ISSN: 0973-7510
E-ISSN: 2581-690X
, Alaeddin O. Abuzant2, Ma’en Al-Odat3, Ola D. Al-Maseimi4, Ziad H. Abu Dieyeh1, Aala R. Banyfadel2, Aseel R. Qanazae2, Fatima K. Othman2 and Nesreen N. Dwikat2Multidrug-resistant (MDR) bacteria have been isolated from a major wastewater pathway. These bacteria harbor several antimicrobial resistance genes that confer resistance to several antibiotics simultaneously. The main aim of this study was to investigate the antibiotic resistance profiles of lactose-fermenting gram-negative bacteria isolated from the main wastewater pathway in the Nablus area of the West Bank, Palestine. A total of 162 lactose-fermenting isolates belonging to the Enterobacteriaceae family were isolated from a sample obtained from the main wastewater pathway. Most of the isolates obtained were identified to the species level using the API-20 E identification system. The proportions of MDR strains among the obtained Escherichia coli and Citrobacter Koseri isolates were 19.1% and 10%, respectively. Among all isolates, six were found to be extended-spectrum beta-lactamase (ESBL) producers. These included three E. coli isolates, one Klebsiella pneumoniae isolate, and two C. Koseri isolates. Approximately 12.3% of the total isolates were MDR and 3.7% were identified as ESBL producers. The prevalence of MDR isolates in our study was concerning, indicating that immediate and decisive measures are needed to halt its escalation and promote its reduction.
Wastewater, Gram-negative, Enterobacteriaceae, Lactose-fermenter, Multidrug-resistant, Extended-spectrum beta-lactamases
The family Enterobacteriaceae includes approximately 250 species of Gram-negative, facultatively anaerobic, catalase-positive, non-spore-forming bacilli, most of which are motile. Although many members of this family inhabit the intestinal tracts of humans and animals, some are also free-living.1,2
Some members of this family are primary pathogens transmitted from infected humans or colonized animals.3 These primary pathogens may cause gastroenteritis and other infections, some of which can be fatal.3-6
However, some members of this family are opportunistic pathogens, such as Proteus, Escherichia coli, and Enterobacter, which may cause various nosocomial and community-acquired infections, including urinary tract and wound infections, pneumonia, septicemia, and meningitis.3,7
Antibiotics are chemicals that target specific components of bacterial cells to either stop replication (bacteriostatic antibiotics) or cause death (bactericidal antibiotics).8
Beta-lactam and glycopeptide antibiotics interfere with bacterial cell wall biosynthesis, resulting in bacterial cell death via osmotic lysis.9,10 Aminoglycoside, macrolide, tetracycline, and chloramphenicol antibiotics target ribosomes, inhibiting protein synthesis and preventing bacterial replication or causing bacterial death.9,10 Sulfonamides and trimethoprim interfere with the biosynthetic pathway of purine nucleotides, halting bacterial replication.11 Quinolones and fluoroquinolones kill bacterial cells by inhibiting DNA gyrase.12,13 Finally, rifampin and rifampicin antibiotics kill bacterial cells by targeting their RNA polymerase.14,15
Antibiotics are used worldwide to treat bacterial infections in both humans and animals.16 The widespread use of antibiotics has promoted the emergence of resistant bacterial strains of different species.16,17 Interestingly, antibiotic resistance is now considered a global crisis.18
Various antibiotic resistance mechanisms have been developed by bacteria. These include decreased permeability, enzymatic inactivation, alteration of their target sites, and increased efflux.19,20
A multidrug-resistant (MDR) bacterium is defined as one that resists three or more antibiotics of different classes simultaneously.21 This occurs because resistant strains can harbor several antibiotic resistance genes that confer resistance to different antibiotics. These genes may co-exist on transmissible genetic elements such as plasmids or transposons.22,23
Although infections caused by MDR Gram-negative bacterial strains were initially associated with nosocomial infections, recent studies have shown a notable increase in the prevalence of community-acquired infections caused by these strains.24,25
MDR bacteria of the family Enterobacteriaceae are increasingly becoming part of the gut microbiota in both humans and animals.26-28 The passage of these MDR bacteria, along with fecal material from humans and animals, results in their environmental spread through both wastewater pathways and accumulation sites.29-32
Interestingly, it is estimated that 40%-90% of an antibiotic administered to humans or animals is excreted in their urine and feces in an active form.33-37 Upon reaching wastewater pathways and accumulation sites, these excreted antibiotics may act as a driving force for the emergence of MDR bacteria.29-32 Thus, wastewater pathways and accumulation sites can be important sources for the spread of MDR bacteria in the environment, increasing the risk of community-acquired infections with these highly resistant pathogens.38-40
Many studies in various countries have investigated the prevalence of MDR bacteria among isolates obtained from wastewater pathways and accumulation sites.41-45 Therefore, the main goal of this study was to assess the prevalence of MDR Gram-negative bacteria of the family Enterobacteriaceae isolated from the main wastewater pathway in the Nablus area, West Bank, Palestine, a study that, to our knowledge, has never been conducted before in Palestine.
