Wastewater microbiology: occurrence and prevalence of antibiotic-resistant extended-spectrum β-lactamase-producing Enterobacteriaceae in the district wastewater system

The worldwide issue of antibiotic resistance, fueled by the overuse and misuse of antibiotics, poses a critical challenge. The United States Centers for Disease Control and Prevention (CDC) along with the World Health Organization (WHO) has considered antimicrobial resistance as a leading health risk for years (CDC 2024). Estimates indicate that antimicrobial resistance caused 1.27 million deaths and added to 5 million other deaths (Murray et al. 2022; WHO 2023). Members of Enterobacteriaceae, such as Escherichia coli and Klebsiella pneumoniae that produce extended spectrum beta lactamases (ESBLs), strongly contribute to this resistance and represent a vital risk to human health worldwide (Ramatla et al. 2023).

ESBL-producing Enterobacteriaceae exhibit resistance to multiple antibiotics commonly used for bacterial infections, leading to challenges in treatment, more extended hospital stays, increased medical expenses, and elevated mortality rates (Zahar et al. 2015; Tschudin-Sutter et al. 2017). ESBLs make bacteria unresponsive to various β-lactam antibiotics such as both penicillin and cephalosporin. The ESBL inactivates the β-lactam antibiotics via cleavage of the beta-lactam ring. Third-generation cephalosporin are cefotaxime and ceftazidime, which are highly affected by the activities of ESBL's. ESBLs are commonly tied to the Enterobacteriaceae family and categorically to E. coli and K. pneumoniae (Paterson & Bonomo 2005; Pitout & Laupland 2008).

In terms of wastewater research, K. pneumoniae and E. coli occupy an important position, serving as key indicator characteristics of fecal contamination alongside antibiotic resistance. Both are members of the Enterobacteriaceae family, making them the usual residents of the human gut microbiome. Just like E. coli, K. pneumoniae is a principal cause of healthcare-associated infections and illustrates a growing resistance to several antibiotics, notably carbapenems (Wyres & Holt 2018). The concurrence of these two factors in wastewater gives us an understanding about the spread of antibiotic-resistance genes in environmental systems (Pärnänen et al. 2019). Navon-Venezia et al. (2017) found that in the context of wastewater treatment, K. pneumoniae shows a heightened ability to obtain and distribute resistance genes, which deserves scrutiny in terms of its contrast with E. coli.

Rapid transmission of ESBLs within healthcare settings, particularly hospitals, raises concerns about outbreaks of infection caused by ESBL-producing bacteria, especially E. coli and K. pneumoniae (Tschudin-Sutter et al. 2016). These outbreaks can, therefore, result in high morbidity, mortality, and increased costs to the healthcare systems. Several factors facilitate resilience in the rapid transmission of ESBL in healthcare settings:

• 1. The presence of ESBL genes on mobile genetic elements like plasmids enables bacteria to acquire resistant genes easily by horizontal gene transfer mechanism (Carattoli 2013).

• 2. The selective pressure from widespread antibiotic use in hospitals promotes the survival of and proliferation of ESBL-producing organisms (Paterson & Bonomo 2005).

• 3. The high density of the patient population and frequent patient-to-patient contact further contribute to infections caused by ESBL-producing bacteria (Hendrik et al. 2015).

These multidrug-resistant Enterobacteriaceae can cause clinical infections such as urinary system infections, bacteremia, pneumonia, and many more (Paterson & Bonomo 2005).

Wastewater is a mixture containing various organic and inorganic chemicals, dissolved and suspended solids, varied microorganisms, and pathogens (Metcalf et al. 2014). Its complexity stems from the distinct sources of contents which is a composition of household sewage, industrial effluent, and stormwater effluent (Water Resources Research Center – College of Agriculture and Life Sciences et al. 2013). This sludge is heterogeneous in nature and consists of nutrients, pathogens, chemicals, and contaminants including pharmaceuticals and personal care products (Tran et al. 2018) and antibiotic-resistant bacteria (Karkman et al. 2018).

Municipal and hospital wastewater pose potential sources of contamination with antibiotic-resistant bacteria, especially Enterobacteriaceae that produce ESBLs (Wang et al. 2016). The emission of wastewater that is either unmanaged or poorly managed into the environment can adversely affect public health. These bacteria's resilience through the treatment process is shown by their ongoing existence in wastewater treatment plants (Cahill et al. 2019).

