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Objectives: Carbapenem resistance mediated by mobile genetic elements has emerged worldwide and has become a major public health threat. To gain insight into the molecular epidemiology of carbapenem resistance in The Netherlands, Dutch medical microbiology laboratories are requested to submit suspected carbapenemase-producing Enterobacterales (CPE) to the National Institute for Public Health and the Environment as part of a national surveillance system. Methods: Meropenem MICs and species identification were confirmed by E-test and MALDI-TOF and carbapenemase production was assessed by the Carbapenem Inactivation Method. Of all submitted CPE, one species/carbapenemase gene combination per person per year was subjected to next-generation sequencing (NGS). Results: In total, 1838 unique isolates were received between 2014 and 2018, of which 892 were unique CPE isolates with NGS data available. The predominant CPE species were Klebsiella pneumoniae (n = 388, 43%), Escherichia coli (n = 264, 30%) and Enterobacter cloacae complex (n = 116, 13%). Various carbapenemase alleles of the same carbapenemase gene resulted in different susceptibilities to meropenem and this effect varied between species. Analyses of NGS data showed variation of prevalence of carbapenemase alleles over time with blaOXA-48 being predominant (38%, 336/892), followed by blaNDM-1 (16%, 145/892). For the first time in the Netherlands, blaOXA-181, blaOXA-232 and blaVIM-4 were detected. The genetic background of K. pneumoniae and E. coli isolates was highly diverse. Conclusions: The CPE population in the Netherlands is diverse, suggesting multiple introductions. The predominant carbapenemase alleles are blaOXA-48 and blaNDM-1. There was a clear association between species, carbapenemase allele and susceptibility to meropenem.

Objectives: Since the introduction of the 13-lactam/13-lactamase inhibitor ceftazidime-avibactam (CZA), rapid evolution of resistance has been reported in different KPC-producing Klebsiella pneumoniae isolates. In this multicenter retrospective study, we describe the emergence of CZA resistance and evaluate the mutations that might be responsible for the restoration of carbapenem susceptibility. Methods: During a study period of 18 months, KPC-producing K. pneumoniae isolates of five hospitalized patients were collected with phenotypic development of CZA resistance. Results: In vitro restoration of carbapenem susceptibility during treatment was observed in 3 isolates. Whole genome sequencing of these isolates showed a D179Y mutation in the KPC gene of 2 variants and a KPC-2 with a A242-GT-243 deletion (KPC-14). Two KPC-3 variants showed CZA resistance with sustained carbapenemase activity without genomic adaptations in the KPC gene. Conclusions: This study confirms the emergence of CZA resistance in KPC K. pneumoniae. The role of carbapenems in treating patients with these variants is unclear and combination therapies warrant further investigation.(c) 2021 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Objective A cornerstone in the management of Staphylococcus aureus bacteraemia (SAB) is the differentiation between a complicated and an uncomplicated SAB course. The ability to early and accurately identify patients with ‐ and without ‐ complicated bacteraemia may optimise the utility of diagnostics and prevent unnecessary prolonged antibiotic therapy. Methods Development and validation of a prediction score in SAB using demographic, clinical, and laboratory data from two independent Dutch cohorts; estimating the risk of complicated disease at the time of the first positive blood culture. Models were developed using logistic regression and evaluated by c‐statistics, ie area under the ROC‐curve, and negative predictive values (NPV). Results The development‐ and validation cohorts included 150 and 183 patients, respectively. The most optimal prediction model included: mean arterial pressure, signs of metastatic infection on physical examination, leucocyte count, urea level and time to positivity of blood cultures (c‐statistic 0.82, 95% CI 0.74‐0.89). In the validation cohort, the c‐statistic of the prediction score was 0,77 (95% CI 0.69‐0.84). The NPV for complicated disease for patients with a score of ≤2 was 0.83 (95% CI 0.68‐0.92), with a negative likelihood ratio of 0.14 (95% CI 0.06‐0.31). Conclusion The early SAB risk score helps to identify patients with high probability of uncomplicated SAB. However, the risk score's lacked absolute discriminative power to guide decisions on the management of all patients with SAB on its own. The heterogenicity of the disease and inconsistency in definitions of complicated SAB are important challenges in the development of clinical rules to guide the management of SAB.

Background: Timely empiric antimicrobial therapy is one of the cornerstones of the management of suspected bloodstream infection (BSI). However, studies about the effects of empiric therapy on mortality have reported inconsistent results. The objective of this study was to estimate the effect of delay of appropriate empiric therapy on early mortality in patients with BSI. Methods: Data for the propensity score matching (PSM) study were obtained from a cohort of patients with BSI. Inadequate empiric treatment was defined as in vitro resistance to the antimicrobial regimen administered &lt, 6 h after blood cultures were taken. The primary outcome measure was 14-day mortality. Thirty-day mortality and median length of stay (LOS) were secondary outcomes. PSM was applied to control for confounding. Results: Of a total of 893 included patients with BSI, 35.7% received inadequate initial empiric treatment. In the PSM cohort (n = 334), 14-day mortality was 9.6% for inadequate antibiotic treatment, compared to. 10.2% in adequate empiric treatment (p = 0.85). No prolonged median LOS was observed in patients who initially received inadequate therapy (10.5 vs. 10.7 days, p = 0.89). Conclusions: In this study, we found no clear effect of inadequate empirical treatment on mortality in a low-risk BSI population. The importance of early empiric therapy compared to other determinants, may be limited. This may not apply for specific subpopulations, e.g., patients with sepsis.

