**Characterization of Extended-Spectrum Cephalosporin (ESC) Resistance in** *Salmonella* **Isolated from Chicken and Identification of High Frequency Transfer of** *bla***CMY-2 Gene Harboring Plasmid In Vitro and In Vivo**

**Bo-Ram Kwon 1,†, Bai Wei 1,†, Se-Yeoun Cha 1, Ke Shang 1, Jun-Feng Zhang 1, Hyung-Kwan Jang 1,2,\* and Min Kang 1,2,\***


**Citation:** Kwon, B.-R.; Wei, B.; Cha, S.-Y.; Shang, K.; Zhang, J.-F.; Jang, H.-K.; Kang, M. Characterization of Extended-Spectrum Cephalosporin (ESC) Resistance in *Salmonella* Isolated from Chicken and Identification of High Frequency Transfer of *bla*CMY-2 Gene Harboring Plasmid In Vitro and In Vivo. *Animals* **2021**, *11*, 1778. https://doi.org/ 10.3390/ani11061778

Academic Editors: Bruno Tilocca and Paola Roncada

Received: 1 May 2021 Accepted: 10 June 2021 Published: 14 June 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Simple Summary:** The prevalence of extended-spectrum cephalosporin (ESC)-resistant *Salmonella* is of great concern, as these strains with the same *β*-lactamase (*bla*) genes were found in human and poultry. The objective is to characterize ESC-resistant *Salmonella* isolated from chicken and to determine the transferability of *β*-lactamase gene-harboring plasmid in vitro and in vivo. ESC resistance genes in *Salmonella* isolated from chickens and presented a comprehensive analysis of the highly frequent transfer of the *bla*CMY-2 gene in vitro and in vivo. In addition, this study has demonstrated the ease with which a *bla*CMY-2 gene-harboring plasmid can be rapidly transferred between *Salmonella* and pathogenic *E. coli* within the intestinal tracts of mice, even without antimicrobial selective pressure. Given the potential risk of the frequent transfer of the *bla*CMY-2 gene via the food chain to the human digestive tract, the molecular mechanism involved in the dissemination and maintenance of ESC resistance genes should be studied as further research in greater detail, and enhanced surveillance should be implemented to prevent the widespread of ESC resistant strains.

**Abstract:** A total of 136 *Salmonella* isolates from chicken feces and meat samples of the top 12 integrated chicken production companies throughout Korea were collected. Among the 17 ESC-resistant *Salmonella*; *bla*CTX-M-15 was the most prevalent gene and two strains carried *bla*TEM-1/*bla*CTX-M-15 and *bla*CMY-2, respectively. The transferable *bla*CTX-M-15 gene was carried by IncFII plasmid in three isolates and the *bla*CMY-2 gene carried by IncI1 plasmid in one isolate. *bla*CMY-2 gene-harboring strain was selected as the donor based on the high frequency of *bla*CMY-2 gene transfer in vitro and its transfer frequencies were determined at 10−<sup>3</sup> transconjugants per recipient. The transfer of *bla*CMY-2 gene-harboring plasmid derived from chicken isolate into a human pathogen; enteroinvasive *Escherichia coli* (EIEC), presented in mouse intestine with about 10−<sup>1</sup> transfer frequency without selective pressure. From the competition experiment; *bla*CMY-2 gene-harboring transconjugant showed variable fitness burden depends on the parent strains. Our study demonstrated direct evidence that the *bla*CMY-2 gene harboring *Salmonella* from chicken could frequently transfer its ESC-resistant gene to *E. coli* in a mouse intestine without antimicrobial pressure; resulting in the emergence of multidrug resistance in potentially virulent EIEC isolates of significance to human health; which can increase the risk of therapeutic inadequacy or failures.

**Keywords:** *Salmonella*; chicken; extended-spectrum cephalosporin; *bla*CMY-2; mouse; frequent transfer

#### **1. Introduction**

Recently, an increasing occurrence of extended-spectrum cephalosporins (ESC)-resistant strains has been recognized as a serious threat to human health [1]. Resistance to *β*-lactam antimicrobials is mainly caused by the production of antimicrobial inactivation enzymes called *β*-lactamases [2]. Extended-spectrum *β*-lactamases (ESBL) and AmpC *β*-lactamase (AmpC) are the major *β*-lactamases detected in ESC-resistant strains worldwide [3]. These enzymes are frequently encoded by genes that are located on a plasmid, which is a mobile genetic element that can transfer horizontally within and between different bacterial species [4]. Various studies have suggested that food-producing animals as a reservoir for ESBL/AmpC-producing strains that could promote the transmission of resistance determinants to humans [2]. Similar or identical ESC-resistant isolates or ESBL/AmpC plasmids were found in chicken meat and patients, suggesting poultry and poultry products play a pivotal role in the spread of ESC resistance genes to humans [2].

The fact that the same plasmid is observed in several bacterial strains isolated from poultry and humans confirms that antimicrobial resistance genes can be transferred from poultry to humans [2]. A previous study observed the possibility using in vitro human gut simulation model that there is a transfer from food-borne ESC resistant isolates to other commensal and pathogenic bacteria [5]. However, there is a lack of actual evidence that ESC resistance genes and particularly the *bla*CMY-2 gene transfer from poultry to human-origin pathogenic isolates in vivo could cause considerable risks, such as the high possibility of inadequate treatment or therapeutic failures. Antimicrobial resistance, by the acquisition of a mobile genetic element or by mutation, is generally thought to induce a competitive fitness disadvantage on host bacteria in the absence of selective pressure for resistance phenotypes [6]. However, few studies have examined the fitness advantage of their host bacteria after acquired resistance plasmids [7].

This study aimed to clarify the characteristic of ESC-resistant *Salmonella* isolated from chicken and to determine the transferability of ESC resistance-determining plasmid in vitro and in vivo. We also examined the ability to donate ESC resistance genes and how frequently they are transferred from chicken isolates to human pathogens in the mammalian mouse intestine. As antimicrobial resistance is a widely acknowledged factor affecting plasmid persistence in the absence of selective pressure [8], we attempted to identify the contribution of ESC-resistant plasmid in in vitro fitness by competition between susceptible and resistant isolates. Our goals were to assess the interspecific horizontal gene transfer (HGT) of ESC resistance from animal-derived *Salmonella* to human-derived bacteria in vitro and in vivo, also to evaluate the impact of ESC resistance genes acquisition on bacteria fitness.

#### **2. Materials and Methods**

#### *2.1. Bacterial Isolates*

A total of 136 *Salmonella* isolates isolated from chicken feces and chicken meat samples from 2017 to 2018 were collected from the top 12 integrated chicken production companies throughout Korea. The isolation and serotyping of *Salmonella* were conducted as described previously [9]. Among 136 *Salmonella* isolates, those showing either ESBL or AmpC phenotype were used in this study. *Salmonella* strains that are resistant to ceftiofur are considered ESBL/AmpC-producing strains. To select a recipient for the in vivo transfer experiment, we obtained a total of 10 strains (Table S1), which were isolated from human patient's stool samples and categorized as pathogenic *Escherichia coli*, from the National Culture Collection for Pathogens (NCCP) South Korea.

#### *2.2. Antimicrobial Susceptibility Test*

The antimicrobial susceptibility of all isolates was evaluated by the minimum inhibitory concentrations (MICs) of the test antimicrobial agents amoxicillin/clavulanic acid (AMC), cefoxitin (FOX), cefepime (FEP), ceftazidime (TAZ), ceftiofur (XNL), trimethoprim/sulfamethoxazole (SXT), sulfisoxazole (FIS), chloramphenicol (CHL), ampicillin

(AMP), ciprofloxacin (CIP), nalidixic acid (NAL), streptomycin (STR), gentamicin (GEN), tetracycline (TET), meropenem (MERO), and colistin (COL) using the KRNV5F (TREK Diagnostic Systems, Korea). *Escherichia coli* ATCC 25922 was used as the reference strain for quality control. The susceptibility breakpoints of most antimicrobials were interpreted according to the CLSI guidelines [10]. Since CLSI breakpoints were not available for colistin, ceftiofur, and streptomycin, MICs were determined according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [11] for colistin and to Centers for Disease Control and Prevention [12] for the ceftiofur and streptomycin.