Wastewater from hospitals (HWW) is especially hazardous, comprising a variety of pathogens and a variety of toxins such as prions, viroids, and toxins, which endanger ecosystems and human health (Kluytmans-van den Bergh et al. 2019). Hospitals contribute to wastewater with unmetabolized medications, disinfectants, and pathogens from medical processes (Ensink et al. 2004). Even though it poses a risk, HWW is usually treated in the same way as residential waste, revealing the need for broad studies on its microbial makeup and its implications for antibiotic resistance (Ensink et al. 2004; Murtaza & Zia 2012).

As one of the most quickly developing cities in Pakistan's Khyber Pakhtunkhwa province, Kohat is suffering severely from a deficiency in wastewater treatment, a challenge exacerbated by the increasing urban and industrial populations. Such neglect is worrisome for water pollution and can create a possible health hazard to the populace (Fida et al. 2023; Natasha et al. 2023). Not much is understood about how frequently and unevenly ESBL-producing Enterobacteriaceae occurs in the wastewater systems of Kohat, which raises alarm regarding antibiotic-resistance patterns in the region. Further exploration is necessary to understand the role of both municipal and hospital wastewater in this problem and the details of their contribution to the growth of antibiotic resistance in Kohat.

Research around the world has revealed that there is a significant amount of antibiotic-resistant bacteria in wastewater. Ahsan et al. (2022) pointed out that hospital wastewater in Pakistan harbored E. coli resistant to ESBLs and examined their antibiotic-resistance levels and molecular profiles. In neighboring India, Diwan et al. (2012) found a considerable abundance of ESBL-producing Enterobacteriaceae in the wastewater from health facilities (Korzeniewska et al. 2013). In Poland, municipal and hospital wastewater revealed ESBL-producing bacteria, whereas, in Brazil, Paschoal et al. (2017) hospital effluents showed a high rate of ESBL-producing Enterobacteriaceae. The studies in unison bring attention to the global issue and the requirement for more local research to define needs and direct disease control operations.

The presence of E. coli and K. pneumoniae in wastewater and showing multidrug resistance presents a peril to public health (Constantinides et al. 2020). To protect and manage public health, one needs inclusive knowledge of antibiotic-resistance patterns and the presence of fecal coliform count (FCC) in wastewater samples (Bürgmann et al. 2018). The study aims to examine the latter factors in a novel area of Kohat. It is postulated that the levels and types of ESBL-producing Enterobacteriaceae will be significantly higher in the hospital wastewater than in the municipal wastewater in Kohat, Pakistan because of greater utilization of antibiotics and the existence of antibiotic resistance in healthcare facilities.

Wastewater samples were collected from 17 different places in Kohat, Pakistan, including areas of hospitals and municipalities (Table 1). The sampling strategy was intended to estimate the frequency and distribution of multidrug-resistant (MDR) bacteria in various urban settings. The study spanned 1 year; however, sample collection was concentrated within the 3-month period of June through August. Sampling was done thrice in each selected site, after a 1-week interval, making up to 51 samples. The above sampling approach of repeated sampling assists in controlling for temporal fluctuations in bacterial populations as noted by Czekalski et al. (2012).

Each wastewater sample consisted of 100 ml, collected in sterile containers. Immediately after collection, samples were placed in coolers with ice packs and transported to the laboratory within 4 h, adhering to standard protocols for environmental sample handling (APHA 2017). Upon arrival, samples were either processed immediately or stored at 4 °C for up to 24 h before analysis to ensure sample integrity, following guidelines established by Rizzo et al. (2013).

Spread plate method and colony isolation wastewater samples were processed using the spread plate technique, a method widely employed in environmental microbiology for isolating and enumerating bacteria. A dilution series as a 10⁻¹–10⁻⁶ serial dilution was made for each of the samples. From each dilution, 0.1 ml was then aseptically streaked onto MacConkey agar plates with the help of a sterile glass spreader. The plates were then incubated aerobically at 37 °C for 18–24 h.

