Composting manure in the poultry farm harbours multidrug-resistance Salmonella

ABSTRACT

Salmonella is one of the leading foodborne pathogens that cause gastroenteritis. It is imperative to monitor Salmonella contamination within poultry farms. This study focuses on the prevalence of Salmonella in composting manure at poultry farms a potential and persistent contamination sources. A batch of manure samples (n = 10) were collected from a local poultry farm, and half of the manure samples were detected to be Salmonella-positive. Serotyping results showed four serotypes among different isolated strains, including Typhimurium, Typhi, Newport, and Dublin. Isolates from manure generally demonstrated a high ciprofloxacin resistance and biofilm formation capacity. Multi-drug resistance was found in isolates from organic manure that had been fermented using high-temperature aerobic technology. Our study indicated that composting manure harboured Salmonella and might facilitate the transmission of Salmonella and the development of antimicrobial resistance. Proper composting strategies are needed to reduce the transmission of antimicrobial resistance and Salmonella.

GRAPHIC ABSTRACT

KEYWORDS:

• Salmonella
• composting manure
• antibiotic resistance
• biofilm

Introduction

Salmonella is one of the leading causes of foodborne gastroenteritis worldwide (Castro-Vargas et al., 2020). According to the report released by the US Food and Drug Administration, Salmonella contamination is responsible for 4 of the total 14 foodborne outbreaks that happened in the United States in 2022 (USDA, 2022). Currently, more than 2500 Salmonella enterica serotypes have been identified. Enteritidis and Typhimurium are among the most relevant serovars to foodborne contamination and diseases (Gantois et al., 2009; Rajan et al., 2017). A trend has been observed that serotypes, like Heidelberg, Javiana, Infantis, and Thompson, are emerging as dominant in the chicken production chain and infecting humans (Coburn et al., 2007).

Salmonella contamination is prevalent among poultry products; the most frequently implicated vehicles in outbreaks are chicken meat and shell eggs (Nair & Kollanoor Johny, 2019). Many Salmonella serovars can colonize the chicken’s digestive tract, with the paired blind sacs known as the ceca at the rear end of the tract showing the highest preference potential. Once infected, the pathogen can be excreted through the chicken manure without any noticeable clinical symptoms (Gutierrez et al., 2020).

For nearly a decade, the development of antibiotic resistance in Salmonella has been an increasing concern for the poultry industry worldwide. According to the research carried out by Parveen et al. (2007), 79.8% of Salmonella from poultry carcasses were antibiotic-resistant, while 53.4% were multi-drug-resistant (Parveen et al., 2007). Contamination of antibiotic-resistant Salmonella in the food chain endangers food safety and public health (Barreto et al., 2016). Antibiotic-resistant Salmonella poses a major public health risk since it restricts treatment options and makes illness control challenging (Pan et al., 2018).

At present, it has been widely believed that poultry farms harbour a high level of Salmonella, serving as a contamination source (Obe et al., 2023), but the specific pollution point remains to be explored. It is estimated that about 75% of antibiotics are not absorbed by animals but excreted as manure (Li et al., 2023). Under this circumstance, composting manures provided a selective environment for the development of antibiotic resistance (Gutierrez et al., 2020). However, relevant information, such as the existence of Salmonella in composting manure at poultry farms and associated antimicrobial profiles, is limited.

In this study, we aim to investigate the prevalence of Salmonella in the manure at a poultry farm and assess its antibiotic resistance and virulence at genomic levels. The findings will provide new insights into the sources of Salmonella contamination and contribute to the prevention of Salmonella transmission.

Materials and methods

Sample collection and interviews

Chicken manure samples were obtained from a chicken farm in Taihe, Jiangxi Province, China (sampling time: August 6 2023). Total 10 samples were collected at different phases, the collection proceeded as illustrated in Figure 1: first defecation manure from the hatchlings, manure from chickens in 40 days stage, 108 days stage, 110 days of first-laying stage, 185 days at the peak-laying period, 230 days at ceased-laying period, sick chickens, and organic manure after 30 days of fermentation. In addition, chickens fed terramycin and oxytetracycline were sampled for 6 consecutive days. Fresh manure was collected using sterile spatulas and stored in sterile sampling vials (Shengkesi Technology Ltd, China), the droppings were taken from the litter at random locations and brought to the lab on ice. The drug usage information, including type of drugs, frequency, and lasting periods, were recorded.

Figure 1. Sample acquisition time point.

