Molecular characterization and prevalence of β-lactamase-producing Enterobacterales in livestock and poultry slaughterhouses wastewater in Iran

Enterobacterales constitute the main group of Gram-negative bacteria (GNB) that cause several different nosocomial and community-acquired diseases (González 2022). The bacteria included in the Enterobacterales cause severe infections, such as pneumonia, bloodstream infections, wound infections, urinary tract infections, gastroenteritis, and meningitis (González 2022). The use of various antibiotics especially β-lactam agents is the mainstay treatment of these infections (Arzanlou et al. 2017). However, in recent years, the emergence of antibiotic resistance among Enterobacterales has complicated the treatment of infections all over the world (Lavakhamseh et al. 2016; De Angelis et al. 2020). It is demonstrated that overuse or misuse of different antibiotics in medical clinics and animal husbandry has led to the emergence and dissemination of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which can be transmitted in animals and humans (Nabovati et al. 2021; Rahman & Hollis 2023).

Given that various β-lactam agents, especially extended-spectrum cephalosporins and carbapenems are used in human and veterinary medicine; therefore, it is predictable that high-level β-lactam-resistant Enterobacterales have emerged (Arzanlou et al. 2017; Habibzadeh et al. 2022). β-lactam agents bind to the penicillin-binding proteins of the bacterial cell wall and inhibit the synthesis of peptidoglycan, which leads to the lysis of the bacteria cells (Arzanlou et al. 2017). The GNB belonging to the Enterobacterales become resistant against β-lactams through multiple mechanisms including the production of various β-lactamase enzymes such as plasmid-mediated AmpC (pAmpC), extended-spectrum β-lactamases (ESBLs), and metallo-β-lactamase (MBL) (Dantas Palmeira & Ferreira 2020). Based on the World Health Organization (WHO) reports, emerging ESBL-producing Enterobacterales (ESBL-PE) are considered the most serious and life-threatening threats of the 21st century (Tacconelli et al. 2018). AmpC β-lactamases are enzymes encoded on the chromosomes or plasmids and mediate resistance to cephalosporins, penicillins, β-lactamase inhibitor combinations, and cephamycins (Tamma et al. 2023). These two enzymes are the most important resistance mechanisms against β-lactams among Enterobacterales members that can hydrolyze various β-lactam antibiotics (Sheng et al. 2013). During the last 20 years, ESBL-PE and AmpC-producing Enterobacterales (AmpC-PE) have emerged in human health care and animals worldwide. Depending on geographical regions, the global prevalence of ESBL-PE isolates varies widely (2–70%) (Telling et al. 2020). It is revealed that the meat, digestive tract of domestic livestock and poultry, and the wastewater of slaughterhouses are important reservoirs of ESBL-/AmpC-PE isolates (Pormohammad et al. 2019; Gregova & Kmet 2020). These resources may lead to the dissemination of ABR Enterobacterales into the environment and serve as a possible source of human colonization (Wu et al. 2009). Therefore, surveillance for the presence of β-lactamase-producing Enterobacterales in the wastewater of livestock and poultry slaughterhouses is important. However, there is limited data on antibiotic resistance profiles of Enterobacterales in wastewater samples from livestock and poultry slaughterhouses wastewater in Iran.

This research aimed to achieve an understanding of the epidemiology and molecular characterization of ESBL-PE and AmpC-PE isolates from livestock and poultry slaughterhouse wastewater in Iran.

In the current study, a total of 12 raw sewage samples were collected in 500 mL sterile bottles from livestock and poultry slaughterhouses in Ardabil, Iran between June 2020 and June 2021. The samples were transported to the microbiology laboratory in ice-cold containers and kept at 4 °C until microbiological analysis. The analysis was carried out within 2 h after sample collection.

