Chronic infections are often attributed to bacterial biofilms. These biofilms are extremely tolerant to antimicrobial treatment due to the presence of dormant persister cells. Whilst a number of persister genes and pathways have been identified, it is likely that others remain. Investigating persistence of S. Typhimurium was therefore undertaken. A csp null mutant of Salmonella enterica sv. Typhimurium, lacking all six cold shock protein (CspA) paralogues was previously constructed (Hutchinson 2005). At 10°C, this strain is unable to divide, but remains viable for several weeks. However it remains capable of growth at 37°C and thus is conditionally dormant. Using this strain, the link between dormancy and persistence was investigated. Treatment of stationary phase planktonic cultures with fluoroquinolones revealed persister cells in SL1344. In contrast the csp null mutant was completely eliminated by treatment at 37°C; this could be prevented by cspC or cspE expression, implicating a role for cspA paralogues in persistence. Cold shock (10°C) substantially increased persister levels, although csp null cultures remained hypersensitive. Chloramphenicol pre-treatment also reduced elimination of the csp null mutant, linking translation with the persister phenotype. Mutations in 5 genes affecting chromosomal structure and function were investigated, 3 of which (hns, hfq, rpoS) were found to reduce persister levels, suggesting a possible role for DNA supercoiling in persistence. Plasmid topologies in the csp null mutant were highly supercoiled compared to SL1344, a phenotype prevented by cspC or cspE expression. Altered supercoiling is therefore proposed as a mechanism for fluoroquinolone sensitivity in the csp null mutant. Persister levels were also characterised in biofilms of SL1344 and the csp null mutant. In contrast to stationary phase planktonic cultures, the CspA paralogues did not appear to play a role in biofilm persistence under the experimental conditions tested. However, the study revealed a novel role for CspA paralogues in pellicle formation at the air-liquid interface. A plasmid library was used to identify chromosomal regions capable of rescuing the planktonic persister phenotype of the csp null mutant. One region which delayed fluoroquinolone elimination of the csp null mutant, contained components of the hpa gene cluster, replicated in 11 isolates. This locus is involved in hydroxyphenylacetate (HPA) catabolism, indicating a possible role of cellular metabolism in the persistence. Overall this study has revealed novel information about antibiotic persistence in S. Typhimurium and the involvement of the CspA paralogues. These results provide an important foundation for further investigations and contribute towards knowledge of the complex processes of dormancy, persistence and biofilm formation in bacteria.

Rhodococcus, a species of bacteria commonly found in the soil, is an under-explored producer of small bioactive compounds including siderophores, pigments and antibiotics. MTM3W5.2 is a strain of Rhodococcus that was previously discovered to produce an antibiotic-like compound that has inhibitory effects on other Rhodococcus strains, including the veterinary pathogen, R. equi. The biosynthetic gene cluster responsible for production of the antibiotic has been identified, and a small gene, BTZ20_3964 at the start of the operon is believed to be a luxR solo regulator of the gene cluster. The goal of this project was to determine this gene’s status as a regulator for the gene cluster. Merodiploids were constructed using the deletion construct, pEX18Km3964AD to obtain a double crossover recombination event to replace the functional gene with the deletion construct. However, evidence indicates that an illegitimate recombination event occurred to produce a merodiploid strain.