After incubation, an equivalence of 30–300 colonies per plate was picked for further digestion. In the cumulative plates, 22 morphologically different colonies were randomly picked on all sampled sites. For confirmed purity, each of all chosen colonies was passed through three subsequent cycles of sub-culturing on the fresh MacConkey agar. This isolation strategy enables the characterization of a wide array of possible antibiotic-resistant bacteria while at the same time avoiding highly numerous isolates for a detailed analysis (Ahmed et al. 2022).

The biochemical test is essential in the identification of the members of the Enterobacteriaceae family, especially K. pneumoniae and E. coli. All these bacteria are characterized by creating distinct metabolic profiles by using a series of tests called the IMViC series (indole, methyl red (MR), Voges–Proskauer (VP), and citrate) and other assays as well. The ability to produce indole as an indicator of the ability to degrade tryptophan is usually positive for E. coli and is negative for K. pneumoniae. On the other hand, the citrate utilization test, which determines the ability of an organism to use citrate whenever it is the only source of carbon, is usually positive for K. pneumoniae, although it is negative for E. coli. The Voges–Proskauer test, which analyses acetoin production, is always positive for K. pneumoniae and never for E. coli, while the methyl red test, for mixed acid fermentation, is always negative for K. pneumoniae and positive for E. coli. Other tests, like urease production, are positive for K. pneumoniae while negative for E. coli, and motility is negative for K. pneumoniae while variable for E. coli. These biochemical properties make it possible to presumptively identify these clinically relevant bacteria in the family Enterobacteriaceae.

The application of this set of biochemical characterization techniques is most appropriate when it comes to the analysis of wastewater samples containing a broad spectrum of potentially pathogenic and antibiotic-resistant microorganisms. When we can identify the isolates to the genus or species level then, it will be easier for us to see the distribution of various bacterial groups that are present in the wastewater samples and make some rational assumptions about the connected health hazards and antibiotic resistance (Cheesbrough 2000; Karim et al. 2020; Masi et al. 2021).

To analyze the wastewater samples for fecal contamination, the Most Probable Number (MPN) method for fecal coliform was used in relation to standard methods of analyzing water, and wastewater (American Public Health Association, American Water Works Association, Water Environment Federation 2012).

The MPN technique involves a series of steps: Presumptive test: Samples from the wastewater treatment were diluted in a series of 10, 1, and 0.1 ml and then were poured into lactose broth tubes with Durham tubes. Five tubes were used for each dilution and at least four concentrations were tested. The tubes were incubated at 35 ± 0.5 °C for 24–48 h.

Confirmed test: From each positive presumptive tube, where gas has been produced, one loopful of culture was then cultured in the tubes of Brilliant Green Lactose Bile broth. These were incubated at 44.5 ± 0.2 °C for 24 h

Completed test: The positive tubes from the confirmed test were struck on eosin methylene blue agar plates that were incubated at a temperature of 35 ± 0. 5 °C for 24 h.

Positive reactions were confirmed by the production of acid, which resulted in a change of color in the medium as well as the presence of gases in the Durham tubes. The number of positive tubes for each dilution was recorded and used to calculate the MPN value per 100 ml of sample using standard MPN tables (American Public Health Association, American Water Works Association, Water Environment Federation 2012).

It affords an opportunity to quantify fecal coliform in wastewater samples and to delineate the level of fecal pollution and possible health hazards of each sampling point (Tallon et al. 2005).

The detection of ESBL-producing Enterobacteriaceae was carried out in the first step by using the disc diffusion method on Mueller–Hinton agar (MHA) plates, according to the protocol of Clinical and Laboratory (CLSI) standards guidelines (Gaur et al. 2023). Two third-generation cephalosporins were used as indicator antibiotics: cefotaxime (30 μg) and ceftazidime (30 μg). Isolates were regarded as possible ESBL producers when they showed decreased susceptibilities, in this case, reduced susceptibility was judged by zone diameters of ≤ 27 mm for cefotaxime, and/or ≤ 22 mm for ceftazidime (Shakya et al. 2017; Cusack et al. 2019; Giske et al. 2022; Gaur et al. 2023).

