Untreated Wastewater as a Reservoir of Carbapenem-Resistant Escherichia Coli and Klebsiella Pneumoniae Isolates

I. INTRODUCTION

Untreated wastewaters from hospitals, agriculture, slaughterhouses, industry and, together with domestic wastewaters contribute to the contamination and degradation of aquatic environments (Oliveira et al., 2021). The release of untreated or poorly treated wastewater into the environment is being increasingly recognized as the major factor in spreading clinically relevant antimicrobial resistant bacteria and genes (Obayiuwana and Ibekwe, 2020). Antimicrobials in wastewater promote the emergence of antibiotic resistance, facilitated by selective pressure and transfer of resistant genes. The widespread use of antimicrobials in clinical practice to control infectious diseases, their application in veterinary medicine coupled with the discharge of non-treated pharmaceutical effluent into the environment, results in the selective pressure which is associated with the emergence and subsequent evolution of bacterial resistant to antibiotics (Tiwari et al., 2022).

Contamination of water bodies is dependent on the amount, type and degradation potential of contaminants, and the self-purification ability of the recipient water body (Obasi et al 2017). Human and animal pathogens are considered to be important water contaminants; this is possible because, together with human and animal pathogens from all infected individuals and animals (symptomatic, asymptomatic, pre-symptomatic, and post-symptomatic), is excreted through feces, urine, nasal mucus, and sputum from veterinary clinics, slaughterhouses, households, hospitals, and nursing homes, end up in the municipal sewage system. Within the concept of one health perspective, such discharges of clinically relevant antibiotic-resistant bacteria pose a significant health risk to both humans and animals. Therefore, determining the prevalence of clinically important antibiotic resistant bacteria in wastewater may help in controlling their circulation in the environment and saving human and environmental health.

Carbapenems are broad range -lactam antibiotics used as a last resort in treating infections with multi-drug resistant Enterobacteriaceae (MDR-E). In the past few decades, the emergence of strains resistant to carbapenems has been a growing concern (Meletis, 2016). In February 2017, the World Health organization (WHO) published a list of the most dangerous pathogens for human health. At the top of that list are carbapenem-resistant Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae (WHO, 2017). There have been several reports of bacteria with acquired carbapenem resistance isolated from hospital wastewaters (Zhang et al., 2013; Chandran et al., 2014; Seruga-Music et al., 2017), as well as raw and secondary treated municipal wastewaters (Hrenovic et al., 2017). However, data on the prevalence of bacteria of clinical importance from wastewater sources in southeast Nigeria is scarce; most studies are centered on the clinical environment and there is no information about prevalence of carbapenem-resistant E. coli and K. pneumoniae in environmental sources in the region. This study was designed to bridge this knowledge gap and was therefore, aimed to investigate the prevalence of E. coli and K. pneumoniae isolated from hospital and slaughterhouse wastewater sources in Owerri, a city located in southeast Nigeria, during a regional surveillance program conducted in March to September 2023.

II. METHODOLOGY

2.1 Sample Collection and Analysis

Untreated wastewater samples were drawn from the Federal University Teaching Hospital Owerri wastewater discharge points and a discharge point of Owerri slaughterhouse. Samples were collected from the upper 5 cm portion of each of the two wastewater sources in triplicate and pooled to form a composite sample. The samples were taken aseptically in sterile plastic bottles under refrigerated conditions and processed in the laboratory within 4 h after collection. Wastewater samples were serially diluted with peptone water and a 100 l volume of each dilution concentration (original, 1/10, 1/100, and 1/1000) was inoculated on MacConkey agar with a spread plate technique, following the manufacturer's instructions. The plates were incubated at 37°C for 18–24 h, after which colonies were counted and expressed as colony forming units (CFU) per milliliter (ml). Colonies resembling E. coli and K. pneumoniae were sub-cultured in nutrient agar and the isolates were identified using conventional microbiological techniques.

2.2 Antibiotics Susceptibility Testing

The antibiotic susceptibility pattern of the isolates was determined using the Kirby-Bauer Disk diffusion method on Mueller Hinton agar (Oxoid, England). Inhibition zone diameter values were interpreted using standard recommendations of the Clinical Laboratory Standard Institute (CLSI, 2012). Susceptibility was tested against ertapenem, imipenem, meropenem, amoxicillin/clavulanic acid, ceftazidime, and cefuroxime (Oxoid, England). E. coli ATCC 25922 was included as a reference strain. Isolates found to have an ertapenem, meropenem or imipenem ≤22mm inhibition zone diameter were classified as carbapenem-resistant isolates.

III. RESULTS

3.1 Prevalence of Bacterial Isolates

In this study, 269 wastewater samples from Owerri Nigeria were processed for carbapenem-resistant E. coli and K. pneumoniae isolation. The samples included 135 hospital wastewater and 134 slaughterhouse wastewater respectively. In all, a total of 193 bacteria comprising of consecutive 92 (46.7%) E. coli and 101 (52.3%) K. pneumoniae was isolated. One bacteria isolate was selected from one wastewater sample and one sample;

either hospital or slaughterhouse wastewater was collected and not both. Sample collection was based on availability and we could not compare isolation rate from hospital or slaughterhouse wastewater. The distribution of the 92 E. coli isolates according to sample sources showed that 59 (64.1%) came from hospital wastewater while

33 (35.9%) came from slaughterhouse wastewater. Similarly, 61.4% (62/101) of the 101 K. pneumoniae isolates were from hospital wastewater while 38.6% (39/101) of the isolates came from slaughterhouse wastewater respectively Table 1.

