Einführung: Enterobacter (E.) cloacae subsp. cloacae sind wichtige Humanpathogene, insbesondere bei stationär untergebrachten Patienten. Sie sind in der Lage, Medizinprodukte zu kontaminieren, und es wurde über nosokomiale Krankheitsausbrüche in Verbindung mit der Kolonisation von chirurgischen Utensilien berichtet. Es ist daher wichtig, die Wirksamkeit und Effizienz von Desinfektionsmitteln gegenüber dieser Bakterienart zu bestimmen. Ziele: Die aktuelle Studie wurde durchgeführt, um nachzuweisen, ob Peressigsäure, Ethanol, Benzalkoniumchlorid und Natriumhypochlorit, welche weit verbreitet in kommerziellen Desinfektionsmitteln enthalten sind, eine ausreichende Wirksamkeit gegen multiresistente, von Patienten im Krankenhaus isolierte, E. cloacae aufweisen. Material und Methoden: Sechs multiresistente E. cloacae Isolate, die von Patienten in einem klinischen Umfeld gewonnen wurden, wurden getestet und mit dem E. cloacae Typstamm verglichen. Die Studien wurden in vitro mit Peressigsäure, Ethanol, Benzalkoniumchlorid und Natriumhypochlorit nach den Richtlinien der Desinfektionsmittel-Kommission des Verbundes für Angewandte Hygiene e.V. durchgeführt. Die Tests umfassten qualitative und quantitative Suspensionstests zur Bestimmung der bakteriziden Wirkung, den sogenannten Keimträgertest und die Bestimmung der minimalen Hemmkonzentrationen. Ergebnisse: Die Studienergebnisse zeigten, dass multiresistente E. cloacae Stämme genauso empfindlich gegenüber Desinfektionsmitteln waren wie der Typstamm. Organische Belastung interagierte stark mit Natriumhypochlorit und minderte dadurch seine Wirksamkeit, während Peressigsäure und Ethanol nicht durch organische Verunreinigung beeinflusst wurden. Die Kontaktzeit hatte nur einen geringen Einfluss auf die bakterizide Wirkung. Im Gegensatz dazu spielten bei Benzalkoniumchlorid organische Verunreinigung und die Kontaktzeit eine wichtige Rolle. Insgesamt waren die minimalen Hemmkonzentrationen und die bakterizid wirksamen Konzentrationen niedriger als die für kommerzielle Produkte gebräuchlichen Konzentrationen. In den Keimträgertests hatte das Trocknen auf einer glatten Oberfläche einen Einfluss auf das Überleben eines Stammes von E. cloacae. Die Ergebnisse zeigten auch, dass sich die Wirksamkeit der Desinfektionsmittel in den verschiedenen verwendeten Tests deutlich unterscheiden kann. Die Ergebnisse waren schwer mit anderen Studien zu vergleichen, da eine internationale Durchführungsrichtlinie für die Prüfung der Wirksamkeit von Desinfektionsmitteln gegen multiresistente Bakterien fehlt. Fazit: Peressigsäure, Ethanol, Benzalkoniumchlorid und Natriumhypochlorit eignen sich zur Desinfektion von multiresistenten E. cloacae. Die Wirksamkeit von Natriumhypochlorit und Benzalkoniumchlorid wird jedoch stark durch organische Stoffe beeinflusst. Dies unterstreicht die Bedeutung geeigneter Reinigungsmaßnahmen vor der Desinfektion. Wenn dies erfolgt ist, erweisen sich die getesteten Desinfektionsmittel gegen multiresistente E. cloacae genauso effektiv wie gegen den Typstamm.:1. Introduction ............................................................ 1 2. Literature Review ....................................................... 3 2.1 Enterobacteriaceae ..................................................... 4 2.1.1 General properties ................................................... 4 2.1.2 Enterobacter cloacae complex ......................................... 4 2.2 Multidrug-resistant bacteria and disinfectant “resistance” ............. 6 2.3 Disinfectant testing ................................................... 7 2.4 Active substances investigated in this study ........................... 8 2.4.1 Peracetic acid (PAA) ................................................. 8 2.4.2 Ethanol (ETH) ........................................................ 9 2.4.3 Benzalkonium chloride (BKC) .......................................... 9 2.4.4 Sodium hypochlorite (NaOCl) .......................................... 10 3. Materials and Methods ................................................... 11 3.1 Materials .............................................................. 11 3.2 Methods ................................................................ 14 3.2.1 Culture and storage of bacteria ...................................... 14 3.2.2 Preparation of disinfectants and the neutralizing agent .............. 14 3.2.3 Minimum inhibitory concentration (MIC) ............................... 15 3.2.4 Qualitative suspension test .......................................... 15 3.2.5 Quantitative suspension test.......................................... 16 3.2.6 Surface disinfection without mechanical action (germ carrier test) ... 16 3.2.7 Statistical analysis ................................................. 17 4. Results ................................................................. 18 4.1 Minimum inhibitory concentrations ...................................... 18 4.2 Qualitative suspension tests ........................................... 18 4.3 Quantitative suspension tests .......................................... 21 4.4 Surface disinfection without mechanical action (germ carrier test) ..... 24 5. Discussion .............................................................. 28 6. Summary ................................................................. 31 7. Zusammenfassung ......................................................... 