ESBL production was confirmed using the combination disc method. This method involves comparing the inhibition zones of cephalosporin discs alone and in combination with clavulanic acid. The following disc pairs were used:

• 1. Cefotaxime (30 μg) and cefotaxime-clavulanic acid (30/10 μg)

• 2. Ceftazidime (30 μg) and ceftazidime-clavulanic acid (30/10 μg)

An increase in the inhibition zone diameter of ≥5 mm for either antimicrobial agent in combination with clavulanic acid compared to the agent alone was interpreted as positive for ESBL production (Kebede et al. 2022).

Following initial screening and ESBL confirmation, isolates were subjected to AST using the Kirby–Bauer disc diffusion method on MHA, as per CLSI guidelines. The following 12 antibiotics were tested: ampicillin (30 μg), cefotaxime (30 μg), cefoxitin (30 μg), penicillin G (10 μg), amoxicillin–clavulanate (30 μg), cefoperazone–sulbactum (105 μg), ciprofloxacin CIP(5 μg), trimethoprim–sulfhamethoxzole (TS) (25 μg), chloramphenicol (C) (30 μg), gentamycin (CN) (10 μg), and meropenem (MEM) (10 μg) chosen based on their clinical relevance and representation of different antibiotic classes (Humphries et al. 2021).

For every isolate that was positive for ESBL, the original colony from the MacConkey agar plate was preserved and used for further testing. This approach helps in maintaining a standard in the ability to preserve the features of the original colonies throughout. A suspension equivalent to the 0.5 McFarland standard was prepared from this preserved isolate. This suspension was then used to inoculate MHA plates for both the ESBL confirmation test and the additional antibiotic susceptibility testing.

Solidified growth media were streaked with bacterial isolates and incubated at 35 ± 2 °C for 16–18 h under an aerobic environment. When incubation was complete, the diameters of the inhibition zones were determined and considered according to the CLSI breakpoints (Gaur et al. 2023). The isolates were interpreted as susceptible, intermediate, or resistant to each of the antimicrobial agents screened.

We defined the isolates with infection resistance to three or more antibiotics as MDR based on the classification of Humphries et al. (2021). For AST and ESBL confirmation, we used E. coli ATCC 25922 as a quality control strain for antimicrobial susceptibility and K. pneumoniae ATCC 700603 as the ESBL-positive control strain recommended by the CLSI.

The spread plate method revealed two distinct bacterial groups: one of which is with pink-yellow colonies and the other with red-pink colonies. Bacterial isolates were isolated on streak plates, and the bacterial species were differentiated and identified based on their morphological characteristics, including shape, color, and size, as described by Sanders (2012).

Additional biochemical tests were then performed to clarify the isolate's characteristics. The colonies, which are pink-yellow colonies (Type 1), were non-motile and negative for the indole and methyl red tests. Unlike the red-pink colonies (Type 2) were motile and were positive for both the indole and methyl red tests. Further biochemical tests, such as the VP test, TSI test, catalase test, oxidase test, and citrate utilization test, were carried out as per the standard methods. The outcome of these tests is presented in Table 1 (Paul et al. 2010; Sikarwar & Batra 2011). From biochemical characterization, Type 1 bacteria were found to be: K. pneumoniae, while Type 2 was E. coli. These results are consistent with other studies done regarding the identification of these two species in samples collected from wastewater sources (Paul et al. 2010; Sikarwar & Batra 2011).

The FCC also proved the above results and Frontier Medical Centre (FRMC), W2 had the highest count (93 × 10⁶ cfu/ml), LIMH, W3 had 90 × 10⁶ cfu/ml. Such high FCCs have a possibility of posing a high public health risk as observed in other related research.

FCC values represent fecal coliform bacteria concentration, indicating fecal contamination and potential pathogen presence that can cause health emergency outbreaks. Table 2 presents the bacterial isolates, collection sites, and related FCC values (in ×10⁶ cfu/ml) for examining FCC in wastewater samples. Higher FCC values, such as FRMC, wastewater sample 2 (93 × 10⁶ cfu/ml) and LIMH, wastewater sample 3 (90 × 10⁶ cfu/ml), suggest elevated concentrations of fecal coliform bacteria, indicating a higher possibility of infections and potential ESBL pathogenic presence in these samples (Patel et al. 2014; Makuwa et al. 2020).