For isolation of Enterobacterales, 5 mL of raw sewage samples were cultured into 5 mL double concentration Enterobacterales enrichment (EE) broth (Merck, Germany). The cultures were incubated at 37 °C for 24 h, then 100 μL of each enriched EE-broth media was subcultured onto MacConkey agar (Merck, Germany) plates and additionally incubated at 37 °C overnight. Raised colonies were examined based on Gram staining and colonial characteristics. Three colonies with the same appearance per colony morphology in each sample were selected and isolated on nutrient agar (Merck, Germany) plates (Ojer-Usoz et al. 2014; Miyagi & Hirai 2019). Definitive identification of the genus and species of Enterobacterales members was conducted using conventional biochemical tests. Selected bacteria were isolated on Trypticase Soy Agar (Merck, Germany) and initially screened using three tests: the oxidase test, the oxidative/fermentative glucose test (OF medium), and the nitrate reduction test (Nitrate Broth). Bacteria identified as oxidase-negative, nitrate-positive, and glucose fermenters underwent further characterization to the species level using a battery of biochemical tests based on standard manuals (Hasani et al. 2023; Mahon et al. 2014). This battery included tests for carbohydrate fermentation (lactose fermentation, H₂S, and gas production on Triple Sugar Iron Agar), enzyme and metabolite production (indole and motility on Sulfide Indole Motility medium, urease on urease agar, citrate utilization on Simmons Citrate Agar, Voges-Proskauer and methyl red tests on MR/VP medium), amino acids (lysine, arginine, and ornithine) decarboxylase test, and additional tests like phenylalanine deaminase on Phenylalanine Agar. All differential culture media were purchased from Himedia Laboratories Pvt. Ltd, India. The validity of the biochemical tests was ensured by incorporating established reference strains: Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Enterobacter aerogenes ATCC 13049, Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 27853, and Proteus mirabilis ATCC 43071.

To prevent biases that may have been introduced by the enrichment process, duplicate isolates of each species and each sample showing the same phenotypic and genotypic antimicrobial resistance characteristics were excluded from the study. The isolates were stored in Tryptic Soy Broth (TSB; Merck, Germany) with 15% glycerol in the deep freezer (−70 °C) until use.

Antibiotic susceptibility of the isolates was determined by the Kirby–Bauer disk diffusion method (DDM) on Mueller Hinton agar (Merck, Germany). Ampicillin (AM,10 μg), cefotaxime (CTX, 30 μg), ceftazidime (CAZ, 30 μg), cefepime (FEP, 30 μg), imipenem (IMP, 10 μg), gentamicin (GM,10 μg), amikacin (AN, 30 μg), nitrofurantoin (FM, 300 μg), ciprofloxacin (CP, 5 μg), nalidixic acid (NA, 30 μg), cephalexin (CN, 30 μg), trimethoprim-sulfamethoxazole (SXT, 1.25 + 23.75 μg), and tetracycline (TET, 30 μg) were used. The antibiotic susceptibility testing was carried out and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) criteria (CLSI 2023). The E. coli ATCC 25922 was used as a quality control strain for DDM. The bacteria that were resistant to at least one antibiotic among at least three or more drug categories were considered multidrug-resistant (MDR).

Initially, all the Enterobacterales isolates were subjected to susceptibility testing using CTX (30 μg) and CAZ (30 μg) disks. The isolates with reduced inhibition zone diameter for CTX (≤27 mm) and/or CAZ (≤22 mm) were considered as suspected ESBL-producing isolates. For definitive identification of ESBL-producing isolates, double-disk synergy test (DDST) was used as described with CLSI (CLSI 2023). The CAZ and CAZ/clavulanic acid (30 μg/10 μg, PadtanTeb, Iran), CTX, and CTX/clavulanic acid (30 μg/10 μg, PadtanTeb, Iran) disks were placed on the cultured Muller–Hinton agar plate with 30 mm apart from each other and incubated at 37 °C overnight. Isolates were considered ESBL producers when the inhibition zone diameter produced by the combined effects of either CAZ- or CTX-plus clavulanic acid disks was at least 5 mm larger than that produced by either CAZ or CTX disks individually. K. pneumoniae ATCC 700603 and E. coli ATCC 25922 were used as the ESBL positive and negative controls, respectively.