Sample processing and identification of the obtained bacterial isolates
A single 300 mL wastewater sample was obtained in June 2023 from the main wastewater pathway in the Nablus city, West Bank, Palestine. The sample was filtered through a sterile cotton cloth to remove solid waste material and collected in a sterile container. Approximately 10 mL of the drained sample was transferred into a sterile 50 mL screw-capped conical tube (obtained from Fisher Scientific (Loughborough, Leicestershire, UK). The tubes were then centrifuged at 1,958 × g for approximately 10 min. The supernatant was then removed, and the pellet was resuspended in 10 mL of sterile nutrient broth. The tubes were then incubated for 1 h in a shaker incubator at 37 °C under aerobic conditions. At the end of the incubation period, 10-fold serial dilutions were prepared from the cultures. The tube with the 104 dilution was used to inoculate 20 plates of MacConkey culture medium obtained from Oxoid (Basingstoke, Hampshire, UK). Each of the MacConkey agar plates was inoculated with 50 µL, which was spread all over the agar surface using a disposable sterile plastic spreader. The plates were incubated at 37 °C for 24 h under aerobic conditions. After incubation, 200 individual pink colonies (lactose fermenters) with smooth surfaces were randomly selected and then sub-cultured separately on MacConkey agar plates (Oxoid). The plates were incubated at 37 °C for approximately 24 h under aerobic conditions.
Subsequently, each bacterial isolate was identified using the API-20 E identification system obtained from BioMerieux (Marcy-I’Étoile, France) as described by the manufacturer.
Antibiotic susceptibility testing
Antibiotic susceptibility testing and interpretation of the obtained results were performed using the disc diffusion method according to the guidelines of the Clinical Laboratory Standards Institute (CLSI).46 Each isolate belonging to the family Enterobacteriaceae was examined for susceptibility to ampicillin, cefepime, cefotaxime, ceftazidime, trimethoprim-sulfamethoxazole, gentamicin, tetracycline, ciprofloxacin, meropenem, aztreonam, and chloramphenicol. The antibiotic discs were obtained from Oxoid (Basingstoke, Hampshire, UK), and the E. coli ATCC strain 25922 obtained from the American Type Culture Collection (Manassas, VA, USA), was used as a control.
Phenotypic characterization of ESBLs
Extended-spectrum beta-lactamase (ESBL) production was phenotypically characterized for each isolate that demonstrated resistance or intermediate resistance to cefotaxime and/or ceftazidime. This was conducted using the combination disc procedure with ceftazidime, ceftazidime-clavulanic acid, cefotaxime, and cefotaxime-clavulanic acid, as recommended by the CLSI.46 An isolate was considered an ESBL producer if the inhibition zone diameter around ceftazidime-clavulanic acid or cefotaxime-clavulanic acid discs increased by more than 5 mm relative to the diameter around discs containing only cefotaxime and/or ceftazidime.
