Antimicrobial Susceptibility and Distribution of β-Lactamase A ( blaA ) and β-Lactamase B ( blaB ) Genes in Enteropathogenic Yersinia Species

Abstract

One hundred eighty-six strains of enteropathogenic Yersinia (Y.) enterocolitica of bioserotypes 2/O:5,27, 2/O:9, 3/O:3, and 4/O:3 and 12 strains of Yersinia pseudotuberculosis of bioserotypes 1/O:1, 1/O:2, and 2/O:1 from different human (feces) and nonhuman (pig, pork, wild boar, monkey, chinchilla, mara, capybara, salad) sources collected in the years 1995–2009 were examined. Antimicrobial resistance patterns for 12 antimicrobial agents were generated using broth microdilution. The presence and characterization of the β-lactamase genes blaA and blaB were studied using polymerase chain reaction (PCR) and PCR–restriction fragment length polymorphism (RFLP), respectively. The expression of β-lactamase BlaA and BlaB was detected using double-disc diffusion. Y. enterocolitica strains showed resistance to ampicillin (92%), streptomycin (13%), and sulfamethoxazole (2%). Intermediate susceptibility to tetracycline was shown by two Y. enterocolitica strains. All Y. pseudotuberculosis strains were susceptible to all tested antimicrobial agents. Most (99%) of the Y. enterocolitica strains carried both β-lactamase genes. One strain of bioserotype 3/O:3 lacked both genes. In contrast, all Y. pseudotuberculosis strains carried neither of the β-lactamase genes. Homogeneity was detected in all blaA and blaB genes of Y. enterocolitica using PCR-RFLP. The majority (89%) of Y. enterocolitica strains expressed both β-lactamase enzymes, whereas none of the Y. pseudotuberculosis strains showed expression of either enzyme. Also, it seems that the resistance of Y. enterocolitica has not changed during the last years.

Introduction

Y ersinia (Y.) enterocolitica and Yersinia pseudotuberculosis are important food-borne pathogens that cause human yersiniosis with gastroenteritis, terminal ileitis, and mesenteric lymphadenitis.⁵ Y. enterocolitica can be classified into six biotypes, namely 1A, 1B, 2, 3, 4, and 5. Y. enterocolitica strains belonging to biotypes 2, 3, and 4 are commonly associated with human infections.¹² The serotypes O:3 and O:9 and, in rare cases, O:5,27 and O:8 are associated with human yersiniosis in Europe, with serotype O:3 being the most well-known clinical serotype.^5,38 Y. pseudotuberculosis can be classified into 4 biotypes (1, 2, 3, and 4) and 15 O-serotypes (O:1–O:15).⁴² The most commonly isolated strains in Europe belong to the serotypes O:1–O:3, whereas serotypes O:4–O:15 occur mainly in Asia.^1,17 All Y. pseudotuberculosis strains are considered pathogenic.¹²

Food-borne yersiniosis has a major impact on public health in many European countries,¹³ including Germany.³⁴ Although most of the clinical cases result in a self-limiting gastroenteritis in humans, severe invasive disease including septicemia or prolonged illness can occur. Antimicrobial treatment of human yersiniosis is not indicated in most cases; however, trimethoprim–sulfamethoxazole, tetracycline, fluoroquinolones, and gentamicin have been recommended as therapy drugs in severe cases.³¹ Although antimicrobial resistance among human Y. enterocolitica and Y. pseudotuberculosis strains has shown to be low, multiresistant strains of Y. enterocolitica have also been reported.^2,31

Y. enterocolitica produces two chromosome-mediated β-lactamases, namely β-lactamase A (BlaA, a broad-spectrum constitutively expressed class A, group 2b enzyme) and β-lactamase B (BlaB, an inducibly expressed class C, group 1 cephalosporinase enzyme).^9,28,38 The presence and the differential expression of β-lactamases in Y. enterocolitica have been reported to vary among biotypes as well as in the geographical distribution of the Y. enterocolitica strains.^{11,28,30,36,39} Strains of the globally distributed biotype 4 isolated in Europe, Asia, Brazil, and South Africa expressed both enzymes, whereas strains of Australian and New Zealand origin showed BlaA only.^26,28 Previous studies from Switzerland, the United States, Germany, and Austria have shown high susceptibility of Y. enterocolitica strains to other antimicrobial agents than β-lactams.^2,4,7,24 The occurrence of β-lactamase in Y. pseudotuberculosis has not been yet reported.

