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Rationale: Selective digestive tract decontamination (SDD) and selective oropharyngeal decontamination (SOD) eradicate gram-negative bacteria (GNB) from the intestinal and respiratory tract in intensive care unit (ICU) patients, but their effect on antibiotic resistance remains controversial. Objectives: We quantified the effects of SDD and SOD on bacterial ecology in 13 ICUs that participated in a study, in which SDD, SOD, or standard care was used during consecutive periods of 6 months (de Smet AM, Kluytmans JA, Cooper BS, Mascini EM, Benus RF, van der Werf TS, van der Hoeven JG, Pickkers P, Bogaers-Hofman D, van der Meer NJ, et al. N Engl J Med 2009;360:20-31). Methods: Point prevalence surveys of rectal and respiratory samples were performed once monthly in all ICU patients (receiving or not receiving SOD/SDD). Effects of SDD on rectal, and of SDD/SOD on respiratory tract, carriage of GNB were determined by comparing results from consecutive point prevalence surveys during intervention (6 mo for SDD and 12 mo for SDD/SOD) with consecutive point prevalence data in the pre- and postintervention periods. Measurements and Main Results: During SDD, average proportions of patients with intestinal colonization with GNB resistant to either ceftazidime, tobramycin, or ciprofloxacin were 5, 7, and 7%, and increased to 15, 13, and 13% postintervention (P < 0.05). During SDD/SOD resistance levels in the respiratory tract were not more than 6% for all three antibiotics but increased gradually (for ceftazidime; P < 0.05 for trend) during intervention and to levels of 10% or more for all three antibiotics postintervention (P < 0.05). Conclusions: SOD and SDD have marked effects on the bacterial ecology in an ICU, with rising ceftazidime resistance prevalence rates in the respiratory tract during intervention and a considerable rebound effect of ceftazidime resistance in the intestinal tract after discontinuation of SDD. [ABSTRACT FROM AUTHOR]

Abnormal vaginal discharge may be caused by bacterial vaginosis, vulvovaginal candidiasis, trichomoniasis and/or aerobic vaginitis. For the development of a diagnostic algorithm, tree-based classification analysis was performed on symptoms, signs and bedside test results of 56 patients, and laboratory tests (culture, Nugent score, qPCRs) were compared. Amplicon sequencing of the 16S rRNA gene was used as reference test for bacterial vaginosis and aerobic vaginitis, culture for vulvovaginal candidiasis and qPCR for trichomoniasis. For bacterial vaginosis, the best diagnostic algorithm was to screen at the bedside with a pH and odour test and if positive, to confirm by qPCR (sensitivity 94%; specificity 97%) rather than Nugent score (sensitivity of 59%; specificity 97%; P = 0.031). The analysis for the other infections was less conclusive due to the low number of patients with these infections. For bacterial vaginosis, the developed algorithm is sensitive, specific, and reduces the need for laboratory tests in 50% of the patients., Competing Interests: Declaration of competing interest WQ is a shareholder of DDL Diagnostic Laboratory. The other authors declare that they have no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bacterial vaginosis (BV) is perceived as a condition of disrupted vaginal microbiota, but remains of unknown aetiology. In this study, vaginal microbiota composition was determined in twenty-one women with BV, before and after treatment with metronidazole or clindamycin. Microbiota composition varied greatly between women and defining a (un)healthy vaginal microbiota state remains elusive, challenging BV diagnosis and treatment. While relative abundance of Lactobacillus increased after antibiotic treatment in two-third of women, its abundance was not associated with treatment outcome. Instead, remaining complaints of abnormal vaginal discharge were more common after metronidazole treatment and associated with increased relative abundance of Ureaplasma.

