In vitro adhesion to human cells by viable but nonculturable Enterococcus faecalis

The ability of viable but nonculturable (VBNC) Enterococcus faecalis to adhere to Caco-2 and Girardi heart cultured cells and to urinary tract epithelial cells (ECs) was studied. Enterococci were harvested during the vegetative growth phase (early exponential and stationary), in the VBNC state, and after recovery of the ability to divide. VBNC bacteria maintained their adherence capability but the efficiency of attachment was reduced by about 50 to 70%, depending on the target cell employed. The decrease was transient, since enterococci that regained their culturability showed adherence values similar to those observed for actively growing cells. Analysis of the invasive properties of E. faecalis revealed that the VBNC state caused a decrease in the number of bacteria that entered the cultured HEK cells as a result of the reduction in the number of adhering bacteria. These results highlight the importance of studies of the VBNC phenomenon, with respect to both microbial survival in the environment and the impact on human health. Received: 26 September 2001 / Accepted: 4 December 2001     

Cell wall chemical composition of Enterococcus faecalis in the viable but nonculturable state

The viable but nonculturable (VBNC) state is a survival mechanism adopted by many bacteria (including those of medical interest) when exposed to adverse environmental conditions. In this state bacteria lose the ability to grow in bacteriological media but maintain viability and pathogenicity and sometimes are able to revert to regular division upon restoration of normal growth conditions. The aim of this work was to analyze the biochemical composition of the cell wall of Enterococcus faecalis in the VBNC state in comparison with exponentially growing and stationary cells. VBNC enterococcal cells appeared as slightly elongated and were endowed with a wall more resistant to mechanical disruption than dividing cells. Analysis of the peptidoglycan chemical composition showed an increase in total cross-linking, which rose from 39% in growing cells to 48% in VBNC cells. This increase was detected in oligomers of a higher order than dimers, such as trimers (24% increase), tetramers (37% increase), pentamers (65% increase), and higher oligomers (95% increase). Changes were also observed in penicillin binding proteins (PBPs), the enzymes involved in the terminal stages of peptidoglycan assembly, with PBPs 5 and 1 being prevalent, and in autolytic enzymes, with a threefold increase in the activity of latent muramidase-1 in E. faecalis in the VBNC state. Accessory wall polymers such as teichoic acid and lipoteichoic acid proved unchanged and doubled in quantity, respectively, in VBNC cells in comparison to dividing cells. It is suggested that all these changes in the cell wall of VBNC enterococci are specific to this particular physiological state. This may provide indirect confirmation of the viability of these cells.     

Peptidoglycan O acetylation and autolysin profile of Enterococcus faecalis in the viable but nonculturable state

The O acetylation of peptidoglycan occurs specifically at the C-6 hydroxyl group of muramoyl residues. Using a combination of high-performance liquid chromatography-based organic acid analysis and carbohydrate analysis by high-pH anion-exchange chromatography, we determined that strains of Entercoccus durans, E. faecalis, E. faecium, and E. hirae produce O-acetylated peptidoglycan. The levels of O acetylation ranged from 19% to 72% relative to the muramic acid content, and they were found to vary with the growth phase of the culture. Increases of 10 to 40% in O acetylation were observed with cultures entering the stationary phase. Cells of E. faecalis in the viable but nonculturable (VBNC) state had the highest levels of peptidoglycan O acetylation. The presence of this modification to peptidoglycan was shown to inhibit the action of hen egg white lysozyme in a concentration-dependent manner. Zymography using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing either O-acetylated or chemically de-O-acetylated peptidoglycan was used to monitor the production of specific autolysins in E. faecalis. Differences in the expression of specific autolysins were observed with the age of the culture, and VBNC E. faecalis produced the highest levels of these enzymes. This technique also permitted classification of the enterococcal autolysins into enzymes that preferentially hydrolyze either O-acetylated or non-O-acetylated peptidoglycan and enzymes that show no apparent preference for either substrate type.     

The viable but nonculturable state and starvation are different stress responses of Enterococcus faecalis,as determined by proteome analysis

The protein expression patterns of exponentially growing, starved, and viable but nonculturable (VBNC) Enterococcus faecalis cells were analyzed to establish whether differences exist between the VBNC state and other stress responses. The results indicate that the protein profile of VBNC cells differs from that of either starved or exponentially growing bacteria. This demonstrates that the VBNC state is a distinct physiological phase within the life cycle of E. faecalis, which is activated in response to multiple environmental stresses.     

Entry of Yersinia pestis into the viable but nonculturable state in a low-temperature tap water microcosm

Yersinia pestis, the causative agent of plague, has caused several pandemics throughout history and remains endemic in the rodent populations of the western United States. More recently, Y. pestis is one of several bacterial pathogens considered to be a potential agent of bioterrorism. Thus, elucidating potential mechanisms of survival and persistence in the environment would be important in the event of an intentional release of the organism. One such mechanism is entry into the viable but non-culturable (VBNC) state, as has been demonstrated for several other bacterial pathogens. In this study, we showed that Y. pestis became nonculturable by normal laboratory methods after 21 days in a low-temperature tap water microcosm. We further show evidence that, after the loss of culturability, the cells remained viable by using a variety of criteria, including cellular membrane integrity, uptake and incorporation of radiolabeled amino acids, and protection of genomic DNA from DNase I digestion. Additionally, we identified morphological and ultrastructural characteristics of Y. pestis VBNC cells, such as cell rounding and large periplasmic spaces, by electron microscopy, which are consistent with entry into the VBNC state in other bacteria. Finally, we demonstrated resuscitation of a small number of the non-culturable cells. This study provides compelling evidence that Y. pestis persists in a low-temperature tap water microcosm in a viable state yet is unable to be cultured under normal laboratory conditions, which may prove useful in risk assessment and remediation efforts, particularly in the event of an intentional release of this organism.     

Use of the polymerase chain reaction and fluorescent-antibody methods for detecting viable but nonculturable Shigella dysenteriae type 1 in laboratory microcosms.

Epidemiological studies of shigellosis in Bangladesh have demonstrated that surface-water sources can act as foci of infection. Studies of laboratory microcosms have shown that shigellae become nonculturable but remain viable when exposed to environmental samples of water. The present study was carried out to detect viable but nonculturable Shigella dysenteriae 1 from laboratory microcosms by the polymerase chain reaction and the fluorescent-antibody techniques. S. dysenteriae 1 was inoculated into laboratory microcosms consisting of water samples collected from ponds, lakes, rivers, and drains in Bangladesh. The survival of S. dysenteriae in microcosms was assessed by viable counting on MacConkey agar. After 2 to 3 weeks, S. dysenteriae 1 became nonculturable but remained viable. After 6 weeks, this nonculturable but viable S. dysenteriae 1 was detected by both the polymerase chain reaction and the fluorescent-antibody methods. The viable but nonculturable state of S. dysenteriae 1 demonstrated in this study may be important for understanding the epidemiology of shigellosis.     

Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment.

Using plate counts, total cell counts, and direct viable counts, we examined the fate of cells of Vibrio vulnificus placed into natural estuarine waters during both winter and summer months. Cells inoculated into membrane diffusion chambers and placed into estuarine waters entered into a viable but nonculturable (VBNC) state in January and February, when the water temperatures were low (average, < 15 degrees C). In contrast, when cells in the VBNC state were placed into the same waters in the warmer months of August through November (average water temperature of ca. 21 degrees C), the cells appeared to undergo a rapid (typically, within 24 h) resuscitation to the fully culturable state. These results were independent of whether the cells were in the logarithmic or stationary phase and whether they were encapsulated or not. This study indicates that the inability to isolate V. vulnificus from cold estuarine sites may be accounted for by entrance of the cells into a VBNC state and that recovery from this state in natural environments may result from a temperature upshift.     

Optimal schedule for monitoring a plant incursion when detection and treatment success vary over time

Management of an invasive plant species can be viewed as two separate and successive processes. The first, survey, aims to find infested areas and remove individuals. The second, monitoring, consists of repeated visits to these areas in order to prevent possible re-emergence. As detection probability may vary over time, the timing and number of monitoring visits can dramatically impact monitoring efficacy. We explore the optimal timing and number of monitoring visits, by focusing on one infested site. Our decision-analysis framework defines an optimal monitoring schedule which accounts for a time-dependent probability of detection, based on the presence/absence of a flower. We use this framework to investigate the optimal monitoring schedule for Hieracium aurantiacum, an invasive species in the Australian Alps and many other countries. We also perform a sensitivity analysis to draw more general conclusions. For H. aurantiacum eight monitoring visits (compared to 12 visits in the current program) are sufficient to obtain a 99% monitoring efficacy. When four or fewer visits to a site are allowed, it is optimal to visit during the high season, when the weed is likely to initiate flowering. Any extra visits should be scheduled in the early season, before the plants flower. The sensitivity analysis shows that increasing the detection probability early in the season has a greater impact than increasing it late in the season. An effective treatment method increases the value of site visits late in the season, when the detection probability is higher. Our decision-analysis framework can assist invasive species managers to reduce or reallocate management resources by determining the minimum number of monitoring visits required to satisfy an acceptable risk of re-emergence.     

Nosocomial infection caused by vancomycin‐susceptible multidrug‐resistant Enterococcus faecalis over a long period in a university hospital in Japan

Compared with other developed countries, vancomycin‐resistant enterococci (VRE) are not widespread in clinical environments in Japan. There have been no VRE outbreaks and only a few VRE strains have sporadically been isolated in our university hospital in Gunma, Japan. To examine the drug susceptibility of Enterococcus faecalis and nosocomial infection caused by non‐VRE strains, a retrospective surveillance was conducted in our university hospital. Molecular epidemiological analyses were performed on 1711 E. faecalis clinical isolates collected in our hospital over a 6‐year period [1998–2003]. Of these isolates, 1241 (72.5%) were antibiotic resistant and 881 (51.5%) were resistant to two or more drugs. The incidence of multidrug resistant E. faecalis (MDR‐Ef) isolates in the intensive care unit increased after enlargement and restructuring of the hospital. The major group of MDR‐Ef strains consisted of 209 isolates (12.2%) resistant to the five drug combination tetracycline/erythromycin/kanamycin/streptomycin/gentamicin. Pulsed‐field gel electrophoresis analysis of the major MDR‐Ef isolates showed that nosocomial infections have been caused by MDR‐Ef over a long period (more than 3 years). Multilocus sequence typing showed that these strains were mainly grouped into ST16 (CC58) or ST64 (CC8). Mating experiments suggested that the drug resistances were encoded on two conjugative transposons (integrative conjugative elements), one encoded tetracycline‐resistance and the other erythromycin/kanamycin/streptomycin/gentamicin‐resistance. To our knowledge, this is the first report of nosocomial infection caused by vancomycin‐susceptible MDR‐Ef strains over a long period in Japan.     

Health centre surveys as a potential tool for monitoring malaria epidemiology by area and over time

Background

Presently, many malaria control programmes use health facility data to evaluate the impact of their interventions. Facility-based malaria data, although useful, have problems with completeness, validity and representativeness and reliance on routinely collected health facility data might undermine demonstration of the magnitude of the impact of the recent scaleups of malaria interventions. To determine whether carefully conducted health centre surveys can be reliable means of monitoring area specific malaria epidemiology, we have compared malaria specific indices obtained from surveys in health centres with indices obtained from cross-sectional surveys conducted in their catchment communities.

Methods

A series of age stratified, seasonal, cross-sectional surveys were conducted during the peak malaria transmission season in 2008 and during the following dry season in 2009 in six ecologically diverse areas in The Gambia. Participants were patients who attended the health centres plus a representative sample from the catchment villages of these health facilities. Parasitaemia, anaemia, attributable proportion of fever and anti-MSP1-₁₉ antibody seroprevalence were compared in the health facility attendees and community participants.

Results

A total of 16,230 subjects completed the study; approximately half participated in the health centre surveys and half in the wet season surveys. Data from both the health centre and community surveys showed that malaria endemicity in The Gambia is now low, heterogeneous and seasonal. In the wet season, parasitaemia, seroprevalence and fever prevalence were higher in subjects seen in the health centres than in the community surveys. Age patterns of parasitaemia, attributable proportions of fever and seroprevalence rates were similar in subjects who participated in the community and health centre surveys.

Conclusion

Health centre surveys have potential as a surveillance tool for evaluating area specific malaria control activities and for monitoring changes in local malaria epidemiology over time.     

Construction and characterization of Enterococcus faecalis CG110/gfp/pRE25*, a tool for monitoring horizontal gene transfer in complex microbial ecosystems

Enterococci are among the most notorious bacteria involved in the spread of antibiotic resistance (ABR) determinants via horizontal gene transfer, a process that leads to increased prevalence of antibiotic-resistant bacteria. In complex microbial communities with a high background of ABR genes, detection of gene transfer is possible only when the ABR determinant is marked. Therefore, the conjugative multiresistance plasmid pRE25, originating from a sausage-associated Enterococcus faecalis, was tagged with a 34-bp random sequence marker spliced by tet(M). The plasmid constructed, designated pRE25(*) , was introduced into E. faecalis CG110/gfp, a strain containing a gfp gene as chromosomal marker. The plasmid pRE25(*) is fully functional compared with its parental pRE25, occurs at one to two copies per chromosome, and can be transferred to Listeria monocytogenes and Listeria innocua at frequencies of 6 × 10(-6) to 8 × 10(-8) transconjugants per donor. The markers on the chromosome and the plasmid enable independent quantification of donor and plasmid, even if ABR genes occur at high numbers in the background ecosystem. Both markers were stable for at least 200 generations, permitting application of the strain in long-running experiments. Enterococcus faecalis CG110/gfp/pRE25(*) is a potent tool for the investigation of horizontal ABR gene transfer in complex environments such as food matrices, biofilms or colonic models.     

Increase of genetic variation over time in a recently founded population of great reed warblers (Acrocephalus arundinaceus) revealed by microsatellites and DNA fingerprinting

Genetic similarity within pairs of individuals was examined using both 10 polymorphic microsatellite loci and multi-locus DNA fingerprinting profiles in a semi-isolated population of great reed warblers at Lake Kvismaren, south Central Sweden, in 1987-1993. The population was founded by a few individuals in 1978, followed by a gradual increase in numbers until 1988, since when the population has remained relatively stable with about 60 breeding birds. We have previously found that high genetic similarity between pair-mates in the population during the early part of the study period reduced egg hatching success, and hence reproductive success. The measures of pairwise genetic similarity, microsatellite allele sharing and DNA fingerprinting band sharing, were highly correlated with pedigree-based relatedness. Both microsatellite and DNA fingerprinting similarities between pair-mates declined significantly over the study period, and the pattern was most pronounced in the DNA fingerprinting data. Analyses restricted to the microsatellite data showed that the average annual microsatellite similarity between pairwise combinations of individuals, as well as individual homozygosity in males, declined significantly over the study period, and that several immigrants carrying novel alleles entered the population during the study. Hence, the temporal decline in genetic similarity of mates in the population is probably a consequence of increased immigration, facilitated by the recent expansion of the species in the region. These results suggest that the population has now recovered genetically, or is in the process of recovering, from a recent founder event.