Identification of the isolated bacterial species
Out of 200 Gram-negative lactose-fermenting isolates, 162 were identified as members of the family Enterobacteriaceae, including the following species: E. coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri, Ewingella americana, Providencia rettgeri, and Enterobacter cloacae. The number and percentage of each species among the 162 isolates are shown in Table 1. Each of these species is an opportunistic pathogen that can both localized and potentially fatal systemic infections.47-53
Table (1):
The number and the percentage of each of the obtained bacteria out of the obtained 162 isolates
Bacterial isolate |
Out of the 162 isolates |
% out of the 162 isolates |
|---|---|---|
E. coli |
94 |
58.0 |
Klebsiella pneumoniae |
31 |
19.1 |
Klebsiella oxytoca |
8 |
4.9 |
Citrobacter koseri |
21 |
13.0 |
Ewingella americana |
3 |
1.9 |
Enterobacter spp. |
3 |
1.9 |
Providencia rettgeri |
2 |
1.2 |
Total number |
162 |
100% |
Antibiotic susceptibility testing
Antibiotic susceptibility analysis was conducted for each of the obtained isolates of the family Enterobacteriaceae against eleven different antibiotics. The numbers and percentages of isolates that exhibited susceptibility, intermediate resistance, or resistance to each tested antibiotic are presented in Table 2. The highest rates of resistance were observed for ampicillin, tetracycline, chloramphenicol, and trimethoprim/sulfamethoxazole, at 26.5%, 24.7%, 12.3%, and 9.3%, respectively. Conversely, the lowest rates of resistance were observed for ciprofloxacin, gentamicin, and cefotaxime, at 6.8%, 3.7%, and 5.6%, respectively. Only 0.6% of the total isolates were resistant to ceftazidime and aztreonam, and none of the isolates were resistant to meropenem or cefepime (Table 2).
Table (2):
The numbers and the percentage rates of all the obtained isolates in terms of their susceptibility profiles to the tested antibiotics
| Antibiotic | Out of obtained 162 isolates | |||||
|---|---|---|---|---|---|---|
| S | IR | R | ||||
| No. of isolates | % | No. of isolates | % | No. of isolates | % | |
| Ampicillin | 74 | 45.7 | 45 | 27.8 | 43 | 26.5 |
| Cefepime | 162 | 100 | 0 | 0 | 0 | 0 |
| Cefotaxime | 138 | 85.2 | 15 | 9.3 | 9 | 5.6 |
| Ceftazidime | 157 | 96.9 | 4 | 2.5 | 1 | 0.6 |
| Meropenem | 162 | 100 | 0 | 0 | 0 | 0 |
| Aztreonam | 158 | 97.5 | 3 | 1.9 | 1 | 0.6 |
| Gentamicin | 155 | 95.7 | 1 | 0.6 | 6 | 3.7 |
| Tetracycline | 120 | 74.1 | 2 | 1.2 | 40 | 24.7 |
| Ciprofloxacin | 81 | 50.0 | 70 | 43.2 | 11 | 6.8 |
| Trimethoprim-Sulfamethoxazole | 144 | 88.9 | 3 | 1.9 | 15 | 9.3 |
| Chloramphenicol | 142 | 87.7 | 0 | 0 | 20 | 12.3 |
(S: susceptible, IR: Intermediate-resistant or R: Resistant)
Concerning the E. coli isolates, the highest rates of resistance were observed for tetracycline, ampicillin, chloramphenicol, and trimethoprim/sulfamethoxazole, at 36.2, 26.6, 20.2, and 14.9% respectively. The resistance rate to ciprofloxacin was 7.4% and the resistance rates to Gentamicin and Cefotaxime were 6.4% for both. The lowest rate of resistance was observed for ceftazidime and aztreonam, at 1.1% for both (Table 3).
Interestingly, 18 (19.1%) of the obtained 94 E. coli isolates were MDR, meaning they were resistant to three or more antibiotics of different classes (21) (Table 3).