#### *2.3. Identification of β-Lactamases*

After phenotypic screening, PCR were implemented regarding ceftiofur resistant isolates for detecting the presence of *β*-lactamase genes encoding CTX-M, TEM, and CMYtype following a previous protocol [13,14]. Genomic DNA templates for PCR were prepared using fresh *Salmonella* colonies on MacConkey agar (Difco laboratories, Sparks, Maryland, USA) plates by adding 100 μL of sterile distilled water and boiling in a heater block at 100 ◦C for 15 min. The sequencing reactions were performed by an external company (Solgent, Daejeon, Korea). The obtained amino acid sequences were compared with those in the GenBank nucleotide database using the BLAST online service, provided by the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/BLAST, accessed on 21 March 2020), to determine the specific types of *β*-lactamase genes.

#### *2.4. Plasmid Replicon Typing*

Plasmid DNA was extracted using HiYield™ Plasmid Mini Kit (RBC Bioscience, Taipei, Taiwan) according to the manufacturer's instructions. Plasmid incompatibility groups were determined by the PCR-based replicon typing (PBRT) method [4]. For plasmids such as IncF, IncI1, IncHI2, and IncHI plasmids were subtyped by plasmid MLST (pMLST) (http://pubmlst.org/plasmid/, accessed on 10 April 2020).

#### *2.5. In Vitro Conjugation Experiment*

A conjugation experiment was performed according to previously reported methods with some modification [15]. In vitro mating was performed in liquid media, and cephalosporin-susceptible *Escherichia coli* J53 (sodium azide-resistance) and selected *E. coli* NCCP isolate (certain antimicrobial resistance) were used as recipients. Briefly, overnight cultures of donor and recipient strains were re-cultured in logarithmic phase (OD 600 nm of 0.5) at 37 ◦C in fresh tryptic soy broth (Difco Laboratories, Detroit, MI, USA) medium for 4 h. Next, 1 mL of the donor and 4 mL of the recipient were mixed and incubated without shaking for 1 h at 37 ◦C. The culture was spread on MacConkey agar plates containing sodium azide (200 μg/mL) and ceftiofur (8 μg/mL) for detecting *E. coli* J53-derived transconjugants. MacConkey agar plates containing certain antimicrobial and ceftiofur (8 μg/mL) were used for detecting *E. coli* NCCP isolate-derived transconjugants. The experiment was repeated three times, and three putative transconjugant colonies were randomly selected from each experiment. For verifying the transconjugant, the transconjugant was evaluated by the MICs with the method described above, and the presence of a marker gene of an ESC-resistant plasmid was confirmed by PCR, as previously described [14]. Conjugation frequency was calculated as the ratio of the number of transconjugants per recipient (Tc/R). Recipient isolate counts were calculated by subtracting transconjugant colony counts from the number of colonies obtained on agar plates, which included both recipients and transconjugants.

#### *2.6. In Vivo Transfer Experiment*

When selecting the recipient for the transfer experiment in vivo, one recipient for the in vivo transfer experiment was selected based on the results of the conjugation frequency test. Enteroinvasive *E. coli* (EIEC) NCCP 13719 carried the virulence gene of *ipa*H [16], which was ceftiofur-susceptible but tetracycline-resistant and showed the highest frequency of transfer, was selected as the recipient for the in vivo transfer experiment.

The animal experiment was conducted in accordance with the requirements of the Animal Care and Ethics Committees of Jeonbuk National University and were approved by the National Association of Laboratory Animal Care (JBNU 2021-06). Female SPF 6 week-old BALB/c mice (Samtako, Osan, Korea) were randomly housed in four groups of five animals each, and each group was kept in a separated isolator (Three-Shine, Daejeon, Korea). Before the inoculation of donor and recipient, fecal samples from all mice in each group were pooled, and the absence of resistant strains was confirmed by spreading onto a plate that we used in this study. In addition, fecal samples were also checked to be free of the *bla*CMY-2 gene and *ipa*H gene. The experimental groups were as follows: streptomycintreated control group (G1), streptomycin-treated and then donor-inoculated group (G2) for monitoring donor strain colonization, streptomycin-treated and then recipient-inoculated group (G3) for monitoring recipient strain colonization, and streptomycin-treated and then donor and recipient simultaneously inoculated group (G4). Before inoculation, streptomycin at a dose of 20 mg per mouse was pretreated to eliminate the circumstance of microbial competition and induce the colonization of inoculating isolates (Figure 1) [17]. Food and water were discontinued 4 h before oral administration of streptomycin. Then, food and water were made available to be consumed ad libitum. At 20 h after streptomycin administration, food and water were ceased again for 4 h before the mice were inoculated orally by gavage with 0.2 mL of 108 CFU/mL of donor and recipient. As for G4, the recipient was inoculated 30 min after inoculating the donor. Then, water was offered immediately and food was made available 2 h after infection ad libitum. On 1, 2, 4, and 7 days after infection, fresh fecal samples were collected from each mouse. The samples were weighed and diluted five-fold and then finally homogenized by vortexing in phosphate buffer saline (PBS). For colony counting, 10-fold serially diluted samples were inoculated onto appropriate agar plates, including antimicrobials for each group. For verification of transconjugant isolates, putative colonies were sub-cultured onto antimicrobial selective agar plates, and genomic DNA was extracted according to the boiling method as described above for using PCR analysis to test for the possession of marker gene (*β*-lactamase gene from donor and virulence gene from the selected recipient). As for G4, transfer frequency was calculated as the ratio of the number of transconjugants per recipient (Tc/R).

**Figure 1.** Schematic representation of in vivo transfer experiment set up.

#### *2.7. Competition Experiment In Vitro*

To assess the fitness effect of resistance plasmid carriage, competition assays between resistance plasmid-harboring transconjugant and its parental isolates, *E. coli* J53 and *E. coli* NCCP 13719, were conducted. The competition experiment was carried out as previously described [18] and repeated five times. Briefly, parental isolates were incubated overnight at 37 ◦C with shaking at 200× *g* rpm in 10 mL of lysogeny broth (LB). Transconjugants were cultured in LB with the addition of 8 μg/mL ceftiofur to ensure the expression of ESC-resistant genes. Overnight cultures of two pairwise strains (*Escherichia coli* J53/transconjugant of *E. coli* J53, *E. coli* NCCP 13719/transconjugant of *E. coli* NCCP 13719) were adjusted to OD 600 nm of 0.5, were diluted 10−4, and were then mixed 1:1 in

LB broth at 0 h. After 24 h of incubation at 37 ◦C, the mixed isolates were again 10−<sup>4</sup> diluted into a fresh LB medium. This procedure was repeated every 24 h until the competition experiment had lasted for 72 h. The total number of isolates were determined by dropping 10 μL of properly diluted samples onto antimicrobial-free and antimicrobial-supplemented selective MacConkey agar plates in triplicate at 24 h, 48 h, and 72 h. The number of CFUs growing on the MacConkey agar plate including ceftiofur (8 μg/mL) was subtracted from the number of CFUs growing on the antimicrobial-free plate to determine the number of ESC-resistant gene-free isolates in the mixed population. To assess the relative fitness of transconjugants compared with its parental strains, an in vitro competitive ratio was calculated using a previously described method [17]. The competitive ratio was defined as the ratio of the number of CFU of the transconjugants vs. the parental strain at 24, 48, and 72 h.

#### **3. Results**

#### *3.1. Characterization of Bacterial Strains*

Based on the results of antimicrobial susceptibility assessment of 136 isolates, 17 out of 136 (12.5%) *Salmonella* spp. were consistent with an ESBL/AmpC phenotype and genotype (Table 1). The frequency of ESBL *β*-lactamase production with CTX-M gene was 11.8% (16/136) in *Salmonella* isolates, which was significantly (*p* < 0.05) higher than AmpC *β*lactamase production (0.7%, 1/136) with CMY gene in *Salmonella* isolates. The four serovars isolated were *Salmonella* Enteritidis (52.9%), *Salmonella* Virchow (35.5%), *Salmonella* Albany (5.9%), and *Salmonella* spp. (5.9%). CTX-M (94.1%) was the most commonly detected *β*lactamase family, and *S. enteritidis* had CTX-M and TEM gene combination while *S.* Albany was CMY-positive. All strains showed multidrug resistance. For all 17 strains, a conjugation experiment was implemented regarding the transfer of the ESC-resistant gene, and seven (41.2%) out of 17 strains were successfully conjugated in a wide range of frequencies from <10−<sup>7</sup> to ≥10−<sup>3</sup> (transconjugant/recipient). Among the transconjugants, one strain, which the harbored *bla*CMY-2 gene, showed high transfer frequency (≥10−3) [19,20]. On analysis using PCR-based replicon typing for conjugative plasmids, IncFIIS plasmids harboring CTX-M-15 were found in three *S.* Enteritidis isolates, and IncI1 plasmid harboring CMY-2 was found in one *S.* Albany isolate. IncI1 plasmids were further submitted to pMLST and assigned to a sequence type of ST12. The remaining three isolates were indicated as non-typeable plasmids.