33 8. References .............................................................. 35 9. Appendix ................................................................ 49 Acknowledgments ............................................................ 50 / Introduction: Enterobacter (E.) cloacae subsp. cloacae are important human pathogens, particularly in hospitalized patients. They tend to contaminate various medical devices and nosocomial outbreaks have been reported to be associated with the colonization of surgical equipment. Therefore, it is critical to determine the efficacy and effectiveness of disinfectants against this bacterial species. Objectives: The current study was undertaken to prove whether single active ingredients (i.e. peracetic acid, ethanol, benzalkonium chloride, and sodium hypochlorite) of widely used commercial disinfectants provide proper efficacy against multidrug-resistant human isolates of E. cloacae. Material and Methods: Six multidrug-resistant E. cloacae isolates obtained from patients in a clinical setting were tested and compared to the E. cloacae type strain. The studies were performed in vitro using peracetic acid, ethanol, benzalkonium chloride and sodium hypochlorite following the guidelines specified by the Disinfectants Commission within the Association of Applied Hygiene. Tests included determination of minimum inhibitory concentrations, bactericidal values by qualitative and quantitative suspension tests, and so-called germ carrier tests. The influence of exposure time and organic load on bacteriostatic and bactericidal concentrations was evaluated for each disinfectant using the two-tailed Mann-Whitney U-test. Results: Study results showed that multidrug-resistant E. cloacae strains were equally susceptible to disinfectants as the type strain. Organic matter highly interfered with sodium hypochlorite thereby decreasing its efficacy whereas peracetic acid and ethanol were not influenced by organic soiling. Contact time had only a minor effect on bactericidal values. This was in contrast to benzalkonium chloride where organic soiling and contact time played an important role. On the whole, minimum inhibitory concentrations and bactericidal concentrations were lower than in-use concentrations of commercial products. Drying on smooth surfaces in the carrier tests had an effect on the survival of one E. cloacae strain. Results also showed that efficacious values determined by the different tests used may differ distinctly. Results were difficult to compare with other studies because an international practical standard for testing disinfectant efficacy against multidrug-resistant bacteria is missing. Conclusion: Peracetic acid, ethanol, benzalkonium chloride and sodium hypochlorite are suitable to disinfect multidrug-resistant E. cloacae but the effectiveness of sodium hypochlorite and benzalkonium chloride is strongly influenced by organic matter. This underlines the importance of proper cleaning measures before disinfection. When this is done, the tested disinfectants proved to be as efficient against multidrug-resistant E. cloacae as against the type strain.:1. Introduction ............................................................ 1 2. Literature Review ....................................................... 3 2.1 Enterobacteriaceae ..................................................... 4 2.1.1 General properties ................................................... 4 2.1.2 Enterobacter cloacae complex ......................................... 4 2.2 Multidrug-resistant bacteria and disinfectant “resistance” ............. 6 2.3 Disinfectant testing ................................................... 7 2.4 Active substances investigated in this study ........................... 8 2.4.1 Peracetic acid (PAA) ................................................. 8 2.4.2 Ethanol (ETH) ........................................................ 9 2.4.3 Benzalkonium chloride (BKC) .......................................... 9 2.4.4 Sodium hypochlorite (NaOCl) .......................................... 10 3. Materials and Methods ................................................... 11 3.1 Materials .............................................................. 11 3.2 Methods ................................................................ 14 3.2.1 Culture and storage of bacteria ...................................... 14 3.2.2 Preparation of disinfectants and the neutralizing agent .............. 14 3.2.3 Minimum inhibitory concentration (MIC) ............................... 15 3.2.4 Qualitative suspension test .......................................... 15 3.2.5 Quantitative suspension test.......................................... 16 3.2.6 Surface disinfection without mechanical action (germ carrier test) ... 16 3.2.7 Statistical analysis ................................................. 17 4. Results ................................................................. 18 4.1 Minimum inhibitory concentrations ...................................... 18 4.2 Qualitative suspension tests ........................................... 18 4.3 Quantitative suspension tests .......................................... 21 4.4 Surface disinfection without mechanical action (germ carrier test) ..... 24 5. Discussion .............................................................. 28 6. Summary ................................................................. 31 7. Zusammenfassung ......................................................... 33 8. References .............................................................. 35 9. Appendix ................................................................ 49 Acknowledgments ............................................................ 50

