Antimicrobial Resistance in Corynebacterium spp., Arcanobacterium spp., and Trueperella pyogenes

ABSTRACT

There is currently only limited information on the antimicrobial susceptibility and resistance of Corynebacterium spp., Arcanobacterium spp., and Trueperella pyogenes from animals. The comparability of the data is hampered by the use of different antimicrobial susceptibility testing methods and interpretive criteria. To date, standard broth microdilution methods and clinical breakpoints that are approved by the Clinical and Laboratory Standards Institute and are applicable to Corynebacterium spp., Arcanobacterium spp., and T. pyogenes are available. The lack of species-specific clinical breakpoints for the different animal species reduces the explanatory power of the data. Among the isolates of the three genera, elevated MICs for different classes of antimicrobial agents (e.g., β-lactams, macrolides, lincosamides, tetracyclines, aminoglycosides, phenicols, sulfonamides/diaminopyrimidines, and fluoroquinolones) have been described. The most comprehensive data set is available for T. pyogenes, which also includes information about genes and mutations involved in antimicrobial resistance. In T. pyogenes isolates, the macrolide-lincosamide-streptogramin B resistance genes erm(B) and erm(X) were identified. Tetracycline resistance in T. pyogenes was based on the resistance genes tet(W), tet(Z), and tet(33), whereas the aminoglycoside resistance genes aacC, aadA1, aadA2, aadA5, aadA24, and aadB have been described in T. pyogenes. So far, only single genes conferring either phenicol resistance (cmlA6), trimethoprim resistance (dfrB2a), or β-lactam resistance (blaP1) are known to occur in T. pyogenes isolates. Various 23S rRNA mutations, including A2058T, A2058G, and G2137C, were identified in macrolide/lincosamide-resistant T. pyogenes.

THE GENERA CORYNEBACTERIUM, ARCANOBACTERIUM, AND TRUEPERELLA

The genus Corynebacterium was introduced in 1896 by Lehmann and Neumann. It belongs to the family Corynebacteriaceae and was listed with 30 species in the “approved lists of bacterial names” from 1980 (1) including also later reclassified species. To date, the genus Corynebacterium includes more than 90 species (www.bacterio.net), some of which play a role as pathogens in veterinary medicine. Corynebacteria are Gram-positive, pleomorphic rods that grow as small white colonies on blood agar after 24 to 48 h of incubation (2). In veterinary medicine, Corynebacterium pseudotuberculosis and the Corynebacterium renale complex are the most widespread bacterial pathogens. C. pseudotuberculosis causes caseous lymphadenitis in goats and sheep; ulcerative lymphangitis in horses, cattle, and sheep; as well as mastitis in cattle (2, 3). C. renale is associated with urinary tract infections in ruminants and pigs (2).

The genus Arcanobacterium was introduced in 1982 by Collins et al. (4) and belongs to the family Actinomycetaceae, which includes eight species: Arcanobacterium canis, Arcanobacterium haemolyticum, Arcanobacterium hippocoleae, Arcanobacterium phocae, Arcanobacterium phocisimile, Arcanobacterium pinnipediorum, Arcanobacterium pluranimalium (www.bacterio.net), and the recently described Arcanobacterium wilhelmae sp. nov. (5). Arcanobacteria are facultatively anaerobic, and their growth is enhanced by blood or serum (4).

The comparatively new genus Trueperella also belongs to the family Actinomycetaceae. In 2011, Yassin et al. (6) published a comparative chemotaxonomic and phylogenetic study of the genus Arcanobacterium and showed that it is not monophyletic. As a consequence, the taxonomy was revised and the new genus Trueperella established. It currently comprises five species—namely, Trueperella abortisuis, Trueperella bernardiae, Trueperella bialowiezensis, Trueperella bonasi, and Trueperella pyogenes (www.bacterio.net). Bacteria of the genus Trueperella are Gram-positive, pleomorphic rods that grow under facultatively anaerobic conditions on blood agar and produce hemolytic colonies of 0.5 to 1 mm after 48 h (2). Among the genus Trueperella, T. pyogenes is the most important veterinary pathogen, being commonly involved in a wide variety of diseases in domestic animals, including mastitis, pneumonia, metritis, arthritis, lymphadenitis, otitis, peritonitis, pyodermatitis, endocarditis, abscesses, osteomyelitis, and urinary and genital tract infections, with bovine mastitis being the most common disease in livestock (7).

SUSCEPTIBILITY TESTING OF CORYNEBACTERIUM, ARCANOBACTERIUM, AND TRUEPERELLA

As outlined in reference 8, antimicrobial susceptibility testing (AST) has to follow an internationally accepted performance standard. For bacteria of animal origin, the standards of the Clinical and Laboratory Standards Institute (CLSI) are most frequently used worldwide. So far, there is only a standard broth microdilution method approved for “Corynebacterium spp. (including Corynebacterium diptheriae) and related coryneform genera” from humans, which is described in the human-specific CLSI document M45 (9). For isolates of “Corynebacterium spp. and Coryneforms” from animals, there is also only a broth microdilution method available in the recently published veterinary document VET06 (10). The “related coryneform genera” or “Coryneforms” include the genera Arcanobacterium, Arthrobacter, Brevibacterium, Cellulomonas, Cellulosimicrobium, Dermabacter, Leifsonia, Microbacterium, Oerskovia, Rothia (excluding Rothia mucilaginosa), Trueperella, and Turicella (9, 10). It should be noted that all clinical breakpoints listed for these bacteria in the aforementioned documents are from human medicine. Since pharmacological properties of antimicrobial agents may differ between humans and animals, the use of breakpoints adopted from human medicine may result in misclassifications of veterinary isolates (11). A specific method for AST of T. pyogenes of animal origin has been developed (12) and included in the VET06 document. Based on MIC distributions (12), breakpoints for the category “susceptible” have been proposed for penicillin, ampicillin, erythromycin, and trimethoprim-sulfamethoxazole (10).

Since studies of the susceptibility of coryneform bacteria date back to the 1960s, a variety of susceptibility testing methods and interpretive criteria has been used, making it difficult—if not impossible—to compare the results of the different studies (Table 1) (11). Another problem arises with studies that only report the classification as susceptible, intermediate, or resistant without indicating quantitative values or the interpretive criteria used. These studies were not included in the description of resistance properties given below. Moreover, an evaluation of data obtained by agar disk diffusion is not possible because no interpretive criteria for this method are available in the current CLSI documents M45 and VET06 (9, 10). Therefore, these studies have also been excluded from the analysis of the resistance properties.