The aim of this study was to examine (1) antimicrobial susceptibility, (2) distribution of blaA and blaB genes, and (3) expression of BlaA and BlaB among enteropathogenic Y. enterocolitica and Y. pseudotuberculosis strains isolated from different human and nonhuman sources from different European countries from 1995 to 2009.

Materials and Methods

Bacterial strains

In total, 186 strains of enteropathogenic Y. enterocolitica from different sources, namely 77 human feces (45 Sweden, 25 Germany, 7 Croatia) and 109 nonhuman sources [61 pigs (27 Germany, 27 Switzerland, 7 Finland), 30 pork (17 Germany, 13 Sweden), 14 wild boars (Switzerland), 2 monkeys (Croatia), and 2 chinchilla (Germany)], collected during the years 1995 to 2008 from different European countries were examined (Table 1). These Y. enterocolitica strains were identified as bioserotypes 2/O:5,27 (9/186), 2/O:9 (8/186), 3/O:3 (2/186), and 4/O:3 (167/186). Additionally, 12 Y. pseudotuberculosis strains from animal and vegetable sources, namely from 4 wild boars (Switzerland), 4 mara (Germany), 3 capybara (Germany), and 1 salad (Finland), collected from 2000 to 2009 were examined. These strains belonged to bioserotypes 1/O:1 (8/12), 1/O:2 (3/12), and 2/O:1 (1/12). Escherichia (E.) coli ATCC^® 25922 was used as control strain in antimicrobial susceptibility testing. A β-lactamase-producing Y. enterocolitica strain of bioserotype 4/O:3 was kindly provided by J. Heesemann (Munich, Germany).

Table 1.

Number and Sources of Yersinia enterocolitica and Yersinia pseudotuberculosis Strains Studied

	Number of strains from different sources
Country (year)	Human	Pig	Pork	Wild boar	Monkey	Chinchilla	Capybara	Mara	Salad
Yersinia enterocolitica 2/O:5,27
SUI^a (2006–2008)	–	6	–	3	–	–	–	–	–
Y. enterocolitica 2/O:9
GER^b (2002)	2	–	–	–	–	–	–	–	–
SUI (2006–2008)	–	1	–	5	–	–	–	–	–
Y. enterocolitica 3/O:3
GER (2002)	–	–	–	–	–	2	–	–	–
Y. enterocolitica 4/O:3
GER (2002)	23	27	17	–	–	–	–	–	–
SUI (2006–2008)	–	20		6	–	–	–	–	–
SWE^c (1999–2004)	45	–	13	–	–	–	–	–	–
FIN^d (1995–2000)	–	7	–	–	–	–	–	–	–
CRO^e (2004)	7	–	–	–	2	–	–	–	–
Yersinia pseudotuberculosis 1/O:1
GER (2002–2003)	–	–	–	–	–	–	3	3	–
SUI (2008–2009)	–	–	–	2	–	–	–	–	–
Y. pseudotuberculosis 1/O:2
GER (2003)	–	–	–	–	–	–	–	1	–
SUI (2008)	–	–	–	1	–	–	–	–	–
FIN (2000)	–	–	–	–	–	–	–	–	1
Y. pseudotuberculosis 2/O:1
SUI (2008)	–	–	–	1	–	–	–	–	–

Switzerland.

Germany.

Sweden.

Finland.

Croatia.