Table (3):
The numbers and the percentage rates out of the 94 E. coli isolates
| Antibiotic | E. coli |
|||||
|---|---|---|---|---|---|---|
| S | IR | R | ||||
| No. of isolates | % | No. of isolates | % | No. of isolates | % | |
| Ampicillin | 49 | 52.1 | 20 | 21.3 | 25 | 26.6 |
| Cefepime | 94 | 100 | 0 | 0 | 0 | 0 |
| Cefotaxime | 80 | 85.1 | 8 | 8.5 | 6 | 6.4 |
| Ceftazidime | 91 | 96.8 | 2 | 2.1 | 1 | 1.1 |
| Meropenem | 94 | 100 | 0 | 0 | 0 | 0 |
| Aztreonam | 91 | 96.8 | 2 | 2.1 | 1 | 1.1 |
| Gentamicin | 87 | 92.6 | 1 | 1.1 | 6 | 6.4 |
| Tetracycline | 58 | 61.7 | 2 | 2.1 | 34 | 36.2 |
| Ciprofloxacin | 48 | 52.1 | 39 | 41.5 | 7 | 7.4 |
| Trimethoprim-Sulfamethoxazole | 77 | 81.9 | 3 | 3.2 | 14 | 14.9 |
| Chloramphenicol | 75 | 79.8 | 0 | 0 | 19 | 20.2 |
S: Susceptible, IR: Intermediate-Resistant or R: Resistant to each of the tested antibiotics
Of these 18 E. coli isolates, 10 were resistant to ampicillin, tetracycline, and Chloramphenicol, 1 was resistant to tetracycline, trimethoprim/sulfamethoxazole, and chloramphenicol, 1 was resistant to ampicillin, tetracycline, trimethoprim/sulfamethoxazole, and chloramphenicol, 1 was resistant to gentamicin, tetracycline, ciprofloxacin, and chloramphenicol, 1 was resistant to gentamicin, tetracycline, trimethoprim/sulfamethoxazole, and chloramphenicol, 1 was resistant to ampicillin, gentamicin, tetracycline, and trimethoprim/sulfamethoxazole, 1 was resistant to ampicillin, gentamicin, tetracycline, and ciprofloxacin, 1 was resistant to gentamicin, tetracycline, and ciprofloxacin, and 1 final isolate was resistant to ampicillin, cefotaxime, gentamicin, tetracycline and ciprofloxacin (Table 4).
Table (4):
The antibiotic resistant profiles of the multidrug-resistant E. coli isolates
Number of the E. coli isolates that were/was multidrug resistant |
Resistant to |
|---|---|
10 |
Ampicillin, Tetracycline and Chloramphenicol |
1 |
Tetracycline, Trimethoprim/Sulfamethoxazole and Chloramphenicol |
1 |
Ampicillin, Tetracycline, Trimethoprim/Sulfamethoxazole and Chloramphenicol |
1 |
Gentamicin, Tetracycline, Ciprofloxacin and Chloramphenicol |
1 |
Gentamicin, Tetracycline, Trimethoprim/Sulfamethoxazole, and Chloramphenicol |
1 |
Ampicillin, Gentamicin, Tetracycline, and Trimethoprim/Sulfamethoxazole |
1 |
Ampicillin, Gentamicin, Tetracycline and Ciprofloxacin |
1 |
Gentamicin, Tetracycline and Ciprofloxacin |
1 |
Ampicillin, Cefotaxime, Gentamicin, Tetracycline and Ciprofloxacin |
Regarding the obtained 31 K. pneumoniae isolates, 29% of them were resistant to ampicillin and 3.2% to cefotaxime and ciprofloxacin (Table 5). Conversely, about 29.2%, 16.1%, 3.2%, and 45.2% of the K. pneumoniae isolates exhibited intermediate resistance to ciprofloxacin, ampicillin, cefotaxime, and aztreonam, respectively (Table 5). None of the obtained K. pneumoniae isolates showed resistance to chloramphenicol, trimethoprim/sulfamethoxazole, ceftazidime, meropenem tetracycline, or gentamicin (Table 5). None of the obtained K. pneumoniae isolates were MDR.
