Abstract
Resistant bacteria may leave the hospital environment through wastewater. The opportunistic pathogen Pseudomonas aeruginosa, due to its intrinsic resistance to many antibiotics and its ability to easily acquire antibiotic resistance determinants, poses a significant threat to public health. The aim of this study was to evaluate the antibiotic resistance profiles of cultivated P. aeruginosa in untreated hospital effluents in the Czech Republic. Fifty-nine P. aeruginosa strains isolated from six hospital wastewaters were tested for antimicrobial susceptibility through the disc diffusion method against seven antimicrobial agents. Resistance was found in all antibiotics tested. The highest resistance values were observed for ciprofloxacin (30.5%), gentamicin (28.8%), and meropenem (27.2%). The P. aeruginosa isolates also exhibited resistance to ceftazidime (11.5%), amikacin (11.5%), piperacillin-tazobactam (11.5%), and aztreonam (8.5%). Seventeen strains of P. aeruginosa (28.8%) were classified as multidrug-resistant (MDR). The results of this study revealed that antibiotic-resistant strains are commonly present in hospital wastewater and are resistant to clinically relevant antipseudomonal drugs. In the absence of an appropriate treatment process for hospital wastewater, resistant bacteria are released directly into public sewer networks, where they can serve as potential vectors for the spread of antibiotic resistance.
HIGHLIGHTS
Pseudomonas aeruginosa is commonly present in untreated hospital wastewater.
The isolates presented resistance to clinically relevant antipseudomonal drugs.
28.8% of P. aeruginosa strains were identified as multidrug-resistant (MDR).
Resistant strains are released with untreated hospital effluents into public sewer networks where antibiotic-resistant strains can easily spread.
Graphical Abstract
INTRODUCTION
Healthcare facilities, where the use of antibiotics is more frequent and intensive and where there is high selective pressure in the bacterial community, are regarded as important hotspots for the selection of resistant bacteria (Varela et al. 2014; Tesfaye et al. 2019). Hospitals also play a significant role in the dissemination of antibiotic-resistant bacteria into the environment. Antibiotic-resistant bacteria may leave hospitals on colonized patients, but also via wastewater (Hocquet et al. 2016). Hospital effluents pose a special category of waste that is highly hazardous to public health and ecological balance due to their infectious and toxic characteristics (Fuentefria et al. 2011; Rodriguez-Mozaz et al. 2015; Proia et al. 2018). Hospital effluents contain large amounts of pathogenic bacteria and a variety of substances with antimicrobial activity, including not only antibiotics, but also disinfectants, heavy metals, and nonmetabolized pharmaceuticals (Chagas et al. 2011; Miranda et al. 2015; Santoro et al. 2015; Hocquet et al. 2016). Large amounts of antimicrobial compounds present in hospital wastewater exert a continuous selective pressure able to promote the development of antibiotic resistance (Hocquet et al. 2016; Lien et al. 2017). The effect of selective pressure consists in the progressive elimination of antibiotic susceptible bacteria and favoring the proliferation of resistant strains (Varela et al. 2014; Hocquet et al. 2016; Pazda et al. 2019). Resistant bacteria can react to selective pressure and adapt to new environmental conditions and are potential vectors for the spread of antibiotic resistance (Moges et al. 2014; Osińska et al. 2017). Moreover, selective pressure may also induce innate organisms to a rapid adaptation to selective conditions by acquiring new genetic traits (Santoro et al. 2015).
