Standard Microbiological Media Research Articles

The chronic wound milieu changes essential oils' antibiofilm activity—an in vitro and larval model study

Essential Oils (EOs) are currently being researched as potential antibiofilm agents to combat infections related to chronic wound biofilms. As documented in the literature, EOs’ in vitro antibacterial properties are often assessed using standard microbiological media and conditions that do not accurately reflect the actual environment of a chronic wound. To address this issue, In vitro Wound Milieu (IVWM) medium, which closely resembles the environment of a chronic wound, was applied for culturing S. aureus biofilms (n = 12) in this research. Biofilms cultivated in the standard Tryptic Soy Broth (TSB) medium served as a control for the experiment. Key biofilm features were analyzed and compared. Subsequently, staphylococci were exposed to the activity of thyme or rosemary EOs (T-EO and R-EO, respectively). As proof of concept, the cytotoxicity of T-EO and its antimicrobial in vivo activity were assessed using a G. mellonella larvae model. Key features of biofilm-forming cells were lower in the IVWM than in the TSB medium: biomass (up to 8 times), metabolic activity (up to 9 times), cell number (up to 100 times), and the live/dead cells ratio. Conversely, biofilm thickness was higher (up to 25%) in IVWM. These differences translated into varied responses of the biofilms to EOs exposure. The application of T-EO led to a greater reduction (up to 2 times) in 67% of biofilm-forming strains in IVWM compared to the TSB medium. Conversely, exposure to R-EO resulted in a higher reduction (up to 2.6 times) of 83% of biofilm-forming strains in TSB than in IVWM. The application of T-EO was not only non-toxic to G. mellonella larvae but also increased the survival of larvae infected with staphylococci (from 48 to 85%). Our findings suggest that EOs not only show promise as agents for treating biofilm-related wound infections but also that providing conditions reflecting the specific niche of the human body is of paramount importance in influencing the results obtained. However, before clinical application, challenges related to the methods of assessing their activity, microbial intra-species variability, and different levels of activity of various EOs should be analyzed and standardized.

Cyclitol metabolism is a central feature of Burkholderia leaf symbionts.

The symbioses between plants of the Rubiaceae and Primulaceae families with Burkholderia bacteria represent unique and intimate plant-bacterial relationships. Many of these interactions have been identified through PCR-dependent typing methods, but there is little information available about their functional and ecological roles. We assembled 17 new endophyte genomes representing endophytes from 13 plant species, including those of two previously unknown associations. Genomes of leaf endophytes belonging to Burkholderia s.l. show extensive signs of genome reduction, albeit to varying degrees. Except for one endophyte, none of the bacterial symbionts could be isolated on standard microbiological media. Despite their taxonomic diversity, all endophyte genomes contained gene clusters linked to the production of specialized metabolites, including genes linked to cyclitol sugar analog metabolism and in one instance non-ribosomal peptide synthesis. These genes and gene clusters are unique within Burkholderia s.l. and are likely horizontally acquired. We propose that the acquisition of secondary metabolite gene clusters through horizontal gene transfer is a prerequisite for the evolution of a stable association between these endophytes and their hosts.

Salmonella enterica Infections Are Disrupted by Two Small Molecules That Accumulate within Phagosomes and Differentially Damage Bacterial Inner Membranes.

ABSTRACTGram-negative bacteria have a robust cell envelope that excludes or expels many antimicrobial agents. However, during infection, host soluble innate immune factors permeabilize the bacterial outer membrane. We identified two small molecules that exploit outer membrane damage to access the bacterial cell. In standard microbiological media, neither compound inhibited bacterial growth nor permeabilized bacterial outer membranes. In contrast, at micromolar concentrations, JAV1 and JAV2 enabled the killing of an intracellular human pathogen, Salmonella enterica serovar Typhimurium. S. Typhimurium is a Gram-negative bacterium that resides within phagosomes of cells from the monocyte lineage. Under broth conditions that destabilized the lipopolysaccharide layer, JAV2 permeabilized the bacterial inner membrane and was rapidly bactericidal. In contrast, JAV1 activity was more subtle: JAV1 increased membrane fluidity, altered reduction potential, and required more time than JAV2 to disrupt the inner membrane barrier and kill bacteria. Both compounds interacted with glycerophospholipids from Escherichia coli total lipid extract-based liposomes. JAV1 preferentially interacted with cardiolipin and partially relied on cardiolipin production for activity, whereas JAV2 generally interacted with lipids and had modest affinity for phosphatidylglycerol. In mammalian cells, neither compound significantly altered mitochondrial membrane potential at concentrations that killed S. Typhimurium. Instead, JAV1 and JAV2 became trapped within acidic compartments, including macrophage phagosomes. Both compounds improved survival of S. Typhimurium-infected Galleria mellonella larvae. Together, these data demonstrate that JAV1 and JAV2 disrupt bacterial inner membranes by distinct mechanisms and highlight how small, lipophilic, amine-substituted molecules can exploit host soluble innate immunity to facilitate the killing of intravesicular pathogens.