Bacterial vaginosis (BV) is a common gynaecological condition. Diagnosis of BV is typically based on Amsel criteria, Nugent score and/or bacterial culture. In this study, these conventional methods and two CE-IVD marked quantitative real-time (q)PCR assays were compared with microbiota analysis for the diagnosis of BV. Eighty women were evaluated for BV during two sequential hospital visits by Amsel criteria, Nugent score, culture, the AmpliSens® Florocenosis/Bacterial vaginosis-FRT PCR kit (InterLabService, Moscow, Russia), and the BD MAX™ Vaginal Panel (BD Diagnostics, MD, USA). Microbiota analysis based on amplicon sequencing of the 16S ribosomal RNA gene was used as reference test. The microbiota profile of 36/115 (31%) included cases was associated with BV. Based on microbiota analysis, the sensitivity of detecting BV was 38.9% for culture, 61.15% for Amsel criteria, 63.9% for Nugent score and the BD MAX assay, and 80.6% for the AmpliSens assay, while the specificity of all methods was ≥ 92.4%. Microbiota profiles of the cases with discrepant results between microbiota analysis and the diagnostic methods were variable. All five diagnostic methods missed BV positive cases with a relatively high abundance of the genus Alloscardovia, Bifidobacterium, or Dialister, which were categorised as unspecified dysbiosis by the AmpliSens assay. Compared to Amsel criteria, Nugent score, culture, and the BD MAX assay, the AmpliSens assay was most in agreement with microbiota analysis, indicating that currently, the AmpliSens assay may be the best diagnostic method available to diagnose BV in a routine clinical setting.

In clinical practice, the diagnosis of lower respiratory tract infections (LRTIs) is based on culture. The aim of this study was to evaluate whether a stepwise approach using microbiota analysis, species-specific quantitative real-time (q)PCRs and culture has the potential to be a more accurate and efficient diagnostic approach than culture alone. Sixty-two sputa obtained in a routine clinical setting from patients with a suspected LRTI were included. All sputa were analysed by culture, microbiota analysis based on the 16S ribosomal RNA gene and multiple species-specific qPCRs. Microbiota and culture data were compared to investigate whether cut-off values for microbiota analysis could be determined. For microbiota analysis, a relative abundance of 25% was identified as the cut-off value for the detection of both genera Streptococcus and Haemophilus. Microbiota analysis combined with species-specific qPCRs resulted in a significant increase in the number of positive sputa (73% vs 58%; p = 0.003) as well as in the number of identified pathogens (51 vs 37; p = 0.049) compared to culture. A stepwise approach using microbiota analysis, species-specific qPCRs and culture has the potential to be used in clinical settings for the diagnosis of LRTIs in the near future.

