Carbapenem-resistant Enterobacterales in wastewater resources and healthy carriers: A survey in Iran

Enterobacterales is a large order of gram-negative bacteria that contains seven families with diverse groups of species occupying distinct ecological niches including human and animal intestines and other environments (Adeolu et al. 2016; Janda & Abbott 2021; Habibzadeh et al. 2022; Hasani et al. 2023). Many genera in this order, including the Enterobacteriaceae family members, are significant human pathogens responsible for several life-threatening true and opportunistic nosocomial infections (Janda & Abbott 2021). The epidemiology of infections caused by Enterobacterales has been significantly altered by the emergence and spread of extended-spectrum beta-lactamase (ESBL) and AmpC-type beta-lactamase-producing strains (Castanheira et al. 2021). Currently, a significant proportion of Enterobacterales bacteria are globally ESBL producers (Raphael et al. 2021). These strains are resistant to third-generation cephalosporins and cephamycins (Bush 2023). Additionally, they are often resistant to multiple other antibiotic classes, including fluoroquinolones (Khademi et al. 2020; Neyestani et al. 2023). However, ESBL and AmpC-type enzymes are not capable of hydrolyzing carbapenem antibiotics (Raphael et al. 2021). Therefore, the Infectious Diseases Society of America (IDSA) has introduced carbapenems as a reliable reserve and the last resort to effectively treat severe infections by multidrug-resistant (MDR) gram-negative nosocomial pathogens (Tamma et al. 2023). Unfortunately, resistance to carbapenems has also increased significantly in recent years due to non-standard prescriptions and the excessive usage of these antibiotics, particularly in developing countries (Khavandi et al. 2022; Ma et al. 2023). Infections caused by carbapenem-resistant Enterobacterales (CRE) are associated with high rates of mortality compared to carbapenem-susceptible counterparts (Zhou et al. 2021). The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) have highlighted CRE as an important public health threat requiring immediate action (Tacconelli et al. 2018). Resistance to carbapenems in gram-negative bacteria is mediated by several mechanisms, including (i) reduction of drug uptake due to reduced membrane permeability caused by mutation or loss of the outer membrane proteins, (ii) increase in the expression of efflux pumps which extrude antibiotics out of the cell, (iii) alterations in penicillin-binding proteins which limit the antibiotics' access to the target sites, and (iv) production of carbapenemase enzymes that inactivate carbapenems together with almost all β-lactam antibiotics by breaking the amide bond in the β-lactam ring (Ma et al. 2023). Production of carbapenemase enzymes is the most efficient mechanism of carbapenem resistance mainly among Enterobacteriaceae (Nordmann & Poirel 2019). Based on the Ambler classification system and according to molecular structures, the β-lactamases are classified into four main classes (classes A–D) (Bush 2023). The carbapenemase enzymes responsible for carbapenem resistance in Enterobacteriaceae family include Klebsiella pneumoniae carbapenemases (KPCs), Serratia marcescens enzyme (SME), Serratia fonticola carbapenemase (SFC), Guiana extended-spectrum β-lactamase (GES), imipenem-hydrolyzing β-lactamases (IMI), and non-metallo-carbapenemases-A (NMC-A), II) (class A β-lactamases), Verona integron-encoded metallo-β-lactamase (VIM), imipenemase metallo-β-lactamase (IMP), New Delhi metallo-β-lactamase (NDM) (class B β-lactamases), and OXA-48-type enzymes (class D β-lactamases) (Logan & Weinstein 2017). Carbapenemase genes are mainly carried on mobile genetic elements, which is a key advantage for the rapid spread of CRE (Nordmann & Poirel 2019). Studies in the literature show that CRE bacteria can be found beyond clinical sources among colonized healthy people (Kelly et al. 2017), food-producing animals (Huang et al. 2023), and environmental resources such as hospitals (Cahill et al. 2019), and municipal wastewater (Urase et al. 2022). These could act as environmental reservoirs for CRE and contaminate humans (Mills & Lee 2019). The epidemiology of CRE bacteria in clinical specimens has been investigated intensively worldwide (Ma et al. 2023), while the information on the epidemiology of CRE in non-clinical settings is still limited in many regions of the world, including Iran. This study aimed to assess the prevalence of CRE in non-clinical settings, including healthy carriers, hospital wastewater, municipal wastewater, and poultry and livestock slaughterhouse wastewater in the northwest region of Iran. The study also aimed to discover the major carbapenemase-encoding genes responsible for carbapenem resistance. Additionally, the molecular epidemiology of carbapenemase-producing Escherichia coli isolates was investigated.