TABLE 1.

Examples of different AST methods and test conditions

Method	Bacterial species	Medium	Temperature	Incubation time	Reference
Broth microdilution	Arcanobacterium spp., Corynebacterium spp., Trueperella spp. (except T. pyogenes)	Cation-adjusted Mueller-Hinton broth + 2.5 to 5% lysed horse blood	35 ± 2°C	24–48 h ambient air	9, 10
	T. pyogenes	Cation-adjusted Mueller-Hinton broth + 2.5 to 5% lysed horse blood	35 ± 2°C	20–24 h with 5% CO2	10
	C. camporealensis, C. bovis, C. mastitidis. C. pseudotuberculosis, C. pseudodiptheriticum, T. pyogenes	Mueller-Hinton broth + 1% Tween 80	37°C	48 h	14
	T. pyogenes	Mueller-Hinton broth + TES biological buffer + lysed horse blood and 10% fetal calf serum	35–37°C	18–24 h	46
	T. pyogenes	Serum-free medium	39°C	36 h with 5% CO2	38
	T. pyogenes	Modified chopped meat medium	37°C	24 h anaerobically	37
Agar dilution	C. pseudotuberculosis	Mueller-Hinton agar + 5% laked horse blood	37°C	Overnight	19
	T. pyogenes	Mueller-Hinton agar + 5% defibrinated horse blood	37°C	48 h	55
	T. pyogenes	Mueller-Hinton agar + 5% fetal calf serum	37°C	24 h with 5% CO2	57
	C. pseudotuberculosis, T. pyogenes	Mueller-Hinton agar + 0.001% NAD + 5% chocolatized calf blood	37°C	24 or 48 h in air	20
	T. pyogenes	Iso-Sensitest agar + 7% hemolyzed sheep blood	36°C	1–2 days	53
Agar disk diffusion	A. haemolyticum, A. pluranimalium	Mueller-Hinton agar + 5% sheep blood	37°C	48 h (candle jar)	34, 35
	T. pyogenes	Mueller-Hinton agar with 0.5% sheep blood and 0.5% Tween 80		48 h	7
	T. pyogenes	Columbia agar with 5% sheep blood	37°C	Overnight 5% CO2	64
	C. ovis, T. pyogenes	Bacto tryptose blood agar plates	37°C	24 h	22
Calgary Biofilm Device	C. renale, C. pseudotuberculosis, T. pyogenes	Tryptic soy broth + 2% fetal calf serum	37°C	24 h + 10% CO2	21

RESISTANCE PROPERTIES OF CORYNEBACTERIUM SPP.

AST studies of Corynebacterium spp. are available for at least the past 50 years, but varying AST methods, test media, incubation times and conditions, as well as interpretive criteria have been used. Antimicrobial susceptibility data have been obtained by broth microdilution in six studies (13–18), with five of them referring to a methodology approved by CLSI or its previous organization, the National Committee for Clinical Laboratory Standards (NCCLS) (Table 2) (13–15, 17, 18). One of these studies reported all 45 canine Corynebacterium ulcerans isolates as susceptible to 14 antimicrobial agents without giving exact MIC values (17). The same situation was seen in a study of equine Corynebacterium pseudotuberculosis, which described all isolates as susceptible to a panel of 16 antimicrobial agents (15). Agar dilution tests were performed in three studies, conducted by Adamson et al. (19) using Mueller-Hinton agar supplemented with 5% laked horse blood, Judson and Songer (3) using blood agar base supplemented with 3% citrated bovine blood, and Prescott and Yielding (20) using Mueller-Hinton agar supplemented with 0.001% NAD and 5% chocolatized calf blood. Moreover, Olson et al. (21) measured MICs and minimum bactericidal concentrations using the Calgary Biofilm Device. Ten studies (22–31) described the use of agar disk diffusion. Although five studies referred to NCCLS or CLSI methods (24, 28–31), to date there is no approved agar disk diffusion method for corynebacteria, resulting in a lack of approved interpretive criteria.

TABLE 2.

Corynebacterium spp. AST data determined by broth microdilution according to CLSI/NCCLS standards

Animal origin	Bacterial species	Disease	Years of isolation	Number of isolates tested	MICs (in mg/liter) of selected antimicrobial agentsa									Reference
					PEN	AMP	ERY	CLI	TET	CHL	ENR	SXT	GEN
Cattle	C. bovis	Mastitis		46		≤0.06–0.25	≤0.06–≥128	0.125–0.5	0.125–≥64		≤0.03–≥64			13
Cattle	C. amycolatum	Mastitis		13		≤0.06–0.25	≤0.06–≥128	0.25–64	0.125–32		0.06–0.25			13
Sheep	C. camporealensis	Mastitis		4	0.06		0.125		2	4			0.008–0.125	14
Sheep	C. bovis	Mastitis		4	0.05		0.06–4		1	4			0.008–0.125	14
Sheep	C. pseudotuberculosis	Mastitis		10	0.125		0.25–2		1	2			0.5	14
Sheep	C. pseudodiptheriticum	Mastitis		13	0.5		0.06–1		0.5–8	2			0.07–0.5	14
Sheep	C. mastitidis	Mastitis		14	0.06–0.5		0.06–16		1	0.03–2			0.008–0.125	14
Horses (n = 49), cattle (n = 4), sheep (n = 1)	C. pseudotuberculosis	Infections (internal, external abscesses)	2000–2003	54										15
Dogs	C. ulcerans	Throat swabs	2007–2008	45				2						17
Horses	C. pseudotuberculosis	Naturally infected	1996–2012	b	≤0.06–4 (n = 178)	≤0.25–16 (n = 204)	≤0.12–2 (n = 146)		≤0.25–2 (n = 148)	≤0.25–4 (n = 203)	≤0.06–4 (n = 182)	≤0.25–4 (n = 203)	≤0.25–8 (n = 206)	18

^a PEN, penicillin; AMP, ampicillin; ERY, erythromycin; CLI, clindamycin; TET, tetracycline; CHL, chloramphenicol; ENR, enrofloxacin; SXT, trimethoprim/sulfamethoxazole; GEN, gentamicin.

^b In this study, not all isolates were tested for susceptibility to all antimicrobial agents; thus, the numbers of isolates tested are given for each antimicrobial agent.