Despite their specific nature, hospital effluents are very often directly discharged into the public sewer network, collected at municipal wastewater treatment plants (WWTPs) and co-treated with urban wastewater (Verlicchi et al. 2010; Santos et al. 2013). In WWTPs, antibiotic-resistant bacteria, antibiotic-resistant genes, and the environmental microbiota are continuously mixed with antibiotics and their residues, and other substances with potential selective pressure that can come from various sources (Rizzo et al. 2013; Santoro et al. 2015; Manaia et al. 2016; Proia et al. 2018). Furthermore, the biological wastewater treatment process based on activated sludge, widely used in municipal WWTPs, provides optimal conditions for the transfer of antibiotic resistance genes, the development of new antibiotic resistance bacteria, and for the creation of hotspots for the spread of resistant bacteria and genes into the environment (Manaia et al. 2016; Nnadozie et al. 2017; Osińska et al. 2017). Wastewater offers an abundance of nutrients, high microbial concentration, and close interaction between bacteria, capable of facilitating the transfer of antibiotic-resistant genes between bacterial communities (Manaia et al. 2018). The final effluents, which are discharged from municipal WWTPs, contain antibiotic-resistant bacteria, sometimes at higher percentages than in raw wastewater (Novo et al. 2013). The worst fear apprehended for public health is the transfer of resistance genes between environmental bacteria and human pathogens (Moges et al. 2014; Asfaw et al. 2017). In addition, the accumulation of resistance features after exposure to various antibiotics and cross-resistance between agents may result in multidrug-resistant (MDR) bacteria (Israel Falodun et al. 2019; Krzeminski et al. 2019). MDR pathogens present a significant threat to global public health and are of intense clinical concern (McLain et al. 2016; Nnadozie et al. 2017). The occurrence and spread of multidrug antibiotic resistance among bacterial pathogens may have serious consequences for human health (Pazda et al. 2019). In the future, most of the presently available antibiotics in medicinal practise may be ineffective against resistance bacteria and the infections caused by MDR pathogens will be completely untreatable (Asfaw et al. 2017; Pazda et al. 2019).
Although hospital effluents usually constitute a minor part of the raw influent that is drained into municipal WWTPs, they are the main source of antibiotic residues. Hospital wastewater also brings with it an enormous number of antibiotic-resistant bacteria and their gene pool (Korzeniewska & Harnisz 2012). For this reason, hospital effluents represent one of the most serious pollutants that discharge into the environment (Asfaw et al. 2017).
The opportunistic human pathogen Pseudomonas aeruginosa is a ubiquitous microorganism, able to persist in many niches but preferring moist environments. It is a common hospital-acquired pathogen responsible for severe nosocomial infections, especially in critically ill and immunosuppressed patients. P. aeruginosa is the major pathogen of cystic fibrosis and is also involved in a variety of infections, including respiratory and urinary tract infections, wound and soft tissue infections, and infection of patients with thermal injuries (Fuentefria et al. 2011; Slekovec et al. 2012; Santoro et al. 2015; Rostami et al. 2018).
P. aeruginosa is intrinsically resistant to many antibiotics and is capable of easily acquired antibiotic resistance determinants (Feng et al. 2017; Imanah et al. 2017). Furthermore, P. aeruginosa has a high potential to evolve multidrug resistance phenotypes (Golle et al. 2017). The presence of different resistance mechanisms has a significant clinical impact, since it limits the therapeutic options for P. aeruginosa infections, compromises the efficacy of antipseudomonal agents, and makes P. aeruginosa infection very difficult to treat (Feng et al. 2017; Golle et al. 2017; Azam & Khan 2019; Rocha et al. 2019). P. aeruginosa strains have been reported to be resistant to a wide range of currently available antimicrobial agents, such as fluoroquinolones, but also carbapenems and third-generation cephalosporins, which are preferred options in the therapy of serious infections caused by MDR strains (Imanah et al. 2017; Azam & Khan 2019).
Resistant strains of P. aeruginosa originating from patients and the hospital environment can be discharged with untreated hospital effluents into municipal sewer networks. This distribution of P. aeruginosa from hospitals to the environment can increase the occurrence of antibiotic resistance and contribute to the transfer of antibiotic resistance genes in the bacterial community. The aim of this study was to evaluate the occurrence and diversity of antibiotic resistance of P. aeruginosa strains isolated from untreated hospital wastewater. Monitoring and characterization of antibiotic-resistant pathogenic bacteria in hospital effluents is necessary to assess their potential threat to human health. In addition, knowledge of the resistance of pathogenic bacteria to current antibiotics is useful for improving the therapeutic efficacy of antibiotics in clinical practice.