#### *3.2. In Vitro Transfer*

Using the liquid mating method, transconjugant with its parental strains, *E. coli* J53 (Tc.J53), and *E. coli* NCCP 13719 (Tc.13719) displayed ESC resistance profile corresponding to the acquisition of the *bla*CMY-2 gene (Table 2). Tc.J53 and Tc.13719 expressing of *the bla*CMY-2 gene were resistant to ampicillin, cefoxitin, ceftiofur, ceftazidime, and amoxicillin/clavulanic acid (AMC), however, remind to susceptive to fourth-generation cephalosporins of cefepime, which is consistent with an AmpC phenotype. The *bla*CMY-2 carrying transconjugants of Tc.J53 did not show any resistance to non–*β*-lactam antibiotics, suggesting that no other resistance genes were located on this IncI1 plasmid.

The conjugation frequency of *bla*CMY-2 IncI1 plasmid from *Salmonella* to *E. coli* J53 and *E. coli* NCCP 13719 was determined to be 1 × <sup>10</sup>−<sup>3</sup> ± <sup>1</sup> × <sup>10</sup>−<sup>7</sup> and 2 × <sup>10</sup>−<sup>3</sup> ± <sup>1</sup> × <sup>10</sup>−<sup>3</sup> on an agar plate with 200 μg/mL sodium azide and 8 μg/mL ceftiofur, 100 μg/mL tetracycline and 8 μg/mL ceftiofur, respectively.



**Table 1.** Information on 17 cephalosporin-resistant *Salmonella* spp. isolated from chicken-related sources.

AMC, Amoxicillin/clavulanic acid; AMP, Ampicillin; FOX, cefoxitin; TAZ, Ceftazidime; XNL, Ceftiofur; FEP, cefepime; SXT, Trimethoprim/sulfamethoxazole; FIS, sulfisoxazole; CHL, chloramphenicol; CIP, ciprofloxacin; NAL, nalidixic acid; STR, streptomycin; GEN, gentamicin; TET, tetracycline; COL, colistin. a, Conjugation frequency (Transconjugant/recipient) with ≥10−3; b, Conjugation frequency with <10−7. NA, not available; ND, not done.



FOX, cefoxitin; XNL, ceftiofur; TAZ, ceftazidime; FEP, cefepime; AMP, ampicillin; AMC, amoxicillin/clavulanic acid; TET, tetracycline. a MNK, *Salmonella* Albany A17-KCI-MNK-002-2S; J53, *Escherichia coli* J53; Tc.J53, transconjugant of *E. coli* J53; 13719, *E. coli* NCCP 13719; Tc.13719, transconjugant of *E. coli* NCCP 13719; EIEC, enteroinvasive *Escherichia coli*. b Transconjugant per recipient (Tc/R).

#### *3.3. In Vivo Transfer*

To get the information on the efficiency of bacterial intergenic plasmid transfer in the mammalian intestine and to better mimic the in vivo situation, streptomycin-treated mice were used in this study. One-day after inoculation with donor and recipient (G4), the concentration of donor from each mouse ranged from 10<sup>7</sup> CFU/g to 109 CFU/g and that of recipients ranged from 102 CFU/g to 104 CFU/g (Figures 2 and S1). The frequency of plasmid transfer at 1-day post-infection (dpi) from G4 was estimated at an average of <sup>2</sup> × <sup>10</sup>−<sup>1</sup> ± <sup>4</sup> × <sup>10</sup>−<sup>1</sup> with the ratio of transconjugants per recipient. This showed that the *bla*CMY-2 IncI1 plasmid was indeed efficiently transferred from the *Salmonella* isolate to the EIEC in the gut of streptomycin-treated mice. The number of transconjugants did not reach detectable levels at 7 dpi in four out of five mice.

**Figure 2.** Bacterial counts of the donor, recipient, and transconjugant from mouse fecal samples in group 4 (G4), expressed as the log number of CFU per gram of feces.

#### *3.4. Fitness Cost Assessment by Competition Experiment*

The impact of harboring the *bla*CMY-2 gene on host fitness was evaluated by a pairwise competition experiment (Figure 3). Our results showed that the ceftiofur-sensitive strains were out-competing resistant strains in the absence of selective pressure with the value of transconjugant per parent strain at below 0, indicating that the *bla*CMY-2 gene-harboring plasmid-free strain dominated. In *E. coli* J53 with the *bla*CMY-2 gene-harboring plasmid, a slight fitness decrease was observed, and the fitness was stable following continuous passage until 72 h. A greater reduction in fitness was observed in *E. coli* NCCP 13719 compared with *E. coli* J53 in the serial passage, with a reduction of more than 3 log units for lasting 72 h. Regarding the value of the log ratio of resistant versus susceptible strains, the *bla*CMY-2 gene-harboring plasmid imposed a slight fitness cost to *E. coli* J53 from about log −0.89 at 24 h to −0.97 at 72 h. In contrast, the transconjugant from *E. coli* NCCP 13719 showed quite a high fitness cost from about log −2.02 at 24 h to −5.58 at 72 h.

**Figure 3.** Competitive growth kinetics. Dynamics of replicate competition experiments for parent strains, *E. coli* J53 and *E. coli* NCCP 13719, and their transconjugant containing the *bla*CMY-2 gene.

#### **4. Discussion**

Since the first finding of the CTX-M-type gene from Korea in 2001, the prevalence of the *bla*CTX-M-15-producing *Salmonella* in humans and chickens has rapidly increased over the years in Korea [3]. In the present study, the *bla*CTX-M-15 gene was the most frequently detected but showed low frequencies at <10−<sup>7</sup> transconjugants per recipient, which is consistent with a previous study [21]. The *bla*CTX-M-15 genes belonging to IncFII plasmid are known to be highly prevalent and involved in the concurrent transfer of antimicrobial resistance and virulence genes, which increases co-selection and probably leads to the emergence or outbreaks of virulent and multidrug-resistant (MDR) clones [22].

Conversely, the *bla*CMY-2 gene was observed from one strain in this study. The first report of the *bla*CMY-2 gene was described in the 1990s [23], and now, it is one of the most common and widely disseminated genes by plasmid-mediated AmpC *β*-lactamase from humans and chickens [2]. Regarding the transfer frequency of the *bla*CMY-2 gene between bacteria, the results of this study explain how frequently their resistance gene gets transferred to other bacterial species. Frequent transfer of the IncI1 plasmid carrying the *bla*CMY-2 gene was measured with the ratio of over 10−<sup>3</sup> transconjugants per recipient in this study. This result is higher than previous findings wherein the transfer frequencies of *bla*CMY-2 and *bla*TEM-1 genes from the *Salmonella* isolated from poultry meat were in the ratio of 6.0 × <sup>10</sup>−<sup>8</sup> to 2.4 × <sup>10</sup>−<sup>4</sup> transconjugants per recipient [24]. The high frequency of transfer and their possibility to exchange genes within and between species might have resulted in the increasing prevalence of the *bla*CMY-2 gene in animals and humans, and its rapid dissemination may constitute a significant risk to public health. To our knowledge, this is the first description of the transfer of a *bla*CMY-2 gene-harboring plasmid from chicken-origin *Salmonella enterica* to pathogenic *E. coli* isolated from a human patient in a mammalian model. Identifying the transfer of antimicrobial-resistant plasmids and their frequency in a mouse model, which is an adequate way to predict the risk of the dissemination of antimicrobial resistance genes with a perspective of food safety. From this point of view, we used a streptomycin-pretreated mouse model, which provides more realistic results than any in vitro or gnotobiotic study because the normal microflora barrier and the present immune system give the tested animal model advantages in mimicking the human gut [25]. In this study, *E. coli* transconjugant appeared in all mice fecal samples 1 dpi in G4, and the high transfer frequency observed with the mean ratio of transconjugants per recipient was about 2 × <sup>10</sup>−<sup>1</sup> and per donor was 4 × <sup>10</sup>−6, which support statements on the rapid transfer of the *bla*CMY-2 gene. Although there is a lack of in vivo studies focused on the *bla*CMY-2 gene, several studies for conjugal transfer of ESBL genes have been reported. *bla*TEM gene from *Salmonella* was transferred to *E. coli* recipient with the ratio of transconjugant per donor being 6.5 × <sup>10</sup>−<sup>5</sup> in mice without selective pressure [26], and *bla*CTX-M-9 gene derived from chicken-origin *Salmonella* to *E. coli* at a frequency range of about 5.4 × <sup>10</sup>−<sup>5</sup> in gnotobiotic rats [27]. It is important to emphasize that it demonstrated not only the capability of transfer of *bla*CMY-2 gene with high frequency but also showed that the ratio of transconjugants per recipient in vivo was 2 log units higher than in vitro. Similar findings were reported wherein the rate of plasmid transfer between *Enterococcus faecium* strains was up to 8 log units higher in the germ-free mice model than in vitro [28]. The high frequencies of plasmid transfer in vivo may be due to the constant mixing of bacteria by the peristaltic movements in the gastrointestinal tract, stimulating a donor with more access to recipients than during in vitro mating, wherein the bacterial movement is much lesser [29]. These results emphasized the necessity of in vivo test for transferability and transfer frequency to figure out the potential risk of the presence of resistant strains in the digestive tract to humans.