Background: A high prevalence of colistin resistance among E. cloacae isolates in two intensive care units (ICU) (of 16 and 6 beds) using selective digestive decontamination (SDD) since 1990 instigated a retrospective and prospective investigation to quantify the role of clonal transmission. SDD is topical application of colistin and tobramycin and systemic use of cefotaxime during the first days of ICU-admission., Methods: Multi-resistant E. cloacae (MREb) was defined as ESBL production and/or tobramycin non-susceptibility and/or colistin non-susceptibility. Incidence of acquisition and prevalence of carriage with MREb was determined from microbiological culture results., Results: Colistin-resistant E. cloacae was first detected in November 2009 and carriage was demonstrated in 141 patients until October 2014. Mean incidence of MREb acquisition was 4.61 and 1.86 per 1000 days at risk in ICUs 1 and 2, respectively, and the mean monthly prevalence of MREb in both ICUs was 7.0 and 3.1%, respectively, without a discernible trend in time. Conversion rates from carriage of colistin-susceptible to resistant E. cloacae were 0.20 and 0.13 per 1000 patient days, respectively. Whole genome sequencing of 149 isolates revealed eight clusters, with the number of SNPs of the largest two clusters ranging between 0 and 116 for cluster 1 ( n  = 49 isolates), and 0 and 27 for cluster 2 ( n  = 36 isolates), among isolates derived between 2009 and 2014., Conclusions: This study demonstrates a stable low-level endemicity of MREb in two Dutch ICUs with prolonged use of SDD, which was characterized by the persistent presence of two clusters, suggesting incidental clonal transmission., Competing Interests: Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Objectives: Patients can acquire extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae during hospitalization, and colonized patients may transmit these bacteria after discharge, most likely to household contacts. In this study, ESBL transmission was quantified in households., Methods: Faecal samples were longitudinally collected from hospitalized patients colonized with ESBL-producing bacteria and from their household members during hospitalization of the index patient and at 3, 6, 12 and 18 months. A mathematical household model was developed, which allowed for person-to-person transmission, acquisition from other sources (background transmission), and losing carriage. Next, a deterministic population model with a household structure was created, informed by parameter values found in the household model., Results: In all, 74 index patients and 84 household members were included. In more than half of the household members ESBL-producing bacteria were demonstrated at some time during follow up. Person-to-person transmission occurred at a rate of 0.0053/colonized person/day (0.0025-0.011), background transmission at 0.00015/day (95% CI 0.00002-0.00039), and decolonization at 0.0026/day (0.0016-0.0040) for index patients and 0.0090/day (0.0046-0.018) for household members. The estimated probability of transmission from an index patient to a household contact was 67% and 37% vice versa., Conclusion: There is frequent transmission of ESBL-producing bacteria in households, which may contribute to the observed endemicity of ESBL carriage in the Netherlands. However, the population model suggests that there is not a single dominant acquisition route in the community., (Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Following the recognition of a measles case in a hospital in The Netherlands, health care workers (HCW) from the premises were screened by a commercial enzyme immunoassay (EIA) for measles IgG to identify persons at risk for measles. At least 10% of the HCW were tested measles IgG-negative. As this was considered an unusually high proportion, we hypothesized suboptimal sensitivity of EIAs, especially in medical personnel that had vaccine-induced immunity rather than antibodies resulting from natural infection., Objectives: To determine (vaccine-induced) measles immunity in HCW, using different EIAs compared to the plaque reduction neutralization (PRN) test, the best surrogate marker for vaccine efficacy and immune protection., Study Design: Sera from HCW were tested for measles IgG antibodies in three commercial EIAs, in a bead-based multiplex immunoassay (MIA) and in the PRN test, and evaluated against age and vaccination history of the HCW., Results: Of the 154 HCW, born between 1960 and 1995, 153 (99.4%) had protective levels of measles antibodies (PRN> 120mIU/ml). The three EIAs failed to detect any measles IgG antibodies in approximately 10% of the HCW, while this percentage was approximately 3% for the MIA. Negative IgG results rose to 19% for individuals born between 1975 and 1985, pointing to an age group largely representing vaccinated persons from the first measles vaccination period in The Netherlands., Conclusion: The results show limitations in the usefulness of current EIA assays for determining protective measles antibodies in persons with a vaccination history., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objectives: The Netherlands is known for a stringent search and destroy policy to prevent spread of MRSA. In the hospital setting, livestock-associated MRSA (LA-MRSA) is frequently found in patients coming from the high density farming area in the south of the Netherlands. The aim of the study was to determine the contribution of LA-MRSA in the epidemiology of MRSA in cases found following the Dutch search and destroy policy., Patients and Methods: From two hospitals serving a population of 550,000 persons all data on MRSA cultures and subsequent control measures from 2008 and 2009 were retrospectively collected and analyzed., Results: A total of 3856 potential index patients were screened for MRSA, 373 (9.7%) were found to be positive, 292 ( 78%) LA-MRSA and 81 (22%) non-LA-MRSA respectively. No secondary cases were found among contact research in persons exposed to LA-MRSA (0/416), whereas similar contact research for non-LA-MRSA resulted in 83 (2.5%) secondary cases. LA-MRSA were rarely found to cause infections., Conclusions: LA-MRSA is more prevalent than non-LA-MRSA in Dutch Hospitals in the South of the Netherlands. However, retrospectively studied cases show that the transmission rate for LA-MRSA was much lower than for non-LA-MRSA. This suggest that infection control practices for LA-MRSA may possibly be less stringent than for non-LA-MRSA.

