Nasal carriage of Staphylococcus aureus, including community-associated methicillin-resistant strains, in Queensland adults

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are emerging in southeast Queensland, Australia, but the incidence of carriage of CA-MRSA strains is unknown. The aim of this study was to assess the nasal carriage rate of S. aureus, including CA-MRSA strains, in the general adult population of southeast Queensland. 396 patients presenting to general practices in two Brisbane suburbs and 303 volunteers randomly selected from the electoral rolls in the same suburbs completed a medical questionnaire and had nasal swabs performed for S. aureus. All isolates of S. aureus underwent antibiotic susceptibility testing and single-nucleotide polymorphism (SNP) and binary typing, including determination of Panton–Valentine leukocidin (PVL). The nasal carriage rate of methicillin-susceptible S. aureus (MSSA) was 202/699 (28%), a rate similar to that found in other community-based nasal carriage studies. According to multivariate analysis, nasal carriage of S. aureus was associated with male sex, young adult age group and Caucasian ethnicity. Only two study isolates (one MSSA and one CA-MRSA) carried PVL. The nasal carriage rate of MRSA was low, at 5/699 (0.7%), and only two study participants (0.3%) had CA-MRSA strains. CA-MRSA is an emerging cause of infection in southeast Queensland, but as yet the incidence of carriage of CA-MRSA in the general community is low.

Protein Synthesis in Mycobacterium tuberculosis H37Rv and the Effect of Streptomycin in Streptomycin-Susceptible and -Resistant Strains

An efficient in vitro amino acid-incorporating system from Mycobacterium tuberculosis H37Rv was standardized. Ribonucleic acid (RNA) isolated from phage-infected M. smegmatis cells served as natural messenger RNA and directed the incorporation of 14C-amino acids into protein. The effects of various antitubercular drugs and “known inhibitors” of protein synthesis on amino acid incorporation were studied. Antibiotics like chloramphenicol and tetracycline inhibited mycobacterial protein synthesis, though they failed to prevent the growth of the organism. This failure was shown to be due to the impermeability of mycobacteria to these drugs by use of “membrane-active” agents along with the antibiotics in growth inhibition studies. Several independent streptomycin-resistant mutants of M. tuberculosis H37Rv were isolated. Streptomycin inhibited the incorporation of 14C-amino acids into proteins by whole cells of a streptomycin-susceptible strain by more than 90%, whereas very little or no inhibition was observed in either high-level or low-level streptomycin-resistant strains.

Estimating Frequencies of Minority Nevirapine-Resistant Strains in Chronically HIV-1-Infected Individuals Naive to Nevirapine by Using Stochastic Simulations and a Mathematical Model

Nevirapine forms the mainstay of our efforts to curtail the pediatric AIDS epidemic through prevention of mother-to-child transmission of HIV-1. A key limitation, however, is the rapid selection of HIV-1 strains resistant to nevirapine following the administration of a single dose. This rapid selection of resistance suggests that nevirapine-resistant strains preexist in HIV-1 patients and may adversely affect outcomes of treatment. The frequencies of nevirapine-resistant strains in vivo, however, remain poorly estimated, possibly because they exist as a minority below current assay detection limits. Here, we employ stochastic simulations and a mathematical model to estimate the frequencies of strains carrying different combinations of the common nevirapine resistance mutations K103N, V106A, Y181C, Y188C, and G190A in chronically infected HIV-1 patients naive to nevirapine. We estimate the relative fitness of mutant strains from an independent analysis of previous competitive growth assays. We predict that single mutants are likely to preexist in patients at frequencies (similar to 0.01% to 0.001%) near or below current assay detection limits (>0.01%), emphasizing the need for more-sensitive assays. The existence of double mutants is subject to large stochastic variations. Triple and higher mutants are predicted not to exist. Our estimates are robust to variations in the recombination rate, cellular superinfection frequency, and the effective population size. Thus, with 10(7) to 10(8) infected cells in HIV-1 patients, even when undetected, nevirapine-resistant genomes may exist in substantial numbers and compromise efforts to prevent mother-to-child transmission of HIV-1, accelerate the failure of subsequent antiretroviral treatments, and facilitate the transmission of drug resistance.

An Expanded Multilocus Sequence Typing Scheme for Propionibacterium acnes: Investigation of ‘Pathogenic’, ‘Commensal’ and Antibiotic Resistant Strains