Our result could be a direct evidence that the ESC resistant *Salmonella* from chickenrelated products can transfer their resistance gene to other pathogens, thus leading to the possibility of inappropriate antimicrobial selection and limited treatment options resulting in therapeutic failure [5]. A case of treatment failure due to the emergence of resistance to ceftriaxone, a 3rd generation of cephalosporin, has been reported. The originally susceptible

pathogen developed ceftriaxone resistance via the acquisition of a plasmid containing the ceftriaxone resistance gene during the 3rd ESC treatment, which finally caused therapeutic failure in the patient [30]. In addition, even if resistant bacteria transiently colonize, it may quickly transfer resistance plasmid into the human gastrointestinal tract; normal microbiota and the nutrient-rich environment make the gastrointestinal tract offer an ideal condition for gene exchange [28]. In this study, two days after inoculation, about >10<sup>4</sup> CFU/g of ceftiofur resistant *E. coli* isolates, regarded as normal flora-derived strain, were observed from one mouse in G2, which was inoculated only with *Salmonella* (donor). Likewise, the intestinal microbiota can act as a massive reservoir of antimicrobial resistance genes, thus prolonging the spread of MDR bacteria and resulting in therapeutic failure. Consequently, secondary infections would occur more often, indicating a serious threat to human health [31].

In vitro direct competition studies of the *bla*CMY-2 gene-harboring plasmid and two recipient *E. coli* showed that a variable fitness cost depends on the parent strains, and we observed that susceptible strains can outcompete resistant strains consistent with a previous study [17]. Normally, the acquisition of a plasmid often imposes a fitness burden on a bacterial cell [6]. Since *E. coli* NCCP 13719 in this study has the virulent gene *ipa*H, which may be encoded by a large plasmid, carrying another plasmid may present an adverse situation for the bacteria [6]. Conversely, the stable inheritance of bacterial plasmids without any selective pressure was also observed from transconjugants from *E. coli* J53 during the 72 h of experiment time. This phenomenon suggests that strains with low fitness costs even with the acquisition of plasmids from other strains may exist. For further studies, the mechanism of sustaining resistance plasmid with low fitness cost is expected to be a key research topic for suggesting the way to control the dissemination of antimicrobial resistance genes.

There are several limitations that we examined the characteristic of *bla*CMY-2 geneharboring bacteria with a single strain; however, it may serve as fundamental data that defined the characteristics, and further studies with a greater number of resistant bacteria harboring the *bla*CMY-2 gene are required due to their increasing trend of emergence recently. In addition, mice are often naturally resistant to non-mice-origin *E. coli* colonization [32] as seen in our results, and thus, decreasing the number of bacteria is an inevitable phenomenon; however, it can be presented as a model that is sufficiently able to establish the transferability and frequency of antimicrobial resistance genes and emphasize that colonizing bacteria may transfer resistance plasmids readily in the intestinal tract [28]. To confirm the persistence of resistant genes through the colonization of antimicrobialresistant bacteria in vivo and subsequent transfer of the gene to normal flora, changes in test strain or replacement of the in vivo model are required.

#### **5. Conclusions**

This study showed the prevalence of ESC resistance genes in *Salmonella* isolated from chickens and presented a comprehensive analysis of the highly frequent transfer of the *bla*CMY-2 gene in vitro and in vivo. In addition, this study has demonstrated the ease with which a *bla*CMY-2 gene-harboring plasmid can be rapidly transferred between *Salmonella* and pathogenic *E. coli* within the intestinal tracts of mice, even without antimicrobial selective pressure. Notably, we observed that once *bla*CMY-2 gene-harboring strains enter the mammalian intestinal tract, their dissemination could be more rapid and frequent than it would be in vitro, and even they could be transferred to the indigenous intestinal microbiota, threatening future treatments of infections. Since the use of cephalosporin in the poultry industry has increased over the last decade in Korea [33], the increasing emergence of ESBL/AmpC producing ESC resistant *Salmonella* spp. isolated from poultry is of concern. There is a risk for consumers related to exposure to ESBL/AmpC genes by contaminated food, so the application of guidelines for prudent antimicrobial usage in the poultry industry is urgently needed. Given the potential risk of the frequent transfer of the *bla*CMY-2 gene via the food chain to the human digestive tract, the molecular mechanism involved in the dissemination and maintenance of ESC resistance genes should be studied as further research in greater detail, and enhanced surveillance should be implemented to prevent the widespread of ESC resistant strains.

**Supplementary Materials:** The following are available online at: https://www.mdpi.com/article/ 10.3390/ani11061778/s1. Table S1. The list of pathogenic *Escherichia coli* isolates was obtained from the National Culture Collection for Pathogen (NCCP), based in Korea, Figure S1. Fecal excretion of the donor in group 2 (G2) and the recipient in group 3 (G3) (a), the donor, recipient, and transconjugant in group 4 (G4) (inoculation of donor and recipient, simultaneously) (b), expressed as the log number of CFU per gram of feces.

**Author Contributions:** B.W., M.K., and H.-K.J. contributed to the conception and design of experiments. S.-Y.C. and B.-R.K. contributed to the acquisition, analysis, and interpretation of data. B.-R.K., B.W., S.-Y.C., K.S., J.-F.Z., M.K. and H.-K.J. drafted and/or revised the article. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Agriculture, Food and Rural Affairs Convergence Technologies Program for Educating Creative Global Leader (716002-7, 320005-4) funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA). And this work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2020R1F1A1065136).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Animal Care and Ethics Committees of Jeonbuk National University, and were approved by the National Association of Laboratory Animal Care (JBNU 2021-06).

**Data Availability Statement:** The data presented in this study are available from the corresponding author on reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Antinematode Activity of Abomasum Bacterial Culture Filtrates against** *Haemonchus contortus* **in Small Ruminants**

**Asfa Nazish 1, Fozia 2, Baharullah Khattak 1, Taj Ali Khan 1,3, Ijaz Ahmad 4,\*, Riaz Ullah 4,\*, Ahmed Bari 4, Majid M. Asmari 5, Hafiz M. Mahmood 6, Muhammad Sohaib 7, Ahmad El Askary 8, Attalla F. El-kott 9,10 and Mohamed M. Abdel-Daim <sup>11</sup>**


**Simple Summary:** *Haemonchus contortus* is an important gastrointestinal nematode parasite of the tropical and sub-tropical regions that cause haemonchosis in small ruminants like goats and sheep. It causes low production, reduced growth and may cause death of the infected animals. Due to the resistance development and environmental issues, the use of anthelmintics can be replaced with biological control, which is an environment friendly alternative. In the present study, three bacteria viz; *Comamonas testosteroni*, *C. jiangduensis* and *Pseudomonas weihenstephanesis* showed significant effect on nematode mortality and egg hatch inhibition. It was also observed that the anthelmintic activity of these bacteria was dose dependent, where 100% bacterial metabolite concentration showed the highest activity. It is suggested that these bacteria may included in the integrated nematode management.

