Vancomycin-resistant Enterococcus faecium: impact of ending screening and isolation in a Danish University hospital.

BACKGROUND: Substantial resources are used in hospitals worldwide to counteract the ever-increasing incidence of vancomycin-resistant and vancomycin-variable Enterococcus faecium (VREfm and VVEfm), but it is important to balance patient safety, infection prevention, and hospital costs. AIM: To investigate the impact of ending VREfm/VVEfm screening and isolation at Odense University Hospital (OUH), Denmark, on patient and clinical characteristics, risk of bacteraemia, and mortality of VREfm/VVEfm disease at OUH. The burden of VREfm/VVEfm bacteraemia at OUH and the three collaborative hospitals in the Region of Southern Denmark (RSD) was also investigated. METHODS: A retrospective cohort study was conducted including first-time VREfm/VVEfm clinical isolates (index isolates) detected at OUH and collaborative hospitals in the period 2015-2022. The intervention period with screening and isolation was from 2015 to 2021, and the post-intervention period was 2022. Information about clinical isolates was retrieved from microbiological databases. Patient data were obtained from hospital records. FINDINGS: At OUH, 436 patients were included in the study, with 285 in the intervention period and 151 in the post-intervention period. Ending screening and isolation was followed by an increased number of index isolates. Besides a change in van genes, only minor non-significant changes were detected in all the other investigated parameters. Mortality within 30 days did not reflect the VREfm/VVEfm-attributable deaths, and in only four cases was VREfm/VVEfm infection the likely cause of death. CONCLUSION: Despite an increasing number of index isolates, nothing in the short follow-up period supported a reintroduction of screening and isolation.

Shiga toxin-producing Escherichia coli: incidence and clinical features in a setting with complete screening of patients with suspected infective diarrhoea.

OBJECTIVES: Shiga toxin-producing Escherichia coli (STEC) causes diarrhoeal disease, bloody diarrhoea, and haemolytic uraemic syndrome. The aim of this study was to describe the incidence of STEC and the clinical features of STEC patients from a well-defined Danish population in which all fecal samples of patients with suspected infective gastroenteritis were analysed for STEC. METHODS: In this population-based cohort study, all stool samples referred to two clinical microbiology laboratories were screened for STEC by culture and/or PCR. Epidemiological (n=170) and clinical (n=209) characteristics were analysed using data from local and national registries. RESULTS: Overall, 75,132 samples from 30,073 patients were screened resulting in 217 unique STEC-isolates. The epidemiological analysis showed an incidence of 10.1 cases per 100,000 person-years, which was more than twofold higher than the incidence in the rest of Denmark (3.4 cases per 100,000 person-years, p <0.001). Three groups were associated with a higher incidence: age <5 years (n=28, p <0.001), age ≥65 years (n=38, p 0.045), and foreign ethnicity (n=27, p 0.003). In the clinical analysis, patients with STEC harbouring only the Shiga toxin 1 gene (stx1-only isolates) showed a lower frequency of acute (n=11, p <0.05) and bloody diarrhoea (n=5, p <0.05) and a higher frequency of gastrointestinal symptoms for ≥3 months (n=8, p <0.05) than the other STEC patients. CONCLUSIONS: We report a more than twofold higher incidence in the project area compared with the rest of Denmark, indicating that patients remain undiagnosed when selective STEC screening is used. We found an association between patients with stx1-only isolates and long-term gastrointestinal symptoms.

Selecting PCR for the Diagnosis of Intestinal Parasitosis: Choice of Targets, Evaluation of In-House Assays, and Comparison with Commercial Kits.

Microscopy of stool samples is a labour-intensive and inaccurate technique for detection of intestinal parasites causing diarrhoea and replacement by PCR is attractive. Almost all cases of diarrhoea induced by parasites over a nine-year period in our laboratory were due to Giardia lamblia, Cryptosporidium species, or Entamoeba histolytica detected by microscopy. We evaluated and selected in-house singleplex real-time PCR (RT-PCR) assays for these pathogens in 99 stool samples from patients suspected of having intestinal parasitosis tested by microscopy. The strategy included a genus-specific PCR assay for C. parvum and C. hominis, with subsequent identification by a PCR that distinguishes between the two species. G. lamblia was detected in five and C. parvum in one out of 68 microscopy-negative samples. The performance of the in-house RT-PCR assays was compared to three commercially available multiplex test (MT-PCR) kit systems in 81 stool samples, collected in 28 microscopy-positive and 27 microscopy-negative samples from individuals suspected of intestinal parasitosis and in 26 samples from individuals without suspicion of parasitic infection. The in-house assays detected parasites in more samples from patients suspected of having parasitosis than did any of the kits. We conclude that commercial kits are targeting relevant parasites, but their performance may vary.

Whole-genome sequencing for identification of the source in hospital-acquired Legionnaires' disease.

Acquisition of Legionnaires' disease is a serious complication of hospitalization. Rapid determination of whether or not the infection is caused by strains of Legionella pneumophila in the hospital environment is crucial to avoid further cases. This study investigated the use of whole-genome sequencing to identify the source of infection in hospital-acquired Legionnaires' disease. Phylogenetic analyses showed close relatedness between one patient isolate and a strain found in hospital water, confirming suspicion of nosocomial infection. It was found that whole-genome sequencing can be a useful tool in the investigation of hospital-acquired Legionnaires' disease.

Identification of CTX-M15-, SHV-28-producing Klebsiella pneumoniae ST15 as an epidemic clone in the Copenhagen area using a semi-automated Rep-PCR typing assay.

Rapid molecular typing methods can be a valuable aid in the investigation of suspected outbreaks. We used a semi-automated repetitive sequence-based polymerase chain reaction (Rep-PCR) typing assay and pulsed field gel electrophoresis (PFGE) to investigate the relationship between local Klebsiella pneumoniae (K. pneumoniae) producing extended spectrum ß-lactamases (ESBLs) and their relation to recognized Danish outbreak strains. PFGE and Rep-PCR produced similar clustering among isolates. Individual isolates from each cluster were further characterized by PCR amplification and sequencing of bla (TEM), bla (SHV), and bla (CTX-M), and multilocus sequence typing (MLST). Thirty-five out of 52 ESBL-producing K. pneumoniae isolates were ST15 and bla (CTX-M15), bla (SHV-28), and bla (TEM-1) positive by PCR. Ten out of 52 were ST16 and tested positive for bla (CTX-M15), bla (SHV-1), and bla (TEM-1). Isolates from previously recognized hospital outbreaks were also ST15 and PCR positive for bla (CTX-M15), bla (SHV-28), and bla (TEM-1), and typed within the main cluster by both Rep-PCR and PFGE. In conclusion, K. pneumoniae ST15 containing bla (CTX-M15) and bla (SHV-28) constitutes an epidemic clone in the Copenhagen area and this clone can be rapidly recognized by semi-automated Rep-PCR.

Transmission of Salmonella between wildlife and meat-production animals in Denmark.

AIMS: To investigate the transmission of Salmonella spp. between production animals (pigs and cattle) and wildlife on production animal farms in Denmark. METHODS AND RESULTS: In the winter and summer of 2001 and 2002, 3622 samples were collected from Salmonella-infected and noninfected herds of pigs and cattle and surrounding wildlife. Salmonella was detected in wildlife on farms carrying Salmonella-positive production animals and only during the periods when Salmonella was detected in the production animals. The presence of Salmonella Typhimurium in wild birds significantly correlated to their migration pattern and food preference. CONCLUSIONS: Salmonella was transmitted from infected herds of production animals (cattle and pigs) to wildlife that lived amongst or in close proximity to them. SIGNIFICANCE AND IMPACT OF THE STUDY: Salmonella in animal food products is associated with the occurrence of Salmonella in primary animal production. Strategies to control the introduction and spread of infection should include wildlife management, as the nearby wildlife may act as reservoirs for Salmonella spp. and/or may be passive carriers of the bacteria.

Occurrence and spread of multiresistant Salmonella Typhimurium DT104 in Danish animal herds investigated by the use of DNA typing and spatio-temporal analysis.

In the investigation we report the occurrence and spread of multiresistant Salmonella Typhimurium phage type (DT) 104 in 174 known Danish animal herds infected between 1996 and 2003. We applied PFGE, plasmid analysis and spatio-temporal analysis for a description of the development and spread of infection among herds. The results showed that in Denmark DT104 are mainly spread by trade of animals; however, horizontal spread of DT104 is also important and the risk of an animal herd becoming infected increased with the proximity in time and space to a DT104-infected herd. Based on these results, it is recommended that specific actions are taken to limit the risk of horizontal spread of DT104 from newly infected herds.

Transmission routes of Salmonella Typhimurium DT 104 between 14 cattle and pig herds in Denmark demonstrated by molecular fingerprinting.

AIMS: Salmonella Typhimurium DT 104 is generally assumed to be spread by contact between live animals, e.g. by trading. The aim of the present study was to assess the importance of other routes of transmission in the dissemination of this bacterium. METHODS AND RESULTS: An outbreak among 14 cattle and pig herds located in a geographically narrow area in Denmark was investigated. Epidemiological information and disease history of the herds was obtained through interviews. Based on this, the hypothesis for horizontal spread was proposed, and these were confirmed by comparison of the pulsed field gel electrophoresis (PFGE) and the plasmid profiles of isolates obtained by continuous sampling over a period of almost 3 years. CONCLUSIONS: The study indicated that other routes might play an important role, than the trading of live animals, in the spread of S. Typhimurium DT 104 among livestock. SIGNIFICANCE AND IMPACT OF THE STUDY: Salmonella Typhimurium DT 104 infected herd might pose a significant risk to herds located within the same geographic area. In advising on how to avoid the spread of this bacterium, factors like person contacts, sharing of equipment and contaminated slurry should be focussed on in addition to infected animals.

Epidemiological characteristics of Salmonella Typhimurium isolated from animals and feed in Poland.

Fifty-seven Salmonella Typhimurium strains isolated from poultry, swine and animal feed in Poland during the years 1979-1998 and 2000-2002 were analysed with conventional and molecular techniques. Antimicrobial resistance as well as multiresistance was found, respectively, in 80.1% and 56.1% of the isolates and most frequently among isolates from 2000-2002. Of several phage types noted, DT104 was prevalent among poultry, swine and feed isolates. DT104, U302 and non-typable strains had a multiple resistant profile (ACSSuT) due to the presence of class I integrons. Pulse-field gel electrophoresis of XbaI and BlnI digest showed high genomic similarity between the strains and confirmed clonal spread of S. Typhimurium infections. Plasmid profiling allowed further differentiation of the strains. We have, therefore, confirmed the appearance of S. Typhimurium DT104 showing genome integrated integron-mediated antimicrobial resistance in Poland. These findings are significant for public and animal health risks and document the dissemination of DT104 epidemic strains into new geographical regions.

The role of litter beetles as potential reservoir for Salmonella enterica and thermophilic Campylobacter spp. between broiler flocks.

We evaluated the role of beetles infesting broiler chicken rearing facilities as potential reservoirs for Salmonella enterica infections between successive broiler flocks. In addition, their role as potential reservoirs for thermophilic Campylobacter spp. was also investigated. Fourteen broiler houses located at 11 different farms were included in the study. The houses were nonrandomly selected on the basis of their salmonella status; nine were persistently contaminated with salmonella whereas five were salmonella negative. For each broiler house, two consecutive broiler flocks (i.e., 28 broiler flocks in all) as well as beetles collected during both rotations of production and in the empty period (after cleaning and disinfection) between these flocks were monitored for the presence of salmonella. Examinations for the presence of campylobacter in the same sample materials were also performed. Beetles sampled during production were positive for salmonella or campylobacter or both. Furthermore, in one house, the occurrence of Salmonella indiana in two consecutive broiler flocks coincided with the presence of S. indiana-contaminated beetles in the empty period between the flocks. The genotype of the identified S. indiana was in all cases identical when analyzed by pulsed-field gel electrophoresis. However, our results also suggest that salmonella from beetles may not always be transmitted to the chickens and that beetles living in contaminated houses can remain free of infection. All cases of campylobacter-positive beetle samples were detected in connection with a positive chicken flock; in no case was campylobacter isolated from beetles taken from the empty period between rotations. Four beetle species were identified during this study. Alphitobius diaperinus was found in all houses and was relatively abundant in most. Typhaea stercorea and Ahasverus advena were found in eight and nine houses, respectively, and were abundant in most of these. Carcinops pumilio was found in small numbers in eight houses. No other insect species was identified. These investigations have shown that beetles in broiler houses infrequently are positive for salmonella. However, transmission of S. indiana between two consecutive broiler flocks can coincide with the presence of salmonella-contaminated beetles in the empty period, indicating that the beetles were the reservoir of S. indiana between the two flocks. Concerning campylobacter, the results suggest that beetles do not play a significant role as a reservoir of campylobacter from one rotation to the next.

The serologic response to Salmonella enteritidis and Salmonella typhimurium in experimentally infected chickens, followed by an indirect lipopolysaccharide enzyme-linked immunosorbent assay and bacteriologic examinations through a one-year period.

Three groups of 100 individually marked salmonella-free chickens were followed for a period of 53 wk. The chickens were infected as day olds by crop instillation of 10(8) colony-forming units: one group with Salmonella enteritidis and a second group with Salmonella typhimurium. A third group was kept uninfected as controls. The groups were monitored bacteriologically by examination of cloacal swabs and organs and serologically by examination of serum and egg yolk by a lipopolysaccharide enzyme-linked immunosorbent assay throughout the period. Within the first week, 100% of birds in both infected groups were excreting salmonella bacteria in the feces. However, the number of fecal excretors declined rapidly with time, down to 6% in 16 wk for S. typhimurium and down to a similar level within the first 8 wk for S. enteritidis. For the latter, relapses with up to 40% positive birds were observed at the onset of egg production. For both S. typhimurium and S. enteritidis, positive bacteriologic cultures were obtained by sampling from internal organs at the end of the experiment, more than 1 yr from the time of infection. At the age of 6-7 wk, 50% of the chickens in the two infected groups showed a measurable serologic response in serum samples. The response persisted throughout the study in both serum and egg yolk samples. The inclusion of serologic methods is a valuable additional tool in the detection of salmonella in poultry, but serology should be used in conjunction with bacteriologic methods in surveillance programs, in particular to detect flocks in early stages of infection before a measurable serologic response has been raised.

Risk factors associated with Salmonella enterica serovar typhimurium infection in Danish broiler flocks.

A retrospective longitudinal study was conducted to identify risk factors associated with Salmonella enterica serovar typhimurium (S. typhimurium) infection in Danish broiler flocks. The data included all broiler flocks slaughtered in 1995, and the epidemiological unit was the individual broiler flock. The S. typhimurium status was determined by microbiological examination of 60 fresh fecal samples. This procedure should detect an infected flock with a probability above 95%, if the prevalence is above 5%, and given that the sensitivity of the test is 100%. Nineteen variables were selected for analysis. Five factors and an interaction term were found significant by multivariate logistic regression analysis. An increased risk for S. typhimurium infection was associated with two parent flocks, one confirmed infected and one suspected of being infected with S. typhimurium, with two of the hatcheries, and with five houses on the farm. An interaction between season and the previously mentioned hatcheries, and a random effect at farm level was also found to be statistically significant. Twelve variables were not found to be associated with S. typhimurium infection: medication, growth promoters, breed of the laying flock, animal density, size of the flock, area of the house, age of the house, geographical location of the farm, observation of beetles, number of days between disinfection and replacement, visual appearance of the bedding, and age of the chickens when they were tested for Salmonella. Three variables (feed mill, slaughterhouse, and Salmonella status of the preceding flock) were not evaluated in the multivariate analysis due to collinearity with other included variables.

Evaluation of sampling methods for the detection of Salmonella in broiler flocks.

The present study compares four different sampling methods potentially applicable to detection of Salmonella in broiler flocks, based on collection of faecal samples (i) by hand, 300 fresh faecal samples (ii) absorbed on five sheets of paper (iii) absorbed on five pairs of socks (elastic cotton tubes pulled over the boots and termed 'socks') and (iv) by using only one pair of socks. Twenty-three broiler flocks were included in the investigation and 18 of these were found to be positive by at least one method. Seven serotypes of Salmonella with different patterns of transmission (mainly horizontal or vertical) were found in the investigation. The results showed that the sock method (five pairs of socks) had a sensitivity comparable with the hand collection method (60 pools of five faecal samples); the paper collection method was inferior, as was the use of only one pair of socks. Estimation of the effective pool sizes for the different methods was also carried out.

Molecular evolution of Vibrio cholerae O1 strains isolated in Lima, Peru, from 1991 to 1995.

Following the emergence of cholera in Lima, Peru, in 1991, isolates of Vibrio cholerae O1 biotype El Tor recovered from patients in various parts of Lima were selected and characterized. Ribotyping and pulsed-field gel electrophoresis (PFGE) revealed four BglI ribotypes and eight NotI PFGE types among 50 V. cholerae O1 strains recovered from patients with cholera in Lima from 1991 to 1995, with certain genotypes appearing to cluster geographically. While differences in ribotype and PFGE type patterns suggest that genetic changes are occurring in the strain responsible for the Latin American cholera epidemic, more frequently than previously reported, 40 (80%) O1 strains showed an identical ribotype pattern and 41 (82%) strains showed closely related PFGE types, types 1, 2, or 3, that differed by less than three restriction fragments. All strains were susceptible to nine antibacterial agents studied. In 1991, more than 95% of the clinical V. cholerae O1 strains were serotype Inaba, whereas from 1992, serotype Ogawa began to predominate, with more than 90% of the isolates being of the Ogawa serotype in 1995. The small differences in genotypes of V. cholerae O1 is remarkable because cholera is highly seasonal in coastal areas of Peru and support the hypothesis that the epidemic strain reemerges from an environmental source. However, the relative high rate of genetic changes within V. cholerae O1 as shown by ribotyping and PFGE should be taken into consideration when typing patterns of V. cholerae O1 associated with cholera in Latin America are evaluated.

Separation of Salmonella typhimurium DT2 and DT135: molecular characterization of isolates of avian origin.

In Denmark, 0.4 and 3.4% of the human Salmonella Typhimurium cases registered between 1988 and 1993 were caused by DT2 and DT135, respectively. Separation of these two phage types was, however, problematic as only minor differences in lysis pattern and lysis strength occurred. Molecular characterization of 23 Danish isolates, 10 German isolates and the two type strains have subsequently been performed. With only minor exceptions, strains examined could be separated by combination of 0.5 agglutination, ribotyping, and PFGE typing into two major groups in conformity with their phage types. The differences between the two groups were, however, very small and it has not been completely clarified whether this grouping is the result of two independent types or of two related lines developing in different environments. It is concluded that the classification of related phage types DT2 and DT135 has to be supported by molecular methods.

Genomic lineage of Salmonella enterica serotype Gallinarum.

Forty-eight strains of Salmonella enterica serotype Gallinarum of biotypes Gallinarum and Pullorum were characterised by three chromosomally based typing methods. The patterns obtained were compared with those of strains of eight other serotypes of Salmonella of O serogroup D. The same PvuII and PstI IS200 patterns were commonly observed among strains of both biotypes and the three SmaI ribotypes of serotype Gallinarum strains differed in only one or two bands, supporting the view that members of these two biotypes are closely related. The same IS200 patterns were also commonly observed among strains of serotype Enteritidis, indicating its evolutionary relationship with serotype Gallinarum. NotI pulsed-field gel electrophoresis (PFGE) patterns divided strains into 24 types. Based on a similarity analysis, two clusters were formed. One contained the majority of biotype Gallinarum strains and two atypical strains of Pullorum; the other contained strains of biotype Pullorum and an otherwise typical strain of biotype Gallinarum. Two atypical strains of biotype Pullorum remained unclustered by PFGE analysis. The grouping of strains differed according to the typing method used, but the majority of strains within each of the biotypes Gallinarum and Pullorum were very similar by the chromosomal markers analysed.

Comparison of pulsed-field gel electrophoresis and ribotyping for subtyping of Vibrio cholerae O139 isolated in Thailand.

Pulsed-field gel electrophoresis (PFGE) of Cpo I-digested genomic DNA and ribotyping (Bgl I) were applied to 60 Vibrio cholerae strains including 48 V. cholerae O139 from Thailand to compare their value in differentiating strains of the present V. cholerae O139 epidemic. PFGE patterns were divided into groups A and B representing five and four subtypes, respectively, while ribotyping showed four different patterns. PFGE group B subtypes were only presented among O139 isolates from Thailand, whereas four O139 strains from Bangladesh and India showed identical PFGE group A subtypes observed in O139 isolates from Thailand. Two nontoxigenic O139 isolates from Thailand showed different and unique PFGE types as did five V. cholerae non-O139 isolates containing a gene virulence complex found in V. cholerae O139. These results indicate that PFGE (Cpo I) can resolve recent evolutionary divergence within V. cholerae O139 and offers a useful supplementary tool for following the progressing V. cholerae O139 epidemic.

Virulence and genotype stability of Salmonella enterica serovar Berta during a natural outbreak.

Strains of Salmonella enterica serotype Berta, collected over a period of 6 years from a well documented natural outbreak in Denmark, have been characterized in order to assess the stability of chromosomal typing systems and virulence properties. Outbreak strains were identical in Pvu II and PSTI IS200 profiles, all but two strains showed the same Sma I ribotype, and all but one strain showed the same Not I pulsed field gel electrophoretic pattern, indicating that these molecular markers remained almost constant during the outbreak. In general, strains of S. Berta were found to be of moderate to low virulence; log VC10 values were found to vary between 3.0 and 4.4 after i.p. challenge of mice, and maximum CFU in internal organs of day-old chicks varied between 2 and 4 log10 units following oral challenge. The minor differences observed between strains in vivo did not correlate with differences in in vitro invasion into cultured MDCK cells, nor with in vitro growth characteristics. A succession of different plasmid profile types was observed during the outbreak but a hierarchical selection of clones based on differences in virulence was unlikely to have caused the succession of types of S. Berta during this outbreak.

Comparison of Pulsed-Field Gel Electrophoresis, Ribotyping, and Plasmid Profiling for Typing of Vibrio anguillarum Serovar O1.

A total of 75 Vibrio anguillarum serogroup O1 strains were studied with respect to their plasmid contents, ribotypes, and pulsed-field gel electrophoresis (PFGE) patterns. Eight plasmid profiles and six ribotypes were demonstrated, and one profile was dominant by both typing methods. In contrast, PFGE had very high discriminatory power, demonstrating 35 profiles. On the basis of PFGE patterns, a similarity matrix and a dendrogram were constructed. The results indicated that Scandinavian strains and southern European isolates (with some exceptions) belong to two different clonal lineages. A few strains from the United States and United Kingdom deviated considerably from each other and from Scandinavian and southern European strains.