Resistance to β-Lactams

Rhodes et al. (18) determined a ceftiofur MIC₉₀ of 2 mg/liter for equine C. pseudotuberculosis isolates, which the authors considered a poor choice for treatment since plasma concentrations of 2 mg/liter could not be achieved for >50% of the dosing interval using the labeled dosage. In comparison, ampicillin (MIC₉₀: 0.5 mg/liter) was considered a good choice for intravenous administration, but the abscess penetrability needs to be considered due to the low lipid solubility of ampicillin (18). For penicillin, Rhodes et al. (18) measured a MIC₉₀ value of 0.25 mg/liter, which is below the plasma concentrations that can be reached via intramuscular injection for adequate duration. The current CLSI breakpoints classify isolates with MICs of 0.25 to 2 mg/liter as intermediate (10). Watts and Rossbach (13) determined MIC₉₀ values for Corynebacterium bovis from bovine mastitis of 0.25 mg/liter (ampicillin), 4 mg/liter (oxacillin), 0.5 mg/liter (cephalothin), and 0.5 mg/liter (ceftiofur). Except for oxacillin with a slightly lower MIC₉₀ of 2 mg/liter, Corynebacterium amycolatum isolates had the same values as the C. bovis isolates (13). Fernández et al. (14) investigated corynebacteria from mastitis in ewes including the species Corynebacterium camporealensis, C. bovis, C. pseudotuberculosis, Corynebacterium pseudodiphteriticum, and Corynebacterium mastitidis. The penicillin MICs ranged from 0.06 to 0.5 mg/liter, resulting in C. pseudodiphteriticum and C. mastitidis isolates, with MICs of 0.5 mg/liter being classified as intermediate according to the CLSI (10, 14). The MIC values ranged from 0.03 to 8 mg/liter for amoxicillin and cephalothin, and from 8 to 16 mg/liter for ceftazidime (14).

Resistance to Macrolides and Lincosamides

In their study of equine C. pseudotuberculosis isolates, Rhodes et al. (18) determined MIC₉₀ values of ≤0.25 mg/liter, ≤1 mg/liter, and ≤0.25 mg/liter for azithromycin, clarithromycin, and erythromycin, respectively. The use of macrolides for treatment was considered appropriate for Corynebacterium abscesses or lymphangitis due to the lipophilicity of the drugs (18). For C. bovis and C. amycolatum, macrolide and lincosamide MIC values are available for bovine mastitis isolates, ranging from ≤0.06 to 64 mg/liter (13). While the MIC₉₀ values for erythromycin, clindamycin, and pirlimycin did not exceed 0.5 mg/liter, the MIC₉₀ values for tilmicosin were ≥64 mg/liter for C. bovis and 32 mg/liter for C. amycolatum (13). In some of the isolates, Fernández et al. (14) found elevated MICs of up to 16 mg/liter and 128 mg/liter for erythromycin and lincomycin, respectively. When applying the CLSI breakpoints for erythromycin, isolates with MICs of ≥2 mg/liter are classified as resistant (10, 14).

Resistance to Tetracyclines

MIC₉₀ values of 2 mg/liter for tetracycline and ≤2 mg/liter for doxycycline were determined for equine C. pseudotuberculosis isolates (18). Despite the lipophilic character of the drugs and the attainable plasma concentration, the authors recommend a treatment only for isolates with MICs of up to 0.25 mg/liter (18). Due to the different test panels used, only part of the collection from Rhodes et al. (18) (isolates from 2007 to 2012) was tested for lower doxycycline concentrations than 2 mg/liter. A MIC₉₀ value of ≤0.25 mg/liter was determined for these isolates (18). For the C. bovis and C. amycolatum isolates from the strain collection investigated by Watts and Rossbach (13), tetracycline MIC₅₀ values of 0.25 mg/liter were determined for both species, whereas tetracycline MIC₉₀ values of 0.25 mg/liter and 16 mg/liter were seen for C. bovis and C. amycolatum, respectively. It should be noted that isolates with MICs of ≥32 mg/liter were seen in both species (13), suggesting the presence of resistant isolates (10). Fernández et al. (14) found a single Corynebacterium pseudodiphtheriticum isolate that was classified as intermediate to tetracycline when applying CLSI breakpoints (10).

Resistance to Aminoglycosides

For gentamicin, Rhodes et al. (18) found a MIC₉₀ value of 2 mg/liter, while the MIC₉₀ value for amikacin was 8 mg/liter. Since hydrophilic characteristics of the drugs complicate the treatment of abscesses, aminoglycosides are not a first choice for the treatment of corynebacterial infections (18). The corynebacterial isolates from mastitis in ewes had kanamycin MICs ranging from 0.06 to 16 mg/liter and gentamicin MICs from 0.008 to 0.5 mg/liter, suggesting a reduced susceptibility for kanamycin in a single C. mastitidis isolate with a kanamycin MIC of 16 mg/liter, whereas all isolates were gentamicin-susceptible when applying the CLSI breakpoints (10, 14).

Resistance to Phenicols

Equine C. pseudotuberculosis isolates had chloramphenicol MIC₅₀ and MIC₉₀ values of 2 mg/liter and ≤4 mg/liter, respectively (18). Among C. bovis isolates, florfenicol MIC₅₀ and MIC₉₀ values of 1 mg/liter and 2 mg/liter, respectively, have been detected (13). In comparison, the C. amycolatum isolates had MIC₅₀ and MIC₉₀ values of 32 mg/liter each for florfenicol (13). The chloramphenicol MICs of the corynebacteria from mastitis in ewes ranged from 0.03 to 4 mg/liter (14).

Resistance to Sulfonamides and Diaminopyrimidines

C. pseudotuberculosis from horses had a MIC₉₀ value of 0.5/9.5 mg/liter for trimethoprim/sulfamethoxazole, and isolates with MICs of up to 4/76 mg/liter were also detected (18). According to the CLSI breakpoints, isolates with trimethoprim/sulfamethoxazole MICs of ≥4/76 mg/liter are classified as resistant (10). Fernández et al. (14) found trimethoprim MICs between 1 and 128 mg/liter, while the MICs for sulfisoxazole ranged from 0.03 to 64 mg/liter.

Resistance to Fluoroquinolones

For equine C. pseudotuberculosis isolates, the achievable plasma concentrations in horses were above the enrofloxacin MIC₉₀ value of 0.25 mg/liter (18). Only 2 of the 92 isolates had MIC values of >0.5 mg/liter (18). Enrofloxacin MIC₉₀ values of 0.25 mg/liter were detected for C. bovis and C. amycolatum isolates from bovine mastitis (13). For the other fluoroquinolones tested—namely, sarafloxacin, danofloxacin, and premafloxacin—the MIC₉₀ values ranged from 0.015 to 0.5 mg/liter (13). Ciprofloxacin MICs of the corynebacterial isolates from ewes ranged between 0.03 and 3 mg/liter (14), indicating the presence of resistant isolates of C. camporealensis, C. bovis, C. pseudotuberculosis, C. pseudodiptheriticum, and C. mastitidis when applying the breakpoints of the human-specific CLSI document M45 (9).

