An in-vitro study of the antimicrobial susceptibilities of Yersinia enterocolitica and the definition of a database

Ingo Stock and Bernd Wiedemann*

Pharmazeutische Mikrobiologie, University of Bonn, Meckenheimer Allee 168, D-53115, Bonn, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The antibiotic susceptibilities of 151 Yersinia enterocolitica strains isolated from humans, animals and the environment, and belonging to biovars 1A (n = 22), 1B (n = 13), 2 (n = 12), 3 (n = 31), 4 (n = 63) and 5 (n = 10) to 71 antibiotics were examined. Biovars could be characterized phenotypically by their similar antibiograms. For the majority of biovars only minor differences in susceptibilities were detected. Ninety-nine percent of all Yersinia strains were resistant to amoxycillin, but biovar-related differences in MICs were demonstrated. Significant differences, characteristic of specific biovars, were detected for fosfomycin, some ß-lactams (in particular amoxycillin/clavulanate, and ticarcillin). Strains of biovar 1A were resistant or of intermediate susceptibility to ticarcillin and had fosfomycin MICs of 16–64 mg/L and amoxycillin/ clavulanate MICs of 4–32 mg/L. Most strains of biovar 1B (n = 11) were sensitive to amoxycillin/clavulanate (MIC = 0.5–2 mg/L). Most strains of biovar 2 (n = 10) showed an antibiotic phenotype resembling that of strains of biovar 1A, but they were more susceptible to fosfomycin (MIC = 4–16 mg/L). Twenty-seven strains of biovar 3 had amoxycillin/clavulanate MICs of 4–32 mg/L; 19 of them were sensitive to ticarcillin and fosfomycin. Biovar 4 strains were uniformly sensitive or of intermediate susceptibility to amoxycillin/clavulanate (MIC = 0.5–4 mg/L), sensitive to fosfomycin (MIC = 1–4 mg/L) but resistant to ticarcillin. The prevailing antibiotic phenotype of biovar 5 (n = 7) was similiar to the phenotype of biovar 4, although these biovar 5 strains were slightly more susceptible to amoxycillin/clavulanate and showed a heterogeneous pattern of susceptibility to fosfomycin (MIC = 1–32 mg/L). The results of this study show that unambiguous statements cannot be made about the natural antibiotic susceptibility to certain ß-lactams and fosfomycin of Y. enterocolitica. The data indicate a complex regulation of ß-lactamases in Y. enterocolitica. Some ß-lactamases are found more frequently or will be expressed predominantly in specific biovars.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Yersinia enterocolitica, which was first described in 1934, 1 is primarily a pathogen of the gastrointestinal tract, causing acute enteritis or enterocolitis; it can also cause lymphadenitis and ileitis. Less commonly, septicaemic spread may result in pneumonia, meningitis, panophthalmititis or endocarditis. Patients may present with a focal abscess or osteomyelitis, Secondary reactive phenomena, e.g. erythema nodosum, glomerulonephritis and myocarditis, can occur. 2

Y. enterocolitica is a genotypically and phenotypically heterogeneous species. On the basis of 16S ribosomal RNA sequence analysis, the species has been subdivided into at least two phylogenetic clusters, A and B. 3,4 Phenotypically, Y. enterocolitica is differentiated into six biovars (biovars 1A, 1B, 2, 3, 4 and 5), characterized by Wauters et al., 5 but several atypical strains also exist. Cluster A strains possibly belong to biovar 1B, whereas cluster B strains can be grouped in biovars 2, 3, 4 and 5. 4 Strains of the former biovars 3A and 3B have been reassigned to new species, namely Yersinia mollaretii and Yersinia bercovieri. 6 The described biovars of Y. enterocolitica differ in their geographic distributions, ecological niches and pathogenic properties.