**Abstract:** Haemonchosis is a parasitic disease of small ruminants that adversely affects livestock production. *Haemonchus contortus* is one of the most prevalent nematode parasites that infect the abomasum of small ruminants. This parasite reduces milk production, overall growth and sometimes causes the death of the infected animals. The evaluation of the biocontrol potential of some abomasum bacterial isolates against *H. contortus* is investigated in this study. Out of which, three isolates— *Comamonas testosteroni*, *Comamonas jiangduensis*, *Pseudomonas weihenstephanesis*—show significant effect against the nematode L3, adult, and egg hatch inhibition assays. Various concentrations of metabolites from these bacteria are prepared and applied in different treatments compared with control. In the case of adult mortality assay, 50% metabolites of *C. testosteroni* and *P. weihenstephanesis* show 46% adult mortality, whereas *C. jiangduensis* shows 40% mortality. It is observed that decreasing the concentration of bacterial metabolite, lowers nematode mortality. The minimum nematode mortality rate is recorded at the lowest filtrates concentration of all the bacterial isolates. The same trend is observed in egg hatch inhibition assay, where the higher concentration of bacterial culture filtrates shows 100% inhibition of *H. contortus* egg. It is concluded that the effect of bacterial culture

**Citation:** Nazish, A.; Fozia; Khattak, B.; Ali Khan, T.; Ahmad, I.; Ullah, R.; Bari, A.; Asmari, M.M.; Mahmood, H.M.; Sohaib, M.; et al. Antinematode Activity of Abomasum Bacterial Culture Filtrates against *Haemonchus contortus* in Small Ruminants. *Animals* **2021**, *11*, 1843. https://doi.org/ 10.3390/ani11061843

Academic Editors: Paola Roncada and Bruno Tilocca

Received: 3 May 2021 Accepted: 14 June 2021 Published: 21 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

filtrates against *H. contortus* is dose-dependent for their activity against nematode L3, adult, and inhibition of egg hatchment.

**Keywords:** small ruminants; *H. contortus*; abomasum; fecal samples; bacterial culture filtrates

#### **1. Introduction**

Gastrointestinal parasites are considered as the main cause of economic losses in the livestock sector. Among gastrointestinal parasitic infections, haemonchosis is important and dominant that greatly destroys livestock production, particularly the small ruminants [1]. This disease is caused by three species of the genus Haemonchus, namely, *H. similis*, *H. placei*, and *H. contortus*. Among these, *H. contortus* is one of the most prevalent nematode parasites that infect the abomasum of small ruminants worldwide [2]. It is commonly known as a red stomach worm, the wire worm, or the barber's pole worm. It belongs to phylum Nematoda, family Trichostrongyloidae, class Secernentea, and the order Strongylida. The highly susceptible part of ruminant's stomach to *H. contortus* is the abomasum, in which adult worms are present. This parasite causes low production, decreased growth, lower body weight, and sometimes, cause the death of the infected host. This parasite is most prevalent in Africa; however, many cases have been reported in North America as well [3].

*Haemonchus contortus* mostly affects young animals, having hypo immunological response, showing low resistance to the parasite [4]. Primary symptoms of haemonchosisinclude pallor, anemia, edema, ill thrift, lethargy, and depression, which may cause sudden death in acute infection. Another prominent symptom of haemonchosis is the accumulation of fluid in the submandibular tissue, a phenomenon commonly called "bottle jaw" [5]. When the L3 larvae resume development in spring, the threat of haemonchosis increases. The young and pregnant or lactating mothers are highly susceptible to *H. contortus*, because of their low immunogenic response against the parasite infection [6]. A heavy infection (20,000–30,000 worms) of Haemonchus species can kill sheep and goats very quickly [7]. Haemonchosis can be diagnosed based upon the characteristic clinical signs of anemia, low Packed Cell Volume (PCV), pale mucous membranes dehydration, weakness, retarded growth, and edema. [8].

*Haemonchus contortus* is distributed throughout the world, where warm and humid climate prevails; hence, haemonchosis is a major threat in tropical and subtropical regions. [9]. Haemonchus species are prevalent in Pakistan and are reported almost from every district of Pakistan by different researchers with varying percent prevalence [10–12]. In Khyber Pakhtunkhwa and central Punjab, 72% prevalence of *H. contortus* was reported, while other researchers recorded its prevalence with varying percentages in many districts of Pakistan [13–15].

It is not advisable to eliminate the parasites from livestock, but to keep the population under a threshold, in a sustainable state [16]. To control the nematode parasites, different management practices, such as the use of chemical anthelmintics, sanitation, vaccination, various plant extracts, and biological control, are in common practice [17]. The helminths infection in man and animals is mostly treated by chemotherapy. Gastrointestinal nematode infections can be managed by using chemical anthelmintics, which are used as prophylactic measures. Due to the over-use of chemical anthelmintics, they reduce their effectiveness and emerge resistance in nematodes [18]. The *H. contortus* infection can also be effectively treated with the wire particles of copper oxide (COWP) and copper sulfate (CuSO4). Using copper oxide wire particles 2.5 g to 5 g in sheep, *H. contortus* eggs number was significantly reduced. These anthelmintics have been found to reduce the parasite population in small ruminants with low resistance development. Presently, it is used along with chemical antihelmintics to combat resistance development in *H. contortus* [19].

Limited information available regarding the anthelminthic activity of bacteria antagonists against parasitic nematodes. Some species, including *Bacillus* sp., have been reported to have nematocidal activity. A soil bacterium, *Bacillus thuringienisis*, is widely used as a biological control agent against different pathogens because of its low mammalian toxicity and the species specificity by particular endotoxin groups [20]. *Bacillus* sp. Produce a variety of toxic proteins that are vigorous against different parasites [21]. A number of toxins produced during vegetative growth show lethal activity; however, this lethal activity mainly results from the production of delta endotoxin that is synthesized during sporulation [22,23]. The production of other factors that might contribute to the toxic effects observed, such as proteases, chitinase, exotoxins, and lipases [24].

Little is known about the microbial diversity in the abomasum of the sheep, an important site of nematode infection, and the correlation between the microbial diversity and GIN resistance. Thus, this work aimed to isolate bacteria from the abomasum of sheep and goats to test the anthelmintic activity of metabolites of these bacterial isolates against *H. contortus*.

#### **2. Materials and Methods**

The present research work was carried out in the Department of Microbiology, Kohat University of Science and Technology Kohat. All the processes were performed in the aseptic environment.

#### *2.1. Collection of Abomasal Content and Fecal Samples*

Abomasum content, as shown in Figure 1a–c, was collected from slaughtered goats and sheep at a slaughterhouse in Kohat, Pakistan. A total of 50 samples of the abomasum and 50 samples of feces were taken from goats and sheep, and the samples were brought to Microbiology Laboratory for further processing.

**Figure 1.** (**a**) Abomasum, (**b**) opened of abomasum, (**c**) adult *Haemonchus contortus* in abomasum.

#### *2.2. Isolation and Identification of Bacteria from Abomasal Contents*

Bacteria were isolated by serial dilution method, taking abomasal fluid in 100 mL of distilled water. After that, 1 mL from the suspension was taken and put in a test tube containing 9 mL of distilled water. Different bacterial dilutions 10−1–10−<sup>6</sup> and sterile distilled water (control) were used. Streaks were made from 10−3, 10−4, 10−5, and 10−<sup>6</sup> bacterial dilutions on petri dishes containing nutrient agar. The plates were incubated at 37 ◦C for 24 h. The bacterial isolates were subcultured and identified by colony morphology and biochemical tests, and finally, through genomic DNA sequencing [25].

#### 2.2.1. Colony Morphology

Size, shape, colony consistency, margins, and elevation are included in the colony morphology.

#### 2.2.2. Gram Staining

Smear from each bacterial isolate was organized on a glass slide, and it was heat-fixed by passing it over the flame. Crystal violet was added drop by drop on the smear and left for 60 s. The slide was washed with tap water. For 45 s, iodine was overlaid on the slide and washed with distilled water. The slide was washed for 10 to 15 s with a decolorizer (alcohol). Smear was rinsed for few seconds with distilled water and counter-stained with safranin and air-dried. Deep violet or purple color emerged for Gram positive bacteria, while Gram negative bacteria appeared purple or red.

