Fruit firmness and skin resistance of processing tomato varieties tested in Badajoz (Spain), related to mechanical harvesting.

Determinations of resistance to damage were carried out in a total of 31 tomato varieties for processing, with the purpose of choosing the most suitable ones for mechanical harvesting. The characteristics studied include: puncture, deformation and rupture of the fruits, the ease of detachment of the fruits also being determined. Seventeen varieties were chosen, for further tests, with values 0.76 to 1.7 2 N of resistance to puncture; 3 to 9 N/mm of resistance to compression and 2.16 to 29.40 N resistance to detachment.

CytA enables CryIV endotoxins of Bacillus thuringiensis to overcome high levels of CryIV resistance in the mosquito, Culex quinquefasciatus

Cry proteins produced by Bacillus thuringiensis are selective biodegradable insecticides used increasingly in bacterial insecticides and transgenic plants as alternatives to synthetic chemical insecticides. However, the potential for development of resistance and cross-resistance in target insect populations to Cry proteins used alone or in combination threatens the more widespread use of this novel pest control technology. Here we show that high levels of resistance to CryIV proteins in larvae of the mosquito, Culex quinquefasciatus, can be suppressed or reduced markedly by combining these proteins with sublethal quantities of CytA, a cytolytic endotoxin of B. thuringiensis. Resistance at the LC95 level of 127-fold for a combination of three CryIV toxins (CryIVA, B, and D), resulting from 60 generations of continuous selection, was completely suppressed by combining sporulated powders of CytA in a 1:3 ratio with sporulated powders of a CryIVA, CryIVB, and CryIVD strain. Combining the CytA strain with a CryIVA and CryIVB strain also completely suppressed mosquito resistance of 217-fold to the latter toxins at the LC95 level, whereas combination of CytA with CryIVD reduced resistance in a CryIVD-selected mosquito strain from greater than 1,000-fold to less than 8-fold. The CytA/CryIV model provides a potential molecular genetic strategy for engineering resistance management for Cry proteins directly into bacterial insecticides and transgenic plants.

A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly

Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the “mutant ali-esterase hypothesis”). In the sheep blowfly, Lucilia cuprina, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly137 → Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the Asp137 substitution alone is responsible for both the loss of E3’s carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp137 in the homologous position in acetylcholinesterase suggests that Asp137 may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.

The epidemiology of antibiotic resistance in hospitals: Paradoxes and prescriptions

A simple mathematical model of bacterial transmission within a hospital was used to study the effects of measures to control nosocomial transmission of bacteria and reduce antimicrobial resistance in nosocomial pathogens. The model predicts that: (i) Use of an antibiotic for which resistance is not yet present in a hospital will be positively associated at the individual level (odds ratio) with carriage of bacteria resistant to other antibiotics, but negatively associated at the population level (prevalence). Thus inferences from individual risk factors can yield misleading conclusions about the effect of antibiotic use on resistance to another antibiotic. (ii) Nonspecific interventions that reduce transmission of all bacteria within a hospital will disproportionately reduce the prevalence of colonization with resistant bacteria. (iii) Changes in the prevalence of resistance after a successful intervention will occur on a time scale of weeks to months, considerably faster than in community-acquired infections. Moreover, resistance can decline rapidly in a hospital even if it does not carry a fitness cost. The predictions of the model are compared with those of other models and published data. The implications for resistance control and study design are discussed, along with the limitations and assumptions of the model.

Enhanced resistance in STAT6-deficient mice to infection with ectromelia virus

We inoculated BALB/c mice deficient in STAT6 (STAT6−/−) and their wild-type (wt) littermates (STAT6+/+) with the natural mouse pathogen, ectromelia virus (EV). STAT6−/− mice exhibited increased resistance to generalized infection with EV when compared with STAT6+/+ mice. In the spleens and lymph nodes of STAT6−/− mice, T helper 1 (Th1) cytokines were induced at earlier time points and at higher levels postinfection when compared with those in STAT6+/+ mice. Elevated levels of NO were evident in plasma and splenocyte cultures of EV-infected STAT6−/− mice in comparison with STAT6+/+ mice. The induction of high levels of Th1 cytokines in the mutant mice correlated with a strong natural killer cell response. We demonstrate in genetically susceptible BALB/c mice that the STAT6 locus is critical for progression of EV infection. Furthermore, in the absence of this transcription factor, the immune system defaults toward a protective Th1-like response, conferring pronounced resistance to EV infection and disease progression.

