Citrobacter spp. and Enterobacter spp. as reservoirs of carbapenemase bla_NDM and bla_KPC resistance genes in hospital wastewater

ABSTRACT

Antibiotic resistance has emerged as a global threat to public health, generating a growing interest in investigating the presence of antibiotic-resistant bacteria in environments influenced by anthropogenic activities. Wastewater treatment plants in hospital serve as significant reservoirs of antimicrobial-resistant bacteria, where a favorable environment is established, promoting the proliferation and transfer of resistance genes among different bacterial species. In our study, we isolated a total of 243 strains from 5 hospital wastewater sites in Mexico, belonging to 21 distinct Gram-negative bacterial species. The presence of β-lactamase was detected in 46.9% (114/243) of the isolates, which belonging to the Enterobacteriaceae family. We identified a total of 169 β-lactamase genes; bla_TEM in 33.1%, bla_CTX-M in 25.4%, bla_KPC in 25.4%, bla_NDM 8.8%, bla_SHV in 5.3%, and bla_OXA-48 in 1.1% distributed in 12 different bacteria species. Among the 114 of the isolates, 50.8% were found to harbor at least one carbapenemase and were discharged into the environment. The carbapenemase bla_KPC was found in six Citrobacter spp. and E. coli, while bla_NDM was detected in two distinct Enterobacter spp. and E. coli. Notably, bla_NDM-1 was identified in a 110 Kb IncFII conjugative plasmid in E. cloacae, E. xiangfangensis, and E. coli within the same hospital wastewater. In conclusion, hospital wastewater showed the presence of Enterobacteriaceae carrying a high frequency of carbapenemase bla_KPC and bla_NDM. We propose that hospital wastewater serves as reservoirs for resistance mechanism within bacterial communities and creates an optimal environment for the exchange of this resistance mechanism among different bacterial strains.

IMPORTANCE

The significance of this study lies in its findings regarding the prevalence and diversity of antibiotic-resistant bacteria and genes identified in hospital wastewater in Mexico. The research underscores the urgent need for enhanced surveillance and prevention strategies to tackle the escalating challenge of antibiotic resistance, particularly evident through the elevated frequencies of carbapenemase genes such as bla_KPC and bla_NDM within the Enterobacteriaceae family. Moreover, the identification of these resistance genes on conjugative plasmids highlights the potential for widespread transmission via horizontal gene transfer. Understanding the mechanisms of antibiotic resistance in hospital wastewater is crucial for developing targeted interventions aimed at reducing transmission, thereby safeguarding public health and preserving the efficacy of antimicrobial therapies.

INTRODUCTION

Antibiotic resistance is a pressing global public health concern, with the potential to become the main cause of deceases in the near future (1). This threat increases due to the extensive use of antibiotics in healthcare settings, where a significant portion (30%–90%) of these antimicrobials are eliminated by patients and subsequently released into environment. The continuous discharge of the antibiotics into the environment, often in sub-lethal concentrations within hospital effluent, has the potential to induce and sustain antibiotic resistance in bacteria (2, 3). The World Health Organization (WHO) has considered carbapenem-resistant and extended-spectrum β-lactamases (ESBL)-producing Gram-negative bacteria as a critical priority for study (1). There is a growing interest in exploring the presence of these bacteria in the environment, especially in relation to human activities. Hospital wastewater is considered a significant reservoir of antibiotic-resistant bacteria and functions as an interface between humans and environmental ecosystems (2, 4–6). Within this environment, bacteria communities interact with themselves and have the potential to acquire, modify, and spread resistant genes. This transmission can occur through different mechanism, most notably through horizontal gene transfer facilitated by plasmids (7). Before wastewater is discharged into the aquatic environment, it undergoes treatment in wastewater treatment plants (WWTPs) to eliminate contaminants and microorganisms. Chlorine is commonly used in the disinfection process, following WHO guidelines and regional regulations. However, despite these measures, treatment failures can result in the presence of antibiotic-resistant bacteria (8). To address this issue, surveillance studies have focused on analyzing the presence of antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARG) in WWTPs. These studies aim to assess the impact of wastewater discharge on water quality and predict the emergence of antimicrobial resistance (9, 10). In the context of severe infections, carbapenems are often the last line of defense. However, the presence of carbapenemases, like bla_NDM, bla_KPC and bla_OXA-48, enzymes capable of hydrolyzing carbapenem, is frequently related to resistance penicillins, monobactams, and cephalosporins. These enzymes have the capacity to spread rapidly between different bacterial species by plasmids (11). Recently, studies have identified the presence of Enterobacteriacea carrying carbapenem resistance on all continents. Hospitals, in particular, have the highest concentrations of these resistant bacteria, which are often disseminated into the environment through hospital wastewater discharges (5, 12–17). In the context of this global issue, our study reports the presence of Enterobacteriaceae with high frequency of bla_KPC and bla_NDM genes and the co-production of bla_NDM and bla_OXA-48 obtained from wastewater samples collected from five hospitals in Mexico during 2020.