Étude de la Sous-Classe B3 et des Inhibiteurs des Métallo β-Lactamases En raison de l'introduction dagents antibactériens, les bactéries ont développé divers moyens de résistance. Le plus commun, déjà fortement développé, est la production denzymes qui hydrolysent la forme la plus largement répandue d'agent antibactérien, les antibiotiques à noyau β-lactame (Frère 1995). Ces enzymes, appelées β-lactamases, ont deux origines. Les β-lactamases à sérine correspondant aux classe A, C et D auraient évolué à partir dune DD-transpeptidase ancestrale, alors que les métallo β-lactamases (MBLs), nont pour linstant aucun ancêtre connu (Ambler 1980) (Matagne et al. 1999). Les MBLs sont importantes médicalement, puisqu'elles peuvent hydrolyser la plupart des β-lactames, y compris les carbapénèmes, qui échappent à l'activité des enzymes à sérine les plus actives (Rasmussen et al. 1997). Un transfert des MBLs entre espèce est également envisageable, du fait que certains gènes codant pour ces enzymes sont présents sur des plasmides (Osano et al. 1994) (Laraki et al. 1999). Les inhibiteurs classiques des β-lactamases à sérine active ont peu ou pas d'effet sur les MBLs et dans certains cas ils peuvent même être hydrolysés par les MBLs. Les MBLs sont produites, comme les β-lactamases à sérine, dans le périplasme des bactéries Gram-négatives ou sont sécrétées par les bactéries Gram-positives, cependant elles ont un mode d'action différent. À la différence des β-lactamases à sérine qui emploient une sérine dans leur site actif pour hydrolyser le noyau β-lactame, les MBLs utilisent lion zinc (Bush et al. 1995). Puisque ces enzymes ne présentent pas le même besoin en ions zinc pour faire preuve dune activité maximale, un débat continuel est mené afin de savoir si les MBLs utilisent un ou deux ions zinc dans leur site actif in vivo. Les MBLs forment un groupe hétérogène qui a été divisé en sous-groupes B1, B2 et B3 par similitude de séquence et spécificité de substrat (Galleni et al. 2001) (Garau et al. 2004). C'est l'hétérogénéité de cette classe qui a rendu la recherche d'un inhibiteur générique difficile. Divers inhibiteurs de MBLs ont été décrits; ceux-ci incluent le captopril, un inhibiteur compétitif des MBLs qui sest avéré efficace contre les enzymes mono-zinc BcII et CphA (Heinz et al. 2003). L'acide thiomandelique s'est avéré un inhibiteur à large spectre pour le composé racémique, avec des valeurs de Ki en-dessous de 1 µM, pour toutes les enzymes testées des sous-classes B1 et B3; bien qu'il ait été inefficace sur CphA du sous-groupe B2 (Mollard et al. 2001). Les hydrazones de sulfonyle inhibent IMP-1, le plus bas Ki étant de 0.7 µM, mais ont un effet limité sur d'autres enzymes B1 telles que BcII (Siemann et al. 2002). Les acides succiniques substitués inhibent également IMP-1 montrant des valeurs dIC50 impressionnantes mais aucune valeur de Ki nest donnée (Toney et al. 2001). L'incubation de CphA avec les β-lactames, moxalactame et céfoxitine, cause l'inactivation de l'enzyme par les produits de la réaction, mais ces inactivateurs sont des substrats pour les enzymes des sous-classes B1 et B3 (Zervosen et al. 2001). La recherche dinhibiteurs s'est tout naturellement concentrée sur les variants IMP et VIM, qui sont des MBLs portées par un plasmide; pour linstant, aucun inhibiteur identifié nest efficace sur tous les sous-groupes (Jin et al. 2004) (Kurosaki et al. 2005) (Siemann et al. 2002). La première partie de cette étude s'est concentrée sur lidentification de molécules modèles potentiellement inhibitrices de MBLs pouvant être développées en inhibiteurs à large spectre des MBLs. Par le criblage nous avons identifié trois modèles différents susceptibles de donner des inhibiteurs efficaces; lun deux est capable de chélater l'espèce mono-zinc, lacide 2,4 pyridine dicarboxylique; un autre est spécifique de FEZ-1, le N,3-Dihydroxy-5-(4-hydroxybenzoyl) benzamide et le dernier est efficace contre toutes les MBLs testées, lacide [(3-Mercaptopropanoyl)amino](phenyl) propanoic. Les résultats obtenus montrent des constantes dinhibition allant du micromolaire au nanaomolaire. La sous-classe B3 contient la MBL L1, dont le spectre daction est le plus large. Cette enzyme peut hydrolyser un éventail de substrats tel que les pénicillines, les céphalosporines et les carbapénèmes (Crowder et al. 1998). Elle partage le repliement αβ/βα et le site di-zinc des autres MBLs mais cest un tétramère, une caractéristique unique parmi les β-lactamases (Saino et al. 1982) (Bicknell et al. 1985). La forme tétramerique de L1 la rend plus difficile à étudier par des techniques telles que la spectroscopie de résonance magnétique nucléaire ou la spectrométrie de masse. Par conséquent, pour la deuxième partie de cette étude, nous avons décidé d'étudier L1, visant à trouver les conditions dans lesquelles l'enzyme était présente comme monomère, sans besoin de mutation. Les résultats que nous avons obtenus étaient imprévus et nous ont menés à examiner une méthode de production d'apo-enzyme pour les MBLs. L'enzyme a pu facilement être dénaturée et le zinc être enlevé. L'activité a été trouvée après renaturation suite à l'addition de zinc. Cette étude pourrait être poursuivie à l'avenir, les résultats préliminaires obtenus ici sont peu concluants, mais présentent un intérêt cinétique ainsi quun bénéfice à l'étude des MBLs commencée dans ce travail. Le nombre de MBLs connues augmente constamment et la caractérisation de chaque enzyme doit être accomplie pour gagner une pleine compréhension des β-lactamases, afin quil reste un espoir d'empêcher la diffusion supplémentaire de la résistance bactérienne. Pour cette raison le but de la troisième partie de cette étude était de caractériser plus profondément la MBL, GOB-1 de Chryseobacterium meningosepticum. L'analyse de sa séquence en acides aminés, par Bellais et al. 2000 place GOB-1 dans la sous-classe B3 en dépit de son unique résidu liant le zinc Gln116. Nous avons produit GOB-1 en utilisant un vecteur d'expression basé sur le système dexpression du phage T7 et avons purifié l'enzyme. Des mutants de GOB-1 ont été créés par mutagénèse dirigée du résidu liant le zinc Gln116. Une étude cinétique détaillée a alors été réalisée en présence et absence de zinc additionnel montrant que GOB-1 est une enzyme très efficace, capable dhydrolyser efficacement tous les β-lactames testés. Les mutants du résidu Gln116 de GOB-1, produits par mutagénèse dirigée ont montré une perte d'activité qui ne peut pas être corrigée par addition de zinc, démontrant ainsi que GOB-1 n'est pas une enzyme hybride des sous-classes B2 et B3, comme cela avait été précédemment suggéré (Garau et al. 2004), mais plutôt une enzyme nouvelle et améliorée de la sous-classe B3, utilisant les dispositifs structuraux précédemment utilisés par les enzymes de la sous-classe B2 mais en améliorant l'effet.

