Assessing the construct validity and reliability of the parental perception on antibiotics (PAPA) scales

Background: The overuse of antibiotics is becoming an increasing concern. Antibiotic resistance, which increases both the burden of disease, and the cost of health services, is perhaps the most profound impact of antibiotics overuse. Attempts have been made to develop instruments to measure the psychosocial constructs underlying antibiotics use, however, none of these instruments have undergone thorough psychometric validation. This study evaluates the psychometric properties of the Parental Perceptions on Antibiotics (PAPA) scales. The PAPA scales attempt to measure the factors influencing parental use of antibiotics in children. Methods: 1111 parents of children younger than 12 years old were recruited from primary schools’ parental meetings in the Eastern Province of Saudi Arabia from September 2012 to January 2013. The structure of the PAPA instrument was validated using Confirmatory Factor Analysis (CFA) with measurement model fit evaluated using the raw and scaled χ2, Goodness of Fit Index, and Root Mean Square Error of Approximation. Results: A five-factor model was confirmed with the model showing good fit. Constructs in the model include: Knowledge and Beliefs, Behaviors, Sources of information, Adherence, and Awareness about antibiotics resistance. The instrument was shown to have good internal consistency, and good discriminant and convergent validity. Conclusion: The availability of an instrument able to measure the psychosocial factors underlying antibiotics usage allows the risk factors underlying antibiotic use and overuse to now be investigated.

Distribution and taxonomy of Enterococci from the Davis Station wastewater discharge, Antarctica

This project assessed the potential impact of untreated sewage release in a near-shore marine environment of Antarctica through the distribution and characterisation of the faecal indicator bacteria Enterococcus. Antibiotic resistance and genome sequencing analyses revealed that enterococci resistant to multiple antibiotics closely related to clinical pathogens were introduced to the pristine Antarctic environment by Australia's Davis station.

The serum resistome of a globally disseminated multidrug resistant uropathogenic Escherichia coli Clone

Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, a representative of the predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E. coli EC958, 231 (73%) of which were also essential in E. coli K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for E. coli EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes, hyxA and hyxR, as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of E. coli ST131, and provides a framework for future research on E. coli and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and cause disease.

Molecular analysis of the acinetobacter baumannii biofilm-associated protein

Acinetobacter baumannii is a multidrug-resistant pathogen associated with hospital outbreaks of infection across the globe, particularly in the intensive care unit. The ability of A. baumannii to survive in the hospital environment for long periods is linked to antibiotic resistance and its capacity to form biofilms. Here we studied the prevalence, expression, and function of the A. baumannii biofilm-associated protein (Bap) in 24 carbapenem-resistant A. baumannii ST92 strains isolated from a single institution over a 10-year period. The bap gene was highly prevalent, with 22/24 strains being positive for bap by PCR. Partial sequencing of bap was performed on the index case strain MS1968 and revealed it to be a large and highly repetitive gene approximately 16 kb in size. Phylogenetic analysis employing a 1,948-amino-acid region corresponding to the C terminus of Bap showed that BapMS1968 clusters with Bap sequences from clonal complex 2 (CC2) strains ACICU, TCDC-AB0715, and 1656-2 and is distinct from Bap in CC1 strains. By using overlapping PCR, the bapMS1968 gene was cloned, and its expression in a recombinant Escherichia coli strain resulted in increased biofilm formation. A Bap-specific antibody was generated, and Western blot analysis showed that the majority of A. baumannii strains expressed an ∼200-kDa Bap protein. Further analysis of three Bap-positive A. baumannii strains demonstrated that Bap is expressed at the cell surface and is associated with biofilm formation. Finally, biofilm formation by these Bap-positive strains could be inhibited by affinity-purified Bap antibodies, demonstrating the direct contribution of Bap to biofilm growth by A. baumannii clinical isolates.

