Wide-scale study of 206 buildings in the Netherlands from 2011 to 2015 to determine the effect of drinking water management plans on the presence of Legionella spp.

Previous analysis of the Dutch National Legionella Outbreak Detection Program 2002-2012 has shown that buildings required to maintain a Legionella control plan for their drinking water installation are more likely to test positive for Legionella spp. Than buildings without such a plan (38% versus 22% of samples). To clarify this discrepancy, we analysed the results of mandatory water sample testing conducted as part of risk assessments in 206 buildings in the Netherlands from 2011 to 2015. Of the 6171 samples analysed, 16.2% exceeded the Dutch drinking water standard for Legionella spp. of 100 CFU/litre. In buildings with ≤50 tap points, the average percentage of samples containing ≥100 CFU/litre was 28.2%, and from buildings with >50 tap points, it was 12.2%. Analysis of serial samples (taken every 6 months) from each building showed that 33.2% of all buildings tested positive for at least one sample every 6 months. The overall increase was 4.4% per year. Analysis of Legionella subgroups showed that while the majority of positive samples contained L. non-pneumophila (96.9%), some samples did contain L. pneumophila serogroup 1 (1.0%) and serogroups 2-14 (2.1%). Our data suggest that the Dutch mandatory risk assessment and drinking water management plan is not sufficiently effective in preventing the proliferation of Legionella spp. and may even contribute to proliferation. This analysis should now be expanded to include other areas of the Netherlands in order to understand the geographical differences that we observed in our results, and why smaller buildings appear to be more likely to test positive for Legionella spp.

Association between rectal colonization with Highly Resistant Gram-negative Rods (HR-GNRs) and subsequent infection with HR-GNRs in clinical patients: A one year historical cohort study.

OBJECTIVE: Rectal colonization with Highly Resistant Gram-negative Rods (HR-GNRs) probably precedes infection. We aimed to assess the association between rectal HR-GNR colonization and subsequent HR-GNR infection in clinical patients during a follow-up period of one year in a historical cohort study design. METHODS: Rectal HR-GNR colonization was assessed by culturing. Subsequent development of infection was determined by assessing all clinical microbiological culture results extracted from the laboratory information system including clinical data regarding HR-GNR infections. A multivariable logistic regression model was constructed with HR-GNR rectal colonization as independent variable and HR-GNR infection as dependent variable. Gender, age, antibiotic use, historic clinical admission and previous (HR-GNR) infections were included as possible confounders. RESULTS: 1133 patients were included of whom 68 patients (6.1%) were colonized with a HR-GNR. In total 22 patients with HR-GNR infections were detected. Urinary tract infections were most common (n = 14, 63.6%), followed by bloodstream infections (n = 5, 22.7%) and other infections (n = 8, 36.4%). Eight out of 68 HR-GNR colonized patients (11.8%) developed a subsequent HR-GNR infection compared to 14 out of 1065 HR-GNR negative patients (1.3%), resulting in an odds ratio (95% CI) of 7.1 (2.8-18.1) in the multivariable logistic regression analyses. CONCLUSIONS: Rectal colonization with a HR-GNR was a significant risk factor for a subsequent HR-GNR infection. This implies that historical colonization culture results should be considered in the choice of empirical antibiotic therapy to include coverage of the cultured HR-GNR, at least in critically ill patients.

Two Community Clusters of Legionnaires' Disease Directly Linked to a Biologic Wastewater Treatment Plant, the Netherlands.

A biologic wastewater treatment plant was identified as a common source for 2 consecutive Legionnaires' disease clusters in the Netherlands in 2016 and 2017. Sequence typing and transmission modeling indicated direct and long-distance transmission of Legionella, indicating this source type should also be investigated in sporadic Legionnaires' disease cases.

Growth of Legionella anisa in a model drinking water system to evaluate different shower outlets and the impact of cast iron rust.