Objective: Colonization of the respiratory tract with Gram-negative bacteria in intensive care patients increases the risk of subsequent infections. Application of systemic antibiotics may prevent colonization with Gram-negative bacteria, but this effect has never been quantified. The objective of this study was to determine associations between systemic antibiotic use and acquisition of respiratory tract colonization with Gram-negative bacteria in ICUs., Design: A nested cohort study., Setting: A university hospital and a teaching hospital., Patients: Patients with ICU stay of more than 48 hours and absence of respiratory tract colonization with Gram-negative bacteria on ICU admission., Interventions: None., Measurements and Main Results: Acquisition was determined through protocolized surveillance. Associations were investigated with Cox regression models with antibiotics as a time-dependent covariate. In all, 250 of 481 patients (52%) acquired respiratory tract colonization with Gram-negative bacteria after a median of 5 days (interquartile range, 3-8 d) (acquisition rate, 77.1/1,000 patient-days at risk). Antibiotic exposure during ICU admission was present in 78% and 72% of the patients with and without acquired Gram-negative bacteria colonization, respectively. In Kaplan-Meier curve analysis, the median times to acquisition of Gram-negative bacteria were 9 days (95% CI, 7.9-10.1) and 6 days (95% CI, 4.8-7.2) in patients receiving and not receiving antibiotics, respectively. In time varying Cox regression analysis, however, the association between acquired colonization and systemic antibiotics was not statistically significant (hazard ratio, 0.90; 95% CI, 0.70-1.16)., Conclusions: Among patients not colonized with Gram-negative bacteria in the respiratory tract at admission to ICU, systemic antibiotics during ICU stay were not associated with a reduction in acquisition of Gram-negative bacteria carriage in the respiratory tract during the ICU stay.

The prevalence of patients colonized with extended-spectrum beta-lactamase (ESBL)-producing bacteria increases, especially in long-term-care facilities (LTCFs). Identification of ESBL carriers at hospital admission is relevant for infection control measures and antibiotic therapy for nosocomial infections. We aimed to develop a prediction rule for ESBL carriage at hospital admission for patients admitted from home and LTCFs, and to quantify incidences of nosocomial infections caused by ESBL-producing bacteria. The ESBL-carrier status was determined of patients admitted from LTCFs and from home settings in four hospitals in the Netherlands using perianal swabs obtained within 48 hours of admission. Risk factors for ESBL carriage were assessed. Infections caused by ESBL-producing bacteria were identified retrospectively. Among 1351 patients, 111 (8.2%) were ESBL carriers at admission: 50/579 (8.6%) admitted from LTCFs and 61/772 (7.9%) from home settings (p 0.63). Previous ESBL carriage and previous hospital admission were risk factors for ESBL carriage in multivariable analysis. The area under the curve of the receiver operating characteristic curve of the model was 0.64 (95% CI 0.58-0.71). Presence of ≥1 risk factor (n = 803; 59%) had sensitivity of 72%. Incidences of nosocomial infections caused by ESBL-producing bacteria were 45.5/10,000 and 2.1/10,000 admission days for ESBL carriers and non-carriers, respectively (p <0.05). In conclusion, prevalence of ESBL carriage at hospital admission was 8.2%, and was comparable among patients admitted from LTCF and home. A clinically useful prediction rule for ESBL carriage at admission could not be developed. The absolute incidence of nosocomial infections by ESBL-producing bacteria was low, but higher among patients carrying ESBL-producing bacteria at the time of hospital admission., (Copyright © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Class A and B carbapenemases in Enterobacteriaceae may be detected using carbapenemase inhibition tests with boronic acid derivatives (BA) and dipicolinic acid (DPA)/EDTA, respectively. However, for OXA-48 (like) carbapenemases, no specific inhibitor is available. Because OXA-48 confers high-level temocillin resistance, a disc diffusion assay using temocillin as well as BA and DPA inhibition tests was evaluated for detection of class A, B and OXA-48 carbapenemases. The test collection included 128 well-characterized non-repeat Enterobacteriaceae isolates suspected of carbapenemase production; that is, with meropenem MICs ≥ 0.5 mg/L, including 99 carbapenemase producers (36 KPC, one GES, 31 MBL, four KPC plus VIM, 25 OXA-48, two OXA-162), and 29 ESBL and/or AmpC-producing isolates. PCR and sequencing of beta-lactamase genes was used as a reference test. Phenotypic carbapenemase detection was performed with discs (Rosco) containing meropenem (10 μg), temocillin (30 μg), meropenem + phenyl boronic acid (PBA), meropenem + DPA, meropenem + BA + DPA, and meropenem + cloxacillin (CL). Absence of synergy between meropenem and BA and/or DPA and a temocillin zone ≤10 mm was used to identify OXA-48. The sensitivity for identification of class A, B and OXA-48 carbapenemases was 95%, 90% and 100%, with 96-100% specificity. In non-Proteus species, the sensitivity for class B carbapenemase detection was 97%. All isolates without PBA or DPA synergy and a temocillin disc zone ≤10 mm were OXA-48 (like) positive. In conclusion, carbapenemase inhibition tests with PBA and DPA combined with a temocillin disc provide a reliable phenotypic confirmation method for class A, B and OXA-48 carbapenemases in Enterobacteriaceae., (© 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.)

