Differences in antimicrobial resistance-related genes of Trueperella pyogenes between isolates detected from cattle and pigs

Abstract

We investigated antimicrobial resistance-related genes in 109 isolates of Trueperella pyogenes that were isolated in cattle and pigs. All 89 tetracycline-resistant T. pyogenes isolates carried the resistance gene harbored either tetW, tetM, tetA(33), tetK, or tetL. The ermX or ermB were detected in 18 of 23 erythromycin-resistant isolates. Streptomycin-resistant aadA1, aadA9, aadA11, aadA24, strA, or strB were detected in 25 of 83 isolates. There were significant differences in the percentages of tetA(33), ermB, aadA1, aadA9, aadA11, or aadA24 carriage between cattle and pig isolates. In addition, the Class 1 gene cassette was detected only in 17 cattle isolates. This suggests that T. pyogenes isolates acquire resistance gene in each environment of cattle and pigs, and that the transmission of the bacteria between cattle and pigs is limited.

Trueperella pyogenes is a Gram-positive irregular rod bacterium and a commensal organism on the mucous membranes of the upper respiratory tract, digestive tract, and urogenital tract in animals, exhibiting opportunistic pathogenicity [11]. This bacterium is known to cause various infections such as mastitis, metritis, pneumonia, abscesses, etc., affecting a wide range of domestic animals including pigs, cattle, goats, and sheep [10, 15]. In the treatment of infections caused by Gram-positive rods, including T. pyogenes, β-lactams, tetracyclines, macrolides, aminoglycosides, and fluoroquinolones are commonly used antibiotics. However, the widespread use of these antibiotics may contribute to the development of antimicrobial resistance in T. pyogenes [9, 10, 13, 15, 18].

In the meat inspections post-mortem examinations reveal verrucous endocarditis, and bacterial testing leads to a diagnosis of septicemia. The bacterial species detected in cases of verrucous endocarditis-type septicemia have characteristics depending on the livestock species. In our meat inspection, T. pyogenes was the only bacteria species detected from verrucous endocarditis in both cattle and pigs between April 2012 and March 2017. Among the bacterial strains isolated from verrucous endocarditis, T. pyogenes accounted for 47% (50 of 106 isolates) in cattle and 3% (22 of 664 isolates) in pigs (Unpublished data).

We have previously investigated the hemolytic properties, biochemical properties, and virulence gene carriage of T. pyogenes from verrucous endocarditis-type septicemia in cattle and pigs, and in doing so we observed differences between isolates from cattle and pigs [4]. Furthermore, T. pyogenes derived from verrucous endocarditis-type septicemia in cattle and pigs showed high rates of resistant to tetracycline, aminoglycoside streptomycin. Veterinary antimicrobial agents are commonly used to treat various infectious diseases of livestock and are important materials for protecting animal health and ensuring safe food production. However, there is always a risk of developing new antimicrobial resistance due to their use. Today, the treatment of bacterial infections in livestock is becoming increasingly complex due to the emergence and spread of various types of bacteria that have acquired resistance.

It has been reported that the tetW gene that confers tetracycline resistance in T. pyogenes is located on a transposon [8]. Two genes associated with the macrolide resistance, ermX and ermB, are carried by genetic elements in T. pyogenes, such as plasmids, transposons, or integrons [8]. It is found that integrons may also play an important role in the resistance of T. pyogenes to trimethoprim, chloramphenicol, aminoglycosides, and β-lactam antibiotics [2, 8, 9, 18]. Although these reports were based on isolates detected from clinical materials such as mastitis, endometritis, and abscesses, there have been few reports using bacterial isolates detected from verrucous endocarditis-type septicemia diagnosed by meat inspection. Therefore, in order to understand the genetic background of antimicrobial resistance in T. pyogenes, we decided to investigate the genetic similarities and differences between cattle and pig isolates detected from verrucous endocarditis-type septicemia in meat inspection. We investigated the presence of resistance genes involved in each antimicrobial agent and their association with the phenotype. In addition, the integrons involved in multidrug resistance were also investigated.

We used 109 T. pyogenes isolates that showed resistance to either tetracycline, erythromycin, or streptomycin as determined by drug susceptibility testing in a previous report [3]. Resistant isolates of each drug were detected in 100 cattle isolates from 88 farms and 67 pig isolates from 51 farms [3]. Forty-two tetracycline-resistant isolates were detected in 34 cattle farms and 47 isolates were detected in 39 pig isolates. Next, 8 erythromycin-resistant isolates were detected in 7 cattle farms and 15 isolates were detected in 13 pig farms. Furthermore, 42 streptomycin-resistant isolates were detected in 37 cattle farms and 41 isolates were detected in 36 pig farms. These isolates were detected from cattle or pigs with verrucous endocarditis-type septicemia during meat inspection between 2006 and 2017. T. pyogenes ATCC^®19411 (American Type Culture Collection, Manassas, VA, USA) was used as a reference strain in this study.

