Bacterial Co-selection and Their Resistance to Antimicrobial Compounds

Some organisms are inherently resistant to an antimicrobial compound or drug due to simply lacking the target site it acts upon or because of an inability for that agent to accumulate to a biologically effective concentration. Intrinsic resistance may occur because of exposure to the compound resulting in a phenotypic adaptation, inductive change, or mutation in gene encoding at the chromosomal level (Gilbert and McBain 2003). Phenotypic changes may result in impermeability or the expression of efflux pumps.

Impermeability

“Recent data indicate that the outer membrane barrier as a resistance mechanism is only significant in the context of additional resistance mechanisms that work synergistically with it to promote resistance” (Poole 2002).

Efflux

Efflux pumps naturally occur amongst bacteria as a defense mechanism to pump out toxicants. Typically, it is associated with the resistance of Gram-positive and Gram-negative bacteria to tetracyclines and fluoroquinolones (Poole 2002). Historically, it has not been the most common means of antibiotic resistance within a clinical setting, however, recent reports suggest an increased incidence of this mechanism (Gilbert and McBain 2003). All bacteria have gene-encoded efflux pumps that may not be expressed until selective pressure is exerted by exposure to a biocidal compound or by the acquisition of additional genes from mobile genetic elements through horizontal gene transfer (Hartmann et al. 2016; Poole 2002).

Horizontal Gene Transfer

Fitness variation models predict that new resistances are most likely to occur on existing multidrug-resistant strains of bacteria (Lehtinen et al. 2019). This is explainable by the presence of resistance genes in a bacterial population prior to the administration of an antimicrobial and the ability for their subsequent mutations to be spread between bacteria from the same generation (Domingues et al. 2012; Zhang et al. 2019). This is known as horizontal gene transfer (HGT) and it occurs via phage transduction, transformation, or conjugation (Marraffini and Sontheimer 2008; Partridge et al. 2018). Conjugation occurs when a plasmid is shared by another bacterial cell by allowing for a bacterium to share its genetic material via a pilin bridge with another bacterium (Zhang et al. 2019). This is widely believed to be the most common mechanism of HGT between bacteria phylum (Matyar 2012); however, mobile genetic elements such as integrons also allow for HGT to occur within and between bacteria species as well as archaea species (Gillings 2014).

Mobile Genetic Elements

Mobile genetic elements (MGEs) allow for DNA within a bacterial cell to be moved from the chromosome to a plasmid or between plasmids (Partridge et al. 2018). MGEs include integrons, transposons, and insertion sequences. Transposons and insertion sequences are sometimes nicknamed “jumping genes” because they are DNA segments that have the ability to spontaneously move, and their associated resistance genes, to new locations within the cell (Partridge et al. 2018). When these resistance genes randomly “jump” from the chromosome to a plasmid or vice versa, they may then be shared or incorporated by a bacterium. Figure 1 illustrates the processes associated with intercellular mobility using transposons. In contrast to transposons and insertion sequences, integrons use site-specific recombination to move genes between defined sites (Partridge et al. 2018). MGEs are important to the spread of antibiotic resistance genes (ARGs) and biocides/metals resistance genes (BMRGs) because they generate a genetic variation within bacteria generations that exerts continuous selection pressure on bacteria to evolve new variants (Pathak et al. 2019).

Evidence of Co-Selection for Co-Resistance and Cross-Resistance

Although exposure to antibiotics may result in the spread of ARGs via HGT, it is not limited to this alone. HGT of ARGs may be promoted with exposure to biocides or heavy metals when ARGs and BMRGs are physically located on the same plasmid (Gullberg et al. 2014; Pal et al. 2015). For example, a study investigating a hospital outbreak of Klebsiella pneumonia and Escherichia coli found that very low concentrations of antibiotics and heavy metals could additively select for a large plasmid conferring resistance to aminoglycosides, β-lactams, tetracycline, macrolides, trimethoprim, sulfonamide, silver, copper, and arsenic (Gullberg et al. 2014).