A small molecule that disrupts S. Typhimurium membrane voltage without cell lysis reduces bacterial colonization of mice.

As pathogenic bacteria become increasingly resistant to antibiotics, antimicrobials with mechanisms of action distinct from current clinical antibiotics are needed. Gram-negative bacteria pose a particular problem because they defend themselves against chemicals with a minimally permeable outer membrane and with efflux pumps. During infection, innate immune defense molecules increase bacterial vulnerability to chemicals by permeabilizing the outer membrane and occupying efflux pumps. Therefore, screens for compounds that reduce bacterial colonization of mammalian cells have the potential to reveal unexplored therapeutic avenues. Here we describe a new small molecule, D66, that prevents the survival of a human Gram-negative pathogen in macrophages. D66 inhibits bacterial growth under conditions wherein the bacterial outer membrane or efflux pumps are compromised, but not in standard microbiological media. The compound disrupts voltage across the bacterial inner membrane at concentrations that do not permeabilize the inner membrane or lyse cells. Selection for bacterial clones resistant to D66 activity suggested that outer membrane integrity and efflux are the two major bacterial defense mechanisms against this compound. Treatment of mammalian cells with D66 does not permeabilize the mammalian cell membrane but does cause stress, as revealed by hyperpolarization of mitochondrial membranes. Nevertheless, the compound is tolerated in mice and reduces bacterial tissue load. These data suggest that the inner membrane could be a viable target for anti-Gram-negative antimicrobials, and that disruption of bacterial membrane voltage without lysis is sufficient to enable clearance from the host.

ERADICATING PSEUDOMONAS PERSISTENCE IN ADULT PATIENTS WITH CYSTIC FIBROSIS WITH A NEW ALGORITHM OF ANTIBIOTIC SELECTION

TOPIC: Chest Infections TYPE: Original Investigations PURPOSE: Pseudomonas aeruginosa is a common pathogen, causing chronic and life-threatening infections in airways of patients with cystic fibrosis (CF). Multidrug-resistant P.aeruginosa (MDR-PA), is an important cause of persistent infections due to its ability to form biofilms which display tolerance and high resistance to antimicrobial agents Traditional antibiotic selection tests take days and fail to select effective antibiotics against MDR-PA because not accounting for critical characteristics of “real-life” infections. AtbFinder – a novel culture-based test system addresses these realities by using a novel principle of antibiotic selection, named “populational response”, and has previously proved to be highly effective in selecting antibiotics effective even against MDR bacteria in biofilms, delivering results in 4 hours. Here we present findings from 3-y long microbiological monitoring of P.aeruginosa persistence in patients with CF, that underwent antimicrobial treatment with antibiotics selected using AtbFinder. METHODS: 30 CF patients aged between 18 and 63, with confirmed P.aeruginosa chronic infection, receiving prophylactic antibiotic therapy with antibiotics selected based on AtbFinder results were monitored over 2 years. Sputum was obtained during yearly check-up hospitalisations and the presence of P.aeruginosa was assessed with (i)AtbFinder or (ii) standard microbiological media. RESULTS: First we analyzed the effect of antibiotic therapy selected with the AtbFinder on eradication of P.aeruginosa. By year 1 and 2 after the switch for the use of antibiotics selected with AtbFinder, eradication of P.aeruginosa were noted in 16 (53%) and 22 (73%) out of 30 subjects who had P.aeruginosa infection respectively (p<0.001). We next studied how antibiotics selected with AtbFinder, affected P.aeruginosa sputum load in patients who were still having P.aeruginosa infection. The mean sputum density of P.aeruginosa following a switch in antibiotic therapy selected based on standard methods to those selected with the AtbFinder from baseline (year 0) decreased by 3.2 and 4.1 log10 CFU/g for P.aeruginosa and at year 1 and 2, respectively. Moreover we were able to switch to narrow-spectrum antibiotics in over than 50% of patients. CONCLUSIONS: Our results represent the first study to evaluate the performance of the AtbFinder system in selecting antibiotics that account for the polymicrobial nature of infections within a routine diagnostic setting. Antibiotic optimization with AtbFinder results in progressive eradication of P.aeruginosa from airways of patients with CF. CLINICAL IMPLICATIONS: We have demonstrated that the antibiotics selected using the AtbFinder among patients with CF were more effective than the antibiotics selected for the same patients using routine ASTs. The antibiotics selected have significantly improved critical microbiological and clinical parameters, allowing for a reduction in unnecessary broad-spectrum antibiotic use. DISCLOSURES: No relevant relationships by Christina Kardava, source=Web Response No relevant relationships by George Tetz, source=Web Response No relevant relationships by Victor Tetz, source=Web Response No relevant relationships by Maria Vecherkovskaya, source=Web Response

Phenotypic Characterization of Bacteria Isolated from the Recreational Sites of Two Rivers in Orashi Region, Rivers State, Nigeria

Recreational water bodies are water bodies used for recreational activities such as swimming, surfing, water skiing, water diving and sailing. They include rivers, lakes, beaches, spas and swimming pools. This work was therefore aimed at determining the bacteriological profile of recreational water bodies in Orashi region of Rivers State, Nigeria. Surface water samples were collected from two different sites (Orashi River, Mbiama and Sombreiro River, Ahoada) using standard microbiological methods. Upstream, midstream and downstream samples were collected for a period of three months at monthly interval. Standard plate counts were used for total heterotrophic and coliform bacterial counts using standard microbiological media. The total heterotrophic bacterial count ranged from 4.1X104 to 9.5X104 for Orashi River and 3.0X103 to 4.0X103 for Sombreiro River. A significant statistical difference (p &lt; 0.05) however, existed between total heterotrophic bacterial counts of the samples collected from Orashi River, while no statistical difference (p &gt; 0.05) was observed in the total heterotrophic bacterial counts of samples from Sombreiro River. In the comparative analysis of the samples from the two water bodies, no statistical difference (p &gt; 0.05) was recorded in the total coliform count in Orashi and Sombreiro Rivers. The phenotypic characterization identified the isolates to include Staphylococcus spp., Klebsiella spp., Pseudomonas spp., Bacillus spp., Enterococcus spp. and Micrococcus spp., with Klebsiella pneumoniae as the most occurring (26.1%). Klebsiella pneumoniae and Staphylococcus aureus are known for their pathogenic potentials, hence their presence in these recreational sites are of public health importance. Provision of standard recreational facilities in localities will however reduce the dependency on river sites for recreational activities, and as well prevent recreational associated illnesses.

A case study of possible health hazards associated with poultry houses

This study on layer and broiler houses is expected to give basic information on the nature of microbes, their occurrence, health hazard they could constitute and the possibilities for disease control measures. Modern husbandry practices, state or local concentration of the industry, high stocking densities, uniform age distribution of birds and continuous feeding may promote the spread of poultry diseases. Illness due to contaminated food, poultry wastes, poultry and poultry by products are one of the most widespread problems of the contemporary world. From the poultry houses investigated, bacteria and fungi were isolated from swabs of window nets dust, feed stock, roof dust, faeces, floors, feeders, drinkers, feathers, cages and egg trays using standard microbiological media and biochemicals procedures.The isolates encountered include: Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus terreus, Streptococcus faecalis, Bacillus sp., Pasteurella gallinarum, Pasteurella multocida, Klebsiella sp., Eschericia coli, Salmonella sp., Pseudomonas aeruginosa, Yeast and Rhodotorula spp., which were nol typed. Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus terreus, Bacillus sp., Pasteurella gallinarum, Klebsiella sp., Eschericia coli, Yeast and Rhodotorula Sp., were present in both layers and broilers houses structures and materials in varying proportions. However, Pasteurella Multocida, Salmonella sp., and Pseudomonas aeruginosa were isolated from egg trays, feathers, faeces, and feeders respectively from poultry house materials only and none from the structures of layers and broilers houses. The layer house structures and materials tend to yield more microbes mir than broiler house structures and materials. There were no acid-fast organisms observed from the sediments of washed swabs materials for culture using Ziehl-Neelsen stain

A small molecule that mitigates bacterial infection disrupts Gram-negative cell membranes and is inhibited by cholesterol and neutral lipids.

Infections caused by Gram-negative bacteria are difficult to fight because these pathogens exclude or expel many clinical antibiotics and host defense molecules. However, mammals have evolved a substantial immune arsenal that weakens pathogen defenses, suggesting the feasibility of developing therapies that work in concert with innate immunity to kill Gram-negative bacteria. Using chemical genetics, we recently identified a small molecule, JD1, that kills Salmonella enterica serovar Typhimurium (S. Typhimurium) residing within macrophages. JD1 is not antibacterial in standard microbiological media, but rapidly inhibits growth and curtails bacterial survival under broth conditions that compromise the outer membrane or reduce efflux pump activity. Using a combination of cellular indicators and super resolution microscopy, we found that JD1 damaged bacterial cytoplasmic membranes by increasing fluidity, disrupting barrier function, and causing the formation of membrane distortions. We quantified macrophage cell membrane integrity and mitochondrial membrane potential and found that disruption of eukaryotic cell membranes required approximately 30-fold more JD1 than was needed to kill bacteria in macrophages. Moreover, JD1 preferentially damaged liposomes with compositions similar to E. coli inner membranes versus mammalian cell membranes. Cholesterol, a component of mammalian cell membranes, was protective in the presence of neutral lipids. In mice, intraperitoneal administration of JD1 reduced tissue colonization by S. Typhimurium. These observations indicate that during infection, JD1 gains access to and disrupts the cytoplasmic membrane of Gram-negative bacteria, and that neutral lipids and cholesterol protect mammalian membranes from JD1-mediated damage. Thus, it may be possible to develop therapeutics that exploit host innate immunity to gain access to Gram-negative bacteria and then preferentially damage the bacterial cell membrane over host membranes.

Genetic and Chemical Screening in Human Blood Serum Reveals Unique Antibacterial Targets and Compounds against Klebsiella pneumoniae

Antibiotics halt the growth of bacteria by targeting core, essential physiology that is required for life on standard microbiological media. Many more biochemical and virulence processes, however, are required for bacteria to cause infection in a host. Indeed, chemical inhibitors of the latter processes are overlooked using conventional antibiotic drug discovery approaches. Here, we use human blood serum as an alternative growth medium to explore new targets and compounds. High-throughput screening of genetic and chemical libraries identified compounds targeting biological activities required by Klebsiella pneumoniae to grow in serum, such as nucleobase biosynthesis and iron acquisition, and showed that serum can chemically transform compounds to reveal cryptic antibacterial activity. One of these compounds, ruthenium red, was effective in a rat bloodstream infection model. Our data demonstrate that human serum is an effective tool to find new chemical matter to address the current antibiotic resistance crisis.

Genetic and Chemical Screening in Human Blood Serum Reveals Unique Antibacterial Targets and Compounds Against &lt;i&gt;Klebsiella pneumoniae&lt;/i&gt;

Antibiotics halt the growth of bacteria in the laboratory by targeting core, essential physiology that is required for life on standard microbiological media. Many more biochemical and virulence processes, however, are required for bacteria to cause infection in a host. Indeed, chemical inhibitors of the latter processes are overlooked using conventional antibiotic drug discovery approaches. Here, we use human blood serum as an alternative growth medium to explore new targets and compounds. High-throughput screening of genetic and chemical libraries identified compounds targeting biological activities required by Klebsiella pneumoniae to grow in serum, such as nucleobase biosynthesis and iron acquisition, and showed that serum can chemically transform compounds to reveal cryptic antibacterial activity. One of these compounds, ruthenium red, was effective in a rat bloodstream infection model. Our data demonstrate that human serum is an effective tool to find new chemical matter to address the current antibiotic resistance crisis.

Bacterial aetiology of chronic otitis media with effusion in children - risk factors

BackgroundOtitis media with effusion (OME) may occur spontaneously because of poor Eustachian tube function or as an inflammatory response following AOM. Bacterial involvement in OME has been widely reported, with various available methods to identify pathogens from middle ear effusion, including traditional culture methods and polymerase chain reaction (PCR).ObjectivesThe primary goal of this study was to evaluate the bacteriological profile of middle ear effusion in OME. Risk factors of the bacterial OME aetiology were also identified.MethodsMiddle ear effusions (MEF) from 50 children, aged 2–8 years, diagnosed by ENT and undergoing routine tympanostomy tube placement were collected. MEF samples were streaked on standard microbiological media. Next, DNA was isolated from MEF samples and analysed with multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Alloiococcus otitidis.ResultsIn multiplex PCR assay 37 (74%) of 50 children were positive for at least one of the four microorganisms. In 27.0% positive children multiple bacterial pathogens were identified. A. otitidis was the most frequently identified in positive MEF children (59.5%). By multiplex PCR, H. influenzae, S. pneumoniae and M. catarrhalis were detected in 24, 18 and 8% of OME patients, respectively. There was significant association between bilateral infection and H. influenzae aetiology of OME.ConclusionsOverall we found OME predominantly a single otopathogen infection caused mainly by A. otitidis, which is difficult in identification using standard culture method, ahead to S. pneumoniae and H. influenzae. However, one third of MEF samples had multiple bacterial pathogens.

Simultaneous Detection of Multiple Wine-Spoilage Organisms Using a PCR-Based DNA Dipstick Assay.

Background: The presence of microbial contaminants such as Brettanomyces in wine can lead to undesirable wine. Therefore, monitoring for the presence of these spoilage organisms is critical for winemakers to ensure the quality of their end product. Objective: To address this problem, Molecular Epidemiology, Inc. (MEI, Seattle, WA) has developed a wine-spoilage organism detection kit consisting of a multiplex PCR DNA dipstick that simultaneously detects these organisms. Methods: Wine samples obtained from local wineries that tested negative by routine microbiological culture were spiked with the target microorganisms, while samples that were designated as spoiled by the wineries were used as-is without spiking for assessing the performance characteristics of the DNA dipstick assay. Microbial enumeration was performed following standard microbiological plating methods. Samples spiked with low cell numbers (<5 cells per 100 mL) were enriched using wine enrichment media (WSE; optional component of the kit) prior to analysis using the DNA dipstick assay. Suitability of WSE medium to support the growth of wine-spoilage microorganisms was compared with standard microbiological media. Results: Testing of 92 diverse bacterial and yeast strains commonly found in winery and food operations and 50 various strains of spoilage organisms isolated from wineries indicated that the dipstick assay can exclusively detect the target wine-spoilage microorganisms. All target spoilage organisms in samples containing low cell numbers (<5 cells per 100 mL) were detected by dipstick assay 48 h postenrichment in WSE, except for a few strains of Brettanomyces bruxellensis that required longer incubation times. Conclusions: The wine-spoilage organism detection kit has a detection limit of 10 cells/mL. Highlights: The kit can be used at different stages of the wine-making process to detect multiple spoilage-causing microorganisms in a single assay, thus offering a convenient test system for winemakers interested in monitoring the quality of their product.

A cell-based infection assay identifies efflux pump modulators that reduce bacterial intracellular load.

Bacterial efflux pumps transport small molecules from the cytoplasm or periplasm outside the cell. Efflux pump activity is typically increased in multi-drug resistant (MDR) pathogens; chemicals that inhibit efflux pumps may have potential for antibiotic development. Using an in-cell screen, we identified three efflux pump modulators (EPMs) from a drug diversity library. The screening platform uses macrophages infected with the human Gram-negative pathogen Salmonella enterica (Salmonella) to identify small molecules that prevent bacterial replication or survival within the host environment. A secondary screen for hit compounds that increase the accumulation of an efflux pump substrate, Hoechst 33342, identified three small molecules with activity comparable to the known efflux pump inhibitor PAβN (Phe-Arg β-naphthylamide). The three putative EPMs demonstrated significant antibacterial activity against Salmonella within primary and cell culture macrophages and within a human epithelial cell line. Unlike traditional antibiotics, the three compounds did not inhibit bacterial growth in standard microbiological media. The three compounds prevented energy-dependent efflux pump activity in Salmonella and bound the AcrB subunit of the AcrAB-TolC efflux system with KDs in the micromolar range. Moreover, the EPMs display antibacterial synergy with antimicrobial peptides, a class of host innate immune defense molecules present in body fluids and cells. The EPMs also had synergistic activity with antibiotics exported by AcrAB-TolC in broth and in macrophages and inhibited efflux pump activity in MDR Gram-negative ESKAPE clinical isolates. Thus, an in-cell screening approach identified EPMs that synergize with innate immunity to kill bacteria and have potential for development as adjuvants to antibiotics.

Identity tags : a vector for cross-infection? : research

Background. Nosocomial infections represent one of the challenging problems of modern medicine. Healthcare providers play an important role in the transmission of these infections on their hands, clothing and equipment. Modern security systems require personnel to wear clearly displayed identity (ID) tags, and to have an easily accessible access disc. These access and ID tags are often worn around the neck on a lanyard, and could possibly harbour bacteria and be a vector for cross-infection. Method. Saline-moistened swabs of the front and back of ID tags of 50 healthcare workers were taken for bacterial culture. Swabs were inoculated onto standard microbiological media. Potential pathogens were subjected to sensitivity testing while organisms resembling normal skin commensals were reported as such. Results. Twenty-eight of the 50 (56%) ID swabs cultured exhibited no bacterial growth. Eighteen (36%) swabs grew primarily skin flora. Neutrophils were observed under microscopy on two (4%) swabs. Seven (14%) swabs grew potentially pathogenic bacteria. Doctors were found to have almost three times the risk of carrying pathogenic bacteria on their ID tags compared with nurses. Recent patient contact also showed a higher incidence of colonisation. There were no statistically significant differences between variables such as ward or area of work, nature of patient contact, time since qualification, level of qualification or length of employment at Red Cross War Memorial Children's Hospital, Cape Town, South Africa. Conclusions. Prevention of hospital-acquired infections is important in any setting. The ID tag has been identified as a possible source of infection spread in this and previous studies. The ID tag has to date been neglected as a potential source of pathogen spread, and efforts to make staff aware of this potential danger should be considered in every institution.

Identity tags: A vector for cross-infection?

Nosocomial infections represent one of the challenging problems of modern medicine. Healthcare providers play an important role in the transmission of these infections on their hands, clothing and equipment. Modern security systems require personnel to wear clearly displayed identity (ID) tags, and to have an easily accessible access disc. These access and ID tags are often worn around the neck on a lanyard, and could possibly harbour bacteria and be a vector for cross-infection. Saline-moistened swabs of the front and back of ID tags of 50 healthcare workers were taken for bacterial culture. Swabs were inoculated onto standard microbiological media. Potential pathogens were subjected to sensitivity testing while organisms resembling normal skin commensals were reported as such. Twenty-eight of the 50 (56%) ID swabs cultured exhibited no bacterial growth. Eighteen (36%) swabs grew primarily skin flora. Neutrophils were observed under microscopy on two (4%) swabs. Seven (14%) swabs grew potentially pathogenic bacteria. Doctors were found to have almost three times the risk of carrying pathogenic bacteria on their ID tags compared with nurses. Recent patient contact also showed a higher incidence of colonisation. There were no statistically significant differences between variables such as ward or area of work, nature of patient contact, time since qualification, level of qualification or length of employment at Red Cross War Memorial Children's Hospital, Cape Town, South Africa. Prevention of hospital-acquired infections is important in any setting. The ID tag has been identified as a possible source of infection spread in this and previous studies. The ID tag has to date been neglected as a potential source of pathogen spread, and efforts to make staff aware of this potential danger should be considered in every institution.

Sphingomonas sp. is a novel cell culture contaminant.

A novel contaminant was isolated from Madin Darby Bovine Kidney (MDBK) cells. The organism was unable to grow on standard microbiological media by conventional techniques, but grew well in Dulbecco's Modified Eagle's Medium (DMEM) containing high glucose concentration. The organism formed a white biofilm on the bottom without any signs of turbidity. Upon genome sequence analysis of 16 S rDNA, the contaminant was identified as Sphingomonas sp. Shah, a member of the group α-Proteobacteria. Neutral red dye uptake method confirmed clear cytotoxic potential of the bacterium on A-549 cells. The organism was capable of invading and infecting different mammalian cell lines: MDBK, ZZ-R, 293-T, A549, and HeLa cells. Infected cells showed a variety of cytopathic effects including vacuolation at perinuclear area, cytoplasmic granulation and membrane blebbing. Microscopic analysis of the infected cells revealed the presence of cytoplasmic vacuoles harboring motile organisms. Apparently local serum preparations seem to be the source of this contamination, which is imperceptibly passed on from one culture passage to the other and ultimately leading to serious cytopathic manifestations.

Oral microflora and their relation to risk factors in HIV+ patients with oropharyngeal candidiasis

The purpose of this study was to determine the prevalence of oral microflora and association of oral candidiasis and multiple risk factors in HIV(+) patients. The present study included 100 HIV-infected patients participated in Imam Khomeini Hospital, Tehran, Iran for Oropharyngeal candidiasis (OPC) and HIV. We assessed the presence or absence of OPC, and samples were obtained from the oral cavity and direct microscopic examination, gram staining and culture on standard microbiological media were performed in all patients. CD4(+) cell count/CD4(+) percentage were also calculated. The demographic characteristics showed that the patients had a mean age of 32.3 years old, 78% male and 22% female. Patients belonging to 'O(+)' blood group (27%) were more prone to develop OPC. A total of 460 bacterial colonies were obtained and Streptococcus mutans (15.4%) was the most frequently isolated species in the HIV(+) patients, followed by Staphylococcus epidermidis (12.8%) and Corynebacterium (8.7%). In addition, 254 yeasts (from four different genera) were isolated from the patient under study. Candida species (94.4%) were the most frequently obtained genera, followed by Saccharomyces (2.4%), Kluyveromyces and Cryptococcus (1.6% for both) species. Candida albicans (37.2%) was the most common species isolated from HIV(+) patients with OPC and its frequency was significantly higher than that of other Candida species (P<0.05). Candida glabrata, C. dubliniensis, C. tropicalis, C. parapsilosis, C. krusei, C. lusitaniae, C. guilliermondii and C. norvegensis were also identified. Forty percent of the patients had angular cheilitis as the most frequent clinical variant. The mean CD4(+) cell counts were 154.5 cells/μL, with a range of 8 to 611 cells/μL. Thirty percent patients had a CD4(+) cell count between 101 and 200 cells/μL (28.7% of total yeasts isolated). Yeast and bacteria counts did not differ statistically among HIV(+) patients' subgroups with different levels of CD4(+) cells counts. Our results showed that yeasts of the genus Candida were isolated at a comparable rate from the oral cavity of HIV(+) patients and there was no significant difference of the variables CD4(+) cell count and yeast counts. The findings of this study would be helpful in any further study, which, if done prospectively on a large cohort, can be confirmatory.

Bedbugs as Vectors for Drug-Resistant Bacteria

To the Editor: Over the past 10 years in North America and western Europe, a resurgence of bedbugs (Cimex lectularius) has occurred (1). Although the basis for this resurgence is unclear, large bedbug infestations have been attributed to increased worldwide travel, altered insecticide management, and increased resistance to pesticides (2). Marginalized populations in large urban centers appear to be disproportionately affected; ≈30% of shelters in Toronto, Ontario, Canada, report bedbugs (1). We report recovery of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) from bedbugs in Vancouver, British Columbia. Three patients, all residents of Vancouver’s Downtown Eastside—an impoverished community in Vancouver with high rates of homelessness, poverty, HIV/AIDS, and injection drug use (3)—were hospitalized and found to be infested with bedbugs. Hypothesizing that these parasites may be vectors for the transmission of antimicrobial drug–resistant pathogens, we collected 5 bedbugs and tested them for drug-resistant organisms. The bedbugs were homogenized and streaked onto standard microbiological media, including 5% sheep blood agar. Bacterial colonies were identified by using conventional and automated microbiological methods, and susceptibility testing was performed in accordance with Clinical Laboratory Standards Institute guidelines (4). For 2 patients, VRE was isolated from 1 bedbug each. These bacterial isolates were also resistant to ampicillin, teicoplanin, and aminoglycosides but susceptible to linezolid, quinupristin/dalfopristin, and tetracycline. For 1 other patient, MRSA was isolated from 3 bedbugs. All MRSA isolates had susceptibility patterns consistent with pulsed-field gel electrophoresis type USA300 (susceptible to vancomycin, clindamycin, trimethoprim/sulfamethoxasole, tetracycline, and rifampin; resistant to erythromycin). Despite investigations of transmissibility of numerous infectious agents, including transmissible blood-borne pathogens such as HIV and hepatitis B and C viruses, to our knowledge, no conclusive evidence has demonstrated disease transmission by bedbugs (5,6). Clinically, bedbug bites have been associated with cutaneous manifestations, most commonly pruritic wheals with a central hemorrhagic punctum (6). Excoriation at the site of a bite can cause further skin abrasion, thereby providing an entry point for colonizing bacteria, which can potentially result in folliculitic or cellulitic superinfections (5). S. aureus, which is commonly found on the skin and can cause cellulitis, has been reported to colonize the salivary glands of bedbugs for as long as 15 days (7). However, although transient and persistent forms of colonization may play a role in disease transmission, we did not differentiate these forms because the clinical bedbug specimens were processed at the time of receipt in the laboratory. Similar to other cities worldwide, Vancouver has seen an alarming increase in bedbugs, particularly in the Downtown Eastside, where 31% of residents have reported bedbug infestation (8). Enterococci commensally occupy the gastrointestinal tract. The recovery of VRE from bedbugs could result from the relatively unhygienic living conditions encountered in the Downtown Eastside and the high level of interconnectedness between St. Paul’s Hospital (where a large pool of patients with VRE colonization/infection reside) and the surrounding community. MRSA is also a substantial problem in the Downtown Eastside; it has been cultured from 54.8% of skin and soft tissue infections of patients seen at the emergency department of St. Paul’s Hospital (9). More recently, among wound infections in injection drug users in this community, 43% were colonized or infected with MRSA consistent with USA300, the predominant community-associated MRSA strain in Vancouver (10). The phenotype of the MRSA recovered from the bedbugs was consistent with community-associated MRSA and identical to that found on antibiograms from patients with MRSA infection who reside in this community. Given the high prevalence of MRSA (particularly USA300) in hotels and rooming houses in Vancouver’s Downtown Eastside, bedbugs may become colonized with community-associated MRSA. Consequently, these insects may act as a hidden environmental reservoir for MRSA and may promote the spread of MRSA in impoverished and overcrowded communities. Bedbugs carrying MRSA and/or VRE may have the potential to act as vectors for transmission. Further studies are needed to characterize the association between S. aureus and bedbugs. Bedbug carriage of MRSA, and the portal of entry provided through feeding, suggests a plausible potential mechanism for passive transmission of bacteria during a blood meal. Because of the insect’s ability to compromise the skin integrity of its host, and the propensity for S. aureus to invade damaged skin, bedbugs may serve to amplify MRSA infections in impoverished urban communities.

An ancient but promiscuous host–symbiont association between Burkholderia gut symbionts and their heteropteran hosts

Here, we investigated 124 stinkbug species representing 20 families and 5 superfamilies for their Burkholderia gut symbionts, of which 39 species representing 6 families of the superfamilies Lygaeoidea and Coreoidea were Burkholderia-positive. Diagnostic PCR surveys revealed high frequencies of Burkholderia infection in natural populations of the stinkbugs, and substantial absence of vertical transmission of Burkholderia infection to their eggs. In situ hybridization confirmed localization of the Burkholderia in their midgut crypts. In the lygaeoid and coreoid stinkbugs, development of midgut crypts in their alimentary tract was coincident with the Burkholderia infection, suggesting that the specialized morphological configuration is pivotal for establishment and maintenance of the symbiotic association. The Burkholderia symbionts were easily isolated as pure culture on standard microbiological media, indicating the ability of the gut symbionts to survive outside the host insects. Molecular phylogenetic analysis showed that the gut symbionts of the lygaeoid and coreoid stinkbugs belong to a β-proteobacterial clade together with Burkholderia isolates from soil environments and Burkholderia species that induce plant galls. On the phylogeny, the stinkbug-associated, environmental and gall-forming Burkholderia strains did not form coherent groups, indicating host-symbiont promiscuity among these stinkbugs. Symbiont culturing revealed that slightly different Burkholderia genotypes often coexist in the same insects, which is also suggestive of host-symbiont promiscuity. All these results strongly suggest an ancient but promiscuous host-symbiont relationship between the lygaeoid/coreoid stinkbugs and the Burkholderia gut symbionts. Possible mechanisms as to how the environmentally transmitted promiscuous symbiotic association has been stably maintained in the evolutionary course are discussed.

FLUORESCENT MEASUREMENTS OF DNA, RNA AND PROTEINS TO PERFORM COMPARATIVE ANALYSES OF MICROBIAL COMMUNITIES FROM THE ENVIRONMENTS

ABSTRACT Simple and rapid methods for the quantification of DNA, RNA and proteins using specific fluorescent dyes are proposed for the comparison and monitoring of microbial communities from the environment. The purpose of this study was the use of straightforward in situ methods which voided the need for preservation of samples and the risk of potential degradation and quantitative changes during transportation. Aside from this, methods used to obtain information on environmental microbial communities are generally time‐consuming and present certain difficulty above all when working on solid substrates such as soils and rocks. New generation fluorescent dyes that bind specifically to DNA, RNA and proteins allow simple and rapid estimates of these biomolecules in crude environmental samples.PRACTICAL APPLICATIONSMonitoring the metabolic state of microbial communities on different substrates and environments is a requirement for comparing samples and assessing the participation of microorganisms in a variety of processes. Solid substrates are not easily analyzed by microscopic techniques and they require long processing times and tedious work. Aside from this, only a minor fraction (&lt;1%) of microorganisms in most natural environments can be cultured in standard microbiological media (Ward et al. 1990). Other studies using incorporation of labeled substrates to approach activity rates or biomolecule extractions represent complex and long procedures during environmental studies.In order to evaluate microbial communities in a variety of substrates and environments, rapid and simple methods are proposed by measuring DNA, RNA and/or proteins using specific fluorescent dyes, without a need for prior purification, from crude solid samples.