RESULTS AND DISCUSSION

Bacterial and β-lactamases identification

In the present study, 243 Gram-negative isolates belonging to 21 different bacterial species were identified. The isolates consisted of 32.9% (80/243) E. coli, 24.6% (60/243) Enterobacter spp., and 14.4% (35/243) Citrobacter spp., making up a total of 71.9% (Table S1). The distribution of isolates by states was as follows: Baja California had the highest number of isolates with 75, followed by Jalisco with 64 isolates, Quintana Roo with 46 isolates, Tamaulipas with 36 isolates, and Morelos with 22 isolates (Table S1).

The presence of β-lactamase was detected in 46.9% (114/243) of the isolates belonging to 12 species, all of which included at least one β-lactamase gene (Table 1). A total of 169 β-lactamase genes were detected among the 114 isolates. Specifically, 84 genes were found in Baja California, 37 in Jalisco, 31 in Tamaulipas, and 17 in Morelos (Table 1). The β-lactamases identified were bla_TEM 33.1% (56/169), bla_CTX-M 25.4% (43/169), bla_KPC 25.4% (43/169), bla_NDM 8.8%(15/169), bla_SHV 5.3% (9/169), and bla_OXA-48 1.1% (2/169). None of the screened β-lactamases

TABLE 1.

Beta-lactamase genes identified in the different species from hospital wastewater^a

	Baja California						Jalisco					Morelos					Tamaulipas
	TEM	SHV	CTXM	KPC	NDM	OXA-48	OKP	TEM	CTXM	KPC	NDM	TEM	SHV	CTXM	KPC	NDM	TEM	SHV	CTXM	KPC	NDM
Species of bacteria (n = 115)	β-Lactamase			CPMase			β-Lactamase			CPMase		β-Lactamase			CPMase		β-Lactamase			CPMase
Citrobacter amalonaticus (5)	4	1		3
Citrobacter braakii (7)	6			5
Citrobacter farmeri (2)	2			2
Citrobacter freundii (4)	3			3													1		1	1	1
Citrobacter gillenii (2)	1			2
Citrobacter murliniae (2)	2			2
Enterobacter bugandensis (7)														6	2
Enterobacter cloacae (17)			9		7	1						3		4	1	0	1		4
Enterobacter kobei (2)			1									1				0
Enterobacter xiangfangensis (7)			4		4	1											2		3
Escherichia coli (53)	8	2	3	5	1			12	5	17	2						6		3
Klebsiella pneumoniae (7)		2					1										4	4
	26	5	17	22	12	2	1	12	5	17	2	4	0	10	3	0	14	4	11	1	1
	48			36			18			19		14			3		29			2
Total β-lactamases	84						37					17					31

^a Among the 46 isolates obtained from Quintana Roo, none were found to have β-lactamases. CPMase, carbapenemase.

were identified in the 46 isolates from Quintana Roo.

From the total of 169 β-lactamases, 35.5% (60/169) were identified as carbapenemases among 58 of the isolates. These isolates were released into the environment by the wastewater of the hospitals and correspond to 50.8% (58/114) of the total isolates producing β-lactamases. The regional distribution of the 60 carbapenemases identified from the total of β-lactamases was as follow: 21.3% (36/169) obtained from Baja California, 11.2% (19/169) from Jalisco, 1.7% (3/169) from Morelos and 1.1% (2/169) from Tamaulipas, comprising 35.5% (60/169) of the total β-lactamases identified (Table 1). The 60 carbapenemases identified among the 58 isolates were as follows: 71.6% (43/60) of bla_KPC was identified in 43 isolates, the 25% (13/60) of bla_NDM was identified in 13 isolates, and the 3.3% (2/60) of co-producing of bla_NDM and bla_OXA-48 were found in two isolates, making a total of 60 carbapenemases identified among 58 isolate (Table 2).

