Staphylococcus aureus na cadeia produtiva de suínos e perfil de resistência a antimicrobianos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Impact of biohybrid magnetite nanoparticles and moroccan propolis on adherence of methicillin resistant strains of staphylococcus aureus

Biofilm bacteria are more resistant to antibiotics than planktonic cells. Propolis possesses antimicrobial activity. Generally, nanoparticles containing heavy metals possess antimicrobial and antibiofilm properties. In this study, the ability of adherence of Methicillin Resistant Strains of Staphylococcus aureus (MRSA) to catheters treated with magnetite nanoparticles (MNPs), produced by three methods and functionalized with oleic acid and a hydro-alcoholic extract of propolis from Morocco, was evaluated. The chemical composition of propolis was established by gas chromatography mass spectrometry (GC-MS), and the fabricated nanostructures characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), Mossbauer spectroscopy and Fourrier transform infrared spectroscopy (FTIR). The capacity for impairing biofilm formation was dependent on the strain, as well as on the mode of production of MNPs. The co-precipitation method of MNPs fabrication using Fe(3+) and Na₂SO₃ solution and functionalized with oleic acid and propolis was the most effective in the impairment of adherence of all MRSA strains to catheters (p < 0.001). The adherence of the strain MRSA16 was also significantly lower (p < 0.001) when the catheters were treated with the hybrid MNPs with oleic acid produced by a hydrothermal method. The anti-MRSA observed can be attributed to the presence of benzyl caffeate, pinocembrin, galangin, and isocupressic acid in propolis extract, along with MNPs. However, for MRSA16, the impairment of its adherence on catheters may only be attributed to the hybrid MNPs with oleic acid, since very small amount, if any at all of propolis compounds were added to the MNPs.

Design, synthesis, antimicrobial activity and molecular modeling studies of novel benzofuroxan derivatives against Staphylococcus aureus

Molecular modi. cation is a quite promising strategy in the design and development of drug analogs with better bioavailability, higher intrinsic activity and less toxicity. In the search of new leads with potential antimicrobial activity, a new series of 14 4-substituted [N`-(benzofuroxan-5-yl) methylene] benzohydrazides, nifuroxazide derivatives, were synthesized and tested against standard and multidrug-resistant Staphylococcus aureus strains. The selection of the substituent groups was based on physicochemical properties, such as hydrophobicity and electronic effect. These properties were also evaluated through the lipophilic and electrostatic potential maps, respectively, considering the compounds with better biological pro. le. Twelve compounds exhibited similar bacteriostatic activity against standard and multidrug-resistant strains. The most active compound was the 4-CF(3) substituted derivative, which presented a minimum inhibitory concentration (MIC) value of 14.6-13.1 mu g/mL, and a ClogP value of 1.87. The results highlight the benzofuroxan derivatives as potential leads for designing new future antimicrobial drug candidates. (C) 2009 Elsevier Ltd. All rights reserved.

Staphylococcus aureus: towards a comprehensive view on epidemiology and clonal spread

Dissertation presented to obtain a PhD degree in Biology/ Molecular Biology by the Universidade Nova de Lisboa, Instituto de Tecnologia Química e Biológica

Tamponamento Cardíaco Secundário a Pericardite por Staphylococcus Aureus Meticilino Resistente: Importância da Imunossupressão

As pericardites bacterianas, apesar da sua baixa incidência e das terapêuticas actuais, apresentam um prognóstico desfavorável, sobretudo quando causadas por Staphylococcus aureus meticilino resistente (MRSA). O Tamponamento cardíaco é uma complicação potencialmente letal nos doentes com pericardites por este agente. Numa Unidade de Cuidados Intensivos, para além da imunossupressão, constituem factores predisponentes para este tipo de infecção, a elevada taxa de colonização nasal e cutânea, assim como a utilização de técnicas invasivas (1) entre as quais a simples colocação de catéteres intravenosos. Relatam-se dois casos clínicos de tamponamento cardíaco em doentes jovens, no contexto de imunossupressão de diferentes etiologias (infecção HIV e pós transplante hepático). Os internamentos foram complicados de quadros sépticos importantes com isolamento de MRSA nos líquidos biológicos e desenvolvimento de pericardite bacteriana e subsequente tamponamento cardíaco. Os autores salientam a importância dos quadros clínicos infecciosos em doentes imunodeprimidos, que constituem uma população cada vez mais numerosa, e a importância da monitorização ecocardiográfica na evolução prognóstica das pericardites bacterianas no contexto de sépsis.

