutants selected after exposure to an inducing agent maintained elevated antibiotic resistance after serial subculture in media void of the inducing agent. Results highlight the importance of monitoring the use of antimicrobial agents to ensure that concentrations capable of inactivating target pathogens are used.

Amino acid residues essential for function of the MexF efflux pump protein of Pseudomonas aeruginosa.

At least four broad-spectrum efflux pumps (Mex) are involved in elevated intrinsic antibiotic resistance as well as in acquired multidrug resistance in Pseudomonas aeruginosa. Substrate specificity of the Mex pumps has been shown to be determined by the cytoplasmic membrane component (MexB, MexD, MexF, and MexY) of the tripartite efflux pump system. Alignment of their amino acid sequences with those of the homologous AcrB and AcrD pump proteins of Escherichia coli showed conservation of five charged amino acid residues located in or next to transmembrane segments (TMS). These residues were mutated in the MexF gene by site-directed mutagenesis and replaced by residues of opposite or neutral charge. MexF proteins containing combined D410A and A411G substitutions located in TMS4 were completely inactive. Similarly, the substitutions E417K (next to TMS4) and K951E (TMS10) also caused loss of activity towards all tested antibiotics. The substitution E349K in TMS2 resulted in a MexF mutant protein which was unable to transport trimethoprim and quinolones but retained partial activity for the transport of chloramphenicol. All mutated MexF proteins were expressed at comparable levels when tested by Western blot analysis. It is concluded that charged residues located in or close to TMS are essential for proper function of MexF.