Streptococcus pneumoniae is a major pathogen that causes Otitis Media infections and bacterial meningitis in children as well as community acquired pneumonia in adults. Clinical isolates of S. pneumoniae exhibiting resistance to Beta-lactam antibiotics are being isolated with increased frequency in many countries. Streptococcus pneumoniae strains resistant to Beta-lactam drugs have modified forms of penicillin-binding proteins that exhibit reduced affinity for binding to chemotherapeutic Beta-lactams. Penicillin binding proteins are membrane-bound enzymes that catalyze the terminal step in cell wall synthesis, and are targets for Beta-lactam drugs. Seventeen clinical isolates and six vaccine strains of Streptococcus pneumoniae were characterized using conventional phenotypic methods, susceptibility to antimicrobial agents, capsular serotyping, and by different biochemical and genotyping methods. One strain, Sp D2, was resistant to penicillin and other Beta-lactams used in the study, to erythromycin, and to Trimethoprim/Sulfamethoxazole. Sp D2 exhibited a unique protein profile in 1D SDS-PAGE gels of whole-cell proteins. Cells of Sp D2 were fractionated, and the cytoplasmic membrane fraction was obtained by ultracentrifugation and analyzed using a 1D SDS-PAGE gel. A protein band with a mass of ~50 kDa was excised and subjected to Trypsin In-Gel Digestion, followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. The resulting MALDI-TOF-MS data (peptide mass fingerprints) did not produce any significant matches with proteins in any of the published S. pneumoniae genome databases. The 50 kDa protein was further subjected to N-terminal and internal sequence analysis and database searching, and the protein could not be identified by significant matches. Sp D2 did not react with any anti-pneumococcal polysaccharide capsular antibodies, and is designated as a non-typeable strain. Sp D2 exhibited a positive reaction in the Bile Solubility Test, the Optochin Test, and also positive reactions in PCR assays for the presence of the pneumococcal surface protein gene (PsaA), the autolysin gene (LytA), and the pneumolysin gene (Ply); which confirms that Sp D2 is a strain of S. pneumoniae.