The association of feeding behavior with the resistance and tolerance to parasites in recently diverged sticklebacks

Divergent natural selection regimes can contribute to adaptive population divergence, but can be sensitive to human-mediated environmental change. Nutrient loading of aquatic ecosystems, for example, might modify selection pressures by altering the abundance and distribution of resources and the prevalence and infectivity of parasites. Here, we used a mesocosm experiment to test for interactive effects of nutrient loading and parasitism on host condition and feeding ecology. Specifically, we investigated whether the common fish parasite Gyrodactylus sp. differentially affected recently diverged lake and stream ecotypes of three-spined stickleback (Gasterosteus aculeatus). We found that the stream ecotype had a higher resistance to Gyrodactylus sp. infections than the lake ecotype, and that both ecotypes experienced a cost of parasitism, indicated by negative relationships between parasite load and both stomach fullness and body condition. Overall, our results suggest that in the early stages of adaptive population divergence of hosts, parasites can affect host resistance, body condition, and diet.

Host and Environmental Correlates of Multi-Drug Resistance in Kenyan Children with Acute Bacterial Diarrhea

Thesis (Master's)--University of Washington, 2016-06

Antimicrobial resistance in Staphylococcus aureus in Australian teaching hospitals, 1989-1999

An annual survey of antimicrobial resistance in clinical isolates of Staphylococcus aureus was conducted in 21 Australian teaching hospital microbiology laboratories in eight major cities from 1989 to 1999. A total of 19,000 isolates were tested for susceptibility to 18 antimicrobials, with 3795 being methicillin-resistant (MRSA). Resistance to ciprofloxacin in MRSA increased from 4.9% to 75.9%. The proportion of MRSA resistant to erythromycin decreased significantly (99.0%-88.9%), as did that to trimethoprim (98.4%-82.4%) and to tetracycline (96.5%-80.1%). The proportion of MRSA isolated increased in Sydney, Melbourne, Canberra, Adelaide, Perth, and Darwin, but not in Brisbane. The proportion in Hobart peaked in 1994. MRSA in Perth were predominantly non-multiresistant (nmMRSA) throughout the survey (i.e., resistant to less than three of eight indicator antibiotics) due mainly to local strains that originated in the community. The proportion of nmMRSA increased to modest levels in the other cities. In eastern cities, this was due to the appearance of strains closely related to nmMRSA seen in other countries of the southwestern Pacific.

A DHA14 drug efflux gene from Xanthomonas albilineans confers high-level albicidin antibiotic resistance in Escherichia coli

Aims: Identification of a gene for self-protection from the antibiotic-producing plant pathogen Xanthomonas albilineans, and functional testing by heterologous expression. Methods and Results: Albicidin antibiotics and phytotoxins are potent inhibitors of prokaryote DNA replication. A resistance gene (albF) isolated by shotgun cloning from the X. albilineans albicidin-biosynthesis region encodes a protein with typical features of DHA14 drug efflux pumps. Low-level expression of albF in Escherichia coli increased the MIC of albicidin 3000-fold, without affecting tsx-mediated albicidin uptake into the periplasm or resistance to other tested antibiotics. Bioinformatic analysis indicates more similarity to proteins involved in self-protection in polyketide-antibiotic-producing actinomycetes than to multi-drug resistance pumps in other Gram-negative bacteria. A complex promoter region may co-regulate albF with genes for hydrolases likely to be involved in albicidin activation or self-protection. Conclusions: AlbF is the first apparent single-component antibiotic-specific efflux pump from a Gram-negative antibiotic producer. It shows extraordinary efficiency as measured by resistance level conferred upon heterologous expression. Significance and Impact of the Study: Development of the clinical potential of albicidins as potent bactericidial antibiotics against diverse bacteria has been limited because of low yields in culture. Expression of albF with recently described albicidin-biosynthesis genes may enable large-scale production. Because albicidins are X. albilineans pathogenicity factors, interference with AlbF function is also an opportunity for control of the associated plant disease.