Table 1: The Overall Growth and Distribution of Escherichia Coli and Klebsiella Pneumoniae Within the Different Wastewater Sampled

3.2 Antimicrobial Susceptibility Testing

As shown in Table 2, high antibiotic resistance rates were observed in the isolates, no isolate was susceptible to all the antibiotics tested. Among the carbapenems, moderate to high resistance rate was observed; ranging from 22.8% in imipenem to 30.6% in ertapenem. As expected, very high resistance rate was also observed in cephalosporins including 55.9% in cefuroxime and 58.0% in ceftazidime respectively. Amoxicillin clavulanic acid, an antibiotic used for phenotypic detection ESBL production was considerably inactive against the isolates with a 41.9% resistance rate.

There was no statistically significant difference in carbapenem resistance rates between the hospital and slaughterhouse wastewater isolates (P > 0.05).

Table 2: Antibiotics Susceptibility Patterns of Escherichia Coli and Klebsiella Pneumoniae Isolates against the 6 Antibiotics Tested

IV. DISCUSSION

Antibiotics resistance profiling of pathogens helps to identify the emergence of rare or new resistance threats and prioritize possible actions to be taken against them. The analysis of wastewater can reveal the circulation of antibiotic resistant bacteria and antibiotics resistance genes among the catchment communities. In this study, we carried out untreated wastewater surveillance for carbapenem resistance traits among E. coli and K. pneumoniae. Our results indicate that carbapenem-resistant E. coli and K. pneumoniae are widely distributed among the wastewater sources. The isolates expressed highest resistance to ertapenem (30.6%) while the least resistance was observed in imipenem with 22.8% resistance rate. To our knowledge, these are the earliest carbapenem-resistant E. coli and K. pneumoniae detected in non-clinical samples in Owerri southeast Nigeria. In agreement with our result, a study conducted in 2020 also reported carbapenem-resistant traits as the most abundant resistant traits in hospital wastewater in Helsinki (Majlander et al., 2021). However, their study included genotypic characterization while ours was based on only phenotypic characterization due to limited resources. Another study from the Netherlands, based on molecular characterization reported carbapenem-resistant genes from both hospital and municipal wastewater, but the abundance in hospital wastewater was higher than in the municipal wastewater (Buelow et al., 2018). As untreated wastewater is considered to provide a glimpse of the antibiotic resistant bacteria and antibiotic resistance genes circulating in a community, our findings indicate that ertapenem resistant E. coli and K. pneumoniae was the most prevalent carbapenem-resistant trait in Owerri during the study period.

The World Health Organization (WHO) has classified carbapenem-resistant Enterobacterales as a critical priority, driving the need to develop new antibiotics (Davies and Simeon, 2017). E. coli and K. pneumoniae isolates identified as carbapenem-resistant in this study have been described in the environment and reported to be potential clinical pathogens (Li et al., 2019).

Perhaps, some carbapenemase genes are related to aquatic environments and only occasionally cause infection. Many of these bacterial species could be asymptomatically carried by humans in their intestines, and thus be found in wastewater.

Clinical isolates can differ from wastewater-based isolates. The clinical isolates are pathogenic, but wastewater-based isolates better represent symbiotic and normal human bacteria. The presence of high concentrations of carbapenem resistant E. coli and K. pneumoniae isolates in both the hospital and slaughterhouse wastewater samples is not only a reflection of the increasing use of carbapenems in Nigeria, but also an additional evidence regarding the inefficiency of the conventionally applied wastewater treatments in the elimination of these microorganisms from the treated effluents, subsequently leading to their release into the environment (Oliveira et al., 2020).

V. CONCLUSION

The analysis of the antibiotic resistance phenotypes of the potentially pathogenic carbapenem resistant E. coli and K. pneumoniae isolated from hospital and slaughterhouse wastewater samples revealed that all were multidrug resistance, showing resistance phenotypes to more than three antibiotic classes.

The findings of this study, together with the clinical reports from the previous studies indicate a steady increase in carbapenem resistant bacteria in Nigeria and support wastewater surveillance as a potential preparedness tool for antimicrobial resistance surveillance. Wastewater surveillance could supplement the clinical surveillance approach in monitoring the possible circulation of antimicrobial resistance in communities and shows potential as a public health tool. Future studies could cover larger geographical areas of Nigeria and include molecular tools to obtain a more representative national picture. The detection of carbapenem resistant E. coli and K. pneumoniae in the two wastewater samples underlines the importance of proper wastewater treatment to avoid the dissemination of antimicrobial resistant bacteria in the environment and reduce public health risks.