The Enterobacterales isolates with the following characteristics in the DDM test were selected for phenotypic screening for AmpC β-lactamases production: (i) the isolates that were resistant to CAZ (30 μg) and/or CTX (30 μg), (ii) the isolates which were susceptible to CPM (30 μg), and (iii) the isolates showing zone of inhibition less than or equal to 18 mm to cefoxitin (30 μg). AmpC production was confirmed by disk potentiation test (DPT) using cefoxitin (30 μg) and cefoxitin/boronic acid (30 μg/400 μg) disks as described by others (Pitout et al. 2010). Isolates were considered AmpC β lactamase producers when the diameter of the inhibition zone produced by the combined effects of cefoxitin plus boronic acid was at least 5 mm larger than that produced by the cefoxitin disk alone.

Genomic DNA was extracted using the boiling method (Habibzadeh et al. 2022; Hasani et al. 2023), and extracted DNA was preserved at −80 °C until further use.

ESBL-encoding genes including bla_TEM, bla_SHV, bla_OXA-1, and bla_CTX-M (bla_CTX-M-1, bla_CTX-M-2, bla_CTX-M-3, bla_CTX-M-8, bla_CTX-M-9, bla_CTX-M-14, bla_CTX-M-15, bla_CTX-M-25) were detected in all ESBL-producing isolates using a conventional PCR assay. The sequence of all specific primers (Ruppé et al. 2009; Afzali et al. 2015) used for PCR reaction is shown in Table 1. The PCR reaction was performed in a final volume of 25 μL using 12.5 μL of Premix Taq^® mix (CinnaGen, Tehran, Iran), 1 μL (5 μg) of template DNA, l μL (10 pmol) of each forward and reverse primers, and 9.5 μL of nuclease-free water. Amplification was performed using a thermocycler (Bio-Rad, Malaysia) with the following conditions: one cycle of 95 °C for 5 min, followed by 35 cycles of 94 °C for 55 s, 52–60 °C (Table 1) for 1 min, and 72 °C for 55 s with a final extension at 72 °C for 8 min. ESBL variants of bla_TEM and bla_SHV genes were identified by sequencing and alignment of the PCR products (Microsynth Company, Switzerland). Sequences were aligned and analyzed using the BLAST program available at the National Center for Biotechnology Information (NCBI).

The frequency of AmpC β-lactamase encoding genes (bla_FOX, bla_MOX, bla_CIT, bla_DHA, bla_ACC, bla_EBC, and bla_CMY) among AmpC-PE isolates was determined by multiplex PCR method using specific primers (Table 1). DNA amplification was performed following a previously published protocol with some modifications (Pérez-Pérez & Hanson 2002). Briefly, 25 μL of Premix Taq^® mix (CinnaGen, Tehran, Iran) was combined with 1 μL (5 μg) of template DNA, 1 μL each of forward and reverse primers (10 pmol each), and nuclease-free water to reach a final volume of 50 μL. Amplification was carried out under the following conditions: initial denaturation at 94 °C for 3 min, followed by 25 cycles of denaturation at 94 °C for 30 s, annealing at 64 °C for 30 s, and extension at 72 °C for 1 min. A final extension step at 72 °C for 7 min was included. PCR products were separated on a 1.5% agarose gel, stained with DNA-safe stain (Sinaclon Co., Iran), and photographed under UV light using a gel documentation system (UVitec-England). Genomic DNA from ESBL-PE and AmpC-PE isolates from the authors' previous studies containing target genes were used as the positive control (Habibzadeh et al. 2022; Hasani et al. 2023) and Staphylococcus aureus ATCC 25212 was used as a negative control for PCR assays. Additionally, the 16 s rRNA gene amplification was used as a positive control in PCR testing to ensure that any amplification failure with the specific primers was not due to poor DNA quality or to failure of the PCR itself.

All data were included in the statistical package SPSS v.23.0 (SPSS Inc., Chicago, IL, USA), the difference in frequency of ESBL-PE and AmpC-PE isolates in livestock and poultry slaughterhouses wastewater was analyzed using the chi-squared test. A p-value of ≤0.05 was considered statistically significant.