Insights into cell wall synthesis and cell division in Staphylococcus aureus

Dissertation presented to obtain the Ph.D degree in Biology

Monoterpene isolated from the essential oil of Trachyspermum ammi is cytotoxic to multidrug-resistant Pseudomonas aeruginosa and Staphylococcus aureus strains

Abstract INTRODUCTION: The aim of this study was to determine whether an herbal extract containing monoterpene exhibited activity against multidrug-resistant Staphylococcus aureus and Pseudomonas aeruginosa isolated from clinical infection samples. METHODS: The essential oil of Trachyspermum ammi (L.) Sprague ex Turrill (Apiaceae) fruit was extracted by hydrodistillation. Fruit residues were treated with hydrochloric acid and re-hydrodistilled to obtain volatile compounds. Compounds in the distilled oil were identified using gas-chromatography (GC) and GC-mass spectrometry (MS). The antibiotic susceptibility of all bacterial isolates was analyzed using both the disc diffusion method and determination of the minimum inhibitory concentration (MIC). The sensitivity of antibiotic-resistant isolates to essential oil was also determined by using the disc diffusion method and MIC determination. RESULTS: Of 26 clinical isolates, 92% were multidrug-resistant (MDR). Aromatic monoterpenes (thymol, paracymene, and gamma-terpinene) were the major (90%) components of the oil. Growth of S. aureus strains was successfully inhibited by the oil, with an inhibitory zone diameter (IZD) between 30-60mm and MIC <0.02μL/mL. The oil had no antimicrobial activity against clinical isolates of P. aeruginosa; rather, it prevented pigment production in these isolates. CONCLUSIONS: This study revealed that the essential oil of Trachyspermum ammi, which contains monoterpene, has good antibacterial potency. Monoterpenes could thus be incorporated into antimicrobial ointment formulas in order to treat highly drug-resistant S. aureus infections. Our findings also underscore the utility of research on natural products in order to combat bacterial multidrug resistance.

Study of in vivo interactions between penicillin-binding proteins of Staphylococcus aureus

Staphylococcus aureus (S. aureus) is a major human pathogen that has acquired resistance to practically all classes of β-lactam antibiotics, being responsible of Multidrug resistant S. aureus (MRSA) associated infections both in healthcare (HA-MRSA) and community settings (CA-MRSA). The emergence of laboratory strains with high-resistance (VRSA) to the last resort antibiotic, vancomycin, is a warning of what is to come in clinical strains. Penicillin binding proteins (PBPs) target β-lactams and are responsible for catalyzing the last steps of synthesis of the main component of cell wall, peptidoglycan. As in Escherichia coli, it is suggested that S. aureus uses a multi-protein complex that carries out cell wall synthesis. In the presence of β-lactams, PBP2A and PBP2 perform a joint action to build the cell wall and allow cell survival. Likewise, PBP2 cooperates with PBP4 in cell wall cross-linking. However, an actual interaction between PBP2 and PBP4 and the location of such interaction has not yet been determined. Therefore, investigation of the existence of a PBP2-PBP4 interaction and its location(s) in vivo is of great interest, as it should provide new insights into the function of the cell wall synthesis machinery in S. aureus. The aim of this work was to develop Split-GFPP7 system to determine interactions between PBP2 and PBP4. GFPP7 was split in a strategic site and fused to proteins of interest. When each GFPP7 fragment, fused to proteins, was expressed alone in staphylococcal cells, no fluorescence was detectable. When GFPP7 fragments fused to different peptidoglycan synthesis (PBP2 and PBP4) or cell division (FtsZ and EzrA) proteins were co-expressed together, fluorescent fusions were localized to the septum. However, further analysis revealed that this positive result is mediated by GFPP7 self-association. We then interpret the results in light of such event and provide insights into ways of improving this system.