METHODS
Sample collection
Untreated hospital wastewater samples were collected from six different hospitals in the Czech Republic. The healthcare facilities differed in capacity. The largest facility is Hospital 1 (1,100 beds), followed by Hospital 6 (750 beds) and Hospital 5 (550 beds). Hospitals 2, 3, and 4 have 200 beds. All hospitals are not equipped with WWTP or other wastewater treatment systems (e.g. chlorination), and untreated hospital wastewater is directly discharged into the municipal sewer network and treated at central municipal WWTP. The samples were collected at the central points of discharge of wastewater into the municipal sewer network and contained wastewater from the entire hospital area. Half-liter wastewater samples were collected by submerging sterile bottles at the sampling points, using a telescopic sampling stick. The pH of the wastewater samples was measured immediately after sampling. A total of six samples were collected from January 2020 to March 2020.
Sample processing, isolation, and identification of P. aeruginosa
Immediately after sampling, the samples were transported to the laboratory in a cooling box and processed within 2 h after collection. A 10 mL aliquot of wastewater was transferred to 90 mL of Tryptone Soya Broth (Soyabean Casein Digest Medium, Himedia, India) and incubated aerobically at 37 °C for 24 h. After incubation, a 100 μL aliquot of serial ten-fold dilutions of bacterial suspension was streaked on Cetrimide agar (Cetrimide Agar Base, HiMedia, India). The plates were incubated aerobically at 37 °C for 24–48 h. After incubation, presumptive large, flat blue-green pigmented colonies with irregular margins were subcultured on non-selective blood agar (Blood Agar Base No. 2, HiMedia, India). From each positive Cetrimide agar plate, five presumptive colonies were transferred on blood agar or all colonies if less than five were present. Suspect colonies of P. aeruginosa on blood agar with typical metallic sheen and hemolysis were identified by Gram staining and biochemical tests, catalase, oxidase, and motility. The final identification was performed using the NEFERMtest® 24 kit (Erba Lachema s.r.o., Czech Republic) based on bacterial biochemical properties, according to the manufacturer's instructions.
Antimicrobial susceptibility testing
A standard disk diffusion method was used to determine the antimicrobial susceptibility profiles of P. aeruginosa isolates according to the recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST 2021a). A 24 h old pure culture of isolates standardized to 0.5 McFarland turbidity standards was swabbed on Mueller-Hinton agar (Mueller-Hinton Agar, HiMedia, India) according to the standard operational procedure. Incubation was performed at 35±2 °C for 18±2 h. The zones of inhibition were interpreted according to the current EUCAST Breakpoint Tables (EUCAST 2021b). The isolates were classified as ‘susceptible, standard dosing regimen’ (S), ‘susceptible, increased exposure’ (I), and ‘resistant’ (R) according to the recommendations of the EUCAST (EUCAST 2019). The panel of seven antimicrobial agents (Oxoid, USA) was selected for P. aeruginosa isolates, including piperacillin-tazobactam (30/6 μg), ceftazidime (10 μg), ciprofloxacin (5 μg), meropenem (10 μg), gentamicin (10 μg), amikacin (30 μg), and aztreonam (30 μg). When an isolate was resistant to at least one antimicrobial agent in three or more different classes of antibiotics, it was considered MDR. The reference strain Pseudomonas aeruginosa (ATCC 27853) was used for quality control.
RESULTS
P. aeruginosa isolates in hospital wastewater
The pH of the hospital wastewater samples analyzed ranged from 6 to 7.5. A total of 96 presumptive Pseudomonas spp. isolates were obtained from six hospital wastewater samples. All isolates were biochemically identified as P. aeruginosa. The number of P. aeruginosa isolates in different hospital wastewater samples is shown in Table 1. The highest occurrence of P. aeruginosa isolates was observed in wastewater from Hospitals 3 and 4 (24 strains), while in wastewater from Hospital 2 only three P. aeruginosa isolates were detected. The pH of the wastewater at this sampling point had the highest value of all wastewater samples (7.5); however, the pH was still within the range that enabled the growth of P. aeruginosa.
Number of Pseudomonas aeruginosa strains isolated from wastewater in different hospitals
Sampling points . | Pseudomonas aeruginosa (n=96) . |
---|---|
Number of strains (%) . | |
Hospital 1 (hospital wastewater 1) | 16 (16.7) |
Hospital 2 (hospital wastewater 2) | 3 (3.1) |
Hospital 3 (hospital wastewater 3) | 24 (25) |
Hospital 4 (hospital wastewater 4) | 24 (25) |
Hospital 5 (hospital wastewater 5) | 21 (21.9) |
Hospital 6 (hospital wastewater 6) | 8 (8.3) |
Total | 96 (100) |
Sampling points . | Pseudomonas aeruginosa (n=96) . |
---|---|
Number of strains (%) . | |
Hospital 1 (hospital wastewater 1) | 16 (16.7) |
Hospital 2 (hospital wastewater 2) | 3 (3.1) |
Hospital 3 (hospital wastewater 3) | 24 (25) |
Hospital 4 (hospital wastewater 4) | 24 (25) |
Hospital 5 (hospital wastewater 5) | 21 (21.9) |
Hospital 6 (hospital wastewater 6) | 8 (8.3) |
Total | 96 (100) |
Antimicrobial susceptibility profiles
Of the 96 P. aeruginosa strains obtained from untreated hospital effluents, 59 strains were selected for antibiotic susceptibility testing against a panel of seven antimicrobial agents. Resistance was found for all antibiotics tested in this study. The highest resistance rates were observed for ciprofloxacin (30.5%, 18/59), followed by gentamicin (28.8%, 17/59) and meropenem (27.2%, 16/59). Resistance to piperacillin-tazobactam was detected in seven of P. aeruginosa isolates (11.9%, 7/59). The same levels of resistance were observed both against ceftazidime (11.9%, 7/59) and amikacin (11.9%, 7/59). Resistance to aztreonam was detected relatively rarely (8.5%, 5/59). A significant proportion of strains were categorized as ‘susceptible, increased exposure’ (I). All P. aeruginosa strains were classified either resistant (R) or susceptible, increased exposure (I) to piperacillin-tazobactam, ciprofloxacin, ceftazidime, and aztreonam. No P. aeruginosa strains were characterized as ‘susceptible, standard dosing regimen’ (S) to these antimicrobial agents. Furthermore, six P. aeruginosa strains were classified as susceptible, increased exposure (I) to meropenem (Table 2).
Resistance of Pseudomonas aeruginosa strains isolated from six hospital wastewater samples
Antibiotic class . | Antibiotic (concentration) . | Number of Pseudomonas aeruginosa strains (%) (n=59) . | ||
---|---|---|---|---|
S . | I . | R . | ||
Penicillins | Piperacillin-tazobactam (30/6 μg) | 0 (0) | 52 (88.1) | 7 (11.9) |
Cephalosporins | Ceftazidime (10 μg) | 0 (0) | 52 (88.1) | 7 (11.9) |
Fluoroquinolones | Ciprofloxacin (5 μg) | 0 (0) | 41 (69.5) | 18 (30.5) |
Carbapenems | Meropenem (10 μg) | 37 (62.7) | 6 (10.2) | 16 (27.1) |
Aminoglycosides | Gentamicin (10 μg) | 42 (71.2) | 0 (0) | 17 (28.8.) |
Amikacin (30 μg) | 52 (88.1) | 0 (0) | 7 (11.9) | |
Monobactams | Aztreonam (30 μg) | 0 (0) | 54 (91.5) | 5 (8.5) |
Antibiotic class . | Antibiotic (concentration) . | Number of Pseudomonas aeruginosa strains (%) (n=59) . | ||
---|---|---|---|---|
S . | I . | R . | ||
Penicillins | Piperacillin-tazobactam (30/6 μg) | 0 (0) | 52 (88.1) | 7 (11.9) |
Cephalosporins | Ceftazidime (10 μg) | 0 (0) | 52 (88.1) | 7 (11.9) |
Fluoroquinolones | Ciprofloxacin (5 μg) | 0 (0) | 41 (69.5) | 18 (30.5) |
Carbapenems | Meropenem (10 μg) | 37 (62.7) | 6 (10.2) | 16 (27.1) |
Aminoglycosides | Gentamicin (10 μg) | 42 (71.2) | 0 (0) | 17 (28.8.) |
Amikacin (30 μg) | 52 (88.1) | 0 (0) | 7 (11.9) | |
Monobactams | Aztreonam (30 μg) | 0 (0) | 54 (91.5) | 5 (8.5) |
S, susceptible, standard dosing regimen; I, susceptible, increased exposure; R, resistant.