Background: Complicated urinary tract infections (c-UTIs) are among the most common nosocomial infections and a substantial part of the antimicrobial agents used in hospitals is for the treatment of c-UTIs. Data from surveillance can be used to guide the empirical treatment choices of clinicians when treating c-UTIs. We therefore used nation-wide surveillance data to evaluate antimicrobial coverage of agents for the treatment of c-UTI in the Netherlands., Methods: We included the first isolate per patient of urine samples of hospitalised patients collected by the Infectious Disease Surveillance Information System for Antibiotic Resistance (ISIS-AR) in 2012, and determined the probability of inadequate coverage for antimicrobial agents based on species distribution and susceptibility. Analyses were repeated for various patient groups and hospital settings., Results: The most prevalent bacteria in 27,922 isolates of 23,357 patients were Escherichia coli (47%), Enterococcus spp. (14%), Proteus mirabilis (8%), and Klebsiella pneumoniae (7%). For all species combined, the probability of inadequate coverage was <5% for amoxicillin or amoxicillin-clavulanic acid combined with gentamicin and the carbapenems. When including gram-negative bacteria only, the probability of inadequate coverage was 4.0%, 2.7%, 2.3% and 1.7%, respectively, for amoxicillin, amoxicillin-clavulanic acid, a second or a third generation cephalosporin in combination with gentamicin, and the carbapenems (0.4%). There were only small variations in results among different patient groups and hospital settings., Conclusions: When excluding Enterococcus spp., considered as less virulent, and the carbapenems, considered as last-resort drugs, empirical treatment for c-UTI with the best chance of adequate coverage are one of the studied beta-lactam-gentamicin combinations. This study demonstrates the applicability of routine surveillance data for up-to-date clinical practice guidelines on empirical antimicrobial therapy, essential in patient care given the evolving bacterial susceptibility.

Successful multidrug-resistant clones are increasing in prevalence globally, which makes the ability to identify these clones urgent. However, adequate, easy-to-perform, and reproducible typing methods are lacking. We investigated whether DiversiLab (DL), an automated repetitive-sequence-based PCR bacterial typing system (bioMérieux), is suitable for comparing isolates analyzed at different geographic centers. A total of 39 Escherichia coli and 39 Klebsiella species isolates previously typed by the coordinating center were analyzed. Pulsed-field gel electrophoresis (PFGE) confirmed the presence of one cluster of 6 isolates, three clusters of 3 isolates, and three clusters of 2 isolates for each set of isolates. DL analysis was performed in 11 centers in six different countries using the same protocol. The DL profiles of 425 E. coli and 422 Klebsiella spp. were obtained. The DL system showed a lower discriminatory power for E. coli than did PFGE. The local DL data showed a low concordance, as indicated by the adjusted Rand and Wallace coefficients (0.132 to 0.740 and 0.070 to 1.0 [E. coli] and 0.091 to 0.864 and 0.056 to 1.0 [Klebsiella spp.], respectively). The central analysis showed a significantly improved concordance (0.473 to 1.0 and 0.290 to 1.0 [E. coli] and 0.513 to 0.965 and 0.425 to 1.0 [Klebsiella spp.], respectively). The misclassifications of profiles for individual isolates were mainly due to inconsistent amplification, which was most likely due to variations in the quality and amounts of the isolated DNA used for amplification. Despite local variations, the DL system has the potential to indicate the occurrence of clonal outbreaks in an international setting, provided there is strict adherence to standardized, reproducible DNA isolation methods and analysis protocols, all supported by a central database for profile comparisons.

