High genetic diversity of Staphylococcus aureus in a tertiary care hospital in Southwest Nigeria.

Phenotypic and genetic characterization of 62 Staphylococcus aureus isolates recovered in Nigeria indicated a high proportion of Panton-Valentine leukocidin-positive isolates and a high genetic diversity among the 22 methicillin-resistant S. aureus. This underlines the need for infection control in Africa to prevent further dissemination of potentially highly virulent and resistant clones.

VARIABILITY OF PHENOTYPIC, PROTEOMIC AND TRANSCRIPTOMIC EXPRESSION OF STAPHYLOCOCCUS AUREUS SURFACE ADHESINS

Staphylococcus aureus is a highly successful pathogen responsible of a wide variety of diseases, from minor skin infection to life-threatening sepsis or infective endocarditis, as well as food poisoning and toxic shock syndrome. This heterogeneity of infections and the ability of S. aureus to develop antibiotic-resistance to virtually any available drugs reflect its extraordinary capacity to adapt and survive in a great variety of environments. The pathogenesis of S. aureus infection involves a wide range of cell wall-associated adhesins and extracellular toxins that promote host colonization and invasion. In addition, S. aureus is extremely well equipped with regulatory systems that sense environmental conditions and respond by fine tuning the expression of metabolic and virulence determinants. Surface adhesins referred to MSCRAMMs - for Microbial Surface Component Recognizing Adherence Matrix Molecules - mediate binding to the host extracellular matrix or serum components, including fibrinogen, fibronectin, collagen and elastin, and promote tissue colonization and invasion. Major MSCRAMMs include a family of surface-attached proteins covalently bound to the cell wall peptidoglycan via a conserved LPXTG motif. Genomic analyses indicate that S. aureus contain up to 22 LPXTG surface proteins, which could potentially act individually or in synergy to promote infection. In the first part of this study we determined the range of adherence phenotypes to fibrinogen and fibronectin among 30 carriage isolates of S. aureus and compared it to the adherence phenotypes of 30 infective endocarditis and 30 blood culture isolates. Overall there were great variations in in vitro adherence, but no differences were observed between carriage and infection strains. We further determined the relation between in vitro adherence and in vivo infectivity in a rat model of experimental endocarditis, using 4 isolates that displayed either extremely low or high adherence phenotypes. Unexpectedly, no differences were observed between the in vivo infectivity of isolates that were poorly and highly adherent in vitro. We concluded that the natural variability of in vitro adherence to fibrinogen and fibronectin did not correlate with in vivo infectivity, and thus that pathogenic differences between various strains might only be expressed in in vivo conditions, but not in vitro. Therefore, considering the importance of adhesins expression for infection, direct measurement of those adhesins present on the bacterial surface were made by proteomic approach. 5 In the second series of experiments we assessed the physical presence of the LPXTG species at the staphylococcal surface, as measured at various time points during growth in different culture media. S. aureus Newman was grown in either tryptic soy broth (TSB) or in Roswell Park Memorial Institute (RPMI) culture medium, and samples were removed from early exponential growth phase to late stationary phase. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa) and clumping factor A (ClfA). Peptides of surface proteins were recovered by "trypsin-shaving" of live bacteria, and semi-quantitative proteomic analysis was performed by tandem liquid-chromatography and mass-spectrometry (LC-MS). We also determined in parallel the mRNA expression by microarrays analysis, as well as the phenotypic adherence of the bacteria to fibrinogen in vitro. The surface proteome was highly complex and contained numerous proteins theoretically not belonging to the bacterial envelope, including ribosomal proteins and metabolic enzymes. Sixteen of the 21 known LPXTG species were detected, but were differentially expressed. As expected, 9 known agr-regulated proteins (e.g. including Spa, FnBPA, ClfA, IsdA, IsdB, SasH, SasD, SasG and FmtB) increased up to the late exponential growth phase, and were abrogated in agr-negative mutants. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr negative mutant, while all other LPXTG proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in in vitro fibrinogen adherence tests during late growth (24h), whereas it remained poorly detected by proteomics. Differential expression was also detected in iron-rich TSB versus iron-poor RPMI. Proteins from the iron-regulated surface determinant (isd) system, including IsdA, IsdB and IsdH were barely expressed in iron-rich TSB, whereas they increased their expression by >10 time in iron-poor RPMI. We conclude that semi-quantitative proteomic analysis of specific protein species is feasible in S. aureus and that proteomic, transcriptomic and adherence phenotypes demonstrated differential profiles in S. aureus. Furthermore, peptide signatures released by trypsin shaving suggested differential protein domain exposures in various environments, which might be relevant for antiadhesins vaccines. A comprehensive understanding of the S. aureus physiology should integrate all these approaches.