In this study, a total of 617 Enterobacterales isolates were included, having been previously collected and characterized from various non-clinical settings in Ardabil City, Iran (Habibzadeh et al. 2022; Hasani et al. 2023; Sardari et al. 2024). Of these, 298 isolates originated from untreated hospital wastewater (n = 72), poultry and livestock slaughterhouse wastewater (n = 80) (Sardari et al. 2024), and municipal wastewater (n = 146) (Hasani et al. 2023). The wastewater samples were obtained from teaching hospitals (Imam, Fatemi, Alavi, and Bouali) affiliated with Ardabil University of Medical Sciences, as well as poultry and livestock slaughterhouses (from June 2020 to June 2021), and municipal wastewater treatment plant (between March 2019 and August 2019) in Ardabil province. Liquid wastewater samples were collected following the U.S. Environmental Protection Agency's (US EPA) standard operating procedure for wastewater sampling (U.S. EPA 2013). Additionally, 319 Enterobacterales isolates were retrieved from fecal specimens of healthy students aged 12–15 years during April and August 2017 in 19 male/female middle schools in Ardabil, Iran (Habibzadeh et al. 2022).

The susceptibility and resistance of bacteria against carbapenem antibiotics were determined using the disk diffusion method on Muller-Hinton agar (MHA) (Merck, Germany) medium (Schwalbe et al. 2007). Carbapenem resistance was evaluated using the following antibiotics (Mast, England): imipenem (IMP, 10 μg), meropenem (MEM, 10 μg), ertapenem (ETP, 10 μg), and doripenem (DOR, 10 μg). The results obtained from the disk diffusion method were interpreted based on the criteria outlined in the 33rd edition of the Clinical and Laboratory Standards Institute (CLSI) (CLSI 2023). Enterobacterales isolates with resistance to at least one of the carbapenem antibiotics were defined as CRE (CLSI 2023). Klebsiella pneumoniae ATCC BAA-1705 and E. coli ATCC 25922 were used as carbapenem-resistant and carbapenem-susceptible control bacteria, respectively.

Screening of carbapenemase genes was performed on CRE isolates. For this purpose, CRE isolates were cultured on blood agar plates and were incubated at 37 °C overnight. In the next step, the total genomic DNA was extracted using the DNP Genomic DNA Extraction Kit (Cinagen Co., Tehran, Iran) according to the manufacturer's instructions. The presence of carbapenemase-encoding genes including bla_KPC, bla_GES (Ambler Class A), bla_VIM-1, bla_VIM-2, bla_NDM, bla_IMP, bla_SPM (Ambler Class B), and bla_OXA-48 (Ambler Class D) was determined by the polymerase chain reaction (PCR) method using specific primers in the CRE isolates. The sequence of the primers with their annealing temperatures is listed in Table 1. The PCR reaction was performed in a thermocycler (Bio-Rad, USA) with a final volume of 25 μL. The PCR mixture consisted of 12.5 μL of Premix Taq^® mix (Sinaclon Co., Tehran, Iran), 2 μL of template DNA, l μL (10 pmol) of each forward and reverse primer, and 8.5 μL deionized nuclease-free water. The thermal cycling protocol for PCR was as follows: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55–60 °C (Table 1) for 35 s, and extension at 72 °C for 1 min, with a final extension at 72 °C for 5 min. The PCR products were screened on 1.5% agarose gels stained with DNA-safe stain (Sinaclon Co., Tehran, Iran). DNA from K. pneumoniae ATCC BAA-1705 (bla_KPC positive) and well-characterized clinical isolates (purchased from the Pasteur Institute, Tehran, Iran) positive for bla_NDM, bla_VIM, bla_IMP, bla_GES, and bla_OXA-48 genes were used as positive controls. DNA from E. coli ATCC 25922 was used as a negative control.