Objectives: The CLSI recommends a fixed 2 : 1 ratio of co-amoxiclav for broth microdilution susceptibility testing of Enterobacteriaceae, while EUCAST recommends a fixed 2 mg/L clavulanate concentration. The aims of this study were: (i) to determine the influence of a switch from CLSI to EUCAST methodology on Escherichia coli susceptibility rates; (ii) to compare susceptibility results obtained using EUCAST-compliant microdilution with those from disc diffusion and the Etest; and (iii) to evaluate the clinical outcome of patients with E. coli sepsis treated with co-amoxiclav in relation to the susceptibility results obtained using either method., Methods: Resistance rates were determined in three laboratories that switched from CLSI to EUCAST cards with the Phoenix system (Becton Dickinson) as well as in 17 laboratories that continued to use CLSI cards with the VITEK 2 system (bioMérieux). In one laboratory, isolates were simultaneously tested by both the Phoenix system and either disc diffusion (n = 471) or the Etest (n = 113). Medical and laboratory records were reviewed for E. coli sepsis patients treated with co-amoxiclav monotherapy., Results: Only laboratories that switched methodology showed an increase in resistance rates - from 19% in 2010 to 31% in 2011 (P < 0.0001). All isolates that tested susceptible by microdilution were also susceptible by disc diffusion or the Etest, but of 326 isolates that tested resistant by microdilution, 43% and 59% tested susceptible by disc diffusion and the Etest, respectively. Among the 89 patients included there was a better correlation between clinical response and measured MICs using the Phoenix system than the Etest., Conclusions: EUCAST methodology resulted in higher co-amoxiclav E. coli resistance rates than CLSI methodology, but correlated better with clinical outcome. EUCAST-compliant microdilution and disc diffusion provided discrepant results.

The increasing prevalence of third-generation cephalosporin-resistant Enterobacteriaceae is a worldwide problem. Recent studies showed that poultry meat and humans share identical Extended-Spectrum Beta-Lactamase genes, plasmid types, and Escherichia coli strain types, suggesting that transmission from poultry meat to humans may occur. The aim of this study was to compare plasmid-encoded Ambler class C beta-lactamase (pAmpC) genes, their plasmids, and bacterial strain types between E. coli isolates from retail chicken meat and clinical isolates in the Netherlands. In total, 98 Dutch retail chicken meat samples and 479 third-generation cephalosporin non-susceptible human clinical E. coli isolates from the same period were screened for pAmpC production. Plasmid typing was performed using PCR-based replicon typing (PBRT). E coli strains were compared using Multi-Locus-Sequence-Typing (MLST). In 12 of 98 chicken meat samples (12%), pAmpC producing E. coli were detected (all blaCMY-2). Of the 479 human E. coli, 25 (5.2%) harboured pAmpC genes (blaCMY-2 n = 22, blaACT n = 2, blaMIR n = 1). PBRT showed that 91% of poultry meat isolates harboured blaCMY-2 on an IncK plasmid, and 9% on an IncI1 plasmid. Of the human blaCMY-2 producing isolates, 42% also harboured blaCMY-2 on an IncK plasmid, and 47% on an IncI1 plasmid. Thus, 68% of human pAmpC producing E. coli have the same AmpC gene (blaCMY-2) and plasmid type (IncI1 or IncK) as found in poultry meat. MLST showed one cluster containing one human isolate and three meat isolates, with an IncK plasmid. These findings imply that a foodborne transmission route of blaCMY-2 harbouring plasmids cannot be excluded and that further evaluation is required., (© 2013.)

The concurrent presence of bla CTX-M-1 and bla TEM-52 genes on similar plasmids of Escherichia coli isolated from poultry, chicken meat and humans supports the occurrence of food-borne transmission of extended-spectrum beta-lactamase (ESBL) genes. ESBL-producing E. coli (ESBL-E. coli) are most frequently detected in hospitalised patients and are known to spread in healthcare settings. We hypothesised that poultry-associated (PA) ESBL genes are predominant in the community, where acquisition is fuelled by food contamination, whereas non-PA ESBL genes are predominant in hospitals, with acquisition fuelled by cross-transmission. Then, differences in antimicrobial selective pressure in hospitals and poultry would create differences in co-resistance between PA and non-PA ESBL-E. coli. We, therefore, determined the prevalence and co-resistance of PA and non-PA ESBL-E. coli in community-acquired and nosocomial urinary tract infections in humans and bla CTX-M-1 and bla TEM-52 isolates from poultry. A total of 134 human ESBL-E. coli urine isolates were included in this study. Isolates containing bla CTX-M-1 or bla TEM-52 were considered to be PA, with the remainder being non-PA. Also, 72 poultry ESBL-E. coli were included. Minimum inhibitory concentration (MIC) values were determined by broth microdilution. The prevalence of PA ESBL genes in isolates obtained in general practice and hospitals was 28 % versus 30 % (n.s.). Human PA ESBL-E. coli were more frequently susceptible to ciprofloxacin (51 % vs. 25 %; p = 0.0056), gentamicin (86 % vs. 63 %; p = .0.0082), tobramycin (91 % vs. 34 %; p = 0.0001) and amikacin (98 % vs. 67 %; p = 0.0001) compared to human non-PA ESBL-E. coli. PA ESBL-E. coli are not more prevalent in community acquired than nosocomial urine samples, but are more often susceptible to ciprofloxacin and aminoglycosides than non-PA ESBL-E. coli. This does not support the existence of different reservoirs of ESBL genes.

