Heterogeneous Bacterial Populations Research Articles

Pulsed-field gel electrophoresis, virulence determinants and antimicrobial susceptibility profiles of type Ia group B streptococci isolated from humans in Brazil

Group B streptococci (GBS) infections occur worldwide. Although serotyping has been used for epidemiologic purposes, this does not accurately characterize enough members of a genetically heterogeneous bacterial population. The aims of this work were to evaluate the genetic diversity of 45 type Ia GBS strains isolated in Brazil by pulsed-field gel electrophoresis as well as to evaluate antimicrobial susceptibility profiles and identify virulence genes. Twenty-four strains were assigned to cluster A. All strains under study contained the hylB and scpB genes. The bca gene was detected in only 10 strains and none of the streptococci carried the bac gene. Thirty-nine strains were resistant to tetracycline.

Decreased Susceptibility to Polymyxin B during Treatment for Carbapenem-Resistant Klebsiella pneumoniae Infection

Decreased Susceptibility to Polymyxin B during Treatment for Carbapenem-Resistant <i>Klebsiella pneumoniae</i> Infection

Functional diversity of the microbial community in the rhizosphere of chickpea grown in diesel fuel-spiked soil amended with Trichoderma ressei using sole-carbon-source utilization profiles

Present study describes chickpea (Cicer arietinum) growth, microbial activity and community composition in a soil samples spiked with 0, 20 (LCD) and 80 g (HCD) diesel/kg soils, amended with Trichoderma ressei. T. ressei had stimulatory effect on the plant growth parameters as compared with un-inoculated control chickpea plant. Root length, shoot length, plant dry weight and chlorophyll content enhanced 128, 31, 46, 79%, respectively, as compared over the un-inoculated control. At LCD in the presence of T. ressei chickpea root length, shoot length, plant dry weight and chlorophyll content was maximum indicating that at this concentration of diesel chickpea plants could grow very well and T. ressei amendment had synergistic effect. Effect on microbial population was most evident at HCD and resulted in 4.84 log unit reduction of heterogeneous bacterial population, as compared with LCD which caused reduction of 2.8 log unit, compared with non-diesel spiked control soil. Impact of diesel on soil was somewhat lessened in the presence of T. ressei. Our results indicated that application of diesel improved the organic matter status of soils which was in turn reflected in the higher dehydrogenase activity. This could be due to diesel being a good source of hydrocarbon readily available for microbial activity. The structure of the microbial community in rhizosphere was analyzed through the sole-carbon-source utilization profiles using ECO Biolog microplates. Significant differences were found among the diversity and evenness indices on effect of diesel on chickpea rhizosphere microflora in presence and absence of T. ressei, based on Tukey’s test (at P = 0.05). Principal component analysis of substrate source utilization pattern on Biolog Eco plates by chickpea rhizosphere microflora in presence and absence of T. ressei was determined. Distinct resolution of soil microbial communities in the presence of either diesel or, T. ressei observed thus revealed differences in the microbial metabolic profiles for the different treatments. Our results demonstrated that characteristics of the dynamics in microbial communities complemented well with organic matter status of soils and dehydrogenase activity. The technique highlighted the usefulness of this parameter for ecological indication of land use change in diesel contaminated ecosystems.

Genetic diversity of major surface protein 1a of Anaplasma marginale in beef cattle

The present study was aimed to demonstrate genotypic diversity of Anaplama marginale in infected beef herds grazing within anaplasmosis endemic regions. The genotypic diversity was identified among different herds, within each herd, and also within single animals. The Israeli strains revealed unique characteristics of MSP1a repeats and, in addition to the published repeats, six new tandem repeats designated Is1–5, and Is9 were identified. The superinfections of individual Anaplama centrale vaccinated animals with two genotypically different A. marginale strains were detected. Six out of 43 vaccinated animals in the G herd were each infected with two A. marginale strains carrying two distinct genotypes; in this herd the follow-up during years 2003–2007 demonstrated that several animals carried different msp1a genotypes at different time points. Coinfection with two different genotypes of A. marginale in A. centrale vaccinated cattle was observed in another herd, as well. It appears that A. marginale is composed of a heterogeneous changing bacterial population that evolves in the host or, the genotypic diversity implies high transmission intensity by the vector, or both. Learning how this diversity is generated and identification of distinct A. marginale strains coupled with high sequence variation of MSP1a will aid in understanding Anaplasma transmission and disease development.