Isolation and identification of Salmonella

The isolation and identification of Salmonella was conducted as previous with modification (Andoh et al., 2016). Twenty-five grams of each manure sample was individually put into sterile plastic lidded containers, and 225 mL of 0.1% buffered peptone water (BPW) (Oxoid Ltd, UK) was added. The samples were incubated for 18 h at 37°C for pre-enrichment culture. Then, 0.1 mL pre-enrichment culture was spread in triplicate onto the selective solid medium xylose-lysine-deoxycholate agar (XLD) (Oxoid Ltd, UK) and incubated at 37°C for 24 h. Salmonella-typhi colonies were chosen from the XLD plates (pink colour with or without black cores), and biochemical tests were used to confirm the presence of suspected colonies. The biochemical tests include dextrose fermentation test, lactose fermentation test, sucrose fermentation test, and triple sugar iron test (TSI). Standard methods were followed to conduct these tests (Islam et al., 2014). The characteristic of Salmonella spp. in the biochemical test is that they can ferment glucose to produce acid and gas or only acid, do not ferment lactose or sucrose, and most produce H₂S.

The identities of the isolates were verified by 16S ribosomal RNA (16S rRNA) amplification and sequencing analysis, 16S rRNA polymerase chain reaction (PCR) was used. The TouchTM Thermal Cycler (Biorad C1000) was used to perform the amplification. The 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 911 R (5’-GCCCCCGTCAATTCMTTTGA–3’) primers (T. Yang et al., 2024) were produced at Inqaba Biotechnical Industries Ltd. A 25 µL reaction mix including 12 µL of master mix (Thermo Scientific PCR Master Mix 2X), 1 µL of oligonucleotides, 4 µL of DNA template, and 7 µL of nuclease-free water was used for the PCR. Amplification was performed on a PCR system (MiniAmp, Thermo Fisher Scientific Co., Ltd., Waltham, MA, U.S.A.) for thermal profile consisted of an initial denaturation at 94°C for 15 s followed by 15 s at 55°C, and 30 s at 72°C for 35 cycles, and a final elongation at 72°C for 7 min. Salmonella Typhimurium SL1344 was the positive control in the PCR tests, whereas aspergillus flavus and water were the negative controls. The amplified product was sequenced using an automated DNA sequencer (SpectruMedix model SCE 2410). The FinchTV software version 1.4.0 (Geospiza Inc.) was utilized to clean the resultant sequences. The Nucleotide Basic Local Alignment Search Tool (BLAST) was then used to compare the sequences to the National Centre for Biotechnology Information Citation database (http://www.ncbi.nlm.nih.gov/BLAST). Sequences were deposited in the NCBI gene bank. Isolates were identified based on the highest percentage of similarity.

The serovars were identified using real-time PCR (RT-PCR). Twelve serovar-specific primer pairs (Table S1 in the online supplemental material) for detecting Salmonella serovars designed by S.-M. Yang et al. (2021) were used in this study. The reaction volume was contained 10 µL of Platinum SYBR Green qPCR Super Mix-UDG with ROX, 1 µL of each primer pair, 2 µL of template DNA, and 6 µL of DNase/RNase free water. Amplification was performed on a Real-Time PCR System (QuantStudio 3, Thermo Fisher Scientific Co., Ltd., Waltham, MA, U.S.A.) for 40 cycles of denaturation at 95°C for 2 min, followed by 95°C for 15 s and 60°C for 30 s. The conditions of the melting curve were as follows: 95°C for 15 s, 60°C for 1 min, and 95°C for 1 s.

Antibiotic resistance assessment of Salmonella using disc diffusion method

Disk diffusion was performed according to CLSI methodology. Zone diameters of ≥21 mm considered susceptible, 16–20 mm in diameter considered intermediate, and zone diameters of 15 mm or less considered resistant. Muller-Hinton agar (MHA) plates were covered with antibiotic discs spaced equilaterally apart, and the plates were cultured for 18 h at 37°C. Using a meter rule graded in millimetres and the zones of inhibition surrounding the antibiotics disc were measured after incubation. The inhibitory zone mean diameter was determined after the test was run three times in duplicate. The antibiotic sensitivity of Salmonella isolates was tested using disc diffusion method with five antibiotics, including tetracycline, neomycin, tylosin, ciprofloxacin, and gentamicin sulfate with a concentration of 10 µg/tablet. Among them, tetracycline, neomycin, and tylosin are the antibiotic used in the farm, while ciprofloxacin and gentamicin sulfate are the antibiotic commonly used in clinic.

