Background In this study, we examined the correlation between excitation-contraction coupling characteristics and skeletal muscle fibre type by (1) localizing the distribution of dihydropyridine receptor (DHPR) protein and (2) comparing the effect of DHPR blocker on muscles with different fibre type composition, in order to better understand the differences between contractile phenotypes of fibres and to explain the contradictory reports to date on the interaction of dihydropyridines with skeletal muscle isoform of DHPR.
Background
In this study, we examined the correlation between excitation-contraction coupling characteristics and skeletal muscle fibre type by (1) localizing the distribution of dihydropyridine receptor (DHPR) protein and (2) comparing the effect of DHPR blocker on muscles with different fibre type composition, in order to better understand the differences between contractile phenotypes of fibres and to explain the contradictory reports to date on the interaction of dihydropyridines with skeletal muscle isoform of DHPR.Results Histochemical experiments revealed that fluorophore conjugated dihydropyridines stain selectively the membranes of muscle fibres. The staining was most evident in type IIA fibres. The major fibre type in gluteus and femoris, revealed by mATPase staining, was IIA (45.0 and 38.1 %, respectively). In gastrocnemius the content of IIA fibres was 22.7 %. Contraction forces before and after the addition of blocker for the three muscles investigated were: gluteus 0.075 ± 0.017 N  vs.  0.052 ± 0.011 N, femoris 0.045 ± 0.005 N  vs.  0.033 ± 0.005 N and gastrocnemius 0.089 ± 0.016 N  vs.  0.075 ± 0.014 N, respectively. The attenuation of contraction force proportional to the cross-sectional area of the muscle was significantly (P = 0.023) higher in gluteus (28.3 ± 3.5 %) and femoris (27.6 ± 3.2 %) as compared to gastrocnemius (16.1 ± 2.5 %). However, no significant change in the control measurements was observed ruling out the possibility of fatigue.
Results
Histochemical experiments revealed that fluorophore conjugated dihydropyridines stain selectively the membranes of muscle fibres. The staining was most evident in type IIA fibres. The major fibre type in gluteus and femoris, revealed by mATPase staining, was IIA (45.0 and 38.1 %, respectively). In gastrocnemius the content of IIA fibres was 22.7 %. Contraction forces before and after the addition of blocker for the three muscles investigated were: gluteus 0.075 ± 0.017 N  vs.  0.052 ± 0.011 N, femoris 0.045 ± 0.005 N  vs.  0.033 ± 0.005 N and gastrocnemius 0.089 ± 0.016 N  vs.  0.075 ± 0.014 N, respectively. The attenuation of contraction force proportional to the cross-sectional area of the muscle was significantly (P = 0.023) higher in gluteus (28.3 ± 3.5 %) and femoris (27.6 ± 3.2 %) as compared to gastrocnemius (16.1 ± 2.5 %). However, no significant change in the control measurements was observed ruling out the possibility of fatigue.vs.vs.vs.Conclusion The results indicate that the attenuation of the contraction force was largest in muscles with a high percentage of type IIA fibres. This supports our finding that the abundance of dihydropyridine receptors of IIA fibres outnumbers that in the other fibre types. The present data show that the correlation of density of dihydropyridine receptors can be one of the important factors influencing the overall contractile properties of the muscle and for its part explain the contradictory results of previous studies on coupling process.
Conclusion
The results indicate that the attenuation of the contraction force was largest in muscles with a high percentage of type IIA fibres. This supports our finding that the abundance of dihydropyridine receptors of IIA fibres outnumbers that in the other fibre types. The present data show that the correlation of density of dihydropyridine receptors can be one of the important factors influencing the overall contractile properties of the muscle and for its part explain the contradictory results of previous studies on coupling process.

Background One of the features characterizing mammalian skeletal muscle tissue is the structural variability of muscle fibres. The inclusive properties of skeletal muscle lead to functional diversity of muscle fibres, which has been related to differences in relative proportions of membrane structures and to different amounts of contractile proteins [ 1 ]. Although the interfibre-type differences are well recognized, very little is known about the characteristics of the process leading from electrical membrane excitation to contraction (E-C coupling) related to different cell types. The first indication of variation in E-C coupling between representatives of different fibre types came from studies using vaseline-gap technique in order to record slow calcium currents and asymmetric charge movement in fast- and slow-twitch muscles [ 2 ]. The results suggested functional and structural differences between fast- and slow-twitch mammalian muscles with respect to dihydropyridine receptor (DHPR) density. Recently Goodman et al [ 3 ] provided the first evidence about E-C coupling characteristics being related to myosin heavy chain based fibre type and the muscle from which the fibre originated. Our finding about the density of DHPR varying between different fibre types [ 4 ] supports the hypothesis that optimum contractile function of skeletal muscle is related to its fibre type composition via differences in E-C coupling. Voltage-dependent dihydropyridine receptors play an important role in the function of skeletal muscle. As well known, contraction is initiated by a depolarization of the transverse tubular (T-tubular) membrane, which in turn causes calcium to be released from the stores of sarcoplasmic reticulum (SR). The DHPR is located in the T-tubule membrane acting as a voltage sensor. Linked to the ryanodine receptor located in the sarcoplasmic reticulum membrane, this channel protein triggers the intracellular Ca 2+ -release via the ryanodine receptor [ 5 ]. The DHPR itself is a heteromultimer composed of 5 subunits; α 1  (170 kDa), α 2  (140 kDa), β (55 kDa), γ (33 kDa) and δ (24–33 kDa) [ 6 , 7 ]. The pore forming α 1  subunit contains the receptor for dihydropyridines (DHPs) and other calcium antagonists. DHPs, such as nifedipine, nitrendipine, and nimodipine, bind to DHP receptor in a specific, saturable and reversible fashion. In addition, receptors show high affinity for DHP ligands. The dissociation constant ( K D ) for skeletal muscle membranes is usually in the range of 1–10 nM [ 8 ]. Dihydropyridines are used clinically as blockers in the treatment of hypertension and coronary heart disease [ 9 ]. Besides being the voltage sensor for Ca 2+  release needed in E-C coupling, the α 1 -subunit of the molecule functions also as a calcium channel [ 10 ] giving rise to a slow inward calcium current [ 11 ]. However, DHPR appears to be unimportant as an ion-conducting channel [ 12 ]. Firstly, the calcium current is activated too slowly to generate contraction of the fibre. Second, the contraction continues even if the extracellular calcium is removed, and finally, blocking the slow calcium current through the channel does not prevent contraction. On the other hand, Beam et al [ 5 ] reported a notable increase in the magnitude of inward calcium current in developing muscle thus implicating an important role of the current in maturation. In the present study we examined the E-C coupling characteristics of muscles with different dihydropyridine receptor expression defined by fibre type composition. To test the hypothesis that muscles with different cell type compositions, i.e. DHPR quantities, react differently to calcium channel blocker, we measured contraction forces of three muscles before and after adding the blocker solution. We found variation in the activities of muscles depending on the cell type composition of the muscles. The findings confirm the previously observed pattern and elucidate the role of dihydropyridine receptor in muscle cell contraction.
Background
One of the features characterizing mammalian skeletal muscle tissue is the structural variability of muscle fibres. The inclusive properties of skeletal muscle lead to functional diversity of muscle fibres, which has been related to differences in relative proportions of membrane structures and to different amounts of contractile proteins [ 1 ]. Although the interfibre-type differences are well recognized, very little is known about the characteristics of the process leading from electrical membrane excitation to contraction (E-C coupling) related to different cell types. The first indication of variation in E-C coupling between representatives of different fibre types came from studies using vaseline-gap technique in order to record slow calcium currents and asymmetric charge movement in fast- and slow-twitch muscles [ 2 ]. The results suggested functional and structural differences between fast- and slow-twitch mammalian muscles with respect to dihydropyridine receptor (DHPR) density. Recently Goodman et al [ 3 ] provided the first evidence about E-C coupling characteristics being related to myosin heavy chain based fibre type and the muscle from which the fibre originated. Our finding about the density of DHPR varying between different fibre types [ 4 ] supports the hypothesis that optimum contractile function of skeletal muscle is related to its fibre type composition via differences in E-C coupling.Voltage-dependent dihydropyridine receptors play an important role in the function of skeletal muscle. As well known, contraction is initiated by a depolarization of the transverse tubular (T-tubular) membrane, which in turn causes calcium to be released from the stores of sarcoplasmic reticulum (SR). The DHPR is located in the T-tubule membrane acting as a voltage sensor. Linked to the ryanodine receptor located in the sarcoplasmic reticulum membrane, this channel protein triggers the intracellular Ca 2+ -release via the ryanodine receptor [ 5 ]. The DHPR itself is a heteromultimer composed of 5 subunits; α 1  (170 kDa), α 2  (140 kDa), β (55 kDa), γ (33 kDa) and δ (24–33 kDa) [ 6 , 7 ]. The pore forming α 1  subunit contains the receptor for dihydropyridines (DHPs) and other calcium antagonists. DHPs, such as nifedipine, nitrendipine, and nimodipine, bind to DHP receptor in a specific, saturable and reversible fashion. In addition, receptors show high affinity for DHP ligands. The dissociation constant ( K D ) for skeletal muscle membranes is usually in the range of 1–10 nM [ 8 ]. Dihydropyridines are used clinically as blockers in the treatment of hypertension and coronary heart disease [ 9 ].KDBesides being the voltage sensor for Ca 2+  release needed in E-C coupling, the α 1 -subunit of the molecule functions also as a calcium channel [ 10 ] giving rise to a slow inward calcium current [ 11 ]. However, DHPR appears to be unimportant as an ion-conducting channel [ 12 ]. Firstly, the calcium current is activated too slowly to generate contraction of the fibre. Second, the contraction continues even if the extracellular calcium is removed, and finally, blocking the slow calcium current through the channel does not prevent contraction. On the other hand, Beam et al [ 5 ] reported a notable increase in the magnitude of inward calcium current in developing muscle thus implicating an important role of the current in maturation.In the present study we examined the E-C coupling characteristics of muscles with different dihydropyridine receptor expression defined by fibre type composition. To test the hypothesis that muscles with different cell type compositions, i.e. DHPR quantities, react differently to calcium channel blocker, we measured contraction forces of three muscles before and after adding the blocker solution. We found variation in the activities of muscles depending on the cell type composition of the muscles. The findings confirm the previously observed pattern and elucidate the role of dihydropyridine receptor in muscle cell contraction.Results Myosin heavy chain analysis and fibre typing In order to analyse the distribution of cell types in the gluteus maximus (GLU), rectus femoris (RF) and gastrocnemius (GAS) muscles, tissue samples were collected and subjected to SDS-PAGE. The different muscles of mouse contained four myosin heavy chain (MHC) isoforms in the increasing electrophoretic mobility, MHCIIa, MHCIId, MHCIIb and MHCI (Fig.  1 ) as compared to those of rat gastrocnemius muscle (in this report MHC isoform expressed in fibre is identified by roman numeral and lower case letter, while the fibre type is identified by a roman numeral and a corresponding capital letter according to Hämäläinen and Pette [ 13 ]). The distribution of these myosin heavy chain isoforms differed among the three muscles studied (Table  1 ). In GAS, the major isoform was MHCIId (35.6 %), expressed together with smaller amounts of IIb (28.1 %). On the other hand, the quantity of MHCIIa was smaller (20.6 %). In contrast to GAS, both GLU and RF were characterized by higher contents of MHCIIa (26.6 % and 27.7 %, respectively), together with MHCd (34.3 %; 23.7 %) and lower amount of MHCIIb (15.9 %; 24.3 %). All the muscles contained also portions of MHCI (GAS: 15.7 %, GLU: 23.2 %, RF: 24.3 %). A statistically significant difference (P < 0.05) was found in the portion of IIb between GAS and GLU as well as in IId between GAS and RF. Four fibre types were also identified in muscle sections stained with mATPase (Table  1 ). The major fibre type in GAS was IID (51.5 %). However, in contrast to the results from SDS-PAGE, IIA fibre type (22.7 %) outnumbered the IIB (18.2 %). The most frequent fibre type in both GLU and RF was IIA (45.0 %; 38.1 %), followed by IIB (31.1 %) in GLU and IID (21.6 %) in RF. There was a statistically significant difference (P < 0.05) in the portion of IIA between GLU and RF. In addition, between GAS and RF a significant difference was found in the proportion of I (P < 0.05) and IID (P < 0.01). Between GAS and GLU a significant difference was found in proportion of IIA (P < 0.05) and IID (P < 0.01). To summarise, the significant difference in proportion of IIA between GAS and RF/GLU is particularly relevant to the present study. DHPR expression and correlation to fibre types We have previously shown that the density of dihydropyridine receptors (L-type calcium channels) in T-tubule membranes increases markedly during the postnatal development of mouse skeletal muscle [ 4 ]. Furthermore, the findings showed that the fast oxidative glycolytic (FOG) fibre type has the most evident appearance of DHPRs. Fig.  2A  shows localization of DHPR in mouse RF muscle. DHPR is strongly expressed in some muscle fibres whereas weakly expressed at the membrane of others. The corresponding control slide, preincubated with nifedipine resulted in a loss of staining (Fig.  2C ). To correlate the DHPR expression with muscle fibre typing, histochemical experiments were performed as described. According to the staining intensity, IIA fibre type corresponds to the muscle fibres that strongly express DHPRs (Fig.  2B ). Force measurements GLU, RF and GAS muscles were weighed and the length and diameter were measured. Table  2  shows some morphological details for the muscles studied. To reveal the function of L-type calcium channels in muscles with different kinds of cell type distributions, the single twitch forces of the muscles were measured before and after the addition of a specific channel blocker nifedipine. Maximum contraction forces in proportion to the cross-sectional area of the muscle, and the average responses to 1 μM nifedipine solution are shown in Fig.  3 . The force production of GAS (0.089 ± 0.016 N) was considerably higher in comparison to the ones of GLU (0.075 ± 0.017 N) and RF (0.045 ± 0.005 N). The effect of nifedipine on contractile force was inhibiting in all the muscles studied. The  in vitro  nifedipine effect on the muscle force production was determined as the percentage of the remaining channel mediated contraction force. The weakening of contraction force proportional to the cross-sectional areas of the muscles was significantly (P = 0.023) higher in GLU (28.3 ± 3.5 %) and RF (27.6 ± 3.2 %) in comparison to GAS (16.1 ± 2.5 %). Experiments performed in increasing concentrations of nifedipine resulted in a clear dose-response curve (Fig.  4 ) and revealed a decreasing force output as the blocker concentration increased. Furthermore, the decrease was significantly smaller in GAS (P < 0.05 at nifedipine concentration of 5 μM), in comparison to the decrease in GLU.
Results
Myosin heavy chain analysis and fibre typing In order to analyse the distribution of cell types in the gluteus maximus (GLU), rectus femoris (RF) and gastrocnemius (GAS) muscles, tissue samples were collected and subjected to SDS-PAGE. The different muscles of mouse contained four myosin heavy chain (MHC) isoforms in the increasing electrophoretic mobility, MHCIIa, MHCIId, MHCIIb and MHCI (Fig.  1 ) as compared to those of rat gastrocnemius muscle (in this report MHC isoform expressed in fibre is identified by roman numeral and lower case letter, while the fibre type is identified by a roman numeral and a corresponding capital letter according to Hämäläinen and Pette [ 13 ]). The distribution of these myosin heavy chain isoforms differed among the three muscles studied (Table  1 ). In GAS, the major isoform was MHCIId (35.6 %), expressed together with smaller amounts of IIb (28.1 %). On the other hand, the quantity of MHCIIa was smaller (20.6 %). In contrast to GAS, both GLU and RF were characterized by higher contents of MHCIIa (26.6 % and 27.7 %, respectively), together with MHCd (34.3 %; 23.7 %) and lower amount of MHCIIb (15.9 %; 24.3 %). All the muscles contained also portions of MHCI (GAS: 15.7 %, GLU: 23.2 %, RF: 24.3 %). A statistically significant difference (P < 0.05) was found in the portion of IIb between GAS and GLU as well as in IId between GAS and RF. Four fibre types were also identified in muscle sections stained with mATPase (Table  1 ). The major fibre type in GAS was IID (51.5 %). However, in contrast to the results from SDS-PAGE, IIA fibre type (22.7 %) outnumbered the IIB (18.2 %). The most frequent fibre type in both GLU and RF was IIA (45.0 %; 38.1 %), followed by IIB (31.1 %) in GLU and IID (21.6 %) in RF. There was a statistically significant difference (P < 0.05) in the portion of IIA between GLU and RF. In addition, between GAS and RF a significant difference was found in the proportion of I (P < 0.05) and IID (P < 0.01). Between GAS and GLU a significant difference was found in proportion of IIA (P < 0.05) and IID (P < 0.01). To summarise, the significant difference in proportion of IIA between GAS and RF/GLU is particularly relevant to the present study.
Myosin heavy chain analysis and fibre typing
In order to analyse the distribution of cell types in the gluteus maximus (GLU), rectus femoris (RF) and gastrocnemius (GAS) muscles, tissue samples were collected and subjected to SDS-PAGE. The different muscles of mouse contained four myosin heavy chain (MHC) isoforms in the increasing electrophoretic mobility, MHCIIa, MHCIId, MHCIIb and MHCI (Fig.  1 ) as compared to those of rat gastrocnemius muscle (in this report MHC isoform expressed in fibre is identified by roman numeral and lower case letter, while the fibre type is identified by a roman numeral and a corresponding capital letter according to Hämäläinen and Pette [ 13 ]). The distribution of these myosin heavy chain isoforms differed among the three muscles studied (Table  1 ). In GAS, the major isoform was MHCIId (35.6 %), expressed together with smaller amounts of IIb (28.1 %). On the other hand, the quantity of MHCIIa was smaller (20.6 %). In contrast to GAS, both GLU and RF were characterized by higher contents of MHCIIa (26.6 % and 27.7 %, respectively), together with MHCd (34.3 %; 23.7 %) and lower amount of MHCIIb (15.9 %; 24.3 %). All the muscles contained also portions of MHCI (GAS: 15.7 %, GLU: 23.2 %, RF: 24.3 %). A statistically significant difference (P < 0.05) was found in the portion of IIb between GAS and GLU as well as in IId between GAS and RF. Four fibre types were also identified in muscle sections stained with mATPase (Table  1 ). The major fibre type in GAS was IID (51.5 %). However, in contrast to the results from SDS-PAGE, IIA fibre type (22.7 %) outnumbered the IIB (18.2 %). The most frequent fibre type in both GLU and RF was IIA (45.0 %; 38.1 %), followed by IIB (31.1 %) in GLU and IID (21.6 %) in RF. There was a statistically significant difference (P < 0.05) in the portion of IIA between GLU and RF. In addition, between GAS and RF a significant difference was found in the proportion of I (P < 0.05) and IID (P < 0.01). Between GAS and GLU a significant difference was found in proportion of IIA (P < 0.05) and IID (P < 0.01). To summarise, the significant difference in proportion of IIA between GAS and RF/GLU is particularly relevant to the present study.DHPR expression and correlation to fibre types We have previously shown that the density of dihydropyridine receptors (L-type calcium channels) in T-tubule membranes increases markedly during the postnatal development of mouse skeletal muscle [ 4 ]. Furthermore, the findings showed that the fast oxidative glycolytic (FOG) fibre type has the most evident appearance of DHPRs. Fig.  2A  shows localization of DHPR in mouse RF muscle. DHPR is strongly expressed in some muscle fibres whereas weakly expressed at the membrane of others. The corresponding control slide, preincubated with nifedipine resulted in a loss of staining (Fig.  2C ). To correlate the DHPR expression with muscle fibre typing, histochemical experiments were performed as described. According to the staining intensity, IIA fibre type corresponds to the muscle fibres that strongly express DHPRs (Fig.  2B ).
DHPR expression and correlation to fibre types
We have previously shown that the density of dihydropyridine receptors (L-type calcium channels) in T-tubule membranes increases markedly during the postnatal development of mouse skeletal muscle [ 4 ]. Furthermore, the findings showed that the fast oxidative glycolytic (FOG) fibre type has the most evident appearance of DHPRs. Fig.  2A  shows localization of DHPR in mouse RF muscle. DHPR is strongly expressed in some muscle fibres whereas weakly expressed at the membrane of others. The corresponding control slide, preincubated with nifedipine resulted in a loss of staining (Fig.  2C ). To correlate the DHPR expression with muscle fibre typing, histochemical experiments were performed as described. According to the staining intensity, IIA fibre type corresponds to the muscle fibres that strongly express DHPRs (Fig.  2B ).Force measurements GLU, RF and GAS muscles were weighed and the length and diameter were measured. Table  2  shows some morphological details for the muscles studied. To reveal the function of L-type calcium channels in muscles with different kinds of cell type distributions, the single twitch forces of the muscles were measured before and after the addition of a specific channel blocker nifedipine. Maximum contraction forces in proportion to the cross-sectional area of the muscle, and the average responses to 1 μM nifedipine solution are shown in Fig.  3 . The force production of GAS (0.089 ± 0.016 N) was considerably higher in comparison to the ones of GLU (0.075 ± 0.017 N) and RF (0.045 ± 0.005 N). The effect of nifedipine on contractile force was inhibiting in all the muscles studied. The  in vitro  nifedipine effect on the muscle force production was determined as the percentage of the remaining channel mediated contraction force. The weakening of contraction force proportional to the cross-sectional areas of the muscles was significantly (P = 0.023) higher in GLU (28.3 ± 3.5 %) and RF (27.6 ± 3.2 %) in comparison to GAS (16.1 ± 2.5 %). Experiments performed in increasing concentrations of nifedipine resulted in a clear dose-response curve (Fig.  4 ) and revealed a decreasing force output as the blocker concentration increased. Furthermore, the decrease was significantly smaller in GAS (P < 0.05 at nifedipine concentration of 5 μM), in comparison to the decrease in GLU.
Force measurements
GLU, RF and GAS muscles were weighed and the length and diameter were measured. Table  2  shows some morphological details for the muscles studied. To reveal the function of L-type calcium channels in muscles with different kinds of cell type distributions, the single twitch forces of the muscles were measured before and after the addition of a specific channel blocker nifedipine. Maximum contraction forces in proportion to the cross-sectional area of the muscle, and the average responses to 1 μM nifedipine solution are shown in Fig.  3 . The force production of GAS (0.089 ± 0.016 N) was considerably higher in comparison to the ones of GLU (0.075 ± 0.017 N) and RF (0.045 ± 0.005 N). The effect of nifedipine on contractile force was inhibiting in all the muscles studied. The  in vitro  nifedipine effect on the muscle force production was determined as the percentage of the remaining channel mediated contraction force. The weakening of contraction force proportional to the cross-sectional areas of the muscles was significantly (P = 0.023) higher in GLU (28.3 ± 3.5 %) and RF (27.6 ± 3.2 %) in comparison to GAS (16.1 ± 2.5 %). Experiments performed in increasing concentrations of nifedipine resulted in a clear dose-response curve (Fig.  4 ) and revealed a decreasing force output as the blocker concentration increased. Furthermore, the decrease was significantly smaller in GAS (P < 0.05 at nifedipine concentration of 5 μM), in comparison to the decrease in GLU.in vitroDiscussion The cell type composition of a muscle is one of the features characterizing the function of the muscle. Type I fibres are most prevalent in muscles involved in the maintenance of posture, whereas type II fibres are used for movements which require high power output. Many vertebrate locomotor muscles are composed of a mixture of fibre types. Additionally, one muscle fibre may contain several myosin heavy chain types indicating heterogeneity at fibril level [ 14 ]. The m. gastrocnemius (whole) used in this study has a high type IIB content whereas both m. rectus femoris and m. gluteus maximus are predominantly composed of types IIA and IID. The results from myosin heavy chain analysis and fibre typing are somewhat different. The identification of four different fibre types using histochemical methods is difficult because of the presence of several types of myosin heavy chains within one single fibre. Therefore, the MHC analysis is more qualitative. The correlation of high density of DHPRs with IIA fibres was stated on the basis of ATPase activity. This was previously also shown by statistical analysis where fibre size was additionally taken into account [ 4 ]. Nifedipine was used as an antagonist in order to specifically block the dihydropyridine receptors. Nifedipine is a dihydropyridine derivative that binds in a specific, stereoselective and saturable fashion to DHPR. The selective calcium channel inhibitor prevents the influx of extracellular calcium through the L-type calcium channel [ 15 ] and also has a clear effect on the contraction activity of the skeletal muscle fibre. The effects of nifedipine on depolarization-induced force responses are inhibitory and dose dependent [ 16 ]. As the half life of the blocker is 2 – 6 h, there is enough time to perform reliable measurements. By blocking the channel with nifedipine, a clear selective decrease in the contraction force of the different muscles studied was observed (Fig.  3 ). The inhibition percentages of RF and GLU were, however, significantly higher as compared to that of GAS. This data indicates that the same concentration of blocker causes a different response between different muscles. A similar variability is noted when compared to the cell type composition of the muscles. The muscles with high IIA fibre type content have strongly reduced contraction force as a result of addition of a calcium channel blocker. On the other hand, the effect of a blocker on the muscle with a lower IIA type content is much weaker. The findings are attributable to the varying densities of dihydropyridine receptors in muscles. In GAS, the amount of DHPRs is reduced, likely due to the low IIA fibre type content. Thus the blocking effect of a receptor antagonist is weaker as compared to the muscles with a higher IIA content. Furthermore, the response of GAS and GLU to the gradual saturation of DHPRs with nifedipine molecules is different (Fig.  4 ). Consistently, the inhibition percentage of the contraction force of GAS decreased less as compared to GLU. In addition, a constant level of the contraction force was reached sooner in GAS indicating a complete saturation of the receptors available. There are several examples of the divergent behaviour of the dihydropyridine receptor in skeletal muscle. First, dihydropyridines are shown to have both stimulatory [ 17 ] and inhibitory [ 18 ] effects on excitation-contraction coupling. Furthermore, unlike in cardiac muscle, calcium release from SR does not require inward current, and as yet is induced by blockade of dihydropyridine receptors [ 19 ]. However, it is clear that DHPRs are essential in excitation-contraction coupling since animals with  mdg/mdg  mutation, which results in a lack of receptor proteins, die at birth because of paralysis of the respiratory muscles [ 20 ]. On the other hand, when the α 1 -subunit is introduced into the nuclei of the dysgenic myotubes, some cells contract upon an electrical stimulation. Despite of this, the recovered influx of calcium ions is not necessary for the contraction since the cells are contracting also even if the current is blocked with cadmium. Hence calcium antagonist drugs seem to have very few pharmacologically relevant actions on skeletal muscle as observed also by Walsh et al [ 21 ]. The role of inward current is still an open question although it has been suggested that the current is needed to maintain the calcium stores inside the cell [ 5 ] or for the conditions of activeness of the voltage sensor [ 22 ]. In the present study we report one plausible explanation for the large variation of the results observed in the behaviour of the L-type calcium channels in skeletal muscle. The uneven density distribution of dihydropyridine receptors indicates a difference in E-C coupling machinery between muscle fibre types. In this study, we showed that the difference is also detectable in the contraction forces between muscles of different cell type composition. Although nifedipine specifically blocks the dihydropyridine receptor, there could also be other differences in addition to the density of receptors between the muscles causing the difference in the contraction forces. A larger number of different muscles and developmental stages might clarify the differences in the present results. Goodman et al [ 3 ] provided the first evidence that E-C coupling characteristics are related to fibre types based on myosin heavy chain. By measuring depolarization-induced force responses of skinned single fibres of rat, they concluded that the optimum force production of a skeletal muscle is related to its MHC isoform composition. Although we found no published data on the E-C coupling phenotype of MHC IIa isoform, the results from previous studies suggest that the parameters describing force and velocity properties of a single muscle fibre are significantly higher in the fast, type IIA and IIB, fibres than in slow, type I fibres.
Discussion
The cell type composition of a muscle is one of the features characterizing the function of the muscle. Type I fibres are most prevalent in muscles involved in the maintenance of posture, whereas type II fibres are used for movements which require high power output. Many vertebrate locomotor muscles are composed of a mixture of fibre types. Additionally, one muscle fibre may contain several myosin heavy chain types indicating heterogeneity at fibril level [ 14 ]. The m. gastrocnemius (whole) used in this study has a high type IIB content whereas both m. rectus femoris and m. gluteus maximus are predominantly composed of types IIA and IID. The results from myosin heavy chain analysis and fibre typing are somewhat different. The identification of four different fibre types using histochemical methods is difficult because of the presence of several types of myosin heavy chains within one single fibre. Therefore, the MHC analysis is more qualitative. The correlation of high density of DHPRs with IIA fibres was stated on the basis of ATPase activity. This was previously also shown by statistical analysis where fibre size was additionally taken into account [ 4 ].Nifedipine was used as an antagonist in order to specifically block the dihydropyridine receptors. Nifedipine is a dihydropyridine derivative that binds in a specific, stereoselective and saturable fashion to DHPR. The selective calcium channel inhibitor prevents the influx of extracellular calcium through the L-type calcium channel [ 15 ] and also has a clear effect on the contraction activity of the skeletal muscle fibre. The effects of nifedipine on depolarization-induced force responses are inhibitory and dose dependent [ 16 ]. As the half life of the blocker is 2 – 6 h, there is enough time to perform reliable measurements.By blocking the channel with nifedipine, a clear selective decrease in the contraction force of the different muscles studied was observed (Fig.  3 ). The inhibition percentages of RF and GLU were, however, significantly higher as compared to that of GAS. This data indicates that the same concentration of blocker causes a different response between different muscles. A similar variability is noted when compared to the cell type composition of the muscles. The muscles with high IIA fibre type content have strongly reduced contraction force as a result of addition of a calcium channel blocker. On the other hand, the effect of a blocker on the muscle with a lower IIA type content is much weaker. The findings are attributable to the varying densities of dihydropyridine receptors in muscles. In GAS, the amount of DHPRs is reduced, likely due to the low IIA fibre type content. Thus the blocking effect of a receptor antagonist is weaker as compared to the muscles with a higher IIA content. Furthermore, the response of GAS and GLU to the gradual saturation of DHPRs with nifedipine molecules is different (Fig.  4 ). Consistently, the inhibition percentage of the contraction force of GAS decreased less as compared to GLU. In addition, a constant level of the contraction force was reached sooner in GAS indicating a complete saturation of the receptors available.There are several examples of the divergent behaviour of the dihydropyridine receptor in skeletal muscle. First, dihydropyridines are shown to have both stimulatory [ 17 ] and inhibitory [ 18 ] effects on excitation-contraction coupling. Furthermore, unlike in cardiac muscle, calcium release from SR does not require inward current, and as yet is induced by blockade of dihydropyridine receptors [ 19 ]. However, it is clear that DHPRs are essential in excitation-contraction coupling since animals with  mdg/mdg  mutation, which results in a lack of receptor proteins, die at birth because of paralysis of the respiratory muscles [ 20 ]. On the other hand, when the α 1 -subunit is introduced into the nuclei of the dysgenic myotubes, some cells contract upon an electrical stimulation. Despite of this, the recovered influx of calcium ions is not necessary for the contraction since the cells are contracting also even if the current is blocked with cadmium. Hence calcium antagonist drugs seem to have very few pharmacologically relevant actions on skeletal muscle as observed also by Walsh et al [ 21 ]. The role of inward current is still an open question although it has been suggested that the current is needed to maintain the calcium stores inside the cell [ 5 ] or for the conditions of activeness of the voltage sensor [ 22 ].mdg/mdgIn the present study we report one plausible explanation for the large variation of the results observed in the behaviour of the L-type calcium channels in skeletal muscle. The uneven density distribution of dihydropyridine receptors indicates a difference in E-C coupling machinery between muscle fibre types. In this study, we showed that the difference is also detectable in the contraction forces between muscles of different cell type composition. Although nifedipine specifically blocks the dihydropyridine receptor, there could also be other differences in addition to the density of receptors between the muscles causing the difference in the contraction forces. A larger number of different muscles and developmental stages might clarify the differences in the present results.Goodman et al [ 3 ] provided the first evidence that E-C coupling characteristics are related to fibre types based on myosin heavy chain. By measuring depolarization-induced force responses of skinned single fibres of rat, they concluded that the optimum force production of a skeletal muscle is related to its MHC isoform composition. Although we found no published data on the E-C coupling phenotype of MHC IIa isoform, the results from previous studies suggest that the parameters describing force and velocity properties of a single muscle fibre are significantly higher in the fast, type IIA and IIB, fibres than in slow, type I fibres.Conclusions Taken together, our results obtained from three different muscles confirmed that the expression of dihydropyridine receptor is a fibre type specific character. In addition, the present data indicate that the density of DHPRs is one important factor influencing the overall contractile properties of the muscle. Furthermore, the results of our experiments point out that when determining the physiological relevance of DHPRs, it is necessary to compare histochemistry and protein analysis to relevant functional properties such as contraction of the muscle. Moreover, it is noted, that differences between developing and adult type of muscle cells may emerge due to the differentiation of phenotypes of muscle fibres. In the future it might be useful to examine the correlation from a larger number of different muscles, and from muscles of different developmental stage.
Conclusions
Taken together, our results obtained from three different muscles confirmed that the expression of dihydropyridine receptor is a fibre type specific character. In addition, the present data indicate that the density of DHPRs is one important factor influencing the overall contractile properties of the muscle. Furthermore, the results of our experiments point out that when determining the physiological relevance of DHPRs, it is necessary to compare histochemistry and protein analysis to relevant functional properties such as contraction of the muscle. Moreover, it is noted, that differences between developing and adult type of muscle cells may emerge due to the differentiation of phenotypes of muscle fibres. In the future it might be useful to examine the correlation from a larger number of different muscles, and from muscles of different developmental stage.Methods Electrophoretic separation of myosin heavy chain isoforms M. rectus femoris (N = 6), m. gastrocnemius (N = 6) and m. gluteus maximus (N = 6) from adult mice (strain CD-1) were removed after the animals were killed by paracervical dislocation in accordance with the Animal Ethics Committee of the University of Oulu (licence no. 046/03). The muscles were homogenized in 6 vol of homogenization buffer [62.5 mM Tris-HCl, pH 6.8] and boiled in sample buffer as previously described for 5 min at a final protein concentration of 0.5 mg ml -1  [ 23 ]. Total protein was assayed according to the method of Bradford [ 24 ]. Myosin heavy chain isoforms were separated by gradient (5–8 %) sodiumdodecylsulfate polyacrylamide gel electrophoresis performed at 5°C for 23 h (120 V constant voltage). The gels were stained with Coomassie Brilliant Blue. The stained gels were scanned and the separated protein bands were analysed with FluorS MultiImager program (Bio-Rad, USA). Fibre typing Serial cross-sections, 8 μm thick, were cut on a cryostat microtome at -25°C, mounted on cover slips and stained for myosinATPase (mATPase) with acid preincubation according to Hämäläinen and Pette [ 25 ]. The sections were preincubated at room temperature for 7 min in sodium acetate (54.3 mM) – sodium barbital (32.6 mM) solution adjusted with HCl to pH 4.6. After washing with CaCl 2  (18 mM) and Tris-HCl (199 mM) the sections were incubated in substrate solution (4.5 mM ATP, 19.5 mM CaCl 2 , 116 mM 2-amino-2-methyl-1-propanol; pH 9.4) at room temperature for 45 min. After incubations in 11 mM CaCl 2 , 2% CoCl 2 , and 10 mM sodium barbital, the colour was developed in 2% (v/v) ammonium sulphide for 45 s. After washing with distilled water, the sections were dehydrated in ethanol, cleared in xylene and mounted with DPX. An alkaline preincubation in a solution containing 34 mM 2-amino-2-methyl-1-propanol, 120 mM CaCl 2 , adjusted to pH 10.3 with HCl was also used as previously described by Guth and Samaha [ 26 ]. After preincubation the sections were processed as above. The percentage of each fibre type in different muscles was calculated with the use of the LSM 5 PASCAL software 3.2 (Leo, Germany) for analyzing images. Fluorescence staining For fluorescence labelling, RF muscle from mouse was cut into 8 μm cryostat sections, dried for 30 min and treated as previously described by Mänttäri et al [ 4 ]. The concentration of the high affinity (-)-enantiomer of dihydropyridine was 20 nM. Control samples were preincubated with 10 μM nifedipine in the phosphate buffer for 10 min prior to the addition of dihydropyridine conjugate. Force measurements Adult mice (weighing 23 – 45 g) were killed by paracervical dislocation, and the pelvic region and right hind limb were skinned. The muscle of interest (m. rectus femoris, m. gastrocnemius or m. gluteus maximus) was dissected out and suspended between a fixed clamp at the base of an organ bath and a Grass FTO3C (USA) force-displacement transducer. Muscles were maintained in an oxygenated (95% O 2  – 5% CO 2 ) physiological saline solution (+37°C, pH 7.4) containing 137.0 mM NaCl, 2.7 mM KCl, 1.0 mM MgSO 4 · 7H 2 O, 1.8 mM CaCl 2 , 0.4 mM NaH 2 PO 4 , 12 mM NaHCO 3  and 5.5 mM glucose. After an equilibration period of 2 min, the muscles were supramaximally stimulated using steel electrode, pulses of 1 ms duration at 80 V (model S44, Grass Instrument Co.) and muscle length selected to elicit maximal twitch force. The maximum force of the muscle was measured in series of three stimulus impulses with one minute equilibration time between the stimulations. Recordings of transducer output were A/D converted and collected on a computer at a sample frequency of 1000 Hz. In order to examine the role of a specific, L-type calcium channel blocker, the muscles first measured in saline solution were mounted in a bath containing 1 μM nifedipine [1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester], equilibrated for 2 min, and assessed as described above i.e. the muscle was a control in itself. In addition, series of measurements were made with m. gastrocnemius (N = 6) and m. gluteus (N = 6) using nifedipine concentration range of 2 – 30 μM. The concentration of the drug was progressively increased by adding the nifedipine solution to the saline solution bath. During the experiment, the nifedipine solutions were maintained in dark to prevent photo-bleaching. To exclude the possibility of fatigue caused by successive stimulations, the protocol control measurement was performed similarly devoid of nifedipine. Statistical analysis The statistical significance of differences between means of contraction forces before and after the addition of calcium channel blocker was evaluated by paired samples t-test. The difference in parameters between the different types of muscle were analysed with independent samples t-test for equality of means. A P value of < 0.05 was accepted as indicative of a significant difference between the two sets of observations. The significance of differences between the muscles was evaluated with one way analysis of variance (normality test passed). The significant difference was stated as mentioned above. The values are presented as the means ± SE. All the statistical analyses were performed with the SPSS for Windows software.
Methods
Electrophoretic separation of myosin heavy chain isoforms M. rectus femoris (N = 6), m. gastrocnemius (N = 6) and m. gluteus maximus (N = 6) from adult mice (strain CD-1) were removed after the animals were killed by paracervical dislocation in accordance with the Animal Ethics Committee of the University of Oulu (licence no. 046/03). The muscles were homogenized in 6 vol of homogenization buffer [62.5 mM Tris-HCl, pH 6.8] and boiled in sample buffer as previously described for 5 min at a final protein concentration of 0.5 mg ml -1  [ 23 ]. Total protein was assayed according to the method of Bradford [ 24 ]. Myosin heavy chain isoforms were separated by gradient (5–8 %) sodiumdodecylsulfate polyacrylamide gel electrophoresis performed at 5°C for 23 h (120 V constant voltage). The gels were stained with Coomassie Brilliant Blue. The stained gels were scanned and the separated protein bands were analysed with FluorS MultiImager program (Bio-Rad, USA).
Electrophoretic separation of myosin heavy chain isoforms
M. rectus femoris (N = 6), m. gastrocnemius (N = 6) and m. gluteus maximus (N = 6) from adult mice (strain CD-1) were removed after the animals were killed by paracervical dislocation in accordance with the Animal Ethics Committee of the University of Oulu (licence no. 046/03). The muscles were homogenized in 6 vol of homogenization buffer [62.5 mM Tris-HCl, pH 6.8] and boiled in sample buffer as previously described for 5 min at a final protein concentration of 0.5 mg ml -1  [ 23 ]. Total protein was assayed according to the method of Bradford [ 24 ]. Myosin heavy chain isoforms were separated by gradient (5–8 %) sodiumdodecylsulfate polyacrylamide gel electrophoresis performed at 5°C for 23 h (120 V constant voltage). The gels were stained with Coomassie Brilliant Blue. The stained gels were scanned and the separated protein bands were analysed with FluorS MultiImager program (Bio-Rad, USA).Fibre typing Serial cross-sections, 8 μm thick, were cut on a cryostat microtome at -25°C, mounted on cover slips and stained for myosinATPase (mATPase) with acid preincubation according to Hämäläinen and Pette [ 25 ]. The sections were preincubated at room temperature for 7 min in sodium acetate (54.3 mM) – sodium barbital (32.6 mM) solution adjusted with HCl to pH 4.6. After washing with CaCl 2  (18 mM) and Tris-HCl (199 mM) the sections were incubated in substrate solution (4.5 mM ATP, 19.5 mM CaCl 2 , 116 mM 2-amino-2-methyl-1-propanol; pH 9.4) at room temperature for 45 min. After incubations in 11 mM CaCl 2 , 2% CoCl 2 , and 10 mM sodium barbital, the colour was developed in 2% (v/v) ammonium sulphide for 45 s. After washing with distilled water, the sections were dehydrated in ethanol, cleared in xylene and mounted with DPX. An alkaline preincubation in a solution containing 34 mM 2-amino-2-methyl-1-propanol, 120 mM CaCl 2 , adjusted to pH 10.3 with HCl was also used as previously described by Guth and Samaha [ 26 ]. After preincubation the sections were processed as above. The percentage of each fibre type in different muscles was calculated with the use of the LSM 5 PASCAL software 3.2 (Leo, Germany) for analyzing images.
Fibre typing
Serial cross-sections, 8 μm thick, were cut on a cryostat microtome at -25°C, mounted on cover slips and stained for myosinATPase (mATPase) with acid preincubation according to Hämäläinen and Pette [ 25 ]. The sections were preincubated at room temperature for 7 min in sodium acetate (54.3 mM) – sodium barbital (32.6 mM) solution adjusted with HCl to pH 4.6. After washing with CaCl 2  (18 mM) and Tris-HCl (199 mM) the sections were incubated in substrate solution (4.5 mM ATP, 19.5 mM CaCl 2 , 116 mM 2-amino-2-methyl-1-propanol; pH 9.4) at room temperature for 45 min. After incubations in 11 mM CaCl 2 , 2% CoCl 2 , and 10 mM sodium barbital, the colour was developed in 2% (v/v) ammonium sulphide for 45 s. After washing with distilled water, the sections were dehydrated in ethanol, cleared in xylene and mounted with DPX. An alkaline preincubation in a solution containing 34 mM 2-amino-2-methyl-1-propanol, 120 mM CaCl 2 , adjusted to pH 10.3 with HCl was also used as previously described by Guth and Samaha [ 26 ]. After preincubation the sections were processed as above. The percentage of each fibre type in different muscles was calculated with the use of the LSM 5 PASCAL software 3.2 (Leo, Germany) for analyzing images.Fluorescence staining For fluorescence labelling, RF muscle from mouse was cut into 8 μm cryostat sections, dried for 30 min and treated as previously described by Mänttäri et al [ 4 ]. The concentration of the high affinity (-)-enantiomer of dihydropyridine was 20 nM. Control samples were preincubated with 10 μM nifedipine in the phosphate buffer for 10 min prior to the addition of dihydropyridine conjugate.
Fluorescence staining
For fluorescence labelling, RF muscle from mouse was cut into 8 μm cryostat sections, dried for 30 min and treated as previously described by Mänttäri et al [ 4 ]. The concentration of the high affinity (-)-enantiomer of dihydropyridine was 20 nM. Control samples were preincubated with 10 μM nifedipine in the phosphate buffer for 10 min prior to the addition of dihydropyridine conjugate.Force measurements Adult mice (weighing 23 – 45 g) were killed by paracervical dislocation, and the pelvic region and right hind limb were skinned. The muscle of interest (m. rectus femoris, m. gastrocnemius or m. gluteus maximus) was dissected out and suspended between a fixed clamp at the base of an organ bath and a Grass FTO3C (USA) force-displacement transducer. Muscles were maintained in an oxygenated (95% O 2  – 5% CO 2 ) physiological saline solution (+37°C, pH 7.4) containing 137.0 mM NaCl, 2.7 mM KCl, 1.0 mM MgSO 4 · 7H 2 O, 1.8 mM CaCl 2 , 0.4 mM NaH 2 PO 4 , 12 mM NaHCO 3  and 5.5 mM glucose. After an equilibration period of 2 min, the muscles were supramaximally stimulated using steel electrode, pulses of 1 ms duration at 80 V (model S44, Grass Instrument Co.) and muscle length selected to elicit maximal twitch force. The maximum force of the muscle was measured in series of three stimulus impulses with one minute equilibration time between the stimulations. Recordings of transducer output were A/D converted and collected on a computer at a sample frequency of 1000 Hz. In order to examine the role of a specific, L-type calcium channel blocker, the muscles first measured in saline solution were mounted in a bath containing 1 μM nifedipine [1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester], equilibrated for 2 min, and assessed as described above i.e. the muscle was a control in itself. In addition, series of measurements were made with m. gastrocnemius (N = 6) and m. gluteus (N = 6) using nifedipine concentration range of 2 – 30 μM. The concentration of the drug was progressively increased by adding the nifedipine solution to the saline solution bath. During the experiment, the nifedipine solutions were maintained in dark to prevent photo-bleaching. To exclude the possibility of fatigue caused by successive stimulations, the protocol control measurement was performed similarly devoid of nifedipine.
Force measurements
Adult mice (weighing 23 – 45 g) were killed by paracervical dislocation, and the pelvic region and right hind limb were skinned. The muscle of interest (m. rectus femoris, m. gastrocnemius or m. gluteus maximus) was dissected out and suspended between a fixed clamp at the base of an organ bath and a Grass FTO3C (USA) force-displacement transducer. Muscles were maintained in an oxygenated (95% O 2  – 5% CO 2 ) physiological saline solution (+37°C, pH 7.4) containing 137.0 mM NaCl, 2.7 mM KCl, 1.0 mM MgSO 4 · 7H 2 O, 1.8 mM CaCl 2 , 0.4 mM NaH 2 PO 4 , 12 mM NaHCO 3  and 5.5 mM glucose. After an equilibration period of 2 min, the muscles were supramaximally stimulated using steel electrode, pulses of 1 ms duration at 80 V (model S44, Grass Instrument Co.) and muscle length selected to elicit maximal twitch force. The maximum force of the muscle was measured in series of three stimulus impulses with one minute equilibration time between the stimulations. Recordings of transducer output were A/D converted and collected on a computer at a sample frequency of 1000 Hz.In order to examine the role of a specific, L-type calcium channel blocker, the muscles first measured in saline solution were mounted in a bath containing 1 μM nifedipine [1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester], equilibrated for 2 min, and assessed as described above i.e. the muscle was a control in itself. In addition, series of measurements were made with m. gastrocnemius (N = 6) and m. gluteus (N = 6) using nifedipine concentration range of 2 – 30 μM. The concentration of the drug was progressively increased by adding the nifedipine solution to the saline solution bath. During the experiment, the nifedipine solutions were maintained in dark to prevent photo-bleaching. To exclude the possibility of fatigue caused by successive stimulations, the protocol control measurement was performed similarly devoid of nifedipine.Statistical analysis The statistical significance of differences between means of contraction forces before and after the addition of calcium channel blocker was evaluated by paired samples t-test. The difference in parameters between the different types of muscle were analysed with independent samples t-test for equality of means. A P value of < 0.05 was accepted as indicative of a significant difference between the two sets of observations. The significance of differences between the muscles was evaluated with one way analysis of variance (normality test passed). The significant difference was stated as mentioned above. The values are presented as the means ± SE. All the statistical analyses were performed with the SPSS for Windows software.
Statistical analysis
The statistical significance of differences between means of contraction forces before and after the addition of calcium channel blocker was evaluated by paired samples t-test. The difference in parameters between the different types of muscle were analysed with independent samples t-test for equality of means. A P value of < 0.05 was accepted as indicative of a significant difference between the two sets of observations. The significance of differences between the muscles was evaluated with one way analysis of variance (normality test passed). The significant difference was stated as mentioned above. The values are presented as the means ± SE. All the statistical analyses were performed with the SPSS for Windows software.List of abbreviations used DHP; dihydropyridine DHPR; dihydropyridine receptor E-C coupling; excitation-contraction coupling FOG; fast oxidative glycolytic GAS;  musculus gastrocnemius GLU;  musculus gluteus maximus mATPase; myosin adenosine triphosphatase MHC; myosin heavy chain RF;  musculus rectus femoris SDS-PAGE; sodium dodecyl sulphate polyacrylamide gel electrophoresis SR; sarcoplasmatic reticulum T-tubule; transverse tubule IIa, IIb, IId; myosin heavy chain type IIA, IIB, IID; fibre type
List of abbreviations used
DHP; dihydropyridineDHPR; dihydropyridine receptorE-C coupling; excitation-contraction couplingFOG; fast oxidative glycolyticGAS;  musculus gastrocnemiusmusculus gastrocnemiusGLU;  musculus gluteus maximusmusculus gluteus maximusmATPase; myosin adenosine triphosphataseMHC; myosin heavy chainRF;  musculus rectus femorismusculus rectus femorisSDS-PAGE; sodium dodecyl sulphate polyacrylamide gel electrophoresisSR; sarcoplasmatic reticulumT-tubule; transverse tubuleIIa, IIb, IId; myosin heavy chain typeIIA, IIB, IID; fibre typeAuthors' contributions SM carried out most of the experiments, assisted in interpretation of results, and participated in writing the manuscript. MJ conceived of the study, participated in its design and coordination, and participated in writing the manuscript.
Authors' contributions
SM carried out most of the experiments, assisted in interpretation of results, and participated in writing the manuscript. MJ conceived of the study, participated in its design and coordination, and participated in writing the manuscript.


Background Enterobacter aerogenes  and  Klebsiella pneumoniae  are common isolates in clinical microbiology and important as producers of extended spectrum β-lactamases (ESBL). The discrimination between both species, which is routinely based on biochemical characteristics, is generally accepted to be straightforward. Here we report that genotypically unrelated strains of  E. aerogenes  can be misidentified as  K. pneumoniae  by routine laboratories using standard biochemical identification and using identification automates.
Background
Enterobacter aerogenes  and  Klebsiella pneumoniae  are common isolates in clinical microbiology and important as producers of extended spectrum β-lactamases (ESBL). The discrimination between both species, which is routinely based on biochemical characteristics, is generally accepted to be straightforward. Here we report that genotypically unrelated strains of  E. aerogenes  can be misidentified as  K. pneumoniae  by routine laboratories using standard biochemical identification and using identification automates.Enterobacter aerogenesKlebsiella pneumoniaeE. aerogenesK. pneumoniaeResults Ten clinical isolates, identified as  K. pneumoniae  or  K. terrigena  with the routinely used biochemical tests and with API-20E, were identified as  E. aerogenes  by tDNA-PCR – an identification that was confirmed by 16S rRNA gene sequencing for five of these isolates. Misidentification also occurred when using the automated identification systems Vitek 2 and Phoenix, and was due to delayed positivity for ornithine decarboxylase and motility. Subculture and prolonged incubation resulted in positive results for ornithine decarboxylase and for motility. It could be shown by RAPD-analysis that the  E. aerogenes  strains belonged to different genotypes.
Results
Ten clinical isolates, identified as  K. pneumoniae  or  K. terrigena  with the routinely used biochemical tests and with API-20E, were identified as  E. aerogenes  by tDNA-PCR – an identification that was confirmed by 16S rRNA gene sequencing for five of these isolates. Misidentification also occurred when using the automated identification systems Vitek 2 and Phoenix, and was due to delayed positivity for ornithine decarboxylase and motility. Subculture and prolonged incubation resulted in positive results for ornithine decarboxylase and for motility. It could be shown by RAPD-analysis that the  E. aerogenes  strains belonged to different genotypes.K. pneumoniaeK. terrigenaE. aerogenesE. aerogenesConclusions Clinical  E. aerogenes  isolates can be easily misidentified as  Klebsiella  due to delayed positivity for ornithine decarboxylase and motility. The phenomenon may be widespread, since it was shown to occur among genotypically unrelated strains from different hospitals and different isolation dates. A useful clue for correct identification is the presence of an inducible β-lactamase, which is highly unusual for  K. pneumoniae . In several instances, the use of genotypic techniques like tDNA-PCR may circumvent problems of phenotypic identification.
Conclusions
Clinical  E. aerogenes  isolates can be easily misidentified as  Klebsiella  due to delayed positivity for ornithine decarboxylase and motility. The phenomenon may be widespread, since it was shown to occur among genotypically unrelated strains from different hospitals and different isolation dates. A useful clue for correct identification is the presence of an inducible β-lactamase, which is highly unusual for  K. pneumoniae . In several instances, the use of genotypic techniques like tDNA-PCR may circumvent problems of phenotypic identification.E. aerogenesKlebsiellaK. pneumoniae

Background Enterobacteriaceae with β-lactam resistance due to the production of Extended-Spectrum β-Lactamases (ESBL) were discovered in the eighties and since that time became epidemic and endemic in hospitals worldwide [ 1 ]. Since two decades, 20 to 40 ESBL-producing strains are isolated monthly in our hospital. Amongst the clinical isolates from our hospital, two new TEM-β-lactamase genes were described [ 2 ]. In Belgium, as well as in other countries, a shift occurred from  Klebsiella pneumoniae  isolates as the predominant ESBL-producers [ 3 ] to predominance of  Enterobacter aerogenes . It is also known that most of the  E. aerogenes  isolates in the Belgian hospitals belong to one of two predominant Belgian clones (BEI and BEII) [ 4 ], a situation which is comparable to that in other countries [ 5 ]. During the last years however, we found that several isolates that were identified as  K. pneumoniae  or  K. terrigena  by conventional biochemical testing were in fact  E. aerogenes  as could be shown by the use of genotypic methods, i.e. tDNA-PCR, validated by 16S rRNA gene sequencing, and by extensive phenotypic testing (including subculture and prolonged incubation).
Background
Enterobacteriaceae with β-lactam resistance due to the production of Extended-Spectrum β-Lactamases (ESBL) were discovered in the eighties and since that time became epidemic and endemic in hospitals worldwide [ 1 ]. Since two decades, 20 to 40 ESBL-producing strains are isolated monthly in our hospital. Amongst the clinical isolates from our hospital, two new TEM-β-lactamase genes were described [ 2 ]. In Belgium, as well as in other countries, a shift occurred from  Klebsiella pneumoniae  isolates as the predominant ESBL-producers [ 3 ] to predominance of  Enterobacter aerogenes . It is also known that most of the  E. aerogenes  isolates in the Belgian hospitals belong to one of two predominant Belgian clones (BEI and BEII) [ 4 ], a situation which is comparable to that in other countries [ 5 ]. During the last years however, we found that several isolates that were identified as  K. pneumoniae  or  K. terrigena  by conventional biochemical testing were in fact  E. aerogenes  as could be shown by the use of genotypic methods, i.e. tDNA-PCR, validated by 16S rRNA gene sequencing, and by extensive phenotypic testing (including subculture and prolonged incubation).Klebsiella pneumoniaeEnterobacter aerogenesE. aerogenesK. pneumoniaeK. terrigenaE. aerogenesResults For ten clinical isolates, i.e. seven ESBL-producing clinical isolates collected during 2001 and three more recently collected isolates (Table  1 ), the API20E codes 5205773 (isolates GA1, GA2, GA3, MN2, MN3 and VGM), 5205753 (isolates DHJ1 and DHJ3) or 5204673 (isolates RA and DBH) were obtained. The first code resulted in a weak identification as either  E. aerogenes ,  K. pneumoniae  or  Raoultella  ( Klebsiella )  planticola , the second code did not yield any identification, and the third code resulted in a weak but acceptable identification as  K. terrigena . The isolates with the codes 5205773 and 5205753 were identified as  K. pneumoniae  by additional biochemical testing due to negative reactions for motility (tested in semi-solid agar) and ornithine decarboxylase. However, these isolates all possessed an inducible cefalosporinase, as detected on the antibiogram using a disk approximation test, a finding which strongly contradicts an identification as  K. pneumoniae  or  Klebsiella  sp. In fact, the first hint that these strains, phenotypically identified as  K. pneumoniae , were actually  E. aerogenes , came from tDNA-PCR based identification. Using this method, all isolates were identified as  E. aerogenes , and this by comparison of the obtained fingerprint – composed of amplified intergenic tRNA spacers of 101, 106, 111, 115, 121, 189, 190, 198 or 289, and 391 bp in length – with a library containing fingerprints of more than 3000 strains belonging to hundreds of species, available at  . Confirmation of this genotypic identification was obtained by 16S rRNA gene sequencing for five isolates (Table  1 ). Analysis yielded a similarity of between 99.8% and 100% to  E. aerogenes  Genbank entries. The presence of a genuine  K. pneumoniae  isolate in patient DHJ (Table  1 ) further complicated the identification. This observation lead us to carry out additional phenotypic testing. Using the hanging drop method for testing motility, a few motile cells were observed, and upon retesting in semi-solid medium, weak migration could be observed. Like most biochemical tests in the routine laboratory, ornithine decarboxylase is read after overnight or 24 hours of incubation, but when the incubation period was prolonged to up to 2–5 days, all isolates tested positive. Because at present automated systems are frequently used for routine identification, a selection of six strains, containing four isolates of the study and two controls, i.e. phenotypically correctly identified  E. aerogenes  (LBV268) and  K. pneumoniae  (BG) isolates, were tested in two different systems, i.e. Vitek 2 (bioMérieux, Marcy l'Etoile, France) and Phoenix (BD Biosciences, Sparks, Md.). Both automated systems yielded the same results as the API20E, i.e. that the  E. aerogenes  isolates, aberrant due to a slow reaction for motility and ornithine decarboxylase, were misidentified as  K. pneumoniae  (Table  1 ). This misidentification by the automated systems is not unexpected, since they are based on biochemical testing only and a reading time of 24 hours or less. The control strains were correctly identified. Disk diffusion antibiotic susceptibility testing, carried out according the NCCLS guidelines, revealed basically the same resistotype for all isolates, characterized by resistance to ceftazidime and susceptibility to ceftriaxone. Additional resistance to aztreonam was observed for some isolates, reflecting the most dominant resistance patterns for the  E. aerogenes  isolates in our hospital. All isolates were also found to carry high-level resistance to cefoxitin, which is highly unusual for  Klebsiella  spp. Furthermore, the disk-approximation test with an amoxycillin-clavulanic acid disk close to β-lactam disks on Mueller-Hinton II agar, showed a combined pattern of synergy (broadening of the inhibition zone in the direction of clavulanic acid) and antagonism (flattening of the inhibition zone), which is suggestive for a combination of an ESBL and an inducible β-lactamase. Again, inducible β-lactamases are very rare in  Klebsiella  spp. but typical for  Enterobacter  spp. It should be noticed that this phenomenon will not be detected by automated MIC-determination systems like Vitek 2 and Phoenix. Using PCR and sequencing as described previously [ 2 ], the presence of TEM-5 could be shown in the isolates of patients GA and MN, and SHV-4 in the isolate of patient DHJ. The genotypic relationship of the phenotypically aberrant isolates was investigated with AP-PCR. Isolates GA1, GA2, GA3, MN2, MN3 and DBH were corresponding to Belgian clone BEI (Figure  1 , pattern A), isolates VGM, DHJ1 and DHJ3 were closely related to Belgian clone BEII (Figure  1 , pattern B, differing from pattern C, characteristic of clone BEII, by a single extra band) while isolate RA was not related to any of the others (Figure  1 , pattern D). This genetic diversity among the phenotypically aberrant strains makes it probable that strains with this kind of aberrant phenotype are not restricted to a single clone within  E. aerogenes .
Results
For ten clinical isolates, i.e. seven ESBL-producing clinical isolates collected during 2001 and three more recently collected isolates (Table  1 ), the API20E codes 5205773 (isolates GA1, GA2, GA3, MN2, MN3 and VGM), 5205753 (isolates DHJ1 and DHJ3) or 5204673 (isolates RA and DBH) were obtained. The first code resulted in a weak identification as either  E. aerogenes ,  K. pneumoniae  or  Raoultella  ( Klebsiella )  planticola , the second code did not yield any identification, and the third code resulted in a weak but acceptable identification as  K. terrigena . The isolates with the codes 5205773 and 5205753 were identified as  K. pneumoniae  by additional biochemical testing due to negative reactions for motility (tested in semi-solid agar) and ornithine decarboxylase. However, these isolates all possessed an inducible cefalosporinase, as detected on the antibiogram using a disk approximation test, a finding which strongly contradicts an identification as  K. pneumoniae  or  Klebsiella  sp.E. aerogenesK. pneumoniaeRaoultellaKlebsiellaplanticolaK. terrigenaK. pneumoniaeK. pneumoniaeKlebsiellaIn fact, the first hint that these strains, phenotypically identified as  K. pneumoniae , were actually  E. aerogenes , came from tDNA-PCR based identification. Using this method, all isolates were identified as  E. aerogenes , and this by comparison of the obtained fingerprint – composed of amplified intergenic tRNA spacers of 101, 106, 111, 115, 121, 189, 190, 198 or 289, and 391 bp in length – with a library containing fingerprints of more than 3000 strains belonging to hundreds of species, available at  .K. pneumoniaeE. aerogenesE. aerogenesConfirmation of this genotypic identification was obtained by 16S rRNA gene sequencing for five isolates (Table  1 ). Analysis yielded a similarity of between 99.8% and 100% to  E. aerogenes  Genbank entries.E. aerogenesThe presence of a genuine  K. pneumoniae  isolate in patient DHJ (Table  1 ) further complicated the identification.K. pneumoniaeThis observation lead us to carry out additional phenotypic testing. Using the hanging drop method for testing motility, a few motile cells were observed, and upon retesting in semi-solid medium, weak migration could be observed. Like most biochemical tests in the routine laboratory, ornithine decarboxylase is read after overnight or 24 hours of incubation, but when the incubation period was prolonged to up to 2–5 days, all isolates tested positive.Because at present automated systems are frequently used for routine identification, a selection of six strains, containing four isolates of the study and two controls, i.e. phenotypically correctly identified  E. aerogenes  (LBV268) and  K. pneumoniae  (BG) isolates, were tested in two different systems, i.e. Vitek 2 (bioMérieux, Marcy l'Etoile, France) and Phoenix (BD Biosciences, Sparks, Md.). Both automated systems yielded the same results as the API20E, i.e. that the  E. aerogenes  isolates, aberrant due to a slow reaction for motility and ornithine decarboxylase, were misidentified as  K. pneumoniae  (Table  1 ). This misidentification by the automated systems is not unexpected, since they are based on biochemical testing only and a reading time of 24 hours or less. The control strains were correctly identified.E. aerogenesK. pneumoniaeE. aerogenesK. pneumoniaeDisk diffusion antibiotic susceptibility testing, carried out according the NCCLS guidelines, revealed basically the same resistotype for all isolates, characterized by resistance to ceftazidime and susceptibility to ceftriaxone. Additional resistance to aztreonam was observed for some isolates, reflecting the most dominant resistance patterns for the  E. aerogenes  isolates in our hospital. All isolates were also found to carry high-level resistance to cefoxitin, which is highly unusual for  Klebsiella  spp. Furthermore, the disk-approximation test with an amoxycillin-clavulanic acid disk close to β-lactam disks on Mueller-Hinton II agar, showed a combined pattern of synergy (broadening of the inhibition zone in the direction of clavulanic acid) and antagonism (flattening of the inhibition zone), which is suggestive for a combination of an ESBL and an inducible β-lactamase. Again, inducible β-lactamases are very rare in  Klebsiella  spp. but typical for  Enterobacter  spp. It should be noticed that this phenomenon will not be detected by automated MIC-determination systems like Vitek 2 and Phoenix. Using PCR and sequencing as described previously [ 2 ], the presence of TEM-5 could be shown in the isolates of patients GA and MN, and SHV-4 in the isolate of patient DHJ.E. aerogenesKlebsiellaKlebsiellaEnterobacterThe genotypic relationship of the phenotypically aberrant isolates was investigated with AP-PCR. Isolates GA1, GA2, GA3, MN2, MN3 and DBH were corresponding to Belgian clone BEI (Figure  1 , pattern A), isolates VGM, DHJ1 and DHJ3 were closely related to Belgian clone BEII (Figure  1 , pattern B, differing from pattern C, characteristic of clone BEII, by a single extra band) while isolate RA was not related to any of the others (Figure  1 , pattern D). This genetic diversity among the phenotypically aberrant strains makes it probable that strains with this kind of aberrant phenotype are not restricted to a single clone within  E. aerogenes .E. aerogenesDiscussion K. pneumoniae  and  E. aerogenes  are taxonomically closely related species [ 6 ] which share many characteristics. Our sequencing results (unpublished) and those of others [ 6 ] confirm that the genus  Enterobacter  is polyphyletic and that  E. aerogenes  should be placed within the genus  Klebsiella . However, differentiation between  E. aerogenes  and  K. pneumoniae  is usually straightforward when based on testing for ornithine decarboxylase and motility, both positive for  E. aerogenes . This is also reflected in the name " Klebsiella mobilis " [ 7 ], which is known as a valid synonym for  E. aerogenes . Apparently, in some  E. aerogenes  isolates, the expression of these characteristics can be weak and/or delayed, and these are therefore scored negative when reading is done after the incubation periods that are routinely applied (overnight – 24 hours). However, the combination of an inducible β-lactamase and/or high-level cefoxitin resistance, which are rare in  Klebsiella  spp., and an identification as  Klebsiella  sp. should warrant further investigation. The phenomenon of  E. aerogenes  misidentified as  K. pneumoniae  or  K. terrigena  due to delayed or negative ornithine decarboxylase and motility was reported previously, and was also discovered because of unexpected imipenem resistance of the so-called  K. pneumoniae  isolates [ 8 ]. Also in this case, subculture and prolonged incubation restored the positivity for these characteristics. The problem of misidentification of  E. aerogenes  as  K. pneumoniae  (or even  K. terrigena ) is probably not uncommon and probably also geographically widespread. This can be deduced from the following considerations: i) this phenomenon of misidentification of  E. aerogenes  was already reported in 1993 [ 8 ], ii) the phenomenon occurred in genotypically different organisms, iii) the isolates were found over an extended period of time – also recently, and finally iv) we received similar strains from other Belgian hospitals (unpublished data). It should be noted that misidentification also occurred when using the newer and automated systems like Vitek2 and Phoenix. On the other hand, the problem seems to be largely unknown. In a recent study, Hansen and colleagues [ 9 ] carried out an interlaboratory comparison of the efficacy of 18 biochemical tests for the identification of 242 strains of different  Klebsiella  species and of  Enterobacter aerogenes , but do not mention the problem of possible delayed activity, possibly also because the study started from validated strains of each species.
Discussion
K. pneumoniae  and  E. aerogenes  are taxonomically closely related species [ 6 ] which share many characteristics. Our sequencing results (unpublished) and those of others [ 6 ] confirm that the genus  Enterobacter  is polyphyletic and that  E. aerogenes  should be placed within the genus  Klebsiella . However, differentiation between  E. aerogenes  and  K. pneumoniae  is usually straightforward when based on testing for ornithine decarboxylase and motility, both positive for  E. aerogenes . This is also reflected in the name " Klebsiella mobilis " [ 7 ], which is known as a valid synonym for  E. aerogenes . Apparently, in some  E. aerogenes  isolates, the expression of these characteristics can be weak and/or delayed, and these are therefore scored negative when reading is done after the incubation periods that are routinely applied (overnight – 24 hours).K. pneumoniaeE. aerogenesEnterobacterE. aerogenesKlebsiellaE. aerogenesK. pneumoniaeE. aerogenesKlebsiella mobilisE. aerogenesE. aerogenesHowever, the combination of an inducible β-lactamase and/or high-level cefoxitin resistance, which are rare in  Klebsiella  spp., and an identification as  Klebsiella  sp. should warrant further investigation.KlebsiellaKlebsiellaThe phenomenon of  E. aerogenes  misidentified as  K. pneumoniae  or  K. terrigena  due to delayed or negative ornithine decarboxylase and motility was reported previously, and was also discovered because of unexpected imipenem resistance of the so-called  K. pneumoniae  isolates [ 8 ]. Also in this case, subculture and prolonged incubation restored the positivity for these characteristics.E. aerogenesK. pneumoniaeK. terrigenaK. pneumoniaeThe problem of misidentification of  E. aerogenes  as  K. pneumoniae  (or even  K. terrigena ) is probably not uncommon and probably also geographically widespread. This can be deduced from the following considerations: i) this phenomenon of misidentification of  E. aerogenes  was already reported in 1993 [ 8 ], ii) the phenomenon occurred in genotypically different organisms, iii) the isolates were found over an extended period of time – also recently, and finally iv) we received similar strains from other Belgian hospitals (unpublished data). It should be noted that misidentification also occurred when using the newer and automated systems like Vitek2 and Phoenix.E. aerogenesK. pneumoniaeK. terrigenaE. aerogenesOn the other hand, the problem seems to be largely unknown. In a recent study, Hansen and colleagues [ 9 ] carried out an interlaboratory comparison of the efficacy of 18 biochemical tests for the identification of 242 strains of different  Klebsiella  species and of  Enterobacter aerogenes , but do not mention the problem of possible delayed activity, possibly also because the study started from validated strains of each species.KlebsiellaEnterobacter aerogenesConclusions Identification in a routine clinical microbiology laboratory of the most commonly encountered Enterobacteriaceae is usually considered to be fast and straightforward, but apparently identification problems may occur due to diminished or delayed expression of some characteristics, even for well-established species like  E. aerogenes  and  K. pneumoniae . Here we showed that  E. aerogenes  isolates exist for which ornithine decarboxylase and motility are negative or delayed positive, and that as such these isolates can be misidentified as  K. pneumoniae . This phenomenon may be quite frequent and geographically widespread. Genotypic identification techniques like tDNA-PCR, which moreover are cheaper than phenotypic testing for many bacterial species, can be semi-automatized, are faster and mostly have a higher discriminatory power, which is also reflected in this study.
Conclusions
Identification in a routine clinical microbiology laboratory of the most commonly encountered Enterobacteriaceae is usually considered to be fast and straightforward, but apparently identification problems may occur due to diminished or delayed expression of some characteristics, even for well-established species like  E. aerogenes  and  K. pneumoniae . Here we showed that  E. aerogenes  isolates exist for which ornithine decarboxylase and motility are negative or delayed positive, and that as such these isolates can be misidentified as  K. pneumoniae . This phenomenon may be quite frequent and geographically widespread. Genotypic identification techniques like tDNA-PCR, which moreover are cheaper than phenotypic testing for many bacterial species, can be semi-automatized, are faster and mostly have a higher discriminatory power, which is also reflected in this study.E. aerogenesK. pneumoniaeE. aerogenesK. pneumoniaeMethods tDNA-PCR tDNA-PCR was carried out using the outwardly directed tRNA-gene consensus primers T5A (5'AGTCCGGTGCTCTAACCAACTGAG) and T3B (5'AGGTCGCGGGTTCGAATCC), thus amplifying the intergenic tRNA-spacers, as described previously [ 10 , 11 ]. Electropherograms were normalized using GeneScan Analysis software, version 2.1 (Applied Biosystems). Transformation of GeneScan tables (ABI310, McIntosh) to tables on IBM, separation into separate digital fingerprints, and comparison of the digital tDNA-PCR fingerprints with a library of tDNA-PCR-fingerprints obtained from a large collection of reference strains, was done using in house software described previously [ 10 ]. 16S rRNA gene sequencing For five of the phenotypically aberrant isolates, the complete 16S rRNA sequence was determined by amplification of the 16S rRNA-gene with the primers 5'-AGTTTGATCCTGGCTCAG and 5'-TACCTTGTTACGACTTCGTCCCA [ 12 ], and sequencing was performed as described previously [ 12 ]. Comparison of the obtained 16S rDNA-sequence with all known sequences in Genbank was carried out using the BLAST software (National Center for Biotechnology Information  ). RAPD-analysis for strain typing The genotypic relationship of the isolates was investigated using arbitrarily primed PCR with RAPD Ready-to-Go beads (Amersham Pharmacia, Uppsala, Sweden) and the ERIC II primer 5'-AAGTAAGTGACTGGGGTGAGCG [ 13 ]. Analysis of the fingerprints was obtained by visual interpretation on ethidium bromide stained electrophoresis gels.
Methods
tDNA-PCR tDNA-PCR was carried out using the outwardly directed tRNA-gene consensus primers T5A (5'AGTCCGGTGCTCTAACCAACTGAG) and T3B (5'AGGTCGCGGGTTCGAATCC), thus amplifying the intergenic tRNA-spacers, as described previously [ 10 , 11 ]. Electropherograms were normalized using GeneScan Analysis software, version 2.1 (Applied Biosystems). Transformation of GeneScan tables (ABI310, McIntosh) to tables on IBM, separation into separate digital fingerprints, and comparison of the digital tDNA-PCR fingerprints with a library of tDNA-PCR-fingerprints obtained from a large collection of reference strains, was done using in house software described previously [ 10 ].
tDNA-PCR
tDNA-PCR was carried out using the outwardly directed tRNA-gene consensus primers T5A (5'AGTCCGGTGCTCTAACCAACTGAG) and T3B (5'AGGTCGCGGGTTCGAATCC), thus amplifying the intergenic tRNA-spacers, as described previously [ 10 , 11 ]. Electropherograms were normalized using GeneScan Analysis software, version 2.1 (Applied Biosystems). Transformation of GeneScan tables (ABI310, McIntosh) to tables on IBM, separation into separate digital fingerprints, and comparison of the digital tDNA-PCR fingerprints with a library of tDNA-PCR-fingerprints obtained from a large collection of reference strains, was done using in house software described previously [ 10 ].16S rRNA gene sequencing For five of the phenotypically aberrant isolates, the complete 16S rRNA sequence was determined by amplification of the 16S rRNA-gene with the primers 5'-AGTTTGATCCTGGCTCAG and 5'-TACCTTGTTACGACTTCGTCCCA [ 12 ], and sequencing was performed as described previously [ 12 ]. Comparison of the obtained 16S rDNA-sequence with all known sequences in Genbank was carried out using the BLAST software (National Center for Biotechnology Information  ).
16S rRNA gene sequencing
For five of the phenotypically aberrant isolates, the complete 16S rRNA sequence was determined by amplification of the 16S rRNA-gene with the primers 5'-AGTTTGATCCTGGCTCAG and 5'-TACCTTGTTACGACTTCGTCCCA [ 12 ], and sequencing was performed as described previously [ 12 ]. Comparison of the obtained 16S rDNA-sequence with all known sequences in Genbank was carried out using the BLAST software (National Center for Biotechnology Information  ).RAPD-analysis for strain typing The genotypic relationship of the isolates was investigated using arbitrarily primed PCR with RAPD Ready-to-Go beads (Amersham Pharmacia, Uppsala, Sweden) and the ERIC II primer 5'-AAGTAAGTGACTGGGGTGAGCG [ 13 ]. Analysis of the fingerprints was obtained by visual interpretation on ethidium bromide stained electrophoresis gels.
RAPD-analysis for strain typing
The genotypic relationship of the isolates was investigated using arbitrarily primed PCR with RAPD Ready-to-Go beads (Amersham Pharmacia, Uppsala, Sweden) and the ERIC II primer 5'-AAGTAAGTGACTGGGGTGAGCG [ 13 ]. Analysis of the fingerprints was obtained by visual interpretation on ethidium bromide stained electrophoresis gels.Authors' contributions GC, GV and AM were responsible for sample collection and initial biochemical identification. PVD and GW carried out automated biochemical identification. GW in addition carried out extended biochemical characterization. TDB and MV carried out the molecular analysis. GC, GV, TDB and MV drafted the manuscript. All authors read and approved the final manuscript.
Authors' contributions
GC, GV and AM were responsible for sample collection and initial biochemical identification. PVD and GW carried out automated biochemical identification. GW in addition carried out extended biochemical characterization. TDB and MV carried out the molecular analysis. GC, GV, TDB and MV drafted the manuscript. All authors read and approved the final manuscript.