Salmonella continues to threaten public health as well as negatively impact dairy producers on multiple levels. Efficacious solutions to control Salmonella among dairy cattle have long been sought to alleviate these problems. A novel vaccine technology has been developed based on purified siderophore receptors and porin proteins (SRP®) derived from Salmonella Newport. When vaccinated with these SRP® cattle are stimulated to produce antibodies which act in concert with host defenses to disrupt iron acquisition of pathogenic bacteria. To evaluate the effectiveness of this technology, a prospective cohort study was designed utilizing herds (n = 11) that practiced whole herd vaccination with the SRP® vaccine (vaccinated cohort) and herds (n = 11) that had not used the SRP® vaccine. Samples were collected during four rounds at approximately six week intervals from June through October 2009. Samples were transported to the laboratory at West Texas A&M University and cultured for the prevalence of Salmonella using selective enrichment methods. Salmonella isolates were evaluated for antimicrobial susceptibility and serotype. Data was analyzed using commercially available software to evaluate the herd-level effects of vaccination. Salmonella was ubiquitous throughout the Texas Panhandle and Eastern New Mexico, within-herd animal level estimates of prevalence ranged from 0.0 – 92%, over the length of the study period. Overall all rounds vaccinated herds had decreased (P = 0.012) Salmonella prevalence (15.3 vs. 27.5%). Vaccinated herds had numerically fewer Salmonella isolates belonging to the Newport serotype. Salmonella Typhimurium isolates were recovered approximately equally from vaccinated and non-vaccinated herds. Isolates from vaccinated herds were resistant to fewer antimicrobials throughout the study period. The ACSSuT(resistant to ampicillin, chloramphenicol, streptomycin, sulphisoxazole, and tetracycline) and MDR-AmpC (ACSSuT resistance plus resistance to ceftiofur and amoxicillin/clavulanate) resistant phenotypes were more frequently observed among non-vaccinated herds and none of the isolates from vaccinated or non-vaccinated herds were resistant to nalidixic acid, gentamicin, ciprofloxacin, or amikacin. These findings indicate vaccine efficacy for the reduction of Salmonella prevalence. Dairy operators along with herd veterinarians are encouraged to utilize this data with other herd specific factors in determining whether to use this specific vaccine.

No description available.

With water scarcity as a pressing issue in Saudi Arabia and other Middle Eastern countries, the treatment and reuse of municipal wastewater is increasingly being used as an alternative water source to supplement country water needs. Standards are in place to ensure a safe treated wastewater quality, however they do not regulate pathogenic bacteria and emerging contaminants. Information is lacking on the levels of risk to public health associated with these factors, the efficiency of conventional treatment strategies in removing them, and on wastewater treatment in Saudi Arabia in general. In this study, a municipal wastewater treatment plant in Saudi Arabia is investigated to assess the efficiency of conventional treatment in meeting regulations and removing pathogens and emerging contaminants. The study found pathogenic bacterial genera, antibiotic resistance genes and antibiotic resistant bacteria, many of which were multi-resistant in plant discharges. It was found that although the treatments are able to meet traditional quality guidelines, there remains a risk from the discussed contaminants with wastewater reuse. A deeper understanding of this risk, and suggestions for more thorough guidelines and monitoring are needed.