Tetracycline, erythromycin, or streptomycin resistance genes were detected by PCR using primers specific for resistance genes reported in Gram-positive bacteria including T. pyogenes (Supplementary Table 1). One of the streptomycin genes (aadA24) was originally designed using the NCBI program.

To prepare a PCR template, a frozen solution containing T. pyogenes was dropped into 1 mL of Mueller-Hinton broth and incubated at 37°C for 24–48 hr to obtain bacterial cultures. Genomic DNA was isolated using InstaGene™ Matrix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The reaction mixtures, in a final volume of 50 μL, contained 0.1 mM of each primer, 0.2 mM of each deoxynucleotide triphosphate (Takara, Kusatsu, Japan), 1.5 mM of MgCl₂ (Promega, Madison, WI, USA), 10X Ex Taq Buffer, 1.0 U of TaKaRa Ex Taq^®DNA Polymerase (Takara), and 1 μL of DNA template. PCR was performed in a TaKaRa PCR Thermal Cycler Dice^® Gradient and distilled water was used as a negative control. Amplification products were electrophoresed on 1.5% (w/v) agarose gel stained with 0.5 mg/mL ethidium bromide and visualized with a 2UV High Performance Transilluminator (Analytik Jena, Jena, Germany). The statistical significance of the results was established using Fisher’s exact test, and the levels of significance were set at P<0.05 and P<0.01.

The presence of class 1 and class 2 integrons was confirmed by detection of integrase genes encoded by IntI1 and IntI2, respectively. PCR was performed in the same manner as for the detection of antimicrobial resistance genes. The presence of gene cassettes was also investigated using the primers listed in Supplementary Table 1. The class 1 gene cassette region was amplified in the isolates that were positive for IntI1 using primers specific for the 5’- and 3’-conserved sequences [16]. PCR was aimed at detecting gene cassettes of a wide range of sizes, and extension time was set to 5 min. The nucleotide sequence inside the integron cassette was analyzed by an Applied Biosystems 3730xl DNA analyzer. All amplicons of the class 1 integron gene cassette were sequenced and a Basic Local Alignment Search Tool (BLAST) analysis was carried out on the National Center for Biotechnology Information (NCBI) Web site (http://blast.ncbi.nlm.nih.gov) [1].

Of the 89 tetracycline-resistant T. pyogenes isolates, 97.8%, 13.5%, 6.7%, 5.6%, and 3.4% carried the tetW, tetA(33), tetM, tetK, and tetL genes, respectively, but not the tetO gene. In comparing the percentage of cattle isolates and pig isolates, only the tetA(33) gene showed a difference in possession between cattle and pig isolates, suggesting that this gene is prevalent in the cattle farm environment, but not in pigs (Table 1).

Table 1. Percentage of Trueperella pyogenes isolates carrying tetracycline, erythromycin, or streptomycin resistance genes.

Antimicrobial	Resistance gene	Total (n)	Cattle (n)	Pigs (n)
Tetracycline Total (n=89) Cattle (n=42) Pigs (n=47)	tetW	97.8% (87)	95.2% (40)	100% (47)
	tetA(33)	13.5% (12)	**28.6% (12)	0.0% (0)
	tetM	6.7% (6)	9.5% (4)	4.3% (2)
	tetK	5.6% (5)	9.5% (4)	2.1% (1)
	tetL	3.4% (3)	4.8% (2)	2.1% (1)
	tetO	0.0% (0)	0.0% (0)	0.0% (0)
Erythromycin Total (n=23) Cattle (n=8) Pigs (n=15)	ermX	52.2% (12)	75.0% (6)	40.0% (6)
	ermB	26.1% (6)	0.0% (0)	*40.0% (6)
	ermA	0.0% (0)	0.0% (0)	0.0% (0)
	mefA/E	0.0% (0)	0.0% (0)	0.0% (0)
Streptomycin Total (n=83) Cattle (n=42) Pigs (n=41)	aadA1	6.0% (5)	*11.9% (5)	0.0% (0)
	aadA9	19.3% (16)	**38.1% (16)	0.0% (0)
	aadA11	6.0% (5)	*11.9% (5)	0.0% (0)
	aadA24	6.0% (5)	*11.9% (5)	0.0% (0)
	strA	3.6% (3)	7.1% (3)	0.0% (0)
	strB	6.0% (5)	4.8% (2)	7.3% (3)