In wastewater samples from 17 collection locations, the frequency of ESBL-producing E. coli and K. pneumoniae isolates is investigated in this study. Twelve (23%) and 10 (19%) of the 51 samples were found to be ESBL-producing E. coli and K. pneumoniae, respectively. In all, 22 samples (43%) contained isolates that produced ESBLs, with E. coli having a greater incidence than other bacteria (Table 4).

Our findings indicated a high frequency of Gram-negative bacteria, particularly focused on E. coli and K. pneumoniae, through 17 sampling sites in Kohat. This is in accord with recent research conducted in corresponding regions (Manyi-Loh et al. 2018; Khan et al. 2020). However, our study is novel in its comprehensive coverage of both municipal and hospital wastewater sources in the Kohat urban setting of Pakistan.

We observed greater fecal coliform bacterial concentrations in the hospital wastewater system compared to municipal wastewater samples. For example, FRMC Wastewater Sample 2 had a high FCC value of 93 × 10⁶ cfu/ml. Similarly, in LIMH, Wastewater Sample 3, the FCC value was also high 90 × 10⁶ cfu/ml. These values are significantly higher than those found in municipal wastewater samples, which averaged around 45 × 10⁶ cfu/ml. This disparity can be attributed to the concentrated use of antibiotics in healthcare settings, leading to increased selection pressure for resistant strains (Korzeniewska et al. 2013; Pruden et al. 2013; Lien et al. 2017). It was interesting to note that a higher FCC value, and hence a greater diversity of antibiotic-resistant strains, emerged in this study from sites closer to hospitals. This spatial variation in antibiotic resistance represents new information in Kohat as the body of evidence grows toward identifying hospitals as hubs for the spread of antibiotic resistance (Hocquet et al. 2016).

Our antibiotic sensitivity profiles revealed high resistance to ampicillin, cefotaxime, cefoxitin, amoxicillin-clavulanic acid, and ciprofloxacin in both E. coli and K. pneumoniae isolates. This multidrug resistance pattern is particularly concerning and aligns with recent global trends in antibiotic resistance (Devi et al. 2021; Mączyńska et al. 2023; WHO 2023). However, our study provides specific data for Kohat, filling a crucial gap in understanding local resistance patterns. Gentamicin exhibited moderate effectiveness in a slightly more extensive zone of inhibition with E. coli. These findings align with prior studies reporting similar antibiotic-resistance patterns in E. coli and K. pneumoniae isolates, including resistance to ciprofloxacin, ampicillin, and third-generation cephalosporins like cefotaxime (Maina et al. 2013; Ahmed et al. 2022).

We observed differential susceptibility between E. coli and K. pneumoniae to certain antibiotics, particularly chloramphenicol and sulfamethoxazole, is noteworthy. E. coli showed higher susceptibility to these antibiotics, with inhibition zones of 25 and 20 mm, respectively, while K. pneumoniae showed complete resistance. However, it is important to note that resistance patterns can vary significantly based on geographical location, specific strains, and local antibiotic use for practices. For instance, Gundran et al. (2019) found that both E. coli and K. pneumoniae isolates from hospital wastewater in the Philippines showed high resistance to sulfamethoxazole-trimethoprim, contradicting the statement about E. coli's higher susceptibility. Similarly Amaya et al. (2012), in Colombia, found that both E. coli and K. pneumoniae isolates from clinical samples showed high resistance to TS. Conversely Moges et al. (2014), in Ethiopia, found that K. pneumoniae isolates were more susceptible to chloramphenicol than E. coli isolates from clinical samples. These contrasting findings highlight the importance of local surveillance in guiding antibiotic stewardship programs.

Our study also revealed a significant difference in ceftazidime resistance between E. coli and K. pneumoniae. E. coli shows higher resistance with MICs of 96 μg/100 μl, compared to K. pneumoniae with lower MICs at 48 μg/100 μl (Nguyen et al. 2020; Carvalho et al. 2021; Khalifa et al. 2021). Although K. pneumoniae is commonly associated with ESBL production, its lower MICs in this study suggest greater susceptibility to ceftazidime. E. coli's higher MICs suggest reduced susceptibility, possibly due to factors like altered cell wall permeability and specific efflux pumps, as noted by Sivaraman et al. (2021).

