The effect of intestinal microbiota dysbiosis on growth and detection of carbapenemase-producing Enterobacterales within an in vitro gut model.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) can colonize the gut and are of major clinical concern. Identification of CPE colonization is problematic; there is no gold-standard detection method, and the effects of antibiotic exposure and microbiota dysbiosis on detection are unknown. AIM: Based on a national survey we selected four CPE screening assays in common use. We used a clinically reflective in vitro model of human gut microbiota to investigate the performance of each test to detect three different CPE strains under different, clinically relevant antibiotic exposures. METHODS: Twelve gut models were seeded with a pooled faecal slurry and exposed to CPE either before, after, concomitant with, or in the absence of piperacillin-tazobactam (358 mg/L, 3 × daily, seven days). Total Enterobacterales and CPE populations were enumerated daily. Regular screening for CPE was performed using Cepheid Xpert® Carba-R molecular test, and with Brilliance™ CRE, Colorex™ mSuperCARBA and CHROMID® CARBA SMART agars. FINDINGS: Detection of CPE when the microbiota are intact is problematic. Antibiotic exposure disrupts microbiota populations and allows CPE proliferation, increasing detection. The performances of assays varied, particularly with respect to different CPE strains. The Cepheid assay performed better than the three agar methods for detecting a low level of CPE within an intact microbiota, although performance of all screening methods was comparable when CPE populations increased in a disrupted microbiota. CONCLUSION: CPE strains differed in their dynamics of colonization in an in vitro gut model and in their subsequent response to antibiotic exposure. This affected detection by molecular and screening methods, which has implications for the sensitivity of CPE screening in healthcare settings.

Impact of PIT tagging on recapture rates, body condition and reproductive success of wild Daubenton's bats (Myotis daubentonii).

A successful and safe methodology for the subcutaneous insertion of passive integrated transponder (PIT) tags in a small- to medium-sized bat (average mass 9 g) under isoflurane-induced anaesthesia is described. Passive integrated transponder (PIT) tagging had no significant impact on the rate of recapture, body condition index (BCI) (bodyweight/forearm length) and reproductive success of tagged individuals, and no visible injuries or health problems were observed in any of the recaptured bats. Tagging success, in terms of retention and function, was 92 per cent (n=61) by the third year of using the method. Sixteen per cent (n=39) of bats tagged during the three-year study period were not producing positive scans with the microchip reader when recaptured after previously successful tag insertion, indicating that the tags were either working their way out of the bats or ceasing to function.

Spatial variation in fast muscle function of the rainbow trout Oncorhynchus mykiss during fast-starts and sprinting.

Fish fast-starts and sprints are rapid kinematic events powered by the lateral myotomal musculature. A distinction can be made between fast-starts and sprint-swimming activity. Fast-starts are kinematic events involving rapid, asymmetrical movements. Sprints involve a series of symmetrical, high-frequency tailbeats that are kinematically similar to lower-frequency, sustained swimming. The patterns of muscle recruitment and strain associated with these swimming behaviours were determined using electromyography and sonomicrometry. Axial patterns of fast muscle recruitment during sprints were similar to those in slow muscle in that the duration of electromyograhic (EMG) activity decreased in a rostro-caudal direction. There was also an axial shift in activity relative to the strain cycle so that activity occurred relatively earlier in the caudal region. This may result in caudal muscle performing a greater proportion of negative work and acting as a power transmitter as well as a power producer. The threshold tailbeat frequency for recruitment of fast muscle differed with location in the myotome. Superficial muscle fibres were recruited at lower tailbeat frequencies and shortening velocities than those deeper in the musculature. During sprints, fast muscle strain ranged from +/- 3.4% l(0) (where l(0) is muscle resting length) at 0.35FL (where FL is fork length) to +/- 6.3% l(0) at 0.65FL. Fast-starts involved a prestretch of up to 2.5% l(0) followed by shortening of up to 11.3% l(0). Stage 1 EMG activity began simultaneously, during muscle lengthening, at all axial locations. Stage 2 EMG activity associated with the major contralateral contraction also commenced during lengthening and proceeded along the body as a wave. Onset of muscle activity during lengthening may enhance muscle power output.