Twenty-five isolates (42.4%, 25/59) were resistant to at least one antibiotic tested. Resistance to three or more antimicrobials was observed in 28.8% (17/59) of P. aeruginosa isolates and all of these P. aeruginosa isolates (28.8%, 17/59) were classified as MDR. Generally, P. aeruginosa isolates exhibited seven multidrug resistance patterns that ranged from three to six antimicrobial agents (Table 3). Most of the MDR isolates were resistant to three antimicrobial agents (52.9%, 9/17). Four isolates (23.5%, 4/17) showed resistance to the six antibiotics tested. Two isolates (11.8%, 2/17) were resistant to five antimicrobial agents and two isolates (11.8%, 2/17) were resistant to four antimicrobial agents. The most common resistance pattern was ciprofloxacin-meropenem-gentamicin, observed in eight isolates (47.1%, 8/17).
Frequency of resistant patterns of multidrug-resistant Pseudomonas aeruginosa strains
Multidrug-resistant pattern . | Multidrug-resistant Pseudomonas aeruginosa strains (n=17) . |
---|---|
Number of strains (%) . | |
MEM-CIP-CN | 8 (47.1) |
CAZ-CIP-CN | 1 (5.9) |
CIP-CN-AK-ATM | 1 (5.9) |
TZP-CAZ-MEM-CIP | 1 (5.9) |
TZP-MEM-CIP-AN-AK | 1 (5.9) |
TZP-CAZ-MEM-CIP-AK | 1 (5.9) |
TZP-CAZ-MEM-CIP-CN-AK | 4 (23.5) |
Multidrug-resistant pattern . | Multidrug-resistant Pseudomonas aeruginosa strains (n=17) . |
---|---|
Number of strains (%) . | |
MEM-CIP-CN | 8 (47.1) |
CAZ-CIP-CN | 1 (5.9) |
CIP-CN-AK-ATM | 1 (5.9) |
TZP-CAZ-MEM-CIP | 1 (5.9) |
TZP-MEM-CIP-AN-AK | 1 (5.9) |
TZP-CAZ-MEM-CIP-AK | 1 (5.9) |
TZP-CAZ-MEM-CIP-CN-AK | 4 (23.5) |
TZP, piperacillin-tazobactam; CAZ, ceftazidime; MEM, meropenem; CIP, ciprofloxacin; CN, gentamicin; AK, amikacin; ATM, aztreonam.
Although resistant strains of P. aeruginosa were detected in all hospital wastewater samples, a significant difference in their presence was observed. High levels of resistant strains were detected in wastewater of Hospital 4, where 63.6% of P. aeruginosa isolates were resistant to at least one antimicrobial agent. Similarly, strains isolated from wastewater of Hospital 3 showed high resistance values. The percentage of isolates resistant to at least one antibiotic was 53.8%. On the contrary, the occurrence of resistance strains in wastewater of Hospitals 5 and 6 was insignificant (Table 4). MDR P. aeruginosa strains were detected in four hospital effluents. The prevalence of MDR patterns was varied in different hospital wastewater samples. Hospital 3 wastewater contained only the MDR pattern ciprofloxacin-meropenem-gentamicin. In addition, the resistant pattern piperacillin-tazobactam-ciprofloxacin-ceftazidime-meropenem-gentamicin-amikacin was found exclusively in wastewater from Hospital 4.