Our study confirms the presence of ESBL-producing strains in both clinical and non-clinical wastewater sources in Kohat. While ESBL production is known to be prevalent in South Asia (Lamba et al. 2017), our study provides the first comprehensive data for the Kohat urban area in Pakistan. Contrary to our initial hypothesis, we found that ESBL-producing bacteria were nearly as prevalent in non-clinical settings as in clinical situations. For instance, we detected ESBL-producing E. coli in 65% of municipal wastewater samples, compared to 72% in hospital wastewater. This narrow gap indicates a potential underestimation of the community reservoir of antibiotic resistance in previous studies.

The overall prevalence of 43% ESBL producers across all samples underscores the significant burden of antibiotic resistance in Kohat. Notably, while some clinical sites such as MABC, COMH, DHQH, BEMC, and LIMH showed a 100% prevalence of ESBL producers, we also detected these bacteria in non-clinical settings, including municipal areas like JA, TA, and USP. This distribution pattern challenges the conventional assumption that ESBL-producing bacteria are primarily associated with healthcare environments and suggests a potential reservoir for community-acquired ESBL infections (Moges et al. 2014; Lamba et al. 2017).

The findings highlight the need for a One Health approach in addressing antibiotic resistance, considering the interconnectedness of human, animal, and environmental health. Future research should focus on understanding the transmission dynamics between clinical and community settings, as well as evaluating the effectiveness of targeted interventions in both spheres to mitigate the spread of antibiotic-resistant bacteria. Additionally, studies examining the genetic basis of resistance and the potential for horizontal gene transfer in these environments would provide valuable insights into the evolution and spread of antibiotic resistance in urban settings like Kohat.

Our research work in Kohat, Pakistan, brings appalling findings of antibiotic-resistant bacteria in urban wastewater, which adds much value to knowledge about antibiotic-resistant bacteria in South Asia. This study also reveals a relatively high level of antibiotic-resistant Enterobacteriaceae with higher fecal coliform concentrations associated with hospital effluent than municipal effluent sources. Both E. coli and K. pneumoniae displayed higher ceftazidime resistance than expected. Most surprisingly, our study contradicts this quasi-accepted dogma by revealing that ESBL-producing bacteria occur at almost clinically comparable frequencies in non-clinical contexts, with an average value of 43% of all our samples testing positive for ESBL-producing bacteria.

These findings, therefore, have significant ramifications on future public health and antibiotic stewardship policies. These indicate a need for better wastewater treatment, especially hospital effluents, and a need for enhanced surveillance in the community. The high level of antibiotic-resistance bacteria in non-clinical settings baseline that community-acquired antibiotic-resistant infection may be more common than previously thought, requiring reassessment of present prevention policies.

With the growing concern about antibiotic resistance in the global context, our research helps to highlight the fact that there is no strict division between the clinic and the community in the process of spreading resistance. To fill these gaps, future research should further characterize the transmission between these settings and assess the effectiveness of specific interventions, such as improved hospital wastewater treatment, community interventions on antibiotic use, and increased sampling.

Preliminary results of this research show high levels of antibiotic resistance, which should be addressed. We call upon the policymakers, healthcare providers, and other community stakeholders to join efforts and work on effective and integrated interventions for dealing with this rising problem. If coordinated action of people all over the world does not occur, there is little hope of preventing the spread of antibiotic resistance and preserving the effectiveness of these essential drugs in the next generations.

The authors extend their appreciation to the Researchers Supporting Project (RSP2024R120), King Saud University, Riyadh, Saudi Arabia.

This work was funded by the Researchers Supporting Project (RSP2024R120), King Saud University, Riyadh, Saudi Arabia.

The ethical approval for this study was obtained from the KUST Ethical Committee at the Kohat University of Science and Technology, Kohat, Pakistan under Reference No. KUST/Ethical Committee/00234 dated 02/06/2022.

Conceptualized by M.M., M.A.A., B.K.. Rendered support in formal analysis of M.M., F.F., I.A., S.S.A.R., Z.Z., M.M.. Rendered support in funding acquisition of M.M., F.F.. Investigated by J.A., M.D.A.. Developed the methodology by J.A.. Provided project administration by B.K., I.A.. Supervised by B.K.. Wrote the original draft by J.A.. Wrote and review and edited by B.K., M.M., M.D.K., I.A., F.F., S.S.A.R., Z.Z., M.M.

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

The authors declare there is no conflict.