Repeated Isolation of Extended-Spectrum-ß-Lactamase-Positive Escherichia coli Sequence Types 648 and 131 from Community Wastewater Indicates that Sewage Systems Are Important Sources of Emerging Clones of Antibiotic-Resistant Bacteria.

Antibiotic resistance in bacteria is an emerging problem globally. Resistant bacteria are found in human and animal microbiota, as well as in the environment. Wastewater receives bacteria from all these sources and thus can provide a measurement of abundance and diversity of antibiotic-resistant bacteria circulating in communities. In this study, water samples were collected from a wastewater pump station in a Norwegian suburban community over a period of 15 months. A total of 45 daily samples were cultured and analyzed for the presence of Escherichia coli Eighty E. coli-like colonies were collected from each daily sample and then phenotyped and analyzed for antibiotic resistance using the PhenePlate-AREB system. During the sampling period, two unique E. coli phenotypes with resistance to cefotaxime and cefpodoxime indicating carriage of extended-spectrum ß-lactamases (ESBL) were observed repeatedly. Whole-genome sequencing of 15 representative isolates from the two phenotypes identified these as two distinct clones belonging to the two globally spread E. coli multilocus sequence types (STs) ST131 and ST648 and carrying blaCTX-M-15 The number of ESBL-positive E. coli strains in the community wastewater pump station was 314 of 3,123 (10%) analyzed E. coli strains. Of the ESBL-positive isolates, 37% belonged to ST648, and 7% belonged to ST131. Repeated findings of CTX-M-15-positive ST648 and ST131 over time indicate that these STs are resident in the analyzed wastewater systems and/or circulate abundantly in the community.

Persistence of Lactobacilli in Postmenopausal Women - A Double-Blind, Randomized, Pilot Study.

AIM: To investigate the ability of lactobacilli to persist in the genital area (vagina and labia) of women after the topical application of an ointment containing Lactobacillus gasseri LN40, L. fermentum LN99 and L. rhamnosus LN113. Secondary objectives were to study the presence of Escherichia coli and other contaminants, as well as subjective symptoms in the genital tract. METHODS: Eighteen healthy postmenopausal women were randomized to use either the study product or placebo for 10 days. Gynecological examinations, labial and vaginal samplings for bacterial cultivation were performed at baseline (visit 1), after treatment (visit 2), and at a 10-day follow-up (visit 3). LN strains were identified by specific cultivation methods. Subjective symptoms were evaluated by a self-administered questionnaire. RESULTS: The presence of LN99 was shown in 7 out of 8 women in the investigational group at visit 2 (p < 0.001 compared to placebo) and in 5 out of 8 at visit 3 (p < 0.05), whereas the presence of LN113 was shown in 2 out of 8 at visit 2 and in 1 out of 8 at visit 3. Subjective symptoms were significantly reduced (p < 0.01) at visits 2 and 3 for both products. CONCLUSION: Topical application of a probiotic ointment is feasible to achieve persistence of lactobacilli for at least 10 days.

Antibiotic resistance in environmental Escherichia coli - a simple screening method for simultaneous typing and resistance determination.

We describe a simple and standardised screening system (AREB) for surveillance of antibiotic resistant bacteria in the environment. The system consists of 96 well microplates containing eight sets of breakpoint amounts of 10 different antibiotics. The incubated microplates are read by a desktop scanner and the plate images are analysed by special software that automatically presents the resistance data. The AREB method is combined with a rapid typing method, the PhenePlate system, which yields information on the diversity of the bacteria in the studied samples, and on the possible prevalence of resistant clones. In order to demonstrate the usage of AREB, a comparative study on the resistance situation among 970 Escherichia coli isolates from sewage and recipient water in Sweden, Norway and Chile, was performed. Resistance rates to all antibiotics were markedly higher in hospital sewage than in other samples. Our data indicate that the AREB system is useful for comparing resistance rates among E. coli and other environmental indicator bacteria in different countries/regions. Simple handling and automatic data evaluation, combined with low cost, facilitate large studies involving several thousands of isolates.

Escherichia coli ST2797 Is Abundant in Wastewater and Might Be a Novel Emerging Extended-Spectrum Beta-Lactamase E. coli.

The increasing prevalence of antibiotic-resistant bacteria is an emerging threat to global health. The analysis of antibiotic-resistant enterobacteria in wastewater can indicate the prevalence and spread of certain clonal groups of multiresistant bacteria. In a previous study of Escherichia coli that were isolated from a pump station in Norway over 15 months, we found a recurring E. coli clone that was resistant to trimethoprim, ampicillin, and tetracycline in 201 of 3,123 analyzed isolates (6.1%). 11 representative isolates were subjected to whole-genome sequencing and were found to belong to the MLST ST2797 E. coli clone with plasmids carrying resistance genes, including blaTEM-1B, sul2, dfrA7, and tetB. A phenotypic comparison of the ST2797 isolates with the uropathogenic ST131 and ST648 that were repeatedly identified in the same wastewater samples revealed that the ST2797 isolates exhibited a comparable capacity for temporal survival in wastewater, greater biofilm formation, and similar potential for the colonization of mammalian epithelial cells. ST2797 has been isolated from humans and has been found to carry extended spectrum ß-lactamase (ESBL) genes in other studies, suggesting that this clonal type is an emerging ESBL E. coli. Collectively, these findings show that ST2797 was more ubiquitous in the studied wastewater than were the infamous ST131 and ST648 and that ST2797 may have similar abilities to survive in the environment and cause infections in humans. IMPORTANCE The incidence of drug-resistant bacteria found in the environment is increasing together with the levels of antibiotic-resistant bacteria that cause infections. The COVID-19 pandemic has shed new light on the importance of monitoring emerging threats and finding early warning systems. Therefore, to mitigate the antimicrobial resistance burden, the monitoring and early identification of antibiotic-resistant bacteria in hot spots, such as wastewater treatment plants, are required to combat the occurrence and spread of antibiotic-resistant bacteria. Here, we applied a PhenePlate system as a phenotypic screening method for genomic surveillance and discovered a dominant and persistent E. coli clone ST2797 with a multidrug resistance pattern and equivalent phenotypic characteristics to those of the major pandemic lineages, namely, ST131 and ST648, which frequently carry ESBL genes. This study highlights the continuous surveillance and report of multidrug resistant bacteria with the potential to spread in One Health settings.

The Staphylococcus aureus alpha-toxin perturbs the barrier function in Caco-2 epithelial cell monolayers by altering junctional integrity.

Increased microvascular permeability is a hallmark of sepsis and septic shock. Intestinal mucosal dysfunction may allow translocation of bacteria and their products, thereby promoting sepsis and inflammation. Although Staphylococcus aureus alpha-toxin significantly contributes to sepsis and perturbs the endothelial barrier function, little is known about possible effects of S. aureus alpha-toxin on human epithelial barrier functions. We hypothesize that S. aureus alpha-toxin in the blood can impair the intestinal epithelial barrier and thereby facilitate the translocation of luminal bacteria into the blood, which may in turn aggravate a septic condition. Here, we showed that staphylococcal alpha-toxin disrupts the barrier integrity of human intestinal epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER) and reduced cellular levels of junctional proteins, such as ZO-1, ZO-3, and E-cadherin. The Caco-2 cells also responded to alpha-toxin with an elevated cytosolic calcium ion concentration ([Ca(2+)](i)), elicited primarily by calcium influx from the extracellular environment, as well as with a significant reduction in TER, which was modulated by intracellular calcium chelation. Moreover, a significantly larger reduction in TER and amounts of the junctional proteins, viz., ZO-3 and occludin, was achieved by basolateral than by apical application of the alpha-toxin. These experimental findings thus support the hypothesis that free staphylococcal alpha-toxin in the bloodstream may cause intestinal epithelial barrier dysfunction and further aggravate the septic condition by promoting the release of intestinal bacteria into the underlying tissues and the blood.

Diarrheagenic Escherichia coli markers and phenotypes among fecal E. coli isolates collected from Nicaraguan infants.

We analyzed the prevalence of diarrheagenic Escherichia coli (DEC) markers and common phenotypes in 2,164 E. coli isolates from 282 DEC-positive samples. Enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC) were very diverse and were not correlated with diarrhea. Enterotoxigenic E. coli (ETEC) estA and enterohemorrhagic E. coli (EHEC) belonged to a few phenotypes and were significantly correlated with diarrhea.

Channel-forming abilities of spontaneously occurring alpha-toxin fragments from Staphylococcus aureus.

Pore formation by four spontaneously occurring alpha-toxin fragments from Staphylococcus aureus were investigated on liposome and erythrocyte membranes. All the isolated fragments bound to the different types of membranes and formed transmembrane channels in egg-phosphatidyl glycerol vesicles. Fragments of amino acids (aa) 9-293 (32 kD) and aa 13-293 (31 kD) formed heptamers, similar to the intact toxin, while the aa 72-293 (26 kD) fragment formed heptamers, octamers, and nonamers, as judged by gel electrophoresis of the liposomes. All isolated fragments induced release of chloride ions from large unilamellar vesicles. Channel formation was promoted by acidic pH and negatively charged lipid head groups. Also, the fragments' hemolytic activity was strongly decreased under neutral conditions but could be partially restored by acidification of the medium. We paid special attention to the 26-kD fragment, which, despite the loss of about one-fourth of the N-terminal part of alpha-toxin, did form transmembrane channels in liposomes. In light of the available data on channel formation by alpha-toxin, our results suggest that proteolytic degradation might be better tolerated than previously reported. Channel opening could be inhibited and open channels could be closed by zinc in the medium. Channel closure could be reversed by addition of EDTA. In contrast, digestion at the C terminus led to premature oligomerization and resulted in species with strongly diminished activity and dependent on protonation.

Biological relevance of natural alpha-toxin fragments from Staphylococcus aureus.

Serine proteases represent an essential part of cellular homeostasis by generating biologically active peptides. In bacteria, proteolysis serves two different roles: a major housekeeping function and the destruction of foreign or target cell proteins, thereby promoting bacterial invasion. In the process, other virulence factors such as exotoxins become affected. In Staphylococcus aureus culture supernatant, the pore-forming alpha-toxin is cleaved by the coexpressed V8 protease and aureolysin. The oligomerizing and pore-forming abilities of five such spontaneously occurring N- and C-terminal alpha-toxin fragments were studied. (3)H-marked alpha-toxin fragments bound to rabbit erythrocyte membranes but only fragments with intact C termini, missing 8, 12 and 71 amino acids from their N-terminal, formed stable oligomers. All isolated fragments induced intoxication of mouse adrenocortical Y1 cells in vitro, though the nature of membrane damage for a fragment, degraded at its C terminus, remained obscure. Only one fragment, missing the first eight N-terminal amino acids, induced irreversible intoxication of Y1 cells in the same manner as the intact toxin. Four of the isolated fragments caused swelling, indicating altered channel formation. Fragments missing 12 and 71 amino acids from the N terminus occupied the same binding sites on Y1 cell membranes, though they inhibited membrane damage caused by intact toxin. In conclusion, N-terminal deletions up to 71 amino acids are tolerated, though the kinetics of channel formation and the channel's properties are altered. In contrast, digestion at the C terminus results in nonfunctional species.

Prevalence of diarrhoeagenic Escherichia coli in children from León, Nicaragua.

Diarrhoeal disease is a public health problem worldwide, mostly affecting children in developing countries. In Nicaragua, diarrhoea is the second greatest cause of infant mortality. During the period March 2005 to September 2006, a total of 526 faecal samples from children aged 0-60 months (381 with and 145 without diarrhoea) from León, Nicaragua, were studied. In order to detect five different diarrhoeagenic Escherichia coli pathotypes simultaneously [enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC) and enteroinvasive E. coli (EIEC)], a mixture of eight primer pairs was used in a single PCR. At least one diarrhoeagenic E. coli pathotype was detected in 205 samples (53.8%) of the diarrhoea group and in 77 samples (53.1%) in the non-diarrhoea group. ETEC was detected significantly more often in children with diarrhoea (20.5%) than in children without diarrhoea (8.3%) (P=0.001). Atypical EPEC, EIEC and EAEC were detected with slightly lower frequencies in children with (16.0, 0.8 and 27.8%, respectively) than in children without (20.7, 1.4 and 33.1%, respectively) diarrhoea. EHEC was only detected in children with diarrhoea (2.1%). In conclusion, ETEC continues to be an important agent associated with diarrhoea in children from León, Nicaragua. Although not very frequent, the only findings that were 100% associated with diarrhoea were ETEC estA (4.7%) and EHEC (2.1%). Nevertheless, EAEC and EPEC were also frequent pathotypes in the population under study. In children with severe diarrhoea, more than half had EAEC, ETEC or EPEC, and EAEC was the most prevalent pathotype.

Prevalence and transmission of antimicrobial resistance among Aeromonas populations from a duckweed aquaculture based hospital sewage water recycling system in Bangladesh.

In order to investigate the influence of a duckweed aquaculture based hospital sewage water recycling plant on the prevalence and dissemination of antibiotic resistance, we made use of an existing collection of 1,315 Aeromonas isolates that were previously typed by the biochemical fingerprinting PhP-AE system. In these treatment plant, hospital raw sewage water is first collected in a settlement pond (referred to as sewage water in this study) and is then transferred to a lagoon, where the duckweed (Lemnaceae) is grown (referred to as lagoon). The duckweed is harvested and used as feed for the fish in a separate pond (referred to as fish pond). From this collection, representatives of 288 PhP types were subjected to antibiotic susceptibility testing for eight antimicrobials by broth microdilution method. The overall resistance rates among Aeromonas isolates from the treatment plant were highest for ampicillin (87%) and erythromycin (79%) followed by cephalothin (58%), nalidixic acid (52%), streptomycin (51%), tetracycline (31%), chloramphenicol (13%) and gentamicin (8%). A significantly lower prevalence of antibiotic resistance was found in Aeromonas from environmental control water, patient stool samples, duckweed and fish compared to sewage water isolates. The prevalence of resistance in the sewage water was not significantly reduced compared to the lagoon water and fish pond. Throughout the treatment system, the frequencies of resistant strains were found to diminish during the sewage water purification process, i.e. in the lagoon where sewage water is used to grow the duckweed. However, the frequency of resistant strains again increased in the fish pond where sewage grown duckweed is used for aquaculture. Among the selected isolates, two multiresistant clonal groups of Aeromonas caviae HG4 were identified that exhibited indistinguishable PhP and amplified fragment length polymorphism fingerprints and shared a common plasmid of approximately 5 kb. Representatives of both groups were recovered from almost every part of the sewage treatment plant but not in the control ponds nor in human samples, which suggests that specific multiresistant Aeromonas clones are able to persist and spread throughout the entire purification process.

Diversity and antibiotic resistance among Escherichia coli populations in hospital and community wastewater compared to wastewater at the receiving urban treatment plant.

Bacterial diversity and antimicrobial resistance patterns among the indicator organism Escherichia coli were monitored in wastewater samples collected over one year from a hospital (HW), a community (CW) and the receiving urban (UW) wastewater treatment plant (WWTP). We compared levels of antibiotic resistance in the different types of wastewater, and identified whether resistant strains were endemic in the wastewater system. If so, implementation of local treatment at certain resistance hotspots (e.g. hospital outlets) could be used to decrease the amount of resistant bacteria in the wastewater. E. coli from HW (n = 2644), CW (n = 2525) and UW (n = 2693) were analyzed by biochemical phenotyping (PhenePlate System) and antimicrobial susceptibility testing to nine antibiotics (AREB System). The phenotypic diversities of the total E. coli populations were similar for all three sites (Simpson's Diversity index, Di = 0.973), however for individual samples, HW showed low diversities (Median Di = 0.800) and the E. coli flora was often dominated by strains that may have originated from the fecal flora of single individuals. The diversities in CW samples was higher (Median Di = 0.936), and UW samples showed similar diversities as the whole collection of isolates (Median Di = 0.971). Resistance to at least one of the nine antibiotics was observed in 45% of the HW isolates, 44% of CW isolates, and 33% of UW isolates. Resistance to gentamicin and chloramphenicol was uncommon (3.2 and 5.3%, respectively), whereas resistance to tetracycline and ampicillin was most common (24% and 31%, respectively). Extended-spectrum beta-lactamase-producing E. coli (ESBL-EC) were more common in HW (11.5%) and in CW (6.9%) compared to UW (3.7%). A high diversity (Di = 0.974) was observed among ESBL-EC isolates from UW (n = 99), indicating absence of any clonal structure among these isolates. Common PhP types of ESBL-EC often dominated in each HW sample, but were not identified across different samples, whereas ESBL-EC in CW showed low diversity (Di = 0.857) and were dominated by a specific PhP type that was found across almost all CW samples. The antibiotic resistance rates were highest in hospital wastewater, but surprisingly they were also high in the studied community wastewater, compared to the urban wastewater. The relative contribution of HW seemed low in terms of dissemination of antibiotic resistant bacteria to the WWTP.

Clonal heterogeneity of clinical isolates of vancomycin-resistant Enterococcus faecium with unique vanS.

OBJECTIVE: To examine the clonal diversity of vancomycin-resistant enterococci (VRE). METHODS: A total of 900 clinical isolates of enterococci were obtained, and VRE isolates were subjected to antimicrobial susceptibility tests, biochemical fingerprinting with the PhPlate system (PhP), ribotyping and pulsed-field gel electrophoresis (PFGE) typing. RESULTS: Forty-nine of all enterococcal isolates were resistant to high levels of vancomycin (MIC >or= 128) and identified as Enterococcus faecium. Biochemical fingerprinting with PhP showed that the VRE isolates were highly diverse (diversity index, D(i) = 0.93) and belonged to 24 PhP-types. The VRE could be separated into 34 and 27 types with PFGE and ribotyping, giving diversity indices of 0.98 and 0.97, respectively. The PFGE method was more discriminatory than ribotyping and PhP system for E. faecium isolates. A combination of either of the two typing methods resulted in at least 44 types. Furthermore, sequencing analysis of vanS of Tn1546 showed one nucleotide mutation (C-->A) at position 5727 in comparison with the prototype BM4147, which was found to be unique in all Iranian VRE isolates. CONCLUSION: The isolated clinical VRE strains were highly diverse in Tehran.

Relationship between endotoxin core, staphylococcal and varicella antibody levels and outcome following aortic valve replacement surgery: a prospective observational study.

BACKGROUND: Morbidity and mortality following cardiac valve surgery is high. Immunity is an important contributor to outcome. This study examines the relationship of staphylococcal and endotoxin antibody levels to outcome following cardiac surgery. METHODS: Using enzyme-linked immunosorbent assays (ELISA), we measured pre-operative levels of antibodies to endotoxin core (EndoCAb); 3 common staphylococcal epitopes and varicella on saved serum of 60 adult patients scheduled to undergo elective primary surgical aortic valve replacement (AVR). Primary outcome measure was post-operative length of stay (LOS) in hospital with secondary outcomes being development of infective complications, length of stay on the intensive care unit (ICU) and 30-day mortality. Patients were quartiled according to antibody levels and outcomes compared between the quartile groups using Mann-Whitney tests for length of stay and Fisher's test for development of infection. RESULTS: Sixty patients (34 M, 26 F) were recruited with mean age 73 years (IQR 66-78), mean body mass index (BMI) 27.7 (IQR 25-31) and EuroSCORE II 1.44 (0.95-1.99). Those patients in the lower quartile for pre-operative antibody level had a longer post-operative stay than the upper quartile. EndoCAb (median IgG level Q1 42.2 MU/ml vs Q4 256 MU/ml) 9 vs 6 days, p = 0.025; alpha-toxin (median IgG level Q1 63 U vs Q4 558 U) 10 vs 7 days, p = 0.034; teichoic acid (median IgG level Q1 14 U vs Q4 419 U) 10 vs 8 days, p = 0.441; staphylococcal enterotoxin A (median IgG level Q1 55 U vs Q4 427 U) 9 vs 7 days, p = 0.865; varicella zoster (median IgG level Q1 1.325 U vs Q4 2.54 U) 8 vs 7 days, p = 1.0; and combined antibody levels 10 vs 6 days, p = 0.017. There were no differences in the number developing post-operative infections for each antibody type. The combined antibody analysis suggested a reduction in proportion of individuals developing infection from the upper vs lower quartile: 0 vs 0.33, p = 0.042. CONCLUSIONS: This study again suggests the inverse relationship between endotoxin core antibody levels and outcome following aortic valve surgery as well as suggesting a similar relationship with antibodies to staphylococcus. There is no such relationship for antibody levels against an organism not providing a peri-operative threat. Understanding this relationship may enable therapeutic manipulation of immune status, re-evaluation of risk and further investigation of the low immune state. TRIAL REGISTRATION: The patients in this study are a sub-group of the RELIEF AS study.ClinicalTrials.gov identifier NCT02174471.

The role of c-di-GMP signaling in an Aeromonas veronii biovar sobria strain.

Aeromonas is a ubiquitous gram-negative bacterium that persists in the environment. It is shown that all isolates of persistent Aeromonas clones show strong biofilm formation ability. C-di-GMP regulates biofilm formation in many bacteria. To investigate the impact of c-di-GMP signaling, we introduced heterologous GGDEF and EAL domain proteins from Salmonella Typhimurium to an Aeromonas veronii biovar sobria strain. Overexpression of the GGDEF domain protein AdrA increased c-di-GMP concentration and biofilm formation and reduced motility. Production of the quorum-sensing signaling molecule C4-homoserine lactone and adhesion to aquatic plant duckweed and amoeba surfaces were enhanced. On the other hand, overexpression of the EAL domain protein YhjH decreased biofilm formation and increased motility.

The Escherichia coli BarA-UvrY two-component system is a virulence determinant in the urinary tract.

BACKGROUND: The Salmonella enterica BarA-SirA, the Erwinia carotovora ExpS-ExpA, the Vibrio cholerae BarA-VarA and the Pseudomonas spp GacS-GacA all belong to the same orthologous family of two-component systems as the Escherichia coli BarA-UvrY. In the first four species it has been demonstrated that disruption of this two-component system leads to a clear reduction in virulence of the bacteria. Our aim was to determine if the Escherichia coli BarA-UvrY two-component system is connected with virulence using a monkey cystitis model. RESULTS: Cystitis was generated in Macaque fascularis monkeys by infecting the bladder with a 1:1 mixture of the uropathogenic Escherichia coli isolate DS17 and a derivative where the uvrY gene had been disrupted with a kanamycin resistance gene. Urine was collected through bladder punctuation at subsequent time intervals and the relative amount of uvrY mutant was determined. This showed that inactivation of the UvrY response regulator leads to a reduced fitness. In similar competitions in culture flasks with Luria Broth (LB) the uvrY mutant rather had a higher fitness than the wild type. When the competitions were done in flasks with human urine the uvrY mutant initially had a lower fitness. This was followed by a fluctuation in the level of mutant in the long-term culture, with a pattern that was specific for the individual urines that were tested. Addition of LB to the different urine competition cultures however clearly led to a consistently higher fitness of the uvrY mutant. CONCLUSION: This paper demonstrates that the BarA-UvrY two-component system is a determinant for virulence in a monkey cystitis model. The observed competition profiles strengthen our previous hypothesis that disruption of the BarA-UvrY two-component system impairs the ability of the bacteria to switch between different carbon sources. The urine in the bladder contains several different carbon sources and its composition changes over time. Inability to efficiently switch between the carbon sources may thus provide an explanation to the reduced fitness of the uvrY mutant in the cystitis model.

NotI passporting to identify species composition of complex microbial systems.

We describe here a new method for large-scale scanning of microbial genomes on a quantitative and qualitative basis. To achieve this aim we propose to create NotI passports: databases containing NotI tags. We demonstrated that these tags comprising 19 bp of sequence information could be successfully generated using DNA isolated from intestinal or fecal samples. Such NotI passports allow the discrimination between closely related bacterial species and even strains. This procedure for generating restriction site tagged sequences (RSTS) is called passporting and can be adapted to any other rare cutting restriction enzyme. A comparison of 1312 tags from available sequenced Escherichia coli genomes, generated with the NotI, PmeI and SbfI restriction enzymes, revealed only 219 tags that were not unique. None of these tags matched human or rodent sequences. Therefore the approach allows analysis of complex microbial mixtures such as in human gut and identification with high accuracy of a particular bacterial strain on a quantitative and qualitative basis.

Restriction site tagged (RST) microarrays: a novel technique to study the species composition of complex microbial systems.

We have developed a new type of microarray, restriction site tagged (RST), for example NotI, microarrays. In this approach only sequences surrounding specific restriction sites (i.e. NotI linking clones) were used for generating microarrays. DNA was labeled using a new procedure, NotI representation, where only sequences surrounding NotI sites were labeled. Due to these modifications, the sensitivity of RST microarrays increases several hundred-fold compared to that of ordinary genomic microarrays. In a pilot experiment we have produced NotI microarrays from Gram-positive and Gram-negative bacteria and have shown that even closely related Escherichia coli strains can be easily discriminated using this technique. For example, two E.coli strains, K12 and R2, differ by less than 0.1% in their 16S rRNA sequences and thus the 16S rRNA sequence would not easily discriminate between these strains. However, these strains showed distinctly different hybridization patterns with NotI microarrays. The same technique can be adapted to other restriction enzymes as well. This type of microarray opens the possibility not only for studies of the normal flora of the gut but also for any problem where quantitative and qualitative analysis of microbial (or large viral) genomes is needed.

New Insights into the Enterococcus faecium and Streptococcus gallolyticus subsp. gallolyticus Host Interaction Mechanisms.

Enterococcus faecium and Streptococcus gallolyticus subsp. gallolyticus (S. gallolyticus) were classically clustered into the Lancefield Group D streptococci and despite their taxonomic reclassification still share a similar genetic content and environment. Both species are considered as opportunistic pathogens. E. faecium is often associated with nosocomial bacteraemia, and S. gallolyticus is sporadically found in endocarditis of colorectal cancer patients. In both cases, the source of infection is commonly endogenous with a translocation process that launches through the intestinal barrier. To get new insights into the pathological processes preceding infection development of both organisms, we used an in vitro model with Caco-2 cells to study and compare the adhesion, invasion and translocation inherent abilities of 6 E. faecium and 4 S. gallolyticus well-characterized isolates. Additionally, biofilm formation on polystyrene, collagen I and IV was also explored. Overall results showed that E. faecium translocated more efficiently than S. gallolyticus, inducing a destabilization of the intestinal monolayer. Isolates Efm106, Efm121 and Efm113 (p < .001 compared to Ef222) exhibited the higher translocation ability and were able to adhere 2-3 times higher than S. gallolyticus isolates. Both species preferred the collagen IV coated surfaces to form biofilm but the S. gallolyticus structures were more compact (p = .01). These results may support a relationship between biofilm formation and vegetation establishment in S. gallolyticus endocarditis, whereas the high translocation ability of E. faecium high-risk clones might partially explain the increasing number of bacteraemia.

Surveillance of antimicrobial resistance among Escherichia coli in wastewater in Stockholm during 1 year: does it reflect the resistance trends in the society?

The resistance patterns of Escherichia coli in untreated (raw) urban wastewater (UW) was monitored by repeated sampling during 1 year. Comparison with data from wastewater samples collected from hospital wastewater (HW) in the same urban area was made. A total of 1326 E. coli isolates from 17 UW samples and 451 isolates from six HW samples were analysed by typing using the PhenePlate™ system, and their susceptibility towards 10 antibiotics was determined. Resistance to at least one antibiotic was observed in 34% of the UW isolates and 55% of the HW isolates. For UW isolates, phenotypic diversity was lower among antibiotic-susceptible than among antibiotic-resistant isolates, indicating a higher presence of clonal groups among susceptible isolates. Total antibiotic resistance measured as the Multiple Antibiotic Resistance (MAR) index was 0.08 for UW compared with 0.19 for HW, and increased over time for UW isolates, indicating increasing resistance among E. coli in the urban population during the studied time period. Resistance to all included ß-lactam antibiotics was detected in 2.4% of UW isolates and 14.0% of HW isolates, and 73/75 (97%) analysed isolates were confirmed to be extended-spectrum ß-lactamase (including plasmid-mediated AmpC)-producing E. coli. Thus, by cultivating samples from wastewater and analysing many independent isolates per sample, increasing frequencies of antibiotic resistance in UW were detected during 1 year that may reflect increasing faecal carriage of resistant bacteria in the society. Surveillance of antibiotic resistance in wastewater could be a valuable tool for screening of resistance trends on a population level.