A decade of neonatal sepsis caused by gram-negative bacilli-a retrospective matched cohort study.

This study is to determine the incidence and outcome of neonatal gram-negative bacilli (GNB) sepsis in Stockholm, Sweden, and describe bacterial characteristics. This is a retrospective cohort study. All infants with GNB-sepsis between 2006 and 2016 were included and matched with two control groups, with suspected sepsis and uninfected neonates, respectively. Outcome was death before discharge, risk of death within 5 days after sepsis onset, and morbidity. The resistance pattern from all GNB was collected, and all available isolates were subjected to genome typing. All neonates with GNB-sepsis (n = 107) were included, and the cumulative GNB-sepsis incidence was 0.35/1000 live born. The in-hospital mortality was 30/107 (28%). GNB late-onset sepsis (LOS) was associated with an increase in mortality before discharge compared to uninfected controls (OR = 3.9; CI 1.6-9.4) but not versus suspected sepsis. The suspected LOS cases did not statistically differ significantly from uninfected controls. The case fatality rate (CFR) at 5 days was 5/33 (15%) in GNB early-onset sepsis (EOS) and 25/74 (34%) in GNB-LOS. The adjusted hazard for 5 days CFR was higher in GNB-LOS versus uninfected controls (HR = 3.7; CI 1.2-11.2), but no significant difference was seen in GNB-LOS versus suspected sepsis or in suspected sepsis versus controls. ESBL production was seen in 7/107 (6.5%) of the GNB isolates. GNB-LOS was associated with a higher 5 days CFR and in-hospital mortality compared to uninfected controls but not versus suspect sepsis. The incidence of both GNB-EOS and GNB-LOS was lower than previously reported from comparable high-income settings. The occurrence of antibiotic resistance was low.

Evaluation of Four Lateral Flow Assays for the Detection of Legionella Urinary Antigen.

Urinary antigen tests (UATs) are often used to diagnose Legionnaires' disease as they are rapid and easy to perform on readily obtainable urine samples without the need for specialized skills compared to conventional methods. Recently developed automated readers for UATs may provide objective results interpretation, especially in cases of weak result bands. Using 53 defined patient urine samples, we evaluated the performance of the BinaxNOW Legionella Antigen Card (Abbott), ImmuView S. pneumoniae and Legionella (SSI Diagnostica), STANDARD F Legionella Ag FIA (SD Biosensor), and Sofia Legionella FIA (Quidel) simultaneously with their respective automated readers. Automatic and visual interpretation of result bands were also compared for the immunochromatography-based BinaxNOW and ImmuView UATs. Overall sensitivity and specificity of Legionella UATs were 53.9-61.5% and 90.0-94.9%, respectively. All four UATs successfully detected all samples from L. pneumophila serogroup 1-positive patients, but most failed to detect samples for Legionella spp., or other serogroups. Automatic results interpretation of results was found to be mostly concordant with visual results reading. In conclusion, the performance of the four UATs were similar to each other in the detection of Legionella urinary antigen with no major difference between automated or visual results reading.

Successful international clones of blaCTX-M-15-producing Klebsiella pneumoniae with coexpression of plasmid-mediated quinolone resistance (PMQR) determinants in Tehran hospitals.

The dissemination of plasmid-mediated multidrug resistance in Enterobacteriaceae is a major public health concern. We investigated the prevalence of plasmid-mediated quinolone resistance (PMQR), 16S rRNA methylases, CTX-M, and acquired AmpC enzymes in ESBL-producing Klebsiella pneumoniae (n=40) from Tehran hospitals. Plasmid replicon typing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were carried out for typing. CTX-M group 1 (confirmed as bla(CTX-M-15) in selected isolates) was found in 35/40 isolates. Thirty-two isolates hosted PMQR genes. Twenty isolates featured aac(6')-Ib-CR only; 9 isolates had aac(6')-Ib-CR and qnrB; 2 isolates had aac(6')-Ib-CR and qnrS; and 1 isolate had aac(6')-Ib-CR, qnrS, and qepA. The 16S rRNA methylase RmtB was found in 1 isolate; and acquired AmpC enzymes, in 6 isolates. PFGE detected 7 pulsotypes, the largest corresponded to sequence type 16. The successful clone ST101 was also found. The emergence of successful clones of K. pneumoniae in Tehran hospitals is concerning.

Four genotyping schemes for phylogenetic analysis of Pseudomonas aeruginosa: comparison of their congruence with multi-locus sequence typing.

Several molecular typing schemes have been proposed to differentiate among isolates and clonal groups, and hence establish epidemiological or phylogenetic links. It has been widely accepted that multi-locus sequence typing (MLST) is the gold standard for phylogenetic typing/long-term epidemiological surveillance, but other recently described methods may be easier to carry out, especially in settings with limited access to DNA sequencing. Comparing the performance of such techniques to MLST is therefore of relevance. A study was therefore carried out with a collection of P. aeruginosa strains (n = 133) typed by four typing schemes: MLST, multiple-locus variable number tandem repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE) and the commercial DiversiLab microbial typing system (DL). The aim of this study was to compare the results of each typing method with MLST. The Simpson's indices of diversity were 0.989, 0.980, 0.961 and 0.906 respectively for PFGE, MLVA, DL and MLST. The congruence between techniques was measured by the adjusted Wallace index (W): this coefficient indicates the probability that a pair of isolates which is assigned to the same type by one typing method is also typed as identical by the other. In this context, the congruence between techniques was recorded as follow: MLVA-type to predict MLST-type (93%), PFGE to MLST (92%), DL to MLST (64.2%), PFGE to MLVA (63.5%) and PFGE to DL (61.7%). Conversely, for all above combinations, prediction was very poor. The congruence was increased at the clonal complex (CC) level. MLST is regarded the gold standard for phylogenetic classification of bacteria, but is rather laborious to carry out in many settings. Our data suggest that MLVA can predict the MLST-type with high accuracy, and even higher when studying the clonal complex level. Of the studied three techniques MLVA was therefore the best surrogate method to predict MLST.

High proportion of intestinal colonization with successful epidemic clones of ESBL-producing Enterobacteriaceae in a neonatal intensive care unit in Ecuador.

BACKGROUND AND AIMS: Neonatal infections caused by Extended-spectrum beta-lactamase (ESBL)-producing bacteria are associated with increased morbidity and mortality. No data are available on neonatal colonization with ESBL-producing bacteria in Ecuador. The aim of this study was to determine the proportion of intestinal colonization with ESBL-producing Enterobacteriaceae, their resistance pattern and risk factors of colonization in a neonatal intensive care unit in Ecuador. METHODS: During a three month period, stool specimens were collected every two weeks from hospitalized neonates. Species identification and susceptibility testing were performed with Vitek2, epidemiologic typing with automated repetitive PCR. Associations between groups were analyzed using the Pearson X (2) test and Fisher exact test. A forward step logistic regression model identified significant predictors for colonization. RESULTS: Fifty-six percent of the neonates were colonized with ESBL-producing Enterobacteriaceae. Length of stay longer than 20 days and enteral feeding with a combination of breastfeeding and formula feeding were significantly associated with ESBL-colonization. The strains found were E. coli (EC, 89%) and K. pneumoniae (KP, 11%) and epidemiological typing divided these isolates in two major clusters. All EC and KP had bla CTX-M group 1 except for a unique EC isolate that had bla CTX-M group 9. Multi-locus sequence typing performed on the K. pneumoniae strains showed that the strains belonged to ST855 and ST897. The two detected STs belong to two different epidemic clonal complexes (CC), CC11 and CC14, which previously have been associated with dissemination of carbapenemases. None of the E. coli strains belonged to the epidemic ST 131 clone. CONCLUSIONS: More than half of the neonates were colonized with ESBL-producing Enterobacteriaceae where the main risk factor for colonization was length of hospital stay. Two of the isolated clones were epidemic and known to disseminate carbapenemases. The results underline the necessity for improved surveillance and infection control in this context.

High prevalence of 16S rRNA methylase RmtB among CTX-M extended-spectrum ß-lactamase-producing Klebsiella pneumoniae from Islamabad, Pakistan.

The aim of this study was to characterise extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae isolated from urinary tract and wound infections from Pakistan (n=25). Isolates were subjected to commercially available microarray analysis to determine the presence of ESBLs and acquired AmpC enzymes. The genetic diversity of the isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmid replicon typing and capsular serotyping were conducted by PCR. Finally, screening for virulence genes, plasmid-mediated quinolone resistance (PMQR) genes, and genes encoding 16S rRNA methylases was done using PCR. All K. pneumoniae isolates hosted blaCTX-M genes and all strains belonged to phylogroup CTX-M-1. Acquired AmpC ß-lactamases (ACT/MIR and CIT group) were found in 16% of isolates. Two clusters were observed with ≥80% similarity among profiles obtained by PFGE, and two sequence types (STs) by MLST, namely ST215 and ST307, were observed in these clusters. Three ST215 isolates carried virulence factor wcaG and three ST215 isolates had capsular type K20. IncFIA, IncFIB, IncFIIK and FrepB replicons were most commonly found in this collection. Among the PMQR determinants, aac(6')-lb-cr was present in 96% (24/25) of the isolates, qnrB was found in 88% (22/25) and qepA was found in 4% (1/25). The 16S rRNA methylase-encoding gene rmtB was found in 60% (15/25) of the isolates. In conclusion, CTX-M-producing ST215 and ST307 K. pneumoniae were the two major clones detected. Of particular concern was the high prevalence of 16S rRNA methylases conferring resistance to all aminoglycosides.

A multiresistant clone of Pseudomonas aeruginosa sequence type 773 spreading in a burn unit in Orumieh, Iran.

Herein, we describe the phenotypic and genotypic characterization of a multiresistant clone of Pseudomonas aeruginosa disseminating in a burn unit in Orumieh, Iran. A total of 58 isolates of P. aeruginosa were collected during August 2007 and June 2008. Minimum inhibitory concentrations (MICs) of P. aeruginosa isolates were determined against 11 antimicrobial agents by E test. Serotyping, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were used for studying the clonal relationship among the isolates. Antibiotic susceptibility testing revealed that most of the isolates were multidrug resistant and colistin was the antibiotic with the highest activity. Pseudomonas aeruginosa isolates fell into nine different serotypes, and O10 and O11 were the most common. PFGE analyses showed 12 different genotypes and 68.1% of isolates showed more than 80% similarity, indicating possible clonal relatedness. These isolates were found to belong to the same sequence type, ST773. This sequence type has earlier been reported from China, and a double locus variant of this ST has been found earlier in France in a PER-1 extended-spectrum ß-lactamase-producing P. aeruginosa.

Prevalence of antibiotic-resistant bacteria in residents of nursing homes in a Swedish municipality: healthcare staff knowledge of and adherence to principles of basic infection prevention.

BACKGROUND: The aims of this study were to investigate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae in residents living in Swedish nursing homes, and if carriage of resistant bacteria was related to antibiotic treatment, other risk factors, and/or staff's adherence to guidelines for infection control. METHODS: Five hundred and sixty residents from 9 nursing homes on a total of 67 wards participated in the study and had microbiological cultures taken. Faecal samples were obtained from 495 residents (88.3%). ESBL-positive residents were followed for 2 y with repeated sampling. Two hundred and ninety-six [corrected] staff members were interviewed and observed regarding familiarity with and adherence to infection control guidelines. RESULTS: No resident was positive for MRSA or VRE. Fifteen of the residents were found to be ESBL-positive. Residents living on wards where ESBL-positive residents were identified had been treated more frequently with antibiotics (42%), compared to those on wards where no residents with ESBL were found (28%; p = 0.02). ESBL-positive Escherichia coli isolates from residents living in adjacent rooms were found to be closely genetically related when analysed by pulsed-field gel electrophoresis, indicating transmission between residents. Staff adherence to infection control guidelines sometimes revealed shortcomings, but no significant differences regarding compliance to the guidelines could be found. CONCLUSION: Carriage of resistant bacteria was uncommon and only ESBL-producing Enterobacteriaceae were identified in Swedish nursing homes. Usage of antibiotics was higher on wards where ESBL-positive residents were detected and there was an indication of transmission of ESBL between residents.

Efficacy of pivmecillinam for treatment of lower urinary tract infection caused by extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae.

To evaluate the clinical and bacteriological efficacy of pivmecillinam against lower urinary tract infection (UTI) caused by extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae, patients treated for lower UTI with pivmecillinam (n=8) were studied. Patients treated with nitrofurantoin (n=3) and trimethoprim (n=3) or a combination of these agents with pivmecillinam (n=3) were included as a control group. Antimicrobial susceptibility was determined with EUCAST methodology. Bacteriologic cure was defined as <10(3) CFU/ml at follow-up (30 days), and clinical cure as resolved UTI symptoms after completed treatment. All patients receiving pivmecillinam had good clinical response (8/8), but bacteriological cure rates were low (2/8). However, none of the patients with persisting bacteriuria had a relapse of UTI symptoms within 6 months. All isolates were susceptible to the given antimicrobial. Most isolates belonged to the CTX-M-1 group (n=11, 65%) or CTX-M-9-group (n=4, 24%). Four E. coli isolates belonged to the international clone O25b-ST131 (25%). In conclusion, pivmecillinam had good clinical activity against lower UTI caused by ESBL-producing Enterobacteriaceae, but bacteriological cure rates were low. The persistent bacteriuria appears to be of little clinical importance, but larger clinical studies are needed to determine the usefulness of pivmecillinam in infections caused by ESBL-producing bacteria.

Antimicrobial susceptibility to parenteral and oral agents in a largely polyclonal collection of CTX-M-14 and CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae.

Activity of oral and parenteral antimicrobials against consecutively isolated extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (n = 149) and Klebsiella pneumoniae (n = 20) was determined, and susceptibility test methods were compared for parenteral ß-lactams. Polymerase chain reaction (PCR) targeting bla(CTX-M), bla(SHV) and bla(TEM), and DNA sequencing and epidemiological typing with pulsed-field gel electrophoresis were performed. PCR targeting pabB was screened for E. coli O25b-ST131. Minimum inhibitory concentrations (MICs) were determined using Etest and broth microdilution. Disc diffusion was performed according to European Committee on Antimicrobial Susceptibility Testing (EUCAST). Dominating genotypes were bla(CTX-M-15) (75%) and bla(CTX-M-14) (23%). Four E. coli clusters (7-18 isolates) were found. Forty-two per cent of E. coli belonged to O25b-ST131. Ciprofloxacin resistance was 72%, trimethoprim resistance was 70%. Among E. coli, resistance to mecillinam (13%), nitrofurantoin (7%) and fosfomycin (3%) was low, although resistance was high in K. pneumoniae (25%, 60%, 85%). Susceptibility to ertapenem was 99%, piperacillin-tazobactam 91%, tigecycline 96% and temocillin 76%. Susceptibility rates obtained with broth microdilution and Etest were in agreement for cefotaxime (2 vs 1%) and ceftazidime (9 vs 11%), but not for piperacillin-tazobactam (59 vs 91%). With disc diffusion major errors occurred with piperacillin-tazobactam (18/169). Several therapeutic alternatives exist for ESBL-producing E. coli, but few exist for K. pneumoniae. Disc diffusion and Etest can accurately predict susceptibility to cefotaxime and ceftazidime, but not to piperacillin-tazobactam with the present breakpoints.

Population structure of Pseudomonas aeruginosa from five Mediterranean countries: evidence for frequent recombination and epidemic occurrence of CC235.

Several studies in recent years have provided evidence that Pseudomonas aeruginosa has a non-clonal population structure punctuated by highly successful epidemic clones or clonal complexes. The role of recombination in the diversification of P. aeruginosa clones has been suggested, but not yet demonstrated using multi-locus sequence typing (MLST). Isolates of P. aeruginosa from five Mediterranean countries (n = 141) were subjected to pulsed-field gel electrophoresis (PFGE), serotyping and PCR targeting the virulence genes exoS and exoU. The occurrence of multi-resistance (≥ 3 antipseudomonal drugs) was analyzed with disk diffusion according to EUCAST. MLST was performed on a subset of strains (n = 110) most of them had a distinct PFGE variant. MLST data were analyzed with Bionumerics 6.0, using minimal spanning tree (MST) as well as eBURST. Measurement of clonality was assessed by the standardized index of association (I(A) (S)). Evidence of recombination was estimated by ClonalFrame as well as SplitsTree4.0. The MST analysis connected 70 sequence types, among which ST235 was by far the most common. ST235 was very frequently associated with the O11 serotype, and frequently displayed multi-resistance and the virulence genotype exoS⁻/exoU⁺. ClonalFrame linked several groups previously identified by eBURST and MST, and provided insight to the evolutionary events occurring in the population; the recombination/mutation ratio was found to be 8.4. A Neighbor-Net analysis based on the concatenated sequences revealed a complex network, providing evidence of frequent recombination. The index of association when all the strains were considered indicated a freely recombining population. P. aeruginosa isolates from the Mediterranean countries display an epidemic population structure, particularly dominated by ST235-O11, which has earlier also been coupled to the spread of ß-lactamases in many countries.

Outbreak of CTX-M-15-producing Klebsiella pneumoniae of sequence type 199 in a Latvian teaching hospital.

Previous studies on the epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in Latvia are lacking. ESBL-producing Klebsiella pneumoniae (n = 32) were subjected to pulsed-field gel electrophoresis (PFGE) and selected isolates to multi-locus sequence typing (MLST). Species identification and susceptibility testing were performed using VITEK2, and sequencing of bla(CTX-M) was performed in selected isolates. PFGE revealed one major clone (n = 23), with most of the isolates derived from the ICU. The clone harboured bla(CTX-M-15), was sequence type 199 and comprised two ertapenem non-susceptible isolates. This is the first report of an ESBL outbreak in Latvia, and calls for increased epidemiological typing of ESBL-producing Enterobacteriaceae, as well as improved infection control routines.

Integrated analysis of established and novel microbial and chemical methods for microbial source tracking.

Several microbes and chemicals have been considered as potential tracers to identify fecal sources in the environment. However, to date, no one approach has been shown to accurately identify the origins of fecal pollution in aquatic environments. In this multilaboratory study, different microbial and chemical indicators were analyzed in order to distinguish human fecal sources from nonhuman fecal sources using wastewaters and slurries from diverse geographical areas within Europe. Twenty-six parameters, which were later combined to form derived variables for statistical analyses, were obtained by performing methods that were achievable in all the participant laboratories: enumeration of fecal coliform bacteria, enterococci, clostridia, somatic coliphages, F-specific RNA phages, bacteriophages infecting Bacteroides fragilis RYC2056 and Bacteroides thetaiotaomicron GA17, and total and sorbitol-fermenting bifidobacteria; genotyping of F-specific RNA phages; biochemical phenotyping of fecal coliform bacteria and enterococci using miniaturized tests; specific detection of Bifidobacterium adolescentis and Bifidobacterium dentium; and measurement of four fecal sterols. A number of potentially useful source indicators were detected (bacteriophages infecting B. thetaiotaomicron, certain genotypes of F-specific bacteriophages, sorbitol-fermenting bifidobacteria, 24-ethylcoprostanol, and epycoprostanol), although no one source identifier alone provided 100% correct classification of the fecal source. Subsequently, 38 variables (both single and derived) were defined from the measured microbial and chemical parameters in order to find the best subset of variables to develop predictive models using the lowest possible number of measured parameters. To this end, several statistical or machine learning methods were evaluated and provided two successful predictive models based on just two variables, giving 100% correct classification: the ratio of the densities of somatic coliphages and phages infecting Bacteroides thetaiotaomicron to the density of somatic coliphages and the ratio of the densities of fecal coliform bacteria and phages infecting Bacteroides thetaiotaomicron to the density of fecal coliform bacteria. Other models with high rates of correct classification were developed, but in these cases, higher numbers of variables were required.

Occurrence and relatedness of vancomycin-resistant enterococci in animals, humans, and the environment in different European regions.

Vancomycin-resistant enterococci (VRE) in Europe are thought to have emerged partly due to the use of the glycopeptide avoparcin in animal husbandry. We compared the occurrence of VRE in geographical regions of Europe in which until 1997 large amounts of avoparcin were used (Spain, United Kingdom, and Denmark) with the occurrence of VRE in Sweden, where avoparcin was banned in 1986. We also studied the relatedness between VRE strains from different regions and habitats. In total, 2,580 samples were collected from humans, animals, and the environment (soil, sewage, recipient water). VRE resistant to 20 microg/ml vancomycin were identified in 8.2% of the samples and were found most frequently in raw and treated urban sewage samples (means, 71% and 36% of the samples, respectively), pig manure (17%), and hospital sewage (16%). The proportions of VRE-positive sewage samples were similar in Sweden, Spain, and the United Kingdom, whereas pig feces and manure were more often positive in Spain than in Sweden (30% versus 1%). Most VRE were Enterococcus faecium carrying vanA, and computerized biochemical phenotyping of the isolates of different ecological origins showed a high degree of polyclonality. In conclusion, it seems that animal-associated VRE probably reflect the former use of avoparcin in animal production, whereas VRE in human-associated samples may be a result of antibiotic use in hospitals. Since there seems to be a reservoir of the resistance genes in all countries studied, precautions must be taken to limit the use of antibiotics and antibiotic-like feed additives.

Evidence for transmission between humans and the environment of a nosocomial strain of Enterococcus faecium.

An ampicillin- and ciprofloxacin-resistant Enterococcus faecium (ARE) strain, named FMSE1, with a characteristic biochemical phenotype, was in a recent study found to dominate among faecal ARE isolates from patients in several Swedish hospitals. In the present study, the prevalence of this strain among 9676 enterococcal isolates from healthy children, hospital sewage, urban sewage, surface water, slaughtered animals (broilers, pigs and cattle) and pig faeces and manure was investigated. Enterococcal isolates having the same biochemical phenotype as the FMSE1 were most common in samples of hospital sewage (50%), surface water (35%), treated sewage (28%) and untreated sewage (17%), but rare in samples from healthy children (0.8%) and animals (2%). PFGE typing of FMSE1-like isolates from hospital sewage indicated that they were closely related to the nosocomial FMSE1 strain. Thus, this study indicated a possible transmission route for nosocomial E. faecium from patients in hospitals to hospital sewage and urban sewage, and further via treatment plants to surface water and possibly back to humans. This proposed route of circulation of drug-resistant enterococci might be further amplified by antibiotic usage in human medicine. In contrast, such transmission from food animals seems to play a negligible role in Sweden.

Tracking the origin of faecal pollution in surface water: an ongoing project within the European Union research programme.

The objectives of this study are to generate knowledge about methods to track the sources of faecal pollution in surface waters, with the aim of having one or a few easy procedures applicable to different geographic areas in Europe. For this, a first field study using already proposed methods (genotypes of F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, phenotypes of faecal coliforms and enterococci, and sterols) has been done in five areas representing a wide array of conditions in Europe. The present faecal indicators (faecal coliforms, enterococci, sulfite reducing clostridia and somatic coliphages) have also been included in this first field study. At the same time some emerging methods have been settled or adapted to water samples and assayed in a limited number of samples. The results of this first field study indicate that no single parameter alone is able to discriminate the sources, human or non-human, of faecal pollution, but that a 'basket' of 4 or 5 parameters, which includes one of the present faecal indicators, will do so. In addition, numerical analysis of the data shows that this 'basket' will allow the successful building of predictive models. Both the statistical analyses and the studied predictive models indicate that genotype II of F-specific RNA bacteriophages, the coprostanol and the ratio coprostanol: coprostanol+epicoprostanol are, out of the studied parameters, those with a greater discriminating power. Either because unsuccessful adaptation of the methods to water samples or because the preliminary assays in water samples indicated low discriminating capability, only three (sorbitol-fermenting bifidobacteria, some species of bifidobacteria detected by PCR with specific primers and phages infecting Bacteroides tethaiotaomicron) of the newly assayed methods have been considered for a second field study, which is currently underway. Expectations are that these new tools will minimize the number of parameters in the 'basket', or at least minimize the difficulty in assaying them.

Comparison of enterococcal populations in animals, humans, and the environment--a European study.

The objectives of the present study were to generate knowledge of enterococcal populations in the food chain, by studying the population structure (in measures of abundance and diversity) among enterococci in different geographical regions and in different parts of the food chain, as well as the similarities between different enterococcal populations. Altogether, 2868 samples were collected from humans (healthy and hospitalised individuals and clinical isolates), animals (slaughterhouse carcasses and farm animals), and the environment (pig farms, sewage, and surface water) in four European countries-Sweden, Denmark, UK, and Spain. The samples were characterised with regard to presence and numbers of enterococci, and eight (for faecal samples) or 24 (for environmental samples) isolates per sample were phenotyped and preliminarily identified with the PhP-RF system. In total, more than 20,000 isolates were typed. A majority of the samples (77%) showed the presence of presumed enterococci. The diversities of enterococci in environmental samples were generally high, and also faecal samples normally showed presence of more than one enterococcal strain. The most common species found were Enterococcus faecium (33%), E. faecalis (29%), and E. hirae (24%), but different enterococcal populations differed in their species distribution. Clinical isolates, hospitalised patients, and hospital sewage in Sweden showed a clear dominance of E. faecalis (80%, 57%, and 54%, respectively) whereas healthy individuals and urban sewage contained less E. faecalis (39% and 40%, respectively). The species distribution among isolates from slaughterhouses varied between animal species and also between countries, but E. faecalis seemed to be mainly associated with broiler, and E. hirae with cattle and pigs. The results from the study have indicated a simplified method to study the diversity of bacterial populations. Instead of collecting many samples and analysing one or a few isolates per sample, it is possible to collect fewer samples and analyse several isolates per sample. Both approaches yielded similar information on the diversity of the populations. Another useful information was that since samples from hospital sewage, urban sewage, and manure contained enterococcal populations that reflected those in faecal samples of hospitalised patients, healthy humans, and animals, respectively, such samples may be used as pooled faecal samples and may replace cumbersome samplings from many individuals.

The PhenePlate system for studies of the diversity of enterococcal populations from the food chain and the environment.

The growing interest in ecological investigations, such as studies of the "flow" of bacteria through the food chain, has resulted in a great need for simple typing techniques for bacteria that can be used when a large number of isolates need to be analysed. The present study describes a simple method for biochemical fingerprinting of enterococci, the PhenePlate RF (PhP-RF) system. The system is based on a 96-well microplate containing eight sets of 11 dehydrated reagents, selected to have a high discriminatory power among enterococcal isolates. The PhP plates are inoculated easily by picking single colonies directly from the primary agar culture and suspending them in the first well of each row in the microplate. The kinetics of each reaction is evaluated by measuring the absorbance value of each well three times during 64 h, whereupon a biochemical fingerprint is calculated as the mean value for each reagent over the three readings. The PhP-RF method was shown to be highly reproducible, even when results were compared between different laboratories. The discriminatory power, measured as Simpson's diversity index (Di), was as high as 0.96 for all enterococci. The PhP-RF method could also be used as a preliminary species identification method, by comparing the biochemical fingerprints of unknown strains to those of a set of reference strains of known species. Most strains of Enterococcus faecalis, Enterococcus faecium and Enterococcus hirae were correctly identified using this method. We conclude that the PhenePlate RF system is useful for rapid typing of enterococcal populations, and it is especially useful as a first screening method for ecological studies, when many isolates per sample need to be analysed.

High prevalence of vancomycin-resistant enterococci in Swedish sewage.

In Europe the use of the growth promoter avoparcin is considered to have selected for vancomycin-resistant enterococci (VRE). Sweden ceased using avoparcin in 1986, and only occasional cases of VRE from hospitals have been reported since 1995. Within the framework of a European study, samples from urban raw sewage, treated sewage, surface water, and hospital sewage in Sweden (n = 118) were screened for VRE. Surprisingly, VRE were isolated from 21 of 35 untreated sewage samples (60%), from 5 of 14 hospital sewage samples (36%), from 6 of 32 treated sewage samples (19%), and from 1 of 37 surface water samples. Thirty-five isolates from 33 samples were further characterized by geno- and phenotyping, MIC determination, and PCR analysis. Most isolates (30 of 35) carried the vanA gene, and the majority (24 of 35) of the isolates were Enterococcus faecium. Most of the VRE were multiresistant. The typing revealed high diversity of the isolates. However, one major cluster with seven identical or similar isolates was found. These isolates came from three different sewage treatment plants and were collected at different occasions during 1 year. All VRE from hospital sewage originated from one of the two hospitals studied. That hospital also had vancomycin consumption that was 10-fold that of the other. We conclude that VRE were commonly found in sewage samples in Sweden. The origin might be both healthy individuals and individuals in hospitals. Possibly, antimicrobial drugs or chemicals released into the sewage system may sustain VRE in the system.