Biofilm formation ability of Salmonella isolates

The 96-well polystyrene microtiter plate assay, as reported by Yin et al. (2018), was used to quantify the formation of biofilms. For 18 h, the isolated strains of 4 serotypes of Salmonella were cultured in fresh Luria-Bertani broth (LB) at 37°C. Overnight growth strains were washed by 0.1% of BPW three times, followed by adding new LB medium to get the final concentration of bacteria suspension of ∼3 log/CFU. The suspensions were inoculated into 96-well plates and cultured at 37°C for 72 h to form mature biofilm. The biofilms were quantified according to the procedure described by Hussain et al. (2019). In brief, the biofilm-covered well was carefully washed three times using 0.1% of BPW. Then, the biofilms were stained with 0.1% crystal violet for 30 min. The stained biofilm was washed three times with 0.1% of BPW and then filled with 3 mL of 70% ethanol for 45 min. The amount of biofilm biomass was quantified by recording absorbance at a wavelength of 595 nm using the SpectraMax i3 plate reader (Molecular Devices Korea, LLC). The average absorbance of six wells at OD 595 nm was used to calculate the quantity of biofilm.

Detection of virulence genes

Three virulence genes (rpoS, avrA, and cdtB) implicated in pathogenicity were molecularly characterized (Table 1). Following the manufacturer’s instructions, a Bacteria Genomic DNA kit (CWBIO, Beijing, China) was used to extract the genomic DNA from Salmonella isolates. In this investigation, the duplex polymerase-chain reaction (PCR) was performed. A 25 µL reaction mix including 12 µL of master mix (Thermo Scientific PCR Master Mix 2X), 1 µL of oligonucleotides, 4 µL of DNA template, and 7 µL of nuclease-free water was used for the PCR. The PCR process comprised 5 min of initial denaturation at 95°C, 40 cycles of denaturation at 95°C for 1 min, 1 min of primer annealing at various temperatures for distinct genes, 1 min of extension at 72°C, and a final 10 min of extension at 72°C. The PCR amplicon products were visualized on 1% agarose gel electrophoresis. 1.5 g of agarose was weighed into a conical flask containing 100 ml of 1×TBE gel buffer solution to make the agarose gel. By heating the agarose for 2 min, allowing it to cool (55°C), and then adding GoldView I Nuclear Staining Dyes (0.1 µL/mL; Solarbio, Beijing, China) to the gel and pouring it onto the plate, the agarose was completely dissolved. The gel was electrophoretic ally run at 120 V for 30 min, which were then observed using a Gel Photosystem P1-1002.

Table 1. Primer sequences for virulence genes in Salmonella spp.

Virulence factor	Gene	Primer sequences (5’-3’)	Annealing temperature (°C)	Amplicon size (bp)	References
Biofilm-related genes	rpoS	F-TTGAGTCAGAATACGCTGAAAGTT	55	993	Yin et al. (2018)
		R-TTACTCGCGGAACAGCG
Effector protein	avrA	F-AGCCTGGCGCTCGCCAAAAA	57	123	Lozano-Villegas et al. 2023
		R-GCGGTCTGCTTTATCGGACGGG
Toxins	cdtB	F-ACAACTGTCGCATCTCGCCCCGTCATT	57	268	Lozano-Villegas et al. (2023)
		R-CAATTTGCGTGGGTTCTGTAGGTGCGAGT

Statistical analysis

Each experiment was repeated twice, and the mean values for all indicators were derived from the independent triplicate experiments. One-way ANOVA was used to determine mean differences, and a significant difference was defined as p < .05.

Results and discussion

Occurrence of presumptive Salmonella

Presumptive Salmonella strains were screened based on the morphological and biochemical characteristics. The pigment morphology of colonies ranged from pink to colourless with/without a black centre on XLD agar plate. All the isolates chosen were gram-negative with the ability to hydrolyze hydrogen peroxide to produce catalase enzyme. And isolates demonstrated acid generation and hydrogen sulphide formation, which is typical characteristics of Salmonella. The identification results are presented in Table 2.

Table 2. Identity of presumptive Salmonella isolates from purebred black hens farm in Taihe, China.

No. of Salmonella isolates	Source	Tracking of Salmonella in the samples	The highest percentage of similarity in the NCBI gene bank
–	First defecation manure (Meconium)	–	–
–	Manure from chickens in 40 days stage	–	–
1	Manure from chickens in 108 days stage	+	Salmonella Typhimurium
–	Manure from chickens in 110 days of first-laying stage	–	–
–	Manure from 185 days at the peak-laying period	–	–
–	Manure from 230 days at ceased-laying period	–	–
2	Organic manure after 30 days of fermentation	+	Salmonella Newport
3			Salmonella Typhimurium
4			Salmonella Dubin
5	Manure from sick chickens	+	Salmonella Dubin
6	Manure from the Chickens that fed terramycin	+	Salmonella Typhimurium
7	Manure from the Chickens that fed oxytetracycline	+	Salmonella Typhi

The biochemical traits found in this study corroborate the earlier finding reported by Hendriksen et al. (2016). However, it is possible that biochemical traits alone will not be sufficient to distinguish a microbial community, which calls for the application of more dependable methods like molecular techniques (Shan et al., 2012). As results indicated in this study, the PCR discrimination approach outperformed the use of biochemical and morphological criteria in the discrimination of Salmonella spp, and may offer a quick, accurate, and trustworthy way to identify pathogens.