Legionella continues to be a problem in water systems. This study investigated the influence of different shower mixer faucets, and the influence of the presence of cast iron rust from a drinking water system on the growth of Legionella. The research is conducted using a model of a household containing four drinking water systems. All four systems, which contained standard plumbing components including copper pipes and a water heater, were filled with unchlorinated drinking water. Furthermore, all systems had three different shower faucets: (A) a stainless-steel faucet, (B) a brass-ceramic faucet, and (C) a brass thermostatic faucet. System 1 was solely filled with drinking water. System 2 was filled with drinking water, and cast iron rust. System 3 was contaminated with Legionella, and system 4 was contaminated with a Legionella, and cast iron rust. During a period of 34 months, 450 cold water samples were taken from 15 sample points of the four drinking water systems, and tested for Legionella according to the Dutch Standard (NEN 6265). In system 4, with added cast iron rust, the stainless-steel mixer faucet (A) had the highest concentration of Legionella at >4.3log10CFU/l (>20,000CFU/l) and was positive in 46.4% of samples. In contrast, the stainless-steel mixer faucet (A) of system 3 without cast iron rust showed 14.3% positive samples with a maximum concentration of 3.9log10CFU/l (7600CFU/l) Legionella. Additionally, both contaminated systems (3 and 4), with the brass thermostatic faucet (C), tested positive for Legionella. System 3 in 85.7% of the samples, with a maximum concentration of 4.38log10CFU/l (24,200CFU/l), and system 4 in 64.3% of the samples with a maximum concentration of 4.13log10CFU/l (13.400CFU/l). These results suggest that both the type of faucet used in a drinking water system and the presence or absence of cast iron rust influence the growth of Legionella.

No nosocomial transmission under standard hygiene precautions in short term contact patients in case of an unexpected ESBL or Q&A E. coli positive patient: a one-year prospective cohort study within three regional hospitals.

BACKGROUND: Many Highly Resistant Gram Negative Rod (HR-GNR) positive patients are found unexpectedly in clinical cultures, besides patients who are screened and isolated based on risk factors. As unexpected HR-GNR positive patients are isolated after detection, transmission to contact patients possibly occurred. The added value of routine contact tracing in such situations within hospitals with standard hygiene precautions is unknown. METHODS: In 2014, this study was performed as a prospective cohort study. Index patients were defined as those tested unexpectedly HR-GNR positive in clinical cultures to diagnose a possible infection and were nursed under standard hygiene precautions before tested positive. After detection they were nursed in contact isolation. Contact patients were still hospitalized and shared the same room with the index patient for at least 12 h. HR-GNR screening was performed by culturing a rectal and throat swab. Clonal relatedness of HR-GNR isolates was determined using whole genome sequencing (WGS). RESULTS: Out of 152 unexpected HR-GNR positive patients, 35 patients (23.0%) met our inclusion criteria for index patient. ESBL E. coli was found most frequently (n = 20, 57.1%), followed by Q&A E. coli (n = 10, 28.6%), ESBL K. pneumoniae (n = 3, 8.5%), ESBL R. ornithinolytica (n = 1, 2.9%) and multi resistant P. aeruginosa (n = 1, 2.9%). After contact tracing, 69 patients were identified as contact patient of an index patient, with a median time between start of contact and sampling of 3 days. None were found HR-GNR positive by nosocomial transmission. CONCLUSIONS: In a local setting within hospitals with standard hygiene precautions, routine contact tracing among unexpected HR-GNR positive patients may be replaced by appropriate surveillance as we found no nosocomial transmission in short term contacts.

Clinical sensitivity and specificity of the Check-Points Check-Direct ESBL Screen for BD MAX, a real-time PCR for direct ESBL detection from rectal swabs.

Objectives: To determine the diagnostic accuracy of the Check-Direct ESBL Screen for BD MAX (ESBL qPCR) and an ESBL culture method to identify ESBLs directly from rectal swabs. Methods: Rectal swabs were obtained from clinical patients by performing cross-sectional (point)prevalence measurements in three regional hospitals. Rectal swabs were analysed by direct culture (ChromID ESBL agar) and with the ESBL qPCR. Suspected ESBL-producing isolates were confirmed with the combination disc method and analysed by WGS. Results: Out of 354 rectal swabs and 351 patients, 21 rectal swabs and 20 patients were positive for ESBL-producing isolates, resulting in a regional ESBL colonization prevalence of 5.7%. One rectal swab was false negative with the ESBL qPCR (blaTEM-12) and not covered by the ESBL qPCR. Eight ESBL qPCR-positive rectal swabs could not be confirmed by culture and were classified as false ESBL qPCR positive. The sensitivity and specificity of the ESBL qPCR were 95.2% (n = 20) and 97.6% (n = 323), respectively. When an optimal cycle threshold cut-off value of 37 was used, the ESBL qPCR displayed a sensitivity and specificity of 95.2% (n = 20) and 98.8% (n = 327), respectively (AUC = 0.975, 95% CI = 0.922-1). Conclusions: This ESBL qPCR offers rapid direct detection of the most prevalent ESBL types (blaCTX-M group and blaSHV group) from rectal swabs. The relatively high false-positive rate renders this test the most suitable as a screening test in high-prevalence regions or in an outbreak setting where a fast result is essential.

[Prevention of Legionella pneumonia in the Netherlands: results from the Legionella Source Identification Unit, 2002-2012]. / Preventie van Legionella-pneumonie in Nederland: resultaten van de Bronopsporings Eenheid Legionella-pneumonie in 2002-2012.

OBJECTIVE: To study the effectiveness of a Legionella pneumonia (LP) prevention programme. DESIGN: Observational study. METHOD: We evaluated the effectiveness of the current LP prevention programme using two outcome measures, genotype match and cluster, for the period 2002-2012. If patients were associated with a source of infection via a matching or as part of a cluster it could be assumed that prevention of LP was achieved by implementing control measures for this source. By comparing genotypes we were given an indirect impression of the validity of the sampling process. RESULTS: Legionella pneumophila serogroup 1 was detected in 97 (7%) of the 1484 sampled sources. A likely source of infection was identified for 41 (2%) of the 1991 LP patients, and confirmed by matching. In more than half of these patients, the source was either a residential house or a hospital. Of the 1991 LP patients, 266 (13%) were part of a cluster. Two L. pneumophila serogroup 1 genotypes, ST47 and ST62, were present in 48% of the LP patients, but these genotypes were seldom detected in source sampling (0.9%). CONCLUSION: The current method of source detection does not adequately contribute to the prevention of LP, because the presence of L. pneumophila serogroup 1 is not often detected in the source. Other sources than those currently known are probably involved in the transmission of these bacteria. Serial infection via a common source is a substantial cause of LP, which emphasises the importance of cluster registration. It is important to identify as yet unknown alternative infection sources.

Prevalence, risk factors and molecular epidemiology of highly resistant gram negative rods in hospitalized patients in the Dutch region Kennemerland.

BACKGROUND: This paper describes (1) the Highly Resistant Gram Negative Rod (HR-GNR) prevalence rate, (2) their genotypes, acquired resistance genes and (3) associated risk factors of HR-GNR colonization among the hospitalized population in the Dutch region Kennemerland. METHODS: Between 1 October 2013 and 31 March 2014, cross-sectional prevalence measurements were performed in three regional hospitals as part of each hospitals infection control program. Rectal swabs were analyzed at the Regional Public Health Laboratory Kennemerland by direct culturing. Genotypes and acquired resistance genes of positive isolates were determined using Whole Genome Sequencing with the MiSeq instrument (Illumina). Association between several independent variables and HR-GNR positivity was examined using logistic regression models. RESULTS: Out of 427 patients, 24 HR-GNR positive isolates were recovered from 22 patients, resulting in a regional HR-GNR colonization prevalence (95 % CI) of 5.2 % (3.6-7.9). Of these 22 positive patients, 15 were Extended Spectrum Beta-Lactamase (ESBL) positive (3.5 % (2.1-5.7)), 7 patients were positive for a Fluoroquinolones and Aminoglycosides (Q&A) resistant Enterobacteriaceae (1.6 % (0.8-3.3)) and from one patient (0.2 % (0-1.3)) a Stenotrophomonas maltophilia resistant towards co-trimoxazole was isolated. No carbapenemase producing Enterobacteriaceae (CPE), multi-resistant Acinetobacter species or multi-resistant Pseudomonas aeruginosa were isolated. The ESBL genes found were bla CTX-M-1 (n = 4, 25.0 %), bla CTX-M-15 (n = 3, 18.8 %), bla CTX-M-27 (n = 2, 12.5 %), bla CTX-M-14b (n = 2, 12.5 %), bla CTX-M-9 (n = 2, 12.5 %), bla CTX-M-14 (n = 1, 6.3 %), bla CTX-M-3 (n = 1, 6.3 %), bla SHV-11 (n = 1, 6.3 %) and bla SHV-12 (n = 1, 6.3 %). Being known HR-GNR positive in the past was the only significant associated risk factor for HR-GNR positivity, odds ratio (95 % CI): 7.32 (1.82-29.35), p-value = 0.005. CONCLUSIONS: Similar ESBL prevalence rates and genotypes (3.5 %) were found in comparison to other Dutch studies. When previously HR-GNR positive patients are readmitted, they should be screened for HR-GNR colonization since colonization with GR-GNRs could be prolonged. We recommend for future studies to include all defined HR-GNRs in addition to ESBLs in prevalence studies, in order to obtain a more comprehensive overview of colonization with HR-GNRs.

Costs and Benefits Associated with the MRSA Search and Destroy Policy in a Hospital in the Region Kennemerland, The Netherlands.

OBJECTIVE: The objective of this study was to analyze the costs and benefits of the MRSA Search and Destroy (S&D) policy between 2008 and 2013 in the Kennemer Gasthuis, a 400 bed teaching hospital in the region Kennemerland, the Netherlands. METHODS: A patient registration database was used to retrospectively calculate costs, including screening, isolation, follow-up, contact tracing, cleaning, treatment, deployment of extra healthcare workers, salary for an infection control practitioner (ICP) and service of isolation rooms. The estimated benefits (costs and lives when no MRSA S&D was applied) were based on a varying MRSA prevalence rate (up to 50%). RESULTS: When no MRSA S&D policy was applied, the additional costs and deaths due to MRSA bacteraemia were estimated to be € 1,388,907 and 33 respectively (at a MRSA prevalence rate of 50%). Currently, the total costs were estimated to be € 290,672 (€ 48,445 annually) and a MRSA prevalence rate of 17.3% was considered as break-even point. Between 2008 and 2013, a total of 576 high risk patients were screened for MRSA carriage, of whom 19 (3.3%) were found to be MRSA positive. Forty-nine patients (72.1%) were found unexpectedly. CONCLUSIONS: Application of the MRSA S&D policy saves lives and money, although the high rate of unexpected MRSA cases is alarming.

Legionnaires' disease after a campervan holiday: a case report.

This case report describes a case of Legionnaires' disease for whom the source of infection was the campervan in which the patient had travelled for 3 months. This case shows that Legionnaires' disease can be acquired by exposure to a relatively new (not previously reported) source that is commonly used as (holiday)transportation vehicle.

Whole-Genome Mapping as a Novel High-Resolution Typing Tool for Legionella pneumophila.

Legionella is the causative agent for Legionnaires' disease (LD) and is responsible for several large outbreaks in the world. More than 90% of LD cases are caused by Legionella pneumophila, and studies on the origin and transmission routes of this pathogen rely on adequate molecular characterization of isolates. Current typing of L. pneumophila mainly depends on sequence-based typing (SBT). However, studies have shown that in some outbreak situations, SBT does not have sufficient discriminatory power to distinguish between related and nonrelated L. pneumophila isolates. In this study, we used a novel high-resolution typing technique, called whole-genome mapping (WGM), to differentiate between epidemiologically related and nonrelated L. pneumophila isolates. Assessment of the method by various validation experiments showed highly reproducible results, and WGM was able to confirm two well-documented Dutch L. pneumophila outbreaks. Comparison of whole-genome maps of the two outbreaks together with WGMs of epidemiologically nonrelated L. pneumophila isolates showed major differences between the maps, and WGM yielded a higher discriminatory power than SBT. In conclusion, WGM can be a valuable alternative to perform outbreak investigations of L. pneumophila in real time since the turnaround time from culture to comparison of the L. pneumophila maps is less than 24 h.

Results from the National Legionella Outbreak Detection Program, the Netherlands, 2002-2012.

In 2002, the National Legionella Outbreak Detection Program was implemented in the Netherlands to detect and eliminate potential sources of organisms that cause Legionnaires' disease (LD). During 2002-2012, a total of 1,991 patients with LD were reported, and 1,484 source investigations were performed. Of those sources investigated, 24.7% were positive for Legionella spp. For 266 patients with LD, 105 cluster locations were identified. A genotype match was made between a strain detected in 41 patients and a strain from a source location. Despite the systematic approach used by the program, most sources of LD infections during 2002-2012 remained undiscovered. Explorative studies are needed to identify yet undiscovered reservoirs and transmission routes for Legionella bacteria, and improved laboratory techniques are needed to detect Legionella spp. in clinical samples with a high background of microbial flora (such as soil).

Confirmed and Potential Sources of Legionella Reviewed.

Legionella bacteria are ubiquitous in natural matrices and man-made systems. However, it is not always clear if these reservoirs can act as source of infection resulting in cases of Legionnaires' disease. This review provides an overview of reservoirs of Legionella reported in the literature, other than drinking water distribution systems. Levels of evidence were developed to discriminate between potential and confirmed sources of Legionella. A total of 17 systems and matrices could be classified as confirmed sources of Legionella. Many other man-made systems or natural matrices were not classified as a confirmed source, since either no patients were linked to these reservoirs or the supporting evidence was weak. However, these systems or matrices could play an important role in the transmission of infectious Legionella bacteria; they might not yet be considered in source investigations, resulting in an underestimation of their importance. To optimize source investigations it is important to have knowledge about all the (potential) sources of Legionella. Further research is needed to unravel what the contribution is of each confirmed source, and possibly also potential sources, to the LD disease burden.

Soil as a source of Legionella pneumophila sequence type 47.

Legionella pneumophila sequence type (ST) 47 was isolated from soil in a garden. We speculate that this strain was transmitted from soil to the whirlpool in the garden where it caused an outbreak of Legionnaires' disease and Pontiac fever. In the Netherlands, ST47 is frequently isolated from patients, but hardly ever from environmental sources. It is possible that human pathogenic Legionella strains, with ST47 as one of the predominant strains, are transmitted to humans from sources such as natural soil that are currently not targeted in outbreak investigations.

Polyclonal spread and outbreaks with ESBL positive gentamicin resistant Klebsiella spp. in the region Kennemerland, The Netherlands.

OBJECTIVE: The objective of this study was to analyze the transmission dynamics of ESBL positive Klebsiella spp. with an additional resistance towards gentamicin (ESBL-G) in a Dutch region of 650,000 inhabitants in 2012. METHODS: All patient related ESBL-G isolates isolated in 2012 were genotyped using both Amplification Fragment Length Polymorphism (AFLP) and High-throughput MultiLocus Sequence Typing (HiMLST). HiMLST was used to analyze the presence of (unidentified) clusters of ESBL-G positive patients. Furthermore, all consecutive ESBL-G isolates within patients were studied in order to evaluate the intra-patient variation of antibiotic phenotypes. RESULTS: There were 38 ESBL-G isolates, which were classified into 18 different sequence types (STs) and into 21 different AFLP types. Within the STs, four clusters were detected from which two were unknown resulting in a transmission index of 0.27. An analysis of consecutive ESBL-G isolates (with similar STs) within patients showed that for 68.8% of the patients at least one isolate had a different consecutive antibiotic phenotype. CONCLUSION: The transmission of ESBL-G in the region Kennemerland in 2012 was polyclonal with several outbreaks (with a high level of epidemiological linkage). Furthermore, clustering by antibiotic phenotype characterization seems to be an inadequate approach in this setting. The routine practice of molecular typing of collected ESBL-G isolates may help to detect transmission in an early stage, which opens the possibility of a rapid response.

Legionnaires' disease after using an industrial pressure test pump: a case report.

INTRODUCTION: Legionnaires' disease is an acute pneumonia caused by inhalation or aspiration of aerosols contaminated with Legionella bacteria. The majority (>90%) of Legionnaires' disease cases are caused by the species Legionella pneumophila, and about 85% more specifically by L. pneumophila serogroup 1 that can be detected by a fast and easy to perform urinary antigen test. Previously reported sources of infection include cooling towers, plumbing systems of hospitals, and whirlpool spas, but for the majority of cases of Legionnaires' disease the source of infection remains unknown. CASE PRESENTATION: A 52-year-old Caucasian man was admitted to a Dutch hospital with pneumonia, where a culture of the available bronchial lavage was found positive for L. pneumophila serogroup 3, confirming the diagnosis of Legionnaires' disease. An environmental investigation identified a manually operated pressure test pump at the metal processing company where he worked as the source of infection: the water sample from the pump contained 9·8×103 colony forming units/L L. pneumophila, and sequence-based typing showed the same sequence type (ST93) for both the clinical and environmental strains. CONCLUSION: This case shows that Legionnaires' disease can be acquired by exposure to relatively rare sources that are not considered in regular control and prevention measures.

Prediction of the origin of French Legionella pneumophila strains using a mixed-genome microarray.

BACKGROUND: Legionella is a water and soil bacterium that can infect humans, causing a pneumonia known as Legionnaires' disease. The pneumonia is almost exclusively caused by the species L. pneumophila, of which serogroup 1 is responsible for 90% of patients. Within serogroup 1, large differences in prevalence in clinical isolates have been described. A recent study, using a Dutch Legionella strain collection, identified five virulence associated markers. In our study, we verify whether these five Dutch markers can predict the patient or environmental origin of a French Legionella strain collection. In addition, we identify new potential virulence markers and verify whether these can predict better. A total of 219 French patient isolates and environmental strains were compared using a mixed-genome micro-array. The micro-array data were analysed to identify predictive markers, using a Random Forest algorithm combined with a logistic regression model. The sequences of the identified markers were compared with eleven known Legionella genomes, using BlastN and BlastX; the functionality for each of the predictive markers was checked in the literature. RESULTS: The five Dutch markers insufficiently predicted the patient or environmental origin of the French Legionella strains. Subsequent analyses identified four predictive markers for the French collection that were used for the logistic regression model. This model showed a negative predictive value of 91%. Three of the French markers differed from the Dutch markers, one showed considerable overlap and was found in one of the Legionella genomes (Lorraine strain). This marker encodes for a structural toxin protein RtxA, described for L. pneumophila as a factor involved in virulence and entry in both human cells and amoebae. CONCLUSIONS: The combination of a mixed-genome micro-array and statistical analysis using a Random Forest algorithm has identified virulence markers in a consistent way. The Lorraine strain and related Dutch and French Legionella strains contain a marker that encodes a RtxA protein which probably is involved in the increased prevalence in clinical isolates. The current set of predictive markers is insufficient to justify its use as a reliable test in the public health field in France. Our results suggest that genetic differences in Legionella strains exist between geographically distinct entities. It may be necessary to develop region-specific mixed-genome microarrays that are constantly adapted and updated.

Correlation of MIC value and disk inhibition zone diameters in clinical Legionella pneumophila serogroup 1 isolates.

OBJECTIVES: Routine use of disk diffusion tests for detecting antibiotic resistance in Legionella pneumophila has not been described. The goal of this study was to determine the correlation of MIC values and inhibition zone diameter (MDcorr) in clinical L. pneumophila isolates. METHODS: Inhibition zone diameter of 183 L. pneumophila clinical isolates were determined for ten antimicrobials. Disk diffusion results were correlated with MICs as determined earlier with E-tests. RESULTS: Overall the correlation of MIC values and inhibition zone diameters (MDcorr) of the tested antimicrobials is good, and all antimicrobials showed a WT distribution. Of the tested fluoroquinolones levofloxacin showed the best MDcorr. All macrolides showed a wide MIC distribution and good MDcorr. The MDcorr for cefotaxim, doxycycline and tigecycline was good, while for rifampicin and moxifloxacin, they were not. CONCLUSION: Overall good correlation between MIC value and disk inhibition zone were found for the fluoroquinolones, macrolides and cefotaxim.