Aina Iversen

High prevalence of vancomycin-resistant enterococci in Swedish sewage

ABSTRACT 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.

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.

Integrated Analysis of Established and Novel Microbial and Chemical Methods for Microbial Source Tracking

ABSTRACT 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

ABSTRACT Vancomycin-resistant enterococcci (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 μg/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.

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 (IA 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.

Comparison of enterococcal populations related to urban and hospital wastewater in various climatic and geographic European regions

Aims: Scarce knowledge about the distribution of enterococci species in wastewaters limits any statement on their reliability as faecal indicators or the implications of antibiotic resistance transmission by these organisms through the water cycle. Enterococci have been involved in nosocomial infections and the spreading of antibiotic resistance through the food chain. The species distribution of enterococci and the presence of resistant strains to vancomycin and erythromycin were analysed in more than 400 raw and treated urban wastewaters, surface waters receiving these treated wastewaters and hospital wastewaters from three European countries.

Epidemiology and ecology of enterococci, with special reference to antibiotic resistant strains, in animals, humans and the environment. Example of an ongoing project within the European research programme.

The objectives of the present study are to generate knowledge of the ecology and epidemiology of enterococci in the food chain by studying the following: (1) the population structure (in measures of abundance, number of vancomycin resistant strains, antibiotic resistance patterns, diversity, and stability) among enterococcal populations in different geographical regions and in different links of the food chain (2) possible transmission of strains through the food chain and between hospital environments and the food chain (3) the association between vancomycin resistance and individual strains of enterococci and (4) the diversity of the drug resistance genes in enterococci. So far, 1578 samples have been collected from different countries within the EU (Sweden, Denmark, UK and Spain), and from different habitats (pig farms, carcasses in slaughter houses, soil, manure, water, sewage, and humans). Total and vancomycin resistant enterococcal populations in each sample have been enumerated and more than 12000 isolates have been characterised by phenotyping. Representative isolates are further species identified and characterised by genotyping and MIC determination and from antibiotic resistant isolates the resistance genes are characterised.

Microplate-based microbial assay for risk assessment and (eco)toxic fingerprinting of chemicals

Abstract We have developed a multi-species microbial assay, MARA, for assessing the (eco)toxic risks of chemical compounds and for the determination of their toxic fingerprints. The main advantages with MARA are (1) the simultaneous testing on several microbial strains; (2) the concept of toxic fingerprinting; (3) the simple and inexpensive handling and reading of the test. The toxic activity is measured in parallel on 11 different micro-organisms lyophilised in a microplate. A concentration gradient of the chemical to be tested is added and growth is indicated through the reduction of tetrazolium red (TTC). The microplates are read by a common flatbed scanner or a microplate spectrophotometer. The array of the 11 different inhibition values constitute a toxic fingerprint, characteristic for each type of chemical compound, and it is shown that the assay can distinguish between 12 standard chemicals. Both the reproducibility (CV≈20%) and the sensitivity are similar to other toxicity tests based on micro-organisms.

Efficacy of Pivmecillinam for Treatment of Lower Urinary Tract Infection Caused by Extended-Spectrum β-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 β-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.

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.