A quadruplex PCR was used for phylogenetic analysis of carbapenem-resistant E. coli isolates according to the study performed by Clermont et al. (2013). Based on a quadruplex PCR assay, an E. coli strain could be assigned to one of the main phylogroups including A, B1, B2, C, D, E, F, and clade I. The phylogenetic analysis was carried out using the chuA, yjaA, TspE4.C2, and arpA primers. The sequences of primers are shown in Table 2. The PCR reaction was carried out in a 25 μL volume containing 12.5 μL of Premix Taq^® mix (Sinaclon Co., Tehran, Iran), 1 μL (10 pmol) for each chuA, yjaA, TspE4.C2, and arpA primers, 2 μL of DNA template, and 2.5 μL deionized nuclease-free water. The amplification of genes was carried out under the following conditions: initial denaturation at 94 °C for 4 min; 30 cycles of denaturation at 94 °C for 5 s, annealing at 59 °C for quadruplex and group C, 57 °C for group E for 20 s, extension at 72 °C for 10 s, and a final extension step at 72 °C for 5 min. PCR products were electrophoresed on the 1.5% agarose gel and screened under UV light.

The Chi-square test was used to evaluate the prevalence of CRE in different resources and resistance to different carbapenem antibiotics among the isolates. The results were considered statistically significant if the P-value was ≤ 0.05.

In this study, all nine Enterobacterales spp. were found to exhibit carbapenem resistance (Table 2). However, the resistance rates varied per species from different resources. Proteus spp. exhibited the highest resistance rate of 40%, followed by Serratia spp. at 36%, and Hafnia spp. at 31% of isolates.

Table 3 illustrates the resistance profile of four carbapenem antibiotics in Enterobacterales isolates. Meropenem had the highest resistance rate compared to other carbapenems (P ≤ 0.05). However, the distribution of resistance to antibiotics varied per species. Hafnia spp. and Serratia spp. exhibited higher rates of resistance to all carbapenems tested.

Table 4 shows the prevalence of carbapenemase-encoding genes among CRE isolates. The bla_OXA-48 gene was detected in 97% of CRE isolates. The bla_NDM and bla_VIM-1 genes were detected in 24 and 3% of isolates, respectively. While bla_OXA-48 and bla_NDM were prevalent among most of the Enterobacterales isolates, the bla_VIM-1 gene was only detected in Citrobacter spp. and Proteus spp. with 5% (n = 1/21) and 27% (n = 3/11) of isolates, respectively. No isolate was positive for bla_KPC, bla_GES, bla_VIM-2, bla_IMP, and bla_SPM genes in the present study.

Table 5 shows the distribution and frequency of carbapenem-resistant E. coli phylogroups. Results revealed that carbapenem-resistant E. coli isolates belong to B2, clade I, F, and E phylogroups. The B2 phylogroup is the most prominent group identified in carbapenem-resistant E. coli isolates from all sources, accounting for 80% of isolates.

Antibiotic overuse in hospitals and poultry and livestock farms has led to the emergence and development of antibiotic-resistant bacteria such as CRE (Pormohammad et al. 2019). CRE have been classified as critical pathogens by the WHO and infections caused by these organisms are associated with high morbidity and mortality due to delays in the administration of effective treatment and limited therapeutic options (Tacconelli et al. 2018). In the current study, CRE isolates were frequently identified beyond the clinical sources from healthy carriers and the wastewater resources from hospitals, poultry slaughterhouses, livestock slaughterhouses, and the community.

The emergence of CRE in healthy carriers is a concern as it can put individuals at risk of indigenous transmission and lethal infections in predisposed people (Chen et al. 2023). In this study, 22% of healthy children were intestinal CRE carriers. The results are in the upper range of the global proportion reported by Kelly et al., which ranges from 0.0 to 29.5% (Kelly et al. 2017).