A total of 80 non-duplicate Enterobacterales isolates including 37 (46.2%) and 43 (54%) were collected from livestock and poultry slaughterhouse wastewater, respectively. The frequency of Enterobacterales in livestock and poultry slaughterhouse wastewater samples is shown in Table 2. Overall, E. coli (n = 21/80; 26.2%), Citrobacter spp. (n = 18/80; 22.5%), Proteus mirabilis (P. mirabilis) (n = 13/80; 16.2%), Proteus vulgaris (P. vulgaris) (n = 8/80; 10%), K. pneumoniae (n = 7/80; 8.7%), Hafnia spp. (n = 5/80; 6.2%), Yersinia enterocolitica (Y. enterocolitica) (n = 5/80; 6.2%), Providencia spp. (n = 2/80; 2.5%), and Enterobacter spp. (n = 1/80; 1.2%) were the most prevalent Enterobacterales species found in wastewater samples, respectively. Among Enterobacterales isolated from livestock slaughterhouse wastewater samples, E. coli (n = 9/37; 24.3%) and Citrobacter spp. (n = 9/37; 24.3%) were the most frequent organisms identified. On the other hand, E. coli (n = 12/43; 27.9%) was the most frequently isolated pathogen in poultry slaughterhouse wastewater samples. Hafnia spp. was not isolated from poultry wastewater samples.

The frequency of ESBL-PE and AmpC-PE isolates is shown in Table 3. The results of the DDST test showed that 18.7% (n = 15/80) of isolates were ESBL producers. Among ESBL-PE, 80% (n = 12/15) of isolates were isolated from poultry slaughterhouse wastewater, and 20% (n = 3/15) of isolates were obtained from livestock slaughterhouse wastewater (p < 0.05). ESBL production was observed in E. coli (n = 9/21; 42.8%) and K. pneumoniae (n = 6/7; 85.7%) isolates.

The results of the DPT test showed that 2.5% (n = 2/80) of isolates were AmpC β-lactamase producers. All AmpC-PE isolates were obtained from poultry slaughterhouse wastewater samples. Moreover, AmpC production was restricted to E. coli (n = 2/21; 9.5%) isolates. No isolate produced ESBL- and AmpC-β-lactamase simultaneously.

In comparison, the resistance rate to CPM, CTX, CAZ, trimethoprim-sulfamethoxazole, and nitrofurantoin among isolates recovered from poultry slaughterhouse wastewater samples was significantly higher than the isolates recovered from livestock slaughterhouse wastewater samples (p ≤ 0.05).

Results showed that 100% ESBL- and AmpC-PE isolates were MDR. At the same time, 47.5% (n = 26/63) of non-ESBL-/non-AmpC-PE isolates were MDR (P ≤ 0.05).

The imipenem (n = 12/12; 100%), amikacin (n = 11/12; 92%), and gentamicin (n = 10/12; 83%) were the most effective antibiotics against ESBL-PE species isolated from poultry slaughterhouse wastewater samples. ESBL-PE isolates from livestock slaughterhouse wastewater samples had higher susceptibility rates to imipenem, TET, NA, and trimethoprim-sulfamethoxazole at 100% (n = 3/3), and to gentamicin and amikacin at 75% (n = 2/3) each. AmpC-PE isolates were more resistant than ESBL-PE isolates, but they were all susceptible to imipenem, CPM, gentamicin, and amikacin (Table 4).

A conventional PCR and multiplex PCR assay were used to investigate the frequency of encoding genes in ESBL-PE and AmpC-PE isolates, respectively. The results are shown in Table 5. Screening for ESBL-encoding genes revealed that 100% (n = 9/9) and 83% (n = 5/6) of ESBL-producing E. coli and K. pneumoniae isolates harbored bla_CTX-M-₁ gene, respectively. All of these isolates were positive for the bla_CTX-M-15 subgroup. ESBL-encoding bla_OXA-1 gene was identified in 16.7% (n = 1/6) of ESBL-producing K. pneumoniae isolates. 44% (n = 4/9) and 50% (n = 3/6) of ESBL-producing E. coli and K. pneumoniae isolates contained the bla_TEM-1 gene, respectively. However, the bla_TEM-1 gene does not code for an ESBL enzyme. Other ESBL-encoding genes (bla_CTX-M-2, bla_CTX-M-3, bla_CTX-M-8, bla_CTX-M-9, bla_CTX-M-14, bla_CTX-M-25, and bla_SHV) were not identified in this study.