Previous studies of the susceptibility patterns of Y. enterocolitica have yielded conflicting results. Stolk-Engelaar et al., examining 335 strains of Y. enterocolitica biovars 1A, 1B, 2, 3 and 4, found no association between susceptibility patterns and biovars or serovars. 7 In contrast, it has been reported that strains of biovar 1B (serovar O:8) were resistant to carbenicillin, ticarcillin and cephalothin but generally sensitive to ampicillin, 8 whereas strains of biovar 1A were resistant to amoxycillin/clavulanate. 9 Ampicillin-sensitive biovar 1B strains were noted as early as 1974. 10 Y. enterocolitica biovar 3 strains have been reported to be sensitive to carbenicillin and ticarcillin, but resistant to amoxycillin/clavulanate and cefoxitin. 11 Preston et al. 8 and Kwaga & Iversen 12 in part confirmed these results, finding that most strains of serovar O:5,27 were sensitive to ticarcillin. Pham et al.found that strains of biovar 4 were resistant to carbenicillin, ticarcillin and ampicillin, but sensitive to amoxycillin/clavulanate and cefoxitin. 11 These data were confirmed by Preston et al., although they found that only three-quarters of all biovar 4 strains were sensitive to amoxycillin/clavulanate. 8

The aims of this study were to investigate whether the antibiotic susceptibility of Y. enterocolitica depends on the biovar, and to create a database containing this information. MICs of 71 antibiotics for a collection of 151 Y. enterocolitica strains were determined using a microdilution technique. All known biovars were represented in this collection.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

A total of 160 Y. enterocolitica strains were biotyped. One hundred of these were clinical isolates, most of which originated from the University Hospital of Bonn, Germany, and were collected in 1996 and 1997. These were all initial isolates from different patients. The remaining clinical strains originated from the culture collection of Merlin-Diagnostika, Bornheim, Germany. Eight-eight `yersinia-like' strains were kindly provided by M. Exner (Bonn, Germany); 16 of these strains were identified as Y. enterocolitica and were also included in this study. These strains were of environmental origin, having been isolated from samples of surface waters and discharges of several filter plants in the area of Bonn and Cologne, Germany (these strains are referred to below as `aquatic isolates'). Eleven Y. enterocolitica strains were isolated from raw milk; these were collected by H. Neubauer (Munich, Germany). A further 33 strains (`Hamburg strains') were kindly provided by S. Aleksic (Hamburg, Germany). These strains, which were of human and mammalian origin, were used as reference strains. They had been biotyped and serotyped at the Hygiene Institute of Hamburg and comprised five strains of biovar 1A, ten of biovar 1B, two of biovar 2, nine of biovar 3 and seven of biovar 5.

Identification

The strains were identified to species level with a commercial identification system for Enterobacteriaceae (Micronaut-E (MCN-E), Merlin-Diagnostika, Bornheim, Germany). The MCN-E identification was carried out using incubation times of 24 and 48 h and incubation temperatures of 37°C and 24°C. Results of xylose and inositol fermentation tests were checked by using bromocresol purple agar (Difco Laboratories, Detroit, MI, USA) supplemented with xylose (inositol) 7 g/L. Incubation was for 24, 48 and 72 h, at 37°C and 24°C. Biovars were categorized according to the revised biogrouping schema of Wauters et al. 5 Tests for lipase activity were carried out at room temperature and read after 24, 48, 72 and 96 h; indole tests were performed in tryptone water using KOVACZ reagent (Difco). Incubation was for 24, 48 and 96 h at 37°C.

Antibiotic susceptibility testing

Antibiotic susceptibility was tested by a microdilution procedure in Isosensitest broth (Oxoid). Antibiotic-containing microtitre plates (Merlin-Diagnostika) were inoculated with 100 µL of the appropiate bacterial suspension (c. 105 cfu/mL) and incubated for 24 h at 36°C ± 1°C. Overnight cultures of the bacteria grown on Isosensitest agar at 36°C were used for the preparation of inocula. Bacteria were suspended in physiological saline to achieve a turbidity equivalent to that of a 0.5 McFarland standard (c. 108 cfu/mL) and diluted in Isosensitest broth. MIC values were read using a photometer (Labsystems Multiskan Multisoft), and the data were evaluted using Excel (Microsoft).