Antibiotic nonsusceptibility was consistently associated with higher risks of recurrent bacteremia, but the estimated number of additional recurrent episodes in the Netherlands (40 per year) was rather limited., Background Direct health effects of antibiotic resistance are difficult to assess. We quantified the risk of recurrent bacteremia associated with resistance. Methods We extracted antimicrobial susceptibility testing data on blood isolates from the Dutch surveillance system for antimicrobial resistance between 2008 and 2017. First and first recurrent (4–30 days) bacteremia episodes were categorized as susceptible, single nonsusceptible, or co-nonsusceptible to third-generation cephalosporins without or with carbapenems (Enterobacteriaceae), ceftazidime without or with carbapenems (Pseudomonas species), aminopenicillins without or with vancomycin (Enterococcus species), or as methicillin-sensitive/-resistant S. aureus (MSSA/MRSA). We calculated risks of recurrent bacteremia after nonsusceptible vs susceptible first bacteremia, estimated the crude population attributable effect of resistance for the Netherlands, and calculated risks of nonsusceptible recurrent bacteremia after a susceptible first episode. Results Risk ratios for recurrent bacteremia after a single- and co-nonsusceptible first episode, respectively, vs susceptible first episode, were 1.7 (95% confidence interval [CI], 1.5–2.0) and 5.2 (95% CI, 2.1–12.4) for Enterobacteriaceae, 1.3 (95% CI, 0.5–3.1) and 5.0 (95% CI, 2.9–8.5) for Pseudomonas species, 1.4 (95% CI, 1.2–1.7) and 1.6 (95% CI, 0.6–4.2) for Enterococcus species, and 1.6 (95% CI, 1.1–2.4) for MRSA vs MSSA. The estimated population annual number of recurrent bacteremias associated with nonsusceptibility was 40. The risk of nonsusceptible recurrent bacteremia after a susceptible first episode was at most 0.4% (Pseudomonas species). Conclusions Although antibiotic nonsusceptibility was consistently associated with higher risks of recurrent bacteremia, the estimated annual number of additional recurrent episodes in the Netherlands (40) was rather limited.

The course of both the bacterial species and load and the incidence of infection during negative pressure wound therapy (NPWT) are unclear, with published studies presenting contradicting results.The aim of the study is to assess the changes in both bacterial species and load, as well as the incidence of infection, before and after NPWT in a patient population with a variety of wounds.Surgical patients 18 years of age or older who needed NPWT were included in this multicenter, prospective cohort study. A wound swab culture was taken before NPWT and either immediately following NPWT or 6 weeks of follow-up. The change of bacterial species, bacterial load, and rate of infection were determined before and after the start of NPWT.In total, 104 patients were analyzed. The number of positive cultures increased from pre- to post-NPWT. The most cultured pathogenic bacterium was Staphylococcus aureus. The bacterial load was moderately higher at the end of NPWT than at the start (P ⟨ .0001). It was noted that 2 swabs contained multidrug-resistant bacteria, 1 pre-NPWT and 1 post-NPWT. Prior to NPWT, 26 patients had a wound infection, 5 of which had a persisting infection at the end of the study. Post-NPWT, 14 patients developed a wound infection.The number of S aureus strains and overall bacterial load increased during NPWT, and the incidence of infection remained the same. Further studies should be conducted to determine whether the increase in bacterial load influences other wound outcome parameters.

We prospectively compared the performance of RESIST-5 O.O.K.N.V. and NG-Test Carba 5 assays directly from blood cultures spiked with 130 characterized Enterobacterales isolates. Overall, both assays yielded 100% sensitivity to detect KPC-type carbapenemases and OXA-48-like carbapenemases. Both assays failed to detect KPC-31 and KPC-33, D179Y point mutation variants of KPC-3 and KPC-2, that are deprived of carbapenemase activity and confer resistance to ceftazidime-avibactam. On blood culture bacterial pellets, NDMand VIM-type carbapenemases were detected in 50.0% and 52.2%, respectively, by RESIST5 O.O.K.N.V. vs 100% by NG-Test Carba 5. The sensitivity of RESIST-5 O.O.K.N.V. improved to 100% and 95.6%, respectively, by performing the assay on 4-h early subculture. (C) 2020 Published by Elsevier Ltd on behalf of The Healthcare Infection Society.

Background Bacteria carry a wide array of genes, some of which have multiple alleles. These different alleles are often responsible for distinct types of virulence and can determine the classification at the subspecies levels (e.g., housekeeping genes for Multi Locus Sequence Typing, MLST). Therefore, it is important to rapidly detect not only the gene of interest, but also the relevant allele. Current sequencing-based methods are limited to mapping reads to each of the known allele reference, which is a time-consuming procedure. Results To address this limitation, we developed BacTag - a pipeline that rapidly and accurately detects which genes are present in a sequencing dataset and reports the allele of each of the identified genes. We exploit the fact that different alleles of the same gene have a high similarity. Instead of mapping the reads to each of the allele reference sequences, we preprocess the database prior to the analysis, which makes the subsequent gene and allele identification efficient. During the preprocessing, we determine a representative reference sequence for each gene and store the differences between all alleles and this chosen reference. Throughout the analysis we estimate whether the gene is present in the sequencing data by mapping the reads to this reference sequence; if the gene is found, we compare the variants to those in the preprocessed database. This allows to detect which specific allele is present in the sequencing data. Our pipeline was successfully tested on artificial WGS E. coli, S. pseudintermedius, P. gingivalis, M. bovis, Borrelia spp. and Streptomyces spp. data and real WGS E. coli and K. pneumoniae data in order to report alleles of MLST house-keeping genes. Conclusions We developed a new pipeline for fast and accurate gene and allele recognition based on database preprocessing and parallel computing and performed better or comparable to the current popular tools. We believe that our approach can be useful for a wide range of projects, including bacterial subspecies classification, clinical diagnostics of bacterial infections, and epidemiological studies. Electronic supplementary material The online version of this article (10.1186/s12864-019-5723-0) contains supplementary material, which is available to authorized users.