Fast muscle function in the European eel (Anguilla anguilla L.) during aquatic and terrestrial locomotion.

Eels are capable of locomotion both in water and on land using undulations of the body axis. Axial undulations are powered by the lateral musculature. Differences in kinematics and the underlying patterns of fast muscle activation are apparent between locomotion in these two environments. The change in isometric fast muscle properties with axial location was less marked than in most other species. Time from stimulus to peak force (T(a)) did not change significantly with axial position and was 82+/-6 ms at 0.45BL and 93+/-3 ms at 0.75BL, where BL is total body length. Time from stimulus to 90% relaxation (T(90)) changed significantly with axial location, increasing from 203+/-11ms at 0.45BL to 239+/-9 ms at 0.75BL. Fast muscle power outputs were measured using the work loop technique. Maximum power outputs at +/-5% strain using optimal stimuli were 17.3+/-1.3W kg(-1) in muscle from 0.45BL and 16.3+/-1.5W kg(-1) in muscle from 0.75BL. Power output peaked at a cycle frequency of 2Hz. The stimulus patterns associated with swimming generated greater force and power than those associated with terrestrial crawling. This decrease in muscle performance in eels may occur because on land the eel is constrained to a particular kinematic pattern in order to produce thrust against an underlying substratum.

Slow muscle power output of yellow- and silver-phase European eels (Anguilla anguilla L.): changes in muscle performance prior to migration.

Eels swim in the anguilliform mode in which the majority of the body axis undulates to generate thrust. For this reason, muscle function has been hypothesised to be relatively uniform along the body axis relative to some other teleosts in which the caudal fin is the main site of thrust production. The European eel (Anguilla anguilla L.) has a complex life cycle involving a lengthy spawning migration. Prior to migration, there is a metamorphosis from a yellow (non-migratory) to a silver (migratory) life-history phase. The work loop technique was used to determine slow muscle power outputs in yellow- and silver-phase eels. Differences in muscle properties and power outputs were apparent between yellow- and silver-phase eels. The mass-specific power output of silver-phase slow muscle was greater than that of yellow-phase slow muscle. Maximum slow muscle power outputs under approximated in vivo conditions were 0.24 W kg(-1) in yellow-phase eel and 0.74 W kg(-1) in silver-phase eel. Power output peaked at cycle frequencies of 0.3--0.5 Hz in yellow-phase slow muscle and at 0.5--0.8 Hz in silver-phase slow muscle. The time from stimulus offset to 90 % relaxation was significantly greater in yellow- than in silver-phase eels. The time from stimulus onset to peak force was not significantly different between life-history stages or axial locations. Yellow-phase eels shifted to intermittent bursts of higher-frequency tailbeats at a lower swimming speed than silver-phase eels. This may indicate recruitment of fast muscle at low speeds in yellow-phase eels to compensate for a relatively lower slow muscle power output and operating frequency.

Slow muscle function of Pacific bonito (Sarda chiliensis) during steady swimming.

The Pacific bonito, Sarda chiliensis, is anatomically intermediate between mackerel and tuna. The specialisations exhibited by tuna are present in the bonito, but to a lesser degree. Slow-twitch muscle strain and activity patterns were determined during steady swimming (tailbeat frequency 1.2-3.2 Hz) at four locations on the body of Sarda chiliensis using sonomicrometry and electromyography. Both strain and the phase of electromygraphic activity were independent of tailbeat frequency. The strain of superficial slow-twitch muscle increased from +/-3.1 % l(0) at 0.35FL to +/-5.8 % l(0) at 0.65FL, where l(0) is muscle resting length and FL is the body length from snout to tail fork. Between 0.35 and 0.65FL, there was a negative phase shift of 16 degrees in the onset of electromygraphic activity in superficial slow-twitch muscle relative to the strain cycle. Muscle activity patterns are comparable with those of tuna. At 0.58FL, the onset of activity in deep slow-twitch muscle was approximately synchronous with the onset of activity in superficial muscle in the same myotome at 0.65FL. The distribution of slow-twitch muscle along the body of Sarda chiliensis and four additional fish species, Anguilla anguilla, Oncorhynchus mykiss, Scomber scombrus and Thunnus albacares, was also measured. Slow-twitch muscle appears to become more concentrated at approximately 0.5FL as swimming kinematics become more thunniform.

Rate-dependent changes in cell shortening, intracellular Ca(2+) levels and membrane potential in single, isolated rainbow trout (Oncorhynchus mykiss) ventricular myocytes.

The effects of increasing stimulation frequency (from 0.2 to 1.4 Hz) on the contractility, intracellular Ca(2+) concentration ([Ca(2+)](i)) and membrane potential of single ventricular myocytes isolated from the heart of rainbow trout (Oncorhynchus mykiss) were measured. Cell shortening, expressed as a percentage of resting cell length, was our index of contractility. The fluorescent Ca(2+) indicator Fura-2 was used to monitor changes in [Ca(2+)](i). Action potentials and L-type Ca(2+) currents (I(Ca)) were recorded using the whole-cell patch-clamp technique. Experiments were performed at 15 degrees C. Increasing the stimulation frequency caused a significant increase in diastolic [Ca(2+)](i) and a significant decrease in diastolic cell length and membrane potential. During systole, there was a significant fall in the amplitude of the [Ca(2+)](i) transient, cell shortening and action potential with a decrease in the duration of the action potential at both 20 % and 90 % repolarisation. Caffeine was used to assess the Ca(2+) content of the sarcoplasmic reticulum. We observed that sarcoplasmic reticulum Ca(2+) load was greater at 1.0 Hz than at 0.6 Hz, despite a smaller electrically evoked [Ca(2+)](i) transient. The amplitude of I(Ca) was found to decrease with increased stimulation frequency. At 0.6 Hz, electrically evoked [Ca(2+)](i) transients in the presence of 10 mmol l(-)(1) caffeine or 10 micromol l(-)(1) ryanodine and 2 micromol l(-)(1) thapsigargin were reduced by approximately 15 %. We have described the changes in contractility, [Ca(2+)](i) and action potential configuration in a fish cardiac muscle system. Under the conditions tested (0.6 Hz, 15 degrees C), we conclude that the sarcoplasmic reticulum contributes at least 15 % of the Ca(2+) associated with the [Ca(2+)](i) transient. The rate-dependent decrease in contraction amplitude appears to be associated with the fall in the amplitude of the [Ca(2+)](i) transient. This, in turn, may be influenced by changes in the action potential configuration via mechanisms such as altered Ca(2+) efflux and Ca(2+) influx. In support of our conclusions, we present evidence that there is a rate-dependent decrease in Ca(2+) influx via I(Ca) but that the Ca(2+) load of the sarcoplasmic reticulum is not reduced at increased contraction frequencies.

Fish swimming: patterns in muscle function.

Undulatory swimming in fish is powered by the segmental body musculature of the myotomes. Power generated by this muscle and the interactions between the fish and the water generate a backward-travelling wave of lateral displacement of the body and caudal fin. The body and tail push against the water, generating forward thrust. The muscle activation and strain patterns that underlie body bending and thrust generation have been described for a number of species and show considerable variation. This suggests that muscle function may also vary among species. This variation must be due in large part to the complex interactions between muscle mechanical properties, fish body form, swimming mode, swimming speed and phylogenetic relationships. Recent work in several laboratories has been directed at studying patterns of muscle power output in vitro under simulated swimming conditions. This work suggests that the way that fish generate muscle power and convert it into thrust through the body and caudal fin does indeed vary. However, despite the differences, several features appear to be common to virtually all species studied and suggest where future effort should be directed if muscle function in swimming fish is to be better understood.

Myotomal slow muscle function of rainbow trout Oncorhynchus mykiss during steady swimming.

Strain and activity patterns were determined during slow steady swimming (tailbeat frequency 1.5-2.5 Hz) at three locations on the body in the slow myotomal muscle of rainbow trout Oncorhynchus mykiss using sonomicrometry and electromyography. Strain was independent of tailbeat frequency over the range studied and increased significantly from +/-3.3 % l0 at 0.35BL to +/-6 % at 0.65BL, where l0 is muscle resting length and BL is total body length. Muscle activation occurred significantly later in the strain cycle at 0.35BL (phase shift 59 degrees) than at 0.65BL (30 degrees), and the duration of activity was significantly longer (211 degrees at 0.35BL and 181 degrees at 0.65BL). These results differ from those of previous studies. The results have been used to simulate in vivo activity in isolated muscle preparations using the work loop technique. Preparations from all three locations generated net positive power under in vivo conditions, but the negative power component increased from head to tail. Both kinematically, and in the way its muscle functions to generate hydrodynamic thrust, the rainbow trout appears to be intermediate between anguilliform swimmers such as the eel, which generate thrust along their entire body length, and carangiform fish (e.g. saithe Pollachius virens), which generate thrust primarily at the tail blade.

Why do tuna maintain elevated slow muscle temperatures? Power output of muscle isolated from endothermic and ectothermic fish.

It has been hypothesised that regional endothermy has evolved in the muscle of some tunas to enhance the locomotory performance of the fish by increasing muscle power output. Using the work loop technique, we have determined the relationship between cycle frequency and power output, over a range of temperatures, in isolated bundles of slow muscle fibres from the endothermic yellowfin tuna (Thunnus albacares) and its ectothermic relative the bonito (Sarda chiliensis). Power output in all preparations was highly temperature-dependent. A counter-current heat exchanger which could maintain a 10 degrees C temperature differential would typically double maximum muscle power output and the frequency at which maximum power is generated (fopt). The deep slow muscle of the tuna was able to operate at higher temperatures than slow muscle from the bonito, but was more sensitive to temperature change than more superficially located slow fibres from both tuna and bonito. This suggests that it has undergone some evolutionary specialisation for operation at higher, but relatively stable, temperatures. fopt of slow muscle was higher than the tailbeat frequency of undisturbed cruising tuna and, together with the high intrinsic power output of the slow muscle mass, suggests that cruising fish have a substantial slow muscle power reserve. This reserve should be sufficient to power significantly higher sustainable swimming speeds, presumably at lower energetic cost than if intrinsically less efficient fast fibres were recruited.

Mechanical properties of rabbit latissimus dorsi muscle after stretch and/or electrical stimulation.

The work loop technique was used to measure the mechanical performance in situ of the latissimus dorsi (LD) muscles of rabbits maintained under fentanyl anesthesia. After 3 wk of incrementally applied stretch the LD muscles were 36% heavier, but absolute power output (195 mW/muscle) was not significantly changed relative to that of external control muscle (206 mW). In contrast, continuous 10-Hz electrical stimulation reduced power output per kilogram of muscle >75% after 3 or 6 wk and muscle mass by 32% after 6 wk. When combined, stretch and 10-Hz electrical stimulation preserved or increased the mass of the treated muscles but failed to prevent an 80% loss in maximum muscle power. However, this combined treatment increased fatigue resistance to a greater degree than electrical stimulation alone. These stretched/stimulated muscles, therefore, are more suitable for cardiomyoplasty. Nonetheless, further work will be necessary to find an ideal training program for this surgical procedure.

The effects of adrenaline on the work- and power-generating capacity of rat papillary muscle in vitro.

The work loop technique was used to examine the effects of adrenaline on the mechanics of cardiac muscle contraction in vitro. The length for maximum active force (Lmax) and net work production (Lopt) for rat papillary muscles was determined under control conditions (without adrenaline). The concentration of adrenaline producing the maximum inotropic effect was determined. This concentration was used in the remainder of the experiments. Sinusoidal strain cycles about Lopt were performed over a physiologically relevant range of cycle frequencies (4-11 Hz). Maximum work and the frequency for maximum work increased from 1.91 J kg-1 at 3 Hz in controls to 2.97 J kg-1 at 6 Hz with adrenaline. Similarly, maximum power output and the frequency for maximum power output (fopt) increased from 8.62 W kg-1 at 6 Hz in controls to 19.95 W kg-1 at 8 Hz with adrenaline. We suggest that the power-frequency relationship, derived using the work loop technique, represents a useful index with which to assess the effects of pharmacological interventions on cardiac muscle contractility.

Fatigue of mouse soleus muscle, using the work loop technique.

The function of many muscles requires that they perform work. Fatigue of mouse soleus muscle was studied in vitro by subjecting it to repeated work loop cycles. Fatigue resulted in a reduction in force, a slowing of relaxation and in changes in the force-velocity properties of the muscle (indicated by changes in work loop shape). These effects interacted to reduce the positive work and to increase the negative work performed by the muscle, producing a decline in net work. Power output was sustained for longer and more cumulative work was performed with decreasing cycle frequency. However, absolute power output was highest at 5 Hz (the cycle frequency for maximum power output) until power fell below 20% of peak power. As cycle frequency increased, slowing of relaxation had greater effects in reducing the positive work and increasing the negative work performed by the muscle, compared with lower cycle frequencies.

The effect of Trichinella spiralis infection on the mechanical properties of the mammalian diaphragm.

Trichinella spiralis larvae infect and develop within skeletal muscle cells causing major changes to their mechanical properties. The aim of this investigation was to determine the effects of T. spiralis on the power output and fatigue resistance of the mammalian diaphragm under conditions simulating in vivo operation and to relate these to respiratory performance. Infection with T. spiralis leads to major reductions in mechanical stress, work, power output and fatigue resistance. These changes are associated with the number of larvae present in the muscle and the duration of infection. However, the initial decline in mechanical performance occurs during the onset of infection when there are few larvae observed within the muscle cells, indicating that T. spiralis may affect the properties of muscle before encapsulation. This may correspond to the host's inflammatory response and the effects of larval excretory/secretory products. The decline in mechanical performance will have a profound effect on respiration both at rest and during exertion. This must influence the behaviour of the host and increase its chance of capture by predators, which is likely to benefit the parasite by facilitating its transmission.

Isometric and isotonic muscle properties as determinants of work loop power output.

The power output of rabbit latissimus dorsi muscle was calculated under isotonic conditions and during oscillatory work. Isotonic shortening studies yielded a maximum power output of 120 W . kg-1 at a P/P0 of 0.4 compared to a maximum power output of 32 W . kg-1 obtained using the work loop technique. This difference can largely be explained by comparing actual work loops with those constructed using force velocity (P/V) and isometric data. At low cycle frequencies, work loop power output is quite close to that predicted from P/V and isometric data. However, at higher frequencies other dynamic muscle properties appear to exert a more marked effect.

The length dependence of work production in rat papillary muscles in vitro.

The influence of length on work production was investigated for rat papillary muscles using the work loop technique. Active and passive length-force relationships were first determined under isometric conditions and the length for maximum force production (Lmax) was derived. Starting from different lengths within the physiological range, a series of work loops was generated using the stimulation phase shift, strain amplitude and cycle frequency previously found to be optimal for power output at 37 degrees C. The relationship between muscle length and net work was used to determine the length at which work output was maximal (Lopt). In order to examine the dynamic passive properties of the muscles, unstimulated muscles were subjected to the same regime of sinusoidal oscillation as used for the active loops. From the hysteresis loops, lengthening work (work done to extend the passive muscle), passive shortening work (work returned during shortening) and net energy loss (hysteresis) could be measured. The decline in net work production at lengths greater than 95% Lmax could largely be attributed to the rapid and non-linear increase in muscle stiffness and the increase in net energy loss over this range of lengths. The physiological significance of the length-work relationship is considered and the mechanical properties of active and passive papillary muscles are discussed with reference to sarcomere length and cardiac muscle ultrastructure.

The effect of cycle frequency on the power output of rat papillary muscles in vitro.

Papillary muscles were isolated from the right ventricles of rats and the length for maximum active force generation (Lmax) was determined isometrically. The work loop technique was used to derive the length for maximum work production (Lopt) at the cycle frequency, strain amplitude and stimulation phase shift found to be optimal for power output. Lopt was typically 7% shorter than Lmax and within the physiological length range (87.5% Lmax to Lmax). Net work and power output were measured during sinusoidal strain cycles around Lopt, over the cycle frequency range 1-9 Hz, strain amplitude and phase shift being optimised for work and power at each frequency. Experiments were performed at 37 degrees C. Distinct optima were found in both the work-frequency and the power-frequency relationships. The optimum cycle frequency for net work production was lower than the frequency for maximum power output. The mean maximum power output at 37 degrees C was 8.62 +/- 0.50 W kg-1 (mean +/- S.E.M., N = 9) and was achieved at a cycle frequency of approximately 6 Hz, close to the estimated resting heart rate of 5.8 Hz for the rats used (mean mass 223 +/- 25 g). The cycle frequency, strain amplitude and stimulation phase shift found to be optimal for power output produced an in vitro contraction closely simulating the basal in vivo contraction.

The mechanical properties of fast and slow skeletal muscles of the mouse in relation to their locomotory function.

The mechanical properties of soleus and extensor digitorum longus (EDL) muscles from the mouse were studied using the work loop technique. Under optimum conditions, the EDL produced a maximum mean power output of 107 W kg-1 at a cycle frequency of 10 Hz. In comparison, the maximum mean power output of the soleus was 34 W kg-1 at 5 Hz cycle frequency. Video analysis of mice determined the stride frequency range to be from 2.87 Hz at a walk to 8.23 Hz at a flat-out gallop, with the trot-to-gallop transition occurring at 5.89 Hz. In vivo EDL electromyogram (EMG) activity is recorded primarily during shortening and the muscle operates in a power-generating mode. The soleus is close to isometric when EMG activity is recorded, but mechanical activity persists into the shortening phase. Both muscles are likely to operate over cycle frequency ranges just below, or at, those yielding maximal power. Soleus and EDL produced maximal power output in vitro when operating at mean sarcomere lengths of 2.58 microns and 2.71 microns respectively. These lengths are slightly above the plateau of the length-force curve predicted for rat leg muscle (2.3-2.5 microns). The sarcomere length ranges used in vivo by the soleus and EDL were determined, by fixing muscles in the extreme active positions predicted from video and cine analysis, to be 2.28-2.57 microns and 2.49-2.88 microns respectively. These ranges are both close to those shown to yield maximum power output in vitro and to the plateau of the sarcomere length-force curve.

The effect of tendon compliance on in vitro/in vivo estimations of sarcomere length

The errors likely to result from using excised rigor muscles to determine in vivo sarcomere length ranges were calculated for mouse extensor digitorum longus muscle (EDL). This muscle was chosen because its very long tendon makes it particularly susceptible to errors arising from tendon compliance. By placing dissected limbs into different locomotory stances, and allowing them to go into rigor, the range of sarcomere lengths over which muscles operate in vivo can be determined, but it is subject to errors due to tendon compliance. A tendon compliance of 0.24 GPa and a muscle rigor stress of 35 kPa were determined, and these were used to correct the estimates of in vivo sarcomere length, under worst case conditions. The error introduced was very small: a reduction in sarcomere length of less than 0.5 %.

Tuning in to fish swimming waves: body form, swimming mode and muscle function

Most fish species swim with lateral body undulations running from head to tail. These waves run more slowly than the waves of muscle activation causing them, reflecting the effect of the interaction between the fish's body and the reactive forces from the water. The coupling between both waves depends on the lateral body shape and on the mechanical properties of the tail. During steady swimming, the length of each myotomal muscle fibre varies cyclically. The phase relationship between the strain (muscle length change) cycle and the active period (when force is generated) determines the work output of the muscle. The muscle power is converted to thrust either directly by the bending body or almost exclusively by the tail, depending upon the body shape of the species and the swimming kinematics. We have compared the kinematics and muscle activity patterns from seven species of fish with different body forms and swimming modes and propose a model which yields a consistent pattern, with at least three extremes. Subtle tuning of the phase relationship between muscle strain and activation cycles can lead to major changes in the way muscles function in different swimming modes.