Antimicrobial activity of enacyloxin IIa and gladiolin against the urogenital pathogens Neisseria gonorrhoeae and Ureaplasma spp.

AIMS: To determine the antimicrobial activity of enacyloxin IIa and gladiolin against Neisseria gonorrhoeae and Ureaplasma spp. METHODS AND RESULTS: The Burkholderia polyketide antibiotics enacyloxin IIa and gladiolin were tested against 14 N. gonorrhoeae and 10 Ureaplasma spp. isolates including multidrug-resistant N. gonorrhoeae isolates WHO V, WHO X and WHO Z as well as macrolide, tetracycline and ciprofloxacin-resistant ureaplasmas. Susceptibility testing of N. gonorrhoeae was carried out by agar dilution, whereas broth micro-dilution and growth kinetic assays were used for Ureaplasma spp. The MIC range for enacyloxin IIa and gladiolin against N. gonorrhoeae was 0·015-0·06 mg l-1 and 1-2 mg l-1 respectively. The presence of resistance to front line antibiotics had no effect on MIC values. The MIC range for enacyloxin IIa against Ureaplasma spp. was 4-32 mg l-1 with a clear dose-dependent effect when observed using a growth kinetic assay. Gladiolin had no antimicrobial activity on Ureaplasma spp. at 32 mg l-1 and limited impact on growth kinetics. CONCLUSIONS: Enacyloxin IIa and gladiolin antibiotics have antimicrobial activity against a range of antibiotic susceptible and resistant N. gonorrhoeae and Ureaplasma isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the potential for a new class of antimicrobial against pathogens in which limited antibiotics are available. Development of these compounds warrants further investigation in the face of emerging extensively drug-resistant strains.

The effect of cationic microbicide exposure against Burkholderia cepacia complex (Bcc); the use of Burkholderia lata strain 383 as a model bacterium.

AIM: The extensive use of microbicides in a wide range of applications has been questioned with regard to their role in the development of bacterial resistance to antimicrobials. This study aims to measure the phenotypic and genotypic changes in Burkholderia lata strain 383 exposed to chlorhexidine gluconate (CHG) and benzalkonium chloride (BZC), two commonly used cationic microbicides. METHODS AND RESULTS: The susceptibility of B. lata strain 383 to CHG and BZC and a range of antibiotics was determined using standardized MIC, MBC and antibiotic susceptibility testing protocols before and after short-term exposure to a low microbicide concentration. Measurements were performed on four separate occasions over a 1-year period. Changes in gene expression were investigated using quantitative real-time PCR. Although the susceptibility profile to CHG and BZC was not altered, a change in antibiotic susceptibility profile was observed for ceftazidime, and for imipenem and ciprofloxacin in 2/4 repeats. An outer membrane protein and ABC transporter were found to be significantly upregulated following treatment with BZC and CHG, respectively. CONCLUSIONS: The comparison of MIC and MBC results following microbicide exposure with baseline data offered a prospective protocol to quantify any change in bacterial susceptibility profile. However, the use of a standardized antibiotic susceptibility protocol with B. lata strain 383 showed some inconsistencies in results between repeats. SIGNIFICANCE AND IMPACT OF THE STUDY: With ever-increasing interest in the impact of microbicides on emerging antimicrobial resistance in bacteria growing, this study demonstrated that comparing susceptibility profile obtained after exposure to microbicides with baseline susceptibility values could play a role in establishing the potential risk of microbicide resistance and cross-resistance development and also in the development of a protocol that allows the prediction of microbicide resistance.

Long-term persistence of a single Legionella pneumophila strain possessing the mip gene in a municipal shower despite repeated cycles of chlorination.