Resistance to Other Antimicrobial Agents

Vancomycin resistance could not be detected in corynebacteria from mastitis in ewes when using CLSI breakpoints (10, 14). Rifampicin resistance was not detected in corynebacteria from ewes with mastitis (14) when using the CLSI breakpoints (10). The rifampicin MIC₉₀ value obtained by Rhodes et al. (18) was ≤1 mg/liter, which would also classify the respective isolates as susceptible (10).

RESISTANCE PROPERTIES OF ARCANOBACTERIUM SPP.

In veterinary medicine, arcanobacteria are of minor importance, and susceptibility data are limited to A. phocae isolates from marine mammals obtained from tissue sites with abnormal discharge or signs of inflammation (32), an odontogenic abscess from a rabbit (33), A. pluranimalium isolates from a dog with pyoderma (34), and A. haemolyticum isolates from diseased horses (35). While the study by Tyrrell and coworkers (33) used agar dilution as the AST method, the latter two studies (34, 35) used agar disk diffusion, another method currently not approved for Arcanobacterium spp. by the CLSI.

During 1994 to 2000, Johnson et al. (32) investigated A. phocae isolates from marine mammals, such as sea lions, harbor seals, elephant seals, sea otters, and a dolphin, from the central California coast and tested 18 A. phocae isolates for their antimicrobial susceptibility by broth microdilution. They followed document M31-A from the NCCLS. However, the breakpoints given in this document are for a variety of livestock and companion animals but not specifically for marine mammals. Consequently, the reliability of the classification of these isolates as being susceptible, intermediate, or resistant is questionable.

The A. phocae isolates from marine mammals were tested for their susceptibility to β-lactams including penicillin (MICs: ≤0.03 to 0.12 mg/liter), ampicillin (MICs: ≤0.25 mg/liter), amoxicillin/clavulanic acid (MICs: ≤2 mg/liter), ticarcillin/clavulanic acid (MICs: ≤8 mg/liter), oxacillin (MICs: ≤2 mg/liter), cefazolin (MICs: ≤2 mg/liter), ceftiofur (MICs: ≤0.06 to 0.5 mg/liter), and ceftizoxime (MICs: ≤0.5 to 4 mg/liter) (32). When re-evaluating the results using the clinical breakpoints for penicillin (10), the isolates would still be classified as susceptible. The erythromycin and tetracycline MICs of all A. phocae isolates from marine mammals were ≤0.12 mg/liter and ≤0.5 mg/liter, respectively (32). They were classified as susceptible to erythromycin and tetracycline, which is in accordance with the current CLSI-approved breakpoints (10). Testing of the aminoglycosides amikacin and gentamicin yielded MIC values of ≤0.5 to 16 mg/liter and ≤0.25 to 4 mg/liter for the A. phocae isolates. All isolates were considered susceptible, based on the NCCLS breakpoints of ≤64 mg/liter for amikacin and ≤16 mg/liter for gentamicin. The recent CLSI document classifies isolates with gentamicin MICs of 8 mg/liter as intermediate and ≥16 mg/liter as resistant (10). The chloramphenicol MIC values of the A. phocae isolates from marine mammals ranged between ≤0.25 and 1 mg/liter, and thus, all isolates were classified as susceptible (32). The A. phocae isolates with MICs of ≤0.25/4.75 mg/liter were classified as susceptible to the combination trimethoprim/sulfamethoxazole (10, 32). The enrofloxacin MICs of the A. phocae isolates varied between ≤0.25 and 1 mg/liter (32). The rifampicin MICs were ≤0.12 mg/liter, which classified the isolates as susceptible (32). This is in accordance with the current clinical breakpoints for corynebacteria and related species (10).

RESISTANCE PROPERTIES OF T. PYOGENES

T. pyogenes is an important pathogen in veterinary medicine, commonly involved in various diseases of domestic animals (7). As already mentioned, the susceptibility testing method for corynebacteria and related species also includes Trueperella spp. (9, 10). However, a broth microdilution susceptibility testing method for T. pyogenes of animal origin has been developed (12) and has been included in CLSI document VET06 (10). Based on the MIC distributions shown (12), clinical breakpoints for penicillin, ampicillin, erythromycin, and trimethoprim/sulfamethoxazole have been proposed (10).

In 17 studies the antimicrobial susceptibility of T. pyogenes was tested using broth microdilution (12, 14, 36–50), with 14 of them referring to NCCLS or CLSI methodology (Table 3) (12, 14, 36, 40–50). Eight studies used agar dilution as the susceptibility testing method (20, 51–57), with three of them referring to NCCLS or CLSI (53, 54, 57), and in another two studies the MICs were determined by the Calgary Biofilm Device or E-test (21, 54). The agar disk diffusion method was used in 14 studies (7, 22, 58–69). Despite the lack of an accepted agar disk diffusion method and the respective interpretive criteria for T. pyogenes, five studies referred to CLSI or NCCLS methods (7, 63, 65, 66, 68).

TABLE 3.

T. pyogenes AST data determined by broth microdilution according to CLSI/NCCLS standards

Animal origin	Disease	Years of isolation	Number of isolates tested	MICs (in mg/liter) of selected antimicrobial agentsa										Reference
				PEN	AMP	ERY	CLI	TET	CHL	ENR	SXT	GEN	STR
Cattle	Mastitis		1	0.125		0.5				0.25				36
Sheep	Mastitis		5	0.008–0.5		0.008–0.25		16	4			2		14
Cattle (n = 27), pigs (n = 17), dogs (n = 2), cat (n = 1), macaw (n = 1)			48			≤0.06–≥128	≤0.06–≥128	≤0.06–16						41
Cattle (n = 76), pigs (n = 24), birds (n = 5), dogs (n = 2), deer (n = 1), sheep (n = 1), cat (n = 1)			11			0.06–1,024								43
Cattle	Urinary, genital tract infections	2004–2006	43	≤0.015	≤0.03–0.06	≤0.015–≥64	≤0.03–≥128	0.12–64	1–2	0.25–8	≤0.015–0.25	0.25–32		12
Cattle	Umbilical cord infections, septicemia	2004–2006	35	≤0.015	≤0.03	≤0.015–≥64	≤0.03–≥128	0.12–64	1–2	0.5–1	≤0.015–0.12	0.5–2		12
Pigs	Infections of the central nervous system, musculoskeletal system	2004–2006	12	≤0.015	≤0.03	≤0.015	≤0.03–0.06	0.12–16	1–2	1	≤0.015–0.06	0.5		12
Cattle	Endometritis	2006	32	0.125–16		0.0625–≥32	0.125–≥32	0.5–32		0.25–1		0.25–≥32	1–≥64	44
Cattle	Infections of the uterus	2008	72	≤0.06–32	≤0.06–≥64			1–≥64	8–≥64				0.5–≥64	45
White-tailed deer	Infections of the lung	2009–2010	29	≤0.12–2	≤0.25–4		≤0.25–16			0.5–2	2			46
Cattle	Mastitis	2008–2011	55	≤0.06	≤0.06–0.125	≤0.125–≥4		≤0.5–≥8						47
Cattle	Endometritis			0.125-16	0.125–64									48
Cattle	Infections of the uterus		35		≤0.06–0.125		≤0.06–0.25			0.5–1				49