In vitro prevention of the emergence of daptomycin resistance in Staphylococcus aureus and enterococci following combination with amoxicillin/clavulanic acid or ampicillin.

Daptomycin is bactericidal against meticillin-resistant Staphylococcus aureus (MRSA), glycopeptide-intermediate-resistant S. aureus (GISA) and vancomycin-susceptible and -resistant enterococci. However, selection for daptomycin-resistant derivatives has occasionally been reported during therapy in humans. Here we evaluate whether selection for daptomycin-resistant S. aureus or enterococci could be prevented in vitro by combining daptomycin with amoxicillin/clavulanic acid, ampicillin, gentamicin or rifampicin. Six strains of S. aureus (four MRSA and two GISA) and four strains of enterococci (two Enterococcus faecalis and two Enterococcus faecium) were serially exposed in broth to two-fold stepwise increasing concentrations of daptomycin alone or in combination with a fixed concentration [0.25x minimum inhibitory concentration (MIC)] of either of the second agents. The daptomycin MIC was examined after each cycle. Exposure to daptomycin alone gradually selected for S. aureus and enterococci with an increased MIC. Gentamicin did not prevent the emergence of daptomycin-resistant bacteria. Rifampicin was also unable to prevent daptomycin resistance, although resistance was slightly delayed. In contrast, amoxicillin/clavulanic acid or ampicillin prevented or greatly delayed the selection of daptomycin-resistant mutants in S. aureus and enterococci, respectively. Addition of amoxicillin/clavulanic acid or ampicillin to daptomycin prevents, or greatly delays, daptomycin resistance in vitro. Future studies in animal models are needed to predict the utility of these combinations in humans.

Bonds between fibronectin and fibronectin-binding proteins on Staphylococcus aureus and Lactococcus lactis.

Bacterial cell-wall-associated fibronectin binding proteins A and B (FnBPA and FnBPB) form bonds with host fibronectin. This binding reaction is often the initial step in prosthetic device infections. Atomic force microscopy was used to evaluate binding interactions between a fibronectin-coated probe and laboratory-derived Staphylococcus aureus that are (i) defective in both FnBPA and FnBPB (fnbA fnbB double mutant, DU5883), (ii) capable of expressing only FnBPA (fnbA fnbB double mutant complemented with pFNBA4), or (iii) capable of expressing only FnBPB (fnbA fnbB double mutant complemented with pFNBB4). These experiments were repeated using Lactococcus lactis constructs expressing fnbA and fnbB genes from S. aureus. A distinct force signature was observed for those bacteria that expressed FnBPA or FnBPB. Analysis of this force signature with the biomechanical wormlike chain model suggests that parallel bonds form between fibronectin and FnBPs on a bacterium. The strength and covalence of bonds were evaluated via nonlinear regression of force profiles. Binding events were more frequent (p < 0.01) for S. aureus expressing FnBPA or FnBPB than for the S. aureus double mutant. The binding force, frequency, and profile were similar between the FnBPA and FnBPB expressing strains of S. aureus. The absence of both FnBPs from the surface of S. aureus removed its ability to form a detectable bond with fibronectin. By contrast, ectopic expression of FnBPA or FnBPB on the surface of L. lactis conferred fibronectin binding characteristics similar to those of S. aureus. These measurements demonstrate that fibronectin-binding adhesins FnBPA and FnBPB are necessary and sufficient for the binding of S. aureus to prosthetic devices that are coated with host fibronectin.

High-dose daptomycin plus fosfomycin is safe and effective in treating methicillin-susceptible and methicillin-resistant Staphylococcus aureus endocarditis.

We describe 3 patients with left-sided staphylococcal endocarditis (1 with methicillin-susceptible Staphylococcus aureus [MSSA] prosthetic aortic valve endocarditis and 2 with methicillin-resistant S. aureus [MRSA] native-valve endocarditis) who were successfully treated with high-dose intravenous daptomycin (10 mg/kg/day) plus fosfomycin (2 g every 6 h) for 6 weeks. This combination was tested in vitro against 7 MSSA, 5 MRSA, and 2 intermediately glycopeptide-resistant S. aureus isolates and proved to be synergistic against 11 (79%) strains and bactericidal against 8 (57%) strains. This combination deserves further clinical study.

The discriminatory power of MALDI-TOF mass spectrometry to differentiate between isogenic teicoplanin-susceptible and teicoplanin-resistant strains of methicillin-resistant Staphylococcus aureus.

To explore the discriminatory power of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for detecting subtle differences in isogenic isolates, we tested isogenic strains of Staphylococcus aureus differing in their expression of resistance to methicillin or teicoplanin. More important changes in MALDI-TOF MS spectra were found with strains differing in methicillin than in teicoplanin resistance. In comparison, very minor or no changes were recorded in pulsed-field gel electrophoresis profiles or peptidoglycan muropeptide digest patterns of these strains, respectively. MALDI-TOF MS might be useful to detect subtle strain-specific differences in ionizable components released from bacterial surfaces and not from their peptidoglycan network.

Efficacy of garenoxacin in treatment of experimental endocarditis due to Staphylococcus aureus or viridans group streptococci.

The activity of garenoxacin was investigated in rats with experimental endocarditis due to staphylococci and viridans group streptococci (VGS). The staphylococci tested comprised one ciprofloxacin-susceptible and methicillin-susceptible Staphylococcus aureus (MSSA) isolate (isolate 1112), one ciprofloxacin-susceptible but methicillin-resistant S. aureus (MRSA) isolate (isolate P8), and one ciprofloxacin-resistant mutant (grlA) of P8 (isolate P8-4). The VGS tested comprised one penicillin-susceptible isolate and one penicillin-resistant isolate (Streptococcus oralis 226 and Streptococcus mitis 531, respectively). To simulate the kinetics of drugs in humans, rats were infused intravenously with garenoxacin every 24 h (peak and trough levels in serum, 6.1 and 1.0 mg/liter, respectively; area under the concentration-time curve [AUC], 63.4 mg. h/liter) or levofloxacin every 12 h (peak and trough levels in serum, 7.3 and 1.5 mg/liter, respectively; AUC, 55.6 mg. h/liter) for 3 or 5 days. Flucloxacillin, vancomycin, and ceftriaxone were used as control drugs. Garenoxacin, levofloxacin, flucloxacillin, and vancomycin sterilized >/=70% of the vegetations infected with both ciprofloxacin-susceptible staphylococcal isolates (P < 0.05 versus the results for the controls). Garenoxacin and vancomycin also sterilized 70% of the vegetations infected with ciprofloxacin-resistant MRSA isolate P8-4, whereas treatment with levofloxacin failed against this organism (cure rate, 0%; P < 0.05 versus the results obtained with the comparator drugs). Garenoxacin did not select for resistant derivatives in vivo. In contrast, levofloxacin selected for resistant variants in four of six rats infected with MRSA isolate P8-4. Garenoxacin sterilized 90% of the vegetations infected with both penicillin-susceptible and penicillin-resistant isolates of VGS. Levofloxacin sterilized only 22 and 40% of the vegetations infected with penicillin-susceptible S. oralis 226 and penicillin-resistant S. mitis 531, respectively. Ceftriaxone sterilized only 40% of those infected with penicillin-resistant S. mitis 531 (P < 0.05 versus the results obtained with garenoxacin). No quinolone-resistant VGS were detected. In all the experiments successful quinolone treatment was predicted by specific pharmacodynamic criteria (D. R. Andes and W. A. Craig, Clin. Infect. Dis. 27:47-50, 1998). The fact that the activity of garenoxacin was equal or superior to those of the standard comparators against staphylococci and VGS indicates that it is a potential alternative for the treatment of infections caused by such bacteria.

Contagem de Staphylococcus coagulase positivo e Escherichia coli nas amostras de queijo fresco da ilha do Fogo comercializado no mercado municipal da cidade da Praia

O presente trabalho monográfico elaborado como parte dos requisitos para obtenção do grau de licenciatura em Análises Clínicas e Saúde Pública, teve como objectivo proceder à contagem de Staphylococcus coagulase positivo e Escherichia coli nas amostras de queijo fresco da ilha do Fogo comercializado no mercado municipal da cidade da Praia. O queijo é um derivado do leite muito apreciado devido ao seu valor nutritivo como também pelo seu sabor, que consegue atender aos mais diferentes paladares. Mas, muitas vezes as condições de processamento, armazenamento e comercialização poderão comprometer as suas características organolépticas, ou torná-lo impróprio para o consumo humano através de contaminação por microrganismos causadores das toxinfecções alimentares ( CORBIA et al., 2000; MELO, 2009 ) . A presença desses microrganismos no queijo poderá ter como fonte de contaminação a utilização do leite cru, utensílios contaminados utilizados durante o processo de fabrico, condições higiénico-sanitária precárias. Para a avaliação da qualidade microbiológica do queijo fresco artesanal comercializado no mercado da cidade da Praia, foram analisadas 40 amostras de queijos provenientes da ilha do Fogo que são comercializados neste estabelecimento. A técnica utilizada para a contagem das bactérias Staphylococcus coagulase positiva foi a de sementeira a superfície e para Escherichia coli fez-se pelo método de incorporação. Dos resultados obtidos para a contagem de Staphylococcus coagulase positiva 87,5% das amostras analisadas estavam contaminadas e para Escherichia coli 82,5% das amostras estavam também contaminadas, o que de acordo com a Resolução - RDC nº 12, de 2 de Janeiro de 2001 da ANVISA, estão fora do limite estabelecido, e estando assim insatisfatórias para o consumo humano. Concluiu-se que os queijos frescos da ilha do Fogo comercializados no mercado municipal da cidade da Praia não são de boa qualidade para o consumo humano. Os produtores do queijo e as vendedeiras precisam de formações sobre as boas práticas de higiene e de fabrico a fim de obtermos um produto de qualidade e isento de microrganismos que são prejudiciais à saúde do consumidor.

