The characteristics of VIM-1-producing Klebsiella pneumoniae from South Africa.

A study was designed to characterize a carbapenem-resistant Klebsiella pneumoniae (KPSA01) isolated from a patient in Gauteng, South Africa without recent travel outside South Africa. Molecular characterization was done using isoelectric focusing, polymerase chain reaction and sequencing for bla(VIM), bla(IMP), bla(NDM), bla(CTX-Ms), bla(OXAs), bla(TEMs), and bla(SHV), plasmid-mediated quinolone resistance determinants, multilocus sequencing typing, plasmid replicon typing, and addiction factors. KPSA01 produced VIM-1 and belonged to the newly described sequence type ST569. The plasmid that harboured bla(VIM) typed within the narrow host range IncF replicon group, contained the aadA1 gene cassette, and tested positive for the vagCD and ccdAB addiction systems. This is the first report of VIM-1-producing K. pneumoniae outside Europe. It is important that surveillance studies be undertaken in Africa to determine if VIM-1-producing K. pneumoniae are present in significant numbers.

Pathogens chipping away at our last line of defence - the rise of the metallo-beta-lactamases.

We describe the first known report of a VIM1 metallo-beta-lactamase-producing Klebsiella pneumonia outside Europe - confirmed by our colleagues in Canada. The patient had no travel history and surveillance failed to identify additional cases. The importance of health care professionals being diligent in identifying these multi-drug resistant isolates is emphasised.

Do binucleate cardiomyocytes have a role in myocardial repair? Insights using isolated rodent myocytes and cell culture.

Neonatal and adult cardiomyocytes were isolated from rat hearts. Some of the adult myocytes were cultured to allow for cell dedifferentiation, a phenomenon thought to mimic cell changes that occur in stressed myocardium, with myocytes regressing to a fetal pattern of metabolism and stellate neonatal shape.Using fluorescence deconvolution microscopy, cells were probed with fluorescent markers and scanned for a number of proteins associated with ion control, calcium movements and cardiac function. Image analysis of deconvoluted image stacks and sequential real-time image recordings of calcium transients of cells were made.All three myocyte groups were predominantly comprised of binucleate cells. Clustering of proteins to a single nucleus was a common observation, suggesting that one nucleus is active in protein synthesis pathways, while the other nucleus assumes a 'dormant' or different role and that cardiomyocytes might be mitotically active even in late development, or specific protein syntheses could be targeted and regulated for reintroduction into the cell cycle.Such possibilities would extend cardiac disease associated stem cell research and therapy options, while producing valuable insights into developmental and death pathways of binucleate cardiomyocytes (word count 183).

Antimicrobial coating agents: can biofilm formation on a breast implant be prevented?

BACKGROUND: Numerous clinical studies have shown that biofilm formation by Staphylococcus epidermidis on the outer surface of a silicone breast implant is strongly associated with capsular contracture formation. Traditional administration of systemic antibiotics and antiseptic washing are not necessarily the most effective methods for the prevention of initial biofilm formation on implants in the clinical scenario. In this study an alternative or supplement was sought for preventing or delaying bacterial colonisation and adherence to the outer surface of a breast implant, by establishing an in vitro model for investigating this complex problem. The in vitro antimicrobial activity of several antimicrobial agents was investigated for inhibitory effects on biofilm formation by S. epidermidis. METHODS: The study consisted of two experiments. The first experiment consisted of two groups (A and B) of seven discs each whilst the second experiment was divided into three groups (C, D and E) of 14 discs each. Each group of 14 consisted of seven smooth and seven textured discs. Discs (biopsies) of each implant were individually coated with one of six different antimicrobial agents. Controls that received no agent were included in the various experimental groups. In the first experiment disc diffusion sensitivity testing was performed and inhibition zone sizes were measured. In the second experiment the discs were cultured in broth. The degree of biofilm formation was evaluated by scanning electron microscopy (SEM). RESULTS: In the first in vitro experiment, all six agents showed a measurable antimicrobial effect against the biofilm-forming strain of S. epidermidis when compared to the effect against the American Type Culture Collection strain. In the second in vitro experiment, discs coated with Chloramex, Fucidin and Terramycin did not allow biofilm formation to take place for at least 7 days. CONCLUSIONS: Staphylococcus epidermidis biofilm formation on the outer surface of a silicone breast implant was prevented in vitro for at least 7 days by coating with an appropriate antimicrobial agent. Further evaluation of the interaction between antimicrobial coating agents and S. epidermidis biofilm formation needs to be made before conclusions regarding the clinical scenario can be drawn.

Antimicrobial susceptibility profile of selected bacteraemic pathogens from private institutions in South Africa.

OBJECTIVES: The National Antimicrobial Surveillance Forum is a continuous surveillance organisation comprising all academic/public and private sector laboratories in South Africa. METHODS: The antibiotic susceptibility of blood culture isolates of Escherichia coli, Klebsiella pneumoniae, Enterobacter species, Pseudomonas aeruginosa, Acinetobacter baumannii and Staphylococcus aureus from patients in private hospitals in five major centres were investigated. Antimicrobial susceptibility tests were performed by 12 participating laboratories according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Extended-spectrum b-lactamase (ESBL) production was determined in selected species of Enterobacteriaceae irrespective of source. RESULTS: The overall prevalence of ampicillin resistance in blood culture isolates of E. coli (N = 471) was 84%, and 20% were resistant to the fluoroquinolones. Considerable geographical differences were noted between the centres with regard to the K. pneumoniae (N = 636) resistance rates for ceftriaxone and/or cefotaxime (39 - 87%). The most active agents in the Enterobacter spp. (N = 244) were imipenem/meropenem, ertapenem, ciprofloxacin, levofloxacin and cefepime, with 100%, 94%, 88%, 87% and 80% susceptibility, respectively. Carbapenem resistance in P. aeruginosa (N = 382) varied between 42% and 45%, and in the case of A. baumannii (N = 190) resistance varied between 32% and 33% for meropenem and imipenem respectively. The nationwide incidence of oxacillin resistance in S. aureus (N = 629) was 36%. Overall, the prevalence of ESBL production among all isolates of K. pneumoniae was 26% (N = 7 514), while in Enterobacter spp. it was 12% (N = 4 031) and in E. coli 5% (N = 28 412). CONCLUSIONS: The data highlight the widespread problem of antibiotic resistance among important bacteraemic pathogens in private institutions in South Africa. Continued surveillance is vital to guide appropriate empirical therapy for invasive infections.

Efficacy of ischaemic preconditioning in the eNOS overexpressed working mouse heart model.

We recently demonstrated that exogenous nitric oxide (NO) acts as a trigger for preconditioning in the isolated rat heart model. There is however little data concerning the effects of elevated cardiac endothelial nitric oxide synthase (eNOS) expression on myocardial tolerance to ischaemia. Similarly, the effects of gender and eNOS overexpression on ischaemic preconditioning is unknown. We hypothesized that: 1) eNOS overexpression increases myocardial tolerance to ischaemia, and, 2) eNOS overexpressed hearts cannot be preconditioned, since the hearts are already maximally protected. Male and female wild-type and transgenic mice that overexpress eNOS exclusively in cardiac myocytes were perfused in the working heart mode with a modified Krebs-Henseleit buffer at a pre-load of 12.5 mm Hg and afterload of 50 mm Hg. Cardiac output, coronary flow, peak aortic systolic pressure and total work were determined before hearts were preconditioned by 4x5 min cycles of ischaemia/reperfusion, and then subjected to 20 min total global ischaemia, followed by reperfusion. Reperfusion function and myocardial infarct size were used as endpoints. Pre-ischaemic mechanical function (rate pressure product and cardiac output) was similar for wild-type and transgenic mice of both sexes. The eNOS overexpressed hearts had smaller infarcts than the hearts from their wild-type littermates (26.9+/-1.4% vs. 37.0+/-2.1% for controls, P<0.05). Preconditioning the eNOS overexpressed hearts resulted in infarct sizes comparable with control non-preconditioned hearts (27.5+/-2.0% vs. 26.9+/-1.4% for controls). Myocardial cGMP levels were elevated during sustained ischaemia in the transgenic hearts when compared with wild-type hearts (22.43+/-1.63 pmol/g ww vs 16.54+/-1.48 pmol/g ww, P<0.05). Preconditioning also elevated myocardial cGMP levels during sustained ischaemia in the wild-type hearts (26.77+/-2.81 pmol/g ww, P<0.05). We conclude that: 1) basal mechanical function is similar for both wild-type and transgenic mice of both sexes, 2) reperfusion function and infarct size was also similar for both sexes under both control conditions and after preconditioning, 3) the transgenic mice are more tolerant of ischaemia as reflected by their smaller myocardial infarcts, and, 4) the eNOS overexpressed mouse heart cannot be preconditioned regardless of whether mechanical function or infarct size is used as an end-point. These hearts may be maximally protected against ischaemia/reperfusion injury by their elevated endogenous NO levels.

Short- and long-term effects of melatonin on myocardial post-ischemic recovery.

Melatonin, the chief secretory product of the pineal gland, has been shown to protect the heart against ischemia-reperfusion injury. This was attributed to its free radical scavenging and broad-spectrum antioxidant properties. The possibility that melatonin may act via its receptor and intracellular signaling, has not yet been addressed in this regard. In all previous studies, only the acute effects of melatonin on the heart, were evaluated. The aims of the present study were to: (i) compare the acute and long-term effects of melatonin on infarct size and functional recovery of the ischemic heart, and (ii) evaluate the role of the melatonin receptor in cardioprotection. For evaluation of the short-term effects of melatonin on contractile recovery and infarct size, the isolated perfused working rat heart was subjected to 20 min global ischemia or 35 min regional ischemia respectively, and melatonin (25-50 microm) administered either before and during reperfusion, or before ischemia or during reperfusion after ischemia. The melatonin receptor was manipulated using luzindole and N-acetyltryptamine. The long-term effects of melatonin were evaluated 24 hr after melatonin administration (2.5 or 5.0 mg/kg, i.p.) or after oral administration for 7 days (20 or 40 microg/mL). Infarct size and mechanical recovery during reperfusion of the working heart were used as endpoints. Melatonin (50 microm), when administered either before and during reperfusion after ischemia or during reperfusion only, significantly improved cardiac output and work performance and reduced infarct size compared with untreated controls. Luzindole (5 microm), a melatonin receptor antagonist, abolished these cardioprotective effects. Long-term administration of melatonin (i.p. or orally for 7 days) caused a significant reduction in infarct size of hearts subjected to 35 min regional ischemia. The cardioprotection persisted for 2-4 days after discontinuation of treatment. In summary, the results obtained suggest that melatonin induces short- as well as long-term protection and that the melatonin receptor is also involved in its cardioprotective actions.

The temporal relationship between p38 MAPK and HSP27 activation in ischaemic and pharmacological preconditioning.

An ischaemic preconditioning protocol and subsequent sustained ischaemia were characterized by activation and attenuation of p38 MAPK phosphorylation, respectively. However, the significance of events downstream of p38 MAPK needs investigation. Therefore the temporal relationship between phosphorylation of p38 MAPK and its downstream substrate HSP27 was studied during either an ischaemic or beta-adrenergic preconditioning protocol and during sustained ischaemia. Isolated rat hearts were preconditioned (with or without a p38 MAPK inhibitor, SB203580) with 1 x 5 min or 3 x 5 min global ischaemia or 5 min beta-adrenergic stimulation (10(-7) M isoproterenol), followed by 25 min sustained ischaemia and 30 min reperfusion. Hearts were freeze-clamped at different time intervals and fractionated to determine p38 MAPK and HSP27 phosphorylation, via Western blotting. Significant phosphorylation of cytosolic p38 MAPK and membrane (myo-fibrillar) HSP27 occurred at the end of the first preconditioning episode. However, p38 MAPK phosphorylation disappeared during subsequent preconditioning episodes, while HSP27 phosphorylation was maintained for the duration of the protocol. Similar changes in p38 MAPK and HSP27 occurred with 5 min beta-adrenergic preconditioning. After 25 min ischaemia, significant phosphorylation of cytosolic and membrane HSP27 was observed, while p38 MAPK phosphorylation was attenuated in ischaemic and beta-adrenergic preconditioned compared to non-preconditioned hearts. SB203580-induced abolishment of p38 MAPK and HSP27 phosphorylation during the triggering phase of both preconditioning protocols reversed the changes in these parameters seen after sustained ischaemia. The results suggest that p38 MAPK activation triggers HSP27 phosphorylation during both the preconditioning protocols and during sustained ischaemia. Protection of preconditioned hearts during sustained ischaemia was characterized by phosphorylation of both cytosolic and myofibrillar HSP27.

Nitric oxide triggers classic ischemic preconditioning.

The role of NO in the classic ischemic preconditioning phenomenon of the myocardium is not well defined, and was investigated by using the isolated perfused rat heart as a model. Hearts were preconditioned with 3 x 5 minute ischemia in the presence and absence of the NOS inhibitors L-NAME (50 microM) and L-NNA (50 microM), and the guanylyl cyclase inhibitor ODQ (20 microM). These inhibitors significantly attenuated the protective effect of preconditioning against 25-min global ischemia (as measured by functional recovery), specifically if administered during the triggering phase. Cyclic infusions (3 x 5 min) of the NO-donors SNAP (50 microM) and SNP (100 microM) elicited protection against both 25-min global or low-flow ischemia. Hearts preconditioned with NO donors displayed significantly superior functional reserve, if stimulated with adrenaline, compared to hearts preconditioned with ischemia. Although the NO donors SNAP and SNP both activated p38 MAPK during the preconditioning protocol, protection was accompanied by significantly decreased p38 MAPK activity during sustained ischemia, as was the case in ischemic preconditioning. We conclude that (1) NO is a trigger for classic preconditioning, (2) cGMP generation plays an important role in its protection, (3) attenuation of p38 MAPK during sustained ischemia accompanies NO preconditioning and may mediate cardiac protection, and (4) preconditioning with NO may be more advantageous than using ischemia.