^a PEN, penicillin; AMP, ampicillin; ERY, erythromycin; CLI, clindamycin; TET, tetracycline; CHL, chloramphenicol; ENR, enrofloxacin; SXT, trimethoprim/sulfamethoxazole; GEN, gentamicin; STR, steptomycin.

Resistance to β-Lactams

Using a breakpoint of ≥2 mg/liter, resistance to penicillin (MICs: 0.125 to 16 mg/liter), amoxicillin (MICs: 0.125 to 16 mg/liter), and oxacillin (MICs: 0.125 to 32 mg/liter) was determined in isolates from bovine uterine samples at dairy farms in Inner Mongolia (44). The finding of resistant isolates is in accordance with the lower CLSI breakpoints for coryneforms classifying isolates with MICs of ≤0.12 mg/liter and T. pyogenes with MICs of ≤0.03 mg/liter as susceptible (10). Cephalosporin resistance has been observed using the breakpoints of ≥16 mg/liter for cefazolin and 4 mg/liter for ceftiofur (44). In another study, de Boer et al. (49) found cloxacillin MICs of ≤0.06 to 4 mg/liter in T. pyogenes of bovine uterine samples, while the MIC values for ampicillin and ticarcillin/clavulanic acid were ≤0.013 mg/liter. For ceftiofur, the MIC values ranged between 0.25 and 4 mg/liter, while the MIC values for cefuroxime and cephapirin were ≤0.5 mg/liter (49). In a study by Santos et al. (45), T. pyogenes isolates from uterine secretions of postpartum dairy cows showed MIC distributions of ≤0.06 to 32 mg/liter for penicillin and amoxicillin, as well as ≤0.06 to ≥64 mg/liter for ampicillin and ceftiofur. With resistance breakpoints of ≥0.125 mg/liter for penicillin and ≥2 mg/liter for the other β-lactams tested, resistant isolates could be detected (45). Resistance would be also detected when applying the current CLSI breakpoints (10). Zastempowska and Lassa (47) described isolates from bovine mastitis being susceptible to the β-lactams, penicillin, ampicillin, ceftiofur, and cephalothin, with all isolates having MIC values of ≤1 mg/liter. For penicillin, all isolates had MICs of ≤0.06 mg/liter (47). Since the T. pyogenes breakpoint of penicillin or ampicillin is ≤0.03 mg/liter, isolates with a MIC of 0.06 mg/liter may be misclassified (10). MICs of up to 0.12 mg/liter result in the detection of ampicillin resistance (10).

In the German BfT-GermVet study, T. pyogenes isolates from bovine infections of the umbilical cord and the urinary and genital tract and septicemia, as well as porcine infections of the central nervous system and the musculoskeletal system revealed MIC₉₀ values of ≤0.015 to 0.5 mg/liter for the β-lactams tested (penicillin, ampicillin, oxacillin, amoxicillin/clavulanic acid, cephalothin, cefazolin, cefoperazone, ceftiofur, and cefquinome) (12). Tell et al. (46) identified T. pyogenes isolates from white-tailed deer with MICs of ≥0.5 mg/liter and ≥0.25 mg/liter as being resistant to ampicillin and penicillin, respectively, while resistance to ceftiofur was not detected (MICs: ≤1 mg/liter). Using the CLSI-proposed penicillin breakpoint (10) resistance would be seen among T. pyogenes from ewe’s mastitis (14). The amoxicillin MIC was 0.25 mg/liter, while the MICs for ceftazidime and cephalothin were 8 mg/liter (14). A single T. pyogenes isolate from dairy heifers had MICs of 0.125 mg/liter, 0.5 mg/liter, 0.5 mg/liter, and 1 mg/liter for penicillin, cloxacillin, cefapirin, and ceftiofur, respectively (36). Applying CLSI breakpoints for penicillin would result in the classification as susceptible using the breakpoint for the coryneforms, but as resistant using the T. pyogenes breakpoints (10). Zhang et al. (48) found high MIC values of up to 64 mg/liter for ampicillin, up to 32 mg/liter for ceftiofur and oxacillin, and up to 16 mg/liter for penicillin, amoxicillin, and cefazolin in isolates from cattle with endometritis. Testing bovine T. pyogenes from mastitis samples in China identified isolates that were resistant to penicillin (MIC: ≥0.25 mg/liter), ampicillin (MIC: ≥0.5 mg/liter), and cefaclor (MIC: ≥32 mg/liter) (10, 50). Zhao et al. (57) investigated T. pyogenes isolates from forest musk deer by agar dilution and found 16 isolates that were resistant to cefazolin and 17 resistant to cefotaxime, based on a breakpoint of 16 mg/liter for both substances. The β-lactam resistance gene blaP1 has been detected in only eight of the resistant isolates (57).