Frequency of resistant strains of Pseudomonas aeruginosa in different hospital wastewater samples
Resistant pattern . | Number of Pseudomonas aeruginosa resistant strains (n=25) . | |||||
---|---|---|---|---|---|---|
HW1 n=12 . | HW2 n=3 . | HW3 n=13 . | HW4 n=11 . | HW5 n=13 . | HW6 n=7 . | |
ATM | 2 | – | – | – | – | 2 |
CIP | 1 | – | – | – | – | – |
CN | 1 | – | – | – | 1 | – |
MEM | – | 1 | – | – | – | – |
MEM-CIP-CN | – | – | 7 | 1 | – | – |
CAZ-CIP-CN | – | – | – | – | 1 | – |
CIP-CN-AK-ATM | – | – | – | 1 | – | – |
TZP-CAZ-MEM-CIP | 1 | – | – | – | – | – |
TZP-MEM-CIP-AN-AK | – | – | – | 1 | – | – |
TZP-CAZ-MEM-CIP-AK | – | – | – | 1 | – | – |
TZP-CAZ-MEM-CIP-CN-AK | 1 | – | – | 3 | – | – |
The percentage of resistant strains | 50 | 33.3 | 53.8 | 63.6 | 15.4 | 28.6 |
Resistant pattern . | Number of Pseudomonas aeruginosa resistant strains (n=25) . | |||||
---|---|---|---|---|---|---|
HW1 n=12 . | HW2 n=3 . | HW3 n=13 . | HW4 n=11 . | HW5 n=13 . | HW6 n=7 . | |
ATM | 2 | – | – | – | – | 2 |
CIP | 1 | – | – | – | – | – |
CN | 1 | – | – | – | 1 | – |
MEM | – | 1 | – | – | – | – |
MEM-CIP-CN | – | – | 7 | 1 | – | – |
CAZ-CIP-CN | – | – | – | – | 1 | – |
CIP-CN-AK-ATM | – | – | – | 1 | – | – |
TZP-CAZ-MEM-CIP | 1 | – | – | – | – | – |
TZP-MEM-CIP-AN-AK | – | – | – | 1 | – | – |
TZP-CAZ-MEM-CIP-AK | – | – | – | 1 | – | – |
TZP-CAZ-MEM-CIP-CN-AK | 1 | – | – | 3 | – | – |
The percentage of resistant strains | 50 | 33.3 | 53.8 | 63.6 | 15.4 | 28.6 |
TZP, piperacillin-tazobactam; CAZ, ceftazidime; MEM, meropenem; CIP, ciprofloxacin; CN, gentamicin; AK, amikacin; ATM, aztreonam; HW, hospital wastewater.
DISCUSSION
In this study, antibiotic-resistant profiles of cultivable P. aeruginosa were analyzed in samples of untreated hospital effluents. P. aeruginosa resistant strains were detected in all samples studied, indicating that these strains can be disseminated from the hospital environment via the wastewater system. However, the different prevalence rates of resistant strains in hospital effluents were revealed. The diversity in the occurrence of P. aeruginosa resistant strains in individual samples was significant. The percentage of resistant strains was independent of the size of the hospital since the highest occurrence rate (63.6%) was observed in Hospital 4 with a capacity of 200 beds. In contrast, the lowest prevalence of resistant strains (15.4%) was observed in Hospital 5, with a capacity of 550 beds. The different occurrence of resistant strains in wastewater from different hospitals may be due to differences in the focus of medicinal activity and the current composition of patients hospitalized at the time of sample collection in the hospital.