Antimicrobial susceptibility testing

Antimicrobial susceptibility was studied using broth microdilution following recommendations of the Clinical and Laboratory Standards Institute,⁸ altering the incubation temperature to 30°C, which is the optimal growth temperature for both plasmid-positive and -negative Yersinia strains. Minimal inhibitory concentrations (MICs) for ampicillin (1–128 mg/l), cefotaxime (0.016–2 mg/l), chloramphenicol (2–64 mg/l), ciprofloxacin (0.008–1 mg/l), florfenicol (4–32 mg/l), gentamicin (0.12–16 mg/l), kanamycin (8–16 mg/l), nalidixic acid (1–128 mg/l), streptomycin (2–256 mg/l), sulfamethoxazole (8–1,024 mg/l), tetracycline (1–128 mg/l), and trimethoprim (0.12–16 mg/l) were used (VetMIC™ GN-mo Version 4; SVA). The microdilution plates containing antimicrobial agents were inoculated with 50 μl of ∼10⁵ cfu/ml bacterial suspension, incubated at 30°C for 16–18 hr, and scored by visual examination. The results were interpreted as susceptible, intermediate, and resistant based on the interpretative criteria recommended for Enterobacteriaceae by CLSI⁸ and the clinical breakpoints for Enterobacteriaceae of the European Committee on Antimicrobial Susceptibility Testing.¹⁴ For streptomycin, the interpretative criteria recommended for Enterobacteriaceae by the British Society for Antimicrobial Chemotherapy⁶ were used.

Detection of blaA and blaB by polymerase chain reaction methods and sequencing of bla genes

The presence of the β-lactamase genes blaA and blaB was screened by a conventional polymerase chain reaction (PCR) method. DNA was extracted from bacterial cultures by boiling. Primers were used for the amplification of blaA and blab, as described by Stock et al.³⁸ The conventional PCR mixture comprised 1× PeqLab Master-Mix S (PeqLab), 200 nM of each primer, and 2 μl of the DNA in a total volume of 25 μl. PCR was performed using iCycler (Bio-Rad) (initial denaturation at 95°C for 5 min, and 35 cycles of denaturation for 30 sec at 95°C, annealing for 60 sec at 54°C, elongation for 120 sec at 72°C, and final extension for 4 min at 72°C). The PCR products were analyzed by electrophoresis in a 1% agarose gel (Agarose; Bio-Rad Molecular Biology, Bio-Rad), which were stained with ethidium bromide and photographed with a Gel Doc EQ system (Bio-Rad). Those strains that were negative using conventional PCR were further examined using a real-time PCR method based on EvaGreen^® (Bio-Rad). The real-time PCR mixture comprised 1 μl of DNA in a total volume of 20 μl. A two-step protocol (denaturation at 98°C for 5 sec and annealing at 54°C for 20 sec) with 35 cycles followed by melt curve analysis was used. The PCR fluorescence was detected using the iQ™ 5 Multicolor Real-Time PCR Detection System (Bio-Rad). A threshold cycle (Ct) under 35 and a specific melting temperature (T_m) of 85.5°C±0.5°C for blaA and 87.0°C±0.5°C for blaB indicated a positive result. The real-time PCR-amplified product for blaA was sequenced (Eurofins Mwg Operon). The nucleotide sequences were analyzed by BLAST, available at the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov/BLAST) and aligned using ClustalW (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalwan.html).

PCR–restriction fragment length polymorphism of blaA and blaB genes

To restrict the amplified PCR products of blaA and blaB, the restriction enzymes HaeIII and RsaI were used. Briefly, 10 μl of amplified PCR product were digested with 1 U each of HaeIII and RsaI enzymes followed by incubation at 37°C overnight in the appropriate buffer as recommended by the supplier (New England Biolabs). The restricted fragments were analyzed by electrophoresis in a 3% agarose gel (Bio-Rad) for 3 hr at 60 V. The gels were stained with ethidium bromide and photographed with a Gel Doc EQ system (Bio-Rad).

Detection of BlaA and BlaB by double-disc diffusion test

The expression of BlaA and BlaB was detected by double-disc diffusion method.^25,29 Mueller-Hinton agar (MHA; Oxoid) plates were inoculated with ∼10⁷ cfu/ml bacterial suspensions. For detection of BlaA, discs containing ticarcillin (75 μg; Oxoid) and amoxicillin/clavulanic acid (2/1 μg; Oxoid) were placed on the plate with their adjacent edges 22 mm apart and were incubated for 20–22 hr at 30°C. Strains expressing BlaA showed a zone of inhibition with an annular radius of 2–8 mm around ticarcillin (75 μg) and an additional zone of inhibition between both discs due to the synergetic effect between ticarcillin and clavulanic acid.²⁹ For detection of BlaB, discs containing imipenem (10 μg; Oxoid) and cefotaxime (5 μg; Oxoid) were placed on the MHA plate with their adjacent edges 22 mm apart and were incubated for 20–22 hr at 30°C. BlaB was expressed when a characteristic flattening of the zone of inhibition around the cefotaxime disc adjacent to the imipenem disc occurred.²⁵