The ability of Legionella pneumophila to colonise domestic water systems is a primary cause of outbreaks of Legionnaire's disease in humans. World Health Organization guidelines recommend that drinking water is chlorinated to between 0.2 and 1mg/L [Chlorine in drinking-water. Guidelines for drinking-water quality, 2nd edn. Geneva: World Health Organization; 1996], but L. pneumophila is repeatedly isolated from chlorinated water systems, indicating that this treatment is not effective at preventing colonisation. Current UK guidelines recommend a one-off treatment of 20-50mg/L of free chlorine to remove the bacteria. In this study we report on the persistence of L. pneumophila serogroup 1 in a domestic shower system despite repeated cycles of chlorination at 50mg/L for 1h exposure time, over the course of two and a half years. Persisting isolates were subjected to in-vitro phenotypic analyses and polymerase chain reaction analysis for the toxin-encoding mip gene. Random amplified polymorphic DNA typing was also performed to determine whether the isolates recovered on different occasions were the same strain. We found that seven isolates of L. pneumophila recovered over a two-and-a-half year period are the same genetically defined strain, indicating that the bacteria can persist despite repeated cycles of chlorination after each successive isolation.

Burkholderia cepacia complex bacteria: opportunistic pathogens with important natural biology.

Interaction with plants around their roots and foliage forms the natural habitat for a wide range of gram-negative bacteria such as Burkholderia, Pseudomonas and Ralstonia. During these interactions many of these bacteria facilitate highly beneficial processes such as the breakdown of pollutants or enhancement of crop growth. All these bacterial species are also capable of causing opportunistic infections in vulnerable individuals, especially people with cystic fibrosis (CF). Here we will review the current understanding of the Burkholderia cepacia complex (Bcc) as a group of model opportunistic pathogens, contrasting their clinical epidemiology with their ecological importance. Currently, the B. cepacia complex is composed of nine formally named species groups which are all difficult to identify using phenotypic methods. Genetic methods such as 16S rRNA and recA gene sequence analysis have proven useful for Bcc species identification. Multilocus sequence typing (MLST) is also emerging as a very useful tool for both Bcc strain and species identification. Historically, Burkholderia cenocepacia was the most dominant Bcc pathogen in CF, however, probably as a result of strict infection control practices introduced to control the spread of this species, its prevalence has been reduced. Burkholderia multivorans is the now the most dominant Bcc infection encountered in the UK CF population, a changing epidemiology that also appears to be occurring in the US CF population. The distribution of Bcc species residing in the natural environment may vary considerably with the type of environment examined. Clonally identical Bcc strains have been found to occur in the natural environment and cause infection. The contamination of medical devices, disinfectants and pharmaceutical formulations has also been directly linked to several outbreaks of infection. In the last 10 years considerable progress has been made in understanding the natural biology and clinical infections caused by this fascinating group of bacteria.

Beware biofilm! Dry biofilms containing bacterial pathogens on multiple healthcare surfaces; a multi-centre study.

BACKGROUND: Wet biofilms associated with medical devices have been widely studied and their link with healthcare-associated infections (HCAIs) is well recognized. Little attention has been paid to the presence of dry biofilms on environmental surfaces in healthcare settings. AIM: To investigate the occurrence, prevalence, and diversity of dry biofilms on hospital surfaces. METHODS: Sixty-one terminally cleaned items were received from three different UK hospitals. The presence of dry biofilm was investigated using culture-based methods and scanning electron microscopy (SEM). Bacterial diversity within biofilms was investigated using ribosomal RNA intergenic spacer analysis (RISA)-polymerase chain reaction and next-generation sequencing. FINDINGS: Multi-species dry biofilms were recovered from 95% of 61 samples. Abundance and complexity of dry biofilms were confirmed by SEM. All biofilms harboured Gram-positive bacteria including pathogens associated with HCAI; 58% of samples grew meticillin-resistant Staphylococcus aureus. Dry biofilms had similar physical composition regardless of the type of items sampled or the ward from which the samples originated. There were differences observed in the dominance of particular species: dry biofilms from two hospitals contained mostly staphylococcal DNA, whereas more Bacillus spp. DNA was found on surfaces from the third hospital. CONCLUSION: The presence of dry biofilms harbouring bacterial pathogens is virtually universal on commonly used items in healthcare settings. The role of dry biofilms in spreading HCAIs may be underestimated. The risk may be further exacerbated by inefficient cleaning and disinfection practices for hospital surfaces.