There is conflicting evidence to explain this phenomenon. On one end, co-selection may occur because ARGs within plasmids display higher transcription probability and abundance than those encoded chromosomally (Liu et al. 2019); however, another large study found that among sequenced bacterial genomes, the presence of biocide/metal resistance genes drove co-selection chromosomally more often than plasmid-mediated HGT (Pal et al. 2015). An explanation of these differences may be due to the presence of MGEs which can affect genetic material on either chromosomes or plasmids. For example, an increase in concentrations of a class 1 integron was associated with the relative abundances of ARGs in upstream wastewater treatment plant (WWTP) effluent in a receiving river (Berglund et al. 2015).

In terms of risk management, this implies WWTPs as an aquatic source ARG and potentially, BMRG proliferation. Thus, stricter regulation of pharmaceutical and personal care products, which include biocides, may help to reduce the incidence of HGT among bacteria in this scenario. As for the implications to their impact on agriculture, limiting the use of heavy metals in feed should reduce the incidence of BMRGs in the soil resistome and associated chromosomal gene transfer either vertically or horizontally. Finally, limiting the application of antibiotic-manure to soils may also reduce the potential of ARG proliferation in the soil resistome by HGT and, alternatively, composting has been found to reduce the effects of prior use (Chen et al. 2019).

The widespread prevalence of the czrC gene among CC398 MRSA isolates in a global strain collection suggests that zinc used in agricultural feed supplements for swine and veal may have contributed to the global emergence of methicillin-resistant Staphylococcus aureus (MRSA) (Cavaco et al. 2011).

A study investigating a hospital outbreak of Klebsiella pneumoniae and Escherichia coli found that very low concentrations of antibiotics and heavy metals could additively select for a large plasmid, pUUH239.2, which confers resistance to aminoglycosides, β-lactams, tetracycline, macrolides, trimethoprim, sulfonamide, silver, copper, and arsenic (Gullberg et al. 2014).

Among sequenced bacterial genomes, the presence of biocide/metal resistance genes drives co-selection chromosomally more frequently than by plasmid-mediated HG (Pal et al. 2015)

Limiting the application of antibiotic-manure to soils may reduce the potential of ARG proliferation in the soil resistome and, alternatively, composting may reduce the effects of prior use (Chen et al. 2019).

Discussion

In my opinion, the evidence reviewed suggests that even the intended use of current antibiotics, biocides, and heavy metals drives the antibiotic resistance of clinical importance. In recent years, there has been a decline in the innovation of newer, improved pharmaceutical antimicrobials. Until novel drug therapies are introduced, public health officials should expect the incidence of resistant infections to increase; however, each drug therapy serves only as a temporary solution to abate the problem. Early evidence suggests that clustered, regularly interspaced, short palindromic repeat (CRISPR) interference could be manipulated to provide a way to impede the spread of antibiotic resistance by preventing plasmid conjugation in pathogens like staphylococci (Marraffini and Sontheimer 2008). Alternatively, a different study found no significant link between CRISPR activity and the presence of plasmids or integrons in E. coli, suggesting that this may only work for some species (Touchon et al. 2012). More research is needed to determine the efficacy of CRISPR interference as a mode of reducing the prevalence of ARGs, and potentially, biocidal or heavy metal resistance genes.

As someone without a background in microbiology, I was unaware of horizontal gene transfer among bacteria or its large role in antibiotic resistance. Although work is being done to communicate the need for individuals to only use pharmaceuticals as directed, I feel there is little being done by environmental health officials to communicate the proper use of personal disinfectants. Although this review suggests that WWTPs and agriculture play a larger role in the dissemination of ARGs and BMRGs, fully sequenced isolates and plasmids still only represent a very small fraction of bacteria in the environment (Pal et al. 2015). As more microbiome research emerges and more bacteria are sequenced, we may find an increased potential of these personal antimicrobials to influence the spread of antibiotic resistance. If this is the case, then greater regulation of these products is warranted at a personal level to reduce the burden on WWTPs.