Which of these means of horizontal gene transfer can bacteria use to become antibiotic

Transmissible Resistance Due to Acquisition of Antibiotic Resistance Genes

1.

Antibiotic resistance genes generally have little effect on the host cell's overall fitness. For example, many of these genes actually destroy the antibiotic and do not alter the target site. Further, the expression of some resistance genes is actually induced by the antibiotic, so there is even less cost to the cell for keeping the gene.

Antibiotic resistance genes are often located on plasmids or transposons and can be transferred from cell to cell by conjugation, transformation, or transduction. This gene exchange allows the resistance to rapidly spread throughout a population of bacteria and among different species of bacteria. This is called horizontal transmission. This type of antibiotic resistance causes major problems for physicians treating infectious diseases.

Transmissible Antibiotic Resistance

Humans or animals exposed to probiotic strains carrying antibiotic resistance genes that might be transferred to the commensal microbiota in vivo are at a risk. This issue has been addressed in the European Union (EU) founded project ‘Biosafety Evaluation of Probiotic Lactic Acid Bacteria for Human Consumption’ and in the ‘Assessment and Critical Evaluation of Antibiotic Resistance Transferability in the Food Chain’ project where the antibiotic resistance character of a large number of strains was analyzed and recommendations on the determination of safety was given. The cut-off values for different antibiotics in different bacterial species were set. Antibiotic susceptibility of other strains belonging to the same species can be compared with those cut-off values to judge the possible acquired antibiotic susceptibility in a specific strain. Special focus on the transfer of antibiotic resistance genes and the presence of virulence genes has been given to the genus Enterococcus. The antibiotic resistance questions have also been in focus in the International Organization for Standardization (ISO) and International Dairy Federation joint Action Team on Probiotics.

3 Potential impacts on human health and in the environment

Fig. 1 shows a schematic diagram of different pathways of antibiotics and ARG dissemination due to CE. As mentioned above, TWW and BS are the principal pathways that promote the dissemination of antibiotics and ARG in the agricultural environment (Voulvoulis, 2018). Despite the numerous studies investigating the occurrence of antibiotics and ARG in TWW and BS, only some authors focused on the determination of the potential impacts of the use of TWW and BS for agricultural purposes (Chen et al., 2015; Hurtado et al., 2017; Östman et al., 2017). The assessment of the potential ecological implications is necessary to make a proper use of these resources (TWW and BS) without risks for human health and the environment. Spataro et al. (2019) evaluated the preliminary potential ecological risks of TWW, determining the risk quotients (RQs) for different antibiotics, such as amoxicillin (AMX), CIP, SMX, and chlorotetracycline (CTC). In this case, the RQs were calculated using the ratio between the measured environmental concentrations (MECs) and the predicted noneffect concentrations (PNECs) of each compound. The overall results of this study highlighted that the antibiotics concentrations detected in TWW pose a potential high environmental risk, with RQs values above 1.

Likewise, Prosser and Sibley (2015) determined the hazard to human health of concentrations of different ECs, including antibiotics, in plant tissue grown in BS-amended soils or irrigated with TWW. For this, they estimated the daily intake values and compared them with acceptable daily intakes. For BS, the hazard quotients associated with CIP and NFL were below 0.1. The same behavior was observed for TWW, where these values associated to erythromycin (ERN), NFL, sulfamethazine (SMZ), SMX, and TC were also less than 0.1. These results indicate low risks for the human health. Despite the hazard quotients of individual compounds not being relevant, a synergetic or additive effect could potentially present a hazard and for this reason, further researches are necessary in this area.

Regarding the risks for human health and for the environment associated with the occurrence of ARG in TWW and BS, to date there is a lack information. Chen et al. (2019b) calculated the resistome risk scores of manure-derived amendments applied to soil that contain antibiotic concentrations. This score is defined as a relative index comparing co-occurrence of sequences corresponding to ARG, mobile genetic elements, and putative pathogens between manure-amended, compost-amended, and control soils (Oh et al., 2018). This study indicated that soil amended with compost reduced the resistome risk score of manure-amended soils and is similar to the control soils. Despite these results, Manaia et al. (2017) considered that a quantitative model is urgently needed for determining the risk of transmission of resistance from the environment to human and it must be based on ARB and ARG that are able to colonize and proliferate in the human body.