Results

Antimicrobial susceptibility testing

All Y. enterocolitica strains were susceptible to 8 of the 12 antimicrobial agents tested (cefotaxime, chloramphenicol, ciprofloxacin, florfenicol, gentamicin, kanamycin, nalidixic acid, and trimethoprim) using broth microdilution. In total, 92% (171/186) of the Y. enterocolitica strains were resistant to ampicillin (MIC: 32–64 mg/l) (Table 2). Six Y. enterocolitica 4/O:3 strains were intermediate susceptible to ampicillin (MIC: 16 mg/l). Resistance to streptomycin was detected in 14% (23/167) of Y. enterocolitica 4/O:3 strains (MIC: 16–256 mg/l), in one Y. enterocolitica 2/O:9 strain (MIC: 16 mg/l), and in one Y. enterocolitica 3/O:3 strain (MIC: 16 mg/l). Resistance to sulfamethoxazole was detected in three (2%) 4/O:3 strains (MIC: 2,048, 2,048, and 1,024 mg/l). Intermediate susceptibility to tetracycline was shown by two bioserotype 4/O:3 strains (MIC: 8 mg/l). All Y. pseudotuberculosis strains were susceptible to all antimicrobials tested.

Table 2.

Antimicrobial Resistance Patterns of Yersinia enterocolitica Strains and Yersinia pseudotuberculosis Strains from Different Sources Using Broth Microdilution

			Number of strains showing resistance against
			Amp ^a			RL ^b			Strep ^c			Tc ^d
Bioserotype	Country	Source (No.)	S ^e	I ^f	R ^g	S	I	R	S	I	R	S	I	R
Y. enterocolitica
2/O:5,27	SUI^h	Pig (6)	3	0	3	6	0	0	6	0	0	6	0	0
		Wild boar (3)	3	0	0	3	0	0	3	0	0	3	0	0
2/O:9	GERⁱ	Human (2)	0	0	2	2	0	0	2	0	0	2	0	0
	SUI	Pig (1)	0	0	1	1	0	0	1	0	0	1	0	0
		Wild boar (5)	0	0	5	5	0	0	4	0	1	5	0	0
3/O:3	GER	Chinchilla (2)	1	0	1	2	0	0	1	0	1	2	0	0
4/O:3	GER	Human (23)	0	0	23	23	0	0	18	0	5	23	0	0
		Pig (27)	1	3	23	27	0	0	20	0	7	25	2	0
		Pork (17)	0	0	17	16	0	1	13	0	4	17	0	0
	SUI	Pig (20)	0	0	20	20	0	0	19	0	1	20	0	0
		Wild boar (6)	0	0	6	6	0	0	6	0	0	6	0	0
	SWE^j	Human (45)	0	2	43	44	0	1	44	0	1	45	0	0
		Pork (13)	0	1	12	13	0	0	12	0	1	13	0	0
	FIN^k	Pig (7)	1	0	6	6	0	1	5	0	2	7	0	0
	CRO^l	Human (7)	0	0	7	7	0	0	5	0	2	7	0	0
		Monkey (2)	0	0	2	2	0	0	2	0	0	2	0	0
Total (186)			9	6	171	183	0	3	161	0	25	184	2	0
Y. pseudotuberculosis
1/O:1	GER	Capybara (3)	3	0	0	3	0	0	3	0	0	3	0	0
		Mara (3)	3	0	0	3	0	0	3	0	0	3	0	0
	SUI	Wild boar (2)	2	0	0	2	0	0	2	0	0	2	0	0
1/O:2	GER	Mara (1)	1	0	0	1	0	0	1	0	0	1	0	0
	SUI	Wild boar (1)	1	0	0	1	0	0	1	0	0	1	0	0
	FIN	Salad (1)	1	0	0	1	0	0	1	0	0	1	0	0
2/O:1	SUI	Wild boar (1)	1	0	0	1	0	0	1	0	0	1	0	0
Total (12)			12	0	0	12	0	0	12	0	0	12	0	0

Ampicillin (1–128 mg/l).