AmpC β-lactamases genes, bla_FOX and bla_CIT, were detected among 100% (n = 2/2) of AmpC-producing E. coli isolates. Other AmpC β-lactamases genes (bla_MOX, bla_DHA, bla_ACC, bla_EBC, and bla_CMY genes were not detected in this study.

Over the last decades, antimicrobials have been frequently used in poultry and livestock, resulting in the emergence and development of ABR bacteria (Pormohammad et al. 2019). The wastewater of farm animals and poultry may act as a reservoir of ESBL-PE and AmpC-PE isolates and dissemination of these bacteria to environments leads to the colonization of humans (Gregova & Kmet 2020).

There are limited data about the frequency, antimicrobial resistance profile, and characterization of Enterobacterales in wastewater samples from livestock and poultry slaughterhouses in Iran. To our knowledge, the current study is the first comprehensive research on the occurrence and characterization of ESBL-PE and AmpC-PE in the wastewater of livestock and poultry slaughterhouses in Iran.

Overall, the results of the present study showed that E. coli and Citrobacter spp. were the most commonly isolated organisms in wastewater samples from both livestock and poultry slaughterhouses. This finding is in agreement with the findings of several studies conducted by Adelowo et al. (2020) from Germany, Ye et al. (2018) from China, Savin et al. (2020) from Germany, and Montso et al. (2019) from South Africa, which stated that the prevalence of E. coli isolates among wastewater samples is high. However, our findings are not consistent with those of a previously published study from Germany (Savin et al. 2021), which reported that the Acinetobacter calcoaceticus–baumannii complex is frequently isolated from wastewater from a poultry slaughterhouse.

In general, bacteria belonging to the Enterobacterales family can colonize the human and animal intestinal tract and are considered a part of normal microbiota in healthy humans and animals (Homeier-Bachmann et al. 2021). Consequently, the occurrence of these bacteria in livestock and poultry slaughterhouse waste is expected. Nevertheless, due to the extensive use of antibiotics in animal farming, various types of ARB are increasingly identified in livestock and poultry waste globally (He et al. 2020). The contamination of slaughterhouse wastewater with ABR bacteria represents a potential risk to human health (Gregova & Kmet 2020).

The phenotypic evaluation of the prevalence of β-lactamase-producing Enterobacterales revealed that 60 and 40% of ESBL-PE were E. coli and K. pneumoniae isolates, respectively. The frequency of ESBL-producing E. coli and K. pneumoniae isolates was 42.8 and 85.7%, respectively. These results are consistent with those of studies by Montso et al. (2019) in South Africa, Ye et al. (2018) in China, and Lim et al. (2015) in Korea. These previously published studies found that the frequency of ESBL-producing E. coli in the wastewater samples was 58.2, 41.8, and 39.7%, respectively. However, our findings were in contrast with those of a previous study from Slovakia, which reported that the prevalence of ESBL-producing E. coli isolates was 20.4% (Gregova & Kmet 2020).

In our study, a significant proportion of ESBL-PE isolates were obtained from poultry slaughterhouse wastewater. This observation suggests a potential link to the intensified use of antibiotics within the poultry industry in our region. Consequently, to prevent the development and spreading of ARB, adopting a One Health approach using suitable treatment methods, antibiotic resistance surveillance programs, and robust wastewater treatment processes are crucial needs in poultry farms.