#### 2.2.3. Biochemical Characterization of the Bacterial Isolates

For the identification of bacterial isolates, various biochemical tests were performed, such as Oxidase test, Indole test, Sugar fermenter test, Motility test, Catalase test.

Catalase Test

A drop of hydrogen peroxide was added to a slide. A loop filled with each of the bacterial isolates was mixed into the droplet. Bubble formation indicated a positive catalase test.

Oxidase Test

A smooth filter paper was put on a petri plate, and a drop of oxidase reagent was added. Bacterial culture was stretched on the droplet of the oxidase reagent, using an inoculating loop. The formation of the reagent's dark purple color confirmed the oxidase positive test [26].

Indole Test

Bacterial colonies were inoculated into individual tubes of 2 mL tryptone water, incubated at 37 ◦C for 24 h, and tested for indole production with Kovac's reagent. If the reagent showed a cherry red color layer, then it confirms the positive test.

Sugar fermenter

The sugar fermentation test was performed by inoculating a loop full of a nutrient broth culture of the organisms into the tubes containing different sugar media (five basic sugars, such as dextrose, sucrose, lactose, maltose, and mannitol) and incubated for 24 h at 37 ◦C. The sugar fermenter was shown by a color change from reddish to yellow and the formation of gas bubbles in the inverted test tubes.

Motility Test

One drop of bacterial culture, grown on nutrient broth, was placed on the coverslip. The same was placed inverted over around the concave depression of the hanging drop slide to make hanging drop preparation and sealed with Vaseline to prevent airflow and evaporation of the fluid. The hanging drop slide was then examined under 100× objective of a compound microscope using immersion oil. The motile and nonmotile organisms were identified by observing motility with to and from the movement of bacteria [27].

#### *2.3. Isolation of Haemonchus Contortus Adults and Larvae from Abomasum*

The abomasum of the freshly slaughtered goats and sheep were taken to pick adult *H. contortus* and larvae in the slaughterhouse of Kohat district, Pakistan. The worms were collected from the abomasum by washing with distilled water, and it was transferred into Phosphate Buffer Saline (PBS) with pH 7.4. The PBS was prepared by dissolving 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g of KH2PO4 in 800 mL of distilled water, and distilled water was added to adjust the volume to one litter.

#### *2.4. Isolation of H. contortus Eggs from Faecal Sample*

In a petri plate (Figure 2), about 4 g of feces were taken, and 60 mL floatation liquid was poured into the container by mixing feces carefully with a moving device. The resultant fecal suspension was poured over a filter of 200 μm into another vessel. The fecal contents were poured into a test tube. Then the test tube was topped gently with the interruption, a curved meniscus was formed at the top of the tube and placed a coverslip carefully. After 20 min, the droplet of liquid sticks to it. Under 10× and 40× of the compound microscope, the slides were observed [28].

**Figure 2.** Fecal Samples of Sheep and Goats Containing *Haemonchus contortus* Eggs.

2.4.1. Estimation of Eggs in the Fecal Samples

The eggs of *H. contortus* in the animal's fecal samples were calculated by using the McMaster counting chamber method [29]. In this procedure, Fresh feces of 3 g were taken and mix them in 42 mL of saturated sodium chloride solution. Through a tea filter or strainer, passed the suspension three times. Then both compartment of the McMaster counting chamber was filled with suspension and wait for 3–5 min and observed the McMaster chambers under the 10× of a light microscope.

Eggs seen in 1st and 2nd chambers were calculated as:

(Eggs seen in chamber 1 + Eggs seen in chamber 2) × 50 = Eggs per Gram (EPG)

The nematode eggs were taken from the feces of sheep and goats. Eggs from the highest EPG were isolated by dissolving 5 g of feces in 10 mL of distill water. The diluted feces were filtered through a 100 mesh and transferred into another flask. Then the saturated salt solution was added into filtrate in the threefold volume of the flask. On top of the container, placed a clear plastic sheet; hence, the surface of the solution could touch the plastic sheet and placed it for 60 min. The eggs were adhered to its lower surface, and it was carefully washed away by tap water in a clean container. By removing the upper layer of the water, the eggs were settled down at the end of the container after one hour [30].

#### 2.4.2. Culture Filtrate Collection from the Bacterial Isolates

The nutrient broth, containing 0.5% Peptone and 0.3% beef extract/yeast extract in distilled water, was autoclaved, and inoculated with bacterial isolates, and incubated in a shaking incubator at 37 ◦C for 7 days. The broth culture was centrifuged at 1000 rpm for 15 min. The pellet was discarded. With the help of Whatman filter paper, the supernatant was filtered and again filter through Miller HA syringe filters (pore size = 0.45 μm). The secondary metabolites were obtained. To check the presence of bacterial cells in metabolites, it was again inoculated on nutrient agar plates at 37 ◦C for 24 h. Extract of metabolites were mixed with phosphate buffer saline and distilled water to make different concentrations.

The extracts were considered as 100% concentrated, while the bacterial extracts with PBS and anthelmintic agent were considered as a positive control (PC), whereas only PBS and worms were considered as a negative control (NC).

#### *2.5. In Vitro Bioassays*

Various concentrations of the bacterial culture filtrates were used to evaluate their anthelmintic effect on adult and larval mortality and egg hatch inhibition of *H. contortus*, by using the standard techniques as per the protocol of Kotze [20].

#### 2.5.1. Adult Mortality Assay (AMA)

Adult worms were taken from the abomasum of freshly slaughtered sheep and goats to perform the adult mortality assay (AMA). Culture filtrate, obtained from three bacterial isolates Abomasum Bacteria with Pinkish Colony (ABP), Abomasum Bacteria with Yellow Colony (ABY), and Abomasum Bacteria with Creamy White Colony (ABCW), were diluted in PBS, as 100%, 50%, 25%, 12.5%, and 6.25% in 48 well plates, into which five adults' nematodes were transferred into each well. The treatments were repeated five times. The bacterial culture filtrates were taken alone with worms, were considered as a 100% concentrated. For negative control, PBS with worms was used, whereas, for positive control, levasole (AgriLabs) 25 μg/mL in PBS with worms was used. Data on the adult nematode mortality were taken after every hour of the treatment, until all the worms were found dead in control. The treated worms were placed in warm PBS and observed their possible motility [31].

The percent nematode mortality was calculated by the following formula:

Percentage mortality = P test/P total × 100

P test: number of dead worms

P total: number of dead worms + number of live worms

#### 2.5.2. Larval Mortality Assay (LMA)

Larvae (L3) of *H. contortus* were taken from the abomasum of freshly slaughtered sheep and goats to perform the Larval Mortality Assay (LMA). Various concentrations of the bacterial metabolites (100%, 50%, 25%, 12.5%, and 6.25%) were prepared in PBS. The concentration was considered as 100% with L3 larvae and with metabolites. Well containing larvae and 2.5 mL phosphate buffer saline were considered as a negative control. The larvae to which an antihelmintic agent of 2.5 mL levasole (25 μg/mL) was added, considered as a positive control. The plates were placed in an incubator for 3 h at 37 ◦C. Under 10× of a stereo microscope, data on the larvae mortality were taken after three hours of treatments, until all the larvae were found dead in control. The treated worms were placed in warm PBS and observed their possible motility [32].

The percent larvae mortality was calculated as:

Percentage mortality of larvae = P check/P total

P check: number of dead larvae

P total: number of dead larvae + number of live larvae

#### 2.5.3. Egg Hatch Inhibition Assay

Egg hatch inhibition assay was performed to evaluate the inhibitory effect of the metabolites of the bacterial isolates ABP, ABY, and ABCW. This assay was repeated in triplicate following the protocol given by Coles et al. [33].

Nematode eggs were placed in 15 mL of sterile distilled water, and by the McMaster technique, their quantity was adjusted to 100–200 eggs in 75 μL of water and was added into each well of a 24 well titration plate. Metabolites of each bacterial isolates were added to each well at various concentrations of 100%, 50%, 25%, 12.5%, 6.25%, and 3.125%. Wells with nematode eggs, having no metabolites, were considered as a negative control, while 0.025 mg/mL Oxfendazole (Zenith Pharma, Karachi, Pakistan), in 0.3% DMSO, served as a positive control. The plates were incubated at 37 ◦C for 24 h. A droplet of Lugol's iodine was added to maintain the process for 24 h. Under 10× of an inverted microscope, the total number of hatched and unhatched eggs were counted. The experiment was replicated five times [34].