TABLE 2.

Beta-lactamase genotype and frequency by species

Genotype profile	Frequency % (n)	Species (n)
bla KPC	19.2% (22)	E. coli (n = 20)
		Citrobacter spp. (n = 2)
bla TEM		E. coli (n = 18)
	19.2% (22)	Citrobacter spp. (n = 3)
		Enterobacter spp. (n = 1)
blaTEM + blaKPC	14% (16)	Citrobacter spp. (n = 15) E. coli (n = 1)
bla CTX-M-1-group	13.1% (15)	Enterobacter spp. (n = 11)
		E. coli (n = 4)
blaTEM + blaCTX-M‐1‐group	9.6% (11)	Enterobacter spp. (n = 6) E. coli (n = 5)
blaCTX-M-1-group + blaNDM	8.7% (10)	Enterobacter spp. (n = 9)
		E. coli (n = 1)
blaTEM + blaSHV	3.5% (4)	K. pneumoniae (n = 3)
		E.coli (n = 1)
bla SHV	2.6% (3)	K. pneumoniae (n = 2)
		Citrobacter spp. (n = 1)
blaCTX-M-1‐group + blaKPC	2.6% (3)	Enterobacter spp. (n = 3)
blaCTX-M-1‐group + blaNDM + blaOXA-48	1.7% (2)	Enterobacter spp. (n = 2)
blaTEM + blaCTX-M-1‐group + blaNDM	1.7% (2)	Citrobacter spp. (n = 1) E. coli (n = 1)
blaSHV + blaKPC	0.87% (1)	E. coli (n = 1)
bla NDM	0.87% (1)	E. coli (n = 1)
blaTEM + blaSHV + blaKPC	0.87% (1)	K. pneumoniae (n = 1)
bla OKP	0.87% (1)	K. pneumoniae (n = 1)
TOTAL	100%	n = 114

Genotypes and antimicrobial susceptibility

From the total of 169 beta-lactamases identified among the 114 isolates, the genotypes of the 60 carbapenemases identified in 58 of the isolates are described in descending order of frequently as follows: bla_KPC in 19.2% (22/114); bla_TEM + bla_KPC in 14% (16/114); bla_{CTX-M-1-group} + bla_NDM in 8.7% (10/114); bla_{CTX-M-1-group} + bla_KPC in 2.6% (3/114); bla_{CTX-M-1-group} + bla_NDM + bla_OXA-48 in 1.7% (2/114); bla_TEM + bla_{CTX-M-1-group} + bla_NDM in 1.7% (2/114); bla_SHV + bla_KPC in 0.87% (1/114); bla_NDM in 0.87% (1/114) and bla_TEM +bla_SHV + bla_KPC in 0.87% (1/114). The remaining genotypes comprise 109 β-lactamases among 56 of the total of 114 isolates (Table 2).

The resistance profiles analysis was conducted on the main genotypes identified which accounted for 79.8% (91/114) of the isolates (Table 3). Among these, seven isolates carrying bla_SHV (include bla_SHV and bla_SHV + bla_TEM genotype) exhibited a 28% and 57% of resistance against levofloxacin and ceftazidime, respectively. The SHV genotype is typically associated with resistance to beta-lactam antibiotics, including penicillins, monobactams, and, on a lesser extent to, cephalosporins.

TABLE 3.

Antibiotic resistance profile of the main genotypes identified of bacteria isolated from hospital wastewater^a

Genotype	No. of isolates	Percentage (%) of resistance isolates
		AMK	LVX	CAZ	FEP	IMI	MEM
bla SHV	7	0	28	57	0	0	0
bla CTX-M‐1‐group	26	0	42	88	77	0	0
bla NDM	1	100	100	100	100	100	100
bla KPC	38	73	44	81	92	100	100
blaSHV + blaKPC	2	50	50	100	50	100	50
blaCTX-M‐1‐group + blaNDM	12	100	33	100	100	100	100
blaCTX-M-1‐group + blaKPC	3	100	100	100	100	100	100
blaCTX-M-1‐group + blaNDM-1 + blaOXA-48	2	100	100	100	100	100	100

^a AMK, amikacin; LEV, levofloxacin; CAZ, ceftazidime; FEP, cefepime; IMI, imipenem; MEM, meropenem.

