Impact of the use of β-lactam antimicrobials on the emergence of Escherichia coli isolates resistant to cephalosporins under standard pig-rearing conditions

The aim of this study was to evaluate if the treatments with ceftiofur and amoxicillin are risk factors for the emergence of cephalosporin resistant (CR) E. coli in a pig farm during the rearing period. One hundred 7-day-old piglets were divided into two groups, a control (n = 50) group and a group parenterally treated with ceftiofur (n = 50). During the fattening period, both groups were subdivided in two. A second treatment with amoxicillin was administered in feed to two of the four groups, as follows: group 1 (untreated, n = 20), group 2 (treated with amoxicillin, n = 26), group 3 (treated with ceftiofur, n = 20), and group 4 (treated with ceftiofur and amoxicillin, n = 26). During treatment with ceftiofur, fecal samples were collected before treatment (day 0) and at days 2, 7, 14, 21, and 42 posttreatment, whereas with amoxicillin, the sampling was extended 73 days posttreatment. CR E. coli bacteria were selected on MacConkey agar with ceftriaxone (1 mg/liter). Pulsed-field gel electrophoresis (PFGE), MICs of 14 antimicrobials, the presence of cephalosporin resistance genes, and replicon typing of plasmids were analyzed. Both treatments generated an increase in the prevalence of CR E. coli, which was statistically significant in the treated groups. Resistance diminished after treatment. A total of 47 CR E. coli isolates were recovered during the study period; of these, 15 contained blaCTX-M-1, 10 contained blaCTX-M-14, 4 contained blaCTX-M-9, 2 contained blaCTX-M-15, and 5 contained blaSHV-12. The treatment with ceftiofur and amoxicillin was associated with the emergence of CR E. coli during the course of the treatment. However, by the time of finishing, CR E. coli bacteria were not recovered from the animals.

Microbial colonisation of human follicular fluid and adverse in vitro fertilisation outcomes