The percent egg hatch inhibition was calculated as:

Inhibition of eggs = P test/P total × 100

P test: number of unhatched or hatched eggs. P total: number of unhatched or hatched eggs + Larvae (L1)

#### *2.6. Genomic DNA Extraction*

DNA from the abomasal bacteria were extracted by the standard protocol of the phenol chloroform method. Fresh bacterial broth cultures were centrifuged at 13,000 rpm for 5 min. The supernatant was discarded, and pellets were resuspended in 550 μL of Tris-EDTA buffer with the addition of 30 μL of 10% SDS and 5 μL Proteinase K. Vortexed properly and incubated at 37 ◦C for 1 h. After incubation, 100 μL of 5 M NaCl and 80 μL of CTAB/NaCl were added and mixed properly and incubated again at 65 ◦C in a water bath for 10 min. An equal volume of phenol, chloroform, and isoamyl alcohol (25:24:1) was added, mixed properly, and centrifuged at 13,000 rpm for 5 min to purify DNA. The supernatant was transferred to a 1.5 mL fresh Eppendorf tube. To this tube, an equal volume of chloroform and isoamyl alcohol, was added and centrifuged at 13,000 rpm for 5 min. The supernatant was transferred to a 1.5 mL Eppendorf tube, and for the precipitation of DNA, 0.6 volume of chilled isopropanol was added and placed at −20 ◦C for 20 min. DNA was pelleted by centrifugation at 13,000 rpm for 5 min and washed the pellet with 70% ethanol, and then dried at room temperature. Finally, DNA was dissolved in 50 μL Tris-EDTA buffer overnight incubation at 37 ◦C. The DNA concentration was measured using a spectrophotometer by taking absorbance at 260 nm and diluted for polymerase chain reaction (PCR).

#### 2.6.1. DNA Confirmation and 16S RNA Amplification

The purified genomic DNA was verified through gel electrophoresis by mixing 4 μL genomic DNA with 2 μL loading dye and then load it in 1% agarose gel. The gel was run in a gel tank for 30 min at 120 volts and observed under UV transilluminator for DNA band. The amplification of the 16S RNA gene, universal primers, as shown in Table 1, were run on genomic DNA samples.

**Table 1.** Primer name and Sequence.


2.6.2. PCR Conditions

The PCR tubes were put in the thermal cycle. The amplification was performed, following the condition given in Table 2.


**Table 2.** PCR condition for 16s RNA universal primers.

#### 2.6.3. Gel Electrophoresis for PCR Products

The PCR products were confirmed on 1.5% agarose gel in TBE buffer containing 3 μL of ethidium bromide and run for 30 min at 100 voltages and 300 milli Ampere current. The bands were visualized by the Gel Doc system [35].

#### *2.7. Statistical Analysis*

The information acquired from the bioassays, i.e., adult nematode mortality, nematode larval mortality, and eggs hatch inhibitions assays, were evaluated by P Test via Statistic version 9.

#### **3. Results**

#### *3.1. Isolation and Identification of Bacterial Isolates from Abomasal Contents*

Three different colonies were subcultured based on different morphological characteristics.

#### 3.1.1. Colony Morphology

The colony morphology of the bacterial isolates are shown in Table 3. The colonial morphology of the ABP bacterial isolate (Figure 3a appeared as irregular with an entire margin, and its elevation was flat and pinkish in color. Similarly, the colony morphology of ABY bacterial isolate was regular with a filamentous margin, and its elevation was flat and yellow. The colony morphology of the ABCW isolate was irregular in shape and creamy white, as shown in Figure 3b.

**Table 3.** Colony Morphology and Gram Staining of Bacterial Isolates.


**Figure 3.** (**a**) Bacterial Isolate ABP colony; (**b**) bacterial Isolate ABY and ABCW colony.

#### 3.1.2. Gram Staining

Gram staining revealed that all the bacterial isolates ABP, ABY, and ABCW, were Gram negative rods, as shown in Table 4.


#### 3.1.3. Biochemical Identification

Selected bacterial isolates ABP, ABY, and ABCW were identified by various biochemical tests as given in Table 4. Bacterial isolate ABP was negative for oxidase, catalase, and indole production, while positive for sugar fermenter and amotility test. The biochemical tests showed that the bacterial strain ABP was identified as *Comamonas testosteroni*. Bacterial isolate ABY was positive for catalase, oxidase test, and negative for motility test, indole, and sugar fermenter. Based on biochemical tests, the isolated bacterial strain ABY was reported as *Comamonas jiangduensis*. Bacterial isolate ABCW was positive for catalase and oxidase, while negative for sugar fermenter, motility test, and indole production. Based on the biochemical tests, the bacterial strain ABCW was considered as *Pseudomonas weihenstephanesis*.

#### *3.2. In Vitro Bioassay*

#### 3.2.1. Adult Mortality Assay (AMA)

Data regarding the mortality rate of *H. contortus* by various concentrations of bacterial culture filtrates are given in Table 5 and Figure 4. Bacterial isolate *C. testosteroni* caused the highest nematode mortality rate (100%) at 100% metabolite concentration. At 50% metabolite concentration, the nematode mortality was recorded as (46%). The lowest mortality rate (26%) was recorded at 6.25% metabolite concentration. Analysis of the data, regarding the adult nematode mortality by *C. jiangduensis*, showed that the bacterial metabolite had a significant effect on nematode mortality. The highest mortality rate (100%) was recorded at 100% concentration, followed by 50% metabolite concentration, where it was noted as 40%. While the minimum mortality rate (6%) was recorded at the metabolite concentration of 6.25%. As for the nematocidal effect of the bacterial isolate *P. weihenstephanesis* is concerned, the maximum adult nematode mortality rate (100%) was also recorded at 100% bacterial metabolite concentration. At 50% metabolite concentration, the mortality rate was recorded as 46%. The lowest adult nematode mortality of 6% was found at the lowest metabolite concentration, as with the case *C. jiangduensis*. The positive control in all cases showed the maximum activity of adult nematode mortality. While negative control shows no activity in all cases. Interestingly it was noted in all the above cases that the adult nematode mortality was found to be metabolite dosage-dependent.


**Table 5.** Adult nematodes mortality by metabolite concentrations of the bacterial isolates.

Note: *p*-value < 0.05 was considered significantly significant. NC: Negative Control; PC: Positive Control.

#### 3.2.2. Larval Mortality Assay (LMA)

Larval Mortality Assay of *H. contortus*, was carried out on 3rd stage larva (L3) by treating with different concentrations of metabolites extract from *C. testosteroni*, *C. jiangduensis*, and *P. weihenstephanesis* with an exposure time of six hours. The results are given in Table 6 and Figure 5. Analysis of the data revealed that the highest 100% metabolite concentration of *C. testosteroni* caused 100% L3 mortality, followed by 60% with 50% metabolite concentration. The minimum nematode larval mortality (13%) was reported to be caused by the bacterial metabolite at a 6.25% concentration.

**Figure 4.** Percent adult nematode mortality by the bacterial isolates, after six hours of treatment.



#### Note: *p*-value < 0.05 was considered significantly significant.

**Figure 5.** Percent nematode larval mortality by the bacterial isolates after six hours of treatment.

*Comamonas jiangduensis* showed 100% larval mortality at 100% bacterial metabolite concentration; at a 50% concentration the mortality was recorded as 46%, while the lowest dose (6.25%) of metabolite concentration caused the lowest (13%) mortality of L3 larvae. Analysis of the data showed that *P. weihenstephanesis* with 100% metabolite concentration caused 100% mortality of *H. contortus'* L3 larvae, which was followed by a 46% mortality rate, where 50% bacterial metabolite concentration was used. The lowest concentration of *P. weihenstephanesis* showed the lowest L3 mortality (20%), as in the case with the other

bacterial isolates. The 100% concentration of all the bacterial isolates and the positive control showed the maximum L3 larva mortality, while the negative control showed no activity at all.

#### 3.2.3. Egg Hatch Inhibition Assay (EHA)

The results of egg hatch inhibition assay are presented in Figure 6. Analysis of the data showed that all the bacterial culture filtrates showed similar (100%) nematode egg hatch inhibition at 100% metabolite concentration. It was noted that the 50% metabolite concentration of *C. testosteroni* and *C. jiangduensis* inhibited 100% *H. contortus* eggs from hatching, while the same concentration of *P. weihenstephanesis* caused 80% nematode's egg hatch inhibition. It was observed that lowering the bacterial metabolite concentration, lowers the egg hatch inhibition, and at the lowest metabolite concentration of 3.125%, showed the minimum (20%) egg hatch inhibition. The positive control (Oxfendazole) produced complete egg hatch inhibition, even at a very low concentration (0.025 mg/mL), while there was no egg hatch inhibition in the negative control.