A gradual increase in resistance profiles was observed among the 26 isolates with the bla_{CTX-M-1-group} (include both bla_CTX-M-1 and bla_CTX-M-1 + bla_TEM genotype), with 42%, 88%, and 77% resistance against levofloxacin, ceftazidime, and cefepime, respectively. Isolates producing the CTX-M beta-lactamase typically exhibit resistance to extended-spectrum cephalosporins, such as cefotaxime, ceftazidime, and ceftriaxone, as observed in ours results.

The following resistance profiles correspond to six genotypes associated with carbapenemases. Among the 13 isolates with bla_NDM (include both bla_NDM and bla_CTX-M-1 + bla_NDM genotypes), 100% resistance was observed against amikacin, levofloxacin, ceftazidime, cefepime, imipenem, and meropenem. Similarly, the two isolates co-producing bla_NDM and bla_OXA-48 exhibited a similar resistance profile (Table 3).

Among the three isolates with (bla_{CTX-M-1-group} + bla_KPC) genotype, 100% resistance against amikacin, levofloxacin ceftazidime, cefepime, imipenem, and meropenem was noted. However, the 40 isolates with bla_KPC and bla_SHV + bla_KPC genotypes (where bla_KPC includes both bla_KPC and bla_TEM + bla_KPC genotype) generally showed 100% resistance against imipenem and meropenem. Nonetheless, resistance against amikacin, levofloxacin, ceftazidime, and cefepime exceeded 50%, in general, although the resistance profile differed between both genotypes (Table 3).

The carbapenem susceptibility results were consistent with the identification of carbapenemase-producing bla_KPC and bla_NDM. Our analysis of resistance profiles revealed that isolates carrying bla_KPC showed 100% resistance to imipenem and meropenem, while those carrying bla_NDM showed 100% resistance to third- and fourth-generation cephalosporins, imipenem and meropenem.

NDM is particularly concerning because it is often found on mobile genetic elements, such as plasmids, which can carry other resistance genes that co-transfer easily between bacteria, spreading the resistance gene among bacterial populations. This horizontal transfer of resistance genes contributes to the dissemination of multidrug-resistant bacteria, making infections caused by NDM-producing bacteria challenging to treat and control.

Carbapenemase-producing isolates

Carbapenemase-producing isolates were identified in various bacterial species from the same hospital wastewater in Baja California. Among these isolates, the prevalence of bla_NDM and bla_KPC genes varied significantly across different species. Notably, 91% (11/12) of isolates carrying bla_NDM were identified in E. cloacae and E. xiangfangensis, with one isolate found in E. coli. Conversely, 77% (17/22) of isolates carrying bla_KPC were identified in various species of Citrobacter: C. amalonaticus, C. braakii, C. farmer, C. freundii, C. gillenii, C. murliniae, while 22% (5/22) were found in E. coli. Additionally, co-occurrence of bla_TEM with bla_KPC, while bla_CTX-M with bla_NDM was within the same isolates, with a distinct prevalence pattern; bla_TEM + bla_KPC predominated in Citrobacter spp., whereas _CTX-M + bla_NDM prevailed in Enterobacter spp. isolates (Table 2).

Further analysis categorized the bla_NDM -producing isolates from hospital wastewater into two groups based on their collection dates. The first group, comprising E. cloacae and E. xiangfangensis, was collected on 18 November 2020, while the second group, including E. cloacae, E. xiangfangensis, and E. coli, was collected on 01 December 2020.

Plasmid mating assays were conducted on the bla_NDM-producing isolates of E. cloacae and E. xiangfangensis from both dates to evaluate the horizontal transfer of bla_NDM. Interestingly, these isolates harbored the same 110 Kb conjugative plasmid carrying bla_NDM with the IncFII incompatibility group. This plasmid is frequently associated with horizontal dissemination among Enterobacteriaceae members. These findings suggest both intra- and inter-species dissemination of the bla_NDM plasmid between two species of the Enterobacter genus, all circulating within the same hospital wastewater.