Resistance to Macrolides and Lincosamides

In T. pyogenes from bovine uterine samples, de Boer et al. (49) determined clindamycin MIC values of ≤0.25 mg/liter, which classify the isolates as susceptible according to the breakpoints for the coryneforms (susceptible: ≤0.5 mg/liter), whereas it cannot be interpreted by using breakpoints for T. pyogenes due to the lack of interpretive criteria for lincosamides (10). Liu et al. (44) found all bovine metritis and endometritis isolates from dairy farms in Inner Mongolia to be susceptible to tilmicosin (MICs: 0.25 mg/liter) and azithromycin (MICs: 0.5 to 2 mg/liter) using ≥2 mg/liter and ≥8 mg/liter as breakpoints for the two macrolides, respectively. Resistance to erythromycin (MICs: 0.0625 to 32 mg/liter) and clindamycin (MICs: 0.125 to 32 mg/liter) was seen when using 1 mg/liter and ≥2 mg/liter as breakpoints, respectively (44). Resistance to clindamycin (resistance breakpoint ≥4 mg/liter) was detected in isolates from white-tailed deer investigated by Tell et al. (46). The detection of erythromycin and clindamycin resistance in the latter two studies is in accordance with the current CLSI breakpoints (10). T. pyogenes isolates were classified as intermediate to tulathromycin (MIC: 64 mg/liter) and either resistant (MIC: ≥32 mg/liter) or intermediate (MIC: 16 mg/liter) to tilmicosin (46). The tylosin MICs of the corresponding isolates ranged from 0.5 to 64 mg/liter (46). AST of 48 T. pyogenes isolates of bovine and porcine origin revealed MIC₅₀ and MIC₉₀ values for erythromycin, tylosin, and clindamycin of ≤0.06 and ≥64 mg/liter, respectively, resulting in resistant isolates for all three antimicrobial agents, when using 8 mg/liter as the resistance breakpoint (41).

Induction tests were performed for isolates with erythromycin MICs of 1 to 8 mg/liter, and inducible macrolide/lincosamide resistance was seen in all isolates tested (41). Three isolates with tylosin MICs of 0.5 to 8 mg/liter were tested, and two showed an inducible phenotype, while the single isolate with a clindamycin MIC of 8 mg/liter was noninducible as confirmed by its tylosin and clindamycin MICs (41). While resistance to erythromycin, tylosin, and clindamycin might point towards a resistance mechanism due to a macrolide-, lincosamide-, and streptogramin B resistance gene, the single noninducible strain had an erythromycin MIC of ≥64 mg/liter and might be resistant via a different mechanism (41). Fernández et al. (14) found erythromycin MICs of 0.008 to 0.25 mg/liter, which indicates the presence of nonsusceptible isolates when using the CLSI-proposed breakpoint for T. pyogenes of ≤0.03 mg/liter (10). The lincomycin MICs were up to 16 mg/liter (14). Watts et al. (36) found an erythromycin MIC of 0.5 mg/liter for the bovine mastitis isolate, which would classify it as susceptible by the breakpoints for coryneforms but as nonsusceptible by the T. pyogenes breakpoints (10). The corresponding pirlimycin MIC was 0.25 mg/liter (36). In bovine T. pyogenes isolates from China, resistance to azithromycin (MICs: ≥8 mg/liter), erythromycin (MICs: ≥8 mg/liter), and clindamycin (MICs: ≥4 mg/liter) was detected (50).

Jost et al. (43) investigated T. pyogenes isolates for the genetic basis of tylosin resistance. In total, 10 of the 32 resistant isolates carried the erm(B) gene. Among the erm(B)-carrying isolates, no inducible resistance was detected, but five bovine strains already had MICs of ≥2,048 mg/liter without induction, and therefore a possible increase of the MICs might not be detectable (43). The constitutive expression of erm(B) in porcine strains with a MIC of 128 mg/liter is in accordance with the missing leader peptide being involved in inducible resistance (43). Differences of the MIC values of bovine and porcine isolates might be due to an additional resistance determinant present in the bovine strains, which could be confirmed by the preparation of erm(B) knockout mutants of one of the bovine strains, which showed an increase of the tylosin MICs from ≤0.06 to ≥2,048 mg/liter after tylosin induction (43). Jost et al. (42) found tylosin-resistant (MICs: ≥64 mg/liter) T. pyogenes harboring the resistance gene erm(X), showing also elevated MICs for erythromycin, oleandomycin, spiramycin, clindamycin, and lincomycin. This finding indicates that the erm(X) gene confers a macrolide-lincosamide-streptogramin B phenotype (42). The erm(X) gene on plasmid pAP2 (AY255627) was colocated with the tetracycline resistance gene tet(33) (42). An inducible phenotype was detected for all but one of the erm(X)-carrying isolates, while for the remaining isolate the MIC was above the detection limit (42). Seven isolates with tylosin MICs of 128 to 1,024 mg/liter showed MICs of ≥2,048 mg/liter after induction, whereas the remaining two isolates with MICs of 2 mg/liter and 8 mg/liter showed MIC values of 128 mg/liter after induction (42). The finding of the different MIC values of the erm(X)-carrying isolates might be due to the presence of additional macrolide resistance determinants or differences in the plasmid copy numbers (42). Cloning of the erm(X) gene in the vector pEP2 in the T. pyogenes strain BBR1 revealed an increase of the tylosin MIC from ≤0.06 mg/liter for the empty vector to 64 mg/liter for the vector carrying erm(X) (42).

The T. pyogenes erm(X) gene had significant identity with the erm(X) genes of C. diptheriae (97.7%), Corynebacterium striatum (97.5%), Corynebacterium jeikeium (94.7%), and Propionibacterium acnes (97.5%) (42). Moreover, the erm(X) gene was also detected in 23 of the 32 T. pyogenes isolates in a subsequent study by Jost et al. (43). In another study, Jost et al. (70) found T. pyogenes isolates with high MICs for erythromycin (>64 mg/liter), oleandomycin (>64 mg/liter), and spiramycin (≥8 mg/liter) and differences in the resistance properties regarding tylosin and clindamycin. These isolates [negative for the erm(B) and erm(X) genes] were investigated for mutations in the 23S rRNA gene, which are known to mediate resistance to different macrolides (70). One strain had the 23S rRNA mutation A2058T; it was considered clindamycin-resistant because of its MIC of 8 mg/liter but had a tylosin MIC of only 0.5 mg/liter (70). Tylosin and clindamycin MICs of 8 mg/liter were seen in an isolate with the 23S rRNA mutations A2058T and G2137C (70). The 23S rRNA mutation A2058G was seen in an isolate with high-level clindamycin resistance (MIC: >64 mg/liter) and a tylosin MIC of 0.25 mg/liter, while the 23S rRNA mutation C2611G was found in an isolate with low MICs for tylosin (0.125 mg/liter) and clindamycin (1 mg/liter) (70). In the BfT-GermVet study the MIC values for erythromycin (≤0.016 to ≥64 mg/liter), tilmicosin (≤0.03 to ≥128 mg/liter), spiramycin (≤0.06 to ≥256 mg/liter), tulathromycin (0.06 to ≥128 mg/liter), and clindamycin (≤0.03 to ≥128 mg/liter) included a wide range of dilution steps (12), and identified erythromycin- and clindamycin-resistant isolates (10). In total, six isolates with erythromycin MICs of ≥64 mg/liter were positive for the erm(X) gene (12). The resistance genes erm(X) and erm(B) were also detected in T. pyogenes from bovine mastitis (47).