A FimH inhibitor prevents acute bladder infection and treats chronic cystitis caused by multidrug-resistant uropathogenic Escherichia coli ST131

Background. Escherichia coli O25b:H4-ST131 represents a predominant clone of multidrug-resistant uropathogens currently circulating worldwide in hospitals and the community. Urinary tract infections (UTIs) caused by E. coli ST131 are typically associated with limited treatment options and are often recurrent. Methods. Using established mouse models of acute and chronic UTI, we mapped the pathogenic trajectory of the reference E. coli ST131 UTI isolate, strain EC958. Results. We demonstrated that E. coli EC958 can invade bladder epithelial cells and form intracellular bacterial communities early during acute UTI. Moreover, E. coli EC958 persisted in the bladder and established chronic UTI. Prophylactic antibiotic administration failed to prevent E. coli EC958–mediated UTI. However, 1 oral dose of a small-molecular-weight compound that inhibits FimH, the type 1 fimbriae adhesin, significantly reduced bacterial colonization of the bladder and prevented acute UTI. Treatment of chronically infected mice with the same FimH inhibitor lowered their bladder bacterial burden by >1000-fold. Conclusions. In this study, we provide novel insight into the pathogenic mechanisms used by the globally disseminated E. coli ST131 clone during acute and chronic UTI and establish the potential of FimH inhibitors as an alternative treatment against multidrug-resistant E. coli.

Host-pathogen checkpoints and population bottlenecks in persistent and intracellular uropathogenic Escherichia coli bladder infection

Bladder infections affect millions of people yearly, and recurrent symptomatic infections (cystitis) are very common. The rapid increase in infections caused by multidrug-resistant uropathogens threatens to make recurrent cystitis an increasingly troubling public health concern. Uropathogenic Escherichia coli (UPEC) cause the vast majority of bladder infections. Upon entry into the lower urinary tract, UPEC face obstacles to colonization that constitute population bottlenecks, reducing diversity, and selecting for fit clones. A critical mucosal barrier to bladder infection is the epithelium (urothelium). UPEC bypass this barrier when they invade urothelial cells and form intracellular bacterial communities (IBCs), a process which requires type 1 pili. IBCs are transient in nature, occurring primarily during acute infection. Chronic bladder infection is common and can be either latent, in the form of the quiescent intracellular reservoir (QIR), or active, in the form of asymptomatic bacteriuria (ASB/ABU) or chronic cystitis. In mice, the fate of bladder infection, QIR, ASB, or chronic cystitis, is determined within the first 24 h of infection and constitutes a putative host–pathogen mucosal checkpoint that contributes to susceptibility to recurrent cystitis. Knowledge of these checkpoints and bottlenecks is critical for our understanding of bladder infection and efforts to devise novel therapeutic strategies.

Escherichia coli isolates causing asymptomatic bacteriuria in catheterized and noncatheterized individuals possess similar virulence properties

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli being responsible for >80% of all cases. Asymptomatic bacteriuria (ABU) occurs when bacteria colonize the urinary tract without causing clinical symptoms and can affect both catheterized patients (catheter-associated ABU [CA-ABU]) and noncatheterized patients. Here, we compared the virulence properties of a collection of ABU and CA-ABU nosocomial E. coli isolates in terms of antibiotic resistance, phylogenetic grouping, specific UTI-associated virulence genes, hemagglutination characteristics, and biofilm formation. CA-ABU isolates were similar to ABU isolates with regard to the majority of these characteristics; exceptions were that CA-ABU isolates had a higher prevalence of the polysaccharide capsule marker genes kpsMT II and kpsMT K1, while more ABU strains were capable of mannose-resistant hemagglutination. To examine biofilm growth in detail, we performed a global gene expression analysis with two CA-ABU strains that formed a strong biofilm and that possessed a limited adhesin repertoire. The gene expression profile of the CA-ABU strains during biofilm growth showed considerable overlap with that previously described for the prototype ABU E. coli strain, 83972. This is the first global gene expression analysis of E. coli CA-ABU strains. Overall, our data suggest that nosocomial ABU and CA-ABU E. coli isolates possess similar virulence profiles.

Uropathogenic Escherichia coli mediated urinary tract infection

Urinary tract infection (UTI) is among the most common infectious diseases of humans and is the most common nosocomial infection in the developed world. They cause significant morbidity and mortality, with approximately 150 million cases globally per year. It is estimated that 40-50% of women and 5% of men will develop a UTI in their lifetime, and UTI accounts for more than 1 million hospitalizations and $1.6 billion in medical expenses each year in the USA. Uropathogenic E. coli (UPEC) is the primary cause of UTI. This review presents an overview of the primary virulence factors of UPEC, the major host responses to infection of the urinary tract, the emergence of specific multidrug resistant clones of UPEC, antibiotic treatment options for UPEC-mediated UTI and the current state of vaccine strategies as well as other novel anti-adhesive and prophylactic approaches to prevent UTI. New and emerging themes in UPEC research are also discussed in the context of future outlooks.

Promiscuity, pheromones and pathogenicity : why all Enterococci are not created equal

Enterococcus faecalis is a Gram-positive, coccus shaped, lactic acid bacterium, with demonstrated ubiquity across multiple anatomical sites. Enterococcus faecalis isolates have been isolated from clinical samples as the etiological agent in patients with overt infections, and from body sites previously thought to be sterile but absent of signs and symptoms of infection. E. faecalis is implicated in both human health and disease, recognized as a commensal, a probiotic and an opportunistic multiply resistant pathogen. E. faecalis has emerged as a key pathogen in nosocomial infections. E. faecalis is well equipped to avert recognition by host cell immune mediators. Antigenic cell wall components including lipotechoic acids are concealed from immune detection by capsular polysaccharides produced by some strains. Thereby preventing complement activation, the pro-inflammatory response, opsonisation and phagocytosis. E. faecalis also produces a suite of enzymes including gelatinase and cytolysin, which aid in both virulence and host immune evasion. The ability of enterococci to form biofilms in vivo further increases virulence, whilst simultaneously preventing detection by host cells. E. faecalis exhibits high levels of both intrinsic and acquired antimicrobial resistance. The mobility of the E. faecalis genome is a significant contributor to antimicrobial resistance, with this species also transferring resistance to other Gram-positive bacteria. Whilst E. faecalis is of increasing concern in nosocomial infections, its role as a member of the endogenous microbiota cannot be underestimated. As a commensal and probiotic, E. faecalis plays an integral role in modulating the immune response, and in providing endogenous antimicrobial activity to enhance exclusion or inhibition of opportunistic pathogens in certain anatomical niches. In this chapter we will review possible mediators of enterococcal transition from commensal microbe to opportunistic pathogen, considering isolates obtained from patients diagnosed with pathogenic infections and those obtained from asymptomatic patients.

General Practitioner Antimicrobial Stewardship Programme Study (GAPS): protocol for a cluster randomised controlled trial

Background There is a strong link between antibiotic consumption and the rate of antibiotic resistance. In Australia, the vast majority of antibiotics are prescribed by general practitioners, and the most common indication is for acute respiratory infections. The aim of this study is to assess if implementing a package of integrated, multifaceted interventions reduces antibiotic prescribing for acute respiratory infections in general practice. Methods/design This is a cluster randomised trial comparing two parallel groups of general practitioners in 28 urban general practices in Queensland, Australia: 14 intervention and 14 control practices. The protocol was peer-reviewed by content experts who were nominated by the funding organization. This study evaluates an integrated, multifaceted evidence-based package of interventions implemented over a six month period. The included interventions, which have previously been demonstrated to be effective at reducing antibiotic prescribing for acute respiratory infections, are: delayed prescribing; patient decision aids; communication training; commitment to a practice prescribing policy for antibiotics; patient information leaflet; and near patient testing with C-reactive protein. In addition, two sub-studies are nested in the main study: (1) point prevalence estimation carriage of bacterial upper respiratory pathogens in practice staff and asymptomatic patients; (2) feasibility of direct measures of antibiotic resistance by nose/throat swabbing. The main outcome data are from Australia’s national health insurance scheme, Medicare, which will be accessed after the completion of the intervention phase. They include the number of antibiotic prescriptions and the number of patient visits per general practitioner for periods before and during the intervention. The incidence of antibiotic prescriptions will be modelled using the numbers of patients as the denominator and seasonal and other factors as explanatory variables. Results will compare the change in prescription rates before and during the intervention in the two groups of practices. Semi-structured interviews will be conducted with the general practitioners and practice staff (practice nurse and/or practice manager) from the intervention practices on conclusion of the intervention phase to assess the feasibility and uptake of the interventions. An economic evaluation will be conducted to estimate the costs of implementing the package, and its cost-effectiveness in terms of cost per unit reduction in prescribing. Discussion The results on the effectiveness, cost-effectiveness, acceptability and feasibility of this package of interventions will inform the policy for any national implementation.

Genetic variability and antimicrobial resistance of Ureaplasma parvum in response to maternal erythromycin treatment : a study in pregnant sheep

Background: Ureaplasmas are the most frequently isolated microorganisms from the amniotic fluid (AF) of pregnant women and can cause chronic infections that are difficult to eradicate with standard macrolide treatment. We tested the effects of erythromycin treatment on phenotypic and genotypic markers of ureaplasmal antimicrobial resistance in sheep. Method: At 50 days of gestation (d, term=145d) 12 pregnant ewes received intra-amniotic injections of U. parvum serovar 3 (erythromycin-sensitive, 2x104 colony-forming-units). At 100d ewes received: erythromycin treatment (500 mg, q3h for 4 days, IM, n=6) or no treatment (n=6). Fetuses were delivered surgically (125d) and AF and chorioamnion were collected for: culture, minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) testing; 23S rRNA sequencing; and detection of macrolide-lincosamide-streptogramin resistance (MLSr) genes. Results: MICs of erythromycin, azithromycin and roxithromycin against AF isolates were low (range = 0.06 mg/L to 1.0 mg/L); however, chorioamnion isolates demonstrated increased resistance to roxithromycin (0.13 – 5.33 mg/L). 62.5% of chorioamnion ureaplasmas formed biofilms in vitro and mutations (125 nucleotides, 29.6%) were found in the 23S rRNA gene (domain V) of chorioamnion (but not AF) ureaplasmas. MLSr genes (ermB, msrC and msrD) were detected in 100% of chorioamnion isolates and only msrD was detected in AF isolates (40%). Conclusions: 23S rRNA mutations and MLSr genes occurred independently of erythromycin treatment, suggesting that the anatomical site of infection and microenvironment may exert selective pressures on ureaplasmas that cause genetic changes and alter antimicrobial sensitivity profiles. These results have serious implications for treatment of in utero infections.

Assessment of antibiotic use in children by their parents in Saudi Arabia

This project assessed the factors influencing the parents' use of antibiotics in children in Saudi Arabia. It was conducted through the development and validation of the Parental Perceptions on Antibiotics (PAPA) Scales, which is the first valid and reliable instrument directed to measure these factors on a population-based level. This is also the first population-based study that measures the patterns of antibiotic use in children in Saudi Arabia, where a very high association was found between the use of antibiotics and the number of cold episodes a year, which suggests a potential of overuse of antibiotics in Saudi Arabia. Also, it was found that antibiotic use decreases significantly with parents having higher scores in knowledge and beleifs, behaviors, and eagerness to seek health-related information.

A conjugative plasmid carrying the carbapenem resistance gene blaOXA-23 in AbaR4 in an extensively resistant GC1 Acinetobacter baumannii isolate

OBJECTIVES: To locate the acquired bla(OXA-23) carbapenem resistance gene in an Australian A. baumannii global clone 1 (GC1) isolate. METHODS: The genome of the extensively antibiotic-resistant GC1 isolate A85 harbouring bla(OXA-23) in Tn2006 was sequenced using Illumina HiSeq, and the reads were used to generate a de novo assembly. PCR was used to assemble relevant contigs. Sequences were compared with ones in GenBank. Conjugation experiments were conducted. RESULTS: The sporadic GC1 isolate A85, recovered in 2003, was extensively resistant, exhibiting resistance to imipenem, meropenem and ticarcillin/clavulanate, to cephalosporins and fluoroquinolones and to the older antibiotics gentamicin, kanamycin and neomycin, sulfamethoxazole, trimethoprim and tetracycline. Genes for resistance to older antibiotics are in the chromosome, in an AbaR3 resistance island. A second copy of the ampC gene in Tn6168 confers cephalosporin resistance and the gyrA and parC genes have mutations leading to fluoroquinolone resistance. An 86 335 bp repAci6 plasmid, pA85-3, carrying bla(OXA-23) in Tn2006 in AbaR4, was shown to transfer imipenem, meropenem and ticarcillin/clavulanate resistance into a susceptible recipient. A85 also contains two small cryptic plasmids of 2.7 and 8.7 kb. A85 is sequence type ST126 (Oxford scheme) and carries a novel KL15 capsule locus and the OCL3 outer core locus. CONCLUSIONS: A85 represents a new GC1 lineage identified by the novel capsule locus but retains AbaR3 carrying genes for resistance to older antibiotics. Resistance to imipenem, meropenem and ticarcillin/clavulanate has been introduced into A85 by pA85-3, a repAci6 conjugative plasmid carrying Tn2006 in AbaR4.

Characteristics of microbial drug resistance and its correlates in chronic diabetic foot ulcer infections

While virulence factors and the biofilm-forming capabilities of microbes are the key regulators of the wound healing process, the host immune response may also contribute in the events following wound closure or exacerbation of non-closure. We examined samples from diabetic and non-diabetic foot ulcers/wounds for microbial association and tested the microbes for their antibiotic susceptibility and ability to produce biofilms. A total of 1074 bacterial strains were obtained with staphylococci, Pseudomonas, Citrobacter and enterococci as major colonizers in diabetic samples. Though non-diabetic samples had a similar assemblage, the frequency of occurrence of different groups of bacteria was different. Gram-negative bacteria were found to be more prevalent in the diabetic wound environment while Gram-positive bacteria were predominant in non-diabetic ulcers. A higher frequency of monomicrobial infection was observed in samples from non-diabetic individuals when compared to samples from diabetic patients. The prevalence of different groups of bacteria varied when the samples were stratified according to age and sex of the individuals. Several multidrug-resistant strains were observed among the samples tested and most of these strains produced moderate to high levels of biofilms. The weakened immune response in diabetic individuals and synergism among pathogenic micro-organisms may be the critical factors that determine the delicate balance of the wound healing process.

Making decisions about antibiotic use in the Australian primary healthcare sector

Background Australia has one of the highest rates of antibiotic use amongst OECD countries. Data from the Australian primary healthcare sector suggests unnecessary antibiotics were prescribed for self-resolving conditions. We need to better understand what drives general practitioners (GPs) to prescribe antibiotics, consumers to seek antibiotics, and pharmacists to fill repeat antibiotic prescriptions. It is also not clear how these individuals trade-off between the possible benefits that antibiotics may provide in the immediate/short term, against the longer term societal risk of antimicrobial resistance. This project investigates what factors drive decisions to use antibiotics for GPs, pharmacists and consumers, and how these individuals discount the future. Methods Factors will be gleaned from published literature and from semi-structured interviews, to inform the development of Discrete Choice Experiments (DCEs). Three DCEs will be constructed – one for each group of interest – to allow investigation of which factors are more important in influencing (a) GPs to prescribe antibiotics, (b) consumers to seek antibiotics, and (c) pharmacists to fill legally valid but old or repeat prescriptions of antibiotics. Regression analysis will be conducted to understand the relative importance of these factors. A Time Trade Off exercise will be developed to investigate how these individuals discount the future. Results Findings from the DCEs will provide an insight into which factors are more important in driving decision making in antibiotic use for GPs, pharmacists and consumers. Findings from the Time Trade Off exercise will show what individuals are willing to trade for preserving the miracle of antibiotics. Conclusion Research findings will contribute to existing national programs to bring about a reduction in inappropriate use of antibiotic in Australia. Specifically, influencing how key messages and public health campaigns are crafted, and clinical education and empowerment of GPs and pharmacists to play a more responsive role as stewards of antibiotic use in the community.