**P<0.01, *P<0.05, significantly different between cattle and pig isolates by Fisher’s exact test.

Two isolates (2.2%) possessed three resistance genes: one isolate had tetW, tetK, tetA(33) and the other isolate had tetW, tetL, tet(33). Twenty isolates (22.5%) possessed the two resistance genes: 9 (10.1%) of the isolates had tetW and tetA(33), 6 isolates (6.7%) had tetW, tetM, 3 isolates (3.4%) had tetW, tetK, and 2 isolates (2.2%) had tetW, tetL. Sixty-seven isolates (75.3%) possessed one resistance gene: 65 isolates (73.0%) had tetW, and 1 isolate (1.1%) each had tetK and tetA(33) (Supplementary Table 2-1). The results showed that all tetracycline-resistant isolates had at least one resistance gene, suggesting a strong association between resistance genes and phenotype. In this study, tetL, tetM, and tetK were detected in cattle and pig isolates. Since resistance genes have been detected in a wider range than in the previous report [7], it is necessary to continue to pay attention to resistance genes other than tetW. In addition, two to three resistance genes were detected in some isolates, suggesting that a variety of tetracycline resistance genes are widely present in the cattle and pig breeding environment. Furthermore, tetM, tetK, and tetL have been detected in pig isolates, although in small numbers. To our knowledge, this is the first time that these three genes have been detected in pig T. pyogenes isolates. In this study, five resistance genes were detected, which is more than previously reported. Although the number of isolates was small, the isolates harboring tetK and tetA(33) alone had low minimum inhibitory concentration (MIC) values of 4 and 8, respectively. However, there was no clear relationship between the number and type of resistance genes possessed by each isolate and MIC values (Supplementary Table 2-1). In their report, Zhang et al. examined the resistance genes possessed and the types of tetracyclines to which they were resistant [17], and it will be necessary to investigate phenotypes with other antimicrobial agents as well.

The ermX gene was identified in 52.2% of the erythromycin-resistant isolates. The ermB gene was identified in 26.1% of erythromycin-resistant isolates (Table 1). In this study, no isolates were found that harbored both ermX and ermB simultaneously. None of the isolates harbored the ermA and mefA/E genes. Of the 23 isolates resistant to erythromycin, 21.7% (5 isolates) were not found to harbor any of the resistance genes investigated in this study. The ermX and ermB genes have been previously reported in T. pyogenes [5, 6], and were frequently detected in this study. Moreover, ermX gene was detected in T. pyogenes isolates from both cattle and pigs, but the ermB gene was detected only in isolates detected from pigs. But, the ermB gene is not unique to pigs, as it has been detected in clinically derived cattle isolates in Poland [16]. The failure to identify the ermB gene in this study may be due to the small number of cattle isolates that were resistant to erythromycin. The percentage of erythromycin resistance was slightly lower in cattle isolates than in previous reports, but was almost the same in pig isolates [9, 11, 16]. Because oral antimicrobials in pigs have been used prominently among macrolide antimicrobials in Japan, it is likely that, erythromycin resistance genes are widespread in the pig farm environment [14]. Some isolates in which none of the four resistance genes were detected showed high MIC values, and a broader range of resistance genes should be investigated for erythromycin resistance in T. pyogenes (Supplementary Table 2-2).

Among the 83 streptomycin-resistant isolates, aadA1, aadA9, aadA11, aadA24, strA, and strB genes were detected from 6.0%, 19.3%, 6.0%, 6.0%, 3.6%, and 6.0% isolates, respectively. The aadA1, aadA9, aadA11, aadA24, and strA genes were detected only in cattle isolates, while the strB gene was detected in both cattle and pig isolates (Table 1). One isolate (1.2%) possessed the four resistance genes: aadA1, aadA9, aadA11, aadA24. Four isolates (4.8%) possessed the three resistance genes: aadA1, aadA11, aadA24. As for two types of gene possession, 2 (2.4%) of the isolates had aadA9 and strA, 1 (1.2%) had aadA9 and strB for a total of 3 (3.6%). Of the 83 resistant isolates, 25 (30.1%) were found to harbor at least one streptomycin resistance gene. However, the remaining 58 isolates (69.9%) did not have the resistance genes investigated in this study (Supplementary Table 2-3). Of the 42 streptomycin-resistant cattle isolates, 54.8% were found to have at least one resistance gene. On the other hand, of the 41 streptomycin-resistant pig isolates, only 7.3% had the resistance gene. Aminoglycoside antimicrobials have been used for a long time, and overuse and misuse are believed to cause resistance [2]. In this study, all six target genes for streptomycin resistance were detected in T. pyogenes isolates from cattle, suggesting that a wide range of resistance genes exists in the cattle farm environment. The percentage of streptomycin-resistant isolates acquired was slightly lower in cattle isolates than in clinical isolates, and almost the same in pig isolates. There were five resistance gene harbors that were detected only in cattle isolates. A large number of isolates did not possess any of the streptomycin-resistant isolates investigated in this study. This suggests that resistance genes other than those investigated may be involved in the streptomycin resistance phenotype. Further investigation of a wider range of resistance genes was considered necessary.