Analysis of the 266 clinical cases reported within the Baja California hospital from January to December 2020 revealed a predominance of various bacterial species, including Acinetobacter baumannii (56.7%), Escherichia coli (13.1%), Pseudomonas aeruginosa (9.3%), Klebsiella pneumoniae (13%), Stenotrophomonas maltophilia (6.3%). Notably, clinical isolates belonging to Enterobacter spp. or Citrobacter spp. were not identified,despite their high prevalence in the hospital wastewater.

Resistance profiles of clinical isolates from the hospital demonstrated high rates of resistance to third- and fourth-generation cephalosporins in 74.8% (199/266), quinolone in 53.7% (143/266), and carbapenems in 19.5% (52/266) (18). Notably, carbapenem resistance from this hospital, it was predominantly observed in A. baumannii in 78.8% (41/52), P. aeruginosa in 11.5% (6/52), S. maltophilia 5.7% (3/52), E. coli in 1.9% (1/52) and Proteus mirabilis in 1.9% (1/52).

Hence, we hypothesize that plasmids carrying bla_NDM identified in isolates from hospital wastewater may originate from bacterial species frequently causing infections in patients within the hospital, such as A. baumannii, P. aeruginosa, S. maltophilia, E. coli, and Proteus mirabilis. Upon entering the hospital wastewater environment, these plasmids may transfer to other bacterial species, such as Citrobacter spp. or Enterobacter spp., commonly found in water and pipes. This transfer could confer an evolutionary advantage and potentially serve as reservoirs for maintaining antibiotic resistance genes. However, further studies are warranted to elucidate the origin, transfer mechanisms, and maintenance of these plasmids.

Conclusions

Citrobacter spp. and Enterobacter spp. are ubiquitous bacteria commonly found in various environments, including hospital wastewater, creating ideal conditions for surface colonization due to the watery environment, and acting as a reservoir for long-term circulation (19). Moreover, they are a source of genes that can confer antibiotic resistance, such as qnr, which confer quinolone resistance. This could provide an evolutionary advantage and increase resistance in different environmental conditions (20). Islam et al. (12) analyzed the dispersion of bla_NDM from hospital to the environment in Bangladesh. They reported that 71% of bla_NDM-carrying bacteria were obtained from a hospital, carrying more than one antibiotic resistance genes, and most were carried on self-transmissible plasmid, mainly in K. pneumoniae and E. coli isolates (12). Interestingly, they reported uncommon species carrying bla_NDM, such as Enterobacter spp. and C. freundii. This finding suggests that its spread to unconventional organism and, in turn, to the environment is greater than previously thought (12).

Hospital wastewater is a pool of a heterogeneous mix of bacteria that converge, and the exchange of resistance genes is favored. These factors might induce the development of new resistance genotypes and the emergence of superbugs. Water cycle is considered an important pathway that accelerate the development, proliferation, and propagation of the antimicrobial resistance (21–24). Moreover, it represents a human health risk through several routes, such as exposure in agriculture, consumption of contaminated food, and the exposure in natural environment (25). Since WWTP can be a containment barrier or encourage dispersion into the environment, the treatment of hospital WWTP is crucial for the protection of aquatic environment and public health. The use of molecular-based methods and epidemiological tools allow to identify ARB and ARG, serving as a useful tool to improve surveillance systems and water quality before being discharged to other environments. Moreover, improving the use of antibiotics in hospitals could have a positive impact on bacterial resistance genes found in hospital wastewater, thereby increasing the selection pressure imposed by human activities.

Our study provides valuable insights into the prevalence and dissemination of antibiotic resistance genes and resistant bacteria in hospital wastewater. By highlighting the role of Citrobacter spp. and Enterobacter spp., as potential reservoirs for antibiotic resistance genes, we underscore the importance of targeted interventions to curb the spread of resistance.

In conclusion, our findings emphasize the urgent need for comprehensive strategies to address antibiotic resistance in hospital wastewater. Our findings shed light on the diverse bacterial species carrying these resistance genes, emphasizing the potential for both inter-species and intra-species dissemination within hospital wastewater.

MATERIALS AND METHODS

Wastewater sampling

As part of a SARS-CoV-2 wastewater surveillance study conducted between September and December 2020, we collected 24 hour flow-adjusted composite wastewater samples using standardized sampling procedures (26). The samples were sourced from hospital wastewater treatment in five different states of Mexico. We obtained 25 raw wastewater samples from 5 hospitals designated for COVID-19 patients located in different states of Mexico: Guadalajara, Jalisco; Mexicali, Baja California; Reynosa, Tamaulipas; Cancún, Quintana Roo; and Cuernavaca, Morelos. The hospital personnel informed about the disinfection process, involving chlorination, applied to the hospital raw wastewater before the sampling point.

Specimen selection

Samples of 50 mL from the hospital wastewater were aseptically filtered through sterile membranes with a pore size of 0.45 µm in a laminar flow cabinet. The collected bacteria cells contained in the filters were eluted in 1 mL of Luria-Bertani (LB) medium mixed with 15% glycerol and stored at −70°C until analysis. From each 1 mL sample, 100 µL of collected bacteria was diluted to concentrations of 1:1,000 and 1:10,000 in a final volume of 1 mL saline solutions (NaCl 0.9%). From each diluted sample, 50 µL was used for seeding LB or MacConkey agar plates supplemented with ceftazidime or imipenem at concentration of 50 µg/mL and 4 µg/mL, respectively. The plates were incubated at 37°C for 18 h, and colonies resistance to the antibiotics were selected for bacterial identification using the Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS, Microflex LT system, Bruker Daltonics, Bremen, Germany) according to the manufacturer’s recommendations.

PCR amplification and DNA sequences

In order to identify the most frequent ESBL and Carbapenem-resistant genes, all the 243 Gram-negative isolates were screened by PCR technique for bla_TEM, bla_SHV, bla_CTX-M, bla_KPC, bla_VIM, bla_IMP, bla_NDM, bla_OXA-58, bla_OXA23-like, bla_OXA-24-like, and bla_OXA-48-like, using specific oligonucleotides as described previously (Table S2) (27–35). DNA extraction from the isolates was performed by heat shock. The PCR mixture used was MgCl₂ (2.5 mM), Buffer (1×), dNTP’s (0.25 mM), oligonucleotides (10 pmol), and Taq Pol (0.5 U) in a final volume of 50 µL. The reaction conditions consist of an initial denaturation of 2 minutes at 92°C, followed by 30 cycles of amplification according to the Tm of the oligonucleotides a final extension step of three minutes at 72°C. All PCR products were purified using a commercial kit from Roche (Roche, USA) and subsequently sequenced. The nucleotide sequences were translated into amino acid sequences using the Translate Tool (https://web.expasy.org/translate/) and then compared using BLASTp in the GenBank database (http://www.ncbi.nlm.nih.gov/).

Antimicrobial susceptibility testing

To determine susceptibility, we conducted broth micro-dilution to determine the minimal inhibitory concentration (MIC) of amikacin, levofloxacin, ceftazidime, cefepime, imipenem, and meropenem. The results of antimicrobial susceptibility testing were then interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (36).

Plasmid analysis

Mating assays for the horizontal transfer of bla_NDM genes were performed using Escherichia coli J53-2 (Met-, Pro-, Rif^r) as the recipient strain in solid-phase mating as described by Miller (37). Transconjugants were selected on LB agar supplemented with rifampin (100 µg/mL) and nalidixic acid (8 µg/mL), cefotaxime (10 µg/mL), or imipenem (1 µg/mL). All transconjugants were verified according to their auxotrophic requirements (Met- and Pro-), and plasmids were analyzed according to the method described by Kieser (38).

Plasmid profiles were obtained from all bla_NDM positive isolates according to the method described by Kieser using plasmids R6K (40 Kb), RP4 (54 Kb), R1 (92 Kb), pMG229 (205 Kb), and pUD21 (275 Kb) as molecular size markers (38).

Plasmid typing

Plasmids were tested to incompatibility groups by PCR replicon typing. Specific primers were used including HI1, HI2, I1γ, X, L/M, N, FIA, FIB, W, P, FIC, Y, FIIA, A/C, T, K, B/O, FrepB, FIIy, FIIk, and FIIs previously described (39, 40).

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aem.01165-24.

Table S1. aem.01165-24-s0001.docx.

Number and percentage of species identified.

Table S2. aem.01165-24-s0002.docx.

Primer information.

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