Table (5):
The numbers and the percentage rates out of the 31 Klebsiella pneumoniae isolates
| Antibiotic | Klebsiella pneumoniae |
|||||
|---|---|---|---|---|---|---|
| S | IR | R | ||||
| No. of isolates | % | No. of isolates | % | No. of isolates | % | |
| Ampicillin | 13 | 41.9 | 9 | 29.0 | 9 | 29.0 |
| Cefepime | 31 | 100 | 0 | 0 | 0 | 0.0 |
| Cefotaxime | 25 | 80.6 | 5 | 16.1 | 1 | 3.2 |
| Ceftazidime | 31 | 100 | 0 | 0 | 0 | 0 |
| Meropenem | 31 | 100 | 0 | 0 | 0 | 0 |
| Aztreonam | 30 | 96.8 | 1 | 3.2 | 0 | 0 |
| Gentamicin | 31 | 100 | 0 | 0 | 0 | 0 |
| Tetracycline | 31 | 100. | 0 | 0 | 0 | 0 |
| Ciprofloxacin | 16 | 51.6 | 14 | 45.2 | 1 | 3.2 |
| Trimethoprim-Sulfamethoxazole | 31 | 100 | 0 | 0 | 0 | 0 |
| Chloramphenicol | 31 | 100 | 0 | 0 | 0 | 0 |
S: Susceptible, IR: Intermediate-resistant or R: Resistant to each of the tested antibiotics
Regarding the eight K. oxytoca isolates, only one showed resistance to ampicillin and another one showed resistance to tetracycline. In contrast, three and six of these isolates showed intermediate resistance to ampicillin and ciprofloxacin, respectively. However, none of the isolates exhibited resistance or intermediate resistance to cefepime, cefotaxime, ceftazidime, meropenem, aztreonam, gentamicin, trimethoprim /sulfamethoxazole, or chloramphenicol (Table 6). None of the obtained K. oxytoca isolates were MDR.
Table (6):
The numbers and the percentage rates out of the 8 Klebsiella oxytoca isolates
| Antibiotic | Klebsiella oxytoca |
|||||
|---|---|---|---|---|---|---|
| S | IR | R | ||||
| No. of isolates | % | No. of isolates | % | No. of isolates | % | |
| Ampicillin | 4 | 50 | 3 | 37.5 | 1 | 12.5 |
| Cefepime | 8 | 100 | 0 | 0 | 0 | 0 |
| Cefotaxime | 8 | 100 | 0 | 0 | 0 | 0 |
| Ceftazidime | 8 | 100 | 0 | 0 | 0 | 0 |
| Meropenem | 8 | 100 | 0 | 0 | 0 | 0 |
| Aztreonam | 8 | 100 | 0 | 0 | 0 | 0 |
| Gentamicin | 8 | 100 | 0 | 0 | 0 | 0 |
| Tetracycline | 7 | 87.5 | 0 | 0 | 1 | 12.5 |
| Ciprofloxacin | 2 | 25 | 6 | 75 | 0 | 0 |
| Trimethoprim-Sulfamethoxazole | 8 | 100 | 0 | 0 | 0 | 0 |
| Chloramphenicol | 8 | 100 | 0 | 0 | 0 | 0 |
S: Susceptible, IR: Intermediate-resistant or R: Resistant to each of the tested antibiotics
Concerning the 21 C. koseri isolates obtained, the highest levels of resistance were observed against ampicillin, tetracycline, cefotaxime, and ciprofloxacin, at 38.1%, 19%, 9.2%, and 14.3%, respectively (Table 7). Only 4.9% of the isolates were resistant to either chloramphenicol or trimethoprim/sulfamethoxazole (Table 7).