Pathogens on surfaces in child care centers can contribute to illness among attendees and may thereby contribute to medical visits as well. This intervention study was conducted to assess the effect of using specific sanitizing products and cleaning protocols in child care centers on the incidences of lower respiratory infections, diarrheal illness, antibiotic use, and medical visits among children attending the centers and on the levels and antibiotic resistance of indicator bacteria in those centers. During the ten-week study period, children from twelve centers were observed. Six of the centers were randomly assigned to the intervention. The other six were controls. Intervention centers were given cleaning protocols and sanitizing products. Control centers were asked to retain their original procedures and products.Acute illness was determined from records kept by the center directors and telephone calls made to parents of ill children. A call was also made to one randomly selected healthy child's parents for every two ill children recorded. Parents were given a questionnaire requesting information including bedroom sharing status, environmental tobacco smoke exposure, and chronic illnesses.After controlling for within-center clustering and zero-inflation, statistically non-significant trends of reduction were seen in the weeks of lower respiratory infections, diarrheal illness, and medical visits. Multivariable zero-inflated Poisson regression revealed that the number of weeks intervention center children were using antibiotics was 32% lower than among the control center children. This was a statistically significant reduction (95% CI = 0.54-0.86; p = 0.001).Bacterial samples were collected from ten sites within each center at the beginning and the end of the study period to determine the effect of the intervention on the microbial population. The study determined the heterotrophic plate count bacteria numbers and the rates of resistance to ampicillin and cephalothin. Neither heterotrophic bacterial concentrations nor antibiotic resistance rates significantly changed over the course of the study.

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium that is also a major opportunistic human pathogen in nosocomial infections and cystic fibrosis chronic lung infections. These P. aeruginosa infections can be extremely difficult to treat due to the high intrinsic antibiotic resistance and broad repertoire of virulence factors, both of which are highly regulated. It was demonstrated here that the psrA gene, encoding a transcriptional regulator, was up-regulated in response to sub-inhibitory concentrations of antimicrobial peptides. Compared to wild-type and the complemented mutant, a P. aeruginosa PAO1 psrA::Tn5 mutant displayed intrinsic super-susceptibility to polymyxin B, a last resort antimicrobial used against multi-drug resistant infections, and indolicidin, a bovine neutrophil antimicrobial peptide; this super-susceptibility phenotype correlated with increased outer membrane permeability. The psrA mutant was also defective in simple biofilm formation, rapid attachment, and normal swarming motility, phenotypes that could be complemented by the cloned psrA gene. The role of PsrA in global gene regulation was studied by comparing the psrA mutant to wild-type by microarray analysis, demonstrating that 178 genes were up or down-regulated by greater than 2-fold (P ≤0.05). Dysregulated genes included those encoding known PsrA targets, the type III secretion apparatus and effectors, adhesion and motility genes and a variety of metabolic, energy metabolism and outer membrane permeability genes. This indicates that PsrA is a central regulator of antimicrobial peptide resistance and virulence. P. aeruginosa containing a mutation in the PhoQ sensor kinase-encoding gene was highly attenuated for persistence in a rat chronic lung infection model. In addition, the polymyxin B hyper-resistant phoQ mutant displayed reduced type IV pili-dependent twitching motility and was less cytotoxic towards human bronchial epithelial cells, indicating that the virulence defect observed could be due at least in part to these phenotypes. Using microarrays it was further demonstrated that PhoQ regulates a large number of genes that are PhoP-independent and that the phoQ mutation leads to up-regulation of PhoP- and PmrA regulated genes as well as other genes consistent with its virulence phenotypes.

Multivariable linear and negative binomial models were produced to assess relationships among socioeconomic and influenza rate data with the use of macrolide and fluoroquinolone antimicrobials by Canadians. Varying results were found both among and between the macrolide and fluoroquinolone groups; however, a pattern of accessibility to care was apparent. Cheaper antimicrobials were used most often in the most disadvantaged populations, and more expensive antimicrobials were used most frequently in advantaged populations. Significant interactions were found between influenza and socioeconomic variables relating to unemployment, education, and degree of poverty in a population. Results suggest that antimicrobials are being prescribed and consumed at inappropriate rates in both disadvantaged and affluent populations in Canada. In order to reduce antimicrobial use and the further development of antimicrobial resistance in Canada, we suggest that responsible antimicrobial stewardship be practiced and promoted by all physicians in community and hospital settings, particularly during the influenza season. / Public Health Agency of Canada