Enhanced Growth of Tetracycline-susceptible Soil Bacteria Treated with Iron and Oxytetracycline

Antibiotic resistance has become an important area of research because of the excessive use of antibiotics in clinical and agricultural settings that are driving the evolution of antibiotic resistant bacteria. However, drug tolerance is a naturally occurring phenomenon in soil communities, and is often linked to those soils that are exposed to heavy metals as well as antibiotics. Resistance to antibiotics maybe coupled with resistance to heavy metals in soil bacteria through efflux pumps that can be regulated by iron. Although considered s a heavy metal, iron is an essential component of life that regulates gene expression through the Ferric Uptake Regulator (Fur) protein. This master regulator protein is known to control siderophore production, and other biological pathways. As a suspected controller of biofilm formation, the role of Fur in environmental antibiotic resistance may be greater than is currently realized. In this study, we sought to explore a potential Fur-regulated drug tolerance pathway by understanding the response of soil bacteria when stressed with oxytetracycline and iron. Bacteria were collected from two locations in Miami Dade County. Isolates were first tested using Kirby-Bauer Disk Diffusion tests for antibiotic resistance/susceptibility and identified by 16S rDNA sequencing. A 96-well growth assay was developed to measure planktonic cell growth with 3 mM FeCl3, Oxytetracycline HCl, and the combination treatments. A Microtiter Dish Biofilm Formation Assay was employed and Fur diversity was evaluated. Tetracycline-susceptible bacterial isolates developed drug resistance with iron supplementation, but iron did not enhance biofilm formation. Development of a Fur-dependent drug resistance may be selected for, but further study is required to evaluate Fur evolution in the studied isolates. Gene expression analysis is also needed to further understand the ecological role of Fur and antibiotic resistance.

Macrophage adaptation leads to parallel evolution of genetically diverseEscherichia colismall-colony variants with increased fitness in vivo and antibiotic collateral sensitivity

Small-colony variants (SCVs) are commonly observed in evolution experiments and clinical isolates, being associated with antibiotic resistance and persistent infections. We recently observed the repeated emergence of Escherichia coli SCVs during adaptation to the interaction with macrophages. To identify the genetic targets underlying the emergence of this clinically relevant morphotype, we performed whole-genome sequencing of independently evolved SCV clones. We uncovered novel mutational targets, not previously associated with SCVs (e.g. cydA, pepP) and observed widespread functional parallelism. All SCV clones had mutations in genes related to the electron-transport chain. As SCVs emerged during adaptation to macrophages, and often show increased antibiotic resistance, we measured SCV fitness inside macrophages and measured their antibiotic resistance profiles. SCVs had a fitness advantage inside macrophages and showed increased aminoglycoside resistance in vitro, but had collateral sensitivity to other antibiotics (e.g. tetracycline). Importantly, we observed similar results in vivo. SCVs had a fitness advantage upon colonization of the mouse gut, which could be tuned by antibiotic treatment: kanamycin (aminoglycoside) increased SCV fitness, but tetracycline strongly reduced it. Our results highlight the power of using experimental evolution as the basis for identifying the causes and consequences of adaptation during host-microbe interactions.

Primary Antibiotic Resistance to Helicobacter pylori Strains Isolated From Children in Northern Iran: A Single Center Study

Background: Initial resistance to antibiotics is the main reason for the failure of Helicobacter pylori (H. pylori) eradication in children. Objectives: As we commonly face high antibiotic resistance rates in children, we aimed to determine the susceptibility of H. pylori to common antibiotics. Patients and Methods: In this cross-sectional in vitro study, 169 children younger than 14 years with clinical diagnosis of peptic ulcer underwent upper gastrointestinal endoscopy. Biopsy specimens from stomach and duodenum were cultured. In isolated colonies, tests of catalase, urease, and oxidase as well as gram staining were performed. After confirming the colonies as H. pylori, the antibiogram was obtained using disk diffusion method. Results: Culture for H. pylori was positive in 12.3% of the specimens, urease test in 21.3%, serological test in 18.9% and stool antigen test was positive in 21.9%. We could show high specificity but moderate sensitivity of both histological and H. pylori stool antigen tests to detect H. pylori. The overall susceptibility to metronidazole was 42.9%, amoxicillin 95.2%, clarithromycin 85.7%, furazolidone 61.9%, azithromycin 81.0%, and tetracycline 76.2% with the highest resistance to metronidazole and the lowest to clarithromycin. Conclusions: In our region, there is high resistance of H. pylori to some antibiotics including metronidazole and furazolidone among affected children. To reduce the prevalence of this antibiotic resistance, more controlled use of antibiotics should be considered in children.

Molecular characterisation of environmental enterococci derived from water samples and assessment of associated public health hazards

Enterococci are versatile Gram-positive bacteria that can survive under extreme conditions. Most enterococci are non-virulent and found in the gastrointestinal tract of humans and animals. Other strains are opportunistic pathogens that contribute to a large number of nosocomial infections globally. Epidemiological studies demonstrated a direct relationship between the density of enterococci in surface waters and the risk of swimmer-associated gastroenteritis. The distribution of infectious enterococcal strains from the hospital environment or other sources to environmental water bodies through sewage discharge or other means, could increase the prevalence of these strains in the human population. Environmental water quality studies may benefit from focusing on a subset of Enterococcus spp. that are consistently associated with sources of faecal pollution such as domestic sewage, rather than testing for the entire genus. E. faecalis and E. faecium are potentially good focal species for such studies, as they have been consistently identified as the dominant Enterococcus spp. in human faeces and sewage. On the other hand enterococcal infections are predominantly caused by E. faecalis and E. faecium. The characterisation of E. faecalis and E. faecium is important in studying their population structures, particularly in environmental samples. In developing and implementing rapid, robust molecular genotyping techniques, it is possible to more accurately establish the relationship between human and environmental enterococci. Of particular importance, is to determine the distribution of high risk enterococcal clonal complexes, such as E. faecium clonal complex 17 and E. faecalis clonal complexes 2 and 9 in recreational waters. These clonal complexes are recognized as particularly pathogenic enterococcal genotypes that cause severe disease in humans globally. The Pimpama-Coomera watershed is located in South East Queensland, Australia and was investigated in this study mainly because it is used intensively for agriculture and recreational purposes and has a strong anthropogenic impact. The primary aim of this study was to develop novel, universally applicable, robust, rapid and cost effective genotyping methods which are likely to yield more definitive results for the routine monitoring of E. faecalis and E. faecium, particularly in environmental water sources. To fullfill this aim, new genotyping methods were developed based on the interrogation of highly informative single nucleotide polymorphisms (SNPs) located in housekeeping genes of both E. faecalis and E. faecium. SNP genotyping was successfully applied in field investigations of the Coomera watershed, South-East Queensland, Australia. E. faecalis and E. faecium isolates were grouped into 29 and 23 SNP profiles respectively. This study showed the high longitudinal diversity of E. faecalis and E. faecium over a period of two years, and both human-related and human-specific SNP profiles were identified. Furthermore, 4.25% of E. faecium strains isolated from water was found to correspond to the important clonal complex-17 (CC17). Strains that belong to CC17 cause the majority of hospital outbreaks and clinical infections globally. Of the six sampling sites of the Coomera River, Paradise Point had the highest number of human-related and human-specific E. faecalis and E. faecium SNP profiles. The secondary aim of this study was to determine the antibiotic-resistance profiles and virulence traits associated with environmental E. faecalis and E. faecium isolates compared to human pathogenic E. faecalis and E. faecium isolates. This was performed to predict the potential health risks associated with coming into contact with these strains in the Coomera watershed. In general, clinical isolates were found to be more resistant to all the antibiotics tested compared to water isolates and they harbored more virulence traits. Multi-drug resistance was more prevalent in clinical isolates (71.18% of E. faecalis and 70.3 % of E. faecium) compared to water isolates (only 5.66 % E. faecium). However, tetracycline, gentamicin, ciprofloxacin and ampicillin resistance was observed in water isolates. The virulence gene esp was the most prevalent virulence determinant observed in clinical isolates (67.79% of E. faecalis and 70.37 % of E. faecium), and this gene has been described as a human-specific marker used for microbial source tracking (MST). The presence of esp in water isolates (16.36% of E. faecalis and 19.14% of E. faecium) could be indicative of human faecal contamination in these waterways. Finally, in order to compare overall gene expression between environmental and clinical strains of E. faecalis, a comparative gene hybridization study was performed. The results of this investigation clearly demonstrated the up-regulation of genes associated with pathogenicity in E. faecalis isolated from water. The expression study was performed at physiological temperatures relative to ambient temperatures. The up-regulation of virulence genes demonstrates that environmental strains of E. faecalis can pose an increased health risk which can lead to serious disease, particularly if these strains belong to the virulent CC17 group. The genotyping techniques developed in this study not only provide a rapid, robust and highly discriminatory tool to characterize E. faecalis and E. faecium, but also enables the efficient identification of virulent enterococci that are distributed in environmental water sources.

ASAP ECMO : antibiotic, sedative and analgesic pharmacokinetics during extracorporeal membrane oxygenation : a multi-centre study to optimise drug therapy during ECMO

BACKGROUND: Given the expanding scope of extracorporeal membrane oxygenation (ECMO) and its variable impact on drug pharmacokinetics as observed in neonatal studies, it is imperative that the effects of the device on the drugs commonly prescribed in the intensive care unit (ICU) are further investigated. Currently, there are no data to confirm the appropriateness of standard drug dosing in adult patients on ECMO. Ineffective drug regimens in these critically ill patients can seriously worsen patient outcomes. This study was designed to describe the pharmacokinetics of the commonly used antibiotic, analgesic and sedative drugs in adult patients receiving ECMO. METHODS: This is a multi-centre, open-label, descriptive pharmacokinetic (PK) study. Eligible patients will be adults treated with ECMO for severe cardiac and/or respiratory failure at five Intensive Care Units in Australia and New Zealand. Patients will receive the study drugs as part of their routine management. Blood samples will be taken from indwelling catheters to investigate plasma concentrations of several antibiotics (ceftriaxone, meropenem, vancomycin, ciprofloxacin, gentamicin, piperacillin-tazobactum, ticarcillin-clavulunate, linezolid, fluconazole, voriconazole, caspofungin, oseltamivir), sedatives and analgesics (midazolam, morphine, fentanyl, propofol, dexmedetomidine, thiopentone). The PK of each drug will be characterised to determine the variability of PK in these patients and to develop dosing guidelines for prescription during ECMO. DISCUSSION: The evidence-based dosing algorithms generated from this analysis can be evaluated in later clinical studies. This knowledge is vitally important for optimising pharmacotherapy in these most severely ill patients to maximise the opportunity for therapeutic success and minimise the risk of therapeutic failure

Effect of vitamin D supplementation on antibiotic use : a randomized controlled trial

BACKGROUND: Observational data suggested that supplementation with vitamin D could reduce risk of infection, but trial data are inconsistent. OBJECTIVE: We aimed to examine the effect of oral vitamin D supplementation on antibiotic use. DESIGN: We conducted a post hoc analysis of data from pilot D-Health, which is a randomized trial carried out in a general community setting between October 2010 and February 2012. A total of 644 Australian residents aged 60-84 y were randomly assigned to receive monthly doses of a placebo (n = 214) or 30,000 (n = 215) or 60,000 (n = 215) IU oral cholecalciferol for ≤12 mo. Antibiotics prescribed during the intervention period were ascertained by linkage with pharmacy records through the national health insurance scheme (Medicare Australia). RESULTS: People who were randomly assigned 60,000 IU cholecalciferol had nonsignificant 28% lower risk of having antibiotics prescribed at least once than did people in the placebo group (RR: 0.72; 95% CI: 0.48, 1.07). In analyses stratified by age, in subjects aged ≥70 y, there was a significant reduction in antibiotic use in the high-dose vitamin D compared with placebo groups (RR: 0.53; 95% CI: 0.32, 0.90), whereas there was no effect in participants <70 y old (RR: 1.07; 95% CI: 0.58, 1.97) (P-interaction = 0.1). CONCLUSION: Although this study was a post hoc analysis and statistically nonsignificant, this trial lends some support to the hypothesis that supplementation with 60,000 IU vitamin D/mo is associated with lower risk of infection, particularly in older adults. The trial was registered at the Australian New Zealand Clinical Trials Registry (anzctr.org.au) as ACTRN12609001063202.

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.

Making decisions about antibiotic use in the Australian primary healthcare sector

Introduction Australia is contributing to the global problem of antimicrobial resistance with one of the highest rates of antibiotic use amongst OECD countries. Data from the Australian primary healthcare sector suggests unnecessary antibiotics were prescribed for conditions that will resolve without it. If left unchecked, this will result in more resistant micro-organisms, against which antibiotics will be useless. There is a lack of understanding about what is influencing decisions to use antibiotics – what factors influences general practitioners (GPs) to prescribe antibiotics, consumers to seek antibiotics, and pharmacists to fill old 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. Method This project will investigate (a) what factors drive decisions to use antibiotics for GPs, pharmacists and consumers, and (b) how these individuals discount the future. Factors will be gleaned from published literature and from a qualitative phase using 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, and whether GPs and pharmacists display the same extent of discounting the future, as consumers. Expected 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 The emergence of antibiotic resistance is inevitable. This research will expand on what is currently known about influencing desired behaviour change in antibiotic use, in the fight against antibiotic resistance. Real World Implications Research findings will contribute to existing national programs to bring about a reduction in inappropriate use of antibiotic in Australia. Specifically, influencing (1) how key messages and public health campaigns are crafted to increase health literacy, and (2) clinical education and empowerment of GPs and pharmacists to play a more responsive role as stewards of antibiotic use in the community.

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.

Genome Sequence of Acinetobacter baumannii Strain A1, an Early Example of Antibiotic-Resistant Global Clone 1

Acinetobacter baumannii isolate A1 was recovered in the United Kingdom in 1982 and belongs to global clone 1 (GC1). Here, we present its complete 3.91-Mbp genome sequence, generated via a combination of short-read sequencing (Illumina), long-read sequencing (PacBio), and manual finishing.

Nutritional impact of antipseudomonas intravenous antibiotic courses in cystic fibrosis

Editor,—In their study of cystic fibrosis patients who were nutritionally assessed at the start and end of a 14 day period of home intravenous antibiotic treatment for chest disease, Vicet al concluded that increased weight was a result of increased fat storage.1 While this may in part be true, their methodology dictates caution in drawing conclusions....

Conformation of antibiotic X-537A (lasalocid-A) and its calcium complex in acetonitrile solvent using circular dichroism spectroscopy

The calcium binding characteristics of antibiotic X-537A (lasalocid-A) in a lipophilic solvent, acetonitrile (CH3CN), have been studied using circular dichroism (CD) spectroscopy. The analysis of the data indicated that in this medium polar solvent, X-537A forms predominantly the charged complexes of stoichiometries 2:1 and 1:1, the relative amounts of the two being dependent on [Ca2+]. The conformation of the complexes, arrived at on the basis of the data, seem to indicate a rigid part encompassing Ca2+, liganded to 3 oxygens of the molecule, viz., the carbonyl, the substituted tetrahydrofuran ring and the substituted pyran ring oxygens (apart from possibly, the liganding provided by nitrogen atoms of the solvent molecules), and a flexible part consisting of the salicylic acid group of the molecule.