Summary CDI and MRSA incidence tables, 1st April 2006 to present

Summary Tables for Clostridium difficile and MRSA (Meticillin Resistant Staphylococcus aureus) cases 2006/07 to 2014/15

Acquisition and cross-transmission of Staphylococcus aureus in European intensive care units

Objective. To study the acquisition and cross-transmission of Staphylococcus aureus in different intensive care units (ICUs). Methods. We performed a multicenter cohort study. Six ICUs in 6 countries participated. During a 3-month period at each ICU, all patients had nasal and perineal swab specimens obtained at ICU admission and during their stay. All S. aureus isolates that were collected were genotyped by spa typing and multilocus variable-number tandem-repeat analysis typing for cross-transmission analysis. A total of 629 patients were admitted to ICUs, and 224 of these patients were found to be colonized with S. aureus at least once during ICU stay (22% were found to be colonized with methicillin-resistant S. aureus [MRSA]). A total of 316 patients who had test results negative for S. aureus at ICU admission and had at least 1 follow-up swab sample obtained for culture were eligible for acquisition analysis. Results. A total of 45 patients acquired S. aureus during ICU stay (31 acquired methicillin-susceptible S. aureus [MSSA], and 14 acquired MRSA). Several factors that were believed to affect the rate of acquisition of S. aureus were analyzed in univariate and multivariate analyses, including the amount of hand disinfectant used, colonization pressure, number of beds per nurse, antibiotic use, length of stay, and ICU setting (private room versus open ICU treatment). Greater colonization pressure and a greater number of beds per nurse correlated with a higher rate of acquisition for both MSSA and MRSA. The type of ICU setting was related to MRSA acquisition only, and the amount of hand disinfectant used was related to MSSA acquisition only. In 18 (40%) of the cases of S. aureus acquisition, cross-transmission from another patient was possible. Conclusions. Colonization pressure, the number of beds per nurse, and the treatment of all patients in private rooms correlated with the number of S. aureus acquisitions on an ICU. The amount of hand disinfectant used was correlated with the number of cases of MSSA acquisition but not with the number of cases of MRSA acquisition. The number of cases of patient-to-patient cross-transmission was comparable for MSSA and MRSA.

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.

Staphylococcus aureus clfB and spa alleles of the repeat regions are segregated into major phylogenetic lineages.

To reliably differentiate among Staphylococcus aureus isolates we recently developed the Double Locus Sequence Typing (DLST) based on the analysis of partial sequences of clfB and spa genes. This method is highly discriminatory and gives unambiguous definition of types. The highly clonal population structure of S. aureus suggests that isolates with identical clfB or spa alleles belong to the same clonal complex (CC) defined by Multi-Locus Sequence Typing (MLST). To test this hypothesis as well as to investigate putative intra-CC genetic structure, we analyzed a total of 289 isolates (186 MSSA and 103 MRSA) with DLST-, spa- and MLST-typing. Among the 289 strains, 242 were clustered into 7 major MLST CCs, 40 into minor CCs and 7 were not grouped into CCs. A total of 205 DLST- and 129 spa-types were observed. With one exception, all DLST-clfB, DLST-spa and spa-type alleles were segregated into CCs. DLST-types sharing an identical allele (clfB or spa) were clustered using eBURST. Except for one strain, all isolates from each DLST cluster belonged to the same CC. However, using both DLST- and spa-typing we were not able to disclose a clear intra-CC structure. Nevertheless, the high diversity of these loci confirmed that they are good markers for local epidemiological investigations.

Staphylococcus aureus host range and human-bovine host shift.

Staphylococcus aureus is a major agent of bovine mastitis. The concomitant emergence of pig-associated methicillin-resistant S. aureus (MRSA) in human carriage and infection requires a reexamination of the host range and specificity of human- and cow-associated S. aureus strains, something which has not been systematically studied previously. The genetic relatedness of 500 S. aureus isolates from bovine mastitis cases, 57 isolates from nasal carriage of farmers, and 133 isolates from nonfarmers was determined by amplified fragment length polymorphism (AFLP) analysis and spa typing. Multilocus sequence typing (MLST) was conducted on a subset of isolates to match AFLP clusters with MLST clonal complexes (CCs). This data set allowed us to study host range and host specificity and to estimate the extent of bovine-to-human transmission. The genotype compositions of S. aureus isolates from farmers and nonfarmers were very similar, while the mastitis isolates were quite distinct. Overall, transmission was low, but specific genotypes did show increased cow-to-human transmission. Unexpectedly, more than one-third of mastitis isolates belonged to CC8, a lineage which has not been considered to be bovine mastitis associated, but it is well known from human carriage and infection (i.e., USA300). Despite the fact that we did detect some transmission of other genotypes from cows to farmers, no transmission of CC8 isolates to farmers was detected, except for one tentative case. This was despite the close genetic relatedness of mastitis CC8 strains to nonfarmer carriage strains. These results suggest that the emergence of the new bovine-adapted genotype was due to a recent host shift from humans to cows concurrent with a loss of the ability to colonize humans. More broadly, our results indicate that host specificity is a lineage-specific trait that can rapidly evolve.

Improved multiple-locus variable-number tandem-repeat assay for Staphylococcus aureus genotyping, providing a highly informative technique together with strong phylogenetic value.

We describe an improved multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) scheme for genotyping Staphylococcus aureus. We compare its performance to those of multilocus sequence typing (MLST) and spa typing in a survey of 309 strains. This collection includes 87 epidemic methicillin-resistant S. aureus (MRSA) strains of the Harmony collection, 75 clinical strains representing the major MLST clonal complexes (CCs) (50 methicillin-sensitive S. aureus [MSSA] and 25 MRSA), 135 nasal carriage strains (133 MSSA and 2 MRSA), and 13 published S. aureus genome sequences. The results show excellent concordance between the techniques' results and demonstrate that the discriminatory power of MLVA is higher than those of both MLST and spa typing. Two hundred forty-two genotypes are discriminated with 14 VNTR loci (diversity index, 0.9965; 95% confidence interval, 0.9947 to 0.9984). Using a cutoff value of 45%, 21 clusters are observed, corresponding to the CCs previously defined by MLST. The variability of the different tandem repeats allows epidemiological studies, as well as follow-up of the evolution of CCs and the identification of potential ancestors. The 14 loci can conveniently be analyzed in two steps, based upon a first-line simplified assay comprising a subset of 10 loci (panel 1) and a second subset of 4 loci (panel 2) that provides higher resolution when needed. In conclusion, the MLVA scheme proposed here, in combination with available on-line genotyping databases (including http://mlva.u-psud.fr/), multiplexing, and automatic sizing, can provide a basis for almost-real-time large-scale population monitoring of S. aureus.

Living with an imperfect cell wall: compensation of femAB inactivation in Staphylococcus aureus.

BACKGROUND: Synthesis of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases FemX, FemA and FemB. Inactivation of the femAB operon reduces the interpeptide to a monoglycine, leading to a poorly crosslinked peptidoglycan. femAB mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making FemAB a potential target to restore beta-lactam susceptibility in methicillin-resistant S. aureus (MRSA). Cis-complementation with wild type femAB only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. This study characterizes the adaptations that ensured survival of the cells after femAB inactivation. RESULTS: In addition to slow growth, the cis-complemented femAB mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. Transcriptional profiling paired with reporter metabolite analysis revealed multiple changes in the global transcriptome. A number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. Striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for ATP production. In addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sarA, and agr. CONCLUSION: Survival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of S. aureus to circumvent cell wall damage. Potential FemAB inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors.