Identification of antibiotic-resistant, gram-negative bacteria in sewage and bioaerosols from a wastewater treatment plant: A genotypic and phenotypic study

Antimicrobial resistance (AMR) is a serious public health problem that causes millions of deaths annually and may become the leading cause of death worldwide by 2030. In 2019, antibiotic-resistant infections were responsible for the death of about 1.3 million people, and this statistic can increase significantly without preventive measures. It is a global health problem comparable to deadly diseases such as cancer (Murray et al. 2022).

Even though it is a natural phenomenon, it can influence the diversity and dissemination of diseases. The AMR relationship with the dissemination of resistant bacteria in humans and animals highlights that it can be intensified by anthropic action (O‘Neill 2016).

As argued by Murray et al. (2022), it is necessary to take effective measures to control and reduce the spread of AMR. Several factors can aggravate the increase in AMR, such as unnecessary prescriptions of antibiotics, self-medication, excessive use in livestock, inadequate control of nosocomial infections, poor hygiene, and sanitation, and, therefore, they should be strictly managed.

Naturally, AMR can be an inherent attribute of the microorganism, or it can be acquired through vertical and horizontal gene transfer, with main resistance mechanisms including drug uptake limitation and drug inactivation, as highlighted by Nadeem et al. (2020).

The presence of antibiotics at low concentrations in wastewater can boost the adaptation of microorganisms and, consequently, promote antimicrobial resistance in different environments. In addition, the presence of other pollutants such as heavy metals and bactericidal cleaning products can create favorable conditions for the development of resistant microorganisms (Langbehn et al. 2021).

Serious bacterial infections are a growing concern because of antibiotic resistance caused by beta-lactamases, enzymes produced by bacteria that reduce the effectiveness of beta-lactam antibiotics (Khan et al. 2020).

Beta-lactam antibiotics are widely used in the treatment of infections in humans, accounting for over 60% of antibiotics prescribed globally. The emergence of the extended-spectrum beta-lactamases (ESBL) producing bacterial group was mainly due to mutations in beta-lactamase (bla) encoded by beta-lactam genes, leading to an increase in the use of carbapenems in clinical medicine (Klein et al. 2018).

Carbapenems are a subclass of antimicrobials used to treat infections caused by Gram-negative bacteria, especially in resistant and multidrug-resistant cases when penicillins and cephalosporins are no longer effective (Aurilio et al. 2022).

ESBL-producing bacteria are associated with serious systemic infections and nosocomial infections and can also be found in non-clinical sources and be a source of community bacterial infections (Khan et al. 2020).

Studies have been conducted to understand the epidemiology of AMR in non-hospital environments, such as domestic sewage, which harbors a wide range of microorganisms (Osman et al. 2019). Metagenomic analyses have demonstrated the probable transmission of ADR determinants from sewage to the environment, in which ADR dissemination is closely related to environmental selective pressure (Zieliński et al. 2022).

Continuous pollution by antimicrobial compounds not metabolized by humans is excreted in feces and urine via domestic sewage, favoring selective pressure on bacteria in sewage and their genetic evolution in response to environmental stress (Felis et al. 2022).

Studies have shown that Wastewater Treatment Plants (WWTPs) can be sources of dissemination of antibiotic resistance genes to the environment since antibiotic-resistant bacteria are found in treated effluents regardless of the WWTP's efficiency or operational conditions (Wang et al. 2020; Zieliński et al. 2022), regardless of efficiency or operating conditions of a WWTP, the result is the production of effluents containing antibiotic-resistant bacteria (Łuczkiewicz et al. 2010).

Some bacteria may show resistance to a variety of antibiotics, including ESBL-producing bacteria that colonize the human digestive tract and are often detected in sewage (Wang et al. 2020; Zieliński et al. 2022).

Sewage treatment in WWTPs with activated sludge systems requires the addition of oxygen to remove organic matter. In general, this is done through aeration in the reactors, which can be carried out with surface or bottom mechanical aerators or with diffuse air aeration using blowers (Metcalf & Eddy 2016).

During the aeration of the reactors in WWTPs, the atmospheric air bubbles transfer oxygen to the sewage and can generate bioaerosols through the rupture of bubbles on the sewage surface, releasing them into the atmosphere (Sanchez-Monedero et al. 2008; Michałkiewicz et al. 2018).

In this way, sewage treatment can emit bioaerosols containing bacteria, viruses, fungi, and allergens, increasing the risk of diseases and infections in communities close to WWTPs and WWTPs workers due to continuous exposure to these particles (Korzeniewska 2011; Zieliński et al. 2022). Thus, bioaerosol particles generated in ETEs may represent a possible source of dissemination of antibiotic-resistant bacteria (Li et al. 2016).