**Figure 6.** Percent Inhibition of eggs hatching, three days after treatment.

#### *3.3. Genomic DNA Extraction*

16S rRNA genes verified the bacterial isolates, and each gene fragment was effectively amplified and sequenced by a polymerase chain reaction from their DNA. On the gel, isolated groups of molecular DNA were noted for separate bacterial isolates. The recognized protein marker below 1 kb size was compared for molecular weight. The pattern of the band, found on agarose gel, revealed that the bacterial isolates ABP, ABY, and ABCW were nearly comparable and about 1000 bp of molecular weight, as shown in the Figure 7. The nucleotide sequences of three isolates were compared with the sequences of nearly linked isolates of 16S rRNA genes. The result revealed that ABP bacterial isolate showed resemblance to *Comamonas testosteroni* was provided by the server and ABY bacterial isolate showed resemblance to *Comamonas jiangduensis*, while ABCW bacterial isolate showed resemblance to *Pseudomonas weihenstephanesis* as provided by the server (Figure 7 and Table 7).

**Figure 7.** DNA bands of the bacterial isolates.


**Table 7.** Molecular Identification of bacteria strain.

#### Phylogenetic Analysis

Two forward and two reverse sequences for each sample were aligned using Bionumerics v3.5 (Applied Maths) to obtain a composite sequence. The quality of each sequence trace was visually assessed, and the poor-quality sequence was edited and removed. Organisms were identified for each assay, by comparing consensus sequences to a database library of known 16S rRNA gene sequences in GenBank (http://www.ncbi.nlm.nih.gov/blast/Blast. cgi, accessed on 3 April 2021) by multiple sequence alignment. The bacterial source of the sequence was identified by matching it with a sequence with the highest maximum identity score from the GenBank database. Where more than one bacterial species had the same highest score, all species were recorded in the results (Figures 8 and 9). Sequences with 96% similarity to hits from the GenBank database were of poor quality and were excluded from this study (Figure 10).

The phylogenetic tree was constructed on the origin of 16S rRNA gene sequences for the bacterial isolates using MEGA 6 software. Phylogenetic analysis showed (Figure 11) that ABP was identified as *Comamonas testosteroni* and ABY *Comamonas jiangduensis*.



**Figure 8.** Sequence similarity of ABP with *Comamonas testosteroni*.



**Figure 9.** Sequence similarity of ABY with *Comamonas jiangduensis*.


**Figure 10.** Sequence similarity less than 96% of ABCW, and hence, excluded.

**Figure 11.** Phylogenetic Tree of *Comamonas testosteroni* (ABP) and *Comamonas jiangduensis* (ABY).

#### **4. Discussion**

*Haemonchus contortus* has a great financial significance causing serious disease and death of cattle and ruminants [2]. Resistance to the available antihelmintic drugs has become a severe threat to livestock production [7]. To decrease the use of chemical anthelmintics, an alternative method is a biocontrol by using bacteria and fungi against the nematode parasites. *Duddingtonia flagrans* is as wide as nematophagous fungi, which is being explored to regulate intestinal nematodes in livestock [32]. *Bacillus thuringiensis* is one of the most commonly used bacterial antagonists in biological control of *H. contortus* that may promote insecticidal crystal proteins, commonly used to control pests and also due to its low mammalian toxicity [23].

The current study tried to explore the bacterial abilities to reduce the population of *H. contortus* at eggs, larval and adult stages. Bacterial isolates were collected from the abomasum of small ruminants. Different bacterial culture filtrate concentrations (100%, 50%, 25%, 12.5%, 6.25%, and 3.125%) were prepared and applied on three life stages of *H. contortus* to observe the mortality of adult, larvae, and egg hatch inhibition. Higher concentration 100% and 50% of *C. testosteroni*, *C. jiangduensis*, and *P. weihenstephanesis* showed 100% nematode eggs hatch inhibition. To our knowledge, these bacterial isolates have never been used against *H. contortus.* Some researchers worked on the effect of *Bacillus*

*thuringiensis* on various life stages of *H. contortus* [30]. Earlier reports of leaf ethyl acetate and methanol extract of *A. squamosa*, *E. prostrata*, *S. torvum*, and *C. roseus* and acetone extract of *T. chebula* showed more consistent results on egg hatch inhibition of *H. contortus* [36].

Previous studies showed the larvicidal effect of various species of Bacillus, such as *Bacillus circulans* (Bcir), *B. thuringienisis* var. osvaldocruzi (Bto), *B. thuringienisis* var. israelensis (Bti), and *B. thuringienisis* var. kurstaki (Btk) on L3 stage of *Haemonchus* sp. among the tested bacteria, *B. circulans* and *B. thuringienisis* var. israelensis showed the best in vitro larvicidal efficiency of 90% and 94%, respectively, against the tested nematodes [37]. In our research studies, *H. contortus* larvae were treated with different bacterial metabolite concentrations, which cause 100% nematode mortality. These results are in line with the earlier reports on the control of nematodes in naturally infected sheep and goats, suggesting that the use of bacteria as an alternative control method for *H. contortus* larvae [21,37].

In the case of egg hatch inhibition assay, the highest bacterial culture filtrates of *C. testosteroni* and *C. jiangduensis* at 100% and 50% bacterial metabolite concentration, resulted in 100% inhibition of *H. contortus* eggs. The extract of the *Annona muricata* plant has also been used to inhibit the eggs hatch and mortality of *H. contortus* larvae and adults [38].

This study has extended the findings by showing that the tested bacteria species are effective against all stages of the nematode parasite. The metabolites of bacterial species *C. testosteroni* and *C. jiangduensis* showed a greater effect than filtrates, obtained from *P. weihenstephanesis*. Similarly, *C. jiangduensis* and *C. testosteroni* showed a higher mortality rate of L3 and adult nematodes as compared to *P. weihenstephanesis*. However, all the bacteria isolates showed a similar impact on eggs hatch inhibition. The positive control in all cases showed the maximum larva mortality, while the negative control exhibited no nematocidal activity at all. It was concluded that the larval and adult nematode mortality, as well as the nematode egg hatch inhibition, have a positive correlation with the doses or concentration of the metabolites, extracted from the bacterial isolates.

#### **5. Conclusion**

The present study was conducted to know the antihelmintic capabilities of metabolites extracted from abomasum bacteria *Comamonas testosteroni*, *C. jiangduensis*, and *Pseudomonas weihenstephanesis* against *H. contortus* eggs, larvae, and adults. It was noted that increasing the concentration of bacterial culture filtrates, increased nematode mortality. The same trend was observed in egg hatch inhibition assay bacterial culture filtrates. The effect of bacterial culture filtrates against *H. contortus* was found as dose-dependent. However, further in vivo bacterial culture filtrates investigation is recommended to seethe anthelmintic activity against various developmental stages of *H. contortus*.

**Author Contributions:** Data curation, B.K., R.U., I.A., A.B.; Formal analysis, F., I.A., T.A.K., A.E.A., A.F.E.-k., M.M.A.-D.; Funding acquisition, R.U., A.B., M.S., A.E.A., A.F.E.-k., M.M.A.-D.; Investigation, A.N., B.K., F., M.S., H.M.M.; Methodology, T.A.K.; Project administration, I.A., B.K., T.A.K., M.M.A.; Writing—review & editing, R.U., B.K., A.E.A., A.F.E.-k., M.M.A., M.S., B.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** The project financially supported by Taif University under Researchers Supporting Project number (TURSP-2020/82), Taif University, Taif, Saudi Arabia and the deanship of Scientific Research at King Khalid University, Abha, KSA, under grant number R.G.P.2/122/42.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All available data incorporated in the MS.

**Acknowledgments:** The authors acknowledge and greatly thankful for the financial support of Taif University Researchers Supporting Project number (TURSP-2020/82), Taif University, Taif, Saudi Arabia. This research was funded by the deanship of Scientific Research at King Khalid University, Abha, KSA, under grant number R.G.P.2/122/42.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