Background Selective decontamination of the digestive tract (SDD) and selective oropharyngeal decontamination (SOD) reduce colonization with antibiotic-resistant Gram-negative bacteria (ARGNB), incidence of nosocomial infections and improve survival in ICU patients. The effect on bacterial gut colonization might be caused by growth suppression by antibiotics during SDD/SOD. We investigated intestinal colonization with ARGNB after discharge from ICU and discontinuation of SDD or SOD. Methods We performed a prospective, observational follow-up study in regular hospital wards of three teaching hospitals in the Netherlands in patients discharged from the ICU, who were participating in a cluster randomized trial comparing SDD with SOD. We determined rectal carriage with ARGNB at ICU discharge (time (T) = 0) and 3, 6 and 10 days after discharge. The primary endpoint was time to first colonization with ARGNB that was not present at T = 0. Bacteria that are intrinsically resistant to antibiotics were not included in the primary analysis, but were included in post-hoc analysis. Results Of 1370 patients screened for inclusion, 996 patients had samples at T = 0 (507 after SDD and 489 after SOD). At ICU discharge, the prevalence of intestinal carriage with any ARGNB was 22/507 (4.3%) after SDD and 87/489 (17.8%) after SOD (p

OBJECTIVES Blood cultures (BCs) are essential in the evaluation of neutropenic fever. Modern BC systems have significantly reduced the time-to-positivity (TTP) of BC. This study explores the probability of bacteraemia when BCs have remained negative for different periods of time. METHODS All adult patients with neutropenia and bacteraemia were included (January 2012-February 2016). Predictive clinical factors for short (≤16 hours) and long (>24 hours) TTP were determined. The residual probability of bacteraemia was estimated for the scenario of negative BC 24 hours after collection. RESULTS The cohort consisted of 154 patients, accounting for 190 episodes of bacteraemia. Median age of 61 years, 60.5% were male. In 123 (64.7%) episodes, BC yielded a single Gram-positive micro-organism and in 49 (25.8%) a Gram-negative micro-organism (median TTP 16.7, 14.5 hours respectively, P

In this paper we aim to provide insight into the complexity of outbreak management in an intensive care unit (ICU) setting. In October 2010 four patients on the ICU of our tertiary care centre were colonized or infected with a multidrug-resistant strain of Pseudomonas aeruginosa (MDR-PA). An outbreak investigation was carried out and infection control measures were taken in an attempt to identify a potential source and stop transmission. The outbreak investigation included descriptive epidemiology, comprising retrospective case finding by reviewing the laboratory information system back to 2004 and prospective case finding by patient screening for MDR-PA. Furthermore, microbiological analysis, environmental screening and a case-control study were carried out. Infection control measures consisted of re-education of healthcare personnel on basic hygiene measures, auditing of hygiene procedures used in daily practice by infection control practitioners, and stepwise up-regulation of isolation measures. From February 2009 to January 2012, 44 patients on our ICU were found to be MDR-PA positive. MDR-PA isolates of the 44 patients showed two distinct AFLP patterns, with homology within each of the AFLP clusters of more than 93%. The VIM metallo-β-lactamasegene was detected in 20 of 21 tested isolates. A descriptive epidemiology investigation identified the rooms with the highest numbers of MDR-PA positive patients. The case-control study showed three factors to be independently associated with MDR-PA positivity: admission to ICU subunit 1 (OR, 6.1; 95% CI, 1.7,22), surgery prior to or during admission (OR, 5.7; 95% CI, 1.6,20) and being warmed-up with the warm-air blanket (OR, 3.6; 95% CI, 1.2, 11). After three environmental screening rounds, with sampling of sinks, furniture and devices in the ICU, without revealing a clear common source, a fourth environmental investigation included culturing of faucet aerators. Two faucets were found to be positive for MDR-PA and were replaced. The occurrence of new cases decreased with the strengthening of infection control measures and declined further with the removal of the common source. With this integrated approach a prolonged outbreak of P. aeruginosa was controlled. Contaminated faucet aerators on the ICU probably served as a persisting source, while interpatient transmission by medical staff was a likely way of spread. Seven months after the last case (January 2012) and 3 months after cessation of extended isolation measures (May 2012), single cases started to occur on the ICU, with a total of seven patients in the past year. No common source has yet been found.

OBJECTIVES To characterize the mechanisms of fluoroquinolone and cephalosporin resistance in Enterobacteriaceae from a Dutch teaching hospital in 2008. METHODS We sequenced gyrA, gyrB, parC and parE. The presence of plasmid-encoded genes qnrA, qnrB, qnrS, aac(6')-Ib, qepA, bla(TEM), bla(SHV,) bla(OXA), bla(CTX-M) and bla(AmpC) was studied by PCR. Escherichia coli isolates were further characterized by AFLP and multilocus sequence typing (MLST). RESULTS In total, 49 E. coli, 16 Klebsiella pneumoniae and 3 Enterobacter cloacae isolates were investigated. Mutations in gyrA were found in all E. coli isolates. Forty-five (92%) E. coli isolates carried at least one point mutation in parC. Most E. coli isolates (59%) also carried mutations in parE, of which I529L was the most prevalent. I529L was unequivocally associated with E. coli sequence type (ST) 131. This single-nucleotide polymorphism (SNP) was later also found in eight out of nine ST131 strains from another collection. Twenty-nine E. coli isolates carried extended-spectrum β-lactamase (ESBL) genes, predominantly bla(CTX-M-15). In E. coli, aac(6')-Ib-cr was the predominant plasmid-mediated resistance mechanism, whereas in K. pneumoniae qnr genes were found mostly. In K. pneumoniae isolates, qnr and aac(6')-Ib-cr co-occurred with ESBL genes (n = 13; bla(CTX-M) and bla(SHV)) and/or bla(AmpC) (n = 3; bla(DHA-1)). CONCLUSIONS E. coli ST131 was the predominant clone, which accumulated a high number of chromosomal mutations. The I529L SNP in parE was a signature of most, but not all, ST131 strains. In contrast to E. coli, fluoroquinolone resistance mechanisms were predominantly plasmid-encoded in K. pneumoniae.

