Microbiological Contamination of Hospital Air: II. Qualitative Studies

Over 10,000 airborne microorganisms, isolated from various areas of two hospitals, were characterized according to colonial and microscopic morphology and certain physiological reactions, including penicillin resistance and hemolysis. On the basis of all isolates examined during a 15-month period, 42.6% were gram-positive cocci, 19.2% were gram-positive rods, 14.0% were gram-negative rods, 17.1% were molds, 2.2% were actinomycetes, 1.2% were yeasts, and the remainder were assorted diphtheroids and coccobacillary types. The distribution of types varied according to hospital area, locations within a given area, and level of gross airborne contamination, but did not vary significantly with season of the year. There appeared to be some relationship between contaminant particle size and type of organism associated with the particle. Distribution of penicillin-resistant types differed markedly in different hospital areas, with proportions ranging from 21.4% in surgery areas to 4.3% in incinerator rooms. Of all gram-positive cocci isolated, 34.9% were hemolytic, and 16.4% were penicillin-resistant.     

Monitoring airborne fungal spores in an experimental indoor environment to evaluate sampling methods and the effects of human activity on air sampling.

Aerobiological monitoring was conducted in an experimental room to aid in the development of standardized sampling protocols for airborne microorganisms in the indoor environment. The objectives of this research were to evaluate the relative efficiencies of selected sampling methods for the retrieval of airborne fungal spores and to determine the effect of human activity on air sampling. Dry aerosols containing known concentrations of Penicillium chrysogenum spores were generated, and air samples were taken by using Andersen six-stage, Surface Air System, Burkard, and depositional samplers. The Andersen and Burkard samplers retrieved the highest numbers of spores compared with the measurement standard, an aerodynamic particle sizer located inside the room. Data from paired samplers demonstrated that the Andersen sampler had the highest levels of sensitivity and repeatability. With a carpet as the source of P. chrysogenum spores, the effects of human activity (walking or vacuuming near the sampling site) on air sampling were also examined. Air samples were taken under undisturbed conditions and after human activity in the room. Human activity resulted in retrieval of significantly higher concentrations of airborne spores. Surface sampling of the carpet revealed moderate to heavy contamination despite relatively low airborne counts. Therefore, in certain situations, air sampling without concomitant surface sampling may not adequately reflect the level of microbial contamination in indoor environments.     

Relationship between mycobiota in wall-to-wall carpet dust and age of carpet

The objective of this study was to evaluate thefungal concentrations within carpets of the same typeand manufacture during their first 2 years of life.The carpets tested came from several different branchoffices of a bank.A total of 60 dust samples from 20 carpets (3samples per carpet) was analysed. The age of thecarpets varied from 50 to 666 days. Analysis of therelationship between fungal concentration and time wasmade by the Pearson test.Fungal concentrations were very different withina same carpet. Moreover, species distribution was alsoheterogeneous; thus the fungi in 1 m² carpet didnot reflect mycobiota of the whole carpet. Very localfungal contaminations (>10 CFU/mg/dust) were builtup in a relatively short time (138 days). Thehighest concentrations (maximum: 200 CFU/mg dust) weremainly due to species of coelomycetes (mitosporicfungi forming conidia in closed bodies) and to variousyeasts. Comparisons of the 20 carpets sampled did notshow significant differences; the fungal level did notincrease with time (P > 0.05). A decrease waseven noticed. The age of carpets had no significanteffect either on the number of CFU/mg dust of the mainfungal taxa (P > 0.10).It was concluded that these carpets, which wereregularly cleaned, in this particular environment, donot favour important fungal growth. Moreover, sincethe species responsible for the highest contaminationobserved produce conidia or cells not easilytransported through the air, their role in respiratoryallergy would be relatively limited.     

Optimisation of a screening procedure for house dust mite numbers in carpets and preliminary application to buildings

The relative performances of the mobility and heat escape methods for measuring house dust mite numbers in carpet has been evaluated. The optimum method was found to be the mobility method for 24 hours at ambient temperatures which exhibited a mite collection efficiency of approximately 30%. Measurements in three dwellings showed that the method should be applied to carpet at several locations in the living room and/or bedrooms as a general sampling procedure, or adjacent to loungeroom seating to determine the worst case scenario. Carpets using different types of fibre within the same dwelling should be assessed separately. For the limited number of dwellings investigated, wool carpets were found to exhibit higher mite numbers than nylon carpets, even when the former had been insect-resist treated. No house dust mites were found in the wool carpets of an office building which was mechanically ventilated and heated and achieved low indoor humidities in winter.     

Comparison of frequency and susceptibility to antimicrobials of bacterial strains of the family Enterobacteriaceae isolated from patients hospitalised in intensive care units

The aim of the study was estimation of frequency and susceptibility to antimicrobial agents of gram-negative rods isolated from clinical specimens obtained from patients requiring intensive care, with emphasis on profile of the unit. The analysis comprised strains of gram-negative rods isolated from patients of two intensive care units (ICUs) of a tertiary care hospital (1200 beds). Identification of cultured isolates was done using automated VITEK and API systems (bioMerieux, France). Susceptibility to antimicrobial agents was tested by a disk-diffusion method according to the NCCLS recommendations. In total the analysis comprised 722 strains of gram-negative rods. In blood cultures predominated strains of Enterobacter spp. (42.5%) and Klebsiella pneumoniae (37.5%). In cultures of clinical specimens other than blood 41.6% comprised strains of Klebsiella pneumoniae, 14.8% Escherichia coli and 14.4% Proteus mirabilis. Frequency of multi-drug resistant strains of bacteria of the family Enterobacteriaceae was much higher among blood isolates in comparison to strains cultured from other clinical specimens. There was a relatively high percentage of strains of Enterobacteriaceae susceptible to piperacillin and tazobactam (69.0%) and ceftazidime (54.6%). Conclusions: 1. All strains were susceptible to carbapenems. 2. There was a relatively high percentage of strains of gram-negative rods susceptible to piperacillin/tazobactam and ceftazidime. 3. Bacteria isolated from blood cultures were characterised by a much higher percentage of resistant strains in comparison to other specimens. 4. Longer stay in ICU promoted selection of strains resistant to antimicrobials.     

Comparison of Microbial Contamination Levels Among Hospital Operating Rooms and Industrial Clean Rooms

Microbial contamination in industrial clean rooms was compared quantitatively and qualitatively with that of hospital operating rooms. The number of aerobic mesophilic microorganisms which accumulated on stainless-steel strips exposed for periods up to 21 weeks to the intramural air of four operating rooms was at least 1 log higher than the accumulation on strips exposed in four clean rooms, and was essentially the same as that found in two factory areas. Volumetric air samplings showed that there were significantly higher numbers of airborne viable particles per cubic foot of air in operating rooms than in industrial clean rooms. In contrast to clean rooms, where most of the airborne contaminants were those associated with human hair, skin, and respiratory tract, the hospital operating rooms showed a very high level of microorganisms associated with dust and soil.     

Hydrophobic properties of gram-negative rods colonizing upper respiratory tract of healthy people

The cell surface hydrophobicity is one of the non specific factors of adhesion influencing the ability of microorganisms to colonize nasopharynx. The aim of this paper was to evaluate via salt aggregation test (SAT) the cell surface hydrophobicity of 150 strains of gram-negative rods isolated from the throat or/and nasal specimens of healthy people. It has been found that among the nonfermenting rods hydrophobic strains were predominant. In contrast, the isolates of Enterobacteriaceae family were characterized by the distinctive features of the cell surface within particular genera or even species. The obtained results show that, despite differences in cell surface hydrophobicity, numerous species of gram-negative rods have the ability to colonize the mucous membrane of upper respiratory tract. This suggests that the cell surface hydrophobicity is rather a feature of species or genus, but it is not related to the ecological niche of microorganisms in human body.     

Enhanced detection of surface-associated bacteria in indoor environments by quantitative PCR

Methods for detecting microorganisms on surfaces are needed to locate biocontamination sources and to relate surface and airborne concentrations. Research was conducted in an experimental room to evaluate surface sampling methods and quantitative PCR (QPCR) for enhanced detection of a target biocontaminant present on flooring materials. QPCR and culture analyses were used to quantitate Bacillus subtilis (Bacillus globigii) endospores on vinyl tile, commercial carpet, and new and soiled residential carpet with samples obtained by four surface sampling methods: a swab kit, a sponge swipe, a cotton swab, and a bulk method. The initial data showed that greater overall sensitivity was obtained with the QPCR than with culture analysis; however, the QPCR results for bulk samples from residential carpet were negative. The swab kit and the sponge swipe methods were then tested with two levels of background biological contamination consisting of Penicillium chrysogenum spores. The B. subtilis values obtained by the QPCR method were greater than those obtained by culture analysis. The differences between the QPCR and culture data were significant for the samples obtained with the swab kit for all flooring materials except soiled residential carpet and with the sponge swipe for commercial carpet. The QPCR data showed that there were no significant differences between the swab kit and sponge swipe sampling methods for any of the flooring materials. Inhibition of QPCR due solely to biological contamination of flooring materials was not evident. However, some degree of inhibition was observed with the soiled residential carpet, which may have been caused by the presence of abiotic contaminants, alone or in combination with biological contaminants. The results of this research demonstrate the ability of QPCR to enhance detection and enumeration of biocontaminants on surface materials and provide information concerning the comparability of currently available surface sampling methods.     

Pupation site selection of cat fleas (Siphonaptera: Pulicidae) in various carpet types and its influence on insecticide efficacy

Pupation sites of cat flea, Ctenocephalides felis (Bouché), larvae were determined in three styles of nylon and one style of wool carpet. Nylon saxony carpet had 59.3% of pupae at the top of the pile and 40% at the base of the pile. In nylon contract carpet, 55.2% of pupae were found at the top, 42.6% in the middle, and only 2.2% at the base of the pile. Nylon loop carpet contained 59.2% of pupae at the base, 25.5% in the middle, and 15.3% in the top of the pile. Wool loop carpet had 92.4% at the base and 3.8% both in the middle and top of the pile. Bioassays comparing the control of pupae manually placed at the base of carpets to that in carpets with natural pupation showed that control of pupae in the latter was 39-68% higher. Pupal control after natural pupation was greatest in nylon saxony and nylon contract carpets and lowest in nylon loop and wool loop carpets. Additional studies demonstrated that vacuuming provided the same level of pupal control on nylon saxony carpet as a spray application of permethrin to the carpet surface. Therefore, pupae that survived chemical and mechanical control treatments in nylon saxony carpet probably pupated away from the surface of the pile. Application of permethrin to the base of nylon saxony carpet did not significantly increase control. Future bioassays with cat flea pupae in carpet should be performed after natural pupation and consider carpet make and style.     

Identification of Zoogloea species and the relationship to zoogloeal matrix and floc formation

Three floc-forming, gram-negative, polarly flagellated rods were isolated and characterized. Our isolates were compared to four similar floc-forming organisms previously isolated in another laboratory and classified as two species of Zoogloea, one of Pseudomonas, and as one unidentified gram-negative rod. Possession of zoogloeal matrix or flocculent growth habit was examined in relation to growth and biochemical patterns of the bacteria. A possible relationship of Zoogloea to other gelatinous matrix-producing bacteria is also discussed.     

Coral frameworks revisited–reefs and coral carpets in the northern Red Sea

 Coral communities were investigated in the northern Red Sea, in the Gulfs of Suez and Aqaba, for their framework building potential. Five types of coral frameworks were differentiated: Acropora reef framework, Porites reef framework, Porites carpet, faviid carpet, and Stylophora carpet. Two non-framework community types were found: the Stylophora-Acropora community, and soft coral communities. Reef frameworks show a clear ecological zonation along depth and hydrodynamic exposure gradients, with clear indicator communities for each zone. By definition, coral carpets build a framework but lack distinct zonation patterns since they grow only in areas without pronounced gradients. In the northern Red Sea they show a gradual change with depth from Porites to faviid dominance. A Stylophora carpet is restricted to shallow water in the northern Gulf of Suez. Although growth rates of carpets may be somewhat less than those of reefs, the carbonate accumulation is considered to be higher in carpet areas due to their significantly higher areal extension. In addition, reefs and carpets have different sediment retention characteristics – the carpet retains, the reef exports. The in situ fossilization potential of coral carpets is expected to be higher than that of reef frameworks. Accepted: 25 May 1999     

Comparison between cultured small-intestinal and fecal microbiotas in beagle dogs

The microbiota of the small intestine is poorly known because of difficulties in sampling. In this study, we examined whether the organisms cultured from the jejunum and feces resemble each other. Small-intestinal fluid samples were collected from 22 beagle dogs with a permanent jejunal fistula in parallel with fecal samples. In addition, corresponding samples from seven of the dogs were collected during a 4-week period (days 4, 10, 14, and 28) to examine the stability of the microbiota. In the jejunal samples, aerobic/facultative and anaerobic bacteria were equally represented, whereas anaerobes dominated in the fecal samples. Despite lower numbers of bacteria in the jejunum (range, 10(2) to 10(6) CFU/g) than in feces (range, 10(8) to 10(11) CFU/g), some microbial groups were more prevalent in the small intestine: staphylococci, 64% versus 36%; nonfermentative gram-negative rods, 27% versus 9%; and yeasts, 27% versus 5%, respectively. In contrast, part of the fecal dominant microbiota (bile-resistant Bacteroides spp., Clostridium hiranonis-like organisms, and lactobacilli) was practically absent in the jejunum. Many species were seldom isolated simultaneously from both sample types, regardless of their overall prevalence. In conclusion, the small intestine contains a few bacterial species at a time with vastly fluctuating counts, opposite to the results obtained for the colon, where the major bacterial groups remain relatively constant over time. Qualitative and quantitative differences between the corresponding jejunal and fecal samples indicate the inability of fecal samples to represent the microbiotas present in the upper gut.     

Adhesion,spreading, and proliferation of cells on protein carpets: Effects of stability of a carpet

Summary In the present report we have investigated the role that the physical properties of substrata play in modulating the effects which components of extracellular matrix (ECM) exert on adhesion, spreading, and growth of retinal pigmented epithelial cells. By simple modifications of conditions for protein adsorption on glass we obtained a set of substrata all coated with proteins of ECM (protein carpets) but with different physical properties. Using these protein carpets we have shown that their stability (desorption rate) in tissue culture conditions varies according to the technique with which they were prepared. Both semiremovable and immobilized carpets are stable, whereas removable protein carpets desorb readily. Therefore, the protein concentration or composition or both may change with time in tissue culture depending on the technique used to prepare the carpet. In addition, efficacy of cell attachment to given protein may vary depending on whether a technique used to prepare the protein carpet involves denaturation of the protein. Adherent cells quickly remove (clear) weakly adsorbed protein carpets and it seems that the carpet removal is a mechanical process. During the carpet removal cells are rounded, which indicates that a spread cell phenotype normally associated with stress fibers and focal contacts occurs when the substratum is rigid enough to sustain cell traction. In addition, substrata lacking the rigidity to support the spread phenotype do not support cell proliferation either.     

Microbiological Profiles of Four Apollo Spacecraft

Selected surfaces from the Command Module, Lunar Module (ascent and descent stages), Instrument Unit, Saturn S-4B engine, and Spacecraft Lunar Module Adapter comprised the various components of four Apollo spacecraft which were assayed quantitatively and qualitatively for microorganisms. In addition, the first Lunar Roving Vehicle was assayed. Average levels of microbial contamination (10(4) per square foot of surface) on the Command Module, Instrument Unit, and Saturn S-4B engine were relatively consistent among spacecraft. The first postflight sampling of interior surfaces of the Command Module was possible due to elimination of the 21-day back-contamination quarantine period. Results of the pre- and postflight samples revealed increases in the postflight samples of 3 logs/inch(2). A total of 5,862 microbial isolates was identified; 183 and 327 were obtained from the Command Module at preflight and postflight sampling periods, respectively. Although the results showed that the majority of microorganisms isolated were those considered to be indigenous to humans, an increase in organisms associated with soil and dust was noted with each successive Apollo spacecraft.     

A model for the morphogenesis of strip reduction patterns in phototrophic euglenids: evidence for heterochrony in pellicle evolution

We propose a general developmental model that explains the evolutionary origin, diversification, and inheritance of pellicle strip patterns in phototrophic euglenids. Dividing cells of Euglena gracilis, E. viridis, and Phacus similis were observed with scanning electron microscopy in order to study the morphogenesis of posterior whorls of strip reduction. We found evidence that constant whorl numbers are maintained through cell division because of organized strip growth before and during cytokinesis. Alternating nascent strips form a new whorl of strip reduction at each of the anterior and posterior ends of daughter cells. Strips that terminated to form posterior whorls in the mother cell change in length during the development of daughter cells. In the mother cells of E. gracilis, the strips forming whorls I and II grow to become whorls II and III, respectively, in the daughter cells; the strips forming whorl III in the mother cell lengthen and meet with other strips already present at the posterior tip of daughter cells. This process of whorl morphogenesis during asexual reproduction is consistent with known variation in pellicle strip patterns and suggests that heterochrony played a major role in the ultrastructural evolution of phototrophic euglenids.     

Isolation of Geobacter species from diverse sedimentary environments.

In an attempt to better understand the microorganisms responsible for Fe(III) reduction in sedimentary environments, Fe(III)-reducing microorganisms were enriched for and isolated from freshwater aquatic sediments, a pristine deep aquifer, and a petroleum-contaminated shallow aquifer. Enrichments were initiated with acetate or toluene as the electron donor and Fe(III) as the electron acceptor. Isolations were made with acetate or benzoate. Five new strains which could obtain energy for growth by dissimilatory Fe(III) reduction were isolated. All five isolates are gram-negative strict anaerobes which grow with acetate as the electron donor and Fe(III) as the electron acceptor. Analysis of the 16S rRNA sequence of the isolated organisms demonstrated that they all belonged to the genus Geobacter in the delta subdivision of the Proteobacteria. Unlike the type strain, Geobacter metallireducens, three of the five isolates could use H2 as an electron donor for Fe(III) reduction. The deep subsurface isolate is the first Fe(III) reducer shown to completely oxidize lactate to carbon dioxide, while one of the freshwater sediment isolates is only the second Fe(III) reducer known that can oxidize toluene. The isolation of these organisms demonstrates that Geobacter species are widely distributed in a diversity of sedimentary environments in which Fe(III) reduction is an important process.     

Enhanced Detection of Surface-Associated Bacteria in Indoor Environments by Quantitative PCR

Methods for detecting microorganisms on surfaces are needed to locate biocontamination sources and to relate surface and airborne concentrations. Research was conducted in an experimental room to evaluate surface sampling methods and quantitative PCR (QPCR) for enhanced detection of a target biocontaminant present on flooring materials. QPCR and culture analyses were used to quantitate Bacillus subtilis (Bacillus globigii) endospores on vinyl tile, commercial carpet, and new and soiled residential carpet with samples obtained by four surface sampling methods: a swab kit, a sponge swipe, a cotton swab, and a bulk method. The initial data showed that greater overall sensitivity was obtained with the QPCR than with culture analysis; however, the QPCR results for bulk samples from residential carpet were negative. The swab kit and the sponge swipe methods were then tested with two levels of background biological contamination consisting of Penicillium chrysogenum spores. The B. subtilis values obtained by the QPCR method were greater than those obtained by culture analysis. The differences between the QPCR and culture data were significant for the samples obtained with the swab kit for all flooring materials except soiled residential carpet and with the sponge swipe for commercial carpet. The QPCR data showed that there were no significant differences between the swab kit and sponge swipe sampling methods for any of the flooring materials. Inhibition of QPCR due solely to biological contamination of flooring materials was not evident. However, some degree of inhibition was observed with the soiled residential carpet, which may have been caused by the presence of abiotic contaminants, alone or in combination with biological contaminants. The results of this research demonstrate the ability of QPCR to enhance detection and enumeration of biocontaminants on surface materials and provide information concerning the comparability of currently available surface sampling methods.     

Penetration of light into carpet, and responses of the European house-dust mite (Dermatophagoides pteronyssinus) to varying light intensity and diurnal cycle

This study examined the question of what effect exposure to light might play in determining the vertical distribution of house-dust mites in carpet, and the degree to which light penetrates worn and unworn carpets of different pile conformation (loop- versus cut-pile), height and colour. The effect on population increase of a diurnal lighting cycle versus continual darkness was also investigated. It was found that the penetration of light into carpets was largely unaffected by pile colour or conformation. Pile height was an important factor, however, and for a given height within the pile, light intensity was higher in carpets subjected to a greater degree of wear. This corresponded to the reduction in effective pile height that occurs with carpet use. Whilst the differences observed were sometimes large (up to two-fold for a given height within the pile), Petri dish studies suggested no mite preference for habitation of areas of low light intensity compared to high intensity. Additionally, culturing mites under a diurnal light cycle was shown to be no more efficient than culturing in complete darkness. These results suggest that exposure to light is not an important determinant of house-dust mite behaviour, or their ability to colonise textile substrates.     

The Microflora of Steam Sterilized Milking Equipment

S ummary : The classification of 1,466 bacteria, isolated on Yeastrel-milk agar incubated at 30° from 68 rinses of steam sterilized dairy equipment taken at five farms, showed that micrococci were dominant, with corynebacteria and aerobic sporeforming rods frequent, representatives of these three groups constituting 73% of the isolates. The microflora was characterized by the dominance of organisms which were relatively inactive in milk. Typical milk souring organisms such as lactic streptococci and coli-aerogenes organisms were rarely isolated, but anaerogenic Gram-negative rods resembling Achromobacter, Pseudomonas and Flavobacterium were not uncommon on milking machine clusters and on a cooling unit. The glass recorder jars contained a relatively high proportion of sporeformers.     

Rapid electrochemical detection and identification of catalase positive micro-organisms

The rapid detection and identification of bacteria has application in a number of fields, e.g. the food industry, environmental monitoring and biomedicine. While in biomedicine the number of organisms present during infection is multiples of millions in the other fields it is the detection of low numbers of organisms that is important, e.g. an infective dose of Escherichia coli O157:H7 from contaminated food is less than 100 organisms. A rapid and sensitive technique has been developed to detect low numbers of the model organism E. coli O55, combining Lateral Flow Immunoassay (LFI) for capture and amperometry for sensitive detection. Nitrocellulose membranes were used as the solid phase for selective capture of the bacteria using antibodies to E. coli O55. Different concentrations of E. coli O55 in Ringers solution were applied to LFI strips and allowed to flow through the membrane to an absorbent pad. The capture region of the LFI strip was placed in close contact with the electrodes of a Clarke cell poised at +0.7 V for the detection of hydrogen peroxide. Earlier research identified that the consumption of hydrogen peroxide by bacterial catalase provided a sensitive indicator of aerobic and facultative anaerobic microorganisms numbers. Modification and application of this technique to the LFI strips demonstrated that the consumption of 8 mM hydrogen peroxide was correlated with the number of microorganisms presented to the LFI strips in the range of 2 x 10(1)-2 x 10(7) colony forming units (cfu). Capture efficiency was dependent on the number of organisms applied and varied from 71% at 2 x 10(2) cfu to 25% at 2 x 10(7) cfu. The procedure was completed in less than 10 min and could detect less than 10 cfu captured from a 200 microl sample applied to the LFI strip. The approached adopted provides proof of principle for the basis of a new technological approach to the rapid, quantitative and sensitive detection of bacteria that express catalase activity.