We studied clinical characteristics, appropriateness of initial antibiotic treatment, and other factors associated with day 30 mortality in patients with bacteremia caused by extended-spectrum-β-lactamase (ESBL)-producing bacteria in eight Dutch hospitals. Retrospectively, information was collected from 232 consecutive patients with ESBL bacteremia (due to Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae) between 2008 and 2010. In this cohort (median age of 65 years; 24 patients were <18 years of age), many had comorbidities, such as malignancy (34%) or recurrent urinary tract infection (UTI) (15%). One hundred forty episodes (60%) were nosocomial, 54 (23%) were otherwise health care associated, and 38 (16%) were community acquired. The most frequent sources of infection were UTI (42%) and intra-abdominal infection (28%). Appropriate therapy within 24 h after bacteremia onset was prescribed to 37% of all patients and to 54% of known ESBL carriers. The day 30 mortality rate was 20%. In a multivariable analysis, a Charlson comorbidity index of ≥ 3, an age of ≥ 75 years, intensive care unit (ICU) stay at bacteremia onset, a non-UTI bacteremia source, and presentation with severe sepsis, but not inappropriate therapy within <24 h (adjusted odds ratio [OR], 1.53; 95% confidence interval [CI], 0.68 to 3.45), were associated with day 30 mortality. Further assessment of confounding and a stratified analysis for patients with UTI and non-UTI origins of infection did not reveal a statistically significant effect of inappropriate therapy on day 30 mortality, and these results were insensitive to the possible misclassification of patients who had received β-lactam-β-lactamase inhibitor combinations or ceftazidime as initial treatment. In conclusion, ESBL bacteremia occurs mostly in patients with comorbidities requiring frequent hospitalization, and 84% of episodes were health care associated. Factors other than inappropriate therapy within <24 h determined day 30 mortality.

Purpose: Topical use of colistin as part of selective digestive decontamination (SDD) and selective oropharyngeal decontamination (SOD) has been associated with improved patient outcome in intensive care units (ICU), yet little is known about the risks of colistin resistance. We quantified effects of selective decontamination on acquisition of colistin-resistant gram-negative bacteria (GNB) using data from a cluster-randomized study and a single-centre cohort., Methods: Acquisition of colistin-resistant GNB and conversion from susceptible to resistance in GNB was determined in respiratory samples [from patients receiving SDD (n = 455), SOD (n = 476), or standard care (SC) (n = 315)], and in rectal swabs from 1,840 SDD-patients. Genotyping of converting isolates was performed where possible., Results: The respiratory tract acquisition rates of colistin-resistant GNB were comparable during SDD, SOD, and SC and ranged from 0.7 to 1.1/1,000 patient-days at risk. Rectal acquisition rates during SDD were <3.3/1,000 days at risk. In patients with respiratory tract GNB carriage, conversion rates were 3.6 and 1.1/1,000 patient-days at risk during SDD and SC, respectively, (p > 0.05). In patients with rectal GNB carriage conversion rates during SDD were 5.4 and 3.2/1,000 days at risk and 15.5 and 12.6/1,000 days at risk when colonized with tobramycin-resistant GNB., Conclusions: Acquisition rates with colistin-resistant GNB in the respiratory tract were low and comparable with and without topical use of colistin. Rates of acquisition of colistin-resistant GNB during SDD were--in ICUs with low endemicity of antibiotic resistance--<2.5/1,000 days at risk, but were fivefold higher during persistent GNB colonization and 15-fold higher during carriage with tobramycin-resistant GNB.

This study aimed to evaluate the routine setting performance of a guