Application of a disposable transparent filtration membrane to the infrared spectroscopic discrimination among bacterial species

This study describes the application of filtration, infrared spectroscopy, and multivariate analysis to the identification of 10 foodborne bacterial species. The bacteria were applied by filtration to a disposable optical membrane that is transparent to infrared radiation. The filtration step was rapid (2 min). Observed cellular infrared spectra were unique and were used to discriminate among the different species. A dataset for the 10 bacterial species investigated was successfully used to correctly identify unknowns included in the dataset.     

Fast and reliable determination of Escherichia coli susceptibility to antibiotics: Infrared microscopy in tandem with machine learning algorithms

Antimicrobial drugs have an important role in controlling bacterial infectious diseases. However, the increasing resistance of bacteria to antibiotics has become a global health care problem. Rapid determination of antimicrobial susceptibility of clinical isolates is often crucial for the optimal antimicrobial therapy. The conventional methods used in medical centers for susceptibility testing are time‐consuming (>2 days). Two bacterial culture steps are needed, the first is used to grow the bacteria from urine on agar plates to determine the species of the bacteria (~24 hours). The second culture is used to determine the susceptibility by growing colonies from the first culture for another 24 hours. Here, the main goal is to examine the potential of infrared microscopy combined with multivariate analysis, to reduce the time it takes to identify Escherichia coli susceptibility to antibiotics and to determine the optimum choice of antibiotic to which the bacteria will respond. E coli colonies of the first culture from patients with urinary tract infections (UTI) were examined for the bacterial susceptibility using Fourier‐transform infrared (FTIR). Our results show that it is possible to determine the optimum choice of antibiotic with better than 89% sensitivity, in the time span of few minutes, following the first culture.      

Optical processing of bacterial libraries for directed evolution

Selection of phenotypically distinct bacterial colonies on a Petri dish is typically performed by one of two methods: chemical or mechanical. Chemical methods (e.g., antibiotic selection) rely on inherent growth advantages of the unique phenotypes desired and thus have limited applicability. Mechanical methods are generally slow and require relatively large colonies (typically hundreds of colonies per plate). Here the use of imaged light to select bacterial colonies is explored, employing either photodynamic therapy agents or a ferrochelatase mutation in combination with porphyrin precursors to sensitize the bacteria to light and a computer-controlled light projection system to illuminate some bacterial colonies while leaving others in the dark. A CCD camera was used to distinguish between bacteria expressing green fluorescent protein (GFP) from nonfluorescent colonies. The fluorescence image from the camera was then used to create a virtual masking image for photoselection. Using a simple commercial projector it was possible to confer a 56-fold selective advantage to colonies expressing GFP. This represents a potentially powerful tool in directed evolution experiments using large libraries.     

Assessment of bacterial community composition within and among Acropora loripes colonies in the wild and in captivity

A growing number of studies have provided insights into the diversity of coral-associated bacteria and their function in the coral holobiont. Yet, information about spatial heterogeneity of bacteria within coral colonies is limited. Using 16S rRNA gene metabarcoding, we analyzed the bacterial community composition across four distinct locations in each of five wild Acropora loripes colonies. Considerable variation within and among colonies was present, which has implications for sampling strategies and data interpretation in coral microbiome research. Bacterial assemblages significantly differed in alpha and beta diversity among colonies, with all corals possessing a high relative abundance of Endozoicomonas. When the same A. loripes colonies were subsequently reared in aquaria over 4 weeks, the relative abundance of Marinobacter initially increased in all colonies. However, no significant alteration in bacterial community composition was observed over time and the colonies maintained distinct bacterial microbiomes.

     

Independent origins and horizontal transfer of bacterial symbionts of aphids

Many insect groups have obligate associations with primary endosymbionts: mutualistic bacteria that are maternally transmitted and derived from an ancient infection. Often, the same insects are hosts to 'secondary' bacterial symbionts which are maternally transmitted but relatively labile within host lineages. To explore the dynamics of secondary symbiont associations in aphids, we characterized bacteria infecting 15 species of macrosiphine aphids using DNA sequencing, diagnostic polymerase chain reaction (PCR), diagnostic restriction digests, phylogenetic analyses, and electron microscopy to examine aphids from nature and from laboratory colonies. Three types of bacteria besides Buchnera were found repeatedly; all three fall within the Enterobacteriaceae. The R-type has a 16S rDNA less than 0.1% different from that of the secondary symbiont previously reported from Acyrthosiphon pisum and is related to Serratia species. The T-type includes a symbiont previously reported from a whitefly; the U-type comprises a new cluster near the T-type. The T-type was found in every one of 40 Uroleucon ambrosiae clones collected throughout the United States. In contrast, A. pisum individuals were infected by any combination of the three symbiont types. Secondary symbionts were maternally transmitted for 11 months within laboratory-reared A. pisum clones and were present in sexually produced eggs. PCR screens for a bacteriophage, APSE-1, indicated its presence in both A. pisum and U. ambrosiae containing secondary symbionts. Electron microscopy of R-type and T-type bacteria in A. pisum and in U. ambrosiae revealed rod-shaped organisms that attain extremely high densities within a few bacteriocytes.     

A new chromogenic agar medium for detection of potentially virulent Yersinia enterocolitica

Several outbreaks of foodborne yersiniosis have been documented and this disease continues to be source of infections transmitted through foods. The selective agars most commonly used to isolate Yersinia enterocolitica in clinical, food and environmental samples, cefsulodin-irgasan-novobiocin (CIN) and MacConkey (MAC) agars, lack the ability to differentiate potentially virulent Y. enterocolitica from other Yersinia that may be present as well as some other bacterial spp. This study proposes the use of an agar medium, Y. enterocolitica chromogenic medium (YeCM), for isolation of potentially virulent Y. enterocolitica. This agar contains cellobiose as the fermentable sugar, a chromogenic substrate and selective inhibitors for suppression of colony formation by many competing bacteria. All strains of potentially virulent Yersinia of biotypes 1B, and biotypes 2-5 formed convex, red bulls-eye colonies on YeCM that were very similar to those described for CIN agar. However, Y. enterocolitica biotype 1A and other related Yersinia formed colonies that were purple/blue on YeCM while they formed typical red bulls-eye colonies on CIN agar. When a mixture of potentially virulent Y. enterocolitica biotype 1B, Y. enterocolitica biotype 1A and 5 other bacterial species was used to artificially contaminate tofu and then spread-plated on three selective agars, Y. enterocolitica biotype 1B colonies were easily distinguished from other strains on YeCM. However, Y. enterocolitica biotype 1B colonies were indistinguishable from many other colonies on CIN and only distinguishable from those of C. freundii on MAC. When colonies were picked and identified from these agars, typical colonies from YeCM were confirmed only as Y. enterocolitica biotype 1B. Typical colonies on CIN and MAC were found to belong to several competing species and biotypes.     

Host-symbiont recognition in the environmentally transmitted sepiolid squid-Vibrio mutualism

Associations between environmentally transmitted symbionts and their hosts provide a unique opportunity to study the evolution of specificity and subsequent radiation of tightly coupled host-symbiont assemblages [3, 8, 24]. The evidence provided here from the environmentally transmitted bacterial symbiont Vibrio fischeri and its sepiolid squid host (Sepiolidae: Euprymna) demonstrates how host-symbiont specificity can still evolve without vertical transmission of the symbiont [1]. Infection by intraspecific V. fischeri symbionts exhibited preferential colonization over interspecific V. fischeri symbionts, indicating a high degree of specificity for the native symbiotic strains. Inoculation with symbiotic bacteria from other taxa (monocentrid fish and loliginid squids) produced little or no colonization in two species of Euprymna, despite their presence in the same or similar habitats as these squids. These findings of host specificity between native Vibrios and sepiolid squids provides evidence that the presence of multiple strains of symbionts does not dictate the composition of bacterial symbionts in the host.     

Bacterial community structure in Apis florea larvae analyzed by denaturing gradient gel electrophoresis and 16S rRNA gene sequencing

This study characterizes the colonization and composition of bacterial flora in dwarf Asian honeybee (Apis florea) larvae and compares bacterial diversity and distribution among different sampling locations. A. florea larvae were collected from 3 locations in Chiang Mai province, Thailand. Bacterial DNA was extracted from each larva using the phenol–chloroform method. Denaturing gradient gel electrophoresis was performed, and the dominant bands were excised from the gels, cloned, and sequenced for bacterial species identification. The result revealed similarities of bacterial community profiles in each individual colony, but differences between colonies from the same and different locations. A. florea larvae harbor bacteria belonging to 2 phyla (Firmicutes and Proteobacteria), 5 classes (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli, and Clostridia), 6 genera (Clostridium, Gilliamella, Melissococcus, Lactobacillus, Saccharibacter, and Snodgrassella), and an unknown genus from uncultured bacterial species. The classes with the highest abundance of bacteria were Alphaproteobacteria (34%), Bacilli (25%), Betaproteobacteria (11%), Gammaproteobacteria (10%), and Clostridia (8%), respectively. Similarly, uncultured bacterial species were identified (12%). Environmental bacterial species, such as Saccharibacter floricola, were also found. This is the first study in which sequences closely related to Melissococcus plutonius, the causal pathogen responsible for European foulbrood, have been identified in Thai A. florea larvae.     

Autoradiographic study of the localization and evolution of growth zones in bacterial colonies.

Incorporation of [3H]leucine in the bacteria of 18 to 48 h-old colonies of Pseudomonas aeruginosa, Pseudomonas putida, Bacillus thuringiensis, Staphylococcus aureus and Escherichia coli enabled the localization of bacterial multiplication sites by means of autoradiography of sagittal sections. In colonies where fast diameter expansion occurred, all the bacteria from the peripheral corona contributed to peripheral growth; in colonies where the expansion was slower, the growth rate of the bacteria in this region was heterogeneous. Besides this peripheral growth, a central region of bacterial multiplication was always found, but with variable localization and extension. In aerobic species, such as P. aeruginosa and P. putida, the central growth site was limited to the zone of oxygen penetration into the bacterial mass. However, in facultatively anaerobic species, bacterial multiplication dependend on nutrient supply. For 48 h-old colonies of S. aureus, a more complex localization of growth seemed to be affected simultaneously by nutrient penetration and accumulation of toxic substances.     

Culture-independent identification of gut bacteria in fourth-instar red imported fire ant, Solenopsis invicta Buren, larvae

Red imported fire ants (RIFA), Solenopsis invicta Buren, are medical, urban, and agricultural pests from South America. They are successful invaders due to their preference for disturbed habitats, high reproductive rates, and the ability to feed on a wide variety of food items (omnivorous). Fourth-instar larvae are used by the colony to digest solid food and then regurgitate it for consumption by workers and queens. Larvae are an ideal source of investigations of endosymbiotic bacteria possibly involved in nutrient distributions. Our study utilized 16S rDNA sequencing to describe the composition of the bacterial community in fourth-instar ant larvae in order to identify possible endosymbiotic bacteria present therein. The 16S rRNA gene was directly amplified from mixed-population DNA of whole fire ant larval guts and cloned into Escherichia coli. Bacterial communities from three geographically separated RIFA colonies were examined. Sequenced bacterial clones from guts were determined to be predominantly from the phylum Proteobacteria and the family Enterobacteriaceae. Our results did not detect the presence of endosymbiotic bacteria in the guts of RIFA larvae among the colonies. In addition, minimal species overlap was found when bacterial inventories were compared among colonies. Thus, bacteria coadapted with red imported fire ant larvae were not detected. Identified bacteria were not closely affiliated with endosymbiotic bacteria common in other insect species. Bacteria communities appeared to be unique to each geographical location and were determined by the foods consumed by the ants.     

Human Fecal Flora: The Normal Flora of 20 Japanese-Hawaiians

Quantitative and qualitative examination of the fecal flora of 20 clinically healthy Japanese-Hawaiian males was carried out by using anaerobic tube culture techniques. Cultural counts were 93% of the microscopic clump counts. Isolated colonies were selected in a randomized manner to give an unbiased sampling of the viable bacterial types. Each isolate was characterized for species identification. From a total of 1,147 isolates, 113 distinct types of organisms were observed. Statistical estimates indicate that these types account for 94% of the viable cells in the feces. The quantitative composition of the flora of this group of people, together with differential characteristics of previously unreported species, is presented for those kinds of bacteria which each represented at least 0.05% of the flora.     

Colony breeding system influences cuticular bacterial load of Formosan subterranean termite (Isoptera: Rhinotermitidae) workers

The goal of this study was to test whether the breeding system and/or the degree of inbreeding of field colonies of the Formosan subterranean termite, Coptotermes formosanus, Shiraki (Isoptera: Rhinotermitidae) influences bacterial load on the cuticle of foraging workers. We enumerated bacterial load on the cuticle of groups of workers foraging in 20 inground monitoring stations surrounding the French Market in New Orleans, LA, and identified bacteria species using 16S rRNA gene sequencing. We used microsatellite genotyping to assign the 20 worker groups to seven simple family colonies (headed by a single pair of reproductives) and four extended family colonies (headed by multiple inbreeding reproductives) with a wide range of degrees of inbreeding. Workers from extended family colonies had a higher bacterial load than those from simple family colonies; however, bacterial load was not significantly correlated to the degree of inbreeding, possibly because of confounding factors in colony life history, such as age and/or size of colonies. Colonies with high bacterial load did not have a higher proportion of entomopathogens, and thus, bacterial load is not necessarily an indicator for disease risk. The majority of bacteria cultured from the cuticle of termites were soil bacteria with no known pathology against termites.     

Applications of fluorophore-containing microbial growth media.

Media containing the fluorogenic compound 8-anilino-1-naphthalene sulfonic acid may be used to discriminate between gram-positive and gram-negative bacteria and to differentiate between various species of bacteria. Fluorescent light emitted from colonies of gram-negative bacteria on 8-anilino-1-naphthalene sulfonic acid-containing agar was visually more intense than that on gram-positive bacteria. The emitted light from the gram-negative bacteria differed in wave-lengths from that of light emitted by colonies of gram-positive bacteria. The fluorescent intensity of colonies on complete 8-anilino-1-napthalene sulfonic acid agar supplemented with 1% of single substrates varied depending on the bacterial species, thus allowing the development of profiles used to identify 12 different species.     

Bacterial population in Russian space station "Mir"

We had the opportunity to investigate the bacterial population in air samples, condensation water, and inner wall swabs from the Russian space station Mir. From the first and second air samples during the mission, 29 and 7 bacterial colonies were collected, respectively. The values were equivalent to 16.8 and 4.0 cfu/100 liter air, respectively. Condensation water was collected from three different sites. The total viable bacterial counts were 2.1 x 10(6), 5.2 x 10(2), and 3.0 x 10(1) cfu/ml. The phylogenetic position of each isolate was determined by total 16S rDNA sequencing. Bacteria from air samples were mainly Gram-positive (35/36 colonies), and staphylococci occupied dominant specifically (23/36 colonies). On the other hand, Gram-negative bacteria were mainly isolated from condensation water samples. Most strains were thought to be opportunistic pathogens or environmental bacteria (such as those that inhabit soil, water, or air) found on earth. However, 6 of 23 isolates were suspected to be new species according to phylogenetic analysis and quantitative DNA-DNA hybridization data. The isolation of the other levels 3 and 2 bacteria, using specific selective media, was unsuccessful because all samples were heavily contaminated with fungi. To overcome this situation, PCR methods were applied to survey most levels 3 and 2 pathogenic bacteria in the condensation water samples. Up to 380 different primers for bacterial pathogens were used in this study. Only Mycobacterium avium 16S DNA sequences, however, could be amplified from the three water samples. The average bacteria count was estimated to be about 10(4) organisms/ml water.     

Specificity of the mutualistic association between actinomycete bacteria and two sympatric species of Acromyrmex leaf-cutting ants

Acromyrmex leaf-cutting ants maintain two highly specialized, vertically transmitted mutualistic ectosymbionts: basidiomycete fungi that are cultivated for food in underground gardens and actinomycete Pseudonocardia bacteria that are reared on the cuticle to produce antibiotics that suppress the growth of Escovopsis parasites of the fungus garden. Mutualism stability has been hypothesized to benefit from genetic uniformity of symbionts, as multiple coexisting strains are expected to compete and, thus, reduce the benefit of the symbiosis. However, the Pseudonocardia symbionts are likely to be involved in Red-Queen-like antagonistic co-evolution with Escovopsis so that multiple strains per host might be favoured by selection provided the cost of competition between bacterial strains is low. We examined the genetic uniformity of the Pseudonocardia symbionts of two sympatric species of Acromyrmex ants by comparing partial sequences of the nuclear Elongation Factor-Tu gene. We find no genetic variation in Pseudonocardia symbionts among nest mate workers, neither in Acromyrmex octospinosus, where colonies are founded by a single queen, nor in Acromyrmex echinatior, where mixing of bacterial lineages might happen when unrelated queens cofound a colony. We further show that the two ant species maintain the same pool of Pseudonocardia symbionts, indicating that horizontal transmission occasionally occurs, and that this pool consists of two distinct clades of closely related Pseudonocardia strains. Our finding that individual colonies cultivate a single actinomycete strain is in agreement with predictions from evolutionary theory on host-symbiont conflict over symbiont mixing, but indicates that there may be constraints on the effectiveness of the bacterial symbionts on an evolutionary timescale.     

A Soil and Rhizosphere Microorganism Isolation and Enumeration Medium That Inhibits Bacillus mycoides

A new solid medium has been developed for the enumeration and isolation of soil and rhizosphere microorganisms. This medium, named rhizosphere isolation medium, contains glucose and 15 of the 20 common amino acids. The absence of five other amino acids, namely, aspartic acid, asparagine, cysteine, proline, and threonine, inhibits the growth of Bacillus mycoides, a commonly encountered bacterium that rapidly spreads on agar media and complicates the isolation and enumeration of other microorganisms. Compared with a similar medium containing Casamino Acids, rhizosphere isolation medium had half as many colonies of B. mycoides, with each colony approximately half the diameter. The two media had similar total numbers of bacterial colonies. Isolates were divided into taxononomic groups, roughly corresponding to species and genus, by fatty acid methyl ester analysis and numerical methods. There were 24 genera and 41 species found in the isolates from rhizosphere isolation medium, while 19 genera and 35 species were found in the isolates from the medium prepared with Casamino Acids. No major group of bacteria was found to occur only on one medium or on the other, indicating that the five missing amino acids had no great effect on organisms other than B. mycoides. This medium may prove useful in soil and rhizosphere studies in which the growth of B. mycoides is undesirable.     

Independent studies using deep sequencing resolve the same set of core bacterial species dominating gut communities of honey bees

Starting in 2003, numerous studies using culture-independent methodologies to characterize the gut microbiota of honey bees have retrieved a consistent and distinctive set of eight bacterial species, based on near identity of the 16S rRNA gene sequences. A recent study [Mattila HR, Rios D, Walker-Sperling VE, Roeselers G, Newton ILG (2012) Characterization of the active microbiotas associated with honey bees reveals healthier and broader communities when colonies are genetically diverse. PLoS ONE 7(3): e32962], using pyrosequencing of the V1-V2 hypervariable region of the 16S rRNA gene, reported finding entirely novel bacterial species in honey bee guts, and used taxonomic assignments from these reads to predict metabolic activities based on known metabolisms of cultivable species. To better understand this discrepancy, we analyzed the Mattila et al. pyrotag dataset. In contrast to the conclusions of Mattila et al., we found that the large majority of pyrotag sequences belonged to clusters for which representative sequences were identical to sequences from previously identified core species of the bee microbiota. On average, they represent 95% of the bacteria in each worker bee in the Mattila et al. dataset, a slightly lower value than that found in other studies. Some colonies contain small proportions of other bacteria, mostly species of Enterobacteriaceae. Reanalysis of the Mattila et al. dataset also did not support a relationship between abundances of Bifidobacterium and of putative pathogens or a significant difference in gut communities between colonies from queens that were singly or multiply mated. Additionally, consistent with previous studies, the dataset supports the occurrence of considerable strain variation within core species, even within single colonies. The roles of these bacteria within bees, or the implications of the strain variation, are not yet clear.     

Enhanced analysis of bacteria susceptibility in connected biofilms

A common method for visualizing bacterial biofilms is through confocal laser scanning microscopy images. Current software packages separate connected-biofilm bacteria from unconnected bacteria, such as planktonic or dispersed bacteria, but do not save both image sequences, making interpretation of the two bacterial populations difficult. Thus we report the development of an algorithm to save separate image sequences and enable qualitative and quantitative evaluation of each bacterial population. To improve bacterial viability assessment using a membrane integrity dye, a colocalization algorithm was also developed. This assigns colocalized pixels to the dead bacteria population, rather than to both the live and dead bacteria groups. Visually, this makes it clearer to distinguish a green live bacteria pixel from a yellow colocalized dead bacteria pixel. This algorithm also aids in the quantification of viability for connected-biofilm bacteria and unconnected bacteria to investigate susceptibility of each population to antimicrobials. The utility of these algorithms was demonstrated with Pseudomonas aeruginosa biofilms treated with ciprofloxacin hydrochloride. Results from this study indicate that quantification with colocalization adjustment can prevent underestimation of dead bacteria. These improvements in image processing will enable researchers to visually differentiate connected-biofilm and unconnected bacteria in a single image and to quantify these populations independently for viability without double counting the colocalized image pixels.     

An oligonucleotide probe to assay lysis and DNA hybridization of a diverse set of bacteria

A computer bank of 16 S rRNA bacterial sequences was searched to determine a consensus sequence expected to hybridize with DNA from a wide variety of bacteria. An oligonucleotide probe, named a panprobe, containing this sequence was used to assay the degree of lysis of bacterial colonies on filter paper heated in a microwave oven and subsequently treated with NaOH. As determined by colony hybridization with the panprobe, lysis was achieved for 51 of 59 different species of bacteria tested. DNA, isolated from the eight bacteria not detected by colony hybridization, did hybridize with the panprobe in slot blot hybridizations.     

A method for the enumeration of bacterial adhesion to epithelial cells using image analysis

Abstract Computerised image analysis was utilised to enumerate the attachment of Staphylococcus epidermidis to HEp2 cell monolayers. A differential staining technique was employed such that individual staphylococcal cells stood out in sharp contrast against the uneven cell surface and granular contents of the epithelial cells. The primary image analysis operation involved subtracting an out-of-focus image from an in-focus image of the bacteria on the monolayer, thereby accentuating the bacterial image. Enumeration, using a particle counting routine, was rapid and reproducible, facilitating counting in excess of 700 bacteria per field at ×500 magnification. The computerised programme compared favourably with manual counting and would provide a rapid, objective and morphologically discriminatory method for evaluating bacterial attachment to various tissues.