A Model To Estimate the Optimal Sample Size for Microbiological Surveys

Estimating optimal sample size for microbiological surveys is a challenge for laboratory managers. When insufficient sampling is conducted, biased inferences are likely; however, when excessive sampling is conducted valuable laboratory resources are wasted. This report presents a statistical model for the estimation of the sample size appropriate for the accurate identification of the bacterial subtypes of interest in a specimen. This applied model for microbiology laboratory use is based on a Bayesian mode of inference, which combines two inputs: (ii) a prespecified estimate, or prior distribution statement, based on available scientific knowledge and (ii) observed data. The specific inputs for the model are a prior distribution statement of the number of strains per specimen provided by an informed microbiologist and data from a microbiological survey indicating the number of strains per specimen. The model output is an updated probability distribution of strains per specimen, which can be used to estimate the probability of observing all strains present according to the number of colonies that are sampled. In this report two scenarios that illustrate the use of the model to estimate bacterial colony sample size requirements are presented. In the first scenario, bacterial colony sample size is estimated to correctly identify Campylobacter amplified restriction fragment length polymorphism types on broiler carcasses. The second scenario estimates bacterial colony sample size to correctly identify Salmonella enterica serotype Enteritidis phage types in fecal drag swabs from egg-laying poultry flocks. An advantage of the model is that as updated inputs from ongoing surveys are incorporated into the model, increasingly precise sample size estimates are likely to be made.     

Identification of bacterial strains by laboratories participating in the Deutsches Krebsforschungszentrum quality assurance programme

The quality assurance programme (QAP) of the Deutsches Krebsforschungszentrum (DKFZ) is a proficiency testing system developed to service the laboratory animal discipline. QAP comprises the quarterly distribution of two bacterial strains originating from various species of animals for identification to the species level and antibiotic susceptibility testing. We compared identification results reported by QAP participants over the years 1996-2004 with those obtained by the Dutch Bacterial Diagnostics reference laboratory on 68 samples comprising 71 bacterial strains and a fungus. Significant differences were found in the frequency of reported and correct identifications when bacteria were assigned to different groups based on morphology by Gram stain and on origin (animal versus environmental, rodent and rabbit versus other animal species, pathogen versus non-pathogens). Rodent and rabbit pathogens yielded 73% correct identifications, and with all bacterial strains only 60% of the identifications were correct. We assume that most QAP participants were from laboratory animal diagnostic laboratories. If this is true, the capabilities of laboratories in the laboratory animal discipline to correctly identify bacterial species are well below what are considered acceptable limits for human diagnostic laboratories. The distribution of cultured bacteria circumvents the most difficult step in the microbiological monitoring of animals, namely primary culture from clinical samples. We propose to set up a QAP that comprises the distribution of specimens mimicking clinical samples normally submitted to laboratory animal diagnostic laboratories.     

Assessing Genetic Heterogeneity within Bacterial Species Isolated from Gastrointestinal and Environmental Samples: How Many Isolates Does It Take?

Strain typing of bacterial isolates is increasingly used to identify sources of infection or product contamination and to elucidate routes of transmission of pathogens or spoilage organisms. Usually, the number of bacterial isolates belonging to the same species that is analyzed per sample is determined by convention, convenience, laboratory capacity, or financial resources. Statistical considerations and knowledge of the heterogeneity of bacterial populations in various sources can be used to determine the number of isolates per sample that is actually needed to address specific research questions. We present data for intestinal Escherichia coli, Listeria monocytogenes, Klebsiella pneumoniae, and Streptococcus uberis from gastrointestinal, fecal, or soil samples characterized by ribotyping, pulsed-field gel electrophoresis, and PCR-based strain-typing methods. In contrast to previous studies, all calculations were performed with a single computer program, employing software that is freely available and with in-depth explanation of the choice and derivation of prior distributions. Also, some of the model assumptions were relaxed to allow analysis of the special case of two (groups of) strains that are observed with different probabilities. Sample size calculations, with a Bayesian method of inference, show that from 2 to 20 isolates per sample need to be characterized to detect all strains that are present in a sample with 95% certainty. Such high numbers of isolates per sample are rarely typed in real life due to financial or logistic constraints. This implies that investigators are not gaining maximal information on strain heterogeneity and that sources and transmission pathways may go undetected.     

A Multinomial Model for the Quality Control of Colony Counting Procedures

The so‐called good‐laboratory‐practice (GLP) test provides an experimental design and appropriate statistical analysis for the problem of analyst performance assessment in microbiological laboratories. For a given sample material multiple dilution series are generated yielding colony counts from several dilution levels. Statistical evaluation is based on the assumption of Poisson‐distributed colony forming units. In this paper a new model based on conditional binomial and multinomial distributions is presented and it is shown how it is related to the standard model which assumes Poisson‐distributed colony counts. The effects of common working errors on the statistical evaluation of the GLP‐test are investigated.     

Indigenous butyric acid-degrading bacteria as surrogate pit latrine odour control: isolation,biodegradability performance and growth kinetics

Butyric acid is one of the volatile organic compounds that significantly contribute to malodour emission from pit latrines. The purpose of this work is to isolate and identify bacterial strains that have the capability to degrade butyric acid, determine their butyric acid degradation efficiencies and estimate their growth pattern parameters of microbiological relevance. Pure cultures of bacterial strains capable of degrading butyric acid were isolated from pit latrine faecal sludge using an enrichment technique and were identified based on 16S rRNA analysis. The bacterial strains were cultured in mineral salt medium (MSM) supplemented with 1000 mg L−1 butyric acid, as a sole carbon and energy source, at 30 ± 1 °C, pH 7 and 110 rpm under aerobic growth conditions. The modified Gompertz model was used to estimate growth pattern parameters of microbiological relevance. Bacterial strains were phylogenetically identified as Alcaligenes sp. strain SY1, Achromobacter animicus, Pseudomonas aeruginosa, Serratia marcescens, Achromobacter xylosoxidans, Bacillus cereus, Lysinibacillus fusiformis, Bacillus methylotrophicus and Bacillus subtilis. The bacterial strains in pure cultures degraded butyric acid of 1000 mg L−1 within 20–24 h. The growth kinetics of the bacterial strains in pure culture utilising butyric acid were well described by the modified Gompertz model. This work highlights the potential for use of these bacterial strains in microbial degradation of butyric acid for deodorisation of pit latrine faecal sludge. This work also contributes significantly to our understanding of bioremediation of faecal sludge odours and informs the development of appropriate odour control technologies that may improve odour emissions from pit latrines.     

A semi-quantitative approach to assess biofilm formation using wrinkled colony development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis, and Gram-negative bacteria, such as Vibrio cholerae, Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Vibrio fischeri. The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect, while strains exhibiting increased biofilm phenotypes are enhanced for colonization. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization. In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.     

Thinking about Bacillus subtilis as a multicellular organism

Initial attempts to use colony morphogenesis as a tool to investigate bacterial multicellularity were limited by the fact that laboratory strains often have lost many of their developmental properties. Recent advances in elucidating the molecular mechanisms underlying colony morphogenesis have been made possible through the use of undomesticated strains. In particular, Bacillus subtilis has proven to be a remarkable model system to study colony morphogenesis because of its well-characterized developmental features. Genetic screens that analyze mutants defective in colony morphology have led to the discovery of an intricate regulatory network that controls the production of an extracellular matrix. This matrix is essential for the development of complex colony architecture characterized by aerial projections that serve as preferential sites for sporulation. While much progress has been made, the challenge for future studies will be to determine the underlying mechanisms that regulate development such that differentiation occurs in a spatially and temporally organized manner.     

Asymptotic analysis of a chemotactic model of bacteria colonies

An estimate of the distance between spots generated by a bacterial colony model is obtained. The model describes the morphogenesis of a spot pattern in colonies of chemotactic strains of Escherichia coli. Asymptotic methods for other cell-chemotaxis models, which have been successfully used by previous researchers, can be applied also to this model. However the calculations and the result is more complicated for this model. The result is verified by comparing it with the results by numerical computations of solutions of the model.     

Bayesian predictive identification and cumulative classification of bacteria

In this paper we give a mathematically precise formulation of an old idea in bacterial taxonomy, namely cumulative classification, where the taxonomy is continuously updated and possibly augmented as new strains are identified. Our formulation is based on Bayesian predictive probability distributions. The criterion for founding a new taxon is given a firm theoretical foundation based on prediction and it is given a clear-cut interpretation. We formulate an algorithm for cumulative classification and apply it to a large database of bacteria belonging to the family Enterobacteriaceae. The resulting taxonomy makes microbiological sense.     

Bacterial communities of diverse Drosophila species: ecological context of a host-microbe model system

Drosophila melanogaster is emerging as an important model of non-pathogenic host-microbe interactions. The genetic and experimental tractability of Drosophila has led to significant gains in our understanding of animal-microbial symbiosis. However, the full implications of these results cannot be appreciated without the knowledge of the microbial communities associated with natural Drosophila populations. In particular, it is not clear whether laboratory cultures can serve as an accurate model of host-microbe interactions that occur in the wild, or those that have occurred over evolutionary time. To fill this gap, we characterized natural bacterial communities associated with 14 species of Drosophila and related genera collected from distant geographic locations. To represent the ecological diversity of Drosophilids, examined species included fruit-, flower-, mushroom-, and cactus-feeders. In parallel, wild host populations were compared to laboratory strains, and controlled experiments were performed to assess the importance of host species and diet in shaping bacterial microbiome composition. We find that Drosophilid flies have taxonomically restricted bacterial communities, with 85% of the natural bacterial microbiome composed of only four bacterial families. The dominant bacterial taxa are widespread and found in many different host species despite the taxonomic, ecological, and geographic diversity of their hosts. Both natural surveys and laboratory experiments indicate that host diet plays a major role in shaping the Drosophila bacterial microbiome. Despite this, the internal bacterial microbiome represents only a highly reduced subset of the external bacterial communities, suggesting that the host exercises some level of control over the bacteria that inhabit its digestive tract. Finally, we show that laboratory strains provide only a limited model of natural host-microbe interactions. Bacterial taxa used in experimental studies are rare or absent in wild Drosophila populations, while the most abundant associates of natural Drosophila populations are rare in the lab.     

Estimation of Occupied and Unoccupied Black-Tailed Prairie Dog Colony Acreage in Colorado

Abstract: Black-tailed prairie dogs (Cynomys ludovicianus) are a species of concern and accurate estimates of occupied area are required to assess their status. We conducted aerial line-intercept surveys to estimate colony areas in Colorado, USA, 2006–2007. Optimal allocation based on results from a previous (2002) survey was used to distribute flight time to sample 28 counties. Uncorrected estimates of active and inactive colony areas from the aerial surveys were 329,529 (SE=16,841) ha and 18,292 (SE=2,366) ha, respectively. We attempted to ground-truth a randomly selected sample of 186 colony intercepts but gained complete access to only 150. Ground-truthing demonstrated that aerial surveys estimated only 96% of the true lengths of colony intercepts but overestimated the proportion of active colonies. Corrected estimates of active and inactive colony areas are 319,165 (SE = 20,105) ha and 42,422 (SE = 11,485) ha, respectively. Because ground-truthing was not conducted in the original 2002 survey, uncorrected estimates from this survey are the appropriate metric to be used for comparison to the 2002 data. Our estimates demonstrated a 29% increase (SE = 6.3) in area occupied since surveys were conducted in 2002. These results are useful to state and federal agencies and other conservation partners in determining the condition of the species when conducting status reviews.     

Relationship between colonial surface and density on agar plate

The size of a colony on an agar plate is influenced by the number of colonies ( N ) on this plate. When N is small, colonies reach a larger size. The relationship between colonial surface and density of bacteria on agar plate was studied for nine bacterial and two yeast strains. A mathematical model describing the relationship between the logarithm base 10 of the number of colonies on the agar plate and the average colonial diameter was built. This model is shown to be adapted to most of the strains studied and could be a tool for media quality control.     

Estimating colony area and population size of little auks <Emphasis Type="Italic">Alle alle</Emphasis> at Northumberland Island using aerial images

This paper presents a technique to estimate little auk population size from the area of the colonies measured by the use of video recordings and aerial photographs. To transform colony area into population size, breeding densities obtained from the literature were used. The study area, Northumberland Island, Thule District, Northwest Greenland, revealed between 6 and 12 million km² inhabited colony area, which was classified into three categories for colony identification. The lower figure of this estimate is equivalent to a little auk population of 4.6 million pairs. An extrapolation to the rest of the Thule District should be treated with care but would amount to at least 33 million pairs. There is no doubt that the methods used would benefit from more fine-scale ground-truth surveys in the Thule District.     

Methods for Determining the Uncertainty of Population Estimates Derived from Satellite Imagery and Limited Survey Data: A Case Study of Bo City,Sierra Leone

This study demonstrates the use of bootstrap methods to estimate the total population of urban and periurban areas using satellite imagery and limited survey data. We conducted complete household surveys in 20 neighborhoods in the city of Bo, Sierra Leone, which collectively were home to 25,954 persons living in 1,979 residential structures. For five of those twenty sections, we quantized the rooftop areas of structures extracted from satellite images. We used bootstrap statistical methods to estimate the total population of the pooled sections, including the associated uncertainty intervals, as a function of sample size. Evaluations based either on rooftop area per person or on the mean number of occupants per residence both converged on the true population size. We demonstrate with this simulation that demographic surveys of a relatively small proportion of residences can provide a foundation for accurately estimating the total population in conjunction with aerial photographs.     

Assessing the density of honey bee colonies at ecosystem scales

1. Information about the density of wild honey bee (Apis spp.) colonies in an ecosystem is central to understanding the functional role of honey bees in that ecosystem, necessary for effective biosecurity response planning, and useful for determining whether pollination services are adequate. However, direct visual surveys of colony locations are not practical at ecosystem scales. Thus, indirect methods based on population genetic analysis of trapped males have been proposed and implemented. 2. In this review, indirect methods of assessment of honey bee colony densities are described, which can be applied at ecosystem scales. The review also describes how to trap males in the field using the Williams drone trap (or virgin queens) the appropriate genetic markers and statistical analyses, and discusses issues surrounding sample size. 3. The review also discusses some outstanding issues concerning the methods and the conversion of estimated colony number to colony density per km². The appropriate conversion factor will require further research to determine the area over which a drone trap draws drones.     

An alternative method for the decontamination of rats carrying Mycoplasma pulmonis without the use of germfree isolators

Two strains of Lewis rat were successfully freed from Mycoplasma pulmonis infection by using a combination of oral treatment with oxytetracycline hydrochloride and obtaining young by hysterectomy. Laminar flow cabinets were used to perform hysterectomies on donor animals and for rearing hysterectomy-derived animals. After thorough microbiological examination the rats were brought to the breeding colony of the Laboratory Animal Centre. Periodic laboratory tests using both cultural and enzyme-linked immunosorbent assay methods showed that the animals have remained free from M. pulmonis for the last 3 years.     

Development and Evaluation of a Potency Index Screen for Detecting Mutants of Penicillium chrysogenum Having Increased Penicillin Yield

U.v.-treated conidia of an industrial strain of Penicillium chrysogenum were spread on a growth-limiting agar medium. Colonies arising from the survivors were surrounded with a spore suspension of Bacillus subtilis to which penicillinase had been added. After appropriate incubation, discrete zones of bacterial inhibition, with sizes limited by the penicillinase, appeared around each colony. Using the criterion of potency index (diameter of inhibition zone divided by diameter of colony) strains were selected that subsequently gave improved penicillin production in shake-flasks. The technique also ranked correctly four industrial strains in order of their known penicillin-producing capacity. Employing three operators, 5000 isolates could be screened in each experiment and approx. 15000 strains could be screened in a month.     

The Influence of Mark-Recapture Sampling Effort on Estimates of Rock Lobster Survival

Five annual capture-mark-recapture surveys on Jasus edwardsii were used to evaluate the effect of sample size and fishing effort on the precision of estimated survival probability. Datasets of different numbers of individual lobsters (ranging from 200 to 1,000 lobsters) were created by random subsampling from each annual survey. This process of random subsampling was also used to create 12 datasets of different levels of effort based on three levels of the number of traps (15, 30 and 50 traps per day) and four levels of the number of sampling-days (2, 4, 6 and 7 days). The most parsimonious Cormack-Jolly-Seber (CJS) model for estimating survival probability shifted from a constant model towards sex-dependent models with increasing sample size and effort. A sample of 500 lobsters or 50 traps used on four consecutive sampling-days was required for obtaining precise survival estimations for males and females, separately. Reduced sampling effort of 30 traps over four sampling days was sufficient if a survival estimate for both sexes combined was sufficient for management of the fishery.     

Experimental evidence for sympatric ecological diversification due to frequency-dependent competition in Escherichia coli

We investigate adaptive diversification in experimental Escherichia coli populations grown in serial batch cultures on a mixture of glucose and acetate. All 12 experimental lines were started from the same genetically uniform ancestral strain but became highly polymorphic for colony size after 1000 generations. Five populations were clearly dimorphic and thus serve as a model for an adaptive lineage split. We analyzed the ecological basis for this dimorphism by studying bacterial growth curves. All strains exhibit diauxie, that is, sequential growth on the two resources. Thus, they exhibit phenotypic plasticity, using mostly glucose when glucose is abundant, then switching to acetate when glucose concentration is low. However, the coexisting strains differ in their diauxie pattern, with one cluster in the dimorphic populations growing better in the glucose phase, and the other cluster having a much shorter lag when switching to the acetate phase. Using invasion experiments, we show that the dimorphism of these two ecological types is maintained by frequency-dependent selection. Using a mathematical model for the adaptive dynamics of diauxie behavior, we show that evolutionary branching in diauxie behavior is a plausible theoretical scenario. Our results support the hypothesis that, in our experiments, adaptive diversification from a genetically uniform ancestor occurred due to frequency-dependent ecological interactions. Our results have implications for understanding the evolution of cross-feeding polymorphism in microorganisms, as well as adaptive speciation due to frequency-dependent selection on phenotypic plasticity.     

Acquisition of iron by Trichodesmium and associated bacteria in culture

Trichodesmium colonies contain an abundant microbial consortium that is likely to play a role in nutrient cycling within the colony. This study used laboratory cultures of Trichodesmium and two genome-sequenced strains of bacteria typical of Trichodesmium-associated microbes to develop an understanding of the cycling of iron, a potentially limiting micronutrient, within Trichodesmium colonies. We found that the ferric siderophores desferrioxamine B and aerobactin were not readily bioavailable to Trichodesmium, relative to ferric chloride or citrate-associated iron. In contrast, the representative bacterial strains we studied were able to acquire iron from all of the iron sources, implying that naturally occurring Trichodesmium-associated bacteria may be capable of utilizing a more diverse array of iron sources than Trichodesmium. From the organism-specific uptake data collected in this study, a theoretical Trichodesmium colony was designed to model whole colony iron uptake. The bacteria accounted for most (>?70%) of the iron acquired by the colony, highlighting the importance of determining organism-specific uptake in a complex environment. Our findings suggest that, although they may share the same micro-environment, Trichodesmium and its colony-associated microbial cohort may differ substantially in terms of iron acquisition strategy.