The identification and detection of mitis group streptococci, which contain Streptococcus pneumoniae , have been hampered by the lack of sensitive and specific assays. In this study, we evaluated several biochemical and molecular assays for the identification of S. pneumoniae and Streptococcus pseudopneumoniae and their distinction from other mitis group streptococci using a collection of 54 isolates obtained by the routine culturing of 53 respiratory specimens from patients with community-acquired pneumonia. The combined results of the biochemical and molecular assays indicated the presence of 23 S. pneumoniae , 2 S. pseudopneumoniae , and 29 other mitis group streptococcal isolates. The tube bile solubility test that is considered gold standard for the identification of S. pneumoniae showed concordant results with optochin susceptibility testing (CO 2 atmosphere) and a real-time multiplex PCR assay targeting the Spn9802 fragment and the autolysin gene. Optochin susceptibility testing upon incubation in an O 2 atmosphere, bile solubility testing by oxgall disk, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and sequence analysis of the tuf and rpoB genes resulted in several false-positive, false-negative, or inconclusive results. The S. pseudopneumoniae isolates could be identified only by molecular assays, and the multiplex real-time PCR assay was concluded to be most convenient for the identification of S. pneumoniae and S. pseudopneumoniae isolates. Using this method, S. pneumoniae and S. pseudopneumoniae DNA could be detected in the respiratory samples from which they were isolated and in an additional 11 samples from which only other streptococci were isolated.

BackgroundBlood cultures are considered the gold standard to distinguish bacteremia from non-bacteremic systemic inflammation. In current clinical practice, bacteraemia is considered unlikely if blood cultures have been negative for 48-72 hours. Modern BC systems have reduced this time-to-positivity (TTP), questioning whether the time frame of 48-72 hrs is still valid. This study investigates the distribution of TTP, the probability of blood culture positivity after 24 hours, and identifies clinical predictors of prolonged TTP.MethodsAdult patients with monomicrobial bacteremia in an academic hospital were included retrospectively over a three-year period. Clinical data were retrieved from the medical records. Predictors of TTP >24 hours were determined by uni- and multivariate analyses. The residual probability of bacteremia was estimated for the scenario of negative BCs at 24 hours after bedside collection.ResultsThe cohort consisted of 801 patients, accounting for 897 episodes of bacteremia. Mean age was 65 years (IQR 54-73), 534 (59.5%) patients were male. Median TTP was 15.7 (IQR 13.5-19.3) hours. TTP was ≤24 hours in 85.3% of episodes. Antibiotic pre-treatment (adjusted OR 1.77; 95%CI 1.14-2.74, pConclusionWith adequate hospital logistics, the probability of positive blood cultures after 24 hours of negative cultures was low. Combined with clinical reassessment, knowledge of this low probability may contribute to prioritization of the differential diagnosis and decisions on antimicrobial therapy. As a potential antibiotic stewardship tool, this strategy warrants further prospective investigation.

The prevalence of the currently known Acinetobacter species and related trends of antimicrobial resistance in a Dutch university hospital were studied. Between 1999 and 2006, Acinetobacter isolates from clinical samples were collected prospectively. Isolates were analyzed by amplified fragment length polymorphism fingerprinting. For species identification, a profile similarity cutoff level of 50% was used, and for strain identification, a cutoff level of 90% was used. Susceptibility for antimicrobial agents was tested by disk diffusion by following the CLSI guideline. The incidences of Acinetobacter isolates ranged from 1.7 to 3.7 per 10,000 patients per year, without a trend of increase, during the study years. Twenty different species were distinguished. Acinetobacter baumannii (27%) and Acinetobacter genomic species (gen. sp.) 3 (26%) were the most prevalent. Other species seen relatively frequently were Acinetobacter lwoffii (11%), Acinetobacter ursingii (4%), Acinetobacter johnsonii (4%), and Acinetobacter junii (3%). One large cluster of A. baumannii , involving 31 patients, and 16 smaller clusters of various species, involving in total 39 patients, with at most 5 patients in 1 cluster, occurred. Overall, 37% of the A. baumannii isolates were fully susceptible to the tested antibiotics. There was a borderline significant ( P = 0.059) trend of decreasing susceptibility. A. baumannii was the Acinetobacter species causing the largest burden of multiple-antibiotic resistance and transmissions in the hospital.

A nested case-control study of 75 patients with cardiac device infections (CDI) and 75 matched controls was conducted to evaluate time course, risk factors, culture results and frequency of CDI.CDI occurred in 75/3410 (2.2%) device implantation and revision procedures, performed between 2000 and 2007. The time delay between device procedure and infection ranged from 0 to 64 months (mean 14 (SD 16)), 21 patients (28%) had an early infection (1 month), 26 (35%) a late infection (1-12 months) and 28 (37%) a delayed infection (12 months). Of interest, 18 (24%) patients presented with an infection24 months after the device-related procedure. Time delay until infection was significantly shorter when cultures were positive for micro-organisms compared to negative cultures (8 (12) vs 18 (18) months, p = 0.03). Pocket cultures in delayed infections remained more often negative (61% vs 23%, p = 0.01). Independent CDI risk factors were: device revision (odds ratio (OR) 3.67; 95% confidence interval (CI), 1.51 to 8.96), renal dysfunction defined as glomerular filtration rate60 ml/min (OR 4.64; CI, 1.48 to 14.62) and oral anticoagulation use (OR 2.83; CI 1.20 to 6.68).CDI occurred in 2.2% of device procedures, with 24% occurring more than two years after the device-related procedure. Renal dysfunction, device revisions and oral anticoagulation are potent risk factors for CDI.

Biofilm formation in wounds and on biomaterials is increasingly recognized as a problem. It therefore is important to focus on new strategies for eradicating severe biofilm-associated infections. The beneficial effects of maggots (Lucilia sericata) in wounds have been known for centuries. We hypothesized sterile maggot excretions and secretions (ES) could prevent, inhibit, and break down biofilms of Pseudomonas aeruginosa (PAO1) on different biomaterials. Therefore, we investigated biofilm formation on polyethylene, titanium, and stainless steel. Furthermore, we compared the biofilm reduction capacity of Instar-1 and Instar-3 maggot ES and tested the temperature tolerance of ES. After biofilms formed in M63 nutrient medium on comb-forming models of the biomaterials, ES solutions in phosphate-buffered saline or M63 were added in different concentrations. PAO1 biofilms adhered tightly to polyethylene and titanium but weakly to stainless steel. Maggot ES prevent and inhibit PAO1 biofilm formation and even break down existing biofilms. ES still had considerable biofilm reduction properties after storage at room temperature for 1 month. ES from Instar-3 maggots were more effective than ES from Instar-1 maggots. These results may be relevant to patient care as biofilms complicate the treatment of infections associated with orthopaedic implants.

Summary Objective A four-fold increase in the incidence of Serratia marcescens occurred in a cardio-thoracic ICU within a 13-month period. Clinical, epidemiological and molecular characteristics were analysed to elucidate the outbreak's origin. Methods Epidemiological data were analysed by mapping clustered cases; isolates were genotyped by AFLP analysis. A case–control study was performed to identify risk factors for the acquisition of S. marcescens . Data were obtained from files and electronic databases of the ICU and Department of Medical Microbiology. The adherence to hygiene protocols on the ICU was reviewed by a medical audit. Results Genotyping showed 16 distinct S. marcescens strains. Twenty-one cases and 39 controls were enrolled in the case–control study. Significant differences found by univariate analysis included the duration of surgery, APACHE-II-score on ICU admission, length of ICU stay, duration of mechanical ventilation, tube feeding and the sum of the number of days per invasive device. In a multivariate logistic regression model, the length of ICU stay and tube feeding were independent risk factors. Outbreak strains were not more frequently resistant to gentamicin, ciprofloxacin, meropenem or trimethoprim–sulfamethoxazole as compared to a reference group. Hygiene protocols, including hand washing, were insufficiently practiced by the ICU's medical staff. Conclusions The heterogeneity of the strains points to transmission from various sources. This outbreak of S. marcescens was most probably caused by reduced hand washing and other breaks in infection prevention protocols in combination with the presence of the identified risk factors, which act by affecting the number and intensity of potential transmission events.

An increase in the number of outbreaks of Acinetobacter infection was notified in The Netherlands during 1999–2001. The present study compared the outbreaks at the species and strain levels, and analysed the epidemiology and control measures at the different locations. For each institute, three representative isolates from three patients were identified to the species and strain levels by genotyping methods. A questionnaire investigated the impact of the outbreak, the control measures that were taken, and the possible effects of the measures. Seven outbreaks were associated with Acinetobacter baumannii (three outbreaks with a strain designated strain A, two outbreaks with a strain designated strain B, and one outbreak each with strains designated C and D). An additional outbreak was caused by genomic species 13TU, which is related closely to A. baumannii. Strains B and D were identified as European clones III and II, respectively. Except for two hospitals with outbreaks caused by strain A, there was no known epidemiological link between the participating hospitals. In all hospitals the outbreak occurred on one or several intensive care units, and spread to other departments was noted in two hospitals. The number of patients affected ranged from six to 66 over a period of 2–22 months. In most outbreaks, patients were the likely reservoir from which spread occurred. In all hospitals, a large panel of measures was required to bring the outbreak to an end. Extensive environmental sampling yielded numerous positive samples in most but not all hospitals.

The ancient method of larval therapy for treatment of acute and chronic infections has become a revival and a new dimension with introduction of the Biobag (Vitapad). With use of this therapy trauma patients suffering infectious complications can be treated very effective, which can reduce the overall time needed for treatment and can result in diminished invalidity.

In total, 226 individuals from the community were investigated for faecal carriage of Acinetobacter spp. by broth enrichment culture, followed by growth on blood agar and/or Leeds Acinetobacter Medium (LAM). Acinetobacter baumannii was isolated on both LAM and blood agar from one of 100 specimens in the UK and one of 126 specimens in The Netherlands. The predominant species were Acinetobactor johnsonii and genomic sp. 11, which were cultured from 22 and five specimens, respectively. A. baumannii did not seem to be widespread in the faecal flora of individuals in the community.

The recent literature is replete with reports on the occurrence of multiresistant epi-demic Acinetobacter baumannii in hospi-tals. Most reports are from Western Euro-pean countries, but publications from other parts of the world including South East Asia and South America are on the in-crease. Relatively little is known about the occurrence of multiresistant

To the Editors: Nontuberculous mycobacteria (NTM) are opportunistic pathogens that are widely present in our environment, i.e. in the soil and in natural and processed water. This group of organisms, with varying biological properties and clinical relevance, has gained notoriety over the past two decades due to their capability to cause severe disease in patients with immunodeficiency and/or chronic lung disease [1]. Despite their rising isolation frequency and growing clinical importance, NTM infections receive little scientific or public health attention. This partly results from the dogma that these NTM infections are not transmitted from male-to-male [1]. During our previous research of Mycobacterium malmoense infection in the Netherlands [2], we studied two patients with M. malmoense pulmonary infection with a strong epidemiological link. Human transmission was suspected because an acid-fast bacilli (AFB) smear-positive patient was in close contact with an immunocompromised patient. Herein, we present details of the cases and the results of the molecular diagnostics that were performed. The patients gave informed consent; ethical approval was not required for this retrospective study. A 38-yr-old male with a medical history of chronic obstructive pulmonary disease and spontaneous pneumothorax reported to the respiratory physician with progressive dyspnoea, productive cough, fever, night sweats, malaise and weight loss. A chest radiograph revealed infiltrates with cavities in the upper lobes. Laboratory diagnostics showed a raised erythrocyte sedimentation rate (90 mm·h−1) and leukocytosis (white blood cell count …

Between19991999 The Hospital Infection SocietyThe Hospital Infection SocietyJanuary 1996 and May 1997, a four-fold increased rate of isolation of Copyright Serratia marcescens was observed amongst patients admitted to the surgical Intensive Care Unit (SICU) of the Leiden University Medical Center compared to the preceding years. Random amplification of polymorphic DNA showed the involvement of genotypically distinct strains, implicating multiple different sources. After improvement of hygienic measures the frequency of isolation of S. marcescens returned to baseline. A case–control study was performed to assess patient-related risk factors for acquisition of S. marcescens . Nineteen cases and 38 controls were included. Hospital- and SICU-stay were significantly longer in case patients than in controls. By univariate analysis, statistically significant differences were found in body weight, the duration of mechanical ventilatory support, the cumulative use of antimicrobial agents, the use of aminoglycosides, parenteral nutrition and tube feeding. The sum of the number of days per invasive device (deep intravenous lines, arterial lines, wound drains and urinary catheters) was higher in cases than in controls ( P =0·08). Categorically, a cumulative number of device-days >25 was a statistically significant risk factor for acquisition of S. marcescens . Multivariable logistic regression analysis showed that body weight, parenteral feeding and mechanical ventilation were independent predictors of acquisition of S. marcescens . As transmission of S. marcescens appears to be by the hands of personnel, the identified risk factors may act by necessitating an increased frequency and intensity of direct contacts. The Hospital Infection Society.

Randomly amplified polymorphic DNA (RAPD), pulsed-field gelelectrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) analyses were used to investigate a possible outbreak of Nocardia farcinica . RAPD and PFGE analyses yielded irreproducible and unsatisfactory results, respectively. AFLP analysis seem to be a promising and welcome addition for molecular analysis of Nocardia isolates.

Resistance to ciprofloxacin in Escherichia coli is increasing parallel to increased use of fluoroquinolones both in The Netherlands and in other European countries. The objective was to investigate the contribution of active efflux and expression of outer membrane proteins (OMPs) in a collection of clinical E. coli isolates collected at a clinical microbiology department in a Dutch hospital. Forty-seven E. coli isolates a wide range of ciprofloxacin minimum inhibitory concentrations and known mutations in the quinolone resistance determining region were included. A fluorometric determination of bisbenzimide efflux was used two different efflux pump inhibitors and compared to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the expression levels of acrA, acrB, tolC, yhiV, and mdfA efflux pump genes and the OMPs ompF and ompX. Six isolates (12.7%) showed increased efflux. Although in 35 isolates (76%), overexpression of ≥1 efflux pump genes using qRT-PCR was present. Only the combined overexpression of acrAB-TolC and mdfA correlated with the phenotypic efflux assay using glucose/carbonyl cyanide m-chlorophenylhydrazone with glucose. Thus, efflux was involved in ciprofloxacin resistance in a limited number of E. coli isolates collected at a clinical microbiology department in a Dutch hospital complementing other resistance mechanisms.

Identification of Acinetobacter spp. to the DNA group level by phenotypic techniques is problematic, and there is a need for an alternative identification method for routine use. The present study validated the suitability of a rapid identification technique based on tRNA spacer (tDNA) fingerprinting in comparison with that of a commercially available assay involving carbon source utilization tests (Biolog MicroStation System) for identifying the 21 DNA-DNA hybridization groups belonging to the genus. For this purpose, 128 strains identified previously by DNA-DNA hybridization were analyzed by both techniques. tDNA fingerprinting was highly reproducible and classified all strains into 17 groups. Six DNA groups belonging to the A. calcoaceticus-A. baumannii complex were grouped into two distinct clusters, indicating the high degree of genetic similarity within this complex. Strains of the more recently described DNA groups BJ13 to BJ16 were ambiguously grouped and displayed three pattern types. The software used with the commercial carbon source utilization method grouped the 128 strains into 12 clusters, explaining the less discriminatory power of this system. We conclude that tDNA fingerprinting offers a quick and reliable method for the routine differentiation of most Acinetobacter spp. at the subgenus level.

A collection of 130 Acinetobacter strains identified by DNA hybridization to 18 different genomic species was used to assess the ability of the API 20NE system (bioMérieux, France) to identify Acinetobacter genomic species and to determine its accuracy. Fifty-eight (87%) of the 67 strains of genomic species defined in the database (version 5.1) were identified to the appropriate genomic species. The Acinetobacter baumannii strains and the Acinetobacter haemolyticus strains were all identified correctly. Three of five Acinetobacter junii strains, three of eight Acinetobacter johnsonii strains, and 11 of 13 Acinetobacter lwoffii strains were also identified correctly. The 58 correctly identified strains represented 45% of the total 130 strains. Thirty-six of the 72 inappropriately identified strains were designated Acinetobacter baumannii. Thirty-one of these 36 strains belonged to genomic species 1 (Acinetobacter calcoaceticus), 3, or 13TU. Analysis of the profiles showed that the API system does not discriminate between genomic species 1, 2, 3, and 13TU. Lumping of these groups into the Acinetobacter calcoaceticus-Acinetobacter baumannii complex in the API 20NE database would make the system considerably more accurate. Incorporation of these data into the database may improve identification of the remaining genomic species, including some that are not defined. However, the discriminative power of the tests in the API galleries is insufficient for correct identification of all Acinetobacter genomic species.

Two outbreaks of multidrug-resistant Acinetobacter baumannii occurred in our hospital. The outbreak strains were eventually isolated from respiratory ventilators, an apparatus used to cool or warm patients, and four continuous veno-venous hemofiltration machines. Removing dust from the machines and replacing all dust filters brought the outbreaks to an end.

Surmmary A collection of 129 Acinetobacter strains belonging to DNA groups (genomic species) 1–14 (1–7 and 10–12 sensu Bouvet and Grimont; 8 and 13–14 sensu Tjernberg and Ursing) were investigated for their ability to oxidise 95 carbon sources in the Biolog system. The strain groupings obtained by cluster analysis with the Biolog software were compared with the results of DNA-DNA hybridisation studies. Strains of DNA groups 1 (A. calcoaceticus), 2 (A. baumannii), 3 and 13 were linked in one cluster, as were DNA groups 4 (A. haemolyticus) and 6, DNA groups 10 and 11, and DNA groups 8 (A. lwoffii) and 12 (A. radioresistens). Strains of DNA group 5 (A. junii) were grouped in a single cluster with one strain of DNA group 4. Strains of DNA groups 7 (A. johnsonii) and 14 formed separate clusters. With the exception of the linkage of DNA groups 8 and 12, these results correlated with classification of reference strains of the DNA groups by DNA-DNA hybridisation, but six strains of four different DNA groups were not allocated to the clusters of their respective DNA groups. In the case of DNA groups-4, 5, 6, 7, 10, 11 and 14, at least one carbon source oxidation test could be used to differentiate them from the other DNA groups.

Item does not contain fulltext BACKGROUND: Previously, we assessed selective digestive tract decontamination (SDD) and selective oropharyngeal decontamination (SOD) on survival and prevention of bacteraemia in patients in intensive-care units. In this analysis, we aimed to assess effectiveness of these interventions for prevention of respiratory tract colonisation and bacteraemia with highly resistant microorganisms acquired in intensive-care units. METHODS: We did an open-label, clustered group-randomised, crossover study in 13 intensive-care units in the Netherlands between May, 2004, and July, 2006. Participants admitted to intensive-care units with an expected duration of mechanical ventilation of more than 48 h or an expected stay of more than 72 h received SOD (topical tobramycin, colistin, and amphotericin B in the oropharynx), SDD (SOD antibiotics in the oropharynx and stomach plus 4 days' intravenous cefotaxime), or standard care. The computer-randomised order of study regimens was applied by an independent clinical pharmacist who was masked to intensive-care-unit identity. We calculated crude odds ratios (95% CI) for rates of bacteraemia or respiratory tract colonisation with highly resistant microorganisms in patients who stayed in intensive-care units for more than 3 days (ie, acquired infection). This trial is registered at http://isrctn.org, number ISRCTN35176830. FINDINGS: Data were available for 5927 (>99%) of 5939 patients, of whom 5463 (92%) were in intensive-care units for more than 3 days. 239 (13%) of 1837 patients in standard care acquired bacteraemia after 3 days, compared with 158 (9%) of 1758 in SOD (odds ratio 0.66, 95% CI 0.53-0.82), and 124 (7%) of 1868 in SDD (0.48, 0.38-0.60). Eight patients acquired bacteraemia with highly resistant microorganisms during SDD, compared with 18 patients (with 19 episodes) during standard care (0.41, 0.18-0.94; rate reduction [RR] 59%, absolute risk reduction [ARR] 0.6%) and 20 during SOD (0.37, 0.16-0.85; RR 63%, ARR 0.7%). Of the patients staying in intensive-care units for more than 3 days, we obtained endotracheal aspirate cultures for 881 (49%) patients receiving standard care, 886 (50%) receiving SOD, and 828 (44%) receiving SDD. 128 (15%) patients acquired respiratory tract colonisation with highly resistant microorganisms during standard care, compared with 74 (8%) during SDD (0.58, 0.43-0.78; RR 38%, ARR 5.5%) and 88 (10%) during SOD (0.65, 0.49-0.87; RR 32%, ARR 4.6%). Acquired respiratory tract colonisation with Gram-negative bacteria or cefotaxime-resistant and colistin-resistant pathogens was lowest during SDD. INTERPRETATION: Widespread use of SDD and SOD in intensive-care units with low levels of antibiotic resistance is justified. FUNDING: None.

Purpose The maggots of Lucilia sericata are successfully used as a treatment for infected wounds. Many articles are published about possible direct antibacterial properties of maggots and their excretions/secretions (ES), but with different results. The present study reinvestigates the susceptibility of six bacterial strains to maggots and their ES. Methods Live maggots were added to a bacterial suspension. After incubation for 16h, the bacterial growth in this suspension was compared with the growth in a suspension without maggots. We tested Instar-1 and Instar-3 maggots and compared nutrient broths. A turbidimetric assay investigated the antibacterial activity of ES. Finally, we compared the bacterial growth of Gram-positive and Gram-negative bacteria. Results The test with live maggots showed an increase of bacterial growth. Instar-1 maggots stimulated more bacterial growth than Instar-3 maggots, as well as the use of a more nutritious broth. The turbidimetric assay showed no inhibition of bacterial growth. For all bacteria, except Pseudomonas aeruginosa , an increase in bacterial growth was shown. Conclusion There is no direct antibacterial effect of maggots and/or ES in vitro , however in clinical observations maggot therapy is successful. More research is needed to focus on possible indirect antibacterial activity, such as an immune-related effect.

In a retrospective, case-control cohort study an assessment was made of the clinical outcome of patients with osteomyelitis treated with a new modality of negative pressure wound therapy, so called negative pressure instillation therapy. In this approach, after surgical debridement, a site of osteomyelitis is treated with negative pressure of at least 300 mmHg applied through polyvinyl alcohol dressing. The polyvinyl alcohol foam is irrigated through the tubes three times a day with a polyhexanide antiseptic solution. In 30 patients (14 males; mean age 52 [range, 26-81]) admitted between 1999 and 2003 with osteomyelitis of the pelvis or lower extremity, we assessed time to wound closure, number of surgical procedures and rate of recurrence of infection as well as need for rehospitalizations. For comparison, a control group of 94 patients (males, 58; mean age 47 [range, 9-85]), matched for site and severity of osteomyelitis, was identified in hospital records between 1982 and 2002. These patients underwent standard surgical debridement, implantation of gentamicin polymethylmethacrylate beads and long-term intravenous antibiotics. In the Instillation group the rate of recurrence of infection was 3/30 (10%), whereas 55/93 (58.5%) of the controls had a recurrence (p

Can Escherichia coli be used as an indicator organism for transmission events in hospitals? Perineal and pharyngeal swabs were obtained from patients admitted to a medical or surgical intensive care unit within 24 h of admission and then twice per week. Escherichia coli isolates were typed by random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) typing. Based on the typing results, transmission rates for RAPD and AFLP typing were 8.5 and 6.6 per 100 patient-days. Requiring in addition to similarity in genotype parity in time and place for a transmission event, the incidence dropped to 3.8 (RAPD) and 1.7 (AFLP) per 100 patient-days. The two typing methods not only differed with respect to numbers of transmissions identified, but also to individuals involved in transmissions. This study identified a number of problems regarding the use of Escherichia coli as indicator organism for transmission events. The use of Escherichia coli for this purpose cannot be recommended at the moment.

Purpose In this report a case of Mycobacterium chelonae keratitis in a patient without any previously described risk factors is described. The only risk factor found was a rheumatoid arthritis related Sjögren's syndrome. Methods Case report. Results A 60-year-old woman was referred to the hospital with an infectious keratitis of the left eye of 3 months duration, unresponsive to empirical therapy with ofloxacin and tobramycin drops. Her medical history included a longstanding rheumatoid arthritis and a secondary ocular surface syndrome. Upon arrival the left eye showed diffuse corneal edema and centrally several large infiltrates with fluffy edges, surrounded by several smaller satellite infiltrates. The cornea was scraped for culture and grew M chelonae and sensitivity testing showed sensitivity to ciprofloxacin, clofazimine, and clarithromycin. Systemically ciprofloxacin 750 mg and clarithromycin 500 mg twice daily were prescribed orally. Topical therapy consisted of topical erythromycin 10 mg/mL and ofloxacin 3 mg/mL every 2 hours. Treatment was continued for a total of 10 months during which the infiltrates cleared completely, but the central cornea remained scarred. Conclusions M chelonae can be a cause of infectious keratitis in patients without known risk factors for rapidly growing mycobacterium keratitis. Especially in the case of ocular infections that show no response to regular antibacterial treatment, mycobacterial infection should be considered. Good communication between the ophthalmologist and the microbiologist is crucial for a rapid diagnosis.

Over a two-week period in November 2006, vancomycin-resistant Bacillus cereus was isolated from respiratory samples from six ventilated paediatric intensive care unit (PICU) patients. To investigate the possibility of a common source and extent of the dissemination, all procedures related to mechanical ventilation were monitored and surveillance cultures performed. B. cereus was isolated from reusable air-flow sensors, before and after on-site disinfection with 70% alcohol. The organism was also isolated from respiratory samples from three other ventilated patients and from two ventilation grids in the ceiling of PICU, as well as from the alcohol solution itself. Using amplified fragment length polymorphism (AFLP) typing, B. cereus strains from the six PICU patients together with isolates recovered from the air-flow sensors and the alcohol solution were shown to be closely related. Isolates from the ventilation grids demonstrated different AFLP patterns to the outbreak strain. Intervening measures, including disinfection by autoclaving all reusable air-flow-guiding parts and the use of disposable non-autoclavable parts, resulted in rapid termination of the outbreak. B. cereus infections can cause significant morbidity, particularly in intensive care patients. Disinfection of all air-flow-guiding reusable parts for mechanical ventilation should be addressed with great care and should include effective autoclaving in order to eradicate spores.

Ist die adjuvante Behandlung mit sterilen Larven (Lucilia Sericata) im Falle einer ernsthaften Infektion der Weichteile oder des Knochens (Osteomyelitis) sinnvoll? Zur Beantwortung dieser Frage wurde dazu ein Patientenkollektiv von 16 Patienten mit Fliegenlarven in Kombination mit chirurgischem Debridement behandelt.

Maggots were used as adjunct treatment for infected wounds that showed no response to the classical approach of wound debridement and antibiotic therapy. We summarize findings for 11 patients with necrotic wounds who received treatment with "surgical" maggots (100-2900 applied in 3-10 changes of dressing) for 11-34 days, which apparently aided in tissue remodeling and cure, and describe 2 typical patients in detail.

Septicaemia caused by Acinetobacter occurred in six infants in the neonatal unit. A total of 18 acinetobacters were isolated from blood cultures, cultures of intravascular catheters, and surveillance cultures. Twelve isolates from the six affected infants were identified as Acinetobacter junii by the use of a novel method, amplified ribosomal DNA restriction analysis (ARDRA). Typing of the organisms using the biochemical profiles of the API 20NE system, antibiogram typing, cell envelope protein electrophoresis, and PCR fingerprinting with two primer sets, ERIC1/ERIC2 and ERIC2/ 1026, showed that these 12 isolates were indistinguishable, whereas the remaining six isolates were different. The six infants recovered after therapy with ciprofloxacin alone in five cases and with a combination of ciprofloxacin and gentamicin in one case. This study showed that A. junii is capable of causing a serious, though non-fatal infection in neonates. The combined use of genotypic and phenotypic methods allowed the rapid separation of epidemic from non-epidemic isolates. It is concluded that for a better understanding of the role of the various Acinetobacter genomic species in human pathology, identification of acinetobacters according to the recent taxonomy is imperative.

AIMS--To investigate the diagnostic value of mucosal IgA and IgG Helicobacter pylori antibodies. METHODS--The study population comprised 209 consecutive patients with severe dyspeptic complaints referred for upper gastrointestinal endoscopy. A positive culture or histological identification of H pylori in gastric biopsy specimens, or both, were used to confirm infection. Specific IgA and IgG H pylori antibodies were determined using a modified ELISA technique. RESULTS--Of the 209 patients, 137 were infected with H pylori. The diagnostic value of systemic IgA and IgG H pylori antibodies was confirmed. Systemic IgA antibodies had a sensitivity of 76.6% (95% confidence interval 69.5-83.7) and a specificity of 94.4% (89.1-99.7). The sensitivity and specificity for systemic IgG antibodies were, respectively, 97.1% (94.3-99.9) and 98.6% (95.9-100). A moderate but clinically important correlation was found between local and systemic IgA and IgG. Mucosal IgA H pylori antibodies had a sensitivity of 98.5% (96.5-100) and a specificity of 91.7% (85.3-98.1), while for IgG these figures were, respectively, 88.3% (82.9-93.7) and 98.6% (95.9-100). As a diagnostic test mucosal IgA H pylori antibodies were comparable with culture and histology. CONCLUSION--Determination of local IgA and IgG H pylori antibody levels is a highly sensitive and specific test for the diagnosis of H pylori infection.

A set of 18 Acinetobacter baumannii isolates, collected prospectively in a Bulgarian hospital during episodes of increased A. baumannii occurrence during 2000–2002, was investigated for genotypic diversity and antibiotic susceptibility. Four genotypes were identified by amplified fragment length polymorphism genomic fingerprinting, one of which (type 1) accounted for 13 isolates, indicating that a specific strain was predominant. The single isolate allocated to type 2 was identified to European clone I. All isolates were resistant to multiple antibiotics, but most retained susceptibility to tobramycin and colistin, and all except one were susceptible to imipenem.