Sulfamethoxazole (8–1,024 mg/l).

Streptomycin (2–256 mg/l).

Tetracycline (1–128 mg/l).

Susceptible.

Intermediate.

Resistant.

Switzerland.

Germany.

Sweden.

Finland.

Croatia.

Detection of blaA and blaB by PCR

The β-lactamase genes blaA and blaB were detected in all examined Y. enterocolitica strains using PCR method, with the exception of one bioserotype 3/O:3 strain (Table 3). This bioserotype 3/O:3 strain isolated from a chinchilla in Germany was negative for both β-lactamase genes. However, a difference between the robustness of the conventional and the real-time PCR methods was detected. Using conventional PCR methods, blaA and blaB were detected in 96% (178/186) and 95% (177/186), respectively, of all Y. enterocolitica strains studied. Yet, using real-time PCR, all of these strains were repeated blaA and blaB positive in each run. All Y. pseudotuberculosis strains were negative for both β-lactamase genes using conventional PCR. However, using real-time PCR, all Y. pseudotuberculosis strains showed a Ct <35 and a T_m of 86.5°C–87.0°C for the detection of blaA, whereas all Y. pseudotuberculosis were negative for the detection of blaB using real-time PCR. Two real-time PCR products for blaA of Y. pseudotuberculosis were sequenced (Eurofins Mwg Operon), and later, they were analyzed using BLAST and aligned using ClustalW. Both products were homologous to each other, but the nucleotide sequence did not match any known sequence for the β-lactamase genes of Y. enterocolitica or any other Enterobacteriaceae in the NCBI database.

Table 3.

Detection of β-Lactamase Genes (blaA, blaB) and Expression of β-Lactamases (BlaA, BlaB) in Yersinia enterocolitica from Different Sources

			Number of Y. enterocolitica strains positive for
Bioserotype	Country	Source (No.)	blaA	blaB	BlaA	BlaB
2/O:5,27	SUI^a	Pig (6)	6	6	0	6
		Wild boar (3)	3	3	0	3
2/O:9	GER^b	Human (2)	2	2	2	2
	SUI	Pig (1)	1	1	1	1
		Wild boar (5)	5	5	5	5
3/O:3	GER	Chinchilla (2)	1	1	1	1
4/O:3	GER	Pig (27)	27	27	26^c	26^d
		Human (23)	23	23	23	21
		Pork (17)	17	17	17	17
	SUI	Pig (20)	20	20	20	19
		Wild boar (6)	6	6	6	6
	SWE^e	Human (45)	45	45	45	41
		Pork (13)	13	13	13	12
	FIN^f	Pig (7)	7	7	6	7
	CRO^g	Human (7)	7	7	7	7
		Monkey (2)	2	2	2	2
Total		186	185	185	174	176

Switzerland.

Germany.

Y. enterocolitica strains without BlaA expression showed expression of BlaB.

Y. enterocolitica strains without BlaB expression showed expression of BlaA.

Sweden.

Finland.

Croatia.

PCR–restriction fragment length polymorphism of blaA and blaB genes of Y. enterocolitica

All strains of the different Y. enterocolitica bioserotypes showed identical patterns. Five fragments of 420, 300, 180, 120, and 70 bp after analysis of blaA and three fragments of 700, 600, and 100 bp for blaB were obtained using HaeIII. Restriction with RsaI gave two fragments of 330 and 160 bp for blaA and two fragments of 460 and 380 bp for blaB.

Detection of BlaA and BlaB by double-disc diffusion test

The majority (89%) of all Y. enterocolitica strains expressed BlaA and BlaB, yet the expression varied within the different bioserotypes. Only two strains (1%) of bioserotype 4/O:3 did not express BlaA but BlaB (Table 3). Additionally, nine (5%) bioserotype 4/O:3 strains did not express BlaB but BlaA. No bioserotype 2/O:5,27 strain expressed BlaA, but BlaB was expressed by all of these nine strains. All strains of bioserotype 2/O:9 expressed both enzymes. One bioserotype 3/O:3 chinchilla strain lacking both β-lactamase genes did not express either of the enzymes. None of the Y. pseudotuberculosis strains showed expression of either β-lactamase.

