Detection and quantification of viable airborne bacteria and fungi using solid-phase cytometry

This protocol describes the use of solid-phase cytometry for the enumeration of airborne bacteria and fungi. In contrast with conventional methods, accurate results can be obtained in real time, especially for air samples with low numbers of microorganisms. Air samples are collected by impaction on a water-soluble polymer that is subsequently dissolved. Part of the sample can be filtered over two membrane filters with different pore sizes. One filter is used to obtain a total count of all viable microorganisms, and a second filter is used to determine the number of airborne fungi. Microorganisms present on the filter are labeled with a viability substrate and subsequently detected and quantified using a solid-phase cytometer. The detected spots are microscopically validated using an epifluorescence microscope to discriminate between bacteria, fungi and fluorescent particles. The whole procedure takes 5 h to complete and results in the accurate quantification of airborne bacteria and fungi for samples with a low or high microbial load

In vitro activity of ceftazidime, ciprofloxacin, meropenem, minocycline, tobramycin and trimethoprim/sulfamethoxazole against planktonic and sessile Burkholderia cepacia complex bacteria

Objectives: The goal of the present study was to obtain a comprehensive overview of the bacteriostatic and bactericidal effects of six commonly used antibiotics on planktonic as well as on sessile Burkholderia cepacia complex cells. Methods: The bacteriostatic and bactericidal activities of ceftazidime, ciprofloxacin, meropenem, minocycline, tobramycin and trimethoprim/sulfamethoxazole were determined against 38 B. cepacia complex strains. MICs and minimal biofilm inhibitory concentrations (MBICs) were determined using a traditional broth microdilution method and a novel resazurin-based viability staining, respectively. The bactericidal effects of the investigated antibiotics (using antibiotic concentrations corresponding to 10 x MIC; except for tobramycin, for which a final concentration of 4 x MIC was tested) on stationary phase planktonic cultures and on 24-h-old biofilms were evaluated using conventional plate count methods. Results: Our results confirm the innate resistance of B. cepacia complex organisms to six first-line antibiotics used to treat infected cystic fibrosis patients. All antibiotics showed similar bacteriostatic activities against exponentially growing B. cepacia complex planktonic cells and freshly adhered sessile cells (4 h). In addition, most of the antibiotics showed similar bactericidal effects on stationary phase planktonic cultures and on young and older biofilms. Conclusions: Despite the general assumption that sessile cells show a decreased susceptibility to antibiotics, our data indicate similar bacteriostatic and bactericidal activity of six selected antibiotics against planktonic and sessile B. cepacia complex bacteria

Transcriptional response of Burkholderia cenocepacia J2315 sessile cells to treatments with high doses of hydrogen peroxide and sodium hypochlorite

Background: Burkholderia cepacia complex bacteria are opportunistic pathogens, which can cause severe respiratory tract infections in patients with cystic fibrosis (CF). As treatment of infected CF patients is problematic, multiple preventive measures are taken to reduce the infection risk. Besides a stringent segregation policy to prevent patient-to-patient transmission, clinicians also advise patients to clean and disinfect their respiratory equipment on a regular basis. However, problems regarding the efficacy of several disinfection procedures for the removal and/or killing of B. cepacia complex bacteria have been reported. In order to unravel the molecular mechanisms involved in the resistance of biofilm-grown Burkholderia cenocepacia cells against high concentrations of reactive oxygen species (ROS), the present study focussed on the transcriptional response in sessile B. cenocepacia J2315 cells following exposure to high levels of H2O2 or NaOCl. Results: The exposure to H2O2 and NaOCl resulted in an upregulation of the transcription of 315 (4.4%) and 386 (5.4%) genes, respectively. Transcription of 185 (2.6%) and 331 (4.6%) genes was decreased in response to the respective treatments. Many of the upregulated genes in the NaOCl- and H2O2-treated biofilms are involved in oxidative stress as well as general stress response, emphasizing the importance of the efficient neutralization and scavenging of ROS. In addition, multiple upregulated genes encode proteins that are necessary to repair ROS-induced cellular damage. Unexpectedly, a prolonged treatment with H2O2 also resulted in an increased transcription of multiple phage-related genes. A closer inspection of hybridisation signals obtained with probes targeting intergenic regions led to the identification of a putative 6S RNA. Conclusion: Our results reveal that the transcription of a large fraction of B. cenocepacia J2315 genes is altered upon exposure of sessile cells to ROS. These observations have highlighted that B. cenocepacia may alter several pathways in response to exposure to ROS and they have led to the identification of many genes not previously implicated in the stress response of this pathogen

Comparison of multiple typing methods for Aspergillus fumigatus

As part of studies on the spread of infections, risk factors and prevention, several typing methods were developed to investigate the epidemiology of Aspergillus fumigatus. In the present study, 52 clinical isolates of A. fumigatus from 12 airway specimens from patients with invasive aspergillosis (hospitalized in three different centres) were characterized by short tandem repeat (STR) typing and multilocus sequence typing (MLST). These isolates were previously typed by random amplified polymorphic DNA (RAPD), sequence-specific DNA polymorphism (SSDP), microsatellite polymorphism (MSP) and multilocus enzyme electrophoresis (MLEE). STR typing identified 30 genotypes and, for most patients, all isolates were grouped in one cluster of the unweighted pair group method with arithmetic mean dendrogram. Using MLST, 16 genotypes were identified among 50 isolates, while two isolates appeared untypeable. RAPD, MSP, SSDP and MLEE allowed identification of eight, 14, nine and eight genotypes, respectively. Combining the results of these methods led to the delineation of 25 genotypes and a similar clustering pattern as with STR typing. In general, STR typing led to similar results to the previous combination of RAPD, SSDP, MSP and MLEE, but had a higher resolution, whereas MLST was less discriminatory and resulted in a totally different clustering pattern. Therefore, this study suggests the use of STR typing for research concerning the local epidemiology of A. fumigatus, which requires a high discriminatory power

Draft genome sequence of methicillin-resistant Staphylococcus epidermidis strain ET-024, isolated from an endotracheal tube biofilm of a mechanically ventilated patient

Staphylococcus epidermidis strain ET-024 was isolated from a biofilm on an endotracheal tube of a mechanically ventilated patient. This strain is resistant to methicillin and the draft genome sequence shares some characteristics with other nosocomial S. epidermidis strains (such as S. epidermidis RP62A)

Fungicidal activity of miconazole against Candida spp. biofilms

Although azole antifungals are considered to be fungistatic, miconazole has fungicidal activity against planktonic Candida albicans cells, presumably associated with the induction of reactive oxygen species (ROS) production. Only few data are available concerning the effect of miconazole against sessile C. albicans cells. In the present study, the fungicidal activity of miconazole against in vitro-grown mature Candida biofilms, and its relationship with the induction of ROS and ROS-dependent apoptosis were examined. The effect of miconazole on mature biofilms formed by 10 C. albicans strains and 5 strains from other Candida species was evaluated by plate counting and measuring the level of ROS induction. MIC tests were performed in the absence and presence of ascorbic acid, a quencher of ROS. The apoptotic population in C. albicans cells was determined using annexin-Cy3. Miconazole showed a significant fungicidal effect against all mature Candida biofilms tested and caused elevated ROS levels, both in planktonic and sessile cells. Addition of ascorbic acid drastically reduced these levels. While ROS quenching decreased the susceptibility to miconazole of planktonic cells of most Candida strains, no reduced fungicidal activity of miconazole against biofilms was observed. Miconazole did not cause a significant increase in apoptosis. ROS levels increased in all Candida biofilms upon addition of miconazole. However, ROS induction was not the only factor that underlies its fungicidal activity, as quenching of ROS did not lead to an enhanced survival of biofilm cells. ROS-induced apoptosis was not observed in C. albicans cells after miconazole treatment