Our analyses regarding the prevalence of beta-lactamase encoding genes among phenotypically resistant organisms showed that the bla_CTX-M-15 subgroup was prevalent among ESBL-producing K. pneumoniae and E. coli isolates (83 and 100%, respectively). Obtained results are in agreement with those of published studies by Gregova et al. (2021) from Slovakia, Franz et al. (2015) from the Netherlands, and Kaesbohrer et al. (2019) from Germany, which reported that the prevalence of bla_CTX-M gene among ESBL-producing K. pneumoniae and E. coli isolates was high. On the other hand, these findings are in contrast to other studies performed worldwide reporting high rates of ESBL-PE harbored other ESBL/AmpC-encoding genes such as bla_TEM, bla_SHV, bla_OXA, bla_EBC, bla_FOX, and bla_CIT (Khan et al. 2020; Savin et al. 2021). In the last decades, bla_TEM and bla_SHV genes were the most prevalent ESBL-encoding genes. However, in recent years, studies revealed that the bla_CTX-M gene has spread worldwide and is the most prevalent ESBL-encoding gene. In most cases, the bla_CTX-M gene is located on large plasmids. Organisms harboring the bla_CTX-M gene have been related to widespread resistance to different classes of antibiotics such as quinolones, aminoglycosides, trimethoprim/sulfamethoxazole, and TET (Abbassi et al. 2008; Sghaier et al. 2019). Among different bla_CTX-M subgroups, the CTX-M-15 subgroup has a high frequency among ESBL-producing E. coli. Globally, CTX-M-15 is one of the most prevalent ESBL genotypes in humans and is related to hospital-acquired infections or community-acquired urinary tract infections (Dolejska et al. 2011). It is presumed that organisms harboring the CTX-M-15 subgroup have different virulence factors and are more virulent than other bacteria (Hassen et al. 2020). The potential link between human and animal CTX-M-15 beta-lactamase underscores the importance of a holistic approach to antimicrobial stewardship, considering both human health and animal welfare.

In Iran, like many developed and developing countries, wastewater generated from livestock and poultry farms and their slaughterhouses, hospitals, and abattoirs do not get suitable treatment and resistant bacteria are present in effluents from wastewater treatment plants discharged to the nearby rivers and streams. The release of untreated wastewater from slaughterhouses and farms to the rivers that contain MDR pathogenic bacteria may lead to the environmental pool of resistant pathogenic bacteria and antimicrobial resistance genes. Moreover, untreated wastewater can act as a source of infection with ABR bacteria (Dolejska et al. 2011). On the other hand, it is presumed that Enterobacterales isolates may horizontally transmit the ABR genes to other Enterobacterales in the wastewater and contribute to the emergence and development of MDR bacteria (Kiros & Workineh 2019).

Our study showed the presence of ABR bacteria in the wastewater of poultry and livestock slaughterhouses in Ardabil, Iran. This contamination poses a significant risk of zoonotic transmission to humans through the consumption of contaminated food products and the spread of bacteria into the environment. Moreover, it is revealed that GNB isolated from wastewater carries different resistance genes and they show various resistance profiles to commonly prescribed antibiotics. Results revealed that among the Enterobacterales family members, the ESBL and AmpC β-lactamases are currently on the increase, especially among E. coli and K. pneumoniae isolates. Therefore, it can be concluded that sufficient sanitation infrastructure and ABR surveillance programs are critically required in livestock and poultry farms and slaughterhouses.

Ethical clearance and approval for the study were obtained from the Institutional Ethics Committee of the Ardabil University of Medical Sciences (IR. ARUMS.1398.369).

All authors have read and agreed to the published version of the manuscript.

M. S. developed the methodology, investigated the data, rendered support in formal analysis, and supported in original draft preparation. H. P. D. conceptualized the whole article, reviewed, and edited the article. M. M. investigated the data and rendered support in formal analysis. K. H. investigated the work. N. H. investigated the work. T. A. supported in original draft preparation. M. A. supervised the work, rendered support in project administration, and revised the manuscript.

The present study was financially supported by the vice-chancellor of research and technology, at Ardabil University of Medical Sciences, Ardabil, Iran.

The authors thank the vice-chancellor of research and technology of Ardabil University of Medical Sciences, Ardabil, Iran for financial support.

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

The authors declare there is no conflict.