Evidence of persistence of Prevotella spp. in the cystic fibrosis lung.

Purpose. Prevotella spp. represent a diverse genus of bacteria, frequently identified by both culture and molecular methods in the lungs of patients with chronic respiratory infection. However, their role in the pathogenesis of chronic lung infection is unclear; therefore, a more complete understanding of their molecular epidemiology is required.Methodology. Pulsed Field Gel Electrophoresis (PFGE) and Random Amplified Polymorphic DNA (RAPD) assays were developed and used to determine the degree of similarity between sequential isolates (n=42) from cystic fibrosis (CF) patients during periods of clinical stability and exacerbation.Results. A wide diversity of PFGE and RAPD banding patterns were observed, demonstrating considerable within-genus heterogeneity. In 8/12 (66.7 %) cases, where the same species was identified at sequential time points, pre- and post-antibiotic treatment of an exacerbation, PFGE/RAPD profiles were highly similar or identical. Congruence was observed between PFGE and RAPD (adjusted Rand coefficient, 0.200; adjusted Wallace RAPD->PFGE 0.459, PFGE->RAPD 0.128). Furthermore, some isolates could not be adequately assigned a species name on the basis of 16S rRNA analysis: these isolates had identical PFGE/RAPD profiles to Prevotella histicola.Conclusion. The similarity in PFGE and RAPD banding patterns observed in sequential CF Prevotella isolates may be indicative of the persistence of this genus in the CF lung. Further work is required to determine the clinical significance of this finding, and to more accurately distinguish differences in pathogenicity between species.

Role of swarming in the formation of crystalline Proteus mirabilis biofilms on urinary catheters.

The care of many patients undergoing long-term bladder catheterization is frequently complicated by infection with Proteus mirabilis. These organisms colonize the catheter, forming surface biofilm communities, and their urease activity generates alkaline conditions under which crystals of magnesium ammonium phosphate and calcium phosphate are formed and become trapped in the biofilm. As the biofilm develops it obstructs the flow of urine through the catheter, causing either incontinence due to leakage of urine around the catheter or retention of urine in the bladder. The aim of this study was to investigate the role of the surface-associated swarming motility of P. mirabilis in the initiation and development of these crystalline catheter biofilms. A set of stable transposon mutants with a range of swimming and swarming abilities were tested for their ability to colonize silicone surfaces in a parallel-plate flow cell. A laboratory model of the catheterized bladder was then used to examine their ability to form crystalline, catheter-blocking biofilms. The results showed that neither swarming nor swimming motility was required for the attachment of P. mirabilis to silicone. Mutants deficient in swarming and swimming were also capable of forming crystalline biofilms and blocking catheters more rapidly than the wild-type strain.

An episodic outbreak of genetically related Burkholderia cepacia among non-cystic fibrosis patients at a university hospital.

OBJECTIVE: To investigate an outbreak of Burkholderia cepacia. DESIGN: Observational study and chart review. PATIENTS: Adult non-cystic fibrosis (CF) patients. SETTING: Intensive care units (ICUs) at a university-affiliated teaching hospital. METHODS: As part of the epidemiological investigation, we conducted a chart review and collected environmental samples. A review of work schedules of healthcare workers also was performed. We used B. cepacia selective agar for preliminary screening for all isolates, which subsequently were confirmed as members of the B. cepacia complex by polyphasic analysis employing conventional biochemical reactions and genus- and species-specific polymerase chain reaction assays. Pulsed-field gel electrophoresis, randomly amplified polymorphic DNA typing, and automated ribotyping were used to genotype the isolates. As part of the intervention, contact isolation precautions were initiated for all patients identified as having had a culture positive for B. cepacia. RESULTS: Between September 1997 and September 1999, B. cepacia was isolated from 31 adult patients without CF in ICUs at a university-affiliated teaching hospital. Based on geographic clustering and genotypic analysis, three distinct clusters were observed involving 20 patients. Isolates from 17 of these patients were available for testing and were found to be of the same strain (outbreak strain). Further taxonomic analysis indicated that the outbreak strain was B. cepacia complex genomovar III. Twelve (71%) of the 17 patients were judged to be infected, and 5 (29%) were colonized with this strain. Six of 200 environmental cultures from multiple sources in the hospital's ICUs yielded B. cepacia. Two of these isolates, both recovered from rooms of colonized patients, were the same genotype as the outbreak strain recovered from patients. CONCLUSION: Despite an extensive investigation, the source of the B. cepacia clone involved in this outbreak remains unknown. The spatial and temporal pattern of cases suggests that cross-transmission of a genetically related strain contributed to clustering among patients. The initiation of contact isolation may have limited the extent of this transmission. Additional studies are needed to elucidate better the epidemiology of nosocomial B. cepacia infection among non-CF adult patients.

Burkholderia cepacia complex in cystic fibrosis and non-cystic fibrosis patients: identification of a cluster of epidemic lineages.

This study was performed in order to compare Burkholderia cepacia complex strains from cystic fibrosis (CF) and non-CF patients at the genomovar, genetic and epidemiological levels. A total of 92 B. cepacia respiratory tract isolates were obtained from patients attending the following CF centres: Catania and Palermo, Sicily; Gualdo Tadino, Central Italy, and Milan, Northern Italy. A total of 23 B. cepacia isolates were obtained from blood, surgical wound, and intravenous catheter sources of patients without CF, hospitalized in Catania and Varese, Northern Italy. Genomovar status identification, clonality and genetic relatedness determination, antibiotic susceptibility pattern determination and electron microscopy were performed. Transmission of infection was shown in both CF and non-CF patients by identifying clonality of responsible strains. In total 13 clones were involved in cross-transmission episodes. No outbreak was described involving both CF and non-CF patients. The present study indicates the existence of a distinct cluster of strains responsible for epidemics in CF and non-CF patients, based on their genetic relatedness, distinct from strains associated with no or negligible transmissibility. This result suggests that transmissibility is not only associated with a specific genomovar in CF patients, but also with a group of genetically related lineages in CF and non-CF patients. A key role is shown for both segregation measures and careful surveillance of infection, based on selective culture, molecular identification and epidemiological characterization of individual isolates.

Burkholderia cenocepacia in cystic fibrosis: epidemiology and molecular mechanisms of virulence.

Burkholderia cepacia complex (Bcc) bacteria have gained notoriety as pathogens in cystic fibrosis (CF) because they are difficult to identify and treat, and also have the ability to spread between CF individuals. Of the 17 formally named species within the complex, Burkholderia multivorans and Burkholderia cenocepacia dominate in CF. Multilocus sequence typing has proven to be a very useful tool for tracing the global epidemiology of Bcc bacteria and has shown that B. cenocepacia strains with high transmissibility, such as the ET-12 strain (ST-28) and the Czech strain (ST-32), have spread epidemically within CF populations in Canada and Europe. The majority of research on the molecular pathogenesis of Bcc bacteria has focused on the B. cenocepacia ET-12 epidemic lineage, with gene mutation, genome sequence analysis and, most recently, global gene expression studies shedding considerable light on the virulence and antimicrobial resistance of this pathogen. These studies demonstrate that the ability of B. cenocepacia to acquire foreign DNA (genomic islands, insertion sequences and other mobile elements), regulate gene expression via quorum sensing, compete for iron during infection, and mediate antimicrobial resistance and inflammation via its membrane and surface polysaccharides are key features that underpin the virulence of different strains. With the wealth of molecular knowledge acquired in the last decade on B. cenocepacia strains, we are now in a much better position to develop strategies for the treatment of pathogenic colonization with Bcc and to answer key questions on pathogenesis concerning, for example, the factors that trigger the rapid clinical decline in CF patients.

Functional foods and paediatric gastro-intestinal health and disease.

The application of molecular methods to gastro-intestinal diseases is giving insight into the way in which the resident intestinal microbiota interacts with the mucosal immune system. Using traditional culture techniques, the importance of mucosally-associated bacterial biofilms in maintaining mucosal integrity has been demonstrated in ways previously impossible. Changes in the balance of organisms at initiation of and during disease provide a rationale for interventions with functional foods which facilitate re-establishment of the homeostasis of healthy gut.

Taxonomy and pathogenesis of the Burkholderia cepacia complex.

Patients with cystic fibrosis (CF) are susceptible to chronic respiratory infection with a number of bacterial pathogens. The Burkholderia cepacia complex bacteria are problematic CF pathogens because (i) they are very resistant to antibiotics, making respiratory infection difficult to treat and eradicate; (ii) infection with these bacteria is associated with high mortality in CF; (iii) they may spread from one CF patient to another, leading to considerable problems for both patients and carers; and (iv) B. cepacia complex bacteria are difficult to identify and nine new species have now been found to constitute isolates originally identified as 'B. cepacia' based on their phenotypic properties. Here we review the changes that have occurred in the taxonomy of the B. cepacia complex and the pathogenic factors these bacteria possess. While the taxonomy of the B. cepacia complex has advanced considerably with the development of accurate methods for their identification, the pathogenic mechanisms employed by these CF pathogens are only just beginning to be explored at the molecular level. Several virulence factors have been defined for B. cenocepacia (the dominant CF pathogen within the complex); however, knowledge of the disease mechanisms employed by other B. cepacia complex species is limited. The recent determination of the complete genome sequences for several of the B. cepacia complex species should greatly enhance our ability to study these problematic CF pathogens.

Biotechnological potential within the genus Burkholderia.

Many species within the genus Burkholderia possess significant biotechnological potential in bioremediation and biological control. Here we provide a description of the Burkholderia strains being investigated for their ability to degrade major xenobiotic pollutants and update information on their taxonomy, metabolic capacity and genomes.

Nonopsonic phagocytosis of Pseudomonas aeruginosa by macrophages and polymorphonuclear leukocytes requires the presence of the bacterial flagellum.

Whereas the mechanism of nonopsonic phagocytosis of Pseudomonas aeruginosa has been described, the bacterial ligands required are poorly understood. To identify the requisite bacterial ligands, studies with isogenic mutants of P. aeruginosa PAK lacking pili, flagella, and the RpoN sigma factor were undertaken. The RpoN mutant, lacking pili, flagella, and nonpilus adhesins, bound poorly and was resistant to ingestion by both macrophages and neutrophils. Pili were not absolutely required for binding or phagocytosis of P. aeruginosa. The presence of a flagellum was not required for binding of P. aeruginosa to macrophages but was critical for the subsequent internalization of the bacterium, suggesting that this factor or a surface ligand associated with its assembly was responsible for stimulation of nonopsonic phagocytosis.

Evaluation of random amplified polymorphic DNA typing of Pseudomonas aeruginosa.

A method for distinguishing among Pseudomonas aeruginosa strains using random amplified polymorphic DNA (RAPD) typing was evaluated for reproducibility and discriminatory power. A total of 200 isolates, blinded in triplicate, were evaluated by RAPD. All 600 samples were typeable; 197 of 200 isolates gave identical results on all three occasions, and 131 distinct RAPD types were identified.

Identification and characterization of a novel DNA marker associated with epidemic Burkholderia cepacia strains recovered from patients with cystic fibrosis.

Burkholderia cepacia is a problematic pathogen that may spread among patients with cystic fibrosis (CF). One highly infectious CF strain that causes epidemics in both the United Kingdom and eastern Canada has been shown to possess both the cable pilin subunit gene (cblA) and a unique combination of insertion sequences. However, no genetic markers linking this strain type with other types epidemic at various centers have been identified. Using a randomly amplified polymorphic DNA (RAPD) typing scheme, we identified an apparently conserved 1.4-kb fragment in the DNA fingerprint of epidemic B. cepacia strains. Conservation of the DNA marker among epidemic strains was demonstrated by Southern hybridization, and its prevalence was assessed in a collection of chromosomal DNAs extracted from 627 isolates representative of 132 RAPD-defined B. cepacia strain types. The marker was specifically associated with seven epidemic CF strains, was absent among nonepidemic strains infecting individual patients with CF, and rare among strains recovered from the natural environment. Only one of the seven epidemic CF strain types possessed DNA homologous to cblA. The RAPD marker was designated the "B. cepacia epidemic strain marker" (BCESM). Sequence analysis of chromosomal DNA corresponding to the 1.4-kb RAPD marker revealed the presence of a putative open reading frame (ORF) with significant homology to several negative transcriptional regulators; the ORF was designated the "epidemic strain marker regulator," or esmR. The BCESM DNA is the first genetic marker that has been identified to be specifically associated with and conserved among several epidemic B. cepacia strains which infect multiple patients with CF.

Site-directed mutagenesis and virulence assessment of the katG gene of Mycobacterium intracellulare.

Mycobacterial catalases have been suggested as acting as virulence factors by protecting intracellular mycobacteria from reactive oxidative metabolites produced by host phagocytes. Mycobacterium intracellulare, like many other mycobacteria, produces two proteins with catalase activity: a heat-stable catalase (KatE) and an inducible, heat-labile catalase peroxidase (KatG). The M. intracellulare katG gene was cloned, and a plasmid derivative with a 4 bp insertion in the katG coding sequence was constructed and used for site-directed mutagenesis of M. intracellulare 1403 (ATCC 35761). The resulting katG mutant was highly resistant to isoniazid (INH), showed an increased sensitivity to H2O2 and had lost peroxidase and heat-sensitive catalase activity but retained heat-stable catalase activity. The plasmid carrying the katG frameshift allele was also used for mutagenesis of the mouse virulent M. intracellulare isolate D673. After intravenous injection into BALB/c mice, D673 and the isogenic katG mutant showed the same growth kinetics in the spleen, liver and lungs of the infected mice. Our results demonstrate that the KatG catalase peroxidase mediates resistance to H2O2 and susceptibility to INH but is not an essential virulence factor for the survival and growth of M. intracellulare in the mouse.

Nonmotility and phagocytic resistance of Pseudomonas aeruginosa isolates from chronically colonized patients with cystic fibrosis.

Although Pseudomonas aeruginosa chronically colonizes most older patients with cystic fibrosis (CF), bacterial features responsible for its persistence are understood poorly. We observed that many P. aeruginosa isolates from chronically colonized patients were nonmotile and resistant to phagocytosis by macrophages. P. aeruginosa isolates were collected from 20 CF patients for up to 10 years. Isolates from early colonization were highly motile and expressed both flagellin and pilin. However, many isolates from chronically colonized patients lacked flagellin expression and were nonmotile; a total of 1,030 P. aeruginosa CF isolates were examined, of which 39% were nonmotile. Moreover, sequential isolates recovered from several of the CF patients were consistently nonmotile for up to 10 years. Lack of motility was rare among environmental isolates (1.4%) and other clinical isolates (3.7%) of P. aeruginosa examined. Partial complementation of motility in nonmotile P. aeruginosa isolates was achieved by introduction of extra copies of the rpoN locus carried on plasmid pPT212, indicating that the alternate sigma factor, RpoN, may be involved in the coordinate regulation of virulence factors during CF infection. We hypothesize that the nonmotile phenotype may provide P. aeruginosa a survival advantage in chronic CF infection by enabling it to resist phagocytosis and conserve energy.