Saskatchewan is the only province in Canada to have routinely tested the antimicrobial susceptibility of all provincially reported human cases of campylobacteriosis. From 1999 to 2006, 1378 human Campylobacter species infections were tested for susceptibility at the Saskatchewan Disease Control Laboratory using the Canadian Integrated Program for Antimicrobial Resistance Surveillance panel and minimum inhibitory concentration (MIC) breakpoints. Of these, 1200 were C. jejuni, 129 were C. coli, with the remaining made up of C. lari, C. laridis, C. upsaliensis and undifferentiated Campylobacter species. Campylobacter coli had significantly higher prevalences of ciprofloxacin resistance (CIPr), erythromycin resistance (ERYr), combined CIPr-ERYr resistance and multidrug resistance (to three or greater drug classes) than C. jejuni. Logistic regression models indicated that CIPr in C. jejuni decreased from 1999 to 2004 and subsequently increased in 2005 and 2006. The risk of CIPr was significantly increased in the winter months (January to March) compared to other seasons. A comparison of logistic regression and Cox proportional hazard survival models found that the latter were better able to detect significant temporal trends in CIPr and tetracycline resistance by directly modeling MICs, but that these trends were more difficult to interpret. Scan statistics detected significant spatial clusters of CIPr C. jejuni infections in urban centers (Saskatoon and Regina) and temporal clusters in the winter months; the space-time permutation model did not detect any space-time clusters. Bernoulli scan tests were computationally the fastest for cluster detection, compared to ordinal MIC and multinomial antibiogram models. eBURST analysis of antibiogram patterns showed a marked distinction between case and non-case isolates from the scan statistic clusters. Multilevel logistic regression models detected significant individual and regional contextual risk factors for infection with CIPr C. jejuni. Patients infected in the winter, that were between the ages of 40-45 years of age, that lived in urban regions and that lived in regions of moderately high poultry density had higher risks of a resistant infection. These results advance the epidemiologic knowledge of CIPr C. jejuni in Saskatchewan and provide novel analytical methods for antimicrobial resistance surveillance data in Canada. / Saskatchewan Disease Control Laboratory (Saskatchewan Ministry of Health); Laboratory for Foodborne Zoonoses (Public Health Agency of Canada); Centre for Foodborne, Environmental and Zoonotic Infectious Diseases (Public Health Agency of Canada); Ontario Veterinary College Blake Graham Fellowship

Background Broth microdilution (BMD) is a gold-standard reference method to determine minimum inhibitory concentration (MIC) of antibiotics. For this, a standardized concentration of bacterial inoculum (2e5–8e5 colony-forming units, CFU/ml) is added to progressively higher concentrations of antibiotics. Bacteria stop growing at a particular antibiotic concentration termed MIC. Like other assays, various biological and/or technical factors can affect BMD results.   Aims To investigate the effects of inoculum concentration (5e4–5e6 CFU/ml), growth-medium concentration (cation-adjusted Mueller-Hinton Broth (CAMHB)), ranging 0.5x to 2x (1x as standard)) and age (<6-months or >1-year old) of fastidious medium on MIC results. And to compare BMD results using 5 different brands of CAMHBs and 1 cation-non-adjusted MH-broth (non-CAMHB).   Methods 12 isolates of bacteria (gram-positive (n=3), gram-negative(n=5), fastidious isolates (n=7)) and custom-made antibiotics-containing plates for gram-positive (11 antibiotics) or gram-negative bacteria (10 antibiotics) were used. Overnight-grown colonies were used to prepare BMD solutions (MH-broth + inoculum +/- fastidious) which were plated on antibiotic-plates as well as diluted prior to plating on agar-plates. Antibiotic- and agar-plates were incubated (18–20hr, 35°C) and used to determine MICs (following European Committee on Antimicrobial Susceptibility Testing instructions) and actual number of viable bacteria in BMD solutions, respectively.   Results Increasing inoculum concentration increased MICs of all antibiotics except cefoxitin. Piperacillin–tazobactam, levofloxacin, benzylpenicillin and ampicillin were especially sensitive to increase in inoculum and showed a 4-fold increase in >50% isolates. MICs for tobramycin, tigecycline and gentamicin increased by 2-fold in >50% isolates every time MH-broth concentration increased. Age of fastidious medium had no decipherable pattern of effects on MIC. All MH-broths gave similar results except when testing daptomycin which gave higher MICs with non-CAMHB compared to CAMHB.    Conclusion This research reveals some technical factors affecting MIC results. These results could help define parameters for automated BMD-performing-systems. However, this research shows only trends as more replicates are needed to determine statistically significant results.