Effective case/infection ratio of poliomyelitis in vaccinated populations.

Recent polio outbreaks in Syria and Ukraine, and isolation of poliovirus from asymptomatic carriers in Israel have raised concerns that polio might endanger Europe. We devised a model to calculate the time needed to detect the first case should the disease be imported into Europe, taking the effect of vaccine coverage - both from inactivated and oral polio vaccines, also considering their differences - on the length of silent transmission into account by deriving an 'effective' case/infection ratio that is applicable for vaccinated populations. Using vaccine coverage data and the newly developed model, the relationship between this ratio and vaccine coverage is derived theoretically and is also numerically determined for European countries. This shows that unnoticed transmission is longer for countries with higher vaccine coverage and a higher proportion of IPV-vaccinated individuals among those vaccinated. Assuming borderline transmission (R = 1·1), the expected time to detect the first case is between 326 days and 512 days in different countries, with the number of infected individuals between 235 and 1439. Imperfect surveillance further increases these numbers, especially the number of infected until detection. While longer silent transmission does not increase the number of clinical diseases, it can make the application of traditional outbreak response methods more complicated, among others.

Quantitative impact of direct, personal feedback on hand hygiene technique.

This study investigated the effectiveness of targeting hand hygiene technique using a new training device that provides objective, personal and quantitative feedback. One hundred and thirty-six healthcare workers in three Hungarian hospitals participated in a repetitive hand hygiene technique assessment study. Ultraviolet (UV)-labelled hand rub was used at each event, and digital images of the hands were subsequently taken under UV light. Immediate objective visual feedback was given to participants, showing missed areas on their hands. The rate of inadequate hand rubbing reduced from 50% to 15% (P < 0.001). However, maintenance of this reduced rate is likely to require continuous use of the electronic equipment.

Trends in Major Lower Limb Amputation Related to Peripheral Arterial Disease in Hungary: A Nationwide Study (2004-2012).

OBJECTIVES: To assess the trends of peripheral arterial disease associated major lower limb amputation in Hungary over a 9 year period (2004-2012) in the whole Hungarian population. METHODS: This was a retrospective cohort study employing administrative health care data. Major amputations were identified in the entire Hungarian population during a 9 year period (2004-2012) using the health care administrative data. Direct standardization was used to eliminate the potential bias induced by the different age and sex structure of the compared populations. For external direct standardization, the ESP 2013 was chosen as reference. RESULTS: 76,798 lower limb amputations were performed. The number of major amputations was 38,200; these procedures affected 32,084 patients. According to case detection, 50.4% of the amputees were diabetic. The overall primary amputation rate was 71.5%. The annual crude and age adjusted major amputation rates exhibited no significant long-term pattern over the observation period. The major lower limb amputation incidence for the overall period was 42.3/10(5) in the total population and 317.9/10(5) in diabetic population. CONCLUSION: According to this whole population based study from Hungary, the incidence of lower limb major amputation is high with no change over the past 9 years. An explanation for this remains to be determined, as the traditional risk factors in Hungary do not account for it. The characteristics of major amputation (the rate of primary amputation, the ratio of below to above knee amputation and the age of the affected population) underline the importance of screening, early detection, improved vascular care and an optimal revascularization policy. Standardization and validation of amputation detection methods and reporting is essential.

The basis of antagonistic pleiotropy in hfq mutations that have opposite effects on fitness at slow and fast growth rates.

Mutations beneficial in one environment may cause costs in different environments, resulting in antagonistic pleiotropy. Here, we describe a novel form of antagonistic pleiotropy that operates even within the same environment, where benefits and deleterious effects exhibit themselves at different growth rates. The fitness of hfq mutations in Escherichia coli affecting the RNA chaperone involved in small-RNA regulation is remarkably sensitive to growth rate. E. coli populations evolving in chemostats under nutrient limitation acquired beneficial mutations in hfq during slow growth (0.1 h(-1)) but not in populations growing sixfold faster. Four identified hfq alleles from parallel populations were beneficial at 0.1 h(-1) and deleterious at 0.6 h(-1). The hfq mutations were beneficial, deleterious or neutral at an intermediate growth rate (0.5 h(-1)) and one changed from beneficial to deleterious within a 36 min difference in doubling time. The benefit of hfq mutations was due to the greater transport of limiting nutrient, which diminished at higher growth rates. The deleterious effects of hfq mutations at 0.6 h(-1) were less clear, with decreased viability a contributing factor. The results demonstrate distinct pleiotropy characteristics in the alleles of the same gene, probably because the altered residues in Hfq affected the regulation of expression of different genes in distinct ways. In addition, these results point to a source of variation in experimental measurement of the selective advantage of a mutation; estimates of fitness need to consider variation in growth rate impacting on the magnitude of the benefit of mutations and on their fitness distributions.

The spread of a beneficial mutation in experimental bacterial populations: the influence of the environment and genotype on the fixation of rpoS mutations.

The spread of beneficial mutations through populations is at the core of evolutionary change. A long-standing hindrance to understanding mutational sweeps was that beneficial mutations have been slow to be identified, even in commonly studied experimental populations. The lack of information on what constitutes a beneficial mutation has led to many uncertainties about the frequency, fitness benefit and fixation of beneficial mutations. A more complete picture is currently emerging for a limited set of identified mutations in bacterial populations. In turn, this will allow quantitation of several features of mutational sweeps. Most importantly, the 'benefit' of beneficial mutations can now be explained in terms of physiological function and how variations in the environment change the selectability of mutations. Here, the sweep of rpoS mutations in Escherichia coli, in both experimental and natural populations, is described in detail. These studies reveal the subtleties of physiology and regulation that strongly influence the benefit of a mutation and explain differences in sweeps between strains and between various environments.

An analysis of multifactorial influences on the transcriptional control of ompF and ompC porin expression under nutrient limitation.

Expression of the major outer-membrane porins in Escherichia coli is transcriptionally controlled during nutrient limitation. Expression of ompF was more than 40-fold higher under glucose limitation than under nitrogen (ammonia) limitation in chemostat cultures at the same growth rate. In contrast, ompC expression was higher under N limitation. The basis of regulation by nutrient limitation was investigated using mutations affecting expression of porin genes. The influence of cyaA, rpoS, ackA and pta, as well as the two-component envZ-ompR system, was studied under glucose and N limitation in chemostat cultures. A major contributor to low ompF expression under N limitation was negative control by the RpoS sigma factor. RpoS levels were high under N limitation and loss of RpoS resulted in a 19-fold increase in ompF transcription, but little change was observed with ompC. Lack of RpoS under glucose limitation had a lesser stimulatory effect on ompF expression. Porin production was minimally dependent on EnvZ under N limitation due to OmpR phosphorylation by acetyl phosphate. Evidence obtained with pta and ackA mutants suggested that the acetyl phosphate level also regulates porins independently and indirectly via RpoS and other pathways. pta-envZ double mutants had a residual level of porin transcription, implicating alternative means of OmpR phosphorylation under nutrient limitation. Another critical factor in regulation was the level of cAMP, as a cyaA mutant hardly expressed ompF under glucose limitation but boosted ompC. In addition, the role of DNA-binding proteins encoded by hns and himA was tested under glucose limitation: the hns mutation reduced the glucose-limitation peak, but the himA mutation suppressed the hns effect, suggesting a complex web of interrelationships between the DNA-binding proteins. Indeed, multiple inputs and no single regulator were responsible for the high peak of ompF expression under glucose limitation.

Hungry bacteria--definition and properties of a nutritional state.

Bacteria are sometimes neither starving nor under nutrient-excess conditions. When growing with suboptimal levels of nutrients, hungry bacteria express appropriate cellular responses. This review discusses approaches to defining the hunger response in both molecular and growth kinetic terms. The gene expression changes unique to hunger conditions are described, using Escherichia coli as the primary example. Metabolite changes with hunger and starvation and the differing role of the stationary phase regulator RpoS also lead to the hypothesis proposed in this review that bacteria undertake distinct approaches to hunger and starvation. Indeed, an understanding of the difference between hunger and starvation and the incompatibility between hunger and starvation responses explains some of the paradoxical mutational adaptations, such as rpoS inactivation, found in natural populations.

Experimental analysis of molecular events during mutational periodic selections in bacterial evolution.

A fundamental feature of bacterial evolution is a succession of adaptive mutational sweeps when fitter mutants take over a population. To understand the processes involved in mutational successions, Escherichia coli continuous cultures were analyzed for changes at two loci where mutations provide strong transport advantages to fitness under steady-state glucose limitation. Three separate sweeps, observed as classic periodic selection events causing a change in the frequency of neutral mutations (in fhuA causing phage T5 resistance), were identified with changes at particular loci. Two of the sweeps were associated with a reduction in the frequency of neutral mutations and the concurrent appearance of at least 13 alleles at the mgl or mlc loci, respectively. These mgl and mlc polymorphisms were of many mutational types, so were not the result of a mutator or directed mutation event. The third sweep observed was altogether distinct and involved hitchhiking between T5 resistance and advantageous mgl mutations. Moreover, the hitchhiking event coincided with an increase in mutation rates, due to the transient appearance of a strong mutator in the population. The spectrum of mgl mutations among mutator isolates was distinct and due to mutS. The mutator-associated periodic selection also resulted in mgl and fhuA polymorphism in the sweeping population. These examples of periodic selections maintained significant genotypic diversity even in a rapidly evolving culture, with no individual "winner clone" or genotype purging the population.

Global adaptations resulting from high population densities in Escherichia coli cultures.

The scope of population density effects was investigated in steady-state continuous cultures of Escherichia coli in the absence of complications caused by transient environmental conditions and growth rates. Four distinct bacterial properties reflecting major regulatory and physiological circuits were analyzed. The metabolome profile of bacteria growing at high density contained major differences from low-density cultures. The 10-fold-elevated level of trehalose at higher densities pointed to the increased role of the RpoS sigma factor, which controls trehalose synthesis genes as well as the general stress response. There was an eightfold difference in RpoS levels between bacteria grown at 10(8) and at 10(9) cells/ml. In contrast, the cellular content of the DNA binding protein H-NS, controlling many genes in concert with RpoS, was decreased by high density. Since H-NS and RpoS also influence porin gene expression, the influence of population density on the intricate regulation of outer membrane composition was also investigated. High culture densities were found to strongly repress ompF porin transcription, with a sharp threshold at a density of 4.4 x 10(8) cells/ml, while increasing the proportion of OmpC in the outer membrane. The density-dependent regulation of ompF was maintained in rpoS or hns mutants and so was independent of these regulators. The consistently dramatic changes indicate that actively growing, high-density cultures are at least as differentiated from low-density cultures as are exponential- from stationary-phase bacteria.

Substrate specificity and signal transduction pathways in the glucose-specific enzyme II (EII(Glc)) component of the Escherichia coli phosphotransferase system.

Escherichia coli adapted to glucose-limited chemostats contained mutations in ptsG resulting in V12G, V12F, and G13C substitutions in glucose-specific enzyme II (EII(Glc)) and resulting in increased transport of glucose and methyl-alpha-glucoside. The mutations also resulted in faster growth on mannose and glucosamine in a PtsG-dependent manner. By use of enhanced growth on glucosamine for selection, four further sites were identified where substitutions caused broadened substrate specificity (G176D, A288V, G320S, and P384R). The altered amino acids include residues previously identified as changing the uptake of ribose, fructose, and mannitol. The mutations belonged to two classes. First, at two sites, changes affected transmembrane residues (A288V and G320S), probably altering sugar selectivity directly. More remarkably, the five other specificity mutations affected residues unlikely to be in transmembrane segments and were additionally associated with increased ptsG transcription in the absence of glucose. Increased expression of wild-type EII(Glc) was not by itself sufficient for growth with other sugars. A model is proposed in which the protein conformation determining sugar accessibility is linked to transcriptional signal transduction in EII(Glc). The conformation of EII(Glc) elicited by either glucose transport in the wild-type protein or permanently altered conformation in the second category of mutants results in altered signal transduction and interaction with a regulator, probably Mlc, controlling the transcription of pts genes.

'Growth of bacterial cultures' 50 years on: towards an uncertainty principle instead of constants in bacterial growth kinetics.

Ever since Monod's efforts to study bacterial cultures in quantitative terms, the growth of Escherichia coli on sugars like glucose has appeared an attractive subject for the mathematical description of nutrient conversion into biomass. But instead of simplicity, it is becoming evident that bacterial adaptations affect 'constants' such as K(s) (growth affinity constant) and are, in turn, a complex function of nutrient concentration. Instead of a single affinity, bacteria exhibit a continuum of nutrient scavenging abilities peaking at micromolar sugar levels; there is lower affinity with millimolar or submicromolar glucose in the medium. Similar problems arise in defining parameters such as Y (growth yield constant), because nutrient-limited growth at low exponential growth rates induces a continuum of hunger and starvation responses. Autocatalytic changes to the environment caused by growth (as well as external factors) ensure that bacteria present an ever-adapting interface to the outside world. The regulatory interaction between the organism and environment means that no universal kinetic constants describe bacterial growth.

Mutational adaptation of Escherichia coli to glucose limitation involves distinct evolutionary pathways in aerobic and oxygen-limited environments.

Mutational adaptations leading to improved glucose transport were followed with Escherichia coli K-12 growing in glucose-limited continuous cultures. When populations were oxygen limited as well as glucose limited, all bacteria within 280 generations contained mutations in a single codon of the ptsG gene. V12F and V12G replacements in the enzyme IIBC(Glc) component of the glucose phosphotransferase system were responsible for improved transport. In stark contrast, ptsG mutations were uncommon in fully aerobic glucose-limited cultures, in which polygenic mutations in mgl, mlc, and malT (regulating an alternate high-affinity Mgl/LamB uptake pathway) spread through the adapted population. Hence the same organism adapted to the same selection (glucose limitation) by different evolutionary pathways depending on a secondary environmental factor. The clonal diversity in the adapted populations was also significantly different. The PtsG V12F substitution under O(2) limitation contributed to a universal "winner clone" whereas polygenic, multiallelic changes led to considerable polymorphism in aerobic cultures. Why the difference in adaptive outcomes? E. coli physiology prevented scavenging by the LamB/Mgl system under O(2) limitation; hence, ptsG mutations provided the only adaptive pathway. But ptsG mutations in aerobic cultures are overtaken by mgl, mlc, and malT adaptations with better glucose-scavenging ability. Indeed, when an mglA::Tn10 mutant with an inactivated Mgl/LamB pathway was introduced into two independent aerobic chemostats, adaptation of the Mgl(-) strain involved the identical ptsG mutation found under O(2)-limited conditions with wild-type or Mgl(-) bacteria.

OmpF changes and the complexity of Escherichia coli adaptation to prolonged lactose limitation.

The disaccharide lactose has no specific diffusion pathway across the outer membrane of Escherichia coli. At least three classes of spontaneous mutation affecting outer membrane permeability arose with each of three independent E. coli populations adapting to prolonged lactose limitation in chemostats. Both structural and regulatory mutations affecting OmpF porin predominated in isolates after 210-280 generations of culture. Six types of ompF mutation were found, including in-frame deletions and substitutions at Arg82 and Asp113, all affecting the channel constriction residues of OmpF. Isolates had increased susceptibility to antibiotics and were affected in the quantity of OmpF, LamB and OmpA proteins. A minimum of three or four mutations was evident in all isolates after 280 generations in a lactose-limited environment, in addition to lac mutations defined in previous studies.

Regulation by nutrient limitation.

Growth with suboptimal nutrient levels elicits adaptations not observed with either starving (resting) or unstressed bacteria. The hunger response results in patterns of gene expression optimising scavenging capabilities through novel control mechanisms. In Escherichia coli, recent results indicate that outermembrane permeability (porin and glycoporin regulation) as well as transport involving the phosphotransferase system and ABC-type high affinity transporters change under glucose limitation. Many other adaptations in expression and metabolic capabilities at subsaturating growth rates are still poorly understood, even in E. coli.

Adaptive mgl-regulatory mutations and genetic diversity evolving in glucose-limited Escherichia coli populations.

The mutational adaptation of E. coli to low glucose concentrations was studied in chemostats over 280 generations of growth. All members of six independent populations acquired increased fitness through the acquisition of mutations at the mgl locus, increasing the binding protein-dependent transport of glucose. These mutations provided a strong fitness advantage (up to 10-fold increase in glucose affinity) and were present in most isolates after 140 generations. mgl constitutivity in some isolates was caused by base substitution, short duplication, small deletion and IS1 insertion in the 1041 bp gene encoding the repressor of the mgl system, mglD (galS). But an unexpectedly large proportion of mutations were located in the short mgl operator sequence (mglO), and the majority of mutations were in mglO after 280 generations of selection. The adaptive mglO substitutions in several independent populations were at exactly the positions conserved in the two 8 bp half-sites of the mgl operator, with the nature of the base changes also completely symmetrical. Either mutations were directed to the operator or the particular operator mutations had a selective advantage under glucose limitation. Indeed, isolates carrying mglO mutations showed greater rates of transport for glucose and galactose at low concentrations than those carrying mglD null mutations. mglO mutations avoid cross-talk by members of the GalR-Lacl repressor family, reducing transporter expression and providing a competitive advantage in a glucose-limited environment. Another interesting aspect of these results was that each adapted population acquired multiple mgl alleles, with several populations containing at least six different mgl-regulatory mutations co-existing after 200 generations. The diversity of mutations in the mglO/mglD region, generally in combination with mutations at other loci regulating glucose uptake (malT, mlc, ptsG), provided evidence for multiple clones in each population. Increased fitness was accompanied by the generation of genetic diversity and not the evolution of a single winner clone, as predicted by the periodic selection model of bacterial populations.

The generation of multiple co-existing mal-regulatory mutations through polygenic evolution in glucose-limited populations of Escherichia coli.

The multicomponent glucose transport system of Escherichia coli was used to study the polygenic basis of increased fitness in prolonged nutrient-limited, continuous cultures. After 280 generations of glucose-limited growth, nearly all bacteria in four independent chemostat populations exhibited increased glucose transport and contained multiple, stable mutations. Fitter bacteria increased outer membrane permeability for glucose through overexpression of the LamB glycoporin. Three classes of mutation influenced LamB levels as well as regulation of other mal genes. Low-level mal/lamB constitutivity resulted from mlc mutations acquired in all populations as well as changes at another uncharacterized locus. Larger increases in transporter content resulted from widespread acquisition of a regulatory malT-con mutation in fit isolates. The malT mutations sequenced from 67 adapted isolates were all single base substitutions resulting in amino acid replacements in the N-terminal third of the MalT activator protein. Analysis of malT-con sequences revealed a mutational spectrum distinct from that found in plate-selected malT mutants, suggesting that mutational pathways were affected by environmental factors. A second major finding was the remarkable allele diversity in malT within a population derived from a single clone, with at least 11 different alleles co-existing in a population. The multiplicity of alleles (as well as those found in adaptive mgl changes in the accompanying study) suggest that the periodic selection events observed previously in such populations are not a major factor in reducing genetic diversity. A simple model is presented for the generation of genetic heterogeneity in bacterial populations undergoing polygenic selection.

Assessing the effect of reactive oxygen species on Escherichia coli using a metabolome approach.

A two-dimensional thin-layer chromatographic analysis of [14C]-labelled metabolites in Escherichia coli was employed to follow metabolic shifts in response to superoxide stress. Steady-state challenge with paraquat at concentrations inducing SoxRS-controlled genes resulted in several alterations in metabolite pools, including a striking increase in valine concentration. Elevated valine levels, together with increased glutathione and alkylperoxidase, are proposed to constitute an intracellular protection mechanism against reactive oxygen species. As shown by this example of metabolome analysis, novel cellular responses to environmental challenge can be revealed by following the total complement of metabolites in a cell.