In the present study, resistance to ciprofloxacin was detected the most frequently. Since ciprofloxacin plays an irreplaceable role in the treatment of P. aeruginosa infection, resistance to this antibiotic can cause serious complications in the therapeutic strategy. Ciprofloxacin is the most effective fluoroquinolone against P. aeruginosa and, for this reason, is also one of the most widely used antibiotics against this bacterium (Rehman et al. 2019). Exposure to high concentrations of fluoroquinolones in the hospital environment can contribute to the development of resistance to ciprofloxacin through overexpression of the efflux pump or by acquisition of ciprofloxacin-resistant genes through horizontal gene transfer (Xu et al. 2021). The occurrence of ciprofloxacin resistance in P. aeruginosa water isolates is closely associated with hospital wastewater. In the present study, resistance to ciprofloxacin was detected in 30.5% of P. aeruginosa strains. Also, Magalhães et al. (2016) reported a high resistance to ciprofloxacin in P. aeruginosa isolates obtained from raw and treated hospital effluents. The percentage of isolates resistant to ciprofloxacin was 78% (7/9) and 63% (6/8), respectively. On the contrary, no ciprofloxacin-resistant P. aeruginosa strains were detected in receiving river water. According to the previous study, Fuentefria et al. (2011) observed resistance to ciprofloxacin only among P. aeruginosa strains isolated from hospital wastewater. In superficial water, no ciprofloxacin resistance was detected. Similarly, resistance to ciprofloxacin was not present in 27 P. aeruginosa strains isolated from municipal wastewater (Luczkiewicz et al. 2015). Another study also reported lower levels of resistance to ciprofloxacin in P. aeruginosa strains obtained from municipal wastewater and surface water. The percentage of isolates resistant to ciprofloxacin was 9.4% (5/53) (Govender et al. 2021).
Resistance to the carbapenem antibiotic meropenem was observed relatively often. Resistance to this antibiotic was determined with a frequency of 27.2%. This finding is consistent with the increasing worldwide occurrence of carbapenem resistance in P. aeruginosa strains (Rostami et al. 2018; El-Mahdy & El-Kannishy 2019). Moreover, these strains are frequently isolated from patients with hospital-acquired infection and could be transferred with hospital effluents to municipal WWTPs (Govender et al. 2021; Pérez-Corrales et al. 2021). Haller et al. (2018) detected significant resistance levels to carbapenems meropenem and ertapenem in Pseudomonas spp. obtained from hospital effluents in Singapore. Similarly, a high percentage of resistance to carbapenems presented P. aeruginosa strains isolated in wastewater from two hospitals located in Rio Grande do Sul, Brazil (Fuentefria et al. 2011). Vaz-Moreira et al. (2016) also found resistance to meropenem in P. aeruginosa strains isolated from hospital effluent. Nevertheless, resistance to meropenem was less common, with prevalence values of 33.3% (12/36). Resistance to carbapenems is a serious threat to global public health, since carbapenems constitute the most effective antibiotics for the treatment of MDR P. aeruginosa infections (Rostami et al. 2018; El-Mahdy & El-Kannishy 2019). Carbapenem-resistant P. aeruginosa was recognized as a critical priority pathogen that poses the utmost threat to human health by the World Health Organization (WHO). This implies that effective therapy is not available and new antibiotics are urgently needed (Balkhair et al. 2019; Govender et al. 2021).
A significant difference in aminoglycoside resistance rates (gentamicin and amikacin) was observed among the tested P. aeruginosa isolates. Resistance to gentamicin was found in 28.8% (17/59) of P. aeruginosa strains and was the second most frequently detected resistance, while resistance to amikacin was observed relatively rarely (11.7%, 7/59). The high resistance to gentamicin in P. aeruginosa strains is consistent with the EUCAST recommendation that this bacterium is not a good target for the therapy of gentamicin. Amikacin can still be used in the therapy of P. aeruginosa infection. Nevertheless, for the treatment of systemic infection, amikacin must be used in combination with other active therapy (EUCAST 2021b). In another study, the diversity of aminoglycoside resistance in P. aeruginosa hospital effluent isolates was also detected. Forty-one P. aeruginosa isolates were obtained from hospital wastewater in Rio de Janeiro. The percentage of isolates resistant to gentamicin was 15%, while resistance to amikacin was the least common and was determined with a frequency of 2% (Miranda et al. 2015). According to a previous study, a difference in aminoglycoside resistance levels was found in P. aeruginosa isolates obtained from raw and treated hospital wastewater. Seven of nine isolates (78%, 7/9) in raw hospital wastewater were resistant to gentamicin, while only four isolates showed resistance to amikacin (44%, 4/9). In treated hospital effluent, the resistance rate to gentamicin was even higher than in raw wastewater (88%, 7/8). Resistance to amikacin in treated hospital wastewater was 25% (2/8) (Magalhães et al. 2016). Resistance to gentamicin was less common in 94 Pseudomonas spp. isolates from hospital effluent in Cluj-Napoca, Romania. Eighteen isolates exhibited resistance to gentamicin (17.02%, 18/94) (Butiuc-Keul et al. 2021). Similarly, low resistance to gentamicin presented P. aeruginosa strains obtained from hospital wastewater in Nigeria. Resistance to this antibiotic was detected in seven isolates (15.9%, 7/44) (Israel Falodun et al. 2019).
Resistance to the third-generation cephalosporin ceftazidime (11.9%, 7/59), as well as to the combination of piperacillin with tazobactam (11.9%, 7/59), was observed rarely. In the case of aztreonam, only five isolates (8.5%, 5/59) were resistant to this antimicrobial agent. Santoro et al. (2015) also found high susceptible values to piperacillin-tazobactam and ceftazidime in 27 P. aeruginosa strains isolated from hospital wastewater in the city of Rio de Janeiro, Brazil. Piperacillin-tazobactam were effective against all isolates and resistance to ceftazidime showed five isolates (18.5%, 5/27). In contrast, the highest proportion of resistance was detected for aztreonam (62.9%, 17/27). Comparable resistance rates to ceftazidime, piperacillin-tazobactam, and aztreonam were detected in 27 P. aeruginosa strains obtained from municipal wastewater in northern Poland. The majority of isolates showed resistance to aztreonam (70.4%, 19/27), while resistance to ceftazidime was observed in five isolates (18.5%, 5/27). No P. aeruginosa strains were resistant to piperacillin-tazobactam (Luczkiewicz et al. 2015). In the other study, in which the resistance profiles of the P. aeruginosa strains from WWTPs effluents were determined, the highest proportion of these isolates was resistant to ceftazidime (37.1%, 23/62). Also, resistance to piperacillin-tazobactam was commonly observed (30.6%, 17/62) (Golle et al. 2017).
Aside from resistance to a single antibiotic, the most significant public health problem is the accumulation of resistance features, which may result in MDR strains. In the present study, 17 P. aeruginosa strains (28.8%, 17/59) were classified as MDR. Israel Falodun et al. (2019) reported the high presence of multidrug P. aeruginosa strains in hospital wastewater in Nigeria. The percentage of MDR strains was 93.2% (41/44). Similarly, a significant proportion of P. aeruginosa strains in hospital wastewater in Rio de Janeiro presented MDR profiles (83%; 34/41) (Miranda et al. 2015). In contrast, only six of the 27 P. aeruginosa strains had multidrug resistance profiles and were classified as MDR (22.2%, 6/27) in hospital wastewater in Rio de Janeiro (Santoro et al. 2015). Nevertheless, as reported by Luczkiewicz et al. (2015), the real prevalence of multidrug resistance in the bacterial community is not well established, due to differences in the definition of this phenomenon.
CONCLUSION
Resistant P. aeruginosa strains were found in all hospital wastewater samples studied. Almost 30% of P. aeruginosa strains were resistant to three or more antibiotics and classified as MDR. The results revealed considerable resistance to important antimicrobial agents such as ciprofloxacin and meropenem, which are the antibiotics of choice for the therapy of P. aeruginosa. Increasing resistance to currently effective antimicrobial agents brings complications to the treatment process of human P. aeruginosa infection. This situation is worsened by the fact that hospital effluents, which commonly contain resistance bacteria, are frequently discharged into public sewer networks without any treatment aimed at reducing bacterial contamination. The release of P. aeruginosa resistant strains through hospital wastewater to municipal wastewater systems could result in extensive genetic exchange by horizontally transferring resistance features to other competent bacteria. For these reasons, it would be appropriate to create reference standards and specific treatment methods to manage hospital effluents.
DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information.