Gabrielle L. Stalder

Characterization of methicillin-resistant Staphylococcus spp. carrying the mecC gene, isolated from wildlife

OBJECTIVES A recently identified mecA homologue, mecC, in methicillin-resistant Staphylococcus aureus (MRSA) has been isolated from humans and different animal hosts. The aim of this study was to determine antimicrobial resistance and provide molecular characterization of MRSA and methicillin-resistant non-Staphylococcus aureus staphylococci (MRnSA) isolated from wildlife that carried the gene mecC. METHODS Five S. aureus and one coagulase-negative Staphylococcus isolate displaying phenotypic oxacillin resistance, but not recognized with conventional PCR for mecA, were further characterized by a polyphasic approach. The presence of mecC in all isolates was determined using specific PCR. PCR targeting Panton-Valentine leucocidin (PVL) genes of MRSA was performed. MRSA isolates were genotyped by spa typing and multilocus sequence typing. All isolates were genotyped by staphylococcal cassette chromosome mec (SCCmec) typing. 16S rDNA sequence analysis for MRnSA identification was performed. Antimicrobial susceptibility testing was performed for all isolates. RESULTS All five MRSA isolates contained the mecC gene, were PVL negative, carried SCCmec type XI and belonged to ST130 (where ST stands for sequence type), with spa types t843, t10513 or t3256, or to ST2620, with spa type t4335. The MRnSA isolate, most closely related to Staphylococcus stepanovicii, carried mecA and blaZ genes related to SCCmec XI. MRSA isolates exhibited resistance to the β-lactams only. CONCLUSIONS The MRSA isolates described in this study represent the first detection of mecC-positive MRSA in a European otter (Lutra lutra) and a European brown hare (Lepus europaeus). The MRnSA isolate represents the first isolation of MRnSA from a Eurasian lynx (Lynx lynx).

Body mass dependent use of hibernation: why not prolong the active season, if they can?

Summary 1. Hibernation is the most effective means for energy conservation during winter in mammals. The drawbacks of deep and prolonged torpor include reduced immunocompetence, and consequently, hibernators should be selected to minimize torpor expression when climatic conditions or energy availability (e.g. food or fat stores) permit. Therefore, it seems surprising that some hibernators employ extraordinary long hibernation seasons, lasting well beyond periods with unfavourable conditions. 2. Because of their extended use of torpor, edible dormice (Glis glis) provide an ideal model for scrutinizing interactions between energy reserves (i.e. body fat stores) and thermoregulatory patterns. We used a multimodel inference approach to analyse body temperature data (i.e. use of torpor) from 42 entire hibernation seasons over 4 years in females in relation to body mass. 3. Body mass prior to hibernation did not affect the duration of the hibernation season, but animals hibernated for c. 8 months, that is, 2 months longer than required by environmental conditions. Fatter individuals aroused significantly more often, had a higher mean minimum body temperature during torpor and remained euthermic for longer periods than leaner animals. 4. Surplus energy was therefore not used to shorten the hibernation season, but to rewarm more frequently, and to allow shallower torpor bouts. These adjustments apparently serve to avoid negative effects of torpor and, perhaps equally importantly, to minimize the time active above-ground. We argue that maintaining a short active season, despite surplus energy reserves, may be explained by known beneficial effects of hibernation on survival rates (via predator avoidance). 5. Our data provide quantitative evidence that hibernation is a flexible tool within life-history strategies. We conclude that, apart from energetic necessities due to harsh environmental conditions, predator avoidance may be an important factor influencing patterns of hibernation and torpor in mammals. Thus, our study indicates that climatic conditions alone are not a good predictor of hibernation patterns or survival in hibernating species during global climate change.

Cooling and freezing of epididymal sperm in the common hippopotamus (Hippopotamus amphibius)

Knowledge concerning reproduction in common hippopotamus is scarce and in particular very little is known about male reproductive physiology and sperm cryopreservation. Testes were obtained from nine castrated bulls and sperm extracted from the epididymides of eight of these individuals. Mean ± SEM values of reproductive parameters were: testicular weight (including epididymis and tunicas)--275.9 ± 54.1 g, total sperm motility--88.1 ± 4.2%, total cells extracted--11.0 ± 3.6 × 10(9), intact acrosome--87.7 ± 1.8%, intact sperm morphology--51.6 ± 4.1%, and, for 3 individuals, hypoosmotic swelling test for membrane integrity-83.3 ± 1.8%. Chilled storage extenders tested were Berliner Cryomedium (BC), Biladyl(®), modification of Kenney modified Tyrodes medium (KMT), and Human Sperm Refrigeration Medium (HSRM). Extender had significant effect on post-dilution motility and motility and intact morphology after 4h and 24h at 4°C (P ≤ 0.007 for all). Berliner Cryomedium and HSRM were superior to Biladyl(®) and KMT. Freezing extenders tested were BC with either 6% dimethyl sulfoxide (Me(2)SO), or 5%, 7%, or 10% glycerol. Post-thaw motility was < 5% in 3/7 bulls in all extenders. When frozen in BC with 6% Me(2)SO, one bull had 15% post-thaw motility and 3/7 had 20 to 60%. In glycerol, 3/7 had 15-30% post-thaw motility in 5%, 2/7 in 7%, and 1/7 in 10%. The extender had significant effect on post-chilling motility (P = 0.008), post-thaw morphology (P = 0.016), and motility 30 min after thawing (P = 0.015). Berliner Cryomedium with 6% Me(2)SO or 7% glycerol were the freezing extenders of choice. Information obtained in this study allows initiation of cryobanking of sperm from the common hippopotamus which is of particular importance for genetically valuable individuals.

Comparison of ESBL--and AmpC producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA) isolated from migratory and resident population of rooks (Corvus frugilegus) in Austria.

In order to test whether rooks (Corvus frugilegus) represent good indicators for the potential circulation of antibiotics in their native habitat, two populations with different migratory behavior were tested for the presence of beta-lactamase producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA). In all, 54 and 102 samples of fresh feces of a migratory and a resident population were investigated. A total of 24 and 3 cefotaxime-resistant enterobacterial isolates were obtained from the migratory and resident population, respectively. In these isolates CTX-M-1 (n = 15), CTX-M-3 (n = 3), and CTX-M-15 (n = 3) genes were detected. TEM-1 and OXA-1 were associated with CTX-M in 3 and 2 isolates, respectively. In two E. coli isolates CMY-2 could be detected, where from one isolate displayed an overexpression of chromosomal AmpC as well. Among E. coli isolates the most common phylogenetic group was A (n = 11) and ST1683 (n = 5). In one E. coli of B2-ST131 the rfbO25b locus was detected. Three Enterobacter isolates were stably derepressed AmpC-producers. In five samples of the migratory population, PVL positive MRSA could be isolated. Two isolates were typed SCCmec IVa, spa type t127, and ST1. Three isolates carried a SCCmec type IVc, with spa type t852 and ST22. The highly significant difference of the occurrence of antibiotic resistance between the migratory population from eastern Europe compared to resident population in our study indicates that rooks may be good indicator species for the evaluation of environmental contamination with antibiotic resistant bacteria, especially due to their ecology, foraging behavior and differing migratory behavior.

Red Deer as Maintenance Host for Bovine Tuberculosis, Alpine Region

To estimate the prevalence of bovine tuberculosis in the Alpine region, we studied the epidemiology of Mycobacterium caprae in wildlife during the 2009–2012 hunting seasons. Free-ranging red deer (Cervus elaphus) were a maintenance host in a hot-spot area, mainly located in Austria.

Hibernation in the pygmy slow loris (Nycticebus pygmaeus): multiday torpor in primates is not restricted to Madagascar.

Hibernation and short daily torpor are states of energy conservation with reduced metabolism and body temperature. Both hibernation, also called multiday torpor, and daily torpor are common among mammals and occur in at least 11 orders. Within the primates, there is a peculiar situation, because to date torpor has been almost exclusively reported for Malagasy lemurs. The single exception is the African lesser bushbaby, which is capable of daily torpor, but uses it only under extremely adverse conditions. For true hibernation, the geographical restriction was absolute. No primate outside of Madagascar was previously known to hibernate. Since hibernation is commonly viewed as an ancient, plesiomorphic trait, theoretically this could mean that hibernation as an overwintering strategy was lost in all other primates in mainland Africa, Asia, and the Americas. However, we hypothesized that a good candidate species for the use of hibernation, outside of Madagascar should be the pygmy slow loris (Nycticebus pygmaeus), a small primate inhabiting tropical forests. Here, we show that pygmy slow lorises exposed to natural climatic conditions in northern Vietnam during winter indeed undergo torpor lasting up to 63 h, that is, hibernation. Thus, hibernation has been retained in at least one primate outside of Madagascar.

Basking hamsters reduce resting metabolism, body temperature and energy costs during rewarming from torpor

ABSTRACT Basking can substantially reduce thermoregulatory energy expenditure of mammals. We tested the hypothesis that the largely white winter fur of hamsters (Phodopus sungorus), originating from Asian steppes, may be related to camouflage to permit sun basking on or near snow. Winter-acclimated hamsters in our study were largely white and had a high proclivity to bask when resting and torpid. Resting hamsters reduced metabolic rate (MR) significantly (>30%) when basking at ambient temperatures (Ta) of ∼15 and 0°C. Interestingly, body temperature (Tb) also was significantly reduced from 34.7±0.6°C (Ta 15°C, not basking) to 30.4±2.0°C (Ta 0°C, basking), which resulted in an extremely low (<50% of predicted) apparent thermal conductance. Induced torpor (food withheld) during respirometry at Ta 15°C occurred on 83.3±36.0% of days and the minimum torpor MR was 36% of basal MR at an average Tb of 22.0±2.6°C; movement to the basking lamp occurred at Tb<20.0°C. Energy expenditure for rewarming was significantly reduced (by >50%) during radiant heat-assisted rewarming; however, radiant heat per se without an endogenous contribution by animals did not strongly affect metabolism and Tb during torpor. Our data show that basking substantially modifies thermal energetics in hamsters, with a drop of resting Tb and MR not previously observed and a reduction of rewarming costs. The energy savings afforded by basking in hamsters suggest that this behaviour is of energetic significance not only for mammals living in deserts, where basking is common, but also for P. sungorus and probably other cold-climate mammals. Summary: Basking substantially modifies thermal energetics in winter-acclimated hamsters, reducing resting body temperature, metabolic rate and rewarming costs. This suggests that this behaviour is of energetic significance not only for desert but also for cold-climate mammals.

Diversity of enterobacteria including β-lactamase producing isolates associated with the Spanish slug (Arion vulgaris).

The aim of this study was to determine diversity in members of the family Enterobacteriaceae including the beta-lactamase producing isolates associated with the Spanish slug (Arion vulgaris). 81 isolates, each representing a distinct colonial morphotype, were obtained from fecal samples of Spanish slugs and analyzed. Genetically heterogeneous or similar groups were assessed among the isolates by enterobacterial repetitive intergenic consensus (ERIC)-PCR and further characterized by 16S rDNA sequence analysis. Representative strains were allocated to the genera Citrobacter, Raoultella, Escherichia, Klebsiella, Kluyvera, and Buttiauxella. Fifteen isolates, most closely related to Citrobacter freundii and Escherichia coli, displayed an AmpC phenotype, one E. coli isolate showed an ESBL phenotype. These isolates were phenotypically and genotypically characterized by their antimicrobial resistance pattern. Phylogenetic background and multi-locus sequence typing (MLST) of the E. coli isolates were determined. The multi-drug resistant TEM-1 E. coli positive isolate belongs to the phylogenetic group A and ST10. The CMY-2 positive E. coli isolate belongs to the phylogenetic group D and ST117. Our results show that the common garden slug represents an important potential vector of β-lactamase producing Enterobacteriaceae.

Use of a medetomidine-ketamine combination for anesthesia in captive common hippopotami (Hippopotamus amphibius).

OBJECTIVE To establish an anesthetic protocol suitable for surgical interventions in hippopotami (Hippopotamus amphibius). DESIGN Prospective case series. ANIMALS 10 adult male hippopotami undergoing castration. PROCEDURES A combination of medetomidine (60 to 80 mg/kg [27.3 to 36.4 mg/lb]) and ketamine (1 mg/kg [0.45 mg/lb]) was administered IM on the basis of mean estimated weights of 1,330 ± 333 kg (2,926 ± 733 lb; median, 1,350 kg [2,790 lb]; range, 900 to 2,000 kg [1,980 to 4,400 lb]). Monitoring included sequential blood gas analyses, pulse oximetry, and capnography. Reversal of anesthesia with atipamezole (0.34 ± 0.06 mg/kg [0.15 ± 0.027 mg/lb]; median, 0.33 mg/kg [0.15 mg/lb]; range, 300 to 500 mg total dose]) was uneventful and rapid in all cases. RESULTS Complete immobilization and a surgical anesthetic plane were achieved 27 ± 11.8 minutes (median, 24.5 minutes [range, 14 to 44 minutes]) after initial injection. Anesthesia (97.3 ± 35.3 minutes; median, 95 minutes [range, 57 to 188 minutes]) was maintained with 3.4 ± 2.2 (median, 3) additional doses of ketamine (0.1 to 0.4 mg/kg [0.045 to 0.18 mg/lb]). Transitory apnea of 4.71 ± 2.87 minutes (median, 4 minutes [range, 1 to 9 minutes]) was documented in 5 animals. Apnea during anesthesia was viewed as a physiologic condition in this semiaquatic mammal because related vital parameters (heart rate, pH, peripheral hemoglobin oxygen saturation as measured by pulse oximetry, venous partial pressure of CO(2), and lactate and HCO(3) concentrations) remained unchanged and did not differ significantly than those parameters for the 5 animals with continuous respiration. CONCLUSIONS AND CLINICAL RELEVANCE Both in captivity and in the wild, common hippopotami are difficult to anesthetize. The combination of medetomidine and ketamine provided an excellent surgical plane of anesthesia and a self-limiting dive response.

Implications of being born late in the active season for growth, fattening, torpor use, winter survival and fecundity

For hibernators, being born late in the active season may have important effects on growth and fattening, hence on winter survival and reproduction. This study investigated differences in growth, fattening, energetic responses, winter survival and fecundity between early-born (‘EB’) and late-born (‘LB’) juvenile garden dormice (Eliomys quercinus). LB juveniles grew and gained mass twice as fast as EB individuals. Torpor use was low during intensive growth, that are, first weeks of body mass gain, but increased during pre-hibernation fattening. LB juveniles showed higher torpor use, reached similar body sizes but lower fat content than EB individuals before hibernation. Finally, LB individuals showed similar patterns of hibernation, but higher proportion of breeders during the following year than EB dormice. These results suggest that torpor is incompatible with growth but promotes fattening and consolidates pre-hibernation fat depots. In garden dormice, being born late in the reproductive season is associated with a fast life history.