As can be seen from Table 2, a total of seven Salmonella strains including four serotypes were isolated from five manure samples. Half of manure samples were detected as Salmonella positive. Serotyping Salmonella isolates include Typhimurium, Typhi, Newport, and Dubin. Notably, three serotypes of Salmonella were detected from the organic manure, one possible explanation is that the organic manure is collected from each henhouse to ferment, which may be the cause of the multiplicity of Salmonella in the organic manure. Meconium is collected immediately after the chicks hatch and is theoretically sterile, therefore no microbes can be detected. During the normal growth phase of chickens (day 0 to day 230), almost no Salmonella is detected in the manure samples, except at the 180-day stage. The 180-day chicken at the peak-laying period, so that extra attention should be paid since that Salmonella detected in the manure is likely to cause egg contamination, which in turn poses a risk to consumers.

The subspecies of all the isolates were Salmonella enterica, which is consistent with report carried by Akinola et al. (2019), they found that Salmonella enterica subsp. enterica is the most prevalent subspecies in the chickens.

Assessment of antibiotic resistance of Salmonella isolates

The antibiotic sensitivity profile of Salmonella isolates from chicken is shown in Table 3. Almost 71% (5/7) of all Salmonella isolates were resistant to tylosin treatment, and 2 isolated strains showed resistance to a commonly used clinical antibiotic (ciprofloxacin). Strains No.3 isolated from organic manure showed resistance to three antibiotics (neomycin, tylosi, and ciprofloxacin). Meanwhile, strain No. 2, isolated from organic manure, was sensitive to all five antibiotics in the study. The only one tetracycline-resistant Salmonella strain among the seven isolates was also derived from organic manure (strain No. 4), this indicates the complexity of drug resistance of the Salmonella isolates of organic manure, different strains evolved different resistance characteristic during the manure fermentation.

Table 3. Antibiotic and multi-drug resistance of Salmonella isolates from purebred black hens.

Strains No.	Source	Antibiotics
		Tetracycline	Neomycin	Tylosin	Ciprofloxacin	Gentamicin sulfate
1	Manure from chickens in 108 days stage	3 mm	2 mm	–	–	9 mm
2	Organic manure after 30 days of fermentation	15 mm	4 mm	9 mm	21 mm	15 mm
3	Organic manure after 30 days of fermentation	5 mm	–	–	–	17 mm
4	Organic manure after 30 days of fermentation	–	–	33 mm	19 mm	13 mm
5	Manure from sick chickens	2 mm	5 mm	–	4 mm	15 mm
6	Manure from the Chickens that fed terramycin	3 mm	5 mm	–	21 mm	11 mm
7	Manure from the Chickens that fed oxytetracycline	5 mm	5 mm	–	25 mm	14 mm

Ciprofloxacin is a member of the class of fluoroquinolone-combined broad-spectrum antibiotic that are frequently used to treat illnesses brought on by gram-negative pathogenic enteric bacteria (Chang et al., 2021). Gentamycin is aminoglycosides, in this investigation, gentamycin proved to be more successful in suppressing Salmonella, this is consistent with Akinola et al.‘s (2019) findings. There were no multi-drug resistance Salmonella strains been detected from the chicken manure that fed antibiotics (No.6 and No.7). According to the interviews with the management of the chicken farm, no antibiotics and drugs are added to the feed and drinking water, and the chickens are only fed medicine for 3–5 days when they are sick. This suggests that bacteria may not evolve multidrug-resistance during a short period of antibiotic administration, whereas multidrug-resistant bacteria can emerge after the organic manure fermentation process. It is reported that aside from antibiotic use, environmental stress such as cold, acid, and disinfectants can also promote resistance to antibiotics in pathogenic bacteria (Liao et al., 2020; Wu et al., 2023). For most of the known pollutants in chicken organic manure, no guidelines have been established especially for it. Kyakuwaire et al. (2019) investigated the theory that, because it contains multi-antibiotics resistant pathogens, chicken manure does not currently meet the requirements to be applied as organic manure (Kyakuwaire et al., 2019). Poultry organic manure is commonly used as crop fertilizer, it may be a key transmission chain for antibiotic-resistant pathogens of avian origin (Kyakuwaire et al., 2019).

Biofilm formation by Salmonella isolates

Salmonella exhibits a variety of environmental adaptations, including the capacity to form biofilms and stick to surfaces (Steenackers et al., 2012). The creation of biofilms is a serious problem, as if they stick on surfaces, bacteria may become resistant to disinfectants and antimicrobials, which would provide a supply of microbes. There were significant differences (p < .05) in the capacity to generate biofilms across the various Salmonella isolates (Figure 2). An interesting finding was that Salmonella isolated from the manure of sick chickens showed the highest biofilm forming properties compared to the isolates from other sources.

Figure 2. Biofilm formation ability of Salmonella isolates from different source. 1: Manure from chickens in 108 days stage; 2, 3, and 4: organic manure after 30 days of fermentation; 5: manure from sick chickens; 6: manure from the chickens that fed terramycin; 7: manure from the chickens that fed oxytetracycline.

As the results shown in Figure 2, all the Salmonella isolates in this study can form biofilms. The U.S.A. National Institutes of Health posted the finding that biofilms are linked to over 80% of all infections (Steenackers et al., 2012). The earliest observation of foodborne bacterial biofilm that was documented and published concerned Salmonella adherence to food surfaces (Giaouris et al., 2012). Research has revealed that elements of bacterial cell surfaces, including cellulose, flagella, and fimbriae, play a crucial role in Salmonella‘s ability to adhere to various surfaces. Salmonella may be able to survive in harsh environments, such chicken farms and slaughterhouses, in large part to biofilms. In addition to the food that is generated, these bacteria can develop biofilms on contact surfaces such stainless steel, aluminum, nylon, rubber, plastic, polystyrene, and glass, as well as on walls, floors, pipes, and drains in the processing sections of chicken farms (Guéneau et al., 2022).

Detection of virulence genes in Salmonella isolates

According to PCR analysis, Salmonella isolated from organic manure contained virulence genes rpoS and avrA (Table 4). The resistance of Salmonella to a variety of environmental stressors is regulated by rpoS gene (Gibbons et al., 2022; Roy et al., 2023). avrA gene, an effector protein gene, was detected in 97.4% of poultry isolates and 100% of human isolates (Lozano-Villegas et al., 2023). During bacterial infections, the avrA protein is essential for controlling epithelial apoptosis, promoting proliferation, and suppressing inflammation (Jones et al., 2008). Only one Salmonella isolate had been detected rpoS gene, while no isolates had the cdtB genes (data do not show). The avrA gene was detected in three isolates, which were three different serotypes (Typhi, Newport, and Typhimurium). The study carried by Abdelhamid and Yousef (2020) showed that rpoS might play a role in their heat resistance in Salmonella Tennessee and Eimsbuettel, which have adapted to desiccation (Abdelhamid & Yousef, 2020). Only a small number of virulence genes were identified in this study, which may not fully reflect the strain’s pathogenicity, and more phenotypic studies on virulence of Salmonella isolates are required.

Table 4. Detection of virulence genes in different Salmonella isolates.

Strains No.	Source	Virulence genes
		rpoS	avrA
1	Manure from chickens in 108 days stage	–	+
2	Organic manure after 30 days of fermentation	–	+
3	Organic manure after 30 days of fermentation	+	+
4	Organic manure after 30 days of fermentation	–	–
5	Manure from sick chickens	–	–
6	Manure from the Chickens that fed terramycin	–	–
7	Manure from the Chickens that fed oxytetracycline	–	–

Conclusion

In this study, seven Salmonella strains were successfully isolated and identified from five manure samples. It was found that the detection rate of Salmonella was low in the fresh manure collected from 0 to 230 days growth stage. However, multiple serotypes and antibiotic resistant Salmonella were detected in the organic fermented manure. The organic manure isolates showed multidrug-resistance and had been detected in virulence genes. Therefore, it is proposed that human needs to be made more aware of the spread of drug-resistant Salmonella in organic manure and suggesting livestock and poultry plant manager that it is necessity to follow recommended guidelines when it comes to using antibiotics. Further studies on the degradation of antibiotics and killing of pathogenic bacteria in organic manure could reduce the evolution of drug-resistance of pathogenic bacteria and hinder the transmission of pathogenic bacteria from livestock to humans.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19476337.2024.2332405.

Additional information

Funding

This research was supported by Taihe Silky Fowl Industrial Technology Joint Research and Application program [518001-I2210H/006] funded by Taihe county government, and this research was also supported by Zhejiang University undergraduate student research training program [Y202304160].