Characteristics of β-lactamases and their genes (blaA and blaB) in Yersinia intermedia and Y. frederiksenii

Background The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India. Results The enzymes, Bla-A (a constitutive class A penicillinase) and Bla-B (an inducible class C cephalosporinase) were found to be present in all the clinical and non-clinical strains of Y. intermedia and Y. frederiksenii by double disc diffusion method. The results showed differential expression of Bla-A as indicated by presence/absence of synergy whereas expression of Bla-B was quite consistent. The presence of these enzymes was also reflected in the high minimum inhibitory concentrations, MIC50 (126–1024 mg/L) and MIC90 (256–1024 mg/L) of β-lactam antibiotics against these species. Restriction fragment length polymorphism (RFLP) revealed heterogeneity in both blaA and blaB genes of Y. intermedia and Y. frederiksenii. The blaA gene of Y. intermedia shared significant sequence identity (87–96%) with blaA of Y. enterocolitica biovars 1A, 1B and 4. The sequence identity of blaA of Y. frederiksenii with these biovars was 77–79%. The sequence identity of blaB gene of Y. intermedia and Y. frederiksenii was more (85%) with that of Y. enterocolitica biovars 1A, 1B and 2 compared to other species viz., Y. bercovieri, Y. aldovae and Y. ruckeri. Isoelectric focusing data further revealed that both Y. intermedia and Y. frederiksenii produced Bla-A (pI 8.7) and "Bla-B like" (pI 5.5–7.1) enzymes. Conclusion Both Y. intermedia and Y. frederiksenii showed presence of blaA and blaB genes and unequivocal expression of the two β-lactamases. Limited heterogeneity was detected in blaA and blaB genes as judged by PCR-RFLP. Phylogenetic relationships showed that the two species shared a high degree of identity in their bla genes. This is the first study reporting characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii isolated from Asian region.


Background
The genus Yersinia, a highly heterogenous organism is cur-rently represented by eleven known species [1]. Y. pestis, Y. pseudotuberculosis and Y. enterocolitica are very well docu-mented human pathogens. Y. pestis is the etiologic agent of plague (black death) while the other two are known to cause a variety of gastrointestinal syndromes [2]. The remaining eight species namely Y. intermedia, Y. frederiksenii, Y. kristensenii, Y. bercovieri, Y. mollaretii, Y. aldovae, Y. rohdei and Y. ruckeri have generally been termed as "Y. enterocolitica-like" species though each is a distinct species. Although most commonly isolated from sources like fresh water and food, these have only been infrequently recovered from human clinical specimens [1]. But, some of these, especially Y. intermedia and Y. frederiksenii may constitute as much as 18-32% of all Yersinia isolated from stools of diarrheic patients [3]. However, not much information is available about these, and Sulakvelidze termed them as the ignored species [1]. Nevertheless, Y. intermedia [4], Y. frederiksenii [5], Y. bercovieri [6] and Y. kristensenii [7] have been strongly implicated in cases of diarrhoea. It is therefore important to know more about these species to understand their public health significance.
The production of β-lactamases is an important mechanism of resistance to β-lactam antibiotics. In Y. enterocolitica, the distribution and production of β-lactamases namely Bla-A (a constitutive class A enzyme) and Bla-B (an inducible class C cephalosporinase or AmpC) has been studied widely and reported to vary with biovars and geographical origin of the strains [8][9][10][11]. However, not much is known about the β-lactamases of other important species like Y. intermedia, Y. frederiksenii and Y. kristensenii. Recently, Y. intermedia, Y. frederiksenii, and some strains of Y. kristensenii and Y. rohdei were reported to produce two β-lactamases [12,13]. On the other hand, Y. bercovieri and Y. mollaretii have been reported to produce only AmpC βlactamase, while Y. aldovae and Y. ruckeri showed either low levels of AmpC or no expression of β-lactamases at all [14]. This information has been inferred from MIC data of β-lactam antibiotics. Schiefer et al [15] recently characterized β-lactamases of Y. frederiksenii, Y. bercovieri, Y. aldovae and Y. ruckeri and reported that except for Y. frederiksenii, all expressed only an AmpC β-lactamase. Mammeri et al [16] cloned and sequenced the complete ampC gene of Y. ruckeri and found that it shared low level of identity with the known chromosomal and plasmid AmpC enzymes of closely related members, viz., Enterobacter cloacae, E. aerogenes and Citrobacter freundii. Much of these data however pertains to strains isolated in Europe. It would be worthwhile to study the β-lactamases and bla genes of strains isolated from other parts of the world to understand the global drug resistance of yersiniae. No data is available on β-lactamases of "Y. enterocolitica-like" species isolated from Asian region. Therefore, the objective of this work was to detect and characterize the β-lactamases and bla genes of Y. intermedia and Y. frederiksenii isolated from clinical and non-clinical sources in India.

Phenotypic detection of Bla-A and Bla-B enzymes
All the clinical and non-clinical isolates of Y. intermedia and Y. frederiksenii showed presence of Bla-A as indicated by the appearance of a small zone of inhibition of 2-8 mm radii (10-22 mm diameter) around ticarcillin 75 μg disc (Table 1). However, synergy, the appearance of a characteristic additional zone of inhibition between ticarcillin and the adjacent clavulanate disc, was detected in only 26% (9/34) of the isolates of Y. intermedia and one isolate of Y. frederiksenii. In an earlier study, similar observations regarding synergy were made for strains of Y. enterocolitica biovar 1A [8]. In the present study, Bla-B, the inducible cephalosporinase, was detected unequivocally based on the characteristic flattening of inhibition zone around cefotaxime disc adjacent to the imipenem disc in all the strains (Table 1). Tzelepi et al [17] also reported detection of a broad spectrum penicillinase (Bla-A) and an inducible cephalosporinase (Bla-B) in all the thirty aquatic isolates of Y. intermedia. Our study extends this information further to indicate that Bla-A was also present in Y. intermedia isolated from clinical and non-clinical (pig/wastewater) sources. In addition, the present study also showed unequivocal presence of Bla-A and Bla-B in strains of Y. frederiksenii, which was inferred earlier on the basis of MIC data only [12,13].
For Bla-A, a larger zone of inhibition (4-8 mm) around ticarcillin disc (75 μg) was observed for wastewater isolates of Y. intermedia compared to that of clinical (1.5-2.0 mm) isolates. The zone of inhibition (1.5-8 mm) around Y. intermedia was nevertheless larger compared to that around the strains of Y. frederiksenii (1.5-3.6 mm). Also, synergy i.e., appearance of an additional zone of inhibition around ticarcillin disc, was observed in the clinical isolates only. As these phenomena are highly dependent on the degree of the expression of enzyme, these observations suggested differential expression of Bla-A as reported earlier for strains of Y. enterocolitica biovar 1A [8].

Minimum Inhibitory Concentration (MIC)
All the strains were resistant to penicillins and cephalosporins tested. When analyzed separately, no difference was observed in the antibiotic susceptibilities of Y. intermedia and Y. frederiksenii. Thus, the combined results are shown in Table 2. The MIC 50 of amoxicillin was 512 mg/ L for the clinical and 1024 mg/L for the non-clinical strains, whereas MIC 90 was 1024 mg/L and 2048 mg/L, respectively. For co-amoxiclav, the MIC 50 ranged from 128-256 mg/L and MIC 90 was 256-512 mg/L for both clinical and non-clinical strains. The strains were equally resistant to cephalosporins. Amongst these, minimum resistance (MIC 50 32-64 mg/L and MIC 90 128 mg/L) was seen against cefotaxime for strains of Y. intermedia and Y. frederiksenii. For ceftazidime, the MIC 50 and MIC 90 were 512 mg/L and 1024 mg/L respectively, and for cefepime, the values ranged from 256-1024 mg/L. Contrary to these, relatively lower MICs of penicillins and cephalosporins have been reported for Y. intermedia and Y. frederiksenii isolated in other parts of the world [13,17,18]. This may be due to difference in the sources from which organisms were isolated or their geographical origin. Interestingly, Tzelepi et al [17] found that the strains of Y. intermedia isolated in Europe were sensitive to both cefotaxime and ceftazidime. The antibiotic resistance data of strains of Y. intermedia and Y. frederiksenii was in accordance with the preliminary reports from our laboratory earlier [19].

PCR amplification and restriction digestion of blaA gene
The β-lactamase genes namely the blaA and blaB were detected by PCR amplification and found to be present in all strains of Y. intermedia and Y. frederiksenii.
Initially, an attempt was made to amplify blaA using published primers (A9-f and A10-r) of Y. enterocolitica biovar 1A [20]. However, though some of the strains of Y. intermedia yielded expected amplicon, none of the Y. frederiksenii strains gave any amplicon. This may be attributed to differences in the gene sequences of blaA of Y. intermedia and Y. frederiksenii compared to that of Y. enterocolitica biovar 1A. Thus, to amplify blaA of Y. intermedia and Y. frederiksenii, new internal primers (A7-f and A8-r) were designed. When these primers were used for amplification of blaA, the expected 450 bp amplicon was obtained for all the forty-nine strains of Y. intermedia and Y. frederiksenii.
Restriction digestion of blaA with NciI revealed that a single site for NciI was present in blaA of only a few strains of Y. intermedia and Y. frederiksenii as either an uncut DNA or two fragments of 350 bp and 100 bp were obtained (Fig.  1). However, restriction with HaeIII gave three types of patterns having molecular weights 210, 190 and 60 bp (pattern I), 210, 190 and 55 bp (pattern II), and 350 and 100 bp (pattern III), for both Y. intermedia and Y. frederiksenii. The phylogenetic relationship and genetic heterogeneity in blaA was studied by constructing a concatenated dendrogram (Fig. 2) of the NciI and HaeIII restriction profiles. The strains of Y. intermedia grouped into three major clusters: A, B and C. Cluster A was formed by three wastewater isolates (T/Y/65, R/Y/59 and O/Y/60, all of serovar O:40). Cluster B was most heterogenous comprising clinical, wastewater and pig throat isolates while the cluster C was represented predominantly by pig throat isolates (Fig.  2a). The clusters B and C were divided further into two subclusters each. The blaA-RFLP grouped strains of Y. frederiksenii into two major clusters -A and B (Fig. 2b). Except for one, all the strains of clinical origin belonged to cluster B. As observed for Y. intermedia, the strains of Y. frederiksenii isolated from pig throat also clustered separately into subgroup BII (Fig. 2b). Earlier, work from our laboratory reported that some clinical and non-clinical strains of Y. enterocolitica biovar 1A formed separate clusters based on blaA-RFLP [20].  Clinical (6) 6 (100) -6 (100) Pig throat (9) 9 (100) 1 (11.1) 9 (100) n number of strains a number of strains with annular radii between 2-8 mm (diameter 10-22 mm) of the zone of inhibition around ticarcillin disc b Synergy -additional zone of inhibition between ticarcillin and co-amoxiclav discs c number of strains that showed characteristic flattening of the zone of inhibition around cefotaxime disc.
Several reports indicate that genetic background of antibiotic resistance genes influence antibiotic susceptibility profiles [21,22]. However, no such unequivocal relationship between blaA-RFLP type and antibiotic susceptibility was observed in this study. The present work however, clearly revealed heterogeneity in blaA gene of Y. intermedia and Y. frederiksenii, which may account for differential expression of Bla-A enzyme as seen in double disc diffusion method. Nevertheless, the heterogeneity was only limited as observed earlier for blaA gene of Y. enterocolitica biovar 1A strains [20]. In Moraxella catarrhalis, the two class A β-lactamase genes namely BRO-1 and BRO-2, have been reported to give different restriction profiles with BcgI [23,24]. Y/60), all the clinical and majority of the wastewater isolates grouped together in cluster B, whereas group C consisted exclusively of pig throat isolates (84%) (Fig. 3a). Cluster analysis of restriction profiles of Y. frederiksenii revealed that except for two, all clinical and pig throat strains grouped together in one major cluster (Fig. 3b). In a previous study, when the same restriction enzymes (HaeIII and RsaI) were used, no heterogeneity was discerned in blaB gene of Y. enterocolitica biovar 1A [20]. This suggested that blaB gene of Y. intermedia and Y. frederiksenii was more heterogenous compared to that of Y. enterocolitica.
The sequence identity of blaA gene of Y. intermedia and Y. frederiksenii with other members of the family Enterobacteriaceae namely Klebsiella oxytoca and Citrobacter koseri was comparatively very low and ranged from 48-50% for Y. intermedia and 62-63% for Y. frederiksenii. Furthermore, identity of blaA of Y. intermedia and Y. frederiksenii with Burkholderia cepacia, a non-enterobacterial species was 47.7% and 60%, respectively.
The deduced amino acid sequences of Bla-A of Y. intermedia and Y. frederiksenii were analyzed to check similarity in β-lactamases at protein level (Fig. 4). The SXXK tetrad, characteristic of β-lactamases possessing a serine active site, was present at position 70-73 [26]. In addition, two structural motifs characteristic of class A β-lactamases, were also found to be present in  [27].
The nucleotide sequences of blaB of Y. intermedia and Y. frederiksenii were found to have 85% identity with that of   (Fig. 5).

Molecular weight determination of Bla-A and Bla-B
Molecular weight determination on SDS-PAGE showed two distinct bands of 37 kDa and 29 kDa for all strains of Y. intermedia and Y. frederiksenii. The band at 37 kDa was characteristic of Bla-A enzyme, as the molecular weight of most class A β-lactamases from other organisms namely Y. enterocolitica [20], Burkholderia pseudomallei [30] and Citrobacter sedlakii [31] has been reported to vary between 29-35 kDa. The identity of this band was also confirmed by specific inhibition with clavulanic acid. The band at 29 kDa corresponded to Bla-B (AmpC) as indicated by its specific inhibition with aztreonam. The molecular weight of inducible cephalosporinases or AmpC of other species namely Y. enterocolitica [32], Y. aldovae, Y. bercovieri, Y. ruckeri and Y. frederiksenii [15], and Serratia marcescens [33], has however been reported to be in the range of 34-40 kDa. The 29 kDa as found in the present study was similar to 29 kDa AmpC of Vibrio fischeri [34] and Y. enterocolitica biovar 1A [20]. Since most studies cited above used SDS-PAGE to determine the molecular weight, the differences cannot be attributed to methodology.

Isoelectric focusing analysis of Bla-A and Bla-B
Isoelectric focusing of β-lactamases of fifteen strains (8 Y. intermedia and 7 Y. frederiksenii) revealed a single band in the alkaline region of the gel at pI 8.7 ( Table 3) that corresponded to Bla-A as indicated by its inhibition by clavulanic acid. Tzelepi et al [35] reported that Bla-A of Y. intermedia focused at pIs 9.0-9.5. The Bla-A of Y. enterocolitica with pI 8.7 has been reported by several investigators [20,36,37].
The inducible cephalosporinase or AmpC β-lactamase focused as multiple bands in the acidic region of the gel at various pI values. The identity of these bands was confirmed both by induction with imipenem that made the bands more prominent, and also by specific inhibition with aztreonam. Two major bands in acidic region of the gel at pI 6.5 and 6.8 were observed for the clinical strains of Y. intermedia. The AmpC of non-clinical strains of Y. intermedia focused at pI ranging from 5.5 to 7.1 (Table 3). Two additional bands (pI 7.8 and 8.0) were observed in the pig throat strains. The only report on Bla-B (AmpC) of Y. intermedia reported pI to be between 5.5 to 6.1 [35]. The Bla-B of all the strains of Y. frederiksenii also appeared as multiple bands at pI 6.0, 6.8 and 8.0. The strains of Y. enterocolitica, for which most information is available in literature [36,37], produced Bla-B with pI 5.3-5.7 except biovar 1A strains, the Bla-B of which focused at pI 6.8 and 7.1. Consequently, the Bla-B of biovar 1A strains has been termed as "Bla-B like" [11]. The present study indicated that, like biovar 1A strains, Y. intermedia and Y. frederiksenii too produced "Bla-B like" enzyme.

Nucleotide sequence accession numbers
The nucleotide sequence data of blaA and blaB has been submitted to NCBI GenBank under accession numbers [GenBank: DQ424965 and GenBank: DQ656113], and [GenBank: DQ424967 and GenBank: DQ424968] respectively.

Conclusion
The two β-lactamases namely Bla-A and Bla-B were detected in all clinical and non-clinical strains of Y. intermedia and Y. frederiksenii isolated from India. Differential expression of Bla-A but not Bla-B was observed by double disc diffusion method. Both Y. intermedia and Y. frederiksenii were highly resistant to β-lactam antibiotics. PCR-RFLP revealed that blaA and blaB genes of both Y. intermedia and Y. frederiksenii were heterogeneous. Phylogenetic relationships also showed that the two species shared high degree of identity in their bla genes. Isoelectric focusing data revealed that both Y. intermedia and Y. frederiksenii produced Bla-A and "Bla-B like" enzymes. This is the first study in which β-lactamases (Bla-A and Bla-B) and βlactamase genes (blaA and blaB) of Y. intermedia and Y. frederiksenii isolated from Asian region have been investigated.

Bacterial strains
Thirty four strains of Y. intermedia and fifteen of Y. frederiksenii isolated in India [38,39] from different sources namely diarrheic human subjects (10 strains), pig throat (31 strains) and wastewater (8 strains) were examined. The strains were authenticated by, and have been deposited with the Yersinia National Reference Laboratory and WHO Collaborating Centre at Institut Pasteur (Paris), France. The details of the strains are given in Table 4.

Phenotypic detection of Bla-A and Bla-B enzymes
Detection of the enzymes Bla-A and Bla-B was carried out by double disc diffusion tests as described previously by Pham et al [9] and Pham and Bell [40] respectively. Briefly, culture grown overnight on TGYE agar was used to prepare a suspension of the test organism containing 10 7 CFU/ml (A 600 = 0.1). MHA plates were inoculated by flooding these with 2.5 ml of this suspension. The excess was removed and plates were allowed to dry. For detection of Bla-A, ticarcillin 75 μg and co-amoxiclav 3 μg discs were placed on the plate, with adjacent edges 22 mm apart. The plates were incubated at 28°C for 20 hours. After incubation, the radii and diameter of the zone of inhibition around ticarcillin disc were recorded. The plates were also observed for presence of synergy (an additional zone of inhibition) between ticarcillin and coamoxiclav discs. For detection of enzyme Bla-B, cefotax-ime 5 μg and imipenem 10 μg discs were placed similarly on MHA plate and incubated. The characteristic flattening of the zone of inhibition around cefotaxime disc adjacent to the imipenem disc was interpreted as presence of Bla-B.

Minimum Inhibitory Concentrations (MIC)
The MICs of five selected antibiotics namely amoxicillin, co-amoxiclav, cefotaxime, ceftazidime and cefepime were determined in Mueller-Hinton broth by microbroth dilution technique using the methodology described by the Working Party of the British Society for Antibacterial Chemotherapy [41].