The IntI1 associated with class 1 integron was detected in 72 (66.1%) T. pyogenes isolates, while the IntI2 associated with class 2 integron was not detected. This suggests that IntI2 is not involved in either cattle or pig T. pyogenes isolates. IntI1 was present in 80.0% (48 of 60) in cattle isolates and 49.0% (24 of 49) in pig isolates; the prevalence in cattle was significantly higher than that in pigs. The class 1 gene cassette was detected in 17 of 48 cattle isolates harboring IntI1, but it was completely absent in pig isolates. Notably, compared to pig isolates, the IntI1 gene was detected in significantly higher rate in cattle isolates. Additionally, the class 1 gene cassette was detected in the cattle isolates, which contained a streptomycin resistance gene inside. This suggested that the streptomycin resistance gene was transmitted via integrons between cattle isolates. On the other hand, the streptomycin resistance genes were detected less frequently in pig isolates than in cattle isolates and no gene cassettes were detected in the pig isolates in this study. This suggests that the spread of streptomycin resistance via integrons is less likely in pigs. In our previous study, the streptomycin resistance rate in pig isolates was significantly higher than in cattle isolates [3]. This is inconsistent with the detection rate of streptomycin resistance genes in cattle and pigs in this study. Further research is needed on the acquisition of streptomycin resistance in pig isolates.

Three types of gene cassettes were identified in this study: 12 strains had gene cassettes with a size of approximately 1.0 Kbp (pattern 1), 4 strains had gene cassettes with a size of approximately 1.5 Kbp (pattern 2), and 1 isolate had gene cassettes with both approximately 1.0 and 1.5 Kbp (Supplementary Fig. 1). The sequence of the gene casette in pattern 1 is homologous to MN171325.1 in GenBank reported by Kwiecien et al [8]. This casette contains the aadA9 gene (837 bp) encoding the ant (3”)-Ia family aminoglycoside nucleotidyltransferase. The nucleotide sequence included in pattern 2 was homologous to aadA24 in T. pyogenes (GenBank: FJ655779.1) reported by Liu et al. [9], and aadA24 in Salmonella enteritidis (GenBnk:AM711129.1) reported in Spain [12]. The 1.5 kb cassette contained an unknown protein in addition to aadA24. This sequence was identified as a “hypothetical protein” by a BLAST search, but its role could not be elucidated (Fig. 1). The gene cassette of T. pyogenes shows the possibility of harboring resistance genes from other bacteria, suggesting that this bacterium is involved in the spread of antimicrobial resistance genes in the field. In this study, the cattle isolates had a greater variety of resistance genes than the pig isolates. In our previous report [3], tetracycline, erythromycin, or streptomycin resistance was found more frequently in pig isolates than in cattle isolates. Because cattle are kept on a smaller scale and have a more diverse breeding environment than pigs, a greater variety of resistance genes may have been detected in the cattle isolates. In particular, mobile class 1 gene cassettes were detected only in cattle isolates, suggesting that resistance genes may spread more frequently in cattle isolates than in pig isolates.

Fig. 1.

Schematic diagram of the class 1 integron structure detected from Trueperella pyogenes. Designations: intI, the integrase gene; attI, the recombination site of integrase; aadA9 and aadA24, the streptomycin resistance genes; attC, the recombination site of cassette; qacEΛ1, the quaternary ammonium compounds resistance gene; sulI, the sulfonamide resistance gene; orf5, a gene of unknown function; P, promoter; Pc, cassette promoter; Pint, integron promoter.

This study revealed differences in antimicrobial resistance-related genes between T. pyogenes isolates detected from cattle and pigs. We confirmed the existence of a resistance gene that had not been previously reported on T. pyogenes. This finding may have contributed to the elucidation of drug resistance mechanisms. The resistance genes in cattle isolates were more diverse than in pig isolates, suggesting that the mechanisms of resistance acquisition are different between cattle and pig isolates. In addition, T. pyogenes isolates acquired resistance genes in both cattle and pig environments, suggesting that transmission of the bacteria between cattle and pigs is limited. Integron structures were only detected in cattle isolates and not in pig isolates, and to our knowledge, this is the first report of a T. pyogenes isolates with two integrons, each containing a different gene cassette. This suggests that T. pyogenes isolates may have the ability to acquire a wide range of resistance genes via integrons. T. pyogenes will continue to require close monitoring for the spread of antimicrobial resistance genes.

CONFLICTS OF INTEREST

This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors.