No description available.

Extended-spectrum beta-lactamases (ESBLs) in Pseudomonas aeruginosa tend to be geographically scattered, such as GES-2, which partially compromises the efficacy of imipenem. The G170N mutation, ascribed to a CC to AA base pair substitution on positions 493-494 of the blaGES-2 coding region, distinguishes this ESBL from blaGES-1 and the blaIBC-type genes, making it an ideal target for developing a novel sequence-specific, peptide nucleic acid (PNA)-based, multiplex-PCR detection method. Utilizing two primer pairs in conjunction with a PNA probe, this novel method delivered accurate identification of blaGES-2 compared to standard PCR and gene sequencing techniques, when tested against one hundred (n = 100) P. aeruginosa clinical isolates as well as previously published, well-described control strains. This method has the potential to be used in large-scale, cost-effective screening programmes for specific or geographically restricted ESBLs. To date, in addition to being only described in South Africa, GES-2 is notoriously difficult to identify in P. aeruginosa, using standard methodology. A real-time PCR method using the LightCycler™ was compared to a two-step nested-PCR assay for the detection of blaGES and blaIBC genes from one hundred P. aeruginosa clinical isolates collected over a four-year period from two teaching hospitals in Pretoria, South Africa. Real-time PCR amplification was monitored through hybridisation of fluorescently labelled probes followed by melting curve analysis to detect the relevant G170N mutation occurring in the omega loop region of blaGES-2. Nested-PCR products were subjected to automated DNA sequencing and compared to melting point (Tm) analyses results obtained from the LightCycler assay. Real time and nested-PCR assays detected a blaIBC gene product from 83 and 88 clinical isolates respectively, with the LightCycler thus exhibiting a sensitivity of 94.3% compared to the nested-PCR assay. Comparison of Tm and gene sequencing data however revealed 100% specificity for sequence specific detection of blaGES-2 with the LightCycler. One clinical isolate was found to harbour a blaGES-1 gene, making this the first report of this specific ESBL from South Africa. Selective antibiotic pressure has recently been implicated as a possible driving force behind point mutations observed in blaGES–type genes. This part of the study subjected two well-characterized clinical isolates with class 1 integron-borne blaGES-type genes to five days incubation in the presence of sub-inhibitory concentrations of 15 different antibiotics, including beta-lactams, aminoglycosides and quinolones. Restriction enzyme analysis and DNA sequencing of blaGES-1, blaGES-2 and their immediate upstream genetic environments failed to demonstrate any changes compared to non-exposed controls. Short-term exposure to a sub-inhibitory level of a single antimicrobial agent is thus unlikely to select significant mutations in these beta-lactamase genes or their regulatory mechanisms. / Thesis (PhD (Medical Microbiology))--University of Pretoria, 2004. / Medical Microbiology / unrestricted

Burkholderia contaminans MS14 shows excellent antimicrobial activities against a wide range of pathogens. Complete sequence analysis reveals that the MS14 genome harbors multiple gene loci that contribute to its antimicrobial activities and lacks key virulence features commonly found in pathogenic Burkholderia species. A mutagenesis study identified the genes required for MS14 antibacterial activities and gene expression profiling targeted a polyketide synthase (PKS) gene cluster. Site-specific mutagenesis confirmed the PKS gene cluster is directly related to MS14 antibacterial activities and the PKS gene product is predicted to be the MS14 antibacterial compound. Strain UFB2 isolated from Mississippi shows significant antifungal and antibacterial activities. UFB2 was classified to be Pseudomonas chlororaphis and its complete genome sequence was reported in this study. Green house trails showed P. chlororaphis strain UFB2 could efficiently reduce the disease severity of bacterial canker of tomato, by significantly inhibiting the growth of the pathogen Clavibacter michiganensis subsp. michiganensis. The research findings of B. contaminans MS14 and P. chlororaphis UFB2 have provided insights into the development of MS14 antibacterial compound for agricultural application and potential use of strain UFB2 as a biocontrol agent.

No description available.

Treatment of enterococcal endocarditis requires up to 6 weeks of intravenous (IV) antimicrobial therapy. When susceptible, an ampicillin-based regimen is preferred. Studies evaluating ampicillin stability utilizing high-pressure liquid chromatography have indicated enhanced stability (greater than 24 hours at room temperature), supporting outpatient administration. Thus, we report the successful treatment of a 30-year-old male with tricuspid valve enterococcal endocarditis in an outpatient setting using continuous infusion ampicillin via an ambulatory infusion pump. The patient received daily gentamicin at an outpatient infusion center with the ampicillin dose to be infused over the next 24 hours. Outpatient ambulatory infusion pumps allow for delivery of ampicillin via continuous infusion or pump-programmed pulse dosing. Preparation and administration in an outpatient infusion center may be a viable option to circumvent stability and delivery issues. Furthermore, 81% (34/42) of treatment days were completed outpatient, supporting that this approach may increase access to treatment and help reduce the economic burden to health care.

In immunodeficient patients, for example with cystic fibrosis (CF), the opportunistic pathogen Staphylococcus aureus causes chronic infections of respiratory tract that are treated with antibiotics (ATB) in the long term. However, exposure to antibiotics can lead to persistence, thereby result a recurrence of infection. The aim of this work was to examine in selected pairs of S. aureus chronological isolates from the respiratory tract of CF patients how their ability to form persisters is changing in time. I have found that the ability to persist within the clonal pair does not change significantly after two years of survival in the host, and that the ability to persist depends on the adaptative mutations of the isolates. Persister formation may depend on mutations in operon of the alternative sigma B factor (sigB) and the major virulence gene regulator (agr). By dual staining with DioC2(3) and To-pro-3, I was able to determine the changes in membrane potential and membrane permeability during the killing curve with ATBs. The distribution into subpopulations according to these parameters depends primarily on the antibiotic used. I conclude that various antibiotics can induce different mechanisms causing a persistent state. Futhermore, I have constructed plasmids with a labeled promoter to determine...

The soil bacterium Rhodococcus has a wide array of secondary metabolic pathways such as production of pigments, siderophores, and antibiotics that makes it an organism of interest for the production of novel natural products. Analysis of the genome sequence of Rhodococcus indicates the presence of 24 non-ribosomal peptide synthases and 7 polyketide synthases possibly involved in production of secondary metabolites. The use of a solid agar extraction method to screen soil isolates of Rhodococcus for compounds with inhibitory activity against other bacteria resulted in the discovery of a promising candidate molecule. The Rhodococcus strain KCHXC3, isolated from eastern Tennessee soil, produces a substance that inhibits the growth of several Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumonia and Gram-positive bacteria such as Micrococcus luteus and, Staphylococcus aureus. After bulk extraction of this compound with ethyl acetate from agar plates, the material was partially purified through different chromatography processes.

To combat widespread infections caused by Staphylococcus aureus, mupirocin was introduced at the Veterans Affairs Medical Center, Mountain Home, Tennessee. Soon after introduction, high-level mupirocin-resistance emerged. The rapid emergence was hypothesized to be due to conjugative transfer of the mupA resistance gene from S. epidermidis to S. aureus. Results have shown that transfer of high-level mupirocin-resistance from S. aureus donors commonly occurs. However, transfer from naturally-occurring S. epidermidis donors was not attainable. Staphylococcus epidermidis transconjugants, however, were capable of serving as donors. Further examination of non-transmissibility included PCR analysis of conjugative transfer genes (tra genes) in capable and non-capable donors. Results confirmed that capable donors possess full-length copies of selected transfer genes. Non-capable donors varied in the presence/absence of full-length copies of transfer genes, but none had all three genes. The genetic differences among non-capable donors suggest that non-transmissibility has arisen independently in different strains via gene deletions and recombinations.

Bacteremia initiated by Staphylococcus aureus infections can be a serious medical problem. Although a number of different antibiotics are used to combat staphylococcal infections, resistance has continued to develop. Combination therapy for certain infections has been used to reduce the emergence of resistance when a single agent has become ineffective. We hypothesize that the use of rifampicin and ciprofloxacin in combination with azithromycin, known for its inhibitory effects on the bacterial ribosome, can create potential synergistic effects resulting from indirect effects on ribosomal subunit synthesis. To determine this we measured the effects of single and multiple antibiotics on cell growth rates, cell viability, and synthesis rates for DNA, RNA, and protein. We then measured synthesis rates of ribosomal subunits and the amounts of gyrase and RNAP. Effects of the antibiotic combinations on 70S ribosomes was assayed and the amounts of RNA and degradation was measured. We lastly studied the effects of these antibiotic combinations on mutation frequency in Staphylococcus aureus. Our data have shown support not only for the use of antibiotic combination therapy but have provided strong evidence of an increase in the inhibition of bacterial ribosome assembly in Staphylococcus aureus. The reduction of 50S ribosomal subunit synthesis and 23S ribosomal RNA in cells grown in the presence of azithromycin, already known for it’s inhibitory effects on the 50S subunit synthesis, in combination with rifampicin or in combination with rifampicin and ciprofloxacin was observed. This also resulted in a reduction or elimination in the frequency of resistant cells when grown in the presence of these combinations. These studies have shed light on the mechanism of action involved and synergistic effects occurring in combination antibiotic treatments and how ribosomal subunit assembly is affected. The insights gained through this research provide necessary information needed for the design of more potent antibiotic combinations. This will create a better understanding and new methods for eliminating the spread of harmful pathogens such as Staphylococcus aureus.

Drug resistance is unfortunately becoming a prevalent issue in the course of patient treatment, ranging from chemotherapy resistance to antimicrobial resistance. The Centers for Disease Control and Prevention (CDC) estimated in 2016 that at least 23,000 people die every year in the United States from an infection with an antibiotic-resistant organism (Munita, et al, 2016). Carl Friedlander was the first scientist to describe Klebsiella pneumoniae in 1882 as an encapsulated bacillus after isolating the bacterium from the lungs of patients who had died from pneumonia (Ashurst and Dawson, 2022). Klebsiella pneumoniae is the type species for the Klebsiella genus and is the bacterium of interest for this project. It is one of the very few Gram-negative bacilli that can cause primary pneumonia, commonly affecting patients with compromised immune systems, alcohol use disorder, or diabetes mellitus (Ristuccia and Burke, 1984). However, microbes are able to produce a wide range of microbial defense systems including classic antibiotics, metabolic byproducts, and lytic agents. Bacteriocins are some of the most common defense mechanisms produced, which are different from antibiotics in that they have a narrow killing spectrum and are toxic only to bacteria that is closely related to the strain that is producing it. It has been estimated that 99% of all bacteria possibly make a minimum of one bacteriocin (Riley and Wertz, 2002). Because of the rapidly growing number of infections that are caused by antibiotic-resistant bacteria along with the harm that broad-spectrum antibiotics can cause to the human microbiome, these bacteriocins are being studied as potential alternatives to tradition antibiotics. In this study, we will assess and characterize a Klebsiella bacteriocin that may work synergistically with antibiotics so that antibiotic dosage might be reduced. In this study, we have isolated the plasmids from a possible Klebsiella bacteriocin and transformed them into E. coli to characterize the plasmid. This potential bacteriocin demonstrates efficacy towards Citrobacter, Enterobacter, and Klebsiella species and could offer an alternative treatment option for the highly drug resistant Enterobacteriaceae family.