Evaluation of natural antibiotic susceptibility

When the MIC of a particular antibiotic for one species is plotted against the number of strains found with the respective MIC, a bimodal distribution is usually obtained: one peak, with relatively low MICs, represents the natural population and one peak with higher MICs represents the strains with acquired (secondary) resistance.

By analysing the MIC distribution for all the test strains of each species for each antibiotic, the biological thresholds were determined. Those strains with MICs higher than the thresholds, indicating secondary (acquired) resistance, were excluded. The correlation of the MIC values for the population examined with standard breakpoints was investigated. The natural population was classified as clinically sensitive, intermediate or resistant by application of French (SFM), Swedish (SIR), German (DIN), American (NCCLS) and British (BSAC) standards.

When the natural population was sensitive or intermediate according to the cited standards, it was described as `naturally sensitive' or `naturally intermediate', respectively. When the natural population was resistant, it was described as naturally (primarily or intrinsically) resistant. 13,14,15


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Identification

The commonest biovar identified among the 100 clinical isolates of Y. enterocolitica was biovar 4 (63% of isolates), followed by biovar 3 (20%). Seven biovar 2 strains and three strains each of biovar 1A, biovar 1B and biovar 5 were found. The frequency of the various biovars in the collection of 16 aquatic isolates was different, with 12 (75%) belonging to biovar 1A, one strain to biovar 2 and two strains to biovar 3. Of the 11 raw milk isolates, seven could not be clearly biotyped. Of the four that could be typed, two strains each belonged to biovar 1A and biovar 2.

One clinical isolate and one `aquatic isolate' could not be biotyped. These strains and the seven untypeable raw milk isolates were not examined further. The clearly characterized strains included 63 of biovar 4, 31 of biovar 3, 22 of biovar 1A, 13 of biovar 1B, 12 of biovar 2 and ten of biovar 5. MICs were determined for these isolates.

Antibiotic susceptibility, natural sensitivity and primary resistance

The MIC distributions for all Y. enterocolitica strains tested are presented in Table I. There were several antibiotics to which different biovars showed different susceptibilities, in particular amoxycillin/clavulanate, ticarcillin, some cephalosporins and fosfomycin. The MICs of these antibiotics are presented separately for each biovar for which a distinctive susceptibility pattern was demonstrated (Table I). The susceptibilities of the natural populations to amoxycillin/clavulanate, ticarcillin and fosfomycin are shown in Table II. The term ` natural population' was used for the most prevalent antibiotic phenotype within a given biovar. The natural antibiotic sensitivity and primary resistances of Y. enterocolitica are summarized in Table III.


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Table I. Antibiotic susceptibility of Yersinia enterocoliticaa
 

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Table II. Susceptibility of natural populationsa of Y. enterocolitica to fosfomycin, ticarcillin and amoxycillin in combination with clavulanic acid
 

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Table III. Natural antibiotic susceptibility of Y. enterocolitica, classified as sensitive, intermediate and resistant according to the standards mentioned in Table I
 

    Discussion
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 Materials and methods
 Results
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 References
 
In this study a correlation between the biovars of Y. enterocolitica and the susceptibility to penicillins, cephalosporins and other antibiotics (e.g. fosfomycin) was detected. Normally, different natural antibiotic susceptibilities below the species level would be predicted between groups of low genetic homogeneity. For each genospecies, consistent susceptibility would be expected. Differences in susceptibility to tetracycline have been described between different genospecies of Proteus vulgaris biovar 3, 13 but not in genospecies of Morganella morganiigenotypes, which are more closely related. 14 The present study clearly demonstrates that the antibiotic susceptibilities of different biovars can be completely different, even when they belong to one genospecies. The data strongly suggest that general statements about the natural susceptibility of the species Y. enterocolitica to penicillins (e.g. ticarcillin and amoxycillin/clavulanate), numerous cephalosporins (e.g. cefazoline, loracarbef, cefoxitin and cefixime), fosfomycin and trimethoprim cannot be made. Within each biovar a prevalent antibiotic phenotype pattern was detected, which was either unique or at least different from one of the other prevalent phenotypes. Therefore for each biovar one natural population was postulated, and determined, according to the most prevalent antibiotic phenotype pattern within the biovar.