Expression of BlaA and ampicillin resistance

Most (97%) Y. enterocolitica strains with expression of BlaA were resistant to ampicillin (Table 4). However, five strains with expression of BlaA were intermediate susceptible and one strain expressing BlaA was susceptible to ampicillin. Two strains of bioserotype 4/O:3 lacked BlaA and were susceptible and intermediate susceptible to ampicillin. Further, one bioserotype 3/O:3 strain lacked BlaA, being susceptible to ampicillin. All nine bioserotype 2/O:5,27 strains carried blaA but BlaA was not expressed by these strains. Nevertheless, three strains were resistant to ampicillin.

Table 4.

Resistance to Ampicillin of Yersinia enterocolitica Strains from Different Origin in Accordance to the Detection of blaA and BlaA

		No. of strains positive for		Number of strains showing resistance against ampicillin
Bioserotype	No. of strains	blaA	BlaA	S ^a	I ^b	R ^c
2/O:5,27	9	9	0	6	0	3
2/O:9	8	8	8	0	0	8
3/O:3	1	1	1	0	0	1
	1	0	0	1	0	0
4/O:3	165	165	165	1	5	159
	2	2	0	1	1	0

Susceptible.

Intermediate susceptible.

Resistant.

Discussion

Resistance of bacterial pathogens to antimicrobial agents is a world-wide problem, as many different antimicrobial agents are used for treatment of animal diseases as well as for human infections.²² In this study, 198 enteropathogenic Yersinia strains were examined upon their resistance to different antimicrobial agents.

All examined Y. enterocolitica strains were susceptible to 8 of the 12 tested antimicrobial agents. Similar susceptibility was observed among strains from porcine origins in the United States, Switzerland, and Southern Germany as well as in human strains.^4,7,15 In the last years, clinical isolates of Y. enterocolitica being resistant to streptomycin³¹ and sulfonamids⁴⁰ have been reported. In this study, many (13%) Y. enterocolitica strains were resistant to streptomycin. Most of these resistant strains belonged to bioserotype 4/O:3. However, varying resistance to streptomycin within Y. enterocolitica bioserotypes has been published.^2,7,15,19,31 One biotype 2/O:9 strain was resistant to streptomycin. This could not be detected before in biotype 2/O:9 strains isolated from human and porcine origin.^2,15 Only a few Y. enterocolitica strains were resistant to sulfamethoxazole (3/186) and intermediate susceptible to tetracycline (2/186). All of those strains belonged to bioserotype 4/O:3. Recently, rare resistance to sulfamethoxazole from clinical samples and from porcine origins in Switzerland, Germany, and Canada has been found.^2,7,15,16,32 However, clearly higher resistance to sulfonamides was reported among pathogenic Y. enterocolitica strains isolated in the United States and in the city of Barcelona.^18,31 This phenomenon might be explained by the varying use of sulfonamides in different countries worldwide. The reported resistance to tetracycline varies in literature. Susceptibility to tetracycline of bioserotype 4/O:3 strains was shown in studies from Switzerland, Southern Germany, Poland, and Barcelona.^2,7,15,31,33 Intermediate susceptibility to tetracycline has also been previously described.^{2,15,19,40,41} Clearly higher resistance was reported among pathogenic Y. enterocolitica strains of porcine origin in the United States (14%), Greece (12%), and Czech Republic (13%).^4,19,37 Tetracycline has been commonly used as treatment for clinical infections in humans and animals.

Most (92%) of the Y. enterocolitica strains were resistant to ampicillin. Similar results have been reported from human clinical and porcine samples in Switzerland, Germany, Greece, Poland, and Barcelona.^{2,7,15,16,19,31,33} However, one study showed high susceptibility to ampicillin of serotype O:3 isolates from pork and beef in Austria using disc diffusion.²⁴ Surprisingly, most (6/9) of the bioserotype 2/O:5,27 strains from Switzerland were susceptible to ampicillin in this study. This has been previously shown only among bioserotype 2/O:5,27 strains from pig tonsils in Switzerland,¹⁵ whereas serotype O:5 strains from clinical samples and samples of porcine origin in Switzerland, the United States, and Canada have been reported to be resistant to ampicillin.^2,4,18,32 Also, one of the two bioserotype 3/O:3 strains examined from chinchillas in Germany was resistant to ampicillin. Only one study found resistance to ampicillin in all 3/O:3 strains examined from human samples in Canada using agar dilution.²⁷