3.8.5 Antibiotic resistance

Antibiotic residues and antibiotic-resistant bacteria find their ways to AD bioreactors. Therefore, the microorganisms (both pathogenic and commensal) in anaerobic digestate are also an important source of antibiotic resistance genes (ARGs). ARGs in municipal wastewater were shown to be transferrable to bacteria in biosludge and crops fertilized with the digestate of human wastes (Zhao and Liu, 2019). Bacterial pathogens are potential hosts of ARGs encoding resistance to multi-drugs and macrolide-lincosamide-streptogramin (MLS). ARGs can spread through mobile genetic elements, such as integron, plasmids, and transposon, with transferrable genes encoding pathogenicity factors. Thermophilic AD has been commonly used to destruct integrons and ARGs in sewage sludge.

Integrons and mobile gene cassettes

Antibiotic genes of Gram-negative bacteria are often situated on plasmids and transposons. In the early 1980s, studies using restriction mapping and heteroduplex analysis showed that different sets of antibiotic-resistance genes were sometimes found in the same location in otherwise closely related plasmids such as the IncW group, or transposons of the Tn21 family (Stokes and Hall, 1989; Recchia and Hall, 1995). Further work showed that this third mechanism of antibiotic-resistance gene dissemination involved naturally-occurring gene expression elements called integrons (Stokes and Hall, 1989). These structures operate as vehicles for the acquisition of resistance genes carried by MGE, termed gene cassettes, by a site-specific recombinational mechanism (Hall and Collis, 1995) as shown in (Fig. 1). While integrons are found on many different plasmids and transposons, one integron, Tn402, has been identified to be itself an active transposon (Bennett, 1999; Domingues et al., 2012). Class 1 integrons are most associated with clinical infections. The presence of integrons has been reported most often in Gram-negative bacteria but they also occur in Gram-positives, including staphylococci (Li and Zhao, 2018) streptococci (Lin et al., 2017) and enterococci (Clark et al., 1999; Yu et al., 2017). Yu et al. (2017) and Lin et al. (2017) experimentally observed class 1 integron mediated excision and integration of various exogenous antibiotic resistance genes using a collection of plasmids and Enterococcus faecalis and Streptococcus pneumoniae, respectively. Interestingly, retrospective analyses have indicated that integrons were associated with resistance genes present in the shigella isolates that characterized the first wave of transferable plasmid-mediated resistance in Japan in the 1950s (Liebert et al., 1999).

4.1 Occurrence of Antibiotics

As mentioned in the introduction of this chapter, data on the occurrence of antibiotics in soils are much more abundant than for other pharmaceuticals, due mainly to the potential spread of antibiotic resistance genes in different microbial communities and so the environmental risk associated. For instance, TCs show high sorption coefficients compared to other antibiotics, indicating not only the strongest sorption tendency but also the potential accumulation and persistence in solid matrices. As shown in Table 3, TCs have been detected frequently and at the highest concentrations in different agricultural soils fertilized with manure. Hamscher et al. [15] detected TC and CTC in soil samples amended with manure at concentrations between 86.2 μg kg− 1 in the first 10 cm and 171.7 μg kg− 1 in the 20–30 cm layer. This apparent increase in the concentration with depth was attributed to the release of bound residues of 4-epitetracycline, a metabolite of this antibiotic that was transferred from the liquid manure into the soil and not to TC translocation. As expected, neither TC nor CTC was detected in deeper soil layers or in soil water or groundwaters located 80–200 cm deep. Furthermore, the authors observed accumulation of TC with successive manure fertilization, corroborating the high retention and persistence of these antibiotics in upper layers of the soil (see Figure 4B). Storage of manure during 6 and 12 months did not seem to decrease the amount of TC that was finally applied to the soil (Figure 4B). Blackwell et al. [53] obtained similar results of OXT and TYL.

Table 3. Pharmaceutical Concentrations (μg kg− 1) Detected in Agricultural Soils

On the contrary, due to their lower KD and high polarity and solubility, sulfonamides are the family of antibiotics that have a greater potential for leaching to groundwater, and several studies have demonstrated their occurrence in aquifers at concentrations up to μg L− 1 level [74–77]. Because of their low KD values, sulfonamides are considered to be very mobile and weakly retained in soil and therefore highly bioavailable and generally nonbioaccumulative. Nevertheless, despite their high potential to leach and run off and that some authors have considered sorption to soils of sulfonamides negligible [22], sulfonamides have been detected in soils together with TCs and macrolides [14] in the top 0–30 cm soil. Regarding SMX, one of the most commonly found antibiotics in wastewater effluents, it has been demonstrated that it is moderately sorbed in soils, [78,79] and different sorption mechanisms have been proposed such as water bridging interaction between neutral SMX molecular and the clay surface, complexation of exchangeable cations through N or the SO2− group, and sorption to natural OM.

Mechanisms of resistance to antibiotics

In the evolution of antibiotic resistance of the genus Corynebacterium most studies conclude that extrachromosomal genetic elements are involved in resistance gene transfer. Horizontal gene transmission plays an important role in their pathogenicity. Antibiotic resistance genes are localized in large plasmids, for example, the pTP10 plasmid is responsible for the resistance to chloramphenicol, tetracycline, streptomycin, and erythromycin described in Corynebacterium xerosis (Oliveira et al., 2017).

Many species such as C. striatum, C. pseudodiphtheriticum, C. jeikeium and C. coyleae (Fernández-Natal et al., 2008) are resistant to macrolides because they possess ermX genes. For this reason, erythromycin, which had traditionally been the treatment of choice, has ceased to be used unless the sensitivity report of the laboratory confirms its sensitivity. It has also been linked to resistance to clindamycin, and trimethoprim-sulfamethoxazole.

Another example is the existence of multidrug-resistant strains, such as C. amycolatum, one of the most common strains of corynebacteria. This property is defined by the existence of resistance to three or more groups of antibiotics commonly used in the treatment of infection such as beta-lactam, aminoglycosides, macrolides, MLSB group and quinolones. The presence of mutations in the gyrA gene is related to resistance to treatment with quinolones (Ortiz-Perez et al., 2010).

7 Summary and conclusions

The antibiotics, metals, and biocides can directly select antimicrobial resistance genes within the AMR pathways that are, i.e., sewage sludge–treated wastewater, rivers, irrigation system, aquaculture, industry, and finally groundwater. Currently, one of the highest drivers of ARGs is WWTP, and moreover besides the effort to treat the wastewater, there are no wastewater treatment technologies in common operation that removes totally ARGs. If regulations on the release of macrolides in effluent would meet the standards, the solutions required to be settled for significantly decreasing levels of antibiotics, biocides, metals, and ARGs, it would significantly increase the price of treating wastewater. The use of antibiotics has led to increased resistance of microorganisms. Currently, applied wastewater treatment systems are not able to effectively disinfect these wastewaters or remove the ARGs. ARB present in surface water can be a serious health problem. Nowadays, the goal is to decrease the spread in ARGs by applying higher hygiene level, avoiding infections, monitoring the nosocomial transmission of bacteria, and appreciating treating of and improving new treatment procedures. Currently, the most important action to be taken is to reduce the use of antibiotics in medicine (especially in outpatient treatment), veterinary medicine, and in agriculture, so as to eliminate the mechanisms associated with the selection pressure.

5.3 Formation and exposure of disinfection by-products in water environment: a new threat for endocrine disrupting chemical and antimicrobial resistance

One of the growing concerns in recent decades is the presence of genotoxic compounds in the effluents discharged from WWTPs. These genotoxic compounds are also frequently encountered during drinking water treatment, posing a risk to both animals and humans (see Table 5.1). The chlorinated hydroxyfuranones are one of the major contributors of mutagenicity in drinking water. The other potential threat is the presence of EDCs in secondary effluents of WWTPs and the emergence of antimicrobial resistance genes. Recent studies describe the presence of a wide range of EDCs in aquatic bodies, some of which are synthetic and others natural (Zhang et al., 2012). Although EDCs appear at low levels, their continuous release into aquatic bodies may increase the environmentally available dose in the long term, increasing the risk for humans, aquatic animals, and other nontarget organisms. Moreover, due to their trace presence in natural and wastewater, detection and treatment is the biggest challenge. In contrast, the posttreatment by-products may be more toxic than the parent compounds in varying order of magnitudes (Postigo et al., 2018).

Table 5.1. Different categories disinfection by-products produced during chemical treatment.

DBP, Disinfection by-product.