The different susceptibilities to ß-lactams indicate a complex regulation and expression of ß-lactamases in Y. enterocolitica. Certain ß-lactamases may be distributed or will be expressed predominantly in some biovars of the species.

As early as 1975, Cornelis & Abraham described two different chromosomally encoded ß-lactamases within the species, ß-lactamase A and B. 16 ß-Lactamase A is a constitutive class A enzyme which is related to other class A enzymes of Gram-negative and Gram-positive bacteria. 17 ß-Lactamase B is an inducible class C enzyme which is related to AmpC ß-lactamases of other species in the Enterobacteriaceae. 18 Matthew et al. 19 found that strains of Y. enterocolitica biovar 4, which is the predominant biovar isolated from clinical specimens, both in this study and worldwide, contained ß-lactamases A and B, whereas Pham et al. 20 found only ß-lactamase A. In contrast to Pham et al., who examined Australian isolates, Matthew et al. investigated European strains. Further studies by Pham et al. 21 revealed that, in 1995, European, Asian, Brazilian and South African biovar 4 isolates contained ß-lactamases A and B, whereas Australian isolates and strains from New Zealand contained only ß-lactamase A. Pham et al. 21 showed that European biovar 4 isolates had amoxycillin/clavulanate and cefoxitin MICs of 8–32 mg/L and 4–32 mg/L, respectively, whereas Australian strains were highly susceptible to amoxycillin/clavulanate (MIC 0.5 mg/L) and cefoxitin (1–2 mg/L). High susceptibility to the combination of amoxycillin with clavulanate was explained by the absence of the enzyme B in biovar 4 strains. It remains to be elucidated why, in this study, all the strains of biovar 4 resembled the Australian rather than the European phenotype.

The second most frequently isolated biovar in this study was biovar 3, which is known to produce only the cephalosporinase enzyme B. 19,20 The natural population of biovar 3 was sensitive to ticarcillin. This could be explained by the lack of the penicillin-hydrolysing enzyme A. However, eight biovar 3 strains were resistant to ticarcillin and their ß-lactamase activity was not affected by clavulanate, while others showed the antibiotic phenotype characteristic of biovar 4.

In the last decade, the ß-lactamases within the `non-pathogenic' biovar 1A have also been investigated. Bottone 2 & Pham et al. 20 showed that strains of this biovar possess ß-lactamases A and B. The latter was renamed `enzyme B-like', 18 because its isoelectric point is slightly different from that originally described by Cornelis & Abraham 16 The biovar 1A genotype described might correspond to the natural population of the tested biovar 1A strains, although clavulanate in some of these strains had almost no effect on ß-lactamase activity, whereas in others ß-lactamase activity was inhibited.

The results of our study form a database of the natural antibiotic susceptibilities of Y. enterocolitica which can be applied to the validation of antibiotic susceptibility tests. Definitive statements about the natural susceptibility of the species to some antibiotics cannot be made, but natural populations with recognized susceptibility patterns for each biovar have been postulated. The molecular backgrounds of the phenomena described remain to be elucidated. Further studies on the distribution and expression of ß-lactamases within the described biovars of Y. enterocolitica are in progress.


    Acknowledgments
 
We gratefully acknowledge the generous support of this study by Merlin-Diagnostika, Bornheim, Germany.


    Notes
 
* Corresponding author. Tel:+49-229-735272; Fax:+49-228-735267. Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 23 March 1998; returned 14 May 1998; revised 26 June 1998; accepted 17 August 1998