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1.
Biochemistry ; 59(46): 4470-4480, 2020 11 24.
Article in English | MEDLINE | ID: mdl-33136372

ABSTRACT

Peptidoglycan is a vital component of the bacterial cell wall, and its dynamic remodeling by NlpC/p60 hydrolases is crucial for proper cell division and survival. Beyond these essential functions, we previously discovered that Enterococcus species express and secrete the NlpC/p60 hydrolase-secreted antigen A (SagA), whose catalytic activity can modulate host immune responses in animal models. However, the localization and peptidoglycan hydrolase activity of SagA in Enterococcus was still unclear. In this study, we show that SagA contributes to a triseptal structure in dividing cells of enterococci and localizes to sites of cell division through its N-terminal coiled-coil domain. Using molecular modeling and site-directed mutagenesis, we identify amino acid residues within the SagA-NlpC/p60 domain that are crucial for catalytic activity and potential substrate binding. Notably, these studies revealed that SagA may function via a catalytic Cys-His dyad instead of the predicted Cys-His-His triad, which is conserved in SagA orthologs from other Enterococcus species. Our results provide key additional insight into peptidoglycan remodeling in Enterococcus by SagA NlpC/p60 hydrolases.


Subject(s)
Bacterial Proteins/metabolism , Enterococcus/metabolism , N-Acetylmuramoyl-L-alanine Amidase/metabolism , Bacterial Proteins/genetics , Catalytic Domain , Cell Division , Enterococcus/cytology , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Molecular Docking Simulation , Mutagenesis, Site-Directed , N-Acetylmuramoyl-L-alanine Amidase/genetics , Peptidoglycan/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Structure-Activity Relationship
2.
Trends Mol Med ; 25(6): 464-466, 2019 06.
Article in English | MEDLINE | ID: mdl-31109795

ABSTRACT

Vancomycin-resistant Enterococcus faecium (VRE) is a major cause of nosocomial infections. A new study by McKenney et al. (Cell Host Microbe 2019;25:695-705.e5) reports that VRE undergo a morphotype switch in response to lithocholic acid (LCA) to facilitate gastrointestinal (GI) tract colonization. This metabolic cue is a potential target to decrease VRE colonization and subsequent transmission of antibiotic resistance.


Subject(s)
Enterococcus/physiology , Gastrointestinal Tract/metabolism , Gastrointestinal Tract/microbiology , Signal Transduction , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Bile Acids and Salts/metabolism , Drug Resistance, Bacterial , Enterococcus/cytology , Enterococcus/drug effects , Enterococcus faecium/drug effects , Enterococcus faecium/physiology , Gastroenteritis/metabolism , Gastroenteritis/microbiology , Gram-Positive Bacterial Infections/metabolism , Gram-Positive Bacterial Infections/microbiology , Humans
3.
Cell ; 169(5): 849-861.e13, 2017 May 18.
Article in English | MEDLINE | ID: mdl-28502769

ABSTRACT

We examined the evolutionary history of leading multidrug resistant hospital pathogens, the enterococci, to their origin hundreds of millions of years ago. Our goal was to understand why, among the vast diversity of gut flora, enterococci are so well adapted to the modern hospital environment. Molecular clock estimation, together with analysis of their environmental distribution, phenotypic diversity, and concordance with host fossil records, place the origins of the enterococci around the time of animal terrestrialization, 425-500 mya. Speciation appears to parallel the diversification of hosts, including the rapid emergence of new enterococcal species following the End Permian Extinction. Major drivers of speciation include changing carbohydrate availability in the host gut. Life on land would have selected for the precise traits that now allow pathogenic enterococci to survive desiccation, starvation, and disinfection in the modern hospital, foreordaining their emergence as leading hospital pathogens.