In addition to humans, livestock and poultry digestive tracts also are colonized with CRE (Köck et al. 2018; Huang et al. 2023). The prevalence of CRE in livestock in Asian countries has been reported to be 1–15% (Köck et al. 2018). So, it can be transmitted to humans by direct contact with animals and contaminated meat products (Li et al. 2019; Huang et al. 2023) and disseminated to the environment via farms and slaughterhouse wastewater (Homeier-Bachmann et al. 2021). In the current study, 34% of Enterobacterales isolates in livestock and poultry slaughterhouse wastewater were carbapenem-resistant. This finding is inconsistent with reports from Germany (Savin et al. 2020), Ethiopia (Tesfaye et al. 2019), and Slovakia (Gregova & Kmet 2020), which found no CRE bacteria from livestock and poultry wastewater and farms. This controversy may be attributed to variations in CRE carriage rates in livestock and poultry by region and also livestock types sacrificed in slaughterhouses (Huang et al. 2023).

In our research, we observed significantly higher rates (33%) of CRE isolates in hospital wastewater. This elevated prevalence of CRE isolates is expected, given that carbapenems are more frequently used in hospitals than in other settings. Similar findings have been reported in studies conducted in Ireland, Germany, and Switzerland (Zurfluh et al. 2017; Cahill et al. 2019; Hoffmann et al. 2023).

In Iran, as in many countries worldwide, hospital effluent is typically released untreated into urban wastewater streams. It then undergoes treatment at urban WWTPs before being discharged into the environment (Mackuľak et al. 2021). Unfortunately, the current treatment procedures employed in municipal wastewater treatment plants are not effective in eliminating antibiotic-resistant bacteria and antibiotic resistance genes (Wang et al. 2015; Osińska et al. 2020; Hasani et al. 2023). Previous research has demonstrated that WWTPs receiving hospital wastewater contribute to the dissemination of clinically relevant CRE clones in superficial water matrices (Oliveira et al. 2023). However, it is worth noting that resistance rates in municipal wastewater tend to be relatively lower than those observed in hospital wastewater (Tiwari et al. 2024). In our specific study, we identified carbapenem resistance in 17% of Enterobacterales isolated from municipal wastewater. This finding aligns with the overall report by Hoffmann et al. (2023) in Germany, Teban-Man et al. (2022) in Romania, and Urase et al. (2022) in Japan.

In this study, a total of nine distinct clinically significant species of CRE were identified. These species include significant pathogens such as E. coli, K. pneumoniae, Proteus spp., and Enterobacter species. Notably, these bacteria have also been frequently detected in wastewater resources across various global regions (Cahill et al. 2019; Hoffmann et al. 2023). Due to limited resources and the divergent species types identified in this study, we did not perform phylogenetic analyses to demonstrate the clonal relatedness of all CRE isolates to corresponding clinical isolates. However, phylogrouping analyses of carbapenem-resistant E. coli isolates, as the most prevalent cause of human disease, revealed that 80% of isolates belonged to phylogroup B2, which are the most clinically significant E. coli isolates from human extraintestinal infections (Clermont et al. 2013). While there is evidence suggesting that geographical location may impact the global distribution of phylogroups (Gordon & Cowling 2003), the literature does not establish a clear association between phylogroups and specific resistance phenotypes. In a related survey conducted by Hoelle et al. (2019) on United States wastewater, it was found that 38 and 15% of imipenem-resistant E. coli isolates belonged to phylogroups D and B2, respectively.

Carbapenem resistance can occur through multiple mechanisms (Ma et al. 2023). Production of carbapenemase enzymes is a major concern as they can confer resistance to carbapenems and other beta-lactam antibiotics, and are often associated with high levels of resistance. Carbapenemase genes can be located on the bacterial chromosome or on mobile genetic elements such as plasmids and transposons, which increases the risk of spreading carbapenem resistance between different bacterial species and genera (Logan & Weinstein 2017; Nordmann & Poirel 2019). These genes have different origins but are not specific to a single host and can be found in various bacteria, especially in the family Enterobacterales (Köck et al. 2018; Huang et al. 2023). The most common carbapenemase genes among Enterobacterales globally are MBLs (IMPs, VIMs, NDMs), KPCs, and OXA-48 enzymes (Logan & Weinstein 2017). In the current study, bla_OXA-48 was the most common carbapenemase gene (97%), followed by bla_NDM (24%) and bla_VIM-1 (3%). The prevalence of these genes can vary depending on the bacterial species and geographic location (Wu et al. 2019). Our results are consistent with previous reports that show bla_OXA-48 has progressively disseminated throughout the Middle East, North African, and European countries and bla_NDM in the Indian subcontinent, the Middle East, and the Balkans. NDM beta-lactamase can hydrolyze almost all beta-lactams except aztreonam (Mairi et al. 2018). The spread of metallo-beta-lactamases (MBLs), such as NDM and VIM enzymes, poses significant health risks. Unfortunately, there are currently no approved beta-lactamase inhibitors specifically targeting MBLs. Avibactam, relebactam, and abrobactam are novel beta-lactamase inhibitors approved for use in combination with ceftazidime, imipenem, and meropenem, respectively. There are many more in development. Avibactam effectively inhibits class A (including ESBLs and most KPCs), class C (AmpC), and certain class D (e.g., OXA-48) enzymes. Relebactam and vaborbactam target classes A and C enzymes; they do not exhibit activity against MBLs, including NDM and VIM variants. Consequently, treatment options remain severely limited for infections caused by MBL-producing organisms (Bush & Bradford 2019).

OXA-48 and its variants are carbapenemases that have high-level hydrolytic activity toward penicillins and low-level hydrolytic activity toward carbapenems but no intrinsic activity against expanded-spectrum cephalosporins (Stewart et al. 2018). However, OXA-48-producing bacteria are commonly resistant to cephalosporins, likely by the production of ESBL and/or AmpC-type beta-lactamases; also, isolates are frequently co-resistant to other classes of antibiotics such as fluoroquinolones and aminoglycosides (Mairi et al. 2018; Stewart et al. 2018). In our previous studies performed on this collection of bacteria, 9 and 16% of Enterobacterales from municipal wastewater (Hasani et al. 2023), 23 and 3% from healthy carriers (Habibzadeh et al. 2022), and 19 and 3% from livestock and poultry slaughterhouse wastewater (Sardari et al. 2024) were positive for ESBL and/or AmpC-type beta-lactamase, respectively. Moreover, a significant portion of E. coli isolates investigated in this study were resistant to ciprofloxacin (Neyestani et al. 2023).

The data indicates that beyond the clinical specimens, CRE bacteria are widely distributed among healthy people and wastewater resources in Ardabil, Iran. This means that they can act as community and environment reservoirs for the evolution and spread of CRE bacteria. The occurrence of divergent clinically significant pathogenic and commensal CRE species harboring transmissible carbapenemase-encoding genes is of great public health concern.

The widespread distribution of CRE across diverse ecological habitats, as highlighted in this study, underscores the critical need for collaborative actions within the One Health framework. These efforts are essential to effectively control the dissemination of CRE bacteria within the interconnected human–animal–environment interface. The One Health approach offers a comprehensive framework that connects integrated surveillance systems for identifying reservoirs, promotes antibiotic stewardship to ensure rational antibiotic use and reduce selection pressure, and emphasizes environmental control through proper waste management and water treatment. Additionally, it prioritizes healthcare provider education and public awareness to encourage prudent antibiotic usage (Velazquez-Meza et al. 2022). By adopting these strategies, we can effectively combat CRE and ensure the protection of public health.

The authors would like to express their gratitude to Dr Sohrab Iranpour from Ardabil University of Medical Sciences, Iran, for his assistance with statistical analysis.

S.H.: Methodology, Investigation, Formal analysis, and Original draft preparation. N.H., K.H., and M.S.: isolate collection and identification. M.A.: Supervision, Project administration, critically reading and revising the manuscript.

The present study was financially supported by the Food and Drug Laboratories Research Center (FDLRC), Iran Food and Drug Administration (IFDA), Tehran, Iran.

The regional ethics committee of the Ardabil University of Medical Sciences approved all experimental protocols in this study under the reference ‘IR.ARUMS.REC.1399.450’. All methods were conducted under relevant guidelines and regulations. Informed consent was obtained from the parents or legal guardians of healthy children to collect samples from them. Permissions were obtained from Ardabil Water and Wastewater Company for municipal wastewater and the Ardabil Provincial Veterinary Organization office for poultry and livestock slaughterhouse wastewater collection.

All relevant data are included in the paper.

The authors declare there is no conflict.