Table (7):
The numbers and the percentage rates out of the 21 Citrobacter koseri isolates
| Antibiotic | Citrobacter koseri | |||||
|---|---|---|---|---|---|---|
| S | IR | R | ||||
| No. of isolates | % | No. of isolates | % | No. of isolates | % | |
| Ampicillin | 5 | 23.8 | 8 | 38.1 | 8 | 38.1 |
| Cefepime | 21 | 100 | 0 | 0 | 0 | 0 |
| Cefotaxime | 17 | 81 | 2 | 9.5 | 2 | 9.5 |
| Ceftazidime | 19 | 90.5 | 2 | 9.5 | 0 | 0 |
| Meropenem | 21 | 100 | 0 | 0 | 0 | 0 |
| Aztreonam | 20 | 95.2 | 1 | 4.8 | 0 | 0 |
| Gentamicin | 21 | 100 | 0 | 0 | 0 | 0 |
| Tetracycline | 17 | 81 | 0 | 0 | 4 | 19 |
| Ciprofloxacin | 11 | 52.4 | 7 | 33.3 | 3 | 14.3 |
| Trimethoprim-Sulfamethoxazole | 20 | 95.2 | 0 | 0 | 1 | 4.8 |
| Chloramphenicol | 20 | 95.2 | 0 | 0 | 1 | 4.8 |
S: Susceptible, IR: Intermediate-resistant and R: Resistant to each of the tested antibiotics
Interestingly, two (12.9%) of the C. koseri isolates were MDR. One was resistant to ampicillin, tetracycline, and ciprofloxacin, while the other was resistant to ampicillin, cefotaxime, ciprofloxacin, and trimethoprim/sulfamethoxazole.
None of the three obtained E. americana or E. cloacae isolates showed resistance to any of the tested antibiotics. Only one of the two P. rettgeri isolates showed resistance to tetracycline. This can be explained by their small number among the total number of isolates obtained. The E. coli ATCC 25922 strain was sensitive to all antibiotics used.
Identification of ESBL producers among the obtained isolates
Eighteen of the obtained isolates were either resistant or intermediately resistant to cefotaxime and/or ceftazidime. These isolates were examined for ESBL production, as described in the Methodology section. Our results confirmed that three E. coli, one K. pneumoniae, and two C. koseri isolates were confirmed to be ESBL producers. This implied that six (3.7%) of the total 162 isolates were ESBL producers.
Nablus City has several wastewater pathways that converge to form the main wastewater pathway from which the sample for this study was collected. Accordingly, the obtained single sample was representative of the city’s wastewater.
In total, 162 lactose-fermenting isolates belonging to the family Enterobacteriaceae were obtained from this sample. E. coli, K. pneumoniae, and C. koseri represented approximately 91% of the total isolates, while K. oxytoca, E. americana, Enterobacter spp., and P. rettgeri represented approximately 8.9%.
An MDR bacterium is one that can resist to three or more different classes of antibiotics. This resistance may occur because of the simultaneous presence of several antibiotic resistance genes, such as those carried on a resistance plasmid.54 Interestingly, a bacterium that shows intermediate resistance to an antibiotic in vitro may exhibit complete resistance in vivo.55 Despite this, only isolates showing full resistance in vitro were considered MDR in this study.
Different mechanisms confer resistance to several antibiotics, such as an efflux pump that can export several antibiotics to the outside of the bacterial cell, thus preventing the antibiotics from reaching an effective concentration in the bacterial cytoplasm.54
The spread of MDR bacterial strains throughout the environment may increase the rate of community-acquired infections with strains that are clinically challenging to treat.18
Many studies from different countries have investigated the prevalence of MDR bacteria in sewage, wastewater pathways, collection areas, treatment plants, sewage sludge, and sewage-contaminated water. In Japan, several MDR bacteria were isolated from public wastewater, including carbapenem-resistant Enterobacteria, ESBL-producing Enterobacteria, MDR Acinetobacter, MDR Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococci.56 In addition, antibiotic-resistant bacteria have been isolated from wastewater in different locations, such as E. coli in South Africa,57 Enterococcus spp., Staphylococcus spp., Pseudomonas spp., and Acinetobacter spp. in Slovakia,58 and Pseudomonas, Aeromonas, and Bacillus in Spain.59 In an Ethiopian study, the prevalence rate of MDR E. coli isolates obtained from a sewage-contaminated river was 78%.60 In an Austrian study, it was found that 16% of the E. coli isolates obtained from sewage sludge were MDR.61 In addition, in a Saudi Arabian study, 7.7% of the E. coli isolates obtained from an urban sewage were MDR.62 In our study, antibiotic-resistant bacteria including E. coli, K. pneumoniae, and C. koseri were isolated from the main wastewater pathway in the Nablus district of the West Bank, Palestine.