Resistance to Tetracyclines

Resistance to tetracyclines was observed in bovine isolates from uterine samples, including resistance to oxytetracycline (breakpoint ≥64 mg/liter), tetracycline (breakpoint ≥4 mg/liter), and doxycycline (breakpoint ≥8 mg/liter) (44). However, the CLSI breakpoints for tetracycline and doxycycline classify isolates with ≤4 mg/liter as susceptible and those with 8 mg/liter as intermediate (10). Tell et al. (46) classified isolates from white-tailed deer as resistant (MIC: ≥8 mg/liter) or intermediate (MIC: 4 mg/liter) to oxytetracycline and chlortetracycline. In a study by de Boer et al. (49), two bovine uterine T. pyogenes isolates revealed elevated oxytetracycline MICs of 16 mg/liter and 128 mg/liter, respectively. MIC ranges of 1 to ≥64 mg/liter for tetracycline and 0.125 to ≥64 mg/liter for oxytetracycline were detected in bovine uterine isolates, suggesting the presence of resistant isolates (10, 45). Tetracycline MICs of 16 mg/liter were seen in T. pyogenes from ewes with mastitis (14). Among the isolates from the BfT-GermVet study, tetracycline MIC₉₀ values of 32 mg/liter and 16 mg/liter were observed for the bovine and porcine isolates, respectively (10, 12). Tetracycline resistance was detected in 70% of the bovine mastitis isolates from four farms in China when using a breakpoint of ≥16 mg/liter (10, 50).

Billington et al. (40) investigated tetracycline-resistant isolates from pigs, cattle, and a macaw for the genetic basis of the resistance and found the tet(W) gene in a bovine strain. This tet(W) gene had 92% sequence identity to tet(W) from Butyrivibrio fibrisolvens (40). Despite the detection of sequences similar to those involved in the regulation of tet(M) in transposon Tn916, an induction of tet(W) was not seen in the T. pyogenes isolates tested (40). The tetracycline-resistant isolates, as well as tetracycline-susceptible control isolates, were tested for the presence of the tet(W) gene via dot blot and a specific PCR assay, confirming the presence of this gene in all resistant isolates, while it was not seen in the susceptible ones (40). Trinh et al. (41) found T. pyogenes MIC₉₀ values of 8 mg/liter for chlortetracycline and oxytetracycline, while the MIC₉₀ for tetracycline was 16 mg/liter. Using 4 mg/liter as the breakpoint, a resistance rate of 41.7% was seen for all three substances, with the same 20 isolates being classified as resistant and carrying the tet(W) gene (41). However, the resistance breakpoint in the most recent CLSI document is ≥16 mg/liter (10). Isolates with MICs in the range of 0.5 to 8 mg/liter were tested for an inducible phenotype, which was not present in any of the isolates (41). Jost et al. (42) detected the tetracycline resistance gene tet(33) colocated with the erm(X) gene on plasmid pAP2. The tet(33) gene was detected in 55.6% of the erm(X)-carrying isolates but in only 5.1% of the erm(X)-negative isolates (42). The tet(33) gene conferred tetracycline MICs of 1 mg/liter, compared to ≤0.06 mg/liter for susceptible strains (42). In contrast to the tetracycline resistance gene tet(W), which confers tetracycline MICs of up to 8 mg/liter, tet(33) apparently only confers low-level resistance, whereas the presence of both genes resulted in a MIC value of up to 16 mg/liter (42). Zastempowska and Lassa (47) found the tet(W) gene in all bovine T. pyogenes isolates that showed a tetracycline MIC of at least 4 mg/liter. In her study, Alešík (71) investigated the tetracycline susceptibility of T. pyogenes isolates and found 36 isolates with tetracycline MICs of ≥8 mg/liter. The resistance gene tet(W) was detected alone or in combination with tet(33) (71). Moreover, the tet(Z) gene, present in a single isolate, has been described for the first time in T. pyogenes (71) but was previously detected in Corynebacterium glutamicum (72).

Resistance to Aminoglycosides

Among the T. pyogenes isolates from ewes with mastitis, gentamicin MICs of 2 mg/liter were determined, while the kanamycin MICs ranged from 0.5 to 8 mg/liter (14). Gentamicin resistance in T. pyogenes isolates from bovine mastitis was detected when applying a breakpoint of ≥16 mg/liter (10, 50). Isolates from white-tailed deer with a MIC of 8 mg/liter were classified as intermediate to gentamicin (10, 46), while all isolates were susceptible to spectinomcin (MIC: ≤16 mg/liter) (46). The neomycin MIC distribution ranged from 4 to 32 mg/liter (46). Testing of T. pyogenes from the BfT-GermVet study for gentamicin, neomycin, and spectinomycin resulted in MIC₉₀ values of 1 mg/liter, 4 mg/liter, and 4 mg/liter for the bovine and 0.5 mg/liter, 8 mg/liter, and 1 mg/liter for the porcine isolates, respectively (12). A single bovine isolate with a MIC of 32 mg/liter was classified as gentamicin-resistant (10, 12). The streptomycin susceptibility testing of bovine uterine isolates revealed a MIC₉₀ of 16 mg/liter (45). For spectinomycin, a MIC₉₀ value of ≥64 mg/liter was determined (45).

Zhao et al. (57) detected kanamycin and amikacin resistance in isolates from forest musk deer by agar dilution, using a breakpoint of 16 mg/liter for both substances. Gentamicin resistance based on a breakpoint of 2 mg/liter, which is below the resistance breakpoint from the CLSI (≥16 mg/liter) (10). Aminoglycoside resistance was observed in 17 isolates (60.7%), while the total detection rate for the resistance genes aacC, aadA1, and aadA2 was 57.1% (57). Liu et al. (44) found MICs of up to ≥64 mg/liter for streptomycin, 0.25 to ≥32 mg/liter for gentamicin, and 0.5 to ≥64 mg/liter for amikacin in isolates from bovine metritis and endometritis. Investigations of the genetic basis of aminoglycoside resistance identified gene cassettes with the resistance genes aadA1, aadA5, aadA24, and aadB (44).

Resistance to Phenicols

High MIC₉₀ values of ≥64 mg/liter for chloramphenicol and 32 mg/liter for florfenicol were detected in T. pyogenes from bovine uterine samples (45). Resistant isolates were detected when using ≥8 mg/liter as the breakpoint (45). Among the isolates from the BfT-GermVet study, the MIC₉₀ values for chloramphenicol and florfenicol were 2 mg/liter and 1 mg/liter for the bovine isolates and 1 mg/liter and 1 mg/liter for the porcine isolates, respectively (12). Fernández et al. (14) found chloramphenicol MICs of 4 mg/liter in ovine T. pyogenes isolates. Florfenicol resistance was detected in three bovine uterine isolates based on a breakpoint of 8 mg/liter, and the phenicol resistance gene cmlA6 was detected (44).

Resistance to Sulfonamides and Diaminopyrimidines

In the BfT-GermVet study, bovine isolates were found to be resistant to sulfamethoxazole based on human breakpoints (≥512 mg/liter), while all porcine isolates were classified as susceptible (12). In comparison, all bovine and porcine isolates had MICs of ≤0.12/2.38 to trimethoprim/sulfamethoxazole (12), which classifies them as susceptible by using the T. pyogenes breakpoint of ≤0.12/2.38 mg/liter (10). Liu et al. (44) classified all T. pyogenes isolates in their study as resistant to sulfadiazine and sulfamethoxydiazine, since the MIC₅₀ and MIC₉₀ values were ≥128 mg/liter for both substances. Zastempowska and Lassa (47) found that all bovine mastitis isolates in their collection also had sulfadimethoxine MICs of ≥128 mg/liter. Tell et al. (46) found all isolates with MICs of 2/38 mg/liter to trimethoprim/sulfamethoxazole and classified them as susceptible. Fernández et al. (14) tested trimethoprim and sulfisoxazole as separate compounds, resulting in MICs of 0.5 mg/liter and 64 mg/liter, respectively. Resistance to trimethoprim/sulfamethoxazole was seen in 90% of the T. pyogenes isolates from four Chinese dairy farms, with MICs of ≥4/76 mg/liter (10, 50).

Trimethoprim resistance was detected by agar dilution in 46.4% of the T. pyogenes isolates from forest musk deer, using 16 mg/liter as breakpoint; the resistance gene dfrB2a was detected in only 28.6% of the isolates (57). This suggests the presence of another resistance determinant in these isolates.

Resistance to Fluoroquinolones

The enrofloxacin MICs obtained for T. pyogenes isolates from bovine uterine samples were 0.5 mg/liter or 1 mg/liter (49). Tell et al. (46) found all isolates from white-tailed deer to be resistant to danofloxacin (MICs: 0.5 mg/liter and 1 mg/liter) and resistant (MIC: 2 mg/liter) or intermediate (MICs: 0.5 mg/liter and 1 mg/liter) to enrofloxacin. Liu et al. (44) reported T. pyogenes from bovine metritis and endometritis exhibiting MIC₉₀ values of 2 mg/liter, 2 mg/liter, 1 mg/liter, and 0.5 mg/liter to ciprofloxacin, ofloxacin, enrofloxacin, and gatifloxacin, respectively. For the first two substances, ≥16 mg/liter and for the latter two, ≥4 mg/liter were used as resistance breakpoints (44). Ciprofloxacin MICs of 2 mg/liter were seen in ovine mastitis isolates (14). The human-specific ciprofloxacin breakpoints available for coryneforms would classify isolates with MICs of 2 mg/liter as intermediate (9). A bovine mastitis isolate showed an enrofloxacin MIC of 0.25 mg/liter (36). Werckenthin et al. (12) described an enrofloxacin MIC₉₀ of 1 mg/liter for both the bovine and the porcine T. pyogenes isolates from the BfT-GermVet study. Resistance to fluoroquinolones was detected among isolates with ciprofloxacin MICs of ≥4 mg/liter and enrofloxacin MICs of ≥1 mg/liter (50).

Resistance to Other Antimicrobial Agents

Rifampicin MICs of up to 2 mg/liter were seen in ovine T. pyogenes isolates (14). In the study conducted by Alkasir et al. (50), resistance to rifampicin has been detected in only two of the 50 bovine mastitis isolates. These isolates had MICs of ≥4 mg/liter, which is in accordance with the CLSI breakpoints for coryneform bacteria (10). Liu et al. (44) found all bovine T. pyogenes isolates from metritis and endometritis to be resistant to Zn-bacitracin, with MICs of ≥32 mg/liter. Fernández et al. (14) found vancomycin MICs of 0.5 mg/liter in isolates from ewes with mastitis, which would be classified as susceptible according to the CLSI breakpoint of ≤2 mg/liter for coryneform bacteria (10). Tell et al. (46) found all isolates from white-tailed deer to be susceptible to tiamulin (MIC: ≤2 mg/liter). A novobiocin MIC of 0.25 mg/liter was determined by Watts et al. (36). Zastempowska and Lassa (47) reported that all 55 bovine mastitis isolates had MICs of 0.5 to 1 mg/liter for the combination of penicillin and novobiocin. The colistin MIC₉₀ value of ≥64 mg/liter detected among all bovine and porcine T. pyogenes isolates (12) confirmed that colistin is not active against Gram-positive bacteria.

CONCLUDING REMARKS

This article has provided an overview of the antimicrobial susceptibility of Corynebacterium spp., Arcanobacterium spp., and T. pyogenes. However, the interpretation and comparability of the results is hampered by the use of different AST methods and test parameters. As described, agar disk diffusion, agar dilution, and broth microdilution using different media, supplements, incubation times, and incubation temperatures have been applied for these bacteria (Table 1). In recent years, the CLSI has provided major improvements with regard to the harmonization of AST of bacteria of the genera Corynebacterium, Arcanobacterium, and Trueperella by approving and publishing standard broth dilution methods accompanied by clinical breakpoints for Corynebacterium spp. and related coryneform genera/coryneforms (9, 10), as well as an additional method with different test conditions for T. pyogenes (10). For the T. pyogenes AST method, which requires a CO₂ atmosphere for incubation, only a few breakpoints are available, with the ones for penicillin, erythromycin, and trimethoprim/sulfamethoxazole differing from those for the coryneform bacteria (10). Moreover, there is a gap of knowledge with regard to the genetic basis of antimicrobial resistance in Corynebacterium spp., Arcanobacterium spp., and T. pyogenes. Expanded studies have been conducted on macrolide/lincosamide- and tetracycline resistance genes in T. pyogenes, while reports of genes and mutations accounting for other resistance properties are rare.