Contagem de Staphylococcus coagulase positivo e Escherichia coli nas amostras de queijo fresco da ilha do Fogo comercializado no mercado municipal da cidade da Praia

O presente trabalho monográfico elaborado como parte dos requisitos para obtenção do grau de licenciatura em Análises Clínicas e Saúde Pública, teve como objectivo proceder à contagem de Staphylococcus coagulase positivo e Escherichia coli nas amostras de queijo fresco da ilha do Fogo comercializado no mercado municipal da cidade da Praia. O queijo é um derivado do leite muito apreciado devido ao seu valor nutritivo como também pelo seu sabor, que consegue atender aos mais diferentes paladares. Mas, muitas vezes as condições de processamento, armazenamento e comercialização poderão comprometer as suas características organolépticas, ou torná-lo impróprio para o consumo humano através de contaminação por microrganismos causadores das toxinfecções alimentares (CORBIA et al., 2000; MELO, 2009). A presença desses microrganismos no queijo poderá ter como fonte de contaminação a utilização do leite cru, utensílios contaminados utilizados durante o processo de fabrico, condições higiénico-sanitária precárias. Para a avaliação da qualidade microbiológica do queijo fresco artesanal comercializado no mercado da cidade da Praia, foram analisadas 40 amostras de queijos provenientes da ilha do Fogo que são comercializados neste estabelecimento. A técnica utilizada para a contagem das bactérias Staphylococcus coagulase positiva foi a de sementeira a superfície e para Escherichia coli fez-se pelo método de incorporação. Dos resultados obtidos para a contagem de Staphylococcus coagulase positiva 87,5% das amostras analisadas estavam contaminadas e para Escherichia coli 82,5% das amostras estavam também contaminadas, o que de acordo com a Resolução - RDC no 12, de 2 de Janeiro de 2001 da ANVISA, estão fora do limite estabelecido, e estando assim insatisfatórias para o consumo humano. Concluiu-se que os queijos frescos da ilha do Fogo comercializados no mercado municipal da cidade da Praia não são de boa qualidade para o consumo humano. Os produtores do queijo e as vendedeiras precisam de formações sobre as boas práticas de higiene e de fabrico a fim de obtermos um produto de qualidade e isento de microrganismos que são prejudiciais à saúde do consumidor.

Efficacy of daptomycin in implant-associated infection due to methicillin-resistant Staphylococcus aureus: importance of combination with rifampin.

Limited treatment options are available for implant-associated infections caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). We compared the activity of daptomycin (alone and with rifampin [rifampicin]) with the activities of other antimicrobial regimens against MRSA ATCC 43300 in the guinea pig foreign-body infection model. The daptomycin MIC and the minimum bactericidal concentration in logarithmic phase and stationary growth phase of MRSA were 0.625, 0.625, and 20 microg/ml, respectively. In time-kill studies, daptomycin showed rapid and concentration-dependent killing of MRSA in stationary growth phase. At concentrations above 20 microg/ml, daptomycin reduced the counts by >3 log(10) CFU/ml in 2 to 4 h. In sterile cage fluid, daptomycin peak concentrations of 23.1, 46.3, and 53.7 microg/ml were reached 4 to 6 h after the administration of single intraperitoneal doses of 20, 30, and 40 mg/kg of body weight, respectively. In treatment studies, daptomycin alone reduced the planktonic MRSA counts by 0.3 log(10) CFU/ml, whereas in combination with rifampin, a reduction in the counts of >6 log(10) CFU/ml was observed. Vancomycin and daptomycin (at both doses) were unable to cure any cage-associated infection when they were given as monotherapy, whereas rifampin alone cured the infections in 33% of the cages. In combination with rifampin, daptomycin showed cure rates of 25% (at 20 mg/kg) and 67% (at 30 mg/kg), vancomycin showed a cure rate of 8%, linezolid showed a cure rate of 0%, and levofloxacin showed a cure rate of 58%. In addition, daptomycin at a high dose (30 mg/kg) completely prevented the emergence of rifampin resistance in planktonic and adherent MRSA cells. Daptomycin at a high dose, corresponding to 6 mg/kg in humans, in combination with rifampin showed the highest activity against planktonic and adherent MRSA. Daptomycin plus rifampin is a promising treatment option for implant-associated MRSA infections.

Impact of recombination on genetic variability within Staphylococcus aureus clonal complexes.

The population structure of Staphylococcus aureus is generally described as highly clonal and is consequently subdivided into several clonal complexes (CCs). Recent data suggested that recombination might occur more frequently within than among CCs. To test this hypothesis as well as to understand how genetic diversity is created in S. aureus, we analyzed a collection of 182 isolates with MLST and five highly variable core adhesion (ADH) genes. As expected the polymorphism of ADH genes was higher than MLST genes. However both categories of genes showed low within CCs diversity with a dominant haplotype and its single nucleotide variants. Several recombination events were detected but none involved intra-CC recombination. This did not confirm the hypothesis of higher recombination within CCs. Nevertheless, molecular analyses of variance indicated that these few recombination events have a significant impact on the genetic diversity within CCs. In addition, although most ADH genes were under purifying selection, signs of positive selection associated with a recombinant group were detected. These data highlight the importance of recombination on the evolution of the highly clonal S. aureus and suggest that recombination when combined with demographic mechanisms as well as selection might favor the rapid creation of new clonal complexes.

Treatment of experimental endocarditis due to erythromycin-susceptible or -resistant methicillin-resistant Staphylococcus aureus with RP 59500.

RP 59500 is a new injectable streptogramin composed of two synergistic components (quinupristin and dalfopristin) which are active against erythromycin-susceptible and -resistant gram-positive pathogens. The present experiments compared the therapeutic efficacy of RP 59500 with that of vancomycin against experimental endocarditis due to either of two erythromycin-susceptible or two constitutively erythromycin-resistant isolates of methicillin-resistant Staphylococcus aureus. RP 59500 had low MICs for the four test organisms as well as for 24 additional isolates (the MIC at which 90% of the isolates were inhibited was &lt; 1 mg/liter) which were mostly inducibly (47%) or constitutively (39%) erythromycin resistant. Aortic endocarditis in rats was produced with catheter-induced vegetations. Three-day therapy was initiated 12 h after infection, and the drugs were delivered via a computerized pump, which permitted the mimicking of the drug kinetics produced in human serum by twice-daily intravenous injections of 7 mg of RP 59500 per kg of body weight or 1 g of vancomycin. Both antibiotics reduced vegetation bacterial titers to below detection levels in ca. 70% of animals infected with the erythromycin-susceptible isolates (P &lt; 0.05 compared with titers in controls). Vancomycin was also effective against the constitutively resistant strains, but RP 59500 failed against these isolates. Further experiments proved that RP 59500 failures were related to the very short life span of dalfopristin in serum (&lt; or = 2 h, compared with &gt; or = 6 h for quinupristin), since successful treatment was restored by artificially prolonging the dalfopristin levels for 6 h. Thus, RP 59500 is a promising alternative to vancomycin against methicillin-resistant S. aureus infections, provided that pharmacokinetic parameters are adjusted to afford prolonged levels of both of its constituents in serum. This observation is also relevant to humans, in whom the life span of dalfopristin in serum is also shorter than that of quinupristin.

Ecological temporal stability of Staphylococcus aureus nasal carriage.

We described the colonization dynamics of Staphylococcus aureus in a group of 266 healthy carriers over a period of approximately 1 year. We used precise genotyping methods, i.e., amplified fragment length polymorphism (AFLP), spa typing, and double-locus sequence typing (DLST), to detect changes in strain identity. Strain change took place rather rarely: out of 89 carriers who had initially been colonized, only 7 acquired a strain different from the original one. Approximately one-third of the carriers eliminated the colonization, and a similar number became newly colonized. Some of these events probably represent detection failure rather than genuine colonization loss or acquisition. Lower bacterial counts were associated with increased probability of eliminating the colonization. We have confirmed a high mutation rate in the spa locus: 6 out of 53 strains underwent mutation in the spa locus. There was no overall change in S. aureus genotype composition.