A case study from Patancheru, sub-urban of Hyderabad, India, reported high level of antibiotics from water bodies that are released from adjacent pharmaceutical industries. Surprisingly their concentration range in water reaches up higher than the concentration that required to kill microorganisms (https://www.reactgroup.org/). The literature reported that emerging micropollutant of industrial origin stands out as extreme when it comes to infiltrating in water; this leads to harboring antibiotic resistance genes (ARGs) and has increased the probability to transfer these resistance genes. If this unsafe disposal continues, there is a risk that 50 million people will die (15 times more than today) due to antimicrobial resistance by the year 2050 (Jim O'Neill, 2016). The World Bank estimated that the societal costs would be increased in many economically marginal countries to deal with this unforeseen situation, which would significantly impact the global economy. The situation is not only confined in developing worlds, but it was reported that around 15 tonnes of antibiotics would end up in Swedish sewage sludge every year (Anonymous, 2019). Water is a suitable environment for the emergence of antimicrobial resistance bacteria (ARB) and ARGs. The indiscriminate use of antibiotics for human and livestock led to the development and dissemination of ARBs and ARGs (Nawaz and Sengupta, 2019). Besides antibiotics, environmental chemicals have been recently implicated to development and transmissions of ARGs. There was substantial evidence confirming the role of DBPs in development of ARBs (Li and Gu, 2018). This is concomitant to use of diverse disinfectants in water treatment processes, increasing the environmental load, culminating in genetic alteration, and ultimate development of ARGs. The residual disinfectants also promote the spread of ARG across strains of bacteria. The ARG may transform competent bacteria present in the water through horizontal transfer. However, these transfers largely depend on the concentration and types of disinfectants used. The horizontal transfer of extracellular DNA of Escherichia coli to Salmonella enterica has been reported elsewhere (Zhang et al., 2017). Disinfectants like free chlorine, chloramines, and hydrogen peroxide were described to accelerate ARGs transfer. Zhang et al. reported a concentration-dependent increase in the conjugative transfer of ARGs. They obtained accelerated ARGs transfer at subinhibitory concentrations of chlorine, chloramines, and hydrogen peroxide. The underlying mechanism responsible for the conjugative transfer has been attributed to intracellular reactive oxygen species formation and SOS responses. SOS response pathways were stimulated by residual disinfectants present in different water systems (Li and Gu, 2018). Earlier studies reported that subinhibitory levels of antibiotics mediate induction of mutants and horizontal transfer of ARGs between bacterial strains (Andersson and Hughes, 2014). Disinfectants modulate membrane proteins of bacteria, increasing membrane permeability, and facilitating horizontal gene transfer between the recipient and the donor bacterium (Sanawar et al., 2017).

The emergence of ARB and ARGs could be attributed to antibiotic-like properties of some DBPs that elicit genetic mutations. ARGs are known to confer resistance to antibiotics, most commonly by altering cell wall permeability or by modification of receptor sites. Recent studies reported the emergence of ARB in aquaculture waters. This may be attributed to uncontrolled use of antibiotics or antibiotic-like substances either for therapeutic or prophylactic uses (Sanawar et al., 2017). Moreover, routine use of selective disinfectants leads to the selective increase of particular microbes exhibiting biological resistance. In contrast, chlorination was reported to inactivate antibiotic-resistant pathogenic microbes and contributes to minimal removal of ARG from water (Sanawar et al., 2017). Till date, no specific guidelines are available about permissible ARG level produced after water treatment processes.

2.4 Integrons

Integrons are genetic systems that allow bacteria to capture and express gene cassettes and they can be found as part of plasmids, chromosomes, and transposons. Integrons are formed by an intI gene, encoding an integrase that is a site-specific recombinase, an attachment site (attI), and one or two strong promoters (P) that drive the expression of inserted gene cassettes [38]. Gene cassettes can be inserted one after the other into the integron insertion site [3], producing the formation of long arrangements of ARGs that can be transferred simultaneously among bacterial populations [39]. This mobile genetic element can be usually found in clinical bacterial strains, possibly because most of the cassettes identified are associated with antibiotic resistance. However, in the last years, several studies have been performed in order to determine the occurrence of integrons in bacteria from aquatic environments.

There are three main classes of integron structures, depending of their integrase, but most resistance integrons conform to a structure known as a class 1 integron [3]. Moura et al. [38] detected genes encoding integrases belonging to classes 1 and 2 integrons among Enterobacteriaceae and Aeromonas spp., in influents and effluents of a WWTP. These integrons harbored different gene cassettes conferring resistance to penicillins, fluoroquinolones, and chloramphenicol, among others. Another recent investigation [40] has demonstrated that suspended aggregates of bacteria in natural aquatic systems (the so-called flocs) contained class 1 integrons with clinically important ARGs.

Industrial activities have also been shown specifically to contribute to the increase of mobile genetic elements. Wright et al. [41] quantified class 1 integrase (intI1) gene abundance in total community DNA extracted from contaminated and reference riverine and estuarine microhabitats and in metal- or antibiotic-amended freshwater microcosms. Results showed that the intI1 gene was more abundant in all contaminant-exposed bacterial communities, indicating that relative gene transfer potential is higher in these communities [41]. Additionally, Rosewarne et al. [42] demonstrated that the abundance of intI1 was increased as a result of ecosystem perturbation, indicated by a strong positive correlation with heavy metals such as zinc, mercury, lead, and copper. Moreover, the abundance of intI1 at sites located downstream from treated sewage outputs was associated with the percentage contribution of the discharge to the basal flow rate [42]. All these studies show that integrons make an important contribution to the dissemination of ARGs.