The suitability of biodegradable poly(dl-lactide-co-glycolide)75:25 microspheres for the sustained release of antibiotics

Post-operative infections resulting from total hip arthroplasty are caused by bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa entering the wound perioperatively or by haemetogenous spread from distant loci of infection. They can endanger patient health and require expensive surgical revision procedures. Gentamicin impregnated poly (methyl methacrylate) bone cement is traditionally used for treatment but is often removed due to harbouring bacterial growth, while bacterial resistance to gentamicin is increasing. The aim of this work was to encapsulate the antibiotics vancomycin, ciprofloxacin and rifampicin within sustained release microspheres composed of the biodegradable polymer poly (dl-lactide-co-glycolide) [PLCG] 75:25. Topical administration to the wound in hydroxypropylmethylcellulose gel should achieve high local antibiotic concentrations while the two week in vivo half life of PLCG 75:25 removes the need for expensive surgical retrieval operations. Unloaded and 20% w/w antibiotic loaded PLCG 75:25 microspheres were fabricated using a Water in Oil emulsification with solvent evaporation technique. Microspheres were spherical in shape with a honeycomb-like internal matrix and showed reproducible physical properties. The kinetics of in vitro antibiotic release into newborn calf serum (NCS) and Hank's balanced salt solution (HBSS) at 37°C were measured using a radial diffusion assay. Generally, the day to day concentration of each antibiotic released into NCS over a 30 day period was in excess of that required to kill St. aureus and Ps. auruginosa. Only limited microsphere biodegradation had occurred after 30 days of in vitro incubation in NCS and HBSS at 37°C. The moderate in vitro cytotoxicity of 20% w/w antibiotic loaded microspheres to cultured 3T3-L1 cells was antibiotic induced. In conclusion, generated data indicate the potential for 20% w/w antibiotic loaded microspheres to improve the present treatment regimens for infections occurring after total hip arthroplasty such that future work should focus on gaining industrial collaboration for commercial exploitation.

Studies on the outer membrane of Pseudomonas aeruginosa and associated resistance properties

The aim of this thesis was to investigate antibacterial agents for use in disinfectant formulation in conjunction with benzalkonium chloride (BKC), and if possible, to synthesise novel agents based upon successful structures. Development of resistance to antibacterial agents following long-term exposure of P. aeruginosa to BKC was also investigated, examining cross-resistance to clinically relevant antibiotics and determining mechanisms of resistance. In this study over 50 compounds were examined for antibacterial action against P. aeruginosa, both alone and in conjunction with BKC. Successful compounds were used to design novel agents, based upon the acridine ring structure, some of which showed synergy with BKC. In 15 of the 16 strains exposed to increasing concentrations of BKC, resistance to the disinfectant arose. Strains PAO1 and OO14 were examined further, each showing stable BKC resistance and a slightly varying profile of cross-resistance. In strain PAO1 alterations in the fatty acids of the cytoplasmic membrane, increase in expression of OprG, decrease in susceptibility to EDTA as an outer membrane permeabilising agent and an increase in negativity of the cell surface charge were observed as cells became more resistant to BKC. In strain OO14 a decrease in whole cell phosphatidylcholine content, a decrease in binding/uptake of BKC and an increase in cell surface hydrophobicity were observed as cells became more resistant to BKC. Resistance to tobramycin in strain OO14 was initially high, but fell as cells were adapted to BKC, this coincided with a quantitative reduction of plasmid DNA in the cells.

The need for continued monitoring of antibiotic resistance patterns in clinical isolates of Staphylococcus aureus from London and Malta

Antibiotic resistance is an increasing problem in isolates of Staphylococcus aureus (S. aureus) worldwide. In 2001 The National Health Service in the UK introduced a mandatory bacteraemia surveillance scheme for the reporting of S. aureus and methicillin-resistant S. aureus (MRSA). This surveillance initiative reports on the percentage of isolates that are methicillin resistant. However, resistance to other antibiotics is not currently reported and therefore the scale of emerging resistance is currently unclear in the UK. In this study, multiple antibiotic resistance (MAR) profiles against fourteen antimicrobial drugs were investigated for 705 isolates of S. aureus collected from two European study sites in the UK (London) and Malta.