E. coli isolates from South Africa showed higher resistance levels to ampicillin (55.6%), gentamicin (0.5%), and tetracycline (60.1%) compared to the resistance level of E. coli in this study, with resistance levels of 26.6%, 6.4%, and 36.2%, respectively.57
In South India, two wastewater samples indicated more than 80% and more than 50% resistance among E. coli isolates to ampicillin, tetracycline, sulfamethoxazole–trimethoprim, and cefotaxime,63 which is higher than resistance levels in this study for the same antibiotics.
The fact that none of the isolates were resistant to meropenem or cefepime can be explained by the fact that these antibiotics are not commonly used in Palestine.64,65
E. coli is an opportunistic pathogen that causes various types of infections. Although it is the most common cause of urinary tract infections, it may also cause wound infections, pneumonia, septicemia, septic shock, and meningitis.66
E. americana was first described by Grimont et al. in 1983.67 Although it may cause plant infections, it mainly affects immunocompromised patients and neonates, and causes various life-threatening infections.68 In our study, only three E. americana isolates were obtained, one of which was susceptible to all antibiotics used, and the other two exhibited intermediate resistance to one of the tested antibiotics (data not shown).
A previous study conducted in Saudi Arabia described an MDR strain of E. americana that caused severe pneumonia in a young patient.51,69
Fecal colonization with MDR Gram-negative bacteria of the Enterobacteriaceae family has been found to occur in both humans and animals. Accordingly, it is not surprising that these MDR Gram-negative bacteria are found in wastewater pathways, collection areas, or treatment plants.
The ability of organisms to share and transfer genetic material not connected to a parental relationship is known as horizontal gene transfer (HGT). HGT has been widely investigated as the cause of adaptation mechanisms that facilitate the transfer of antimicrobial resistance and virulence factors, enhancing the ability of the bacterium to overcome challenging environments.
Wastewater contains a large collection of organisms that interact with one another. The evolution of bacteria relies on HGT between organisms, which results in the spread of resistance genes. Gene transfer between bacteria results in the simultaneous development of resistance to different types of antibiotics. Monitoring these resistances is critical for public health, and appropriate policies must be applied to minimize their impact.
In our study, 12.3% of the total isolates and 19.1% of the E. coli isolates were MDR. Although the prevalence rate in our study was less than that in that mentioned in the Ethiopian study, it is similar to that reported in the Austrian study and it is clearly higher than that reported by the Saudi study mentioned earlier.
This requires prompt measures from the people in charge of the Palestinian Ministry of Health to prevent further progression of the prevalence of MDR bacteria in our environment.
Limitation of the study
Although this study was the first of its kind in Palestine, the number of isolates obtained was relatively small, and this study focused on some lactose-fermenting species of the family Enterobacteriaceae. A more comprehensive study is needed to shed further light on the environmental prevalence of MDR Gram-negative bacteria as well as the prevalence of ESBL producers. In addition, antibiotic resistance genes were not characterized or detected in this study; however, these will be investigated in a future study.
ACKNOWLEDGMENTS
The authors would like to thank the administration of Nablus City Municipality for their help regarding obtaining the sample of the general wastewater pathway used in this study. The authors are also thankful to the technicians of the Microbiology Research Lab of the College of Medicine and Health Sciences of An-Najah National University, for their role in facilitating the conduction of this study.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
AUTHORS’ CONTRIBUTION
MMA and ODA conceptualized the study. MMA, ADA, and NND applied methodology. MMA performed data curation. ZHAD, ARB and FKO performed formal analysis. MA and ARQ performed data investigation. AOA and FKO supervised the study. FKO and ZHAD performed visualization. AOA and MMA wrote the manuscript. MA reviewed and edited the manuscript. All authors read and approved the final manuscript for publication.
FUNDING
None.
DATA AVAILABILITY
All datasets generated or analyzed during this study are included in the manuscript.
ETHICS STATEMENT
Not applicable.
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