Subject(s)
Biological Evolution , Enterococcus/genetics , Animals , Communicable Diseases, Emerging/microbiology , Cross Infection/microbiology , Drug Resistance, Bacterial , Enterococcus/classification , Enterococcus/cytology , Enterococcus/drug effects , Genetic Speciation , Host-Pathogen Interactions , Larva/microbiology , Moths/growth & development , Moths/microbiology , Phylogeny , RNA, Ribosomal, 16S/genetics
4.
São Paulo; s.n; s.n; set. 2015. 112 p. tab, graf, ilus.
Thesis in Portuguese | LILACS | ID: biblio-834104

ABSTRACT

Enterococos são ubíquos no ambiente e fazem parte da microbiota do trato gastrintestinal de humanos e animais. A importância dessas bactérias tem sido associada com infecções hospitalares e resistência a múltiplas drogas, principalmente à vancomicina. O objetivo do presente estudo foi realizar a caracterização molecular de cepas de Enterococcus spp. resistentes à vancomicina (VRE) isoladas a partir de amostras coletadas de pacientes hospitalizados, água superficial de rios urbanos e carne de frango comercializada no Brasil. A presença do gene vanA foi confirmada em 20 cepas multirresitentes isoladas durante 1997-2011. Dentre os isolados VRE, 12 cepas foram identificadas como E. faecium e oito como E. faecalis. Cepas de E. faecium isoladas de amostras clínicas e águas foram classificadas como clonalmente relacionadas pelo PFGE, com perfil virulência predominante (acm+, esp+). Adicionalmente, enquanto cepas de E. faecium isoladas dos rios pertenceram aos ST203, ST412 e ST478 (previamente caracterizados como endêmicos em hospitais brasileiros), novos STs foram identificados entre as cepas de E. faecalis (ST614, ST615 e ST616) e E. faecium (ST953 e ST954) isoladas de alimentos. Sequências completas do transposon Tn1546 das cepas clínicas VREfm 320/07 (ST478) e ambiental VREfm 11 (ST412) mostraram Tn1546-like element de ~12800 pb, com um ponto de mutação no gene vanA na posição 7.698 (substituição do nucleotídeo T pelo C) e uma no gene vanX na posição 8.234 (G pelo T). Além disso, uma deleção na extremidade esquerda do Tn1546, e as sequências IS1251 e IS1216E na região intergênica vanHS e vanYX, respectivamente, também foram detectados. A este respeito, a IS1216E na região intergênica vanXY constitui um conjunto de genes previamente relatado em cepas clínicas de VREfm no Brasil, denotando uma característica regional. IS1216E tem sido associada com os genes tcrB e aadE que conferem resistência ao cobre e aminoglicosídeos, em E. faecium e Streptococcus agalactiae, respectivamente. Portanto, essa IS pode contribuir para a rápida aquisição de resistência antimicrobiana entre as espécies de cocos Gram-positivos clinicamente importantes. Os tipos de Tn1546 indistiguíveis que foram identificados no atual estudo isolados de humano e ambientes aquáticos sugerem uma comum partilha de um pool de genes de resistência à vancomicina


Enterococci are ubiquitous in the environment and in the intestinal tract of humans and animals. The importance of these bacteria has been associated with nosocomial infection and multiple resistance to antimicrobial agents, mainly vancomycin. The aim of the present study was to perform molecular characterization of vancomycin-resistant Enterococcus spp. strains (VRE) isolated from hospitalized patients, surface water of urban rivers and retail chicken meat in Brazil. The presence of the vanA gene was confirmed in 20 multidrug-resistant strains isolated in 1997-2011. Among these VRE isolates, (n = 12) were identified as E. faecium and (n = 8) as E. faecalis. E. faecium strains isolated from water and clinical samples were classified as clonally related by PFGE, the predominant virulence profile being (acm+, esp+). Additionally, while E. faecium strains isolated from rivers belonging to ST203, ST412 and ST478 (previously characterized as endemic in Brazilian hospitals), new STs were identified among strains of E. faecalis (ST614, ST615 and ST616) and E. faecium (ST953 and ST954) isolated from food. Complete sequences of transposon Tn1546 from VREfm clinical strain 320/07 (ST478) and environmental strain VREfm 11 (ST412) showed a Tn1546-like element of ~12800 bp, with T7698C vanA and G8234T vanX mutations. Moreover, deletion of the Tn1546 left extremity, and the IS1251 and IS1216E sequence inside the vanHS and vanYX intergenic region, respectively, were also detected. In this regard, the IS1216E sequence inside the vanXY intergenic region constitutes a gene array previously reported for Brazilian VREfm clinical strains alone, denoting a regional characteristic. IS1216E has been associated with tcrB and aadE genes, which confer resistance to copper and aminoglycosides, in E. faecium and Streptococcus agalactiae, respectively. Therefore, IS1216E should contribute to rapid acquisition of antimicrobial resistance among species of the clinically important Gram-positive cocci. On the other hand, Tn1546-like elements were identical among clinical and environmental VREfm isolates, suggesting sharing of a common vancomycin resistance gene pool


Subject(s)
Humans , Vancomycin/administration & dosage , Enterococcus/cytology , Enterococcus/drug effects , Aquatic Environment/adverse effects , Food/adverse effects , Drug Resistance, Microbial/drug effects , Diagnostic Techniques and Procedures , Genes
5.
J Microbiol Methods ; 116: 15-22, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26122309

ABSTRACT

Despite the powerful potential of fluorescent proteins for labeling bacteria, their use has been limited in multi-species oral biofilm models. Fermentative metabolism by streptococcal species that initiate biofilm colonization results in an acidic, reduced microenvironment that may limit the activities of some fluorescent proteins which are influenced by pH and oxygen availability. The need to reliably distinguish morphologically similar strains within biofilms was the impetus for this work. Teal fluorescent protein (mTFP1) and red fluorescent protein (mCherry) were chosen because their fluorescent properties made them promising candidates. Since tRNA availability has been implicated in efficient translation of sufficient quantities of protein for maximum fluorescence, a streptococcal codon optimization approach was used. DNA was synthesized to encode either protein using codons most frequently used in streptococci; each coding region was preceded by an engineered ribosomal binding site and restriction sites for cloning a promoter. Plasmids carrying this synthesized DNA under control of the Streptococcus mutans lactate dehydrogenase promoter conferred fluorescence to nine representative streptococcal and two Enterococcus faecalis strains. Further characterization in Streptococcus gordonii showed that mTFP1 and mCherry expressions could be detected in cells grown planktonically, in biofilms, or in colonies on agar when expressed on an extrachromosomal plasmid or in single copy integrated into the chromosome. This latter property facilitated counterselection of chromosomal mutations demonstrating value for bacterial strain construction. Fluorescent and non-fluorescent bacteria were distinguishable at acidic pH. These codon-optimized versions of mTFP1 and mCherry have promising potential for use in multiple experimental applications.


Subject(s)
Enterococcus/genetics , Green Fluorescent Proteins/genetics , Luminescent Proteins/genetics , Microscopy, Fluorescence/methods , Streptococcus/genetics , Base Sequence , Biofilms/growth & development , Codon , Enterococcus/cytology , Fluorescent Dyes , Genetic Vectors , Green Fluorescent Proteins/chemistry , Hydrogen-Ion Concentration , Luminescent Agents , Luminescent Proteins/chemistry , Mutation , Promoter Regions, Genetic , Streptococcus/cytology , Streptococcus gordonii/cytology , Streptococcus gordonii/genetics , Streptococcus gordonii/growth & development , Red Fluorescent Protein
6.
Nat Rev Microbiol ; 11(9): 601-14, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23949602

ABSTRACT

Bacteria come in a range of shapes, including round, rod-shaped, curved and spiral cells. This morphological diversity implies that different mechanisms exist to guide proper cell growth, division and chromosome segregation. Although the majority of studies on cell division have focused on rod-shaped cells, the development of new genetic and cell biology tools has provided mechanistic insight into the cell cycles of bacteria with different shapes, allowing us to appreciate the underlying molecular basis for their morphological diversity. In this Review, we discuss recent progress that has advanced our knowledge of the complex mechanisms for chromosome segregation and cell division in bacteria which have, deceptively, the simplest possible shape: the cocci.


Subject(s)
Enterococcus/physiology , Gram-Positive Cocci/physiology , Neisseria/physiology , Cell Division/physiology , Cell Wall/metabolism , Cell Wall/physiology , Chromosomes, Bacterial , Enterococcus/cytology , Enterococcus/growth & development , Gram-Positive Cocci/cytology , Gram-Positive Cocci/growth & development , Neisseria/cytology , Neisseria/growth & development , Peptidoglycan/metabolism
7.
PLoS One ; 7(6): e38514, 2012.
Article in English | MEDLINE | ID: mdl-22701656

ABSTRACT

Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.


Subject(s)
Cytoplasmic Vesicles/physiology , Enterococcus/physiology , L Forms/physiology , Listeria monocytogenes/physiology , Chromosomes, Bacterial/genetics , DNA Primers/genetics , Enterococcus/cytology , L Forms/cytology , Listeria monocytogenes/cytology , Microscopy, Fluorescence , Models, Biological , Reproduction/physiology , Sequence Analysis, DNA , Spectrum Analysis, Raman
8.
Environ Sci Technol ; 45(12): 5380-6, 2011 Jun 15.
Article in English | MEDLINE | ID: mdl-21591688

ABSTRACT

Mycobacteria are waterborne emerging pathogens causing infections in human. Mycobacteria have been previously isolated from wastewater and sludge, but their densities were not estimated due to cultural biases. In order to evaluate the impact of wastewater treatment processes on mycobacteria removal, we used a real time PCR method. First we compared six DNA extraction methods and second we used the more efficient DNA extraction procedure (i.e., enzymatic lysis combined with hexadecyltrimethylammonium bromide-NaCl procedure) in order to quantify Mycobacterium. With the aim to identify parameters that could serve as indicator of mycobacterial behavior, mycobacterial densities were measured in parallel to those of Escherichia coli and enterococci, and to concentrations of chemical parameters usually monitored in wastewater. Mycobacterium reached 5.5 × 105 ± 3.9 × 105 copies/L in the influent, but was not detected in the effluent after decantation and biofiltration. Most mycobacteria (98.6 ± 2.7%, i.e. 2.4 ± 0.7 log10) were removed by the physical-chemical decantation, and the remaining mycobacteria were removed by biofiltration. In contrast, enterococci and E. coli were lightly removed by decantation step and mainly removed by biofiltration. Our results showed that Mycobacterium corresponds to a hydrophobic behavior linked to insoluble compound removal, whereas enterococci and E. coli refer to hydrophilic behaviors linked to soluble compound removals.


Subject(s)
Enterococcus/growth & development , Escherichia coli/growth & development , Models, Biological , Mycobacterium/growth & development , Waste Disposal, Fluid , Water Microbiology , Water Purification , Colony Count, Microbial , DNA, Bacterial/isolation & purification , Enterococcus/cytology , Escherichia coli/cytology , France , Mycobacterium/cytology , Principal Component Analysis
9.
Cell Biochem Biophys ; 57(1): 19-26, 2010 May.
Article in English | MEDLINE | ID: mdl-20352375

ABSTRACT

Enterococcus hirae grow well under anaerobic conditions at alkaline pH (pH 8.0) producing acids by glucose fermentation. Bacterial growth was shown to be accompanied by decrease of redox potential from positive values (approximately +35 mV) to negative ones (approximately -220 mV). An oxidizer copper (II) ions (Cu(2+)) affected bacterial growth in a concentration-dependent manner (within the range of 0.05 mM to 1 mM) increasing lag phase duration and decreasing specific growth rate. These effects were observed with the wild-type strain ATCC9790 and the atpD mutant strain MS116 (with absent beta subunit of F(1) of the F(o)F(1) ATPase) both. Also ATPase activity and proton-potassium ions exchange were assessed with and without N,N'-dicyclohexylcarbodiimide (DCCD), inhibitor of the F(o)F(1) ATPase. In both cases (DCCD +/-), even low Cu(2+) concentrations had noticeable effect on ATPase activity, but with less visible concentration-dependent manner. Changes in the number of accessible SH-groups were observed with E. hirae ATCC9790 and MS116 membrane vesicles. In both strains Cu(2+) markedly decreased the number of SH-groups in the presence of K(+) ions. The addition of ATP increased the amount of accessible SH-groups in ATCC9790 and decreased this number in MS116; Cu(2+) blocked ATP-installed increase in SH-groups number in ATCC9790. H(+)-K(+)-exchange of bacteria was markedly inhibited by Cu(2+), but stronger effects were detected together with DCCD. Moreover, discrimination between Cu(2+) and other bivalent cation--Ni(2+) was shown. It is suggested that Cu(2+) ions inhibit E. hirae cell growth by direct affect on the F(o)F(1) ATPase leading to conformational changes in this protein complex and decrease in its activity.


Subject(s)
Copper/chemistry , Copper/pharmacology , Enterococcus/cytology , Enterococcus/enzymology , Proton-Translocating ATPases/metabolism , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Enterococcus/drug effects , Mutation , Nickel/pharmacology , Oxidation-Reduction/drug effects , Potassium/metabolism , Proton-Translocating ATPases/genetics , Protons , Sulfhydryl Compounds/metabolism
10.
Infect Genet Evol ; 9(6): 1364-70, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19800031

ABSTRACT

This paper reports the first evidence of the presence of bacteria, other than the three previously described as symbionts, Wigglesworthia glossinidia, Wolbachia, and Sodalis glossinidius, in the midgut of Glossina palpalis palpalis, the tsetse fly, a vector of the chronic form of human African trypanosomiasis in sub-Saharan African countries. Based on the morphological, nutritional, physiological, and phylogenetic results, we identified Enterobacter, Enterococcus, and Acinetobacter spp. as inhabitants of the midgut of the tsetse fly from Angola. Enterobacter spp. was the most frequently isolated. The role of these bacteria in the gut, in terms of vector competence of the tsetse fly, is discussed, as is the possibility of using these bacteria to produce in situ trypanolytic molecules.


Subject(s)
Acinetobacter/isolation & purification , Enterobacter/isolation & purification , Enterococcus/isolation & purification , Gastrointestinal Tract/microbiology , Tsetse Flies/microbiology , Acinetobacter/cytology , Acinetobacter/physiology , Angola , Animals , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Enterobacter/cytology , Enterobacter/physiology , Enterococcus/cytology , Enterococcus/physiology , Humans , Insect Vectors/microbiology , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/analysis , RNA, Ribosomal, 16S/genetics , Symbiosis , Trypanosomiasis, African/transmission , Tsetse Flies/physiology
11.
J Appl Microbiol ; 105(5): 1461-9, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18795977

ABSTRACT

AIMS: To measure antibacterial activity of the semi-synthetic flavonoid 3-O-octanoyl-(-)-epicatechin and investigate the mechanism of action. METHODS AND RESULTS: MICs determined by the broth microdilution method were 50 microg ml(-1) for beta-lactam sensitive and resistant Staphylococcus aureus, and 100 microg ml(-1) for vancomycin sensitive and resistant enterococci. In time-kill studies, 100 microg ml(-1) 3-O-octanoyl-(-)-epicatechin reduced colony forming unit numbers of antibiotic sensitive and methicillin-resistant Staph. aureus below detectable levels within 120 min. Bacterial aggregation was not observed when cells exposed to 3-O-octanoyl-(-)-epicatechin were examined by light microscopy. It was also shown that 50 microg ml(-1) 3-O-octanoyl-(-)-epicatechin is capable of reducing colony forming unit numbers of high cell density Staph. aureus populations by 80-fold within 60 min incubation, and inducing leakage of 50% of their internal potassium within just 10 min. CONCLUSIONS: 3-O-Octanoyl-(-)-epicatechin is active against Gram-positive bacteria, has bactericidal activity against both antibiotic sensitive and resistant strains, and is likely to exert its primary antibacterial effect by damaging the cytoplasmic membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: 3-O-Octanoyl-(-)-epicatechin has significant antibacterial activity and additional structural modification and/or formulation studies may allow this to be potentiated.


Subject(s)
Anti-Bacterial Agents/pharmacology , Catechin/analogs & derivatives , Enterococcus/drug effects , Flavonoids/pharmacology , Staphylococcus aureus/drug effects , Catechin/pharmacology , Cell Aggregation/drug effects , Drug Resistance, Bacterial/physiology , Enterococcus/cytology , Microbial Sensitivity Tests , Staphylococcus aureus/cytology
12.
Syst Appl Microbiol ; 31(3): 223-30, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18562146

ABSTRACT

The objective of this work was to investigate the spread of Enterococcus italicus in cheese. For this purpose, a fluorescence whole-cell hybridization protocol (FWCH) with a 16S rRNA probe was optimized to evaluate the presence and abundance of this organism in artisanal Italian cheeses. The FWCH method avoided the quantification problems using classical plate count techniques related to the well-known difficulties to cultivate E. italicus in selective enterococci media. After probe and FWCH optimization, 10 commercially available Italian semi-hard cheeses made with raw ewe or cow milk without starter addition were analyzed. All of them were subjected to FWCH experiments and six of them gave positive results with the probe, i.e. the E. italicus content was >4 log cells/g according to the detection limit of FWCH. Counts showed that E. italicus was present at levels ranging from 5.91+/-0.17 to 7.34+/-0.14 log cells/g; such levels were similar to, or even higher than, the total enterococci counted from the corresponding cheeses using kanamycin aesculin azide agar. The overall reliability of the FWCH method was tested by species-specific PCR. The positive amplification of the expected 323 bp fragment from both a cheese matrix and cell bulks of cheese samples containing high loads of this organism (as determined by FWCH counts) and the successful isolation of E. italicus strains from the above cheeses provided definitive proof of both probe specificity and the presence of this organism in cheeses. Although there is very little available quantitative data on the incidence of E. italicus in cheese, or its role in product quality, this study showed a wide diffusion of this organism in artisanal cheeses, where secondary non-starter lactic acid bacterial microflora, which enterococci belong to, may become dominant during ripening.


Subject(s)
Cheese/microbiology , Enterococcus/cytology , Enterococcus/isolation & purification , In Situ Hybridization, Fluorescence/methods , Bacteriological Techniques , Colony Count, Microbial/methods , DNA Probes , DNA, Bacterial/analysis , DNA, Bacterial/isolation & purification , Enterococcus/classification , Enterococcus/genetics , Italy , Polymerase Chain Reaction , Species Specificity
13.
Water Sci Technol ; 56(3): 175-82, 2007.
Article in English | MEDLINE | ID: mdl-17802853

ABSTRACT

Reduction of pathogenic bacteria: Salmonella, enterococci and Escherichia coli was investigated using the sludge reed bed system. The system at Helsinge was established in 1996 and has a capacity of 630 tonnes dry solids per year and consists of 10 basins. Since 2000 the individual basins have been subjected to an average area-specific loading rate of 46-56 kg dry solids/m2/year. The total sludge residue height in April 2006 was approximately 1.40 m. The sludge (approximately 0.5-0.8% dry solids), with which the individual basins were loaded, contained a large number of bacteria. Salmonella, enterococci and E. coli were found in the sludge in the following quantities: 10-300/100 g (wet weight), 7,000-25,000 CFU/g (wet weight) and 800,000-10,000,000 CFU/100 g (wet weight), respectively. The analysis of the reduction in pathogens in the sludge residue through a period of 3-4 months after the last loading indicated that the pathogen content was reduced down to < 2/100 g (Salmonella), < 10 CFU/g (enterococci) and < 200 number/100 g (E. coli). For enterococci and E. coli the reduction was approximately log 5 and log 6-7, respectively. In the same period the sludge residue achieved a dry solids content of approximately 20-35%.


Subject(s)
Biodegradation, Environmental , Sewage/microbiology , Wetlands , Enterococcus/cytology , Enterococcus/isolation & purification , Escherichia coli/cytology , Escherichia coli/isolation & purification , Humans , Salmonella/cytology , Salmonella/isolation & purification
14.
J Biomed Opt ; 10(4): 44027, 2005.
Article in English | MEDLINE | ID: mdl-16178660

ABSTRACT

We describe the protocol for an inexpensive and nondestructive optical reflectance assay for the measurement of biofilm formation. Reflectance data are obtained using an Ocean Optics (Dunedin, Florida) USB 2000 spectrometer with a polychromatic light source. A fiber optic cable is used both for illumination and collection, and Ocean Optics OOIBase32 Platinum software is used for preliminary processing of the data. Differences in reflectance data collected at times ranging from 2 to 24 h distinguish between cell attachment and volume growth for two strains of Enterococci. Confocal scanning laser microscopy imaging is used to confirm these results. Phase contrast microscopy images are also obtained in conjunction with reflectance measurements for several different biofilm specimens. The experiments consider biofilm formation on glass and polystyrene substrata, but the method can be used for many other abiotic substrata of interest, both opaque and nonopaque.


Subject(s)
Biofilms/growth & development , Colony Count, Microbial/methods , Enterococcus/cytology , Enterococcus/physiology , Image Interpretation, Computer-Assisted/methods , Photometry/methods , Spectrophotometry, Ultraviolet/methods
15.
Bioorg Med Chem ; 12(12): 3357-62, 2004 Jun 15.
Article in English | MEDLINE | ID: mdl-15158804

ABSTRACT

Novel omega-pyridiniumalkylethers of two steroidal phenols were synthesized as compounds with potential antimicrobial activity. 3-Hydroxy-estra-1,3,5(10)-triene-17-one and 1-hydroxy-4-methyl-estra-1,3,5(10)-triene-17-one were reacted with omega,omega'-dibromoalkanes to omega-bromoalkoxy-estra-1,3,5(10)-trienes followed by reaction with pyridine to obtain the desired steroidal omega-pyridiniumalkoxy compounds as bromides. Their antimicrobial activity against strains of multiresistant Staphylococcus aureus (MRSA), a vancomycin resistant Enterococcus faecalis and fast growing mycobacteria depends clearly on the length of the alkyl chain. A strong broadband activity has been found for the compounds with eight or 10 C-atoms; in some cases better than ciprofloxacin or cetylpyridinium salts. In addition, the antiproliferative and cytotoxic activity depends on the chain length, too. The differentiation between antibacterial and cytotoxic activity is better for the steroid hybrid molecules than the cetylpyridinium salts. These new compounds can serve as lead compounds for further optimization.


Subject(s)
Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Ethers/chemistry , Phenazopyridine/chemistry , Phenols/chemistry , Phenols/pharmacology , Steroids/chemistry , Anti-Bacterial Agents/chemistry , Cell Proliferation/drug effects , Enterococcus/cytology , Enterococcus/drug effects , Microbial Sensitivity Tests , Molecular Structure , Staphylococcus aureus/cytology , Staphylococcus aureus/drug effects , Steroids/chemical synthesis
16.
Antonie Van Leeuwenhoek ; 85(3): 237-52, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15031651

ABSTRACT

The purpose of the study was to assess the phenotypic and genotypic taxonomic congruence in order to allow species allocation of dairy enterococci. A total of 364 enterococci isolated from ewes'milk and cheese from four Portuguese Registered Designation of Origin areas and 25 type and reference strains of Enterococcus spp. were characterized by a polyphasic taxonomical approach involving 40 physiological and biochemical tests, whole-cell protein profiles, amplification of 16S-23S intergenic spacer regions (ITS-PCR) and subsequent restriction analysis (ARDRA). Ribotyping was also performed with reference strains and a subset of 146 isolates. Numerical hierarchic data analysis showed that single-technique identification levels increase from the physiological and biochemical tests to the protein approach, being lower with ITS/ARDRA and ribotyping. Cross-analysis confirmed a higher unmatching level in all pairwise combinations involving physiological and biochemical data. Whole-cell protein profiles followed by ITS/ARDRA identified 89% of the enterococci. Reliable identification of enterococci from milk and cheese could be obtained by analysis of whole-cell protein profiles. ITS-PCR can be used to confirm E. durans and E. faecium and ARDRA further confirms E. faecalis. Results revealed E. faecalis, E. durans, E. hirae and E. faecium as the prevalent species, although species prevalence showed some degree of variation among the areas.


Subject(s)
Bacterial Typing Techniques , Cheese/microbiology , Enterococcus/classification , Milk/microbiology , Animals , Bacterial Proteins/analysis , DNA, Ribosomal Spacer/analysis , Enterococcus/chemistry , Enterococcus/cytology , Enterococcus/genetics , Genotype , Phenotype , Phylogeny , Portugal , Ribotyping , Sequence Analysis, DNA/methods , Sheep
17.
New Microbiol ; 23(3): 305-17, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10939046

ABSTRACT

Daptomycin binding proteins (DBPs) are membrane proteins which act as daptomycin targets. Daptomycin is a cyclic lipopeptide antibiotic which is active against Gram-positive bacteria and was shown to be the first inhibitor of lipoteichoic acid (LTA) synthesis. It was found that the antibiotic did not penetrate the bacterial cytoplasm but bound membranes with a non-covalent bond and in particular some proteins which were called DBPs. DBPs were indicated as enzymes involved in LTA synthesis whose binding and inhibition by daptomycin is responsible for the observed effect on bacterial LTA synthesis. The purification of DBPs will make it possible not only to shed light on the biosynthesis of the cell wall polymer but will also provide innovative targets for selection of new antibacterial compounds. In this study, the purification of DBPs is described. Affinity chromatography was used with daptomycin as the ligand. Final elution of DBPs from daptomycin-coupled resin was performed using either 0.1% SDS or 3 M NaCl. Polyacrylamide gel electrophoresis of the eluted protein fractions consistently showed four protein bands (ranging from 55 to 66 kDa) in denaturating conditions and two protein bands (60 and 66 kDa) in non-denaturating conditions. Isoelectrofocusing analysis of the same sample consistently revealed two bands with pIs around 5. That these purified proteins were really the desired DBPs is demonstrated by the retention of daptomycin-binding capability they displayed.


Subject(s)
Bacterial Outer Membrane Proteins/isolation & purification , Bacterial Outer Membrane Proteins/metabolism , Daptomycin/metabolism , Enterococcus/chemistry , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Chromatography, Affinity , Chromatography, High Pressure Liquid , Daptomycin/pharmacology , Daptomycin/therapeutic use , Electrophoresis, Polyacrylamide Gel , Enterococcus/cytology , Isoelectric Focusing , Ligands , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/biosynthesis , Molecular Weight , Osmolar Concentration , Protein Binding , Teichoic Acids/antagonists & inhibitors , Teichoic Acids/biosynthesis
18.
Am J Clin Nutr ; 65(5): 1397-402, 1997 May.
Article in English | MEDLINE | ID: mdl-9129468

ABSTRACT

Constipation is an ailment encountered often in elderly people. A study was initiated to test the effects of lactose or inulin on the bowel habits of constipated elderly patients and to correlate these effects with several variables measured in feces such as microflora composition, concentration of lactate and short-chain fatty acids (SCFAs), pH, and the activities of beta-glucosidase and beta-glucuronidase, Groups of 15 and 10 patients received lactose and inulin, respectively, for a period of 19 d. The dose, 20 g/d from days 1 to 8, was gradually increased to 40 g/d from days 9 to 11 and was kept at this dose from days 12 to 19. There was considerable interindividual variations with this kind of dietary intervention. Inulin increased bifidobacteria significantly from 7.9 to 9.2 log10/g dry feces, but decreased enterococci in number and enterobacteria in frequency. In individuals consuming lactose, a noticeable increase in fecal counts of enterococci and a decrease in lactobacilli and clostridia was detected. Total bacterial counts remained unchanged. No changes in the concentrations of fecal SCFAs and lactate were observed. SCFAs showed a slight trend toward higher molar ratios of acetate to butyrate in response to the intake of lactose or inulin. The fecal pH and the beta-glucosidase and beta-glucuronidase activities were not influenced by sugar intake. Inulin showed a better laxative effect than lactose and reduced functional constipation with only mild discomfort.


Subject(s)
Cathartics/therapeutic use , Constipation/drug therapy , Feces/microbiology , Inulin/therapeutic use , Lactose/therapeutic use , Aged , Aged, 80 and over , Bifidobacterium/cytology , Colony Count, Microbial , Enterobacteriaceae/cytology , Enterococcus/cytology , Fatty Acids/analysis , Feces/chemistry , Female , Glucuronidase/metabolism , Humans , Hydrogen-Ion Concentration , Inulin/administration & dosage , Lactic Acid/analysis , Lactose/administration & dosage , beta-Glucosidase/metabolism
19.
Rev. méd. IMSS ; 32(6): 499-502, nov.-dic. 1994. tab
Article in Spanish | LILACS | ID: lil-173969

ABSTRACT

Con el propósito de conocer la incidencia y el manejo antimicrobiano en la peritonitis posdiálisis, se estudiaron 358 procedimientos dialíticos, encontrando 37 casos de peritonitis con un porcentaje de 10.3, de los cuales 35 cumplían con los criterios de inclusión para el estudio, dividiéndose en dos grupos: grupo I formado por 19 pacientes en tratamiento con pefloxacina y grupo II integrado por 16 pacientes en tratamiento con ceftazidima. Obteniendo en el grupo I una remisión del cuadro en 89.5 por ciento y en el grupo II de 93.8 por ciento, no encontrando diferencia significativa (p=0.56) entre ambos antimicrobianos


Subject(s)
Peritonitis/therapy , Pseudomonas/cytology , Staphylococcus/cytology , Candida/cytology , Urinary Catheterization/adverse effects , Pefloxacin/therapeutic use , Ceftazidime/therapeutic use , Enterococcus/cytology , Escherichia coli/cytology , Antibiosis/drug effects , Peritoneal Dialysis/adverse effects , Renal Insufficiency, Chronic/therapy
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