Lys9 for Glu9 substitution in glucagon-like peptide-1(7-36)amide confers dipeptidylpeptidase IV resistance with cellular and metabolic actions similar to those of established antagonists glucagon-like peptide-1(9-36)amide and exendin (9-39)

The incretin hormone glucagon-like peptide-1(7-36)amide (GLP-1) has been deemed of considerable importance in the regulation of blood glucose. Its effects, mediated through the regulation of insulin, glucagon, and somatostatin, are glucose-dependent and contribute to the tight control of glucose levels. Much enthusiasm has been assigned to a possible role of GLP-1 in the treatment of type 2 diabetes. GLIP-l's action unfortunately is limited through enzymatic inactivation caused by dipeptidylpeptidase IV (DPP IV). It is now well established that modifying GLP-1 at the N-terminal amino acids, His7 and Ala8, can greatly improve resistance to this enzyme. Little research has assessed what effect Glu9-substitution has on GLP-1 activity and its degradation by DPP IV. Here, we report that the replacement of Glu9 of GLP-1 with Lys dramatically increased resistance to DPP IV. This analogue (Lys9)GLP-1, exhibited a preserved GLP-1 receptor affinity, but the usual stimulatory effects of GLP-1 were completely eliminated, a trait duplicated by the other established GLP-1-antagonists, exendin (9-39) and GLP-1 (9-36)amide. We investigated the in vivo antagonistic actions of (Lys9)GLP-1 in comparison with GLP-1(9-36)amide and exendin (9-39) and revealed that this novel analogue may serve as a functional antagonist of the GLP-1 receptor.

Multiplex PCR for Assessment of Tetracycline Resistance (tet) Genes in Antibiotic-Resistant Soil Bacteria and the Ecological Implications

Antibiotics are becoming increasingly prevalent in bacterial communities due to clinical and agricultural misuse and overuse in their environment. As exposure increases, so does the incidence of microbial resistance. Such is the case with bacterial resistance to tetracyclines, a phenotype often acquired through the horizontal gene transfer of tet genes between bacteria. The objective of this project was to analyze the bacterial diversity of tet resistance genes in soil from Miami-Dade County. Bacterial isolates were Gram-stained and the Kirby-Bauer antibiotic disk diffusion test was performed to determine each bacterium’s degree of resistance. The 16S rRNA gene from antibiotic-resistant isolates was amplified by PCR and sequenced to identify the isolates. All isolates’ tet genes were amplified by multiplex PCR, sequenced, and compared. Among eight isolates, three distinct species were positively identified based on their 16S rRNA sequences and four distinct tet genes were identified, though all tested susceptible to tetracycline via the Kirby-Bauer test. This project clarifies some aspects of the ecology of antibiotic resistance genes, their natural ecological function and the potential for the expansion of intrinsic multi-antibiotic resistance into new ecosystems and/or hosts.

Antarctic crustacean grazer assemblages exhibit resistance following exposure to decreased pH

Anthropogenic atmospheric CO2 concentrations are increasing rapidly, resulting in declining seawater pH (ocean acidification). The majority of ocean acidification research to date has focused on the effects of decreased pH in single-species experiments. To assess how decreased pH may influence natural macroalgal-grazer assemblages, we conducted a mesocosm experiment with the common, chemically defended Antarctic brown macroalga Desmarestia menziesii and natural densities of its associated grazer assemblage, predominantly amphipods. Grazer assemblages were collected from the immediate vicinity of Palmer Station (64°46'S, 64°03'W) in March 2013. Assemblages were exposed for 30 days to three levels of pH representing present-day mean summer ambient conditions (pH 8.0), predicted near-future conditions (2100, pH 7.7), and distant-future conditions (pH 7.3). A significant difference was observed in the composition of mesograzer assemblages in the lowest pH treatment (pH 7.3). The differences between assemblages exposed to pH 7.3 and those maintained in the other two treatments were driven primarily by decreases in the abundance of the amphipod Metaleptamphopus pectinatus with decreasing pH, reduced copepod abundance at pH 7.7, and elevated ostracod abundance at pH 7.7. Generally, the assemblages maintained at pH 7.7 were not significantly different from those at ambient pH, demonstrating resistance to short-term decreased pH. The relatively high prevalence of generalist amphipods may have contributed to a net stabilizing effect on the assemblages exposed to decreased pH. Overall, our results suggest that crustacean grazer assemblages associated with D. menziesii, the dominant brown macroalgal species of the western Antarctic Peninsula, may be resistant to short-term near-future decreases in seawater pH.