In the context outlined above, this research focused on monitoring Gram-negative bacteria that produce ESBL enzymes and exhibit antibiotic resistance. The study assessed the genetic and phenotypic profiles of these bacteria in samples obtained from sewage, sludge, and the air within a wastewater treatment plant.

The Wastewater Treatment Plant (WWTP) in question serves a population of approximately 10,000 inhabitants with a flow of 30 L s⁻¹ on average (operational data). The treatment system adopted by the WWTP consisted of a pre-treatment unit, three sequential batch reactors (SBR) with diffuse aeration using activated sludge, a disinfection tank, and a sludge densification and dehydration unit. The effectiveness and satisfaction in the treatment of domestic sewage are ensured by this configuration, which meets the standards set by the Santa Catarina State Environmental Council for WWTPs with flows between 5 and 50 L s⁻¹. This setting aims to ensure efficiency by achieving a BOD (Biochemical Oxygen Demand) less than 70 mg L⁻¹ and phosphorus below 4 mg L⁻¹, in compliance with strict environmental legislation aimed at protecting water quality and public health.

A sampling campaign was carried out over five different days, distributed between December 2021 and August 2022. On each sampling day, samples were collected from four points of interest: raw sewage, treated sewage, sludge, and bioaerosols, totaling 20 samples.

For the identification of the samples, the identification nomenclature ‘ID’ refers to the origin of the sample, the month of collection, and the isolation number. For example, sample 06E10 indicates the collection of a raw sewage sample in June and the isolation number 10. To identify the origin of the sample, we use the letter ‘E’ for raw influent sewage samples, ‘S’ for treated effluent samples, ‘L’ for sludge samples, and ‘B’ for bioaerosol samples.

For the collection of raw sewage samples (E), a refrigerated automatic sampler was employed. This device was set to collect 200 ml of sewage every hour over 24 hours. The collected samples were mixed daily. From each hourly 200 ml sample, a 10 ml aliquot was taken and combined into a single vial. This process resulted in a composite sample that represented the average sewage composition over the 24 hours. This method ensures a comprehensive and representative analysis of the sewage characteristics, facilitating accurate monitoring and assessment of the wastewater treatment process. Sample collections of treated effluent (S) were systematically collected before the disinfection stage, in parallel with the collection of raw sewage, within the same 24-hour cycle. For every batch of treated effluent discharged, a composite sample of 200 ml was collected, ensuring an accurate representation of the pre-disinfection effluent. The collection of sludge samples (L) referred to the sludge discarded from the reactor at each batch on the same day.

The collection of bioaerosol samples (B) was performed on the SBR reactor platform using a liquid-based impactor (gas scrubber bottle with porous cylinder and 100 ml glass base) containing 20 ml of sterilized sodium chloride solution (NaCl 0,9%) and air sectioned by a vacuum pump with a flow rate of 28 L min⁻¹ for 30 minutes. Bioaerosols were collected during the aeration phase of the reactor and the impactor was fixed at a height of 1.5 m on the reactor platform, within the human breathable zone (Niosh 2016), and the sampling times varied on each day. Meteorological data were obtained from the Civil Defense meteorological station, located about 1,200 m from the WWTP.

The collected samples were immediately placed under refrigeration and transported to the laboratory within a timeframe of less than 4 hours, thus ensuring the preservation of their original characteristics and the integrity of the data obtained (CETESB 2011). Upon arrival at the laboratory, the samples were promptly inoculated.

For ESBL screening and to facilitate the isolation of colonies, raw sewage, and sludge samples were diluted in suspensions (10 ml of sample to 90 ml of diluent) and inoculated onto Petri dishes containing selective chromogenic culture media (CROMONEW ESBL, from Newprov), using the spread plate technique. Samples of treated sewage and bioaerosols were seeded directly in the culture medium, without dilution. Plates were incubated at 35 ± 2 °C for 18 to 24 hours, and inoculations were performed in duplicate (Khan et al. 2020).

During the environmental sampling campaign, bacterial colonies from the chromogenic media were selected based on their macroscopic characteristics. Morphologically distinct colonies were randomly chosen, subcultured, and inoculated into tubes with Brain Heart Infusion (BHI) broth, and then incubated at 35 ± 2 °C for 24 hours. After growth in BHI, the bacteria were isolated on Petri dishes with MacConkey Agar, also at 35 ± 2 °C for 24 hours, and sent for identification and antibiotic sensitivity tests (Koneman et al. 2018).

It is important to note that distinct species of bacteria may have the same colony characteristics, which may make identification difficult. However, as the objective of the study was not to quantify the microorganisms present in the sample, the macroscopic selection criteria were adopted.

For the identification of bacterial species, an automated method was used in the MicroScan WalkAlway Plus 96 equipment (Siemens Healthcare Diagnostics, Munich, Germany). This system reads and interprets the biochemical profile of bacteria through comparison with an updated database. Panels containing culture media are automatically incubated for 16–24 hours at 35 °C ± 2 °C. The system also identified through antibiotic susceptibility testing (AST) and ESBL confirmation.

For the extraction of DNA (deoxyribonucleic acid) from the identified Enterobacteriaceae strains, the heat shock method was used, according to the protocol described by Kobs et al. (2020). Initially, a single colony of each strain was suspended in 100 μL of sterile ultrapure water and subjected to three heating sequences at approximately 100 °C for 5 minutes, followed by thermal shock on ice for 5 minutes and centrifugation at 8000 xg for 10 minutes. The resulting supernatant was used for DNA quantification and identification, through spectrophotometric analysis at 260 and 280nm, using the Epoch spectrophotometer from Biotek Instruments (Winooski, USA). It is important to highlight that the isolated strains referred to microorganisms resistant to at least one carbapenem.

For genotypic identification of the isolates, only Gram-negative bacilli resistant to at least one carbapenem antibiotic were selected, excluding non-fermenters (NF-GNB) and the isolate Burkholderia cepacia complex, which is intrinsically resistant to carbapenems.

PCR reactions were performed using the XP Cycler equipment (BIOER Technology, Tokyo, Japan), with an initial denaturation step at 94 °C for 3 minutes, followed by 40 cycles of 94 °C for 1 minute, 37 °C for 1 minute and 72 °C for 2 minutes. A final extension was conducted at 72 °C for 10 minutes (Kobs et al. 2020).

The reactions were performed in a final volume of 50 μL, containing 50–500 ng of extracted DNA, 1 U of Platinum^® Taq DNA Polymerase (Invitrogen, São Paulo, Brazil), 200 μM of dNTPs (GE Healthcare, Little Chalfont, UK (Invitrogen), 1X PCR Buffer (Invitrogen), 50 pmols of each primer specific for the target gene (DNA Express, São Paulo, Brazil) (Table 1) and 1.5 mM MgCl₂ (Invitrogen).

To verify the results of the PCR reactions destined to the genes of interest in the study, the amplified products were submitted to electrophoresis in a 1% agarose gel containing 0.5 ug/mL of ethidium bromide, in TBE buffer, with a standard of electrophoretic run established at 100 V (10 V/cm) and 400 mA, for one hour.

A total of 17 bacterial strains of different species were isolated from samples of sludge, raw sewage, treated sewage (before the disinfection process), and bioaerosols. Species identified included Escherichia coli, Citrobacter farmeri, Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella aerogenes, Burkholderia cepacia complex, and Aeromonas hydrophila complex.

A greater bacterial diversity was observed in raw sewage samples, with isolates of Citrobacter farmeri, Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca. In samples of treated sewage, isolates of Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli were identified. In the sludge, the most frequent species were Escherichia coli and Klebsiella aerogenes, while in the bioaerosols, isolates of Burkholderia cepacia complex and Aeromonas hydrophila complex were found (Table 2).

Table 2

Identification results, AST, and molecular test

ATM (Aztreonam), CAZ (Ceftazidime), CPM (Cefepime), CTX (Cefotaxime), CRX (Cefuroxime), COL (Colistin), ERP (Ertapenem), IPM (Imipenem), MPM (Meropenem). ESBL: MicroScan^® confirmation. Categories: S (Sensitive), I (Intermediate), and R (Resistant), ¹R Intrinsic Resistance EUCAST 2022.

Note: Spaces without results in Table 2 are due to the lack of a cut-off limit for the identified microorganism or the gene not being tested. To facilitate identification, the samples were labeled according to the month of collection and the type of sample. During the collection process, collections were not carried out during periods of rain due to the diluting effect of rainwater in the sewage, which can affect the results of the analyses.

Source: Author, 2023.

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It is important to point out that several of these bacterial species presented genes coding for beta-lactamase (ESBL), conferring resistance to antibiotics commonly used in clinical practice. The detection of bla CTX-M, bla SHV, bla TEM, and bla KPC genes was observed in at least some of the isolates (Table 2). This observation suggests the presence of resistance to these antibiotics in the environmental samples studied, although caution should be exercised in interpreting this information, given that the study does not adopt a quantitative approach.

The predominant detection of the CTX-M gene in the analyzed environmental samples reflected observations documented in the literature, pointing to the widespread dissemination of this resistance gene among different bacteria, especially in Enterobacteriaceae, as discussed by Cantón et al. (2012). This highlights the adaptability and propagation capability of CTX-M genes through mobile genetic vectors, contributing to a concerning scenario of antibiotic resistance.

The presence of different bacterial species and the detection of antibiotic-resistance genes in environmental samples highlight the importance of control and prevention measures to prevent the spread of these microorganisms in the environment and public health. These results underscore the need for continuous monitoring of wastewater and waste treatment to prevent the spread of resistant bacteria and the rise of antibiotic resistance.

The results of this study revealed the presence of bacterial strains resistant to antibiotics in environmental samples of sewage, sludge, and bioaerosols. The predominant bacteria were of the genus Klebsiella spp. and Escherichia coli, common pathogens responsible for infections in humans and often associated with elevated levels of antibiotic resistance. These findings are in line with recent reports, such as the World Health Organization's GLASS (2022), which highlight the growing threat of antimicrobial resistance worldwide.

The molecular detection of resistance genes through PCR is a significant concern, as these genes confer resistance to a broad spectrum of antibiotics, including those frequently utilized in medical practice. Notably, the bla CTX-M, bla SHV, and bla TEM genes were identified in strains of Klebsiella pneumoniae, a pathogen of clinical importance in nosocomial infections. Although only three isolates of K. pneumoniae were detected in this study, the presence of these genes highlights the potential for antibiotic resistance. Additionally, the bla KPC gene was also identified in one of the K. pneumoniae isolates, indicating a further dimension of antibiotic resistance that merits attention.

The presence of antibiotic-resistant strains in the environment represents a threat to public health since exposure to these microorganisms can lead to the spread of antibiotic resistance and make it difficult to treat infections in humans (Steenbeek et al. 2022). Therefore, the results of this study emphasize the importance of preventive measures to reduce the risk of exposure to antibiotic-resistant bacteria in the environment, such as the implementation of adequate hygiene practices and the proper treatment of hospital waste and sewage.

Sewage sludge produced in domestic sewage treatment plants is an important source of nutrients for agriculture. However, its use as a fertilizer requires care due to the presence of antibiotic-resistant bacteria, which pose a risk to human and animal health. Recent studies (Khadra et al. 2019; Stachurová et al. 2021; Major et al. 2022) have shown that the secondary sludge from WWTPs can contain high concentrations of bacteria resistant to beta-lactams, including strains carrying resistance genes such as bla CTX-M. This underscores the need for careful risk assessment and improvement of sludge treatment processes prior to its use as a fertilizer.

The direct application of WWTP sludge to the soil can be a source of contamination of soil and water bodies with bacteria resistant to antibiotics, which can lead to the spread of resistance and make it difficult to treat infections in humans and animals (Khadra et al. 2019; Major et al. 2022). Therefore, the use of WWTP sludge as a fertilizer should be carried out with caution, and stricter controls should be implemented to reduce the risks of spreading antibiotic-resistant bacteria (Piotrowska et al. 2017; Hiruy et al. 2022). It is important to highlight that this is a complex issue that requires a multifaceted approach involving the health, environment, and agriculture sectors to ensure the safe use of WWTP sludge as a fertilizer.

On the other hand, bioaerosols have been recognized as important vectors in the spread of antibiotic-resistant bacteria and pose a risk to human and animal health (Yang et al. 2018). WWTPs are suitable places for the generation and dispersion of these biological agents, as evidenced by studies that highlight the risks of exposure in WWTP environments (Li et al. 2016).

In a recent study by Zieliński et al. (2022), the diversity and abundance of antibiotic resistance genes (ARGs) were analyzed in wastewater samples, identifying genes conferring resistance to various antibiotic classes in microorganisms such as Escherichia coli and Klebsiella pneumoniae, highlighting the presence of mobile genetic elements like ISL3 and IS1447 that facilitate the horizontal transfer of resistance, underscoring the critical role of wastewater treatment plants in the dissemination of ARGs.

Therefore, it is necessary to adopt preventive and mitigating measures concerning the risks of exposure and dissemination of antimicrobial resistance in WWTP environments, including the use of adequate personal protective equipment and the improvement of air quality treatment and control systems (Zhou et al. 2021). The choice of aeration system can also play a significant role in reducing the generation of bioaerosols in WWTP reactors. Studies show that diffusion aeration systems generate fewer bioaerosols than mechanical systems, due to less turbulence and less disruption of air bubbles on the liquid surface, although they generate smaller particles (Michałkiewicz et al. 2018; Wang et al. 2020).

In this study, the presence of bacteria resistant to antibiotics in environmental samples of sewage, sludge, and bioaerosols, highlighting the dissemination of these microorganisms and their implications for public health. The detection of pathogenic bacterial species carrying resistance genes underscores the threat of antimicrobial resistance and the limitation of treatment options. In addition, sewage sludge contamination and the presence of bioaerosols with resistant bacteria pose risks to human and animal health. These results reinforce the need for control and prevention measures to reduce the spread of resistant bacteria by promoting the prudent use of antibiotics, improving sewage treatment systems, and implementing rigorous sludge management protocols.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.