Previous studies have described the presence of blaA and blaB in biotypes 2, 3, and 4 of Y. enterocolitica isolated from clinical specimens and mammals in Germany.^38,39 In the present study, all examined Y. enterocolitica strains except one 3/O:3 strain carried both β-lactamase genes. All analyzed β-lactamase genes showed similar profiles using the restriction enzymes HaeIII and RsaI, suggesting that they are highly homogeneous. This has been previously shown for blaA by de la Prieta et al.¹⁰ using the restriction enzymes EcoRI and HindIII. Also recently, β-lactamase genes of Y. enterocolitica biotype 1A were examined using the restriction enzymes HaeIII and RsaI.³⁶ Here, blaB was highly homogenous, whereas blaA showed heterogeneity, which has been expected as the blaA gene of biotype 1A strains differs from blaA of other biotypes.

The varying sensitivity to β-lactam antimicrobials might be explained by the complex regulation and expression mechanisms of β-lactamases by members of Y. enterocolitica.^27,40 Some Y. enterocolitica strains of different bioserotypes did not express BlaA even though the gene was detected. This has been previously described.^{10,20,23,27–29} de la Prieta et al.¹⁰ identified a 51 bp deletion as the cause of inactivation of blaA. The resistance to ampicillin is due to the expression of BlaA. Nevertheless, examined strains expressing BlaA were susceptible and strains not expressing BlaA were resistant to ampicillin. One explanation might be that some differences in the cell wall are present in different strains.³⁵ Likewise, expression of BlaB could not be detected in all bioserotype 4/O:3 strains, whereas the majority of the published studies has shown that all tested strains express BlaB.^{26,28,30,38,39} Only one study of strains from Polish pigs showed the lack of BlaB.²⁰ The missing expression might be due to the fact that the induction of BlaB in strains with low β-lactamase activity is incomplete using cefotaxime and imipenem.^30,38 Biotype 3 is known to produce BlaB only.^23,28 We were able to show that one bioserotype 3/O:3 strain expressed both enzymes, whereas the other 3/O:3 strain lacked both β-lactamase genes. This has not been previously reported.

All Y. pseudotuberculosis strains were susceptible to all antimicrobial agents tested using broth microdilution. Similar results were also obtained for Y. pseudotuberculosis strains isolated from pigs in Brazil.²¹ One reason for the susceptibility to ampicillin detected in this study could be the lack of blaA of these strains. Another reason could be that the membrane permeability of Y. pseudotuberculosis for hydrophobic agents is much higher than for Y. enterocolitica,³ thus possibly causing susceptibility. However, resistance to ampicillin of Y. pseudotuberculosis strains from porcine origin has also been reported.^19,41 This might be due to acquired resistance among domesticated populations. Neither of the β-lactamase genes was detected in any Y. pseudotuberculosis strain, but some false-positive results occurred. Sequencing of these amplified PCR products gave nonspecific results.

Conclusion

The majority of the Y. enterocolitica strains and all Y. pseudotuberculosis strains were susceptible to the antimicrobial agents tested. This is in accordance to previous studies. Thus, the resistance of Y. enterocolitica does not seem to have changed during the last years/decades, which could be due to genetic stability of Y. enterocolitica in the presence of antimicrobial agents. Additionally, no clear difference in the resistance pattern could be detected between human and nonhuman strains. Surprisingly, one Y. enterocolitica 3/O:3 strain lacked both β-lactamase genes, whereas the second 3/O:3 strain as well as all the other strains of different bioserotypes carried both β-lactamase genes. The expression of BlaA and BlaB was not detected among all Y. enterocolitica strains. Especially bioserotype 2/O:5,27 strains do not seem to express BlaA. Nevertheless, some of these strains are resistant to ampicillin, suggesting a different resistance mechanism among these strains. All Y. pseudotuberculosis strains lacked both β-lactamase genes.

Footnotes

Acknowledgments

The authors thank Ilona Fitzek for technical assistance and Prof. J. Heesemann (Max von Pettenkofer Institute, Munich, Germany) for providing a Y. enterocolitica control strain.

Disclosure Statement

The authors have no competing interests to disclose.

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