This study, investigating 263 women undergoing trans-vaginal oocyte retrieval for in vitro fertilisation (IVF) found that microorganisms colonising follicular fluid contributed to adverse IVF (pre-implantation) and pregnancy (post-implantation) outcomes including poor quality embryos, failed pregnancy and early pregnancy loss (< 37 weeks gestation). Some microorganisms also showed in vitro growth patterns in liquid media that appeared to be enhanced by the hormonal stimulation protocol used for oocyte retrieval. Elaborated cytokines within follicular fluid were also associated with adverse IVF outcomes. This study is imperative because infertility affects 16% of the human population and the numbers of couples needing assistance continues to increase. Despite significant improvements in the technical aspects of assisted reproductive technologies (ART), the live birth rate has not increased proportionally. Overt genital tract infection has been associated with both infertility and adverse pregnancy outcomes (including miscarriage and preterm birth) as a direct result of the infection or the host response to it. Importantly, once inflammation had become established, medical treatment often failed to prevent these significant adverse outcomes. Current evaluations of fertility focus on the ovary as a site of steroid hormone production and ovulation. However, infertility as a result of subclinical colonisation of the ovary has not been reported. Furthermore, identification of the microorganisms present in follicular fluid and the local cytokine profile may provide clinicians with an early indication of the prognosis for IVF treatment in infertile couples, thus allowing antimicrobial treatment and/or counselling about possible IVF failure. During an IVF cycle, multiple oocytes undergo maturation in vivo in response to hormonal hyperstimulation. Oocytes for in vitro insemination are collected trans-vaginally. The follicular fluid that bathes the maturing oocyte in vivo, usually is discarded as part of the IVF procedure, but provides a unique opportunity to investigate microbial causes of adverse IVF outcomes. Some previous studies have identified follicular fluid markers that predict IVF pregnancy outcomes. However, there have not been any detailed microbiological studies of follicular fluid. For this current study, paired follicular fluid and vaginal secretion samples were collected from women undergoing IVF cycles to determine whether microorganisms in follicular fluid were associated with adverse IVF outcomes. Microorganisms in follicular fluid were regarded as either "colonisers" or "contaminants"; colonisers, if they were unique to the follicular fluid sample, and contaminants if the same microorganisms were detected in the vaginal and follicular fluid samples indicating that the follicular fluid was merely contaminated during the oocyte retrieval process. Quite unexpectedly, by these criteria, we found that follicular fluid from approximately 30% of all subjects was colonised with bacteria. Fertile and infertile women with colonised follicular fluid had decreased embryo transfer rates and decreased pregnancy rates compared to women with contaminated follicular fluids. The observation that follicular fluid was not always sterile, but contained a diverse range of microorganisms, is novel. Many of the microorganisms we detected in follicular fluid are known opportunistic pathogens that have been detected in upper genital tract infections and are associated with adverse pregnancy outcomes. Bacteria were able to survive for at least 28 weeks in vitro, in cultures of follicular fluid. Within 10 days of establishing these in vitro cultures, several species (Lactobacillus spp., Bifidobacterium spp., Propionibacterium spp., Streptococcus spp. and Salmonella entericus) had formed biofilms. Biofilms play a major role in microbial pathogenicity and persistence. The propensity of microbial species to form biofilms in follicular fluid suggests that successful treatment of these infections with antimicrobials may be difficult. Bifidobacterium spp. grew, in liquid media, only if concentrations of oestradiol and progesterone were similar to those achieved in vivo during an IVF cycle. In contrast, the growth of Streptococcus agalactiae and Escherichia coli was inhibited or abolished by the addition of these hormones to culture medium. These data suggest that the likelihood of microorganisms colonising follicular fluid and the species of bacteria involved is influenced by the stage of the menstrual cycle and, in the case of IVF, the nature and dose of steroid hormones administered for the maturation of multiple oocytes in vivo. Our findings indicate that the elevated levels of steroid hormones during an IVF cycle may influence the microbial growth within follicular fluid, suggesting that the treatment itself will impact on the microflora present in the female upper genital tract during pre-conception and early post-conception phases of the cycle. The effect of the host immune response on colonising bacteria and on the outcomes of IVF also was investigated. White blood cells reportedly compose between 5% and 15% of the cell population in follicular fluid. The follicular membrane is semi-permeable and cells are actively recruited as part of the normal menstrual cycle and in response to microorganisms. A previous study investigated follicular fluid cytokines from infertile women and fertile oocyte donors undergoing IVF, and concluded that there were no significant differences in the cytokine concentrations between the two groups. However, other studies have reported differences in the follicular fluid cytokine levels associated with infertile women with endometriosis or polycystic ovary syndrome. In this study, elevated levels of interleukin (IL)-1 á, IL-1 â and vascular endothelial growth factor (VEGF) in vaginal fluid were associated with successful fertilisation, which may be useful marker for successful fertilisation outcomes for women trying to conceive naturally or prior to oocyte retrieval for IVF. Elevated levels of IL-6, IL-12p40, granulocyte colony stimulating factor (GCSF) and interferon-gamma (IFN ã) in follicular fluid were associated with successful embryo transfer. Elevated levels of pro-inflammatory IL-18 and decreased levels of anti-inflammatory IL-10 were identified in follicular fluid from women with idiopathic infertility. Successful fertilisation and implantation is dependent on a controlled pro-inflammatory environment, involving active recruitment of pro-inflammatory mediators to the genital tract as part of the menstrual cycle and early pregnancy. However, ongoing pregnancy requires an enhanced anti-inflammatory environment to ensure that the maternal immune system does not reject the semi-allergenic foetus. The pro-inflammatory skew in the follicular fluid of women with idiopathic infertility, correlates with normal rates of fertilisation, embryo discard and embryo transfer, observed for this cohort, which were similar to the outcomes observed for fertile women. However, their pregnancy rate was reduced compared to fertile women. An altered local immune response in follicular fluid may provide a means of explaining infertility in this cohort, previously defined as 'idiopathic'. This study has found that microorganisms colonising follicular fluid may have contributed to adverse IVF and pregnancy outcomes. Follicular fluid bathes the cumulus oocyte complex during the in vivo maturation process, and microorganisms in the fluid, their metabolic products or the local immune response to these microorganisms may result in damage to the oocytes, degradation of the cumulus or contamination of the IVF culture system. Previous studies that have discounted bacterial contamination of follicular fluid as a cause of adverse IVF outcomes failed to distinguish between bacteria that were introduced into the follicular fluid at the time of trans-vaginal oocyte retrieval and those that colonised the follicular fluid. Those bacteria that had colonised the fluid may have had time to form biofilms and to elicit a local immune response. Failure to draw this distinction has previously prevented consideration of bacterial colonisation of follicular fluid as a cause of adverse IVF outcomes. Several observations arising from this study are of significance to IVF programs. Follicular fluid is not always sterile and colonisation of follicular fluid is a cause of adverse IVF and pregnancy outcomes. Hormonal stimulation associated with IVF may influence whether follicular fluid is colonised and enhance the growth of specific species of bacteria within follicular fluid. Bacteria in follicular fluid may form biofilms and literature has reported that this may influence their susceptibility to antibiotics. Monitoring the levels of selected cytokines within vaginal secretions may inform fertilisation outcomes. This study has identified novel factors contributing to adverse IVF outcomes and that are most likely to affect also natural conception outcomes. Early intervention, possibly using antimicrobial or immunological therapies may reduce the need for ART and improve reproductive health outcomes for all women.

Ureaplasma parvum : understanding the complexities of intra-amniotic infection in an ovine model

The human Ureaplasma species are the most frequently isolated bacteria from the upper genital tract of pregnant women and can cause clinically asymptomatic, intra-uterine infections, which are difficult to treat with antimicrobials. Ureaplasma infection of the upper genital tract during pregnancy has been associated with numerous adverse outcomes including preterm birth, chorioamnionitis and neonatal respiratory diseases. The mechanisms by which ureaplasmas are able to chronically colonise the amniotic fluid and avoid eradication by (i) the host immune response and (ii) maternally-administered antimicrobials, remain virtually unexplored. To address this gap within the literature, this study investigated potential mechanisms by which ureaplasmas are able to cause chronic, intra-amniotic infections in an established ovine model. In this PhD program of research the effectiveness of standard, maternal erythromycin for the treatment of chronic, intra-amniotic ureaplasma infections was evaluated. At 55 days of gestation pregnant ewes received an intra-amniotic injection of either: a clinical Ureaplasma parvum serovar 3 isolate that was sensitive to macrolide antibiotics (n = 16); or 10B medium (n = 16). At 100 days of gestation, ewes were then randomised to receive either maternal erythromycin treatment (30 mg/kg/day for four days) or no treatment. Ureaplasmas were isolated from amniotic fluid, chorioamnion, umbilical cord and fetal lung specimens, which were collected at the time of preterm delivery of the fetus (125 days of gestation). Surprisingly, the numbers of ureaplasmas colonising the amniotic fluid and fetal tissues were not different between experimentally-infected animals that received erythromycin treatment or infected animals that did not receive treatment (p > 0.05), nor were there any differences in fetal inflammation and histological chorioamnionitis between these groups (p > 0.05). These data demonstrate the inability of maternal erythromycin to eradicate intra-uterine ureaplasma infections. Erythromycin was detected in the amniotic fluid of animals that received antimicrobial treatment (but not in those that did not receive treatment) by liquid chromatography-mass spectrometry; however, the concentrations were below therapeutic levels (<10 – 76 ng/mL). These findings indicate that the ineffectiveness of standard, maternal erythromycin treatment of intra-amniotic ureaplasma infections may be due to the poor placental transfer of this drug. Subsequently, the phenotypic and genotypic characteristics of ureaplasmas isolated from the amniotic fluid and chorioamnion of pregnant sheep after chronic, intra-amniotic infection and low-level exposure to erythromycin were investigated. At 55 days of gestation twelve pregnant ewes received an intra-amniotic injection of a clinical U. parvum serovar 3 isolate, which was sensitive to macrolide antibiotics. At 100 days of gestation, ewes received standard maternal erythromycin treatment (30 mg/kg/day for four days, n = 6) or saline (n = 6). Preterm fetuses were surgically delivered at 125 days of gestation and ureaplasmas were cultured from the amniotic fluid and the chorioamnion. The minimum inhibitory concentrations (MICs) of erythromycin, azithromycin and roxithromycin were determined for cultured ureaplasma isolates, and antimicrobial susceptibilities were different between ureaplasmas isolated from the amniotic fluid (MIC range = 0.08 – 1.0 mg/L) and chorioamnion (MIC range = 0.06 – 5.33 mg/L). However, the increased resistance to macrolide antibiotics observed in chorioamnion ureaplasma isolates occurred independently of exposure to erythromycin in vivo. Remarkably, domain V of the 23S ribosomal RNA gene (which is the target site of macrolide antimicrobials) of chorioamnion ureaplasmas demonstrated significant variability (125 polymorphisms out of 422 sequenced nucleotides, 29.6%) when compared to the amniotic fluid ureaplasma isolates and the inoculum strain. This sequence variability did not occur as a consequence of exposure to erythromycin, as the nucleotide substitutions were identical between chorioamnion ureaplasmas isolated from different animals, including those that did not receive erythromycin treatment. We propose that these mosaic-like 23S ribosomal RNA gene sequences may represent gene fragments transferred via horizontal gene transfer. The significant differences observed in (i) susceptibility to macrolide antimicrobials and (ii) 23S ribosomal RNA sequences of ureaplasmas isolated from the amniotic fluid and chorioamnion suggests that the anatomical site from which they were isolated may exert selective pressures that alter the socio-microbiological structure of the bacterial population, by selecting for genetic changes and altered antimicrobial susceptibility profiles. The final experiment for this PhD examined antigenic size variation of the multiple banded antigen (MBA, a surface-exposed lipoprotein and predicted ureaplasmal virulence factor) in chronic, intra-amniotic ureaplasma infections. Previously defined ‘virulent-derived’ and ‘avirulent-derived’ clonal U. parvum serovar 6 isolates (each expressing a single MBA protein) were injected into the amniotic fluid of pregnant ewes (n = 20) at 55 days of gestation, and amniotic fluid was collected by amniocentesis every two weeks until the time of near-term delivery of the fetus (at 140 days of gestation). Both the avirulent and virulent clonal ureaplasma strains generated MBA size variants (ranging in size from 32 – 170 kDa) within the amniotic fluid of pregnant ewes. The mean number of MBA size variants produced within the amniotic fluid was not different between the virulent (mean = 4.2 MBA variants) and avirulent (mean = 4.6 MBA variants) ureaplasma strains (p = 0.87). Intra-amniotic infection with the virulent strain was significantly associated with the presence of meconium-stained amniotic fluid (p = 0.01), which is an indicator of fetal distress in utero. However, the severity of histological chorioamnionitis was not different between the avirulent and virulent groups. We demonstrated that ureaplasmas were able to persist within the amniotic fluid of pregnant sheep for 85 days, despite the host mounting an innate and adaptive immune response. Pro-inflammatory cytokines (interleukin (IL)-1â, IL-6 and IL-8) were elevated within the chorioamnion tissue of pregnant sheep from both the avirulent and virulent treatment groups, and this was significantly associated with the production of anti-ureaplasma IgG antibodies within maternal sera (p < 0.05). These findings suggested that the inability of the host immune response to eradicate ureaplasmas from the amniotic cavity may be due to continual size variation of MBA surface-exposed epitopes. Taken together, these data confirm that ureaplasmas are able to cause long-term in utero infections in a sheep model, despite standard antimicrobial treatment and the development of a host immune response. The overall findings of this PhD project suggest that ureaplasmas are able to cause chronic, intra-amniotic infections due to (i) the limited placental transfer of erythromycin, which prevents the accumulation of therapeutic concentrations within the amniotic fluid; (ii) the ability of ureaplasmas to undergo rapid selection and genetic variation in vivo, resulting in ureaplasma isolates with variable MICs to macrolide antimicrobials colonising the amniotic fluid and chorioamnion; and (iii) antigenic size variation of the MBA, which may prevent eradication of ureaplasmas by the host immune response and account for differences in neonatal outcomes. The outcomes of this program of study have improved our understanding of the biology and pathogenesis of this highly adapted microorganism.

A review of the pharmacobiotic regulation of gastrointestinal inflammation by probiotics, commensal bacteria and prebiotics.

The idea that microbes induce disease has steered medical research toward the discovery of antibacterial products for the prevention and treatment of microbial infections. The twentieth century saw increasing dependency on antimicrobials as mainline therapy accentuating the notion that bacterial interactions with humans were to be avoided or desirably controlled. The last two decades, though, have seen a refocusing of thinking and research effort directed towards elucidating the critical inter-relationships between the gut microbiome and its host that control health/wellness or disease. This research has redefined the interactions between gut microbes and vertebrates, now recognizing that the microbial active cohort and its mammalian host have shared co-evolutionary metabolic interactions that span millennia. Microbial interactions in the gastrointestinal tract provide the necessary cues for the development of regulated pro- and anti-inflammatory signals that promotes immunological tolerance, metabolic regulation and other factors which may then control local and extra-intestinal inflammation. Pharmacobiotics, using nutritional and functional food additives to regulate the gut microbiome, will be an exciting growth area of therapeutics, developing alongside an increased scientific understanding of gut-microbiome symbiosis in health and disease.

New approaches to making the microenvironment of the female reproductive tract hostile to HIV

The studies presented in this review explore three distinct areas with potential for inhibiting HIV infection in women. Based on emerging information from the physiology, endocrinology and immunology of the female reproductive tract (FRT), we propose unique 'works in progress' for protecting women from HIV. Various aspects of FRT immunity are suppressed by estradiol during the menstrual cycle, making women more susceptible to HIV infection. By engineering commensal Lactobacillus to secrete the anti-HIV molecule Elafin as estradiol levels increase, women could be protected from HIV infection. Selective estrogen response modifiers enhance barrier integrity and enhance secretion of protective anti-HIV molecules. Finally, understanding the interactions and regulation of FRT endogenous antimicrobials, proteases, antiproteases, etc., all of which are under hormonal control, will open new avenues to therapeutic manipulation of the FRT mucosal microenvironment. By seeking new alternatives to preventing HIV infection in women, we may finally disrupt the HIV pandemic.

Sex hormone regulation of innate immunity in the female reproductive tract : the role of epithelial cells in balancing reproductive potential with protection against sexually transmitted pathogens

The immune system in the female reproductive tract (FRT) does not mount an attack against HIV or other sexually transmitted infections (STI) with a single endogenously produced microbicide or with a single arm of the immune system. Instead, the body deploys dozens of innate antimicrobials to the secretions of the female reproductive tract. Working together, these antimicrobials along with mucosal antibodies attack many different viral, bacterial and fungal targets. Within the FRT, the unique challenges of protection against sexually transmitted pathogens coupled with the need to sustain the development of an allogeneic fetus have evolved in such a way that sex hormones precisely regulate immune function to accomplish both tasks. The studies presented in this review demonstrate that estradiol and progesterone secreted during the menstrual cycle act both directly and indirectly on epithelial cells and other immune cells in the reproductive tract to modify immune function in a way that is unique to specific sites throughout the FRT. As presented in this review, studies from our laboratory and others demonstrate that the innate immune response is under hormonal control, varies with the stage of the menstrual cycle, and as such is suppressed at mid-cycle to optimize conditions for successful fertilization and pregnancy. In doing so, a window of STI vulnerability is created during which potential pathogens including HIV enter the reproductive tract to infect host targets.

Antimicrobial stewardship activities : a survey of Queensland hospitals

Objective: In 2011, the Australian Commission on Safety and Quality in Health Care (ACSQHC) recommended that all hospitals in Australia must have an Antimicrobial Stewardship (AMS) program by 2013. Nevertheless, little is known about current AMS activities. This study aimed to determine the AMS activities currently undertaken, and to identify gaps, barriers to implementation and opportunities for improvement in Queensland hospitals. Methods: The AMS activities of 26 facilities from 15 hospital and health services in Queensland were surveyed during June 2012 to address strategies for effective AMS: implementing clinical guidelines, formulary restriction, reviewing antimicrobial prescribing, auditing antimicrobial use and selective reporting of susceptibility results. Results: The response rate was 62%. Nineteen percent had an AMS team (a dedicated multidisciplinary team consisting of a medically trained staff member and a pharmacist). All facilities had access to an electronic version of Therapeutic Guidelines: Antibiotic, with a further 50% developing local guidelines for antimicrobials. One-third of facilities had additional restrictions. Eighty-eight percent had advice for restricted antimicrobials from in-house infectious disease physicians or clinical microbiologists. Antimicrobials were monitored with feedback given to prescribers at point of care by 76% of facilities. Deficiencies reported as barriers to establishing AMS programs included: pharmacy resources, financial support by hospital management, and training and education in antimicrobial use. Conclusions: Several areas for improvement were identified: reviewing antimicrobial prescribing with feedback to the prescriber, auditing, and training and education in antimicrobial use. There also appears to be a lack of resources to support AMS programs in some facilities. What is known about the topic? The ACSQHC has recommended that all hospitals implement an AMS program by 2013 as a requirement of Standard 3 (Preventing and Controlling Healthcare-Associated Infections) of the National Safety and Quality Health Service Standards. The intent of AMS is to ensure appropriate prescribing of antimicrobials as part of the broader systems within a health service organisation to prevent and manage healthcare-associated infections, and improve patient safety and quality of care. This criterion also aligns closely with Standard 4: Medication Safety. Despite this recommendation, little is known about what AMS activities are undertaken in these facilities and what additional resources would be required in order to meet these national standards. What does the paper add? This is the first survey that has been conducted of public hospital and health services in Queensland, a large decentralised state in Australia. This paper describes what AMS activities are currently being undertaken, identifies practice gaps, barriers to implementation and opportunities for improvement in Queensland hospitals. What are the implications for practitioners? Several areas for improvement such as reviewing antimicrobial prescribing with feedback to the prescriber, auditing, and training and education in antimicrobial use have been identified. In addition, there appears to be a lack of resources to support AMS programs in some facilities.

The ADMIN-ICU survey: A survey on antimicrobial dosing and monitoring in ICUs

Objectives: There is little evidence and few guidelines to inform the most appropriate dosing and monitoring for antimicrobials in the ICU. We aimed to survey current practices around the world. Methods: An online structured questionnaire was developed and sent by e-mail to obtain information on local antimicrobial prescribing practices for glycopeptides, piperacillin/tazobactam, carbapenems, aminoglycosides and colistin. Results: A total of 402 professionals from 328 hospitals in 53 countries responded, of whom 78% were specialists in intensive care medicine (41% intensive care, 30% anaesthesiology, 14% internal medicine) and 12% were pharmacists. Vancomycin was used as a continuous infusion in 31% of units at a median (IQR) daily dose of 25 (25–30) mg/kg. Piperacillin/tazobactam was used as an extended infusion by 22% and as a continuous infusion by 7%. An extended infusion of carbapenem (meropenem or imipenem) was used by 27% and a continuous infusion by 5%. Colistin was used at a daily dose of 7.5 (3.9–9) million IU (MIU)/day, predominantly as a short infusion. The most commonly used aminoglycosides were gentamicin (55%) followed by amikacin (40%), with administration as a single daily dose reported in 94% of the cases. Gentamicin was used at a daily dose of 5 (5–6) mg/day and amikacin at a daily dose of 15 (15–20) mg/day. Therapeutic drug monitoring of vancomycin, piperacillin/tazobactam and meropenem was used by 74%, 1% and 2% of the respondents, respectively. Peak aminoglycoside concentrations were sampled daily by 28% and trough concentrations in all patients by 61% of the respondents. Conclusions: We found wide variability in reported practices for antibiotic dosing and monitoring. Research is required to develop evidence-based guidelines to standardize practices.

Functional correlation of bacterial LuxS with their quaternary associations: interface analysis of the structure networks

Background The genome of a wide variety of prokaryotes contains the luxS gene homologue, which encodes for the protein S-ribosylhomocysteinelyase (LuxS). This protein is responsible for the production of the quorum sensing molecule, AI-2 and has been implicated in a variety of functions such as flagellar motility, metabolic regulation, toxin production and even in pathogenicity. A high structural similarity is present in the LuxS structures determined from a few species. In this study, we have modelled the structures from several other species and have investigated their dimer interfaces. We have attempted to correlate the interface features of LuxS with the phenotypic nature of the organisms. Results The protein structure networks (PSN) are constructed and graph theoretical analysis is performed on the structures obtained from X-ray crystallography and on the modelled ones. The interfaces, which are known to contain the active site, are characterized from the PSNs of these homodimeric proteins. The key features presented by the protein interfaces are investigated for the classification of the proteins in relation to their function. From our analysis, structural interface motifs are identified for each class in our dataset, which showed distinctly different pattern at the interface of LuxS for the probiotics and some extremophiles. Our analysis also reveals potential sites of mutation and geometric patterns at the interface that was not evident from conventional sequence alignment studies. Conclusion The structure network approach employed in this study for the analysis of dimeric interfaces in LuxS has brought out certain structural details at the side-chain interaction level, which were elusive from the conventional structure comparison methods. The results from this study provide a better understanding of the relation between the luxS gene and its functional role in the prokaryotes. This study also makes it possible to explore the potential direction towards the design of inhibitors of LuxS and thus towards a wide range of antimicrobials.

Antimicrobial susceptibility testing of Brachyspira intermedia and Brachyspira pilosicoli isolates from Australian chickens

Susceptibilities of predominantly Australian isolates of the pathogenic intestinal spirochaetes Brachyspira intermedia (n=25) and Brachyspira pilosicoli (n=17) from chickens were tested in agar dilution against four concentrations each of the antimicrobials tiamulin, lincomycin, tylosin, metronidazole, tetracycline and ampicillin. Based on available minimum inhibitory concentration (MIC) breakpoint values for Brachyspira hyodysenteriae or other Gram-negative enteric veterinary pathogens, isolates of both species generally were susceptible to tiamulin, lincomycin, metronidazole and tetracycline. Although not classed as resistant, four isolates of B. intermedia had an elevated MIC range for tiamulin (1 to 4 mg/l), 11 isolates of B. intermedia and five of B. pilosicoli had an elevated MIC range for lincomycin (10 to 50 mg/l), one isolate of B. pilosicoli had an elevated MIC range for tetracycline (10 to 20 mg/l), and one isolate of B. intermedia and five of B. pilosicoli had an elevated MIC range for ampicillin (10 to 50 mg/l). A clear lack of susceptibility to tylosin (MIC >4 mg/l) was seen in 11 isolates each of B. intermedia and B. pilosicoli, and to ampicillin (MIC >32 mg/l) in two isolates of B. pilosicoli. These data suggest that some resistance to common antimicrobials exists among intestinal spirochetes obtained from laying hens and supports the need of MIC data for clinical isolates before any treatment is considered.

Weakening of the Gram-negative bacterial outer membrane - A tool for increasing microbiological safety

Gram-negative bacteria are harmful in various surroundings. In the food industy their metabolites are potential cause of spoilage and this group also includes many severe or potential pathogens, such as Salmonella. Due to their ability to produce biofilms Gram-negative bacteria also cause problems in many industrial processes as well as in clinical surroundings. Control of Gram-negative bacteria is hampered by the outer membrane (OM) in the outermost layer of the cells. This layer is an intrinsic barrier for many hydrophobic agents and macromolecules. Permeabilizers are compounds that weaken OM and can thus increase the activity of antimicrobials by facililating entry of hydrophobic compounds and macromolecules into the cell where they can reach their target sites and inhibit or destroy cellular functions. The work described in this thesis shows that lactic acid acts as a permeabilizer and destabilizes the OM of Gram-negative bacteria. In addition, organic acids present in berriers, i.e. malic, sorbic and benzoic acid, were shown to weaken the OM of Gram-negative bacteria. Organic acids can poteniate the antimicrobial activity of other compounds. Microbial colonic degradation products of plant-derived phenolic compounds (3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 3,4-dihydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid, 3-phenylpropionic acid and 3-hydroxyphenylpropionic acid) efficiently destabilized OM of Salmonella. The studies increase our understanding of the mechanism of action of the classical chelator, ethylenediaminetetra-acetic acid (EDTA). In addition, the results indicate that the biocidic activity of benzalkonium chloride against Pseudomonas can be increased by combined use with polyethylenimine (PEI). In addition to PEI, several other potential permeabilizers, such as succimer, were shown to destabilize the OM of Gram-negative bacteria. Furthermore, combination of the results obtained from various permeability assays (e.g. uptake of a hydrophobic probe, sensitization to hydrophobic antibiotics and detergents, release of lipopolysaccharide (LPS) and LPS-specific fatty acids) with atomic force microscopy (AFM) image results increases our knowledge of the action of permeabilizers.

Combinations of plant-derived compounds against Campylobacter in vitro

Campylobacter occur in fresh retail poultry products as a result of their colonization of the gastro-intestinal tract of chickens during growth. Feed additives could be used for suppression of Campylobacter levels in the chickens prior to slaughter. To address this opportunity, feed manufacturers are targeting natural antimicrobials from plant material as new forms of consumer-accepted feed additives. However, to be practical, these natural antimicrobials must be effective at low concentrations. The current study has validated an improved laboratory method to study minimal inhibitory concentrations of plant compounds and their combinations against Campylobacter. The assay was shown to be valid for testing lipid-soluble and water-soluble plant extracts and byproducts from the food industry. The study screened 29 extracts or plant-derived compounds and their mixtures for anti-Campylobacter activity using a laboratory assay. Combinations of oregano, lactic acid, and sorghum byproduct showed effective synergy in anti-Campylobacter activity. The synergies allowed a large reduction in the concentration of the individual compounds needed to kill the bacteria with an 80% reduction in concentration being achieved for oregano essential oil. The assay gives rise to further opportunities for the testing of a greater range of combinations of plant-derived compounds and other natural antimicrobials. The method is robust, simple, and easily automated, and it could be used to adjust the cost of feed formulations by reducing costs associated with antimicrobial feed additives.

First detection of extended-spectrum cephalosporin- and fluoroquinolone-resistant Escherichia coli in Australian food-producing animals

This study aimed to define the frequency of resistance to critically important antimicrobials (CIAs) [i.e. extended-spectrum cephalosporins (ESCs), fluoroquinolones (FQs) and carbapenems] among Escherichia coli isolates causing clinical disease in Australian food-producing animals. Clinical E. coli isolates (n = 324) from Australian food-producing animals [cattle (n = 169), porcine (n = 114), poultry (n = 32) and sheep (n = 9)] were compiled from all veterinary diagnostic laboratories across Australia over a 1-year period. Isolates underwent antimicrobial susceptibility testing to 18 antimicrobials using the Clinical and Laboratory Standards Institute disc diffusion method. Isolates resistant to CIAs underwent minimum inhibitory concentration determination, multilocus sequence typing (MLST), phylogenetic analysis, plasmid replicon typing, plasmid identification, and virulence and antimicrobial resistance gene typing. The 324 E. coli isolates from different sources exhibited a variable frequency of resistance to tetracycline (29.0–88.6%), ampicillin (9.4–71.1%), trimethoprim/sulfamethoxazole (11.1–67.5%) and streptomycin (21.9–69.3%), whereas none were resistant to imipenem or amikacin. Resistance was detected, albeit at low frequency, to ESCs (bovine isolates, 1%; porcine isolates, 3%) and FQs (porcine isolates, 1%). Most ESC- and FQ-resistant isolates represented globally disseminated E. coli lineages (ST117, ST744, ST10 and ST1). Only a single porcine E. coli isolate (ST100) was identified as a classic porcine enterotoxigenic E. coli strain (non-zoonotic animal pathogen) that exhibited ESC resistance via acquisition of blaCMY-2. This study uniquely establishes the presence of resistance to CIAs among clinical E. coli isolates from Australian food-producing animals, largely attributed to globally disseminated FQ- and ESC-resistant E. coli lineages.