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.

Escherichia coli 83972 expressing a P fimbriae oligosaccharide receptor mimic impairs adhesion of Uropathogenic E. coli

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are a significant health concern, exacerbated by the rapid emergence of multidrug resistant strains refractory to antibiotic treatment. P fimbriae are strongly associated with upper urinary tract colonization due to specific binding to α-D-galactopyranosyl-(1-4)-β-D-galactopyranoside receptors in the kidneys. Thus, inhibiting P-fimbrial adhesion may reduce the incidence of UPEC-mediated UTI. E. coli 83972 is an asymptomatic bacteriuria isolate successfully used as a prophylactic agent to prevent UTI in human studies. We constructed a recombinant E. coli 83972 strain displaying a surface-located oligosaccharide P fimbriae receptor mimic that bound to P-fimbriated E. coli producing any of the 3 PapG adhesin variants. The recombinant strain, E. coli 83972:: lgtCE, impaired P fimbriae–mediated adhesion to human erythrocytes and kidney epithelial cells. Additionally, E. coli 83972::lgtCE impaired urine colonization by UPEC in a mouse UTI model, demonstrating its potential as a prophylactic agent to prevent UTI.

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.

Dapsone-induced agranulocytosis. The role of xenobiotic-metabolizing enzymes demonstrated by a case report [Dapson-induzierte Agranulozytose]

A 34-year-old female patient with a three year history of generalized granuloma annulare was treated systemically with dapsone (DADPS). Six weeks after the onset of treatment, the patient developed an extensive tonsillitis of the base of the tongue with fever and malaise. Routine laboratory work showed a leukocytopenia with agranulocytosis. Further investigation revealed a marked decrease of the enzyme activity of N-acetyltransferase 2, which plays an important role in dapsone metabolism. Treatment included the cessation of dapsone, antibiotic coverage, and G-CSF leading to the rapid improvement of symptoms and normalization of leukocyte counts. Dapsone-induced angina agranulocytotica is a rare event and is interpreted as an idiosyncratic reaction. Depending on genetic polymorphisms of various enzymes, dapsone can be metabolized to immunologically or toxicologically relevant intermediates. Because of the risk of severe hematologic reactions, dapsone should only be employed for solid indications and with appropriate monitoring. [Article in German]

B-lactamase-mediated resistance to antimicrobials : the relationship between genotype and phenotype

This thesis examined the ability to predict the emergence of bacteria resistant to antibiotics using genetic markers in the bacteria. Bacteria containing the genetic markers were able to become resistant to antibiotics, whereas bacteria that did not have the genetic markers remained susceptible. Existing techniques can identify the presence of resistance by looking at the characteristics of the bacteria during growth. However, having the ability to predict antibiotic resistance before it emerges could improve the preservation of currently available antibiotics and minimise treatment failure.

Nanostructured silver-gold bimetallic SERS substrates for selective identification of bacteria in human blood

Surface-enhanced Raman spectroscopy (SERS) is a potentially important tool in the rapid and accurate detection of pathogenic bacteria in biological fluids. However, for diagnostic application of this technique, it is necessary to develop a highly sensitive, stable, biocompatible and reproducible SERS-active substrate. In this work, we have developed a silver–gold bimetallic SERS surface by a simple potentiostatic electrodeposition of a thin gold layer on an electrochemically roughened nanoscopic silver substrate. The resultant substrate was very stable under atmospheric conditions and exhibited the strong Raman enhancement with the high reproducibility of the recorded SERS spectra of bacteria (E. coli, S. enterica, S. epidermidis, and B. megaterium). The coating of the antibiotic over the SERS substrate selectively captured bacteria from blood samples and also increased the Raman signal in contrast to the bare surface. Finally, we have utilized the antibiotic-coated hybrid surface to selectively identify different pathogenic bacteria, namely E. coli, S. enterica and S. epidermidis from blood samples.

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.

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.

Prevalence of methicillin resistance and virulence determinants of Staphylococcus aureus in diabetic foot ulcers

Background Diabetic foot ulceration (DFU) is a multifactorial process and is responsible for considerable morbidity and contributes to the increasing cost of health care worldwide. The diagnosis and identification of these ulcers remains a complex problem. Bacterial infection is promoted in the diabetic foot wound by decreased vascular supply and impaired host immune response. As conventional clinical microbiological methods are time-consuming and only identifies about 1% of the wound microbiota, detection of bacteria present in DFUs using molecular methods is highly advantageous and efficient. The aim of this study was to assess the virulence and methicillin resistance profiles of Staphylococcus aureus detected in DFUs using DNA-based methods. Methods A total of 223 swab samples were collected from 30 patients from March to October 2012. Bacterial DNA was extracted from the swab samples using standard procedures and was used to perform polymerase chain reaction (PCR) using specific oligonucleotide primers. The products were visualized using agarose gel electrophoresis. Results S. aureus was detected in 44.8% of samples. 25% of the S. aureus was methicillin-resistant S. aureus harboring the mecA gene. The alpha-toxin gene was present in 85% of the S. aureus positive samples. 61% of the S. aureus present in DFU samples harbored the exfoliatin factor A gene. Both the fibronectin factor A and fibronectin factor B gene were detected in 71% and 74% of the S. aureus positive samples. Conclusions DNA-based detection and characterization of bacteria in DFUs are rapid and efficient and can assist in accurate, targeted antibiotic therapy of DFU infections. The majority of S. aureus detected in this study were highly virulent and also resistant to methicillin. Further studies are required to understand the role of S. aureus in DFU trajectory.

DNA molecules and human therapeutics

Nucleic acid molecules are championing a new generation of reverse engineered biopharmaceuticals. In terms of potential application in gene medicine, plasmid DNA (pDNA) vectors have exceptional therapeutic and immunological profiles as they are free from safety concerns associated with viral vectors, display non-toxicity and are simpler to develop. This review addresses the potential applications of pDNA molecules in vaccine design/development and gene therapy via recombinant DNA technology as well as a staged delivery mechanism for the introduction of plasmid-borne gene to target cells via the nasal route.

Variation in the complex carbohydrate biosynthesis loci of Acinetobacter baumannii genomes

Extracellular polysaccharides are major immunogenic components of the bacterial cell envelope. However, little is known about their biosynthesis in the genus Acinetobacter, which includes A. baumannii, an important nosocomial pathogen. Whether Acinetobacter sp. produce a capsule or a lipopolysaccharide carrying an O antigen or both is not resolved. To explore these issues, genes involved in the synthesis of complex polysaccharides were located in 10 complete A. baumannii genome sequences, and the function of each of their products was predicted via comparison to enzymes with a known function. The absence of a gene encoding a WaaL ligase, required to link the carbohydrate polymer to the lipid A-core oligosaccharide (lipooligosaccharide) forming lipopolysaccharide, suggests that only a capsule is produced. Nine distinct arrangements of a large capsule biosynthesis locus, designated KL1 to KL9, were found in the genomes. Three forms of a second, smaller variable locus, likely to be required for synthesis of the outer core of the lipid A-core moiety, were designated OCL1 to OCL3 and also annotated. Each K locus includes genes for capsule export as well as genes for synthesis of activated sugar precursors, and for glycosyltransfer, glycan modification and oligosaccharide repeat-unit processing. The K loci all include the export genes at one end and genes for synthesis of common sugar precursors at the other, with a highly variable region that includes the remaining genes in between. Five different capsule loci, KL2, KL6, KL7, KL8 and KL9 were detected in multiply antibiotic resistant isolates belonging to global clone 2, and two other loci, KL1 and KL4, in global clone 1. This indicates that this region is being substituted repeatedly in multiply antibiotic resistant isolates from these clones.

Structure of the K2 capsule associated with the KL2 gene cluster of Acinetobacter baumannii

The repeat unit structure of the K2 capsule from an extensively antibiotic-resistant Acinetobacter baumannii global clone 2 (GC2) strain was determined. The oligosaccharide contains three simple sugars, d-glucopyranose, d-galatopyranose and N-acetyl-d-galactosamine, and the complex sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (Pse5Ac7Ac or pseudaminic acid), which has not previously been reported in any A. baumannii capsule. The strain was found to carry all the genes required for the synthesis of the sugars and construction of the K2 structure. The linkages catalyzed by the initiating transferase, three glycosyltransferases and the Wzy polymerase were also predicted. Examination of publicly available A. baumannii genome sequences revealed that the same gene cluster, KL2, often occurs in extensively antibiotic-resistant GC2 isolates and in further strain types. The gene module responsible for the synthesis of pseudaminic acid was also detected in four other K loci. A related module including genes for an acylated relative of pseudaminic acid was also found in two new KL types. A polymerase chain reaction scheme was developed to detect all modules containing genes for sugars based on pseudaminic acid and to specifically detect KL2.

Variation in the OC locus of Acinetobacter baumannii genomes predicts extensive structural diversity in the Lipooligosaccharide

Lipooligosaccharide (LOS) is a complex surface structure that is linked to many pathogenic properties of Acinetobacter baumannii. In A. baumannii, the genes responsible for the synthesis of the outer core (OC) component of the LOS are located between ilvE and aspS. The content of the OC locus is usually variable within a species, and examination of 6 complete and 227 draft A. baumannii genome sequences available in GenBank non-redundant and Whole Genome Shotgun databases revealed nine distinct new types, OCL4-OCL12, in addition to the three known ones. The twelve gene clusters fell into two distinct groups, designated Group A and Group B, based on similarities in the genes present. OCL6 (Group B) was unique in that it included genes for the synthesis of L-Rhamnosep. Genetic exchange of the different configurations between strains has occurred as some OC forms were found in several different sequence types (STs). OCL1 (Group A) was the most widely distributed being present in 18 STs, and OCL6 was found in 16 STs. Variation within clones was also observed, with more than one OC locus type found in the two globally disseminated clones, GC1 and GC2, that include the majority of multiply antibiotic resistant isolates. OCL1 was the most abundant gene cluster in both GC1 and GC2 genomes but GC1 isolates also carried OCL2, OCL3 or OCL5, and OCL3 was also present in GC2. As replacement of the OC locus in the major global clones indicates the presence of sub-lineages, a PCR typing scheme was developed to rapidly distinguish Group A and Group B types, and to distinguish the specific forms found in GC1 and GC2 isolates.

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.

An Australian native plant: A novel antimicrobial for the treatment of MRSA

Chronic wounds, often associated with venous and arterial ulcers, diabetes and pressure sores, is an area of great concern. In Australia, the cost of treating chronic wounds is conservatively estimated at $285 million/annum for the treatment of pressure ulcers and $654 million annually for the treatment and management of leg ulcers. Current figures indicate that more than seven million people suffer from chronic wounds worldwide with Australians accounting for approximately 600,000 of this number. Bacterial infection of the wound site is a major issue as contamination of a chronic wound with methicillin-resistant Staphylococcus aureus (MRSA) significantly delays wound healing. Further, once systemic, current antibiotic therapies capable of treating the infection are limited. Aboriginal bush medicine has been used for thousands of years for the treatment of wounds and sores. Hence, we selected a native Australian plant to evaluate its bactericidal activity against MRSA.