The Gram-positive bacterium Propionibacterium acnes is a member of the normal human skin microbiota and is associated with various infections and clinical conditions. There is tentative evidence to suggest that certain lineages may be associated with disease and others with health. We recently described a multilocus sequence typing scheme (MLST) for P. acnes based on seven housekeeping genes (http://pubmlst.org/pacnes). We now describe an expanded eight gene version based on six housekeeping genes and two ‘putative virulence’ genes (eMLST) that provides improved high resolution
typing (91eSTs from 285 isolates), and generates phylogenies congruent with those based on whole genome analysis. When compared with the nine gene MLST scheme developed at the University of Bath, UK, and utilised by researchers at Aarhus University, Denmark, the eMLST method offers greater resolution. Using the scheme, we examined 208 isolates from disparate clinical sources, and 77 isolates from healthy skin. Acne was predominately associated with type IA₁ clonal complexes CC1, CC3 and CC4; with eST1 and eST3 lineages being highly represented. In contrast, type IA₂ strains were recovered at a rate similar to type IB and II organisms. Ophthalmic infections were predominately associated with type IA₁ and IA₂ strains, while type IB and II were more frequently recovered from soft tissue and retrieved medical devices. Strains with rRNA mutations conferring resistance to antibiotics used in acne treatment were dominated by eST3, with some evidence for intercontinental spread. In contrast, despite its high association with acne, only a small number of resistant CC1 eSTs were identified. A number of eSTs were only recovered from healthy skin, particularly eSTs representing CC72 (type II) and CC77 (type III). Collectively our data lends support to the view that pathogenic versus truly commensal lineages of P. acnes may exist. This is likely to have important therapeutic and diagnostic implications.

Impact of chlorophenols and heavy metals on soil microbiota: their effects on activity and community composition, and resistant strains with potential for bioremetiation

Pollution by toxic compounds is one of the most relevant environmental damages to ecosystems produced by human activity and, therefore, it must be considered in environmental protection and restoration of contaminated sites. According to this purposes, the main goal of this doctoral thesis has been to analyse the impact of several chlorophenols and heavy metals on the microbial communities of two typical Mediterranean soils. The ecological risk concentrations of each pollutant, which have been determined according to respirometric activity and changes in the microbial community composition, and the factors that influence on their effective toxic concentrations (bioavailable pollutants) have been analysed in order to predict their potential impact on different soil ecosystems and provide scientific data for the regulation of the soil protection policies. Moreover, resistant microorganisms with pollutant removal capacities have been isolated from artificially contaminated soil microcosms and tested in axenic cultures, to infer their potential usefulness for bioremediation.

The Isolation and Characterization of Multiply Antibiotic Resistant Strains of Fish Pathogenic Flavobacterium Species

Since the development of the first antibiotics in the 1940’s, there has been widespread overuse in both clinical and agricultural applications. Antibiotic resistance has become a significant problem as a result of subsequent dissemination of antibiotics into the environment, and multiply-resistant strains of bacteria are now a major pathogenic threat. In this study eight separate strains of Flavobacterium responsible for recent disease outbreaks in fish hatcheries throughout Maine were collected and analyzed. All eight strains were found to be resistant to high levels of a number of different antibiotics, including those used for aquaculture as well as human chemotherapeutic applications. Flavobacterium isolates were also shown phenotypically to transfer antibiotic resistance determinants using a conjugation mating system in which Flavobacterium was the donor and Escherichia coli DH5- alpha was the recipient. This experiment suggests that it may be possible for Flavobacterium strains to transfer their multiple antibiotic resistance determinants to human pathogenic bacterial strains. Importantly, none of the hatcheries from which the Flavobacterium isolates were obtained had ever used antibiotics to treat their fish stock. It is possible that there is another selective agent responsible for the development of antibiotic resistance in the absence of antibiotic pressure. Mercury is one possible candidate, as all of the strains tested were resistant to mercuric chloride and it is known that genes encoding antibiotic resistance can be carried on the same mobile genetic elements that encode for mercury resistance. Preliminary data also suggest that the majority of the Flavobacterium isolates contain genes for mercuric ion reduction, which would confirm the mercury resistance genotype.

The effect of gene tubC on the vegetative growth of benomyl-resistant strains of Aspergillus nidulans

Two benomyl-resistant mutants, benD3 tubC41 and benD4 tubC42, of Aspergillus nidulans were isolated after UV treatment. The tubC mutations permitted good conidiation of these strains in culture media containing benomyl and were responsible for increasing their benomyl resistance levels. This implies that β3-tubulin, a product of the tubC gene, in addition to being involved in fungal conidiation, participates in the vegetative growth of the fungus. The tubC gene was located in linkage group I.

Pefloxacin therapy for experimental endocarditis caused by methicillin-susceptible or methicillin-resistant strains of Staphylococcus aureus

The therapeutic efficacy of pefloxacin in experimental endocarditis caused by methicillin-susceptible or methicillin-resistant Staphylococcus aureus was evaluated. In rabbits infected with a methicillin-susceptible strain, 4 days of pefloxacin therapy significantly reduced both the number of bacteria per gram of vegetation and the mortality rate compared with untreated controls, and pefloxacin was equivalent to cephalothin. Pefloxacin was also as effective as vancomycin in reducing vegetation titers and mortality rate in animals with endocarditis caused by a methicillin-resistant strain. These results suggest that pefloxacin may be an effective agent in the therapy of serious infections caused by either methicillin-susceptible or -resistant strains of S. aureus.

Evolutionary genomics of Staphylococcus aureus: Insights into the origin of methicillin-resistant strains and the toxic shock syndrome epidemic

An emerging theme in medical microbiology is that extensive variation exists in gene content among strains of many pathogenic bacterial species. However, this topic has not been investigated on a genome scale with strains recovered from patients with well-defined clinical conditions. Staphylococcus aureus is a major human pathogen and also causes economically important infections in cows and sheep. A DNA microarray representing >90% of the S. aureus genome was used to characterize genomic diversity, evolutionary relationships, and virulence gene distribution among 36 strains of divergent clonal lineages, including methicillin-resistant strains and organisms causing toxic shock syndrome. Genetic variation in S. aureus is very extensive, with ≈22% of the genome comprised of dispensable genetic material. Eighteen large regions of difference were identified, and 10 of these regions have genes that encode putative virulence factors or proteins mediating antibiotic resistance. We find that lateral gene transfer has played a fundamental role in the evolution of S. aureus. The mec gene has been horizontally transferred into distinct S. aureus chromosomal backgrounds at least five times, demonstrating that methicillin-resistant strains have evolved multiple independent times, rather than from a single ancestral strain. This finding resolves a long-standing controversy in S. aureus research. The epidemic of toxic shock syndrome that occurred in the 1970s was caused by a change in the host environment, rather than rapid geographic dissemination of a new hypervirulent strain. DNA microarray analysis of large samples of clinically characterized strains provides broad insights into evolution, pathogenesis, and disease emergence.

Antimycobacterial activities of antisense oligodeoxynucleotide phosphorothioates in drug-resistant strains.

Strains of Mycobacterium smegmatis, a mycobacterium which shares genetic sequences, grows more rapidly, and is nonpathogenic in man as compared with Mycobacterium tuberculosis, were utilized for the initial development of new antimycobacterial therapy. Drug-resistant strains of M. smegmatis which are known to arise in a manner identical to the emergence of drug-resistant strains of M. tuberculosis were isolated and utilized as models for the antimycobacterial activities of modified and unmodified oligodeoxynucleotide phosphorothioates in broth cultures. Under normal conditions, oligodeoxynucleotide phosphorothioates do not enter mycobacteria, and several strategies were successfully utilized to afford entry of oligonucleotides into the mycobacterial cells. One involved the presence of very low levels of ethambutol, which enables the entry of oligonucleotides into mycobacteria because of its induced alterations in the cell wall, and another involved the utilization of oligonucleotides covalently attached to a D-cycloserine molecule, whereby entry into the mycobacterial cell is achieved by a receptor-mediated process. Another low molecular weight, covalently attached ligand that enabled the entry and subsequent antimycobacterial activities of oligodeoxynucleotide phosphorothioates in the absence of a cell wall modifying reagent was biotin. Significant sequence-specific growth inhibition of wild-type, as well as of drug-resistant, M. smegmatis was obtained by modified oligonucleotides complementary in sequence to a specific region of the mycobacterium aspartokinase (ask) gene when utilized in combinations with ethambutol (as compared to ethambutol alone) or as D-cycloserine or biotin covalent adducts without the presence of any other cytotoxic or cytostatic agent.

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.

Fitness and dissemination of disinfectant-selected multiple-antibiotic-resistant (MAR) strains of Salmonella enterica serovar Typhimurium in chickens

Objectives: The aims of this study were to determine whether strains of Salmonella enterica serovar Typhimurium which had acquired low-level multiple antibiotic resistance (MAR) through repeated exposure to farm disinfectants were able to colonize and transmit between chicks as easily as the parent strain and, if such strains were less susceptible to fluoroquinolones, would high-level resistance be selected after fluoroquinolone treatment. Methods: Two mutants were compared with the isogenic parent. In the first experiment, day-old chicks were co-infected with both the parent and a mutant to determine their relative fitness. In the second experiment, parent and mutant strains (in separate groups of chicks) were assessed for their ability to transmit from infected (contact) to non-infected (naive) birds and with respect to their susceptibility to fluoroquinolone treatment. Birds were regularly monitored for the presence of Salmonella in caecal contents. Replica plating was used to monitor for the selection of antibiotic-resistant strains. Results: The parent strain was shown to be significantly fitter than the two mutants and was more rapidly disseminated to naive birds. Antibiotic treatment did not preferentially select for the two mutants or for resistant strains. Conclusions: The disinfectant-exposed strains, although MAR, were less fit, less able to disseminate than the parent strain and were not preferentially selected by therapeutic antibiotic treatment. As such, these strains are unlikely to present a greater problem than other salmonellae in chickens.

Resistance development in community-acquired strains of methicillin-resistant Staphylococcus aureus: an in vitro study

This study compares in vitro antimicrobial resistance development between strains of Staphylococcus aureus including newly described community-acquired methicillin-resistant strains (CA-MRSA). High-level resistance developed in all strains of S. aureus after exposure to rifampicin and gentamicin and in some strains after fusidic acid exposure, independent of methicillin resistance phenotype. Resistance did not develop after exposure to clindamycin, cotrimoxazole, ciprofloxacin, linezolid, or vancomycin. These results have important implications for therapy of CA-MRSA infections. (C) 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.