Stochastic Receptor Expression Allows Sensitive Bacteria to Evade Phage Attack. Part II: Theoretical Analyses

Stochastic gene expression in bacteria can create a diverse protein distribution. Most of the current studies have focused on fluctuations around the mean, which constitutes the majority of a bacterial population. However, when the bacterial population is subject to a severe selection pressure, it is the properties of the minority cells that determine the fate of the population. The central question is whether phenotype heterogeneity, such as a spread in the expression level of a critical protein, is sufficient to account for the persistence of the bacteria under the selection. A related question is how long such persistence can last before genetic mutation becomes significant. In this work, survival statistics of a bacterial population with a diverse phage-receptor number distribution is theoretically investigated when the cells are subject to phage pressures. The calculations are compared with our experimental observations presented in Part I in this issue. The fundamental basis of our analysis is the Berg-Purcell theoretical result for the reaction rate between a phage particle and a bacterium with a discrete number of receptors, and the observation that most phage-resistant mutants isolated in laboratory cultures are defective in phage binding. It is shown that a heterogeneous bacterial population is significantly more fit compared to a homogeneous population when confronting a phage attack.

Phenotypic heterogeneity in bacterial populations

Genetically uniform bacterial cells exhibit heterogeneity such as intrapopulation differences in metabolism as well as variation in growth rate. Additionally, phenotypic heterogeneity in more complex developmental processes where a portion of a population performs specialized functions has been described. Heterogeneity within populations of bacterial cells ensures that a small fraction of the population is prepared to survive adverse environmental conditions. Phenotypic heterogeneity is mediated by two mechanisms: (i) genotypic alterations such as, mutations and rearrangements of specific DNA fragments or (ii) epigenetic phenomenon. Here examples of genotypic as well as epigenetically regulated phenotypic heterogeneity from several bacterial species are presented.

Profiling of bacterial adhesin — host receptor recognition by soluble immunoglobulin superfamily domains

Several Gram-negative human pathogens recognize members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. Pathogenic Neisseriae employ distinct isoforms of the colony opacity-associated proteins (Opa CEA proteins) to bind to the amino-terminal domains of CEACAMs. Here we present a novel approach to rapidly determine the CEACAM-binding properties of single bacteria. Expression of the isolated amino-terminal domains of various CEACAMs in eukaryotic cells yields soluble probes that selectively recognize Opa CEA-expressing bacteria in a pull-down assay format. Furthermore, by expressing soluble CEACAMs as fusions to green-fluorescent protein (CEACAM-N-GFP), CEACAM-binding bacteria can be decorated with a fluorescent label and analysed by flow cytometry allowing the specific detection of receptor binding events on the level of single bacteria. Besides its potential for rapid and quantitative analysis of pathogen–receptor interactions, this novel approach allows the detection of receptor recognition in heterogeneous bacterial populations and might represent a valuable tool for profiling the host binding capabilities of various microorganisms.

Colonization of mucin by human intestinal bacteria and establishment of biofilm communities in a two-stage continuous culture system.

The human large intestine is covered with a protective mucus coating, which is heavily colonized by complex bacterial populations that are distinct from those in the gut lumen. Little is known of the composition and metabolic activities of these biofilms, although they are likely to play an important role in mucus breakdown. The aims of this study were to determine how intestinal bacteria colonize mucus and to study physiologic and enzymatic factors involved in the destruction of this glycoprotein. Colonization of mucin gels by fecal bacteria was studied in vitro, using a two-stage continuous culture system, simulating conditions of nutrient availability and limitation characteristic of the proximal (vessel 1) and distal (vessel 2) colon. The establishment of bacterial communities in mucin gels was investigated by selective culture methods, scanning electron microscopy, and confocal laser scanning microscopy, in association with fluorescently labeled 16S rRNA oligonucleotide probes. Gel samples were also taken for analysis of mucin-degrading enzymes and measurements of residual mucin sugars. Mucin gels were rapidly colonized by heterogeneous bacterial populations, especially members of the Bacteroides fragilis group, enterobacteria, and clostridia. Intestinal bacterial populations growing on mucin surfaces were shown to be phylogenetically and metabolically distinct from their planktonic counterparts.

Comparison of the Minimum Inhibitory, Mutant Prevention and Minimum Bactericidal Concentrations of Ciprofloxacin, Levofloxacin and Garenoxacin Against Enteric Gram-negative Urinary Tract Infection Pathogens

Acute, uncomplicated urinary tract infections (UTIs) are among the most commonly encountered bacterial infections and management has been made more complicated in the last decade due to the trend toward increasing antimicrobial resistance to ampicillin and trimethoprim/sulfamethoxazole (TMP/SMX). Fluoroquinolones are suggested as alternative antimicrobials for the treatment of UTIs in communities for which TMP/SMX resistance is ≥10%. The mutant-prevention concentration (MPC) is a novel susceptibility parameter designed to minimize the selection of first-step resistant mutants present in large, ≥1010 CFU/mL, heterogeneous bacterial populations and is a distinct measurement from minimum inhibitory concentration testing. We measured MPC results for 80 enteric Gramnegative and 20 Pseudomonas aeruginosa urinary isolates against ciprofloxacin, levofloxacin and garenoxacin. Ciprofloxacin, levofloxacin and garenoxacin MPC results for Escherichia coli, Citrobacter freundii, Enterobacter cloacae, Klebsiella pneumoniae and P. aeruginosa respectively were 0.5, 1, 1, 1 and 4 mg/L; 1, 2, 4, 2 and 16 mg/L; 1, 8, >8, 4 and ≥32 mg/L. By comparison, minimum inhibitory concentration (MIC)90 results for the Enterobacteriaceae organisms ranged from ≤0.06-4 mg/L for the three drugs and 1-4 mg/L against P. aeruginosa. Similarly, MBC90 results ranged from ≤0.06-4 mg/L and 2-8 mg/L respectively. For ciprofloxacin against E.coli, E. cloacae and K. pneumoniae and for levofloxacin against E.coli, C. freundii and K. pneumoniae, MPC results were below susceptible breakpoints and within clinically achievable and sustainable drug concentrations for >24 hours of the dosing interval against. For garenoxacin, urine drug concentrations are expected to be in excess of MPC results for the entire length of the dosing interval for E.coli. Application of MPCs to fluoroquinolones and management of UTIs represents a situation where high levels of in vitro activity, based on low MICs, is reflected in correspondingly low MPC values for most of the organisms tested. Incorporation of MPC strategies into current fluoroquinolone dosing in UTI represents a realistic approach for preventing the further selection of resistant organisms associated with UTIs.

Engineered Polymeric Nanoparticles for Bioremediation of Hydrophobic Contaminants

Sorption of hydrophobic organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs), to soil has been shown to limit their solubilization rate and mobility. In addition, sequestration of contaminants by sorption to soil and by partitioning in nonaqueous phase liquids (NAPLs) reduces their bioavailability. Polymeric nano-network particles have been demonstrated to increase the "effective" solubility of a representative hydrophobic organic contaminant, phenanthrene (PHEN) and to enhance the release of PHEN from contaminated aquifer material. In this study, we investigate the usefulness of nanoparticles made from a poly(ethylene) glycol modified urethane acrylate (PMUA) precursor chain, in enhancing the bioavailability of PHEN. PMUA nanoparticles are shown to increase the mineralization rate of PHEN crystal in water, PHEN sorbed on aquifer material, and PHEN dissolved in a model NAPL (hexadecane) in the presence of aquifer media. These results show that PMUA particles not only enhance the release of sorbed and NAPL-sequestered PHEN but also increase its mineralization rate. The accessibility of contaminants in PMUA particles to bacteria also suggests that particle application may be an effective means to enhance the in-situ biodegradation rate in remediation through natural attenuation of contaminants. In pump-and-treat or soil washing remediation schemes, bioreactors could be used to recycle extracted nanoparticles. The properties of PMUA nanoparticles are shown to be stable in the presence of a heterogeneous active bacterial population, enabling them to be reused after PHEN bound to the particles has been degraded by bacteria.

Bacterial persistence as a phenotypic switch.

A fraction of a genetically homogeneous microbial population may survive exposure to stress such as antibiotic treatment. Unlike resistant mutants, cells regrown from such persistent bacteria remain sensitive to the antibiotic. We investigated the persistence of single cells of Escherichia coli with the use of microfluidic devices. Persistence was linked to preexisting heterogeneity in bacterial populations because phenotypic switching occurred between normally growing cells and persister cells having reduced growth rates. Quantitative measurements led to a simple mathematical description of the persistence switch. Inherent heterogeneity of bacterial populations may be important in adaptation to fluctuating environments and in the persistence of bacterial infections.

The Role of PK/PD Parameters to Avoid Selection and Increase of Resistance: Mutant Prevention Concentration

SummaryThe continuing escalation of antimicrobial resistant human pathogens and the limited number of new antimicrobial agents under development has dictated that our knowledge on the emergence of resistance and any potential strategies to slow the rate at which resistance occurs is of paramount importance. Investigations with fluoroquinolones resulted in the mutant prevention concentration (MPC) concept which represents a novel in vitro measurement of fluoroquinolone potency. In essence, the MPC defines the antimicrobial drug concentration threshold that would require an organism to simultaneously possess two resistance mutations for growth in the presence of the drug. An alternative definition is the drug concentration that prevents the growth of first-step resistant mutants or the minimal inhibitory concentration of the most resistant organism present in the heterogeneous bacterial population when tested against >109 organisms. From in vitro investigations, the new fluoroquinolones (gatifloxacin, gemifloxacin, moxifloxacin) were all found to have lower MPC values than did levofloxacin against clinical isolates of Streptococcus pneumoniae. Ciprofloxacin was found to have lower MPC values than levofloxacin against clinical isolates of Pseudomonas aeruginosa. When MPC data is applied to achievable and sustainable serum drug concentrations in the body, estimation of the time the serum drug concentration exceed both MIC and MPC values can be determined. This data along with kill data allows for an estimate of the amount of time drug concentration needs to exceed MIC/MPC values to not only result in significant kill but also to minimize resistance development. To date, MPC measurements have been determined in in vitro microbiological and pharmacological models and animal and human data are being investigated.The data summarized in this overview detail resistance issues for P. aeruginosa, S. pneumoniae and other pathogens. Also presented is a summary of the MPC concept and investigations completed to date. A brief summary of fluoroquinolone mechanisms of action and resistance is presented. Finally, some preliminary investigations with other classes of compounds are discussed. To date, very limited data is available to conclude if the MPC concept does or does not apply to other classes of antimicrobial agents.

Plasmids spread very fast in heterogeneous bacterial communities.

Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several orders of magnitude. Such donors allow millions of other bacteria to acquire the plasmid in a matter of days whereas, in the absence of such strains, plasmid dissemination would take years. This "amplification effect" could have an impact on the evolution of bacterial pathogens that exist in heterogeneous bacterial communities because conjugative plasmids can carry virulence or antibiotic-resistance genes.

Statistical analysis and biological interpretation of the flow cytometric heterogeneity observed in bacterial axenic cultures

Histogram comparison and meaningful statistics in flow cytometry is probably the most widely encountered mathematical problem in flow cytometry. Ideally, a test for determining the statistical equality or difference of flow cytometric distributions will identify the significant differences or similarities of the obtained histograms. This situation is of particular interest when flow cytometry is used to study the heterogeneity of axenic bacterial populations. We have statistically measured the heterogeneity of successive cytometric measures, the modifications produced after 20 transfers from the same culture, and the differences between 20 subcultures of identical origin. The heterogeneity of the bacterial populations and the similarity of the obtained 360 histograms were analysed by standard statistical methods. We have studied bacterial axenic cultures in order to detect, quantify and interpret their cytometric heterogeneity, and to assess intrinsic differences and differences produced by laboratory manipulations. We concluded that the standard axenic cultures have a considerable intrinsic cellular and molecular heterogeneity. We suggest that the heterogeneity we have detected basically has two origins: cell size diversity and cell cycle variations.

Natural Infestation of Onion Seed by Pantoea ananatis, Causal Agent of Center Rot.

An immunomagnetic separation and polymerase chain reaction (IMS-PCR) assay was used to detect Pantoea ananatis in naturally infested onion seeds. Using species-specific PCR primers and polyclonal antibodies, IMS-PCR consistently demonstrated detection thresholds of 101 to 103 CFU/ml. There was no significant difference between the numbers of CFU recovered from onion seed wash by IMS (after repeated rinses) and by direct plating, indicating that IMS effectively captured P. ananatis cells from heterogeneous bacterial populations. Using IMS-PCR and IMS followed by plating on nutrient agar, P. ananatis was detected in 19.7% of onion seed samples harvested from two onion fields in which center rot developed naturally in 2000. When planted in germination boxes, 53% of the seed samples that tested positive for P. ananatis produced seedlings with symptoms of center rot. There was no significant difference in germination between infested and noninfested seed samples. This is the first report of natural infestation and transmission of P. ananatis in onion seed.

Heterogeneity of bacterial populations and pesticide degradation potentials in the unsaturated zone of loamy and sandy soils

Soil microbial processes play an important role in relation to pesticide pollution of groundwater, and may be strongly influenced by hydrological and geochemical properties. The consequences of such heterogeneous environments on bacterial biomass, enzymatic activities, carbon utilisation patterns, and pesticide mineralisation potentials in the unsaturated zone of a sandy loam and a coarse sandy soil profile were studied. In sandy loam soil profiles the number of bacteria decreased from 109 cells g–1 in the surface layers to about 107 cells g–1 at 1.5–5 m depth. Simultaneously, the hydrolysis of fluorescein diacetate and arylsulfatase activity decreased to below the detection limit at about 1.5 m depth, and carbon utilisation patterns showed that bacterial populations from surface soil were significantly different from those from 4 m depth. Bacterial biomass and activity in macropore soil tended to be slightly higher than in matrix soil, and the carbon utilisation patterns of bacterial populations extracted from macropore soil and from matrix soil seemed to be different. Maximally 3% of 14C-labelled mecoprop and isoproturon was mineralised in soil from the 1–1.5 m depth, and less than 1.5% was mineralised in soil from the 3.5–4 m depth. The macropore soil tended to have a higher degradation potential than the matrix soil. The total number of bacteria in the coarse sandy soil profile decreased from about 108 in the plough layer to 107 cells g–1 at 0.4–2 m. The enzymatic activities and the degradation potentials of 14C-labelled mecoprop and isoproturon were significantly correlated (r2 >0.79) and showed a distinct decrease at about 0.4 m. In addition to the depth variability, a horizontal heterogeneity in this soil was observed as horizons or compartments that differed in colour, i.e. with different chemical composition and concentrations of Fe and organic matter. Counts of viable bacteria and measurements of fluorescein diacetate hydrolysis and arylsulfatase activity confirmed a high variability of microbial biomass and activity in the sandy soil profile.

Rapid flow cytometry--Nile red assessment of PHA cellular content and heterogeneity in cultures of Pseudomonas aeruginosa 47T2 (NCIB 40044) grown in waste frying oil.

The accumulation of cytoplasmic polyhydroxyalkanoates (PHAs) and the heterogeneity ofbacterial populations were analysed by flow cytometry and SYTO-13 and Nile red staining in rhamnolipid-producing Pseudomonas aeruginosa cultures grown in waste frying oil as carbon source. A combination of SYTO-13 and Nile red fluorescence with cytometric forward and side scatter values may allow increases in the final production of polyhydroxyalkanoates (PHA) by two basic mechanisms: (i) rapid assessment of polyhydroxyalkanoate content and (ii) definition of flow cytometric cell sorting protocols to select high polyhydroxyalkanoate (PHA)-producing strains. We report a rapid (less than 30 min) flow cytometric assessment of PHAs in Pseudomonas aeruginosa 47T2 following Nile red staining: (i) to estimate cellular PHAs content; (ii) to study heterogeneity of the batch cultures producing PHAs and (iii) to establish the basis for sorting sub-populations with a high capacity to accumulate PHAs.

Proteomics in medical microbiology

The techniques of proteomics (high resolution two-dimensional electrophoresis and protein characterisation) are widely used for microbiological research to analyse global protein synthesis as an indicator of gene expression. The rapid progress in microbial proteomics has been achieved through the wide availability of whole genome sequences for a number of bacterial groups. Beyond providing a basic understanding of microbial gene expression, proteomics has also played a role in medical areas of microbiology. Progress has been made in the use of the techniques for investigating the epidemiology and taxonomy of human microbial pathogens, the identification of novel pathogenic mechanisms and the analysis of drug resistance. In each of these areas, proteomics has provided new insights that complement genomic-based investigations. This review describes the current progress in these research fields and highlights some of the technical challenges existing for the application of proteomics in medical microbiology. The latter concern the analysis of genetically heterogeneous bacterial populations and the integration of the proteomic and genomic data for these bacteria. The characterisation of the proteomes of bacterial pathogens growing in their natural hosts remains a future challenge.

Proteomics in medical microbiology.

The techniques of proteomics (high resolution two-dimensional electrophoresis and protein characterisation) are widely used for microbiological research to analyse global protein synthesis as an indicator of gene expression. The rapid progress in microbial proteomics has been achieved through the wide availability of whole genome sequences for a number of bacterial groups. Beyond providing a basic understanding of microbial gene expression, proteomics has also played a role in medical areas of microbiology. Progress has been made in the use of the techniques for investigating the epidemiology and taxonomy of human microbial pathogens, the identification of novel pathogenic mechanisms and the analysis of drug resistance. In each of these areas, proteomics has provided new insights that complement genomic-based investigations. This review describes the current progress in these research fields and highlights some of the technical challenges existing for the application of proteomics in medical microbiology. The latter concern the analysis of genetically heterogeneous bacterial populations and the integration of the proteomic and genomic data for these bacteria. The characterisation of the proteomes of bacterial pathogens growing in their natural hosts remains a future challenge.

Assessment of metabolic activity of single bacterial cells — new developments in microcolony and dehydrogenase assays

Some bacteria lose culturability in natural environments but retain measurable metabolic activity and are thus considered viable. Several techniques have been proposed to determine the activity of nonculturable cells. Due to the considerable physiological heterogeneity of bacterial populations in the environment, it is imperative to apply methods which measure cellular activity at the single cell level. This review focuses on two promising methods: the microcolony assay and the respiration assay based on reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). In the microcolony assay, viable cells are identified by their ability to perform a limited number of cell divisions and this approach is thus related to conventional culture techniques. Some recent methodological developments of the technique aiming at improving the incubation conditions and the detection of microcolonies are presented. Results obtained by the microcolony technique are used to introduce its advantages and limitations. The CTC-reduction assay determines a central cellular metabolic activity, but does not measure cell growth. Results of studies using this assay are presented, and it is emphasized that great care should be taken to optimize assay conditions for the studied organisms. Finally, the results obtained by different viability assays are compared. For a specific bacterium, several assays, addressing different aspects of cell metabolism, can provide comparable results suggesting that they provide meaningful viability estimates. On the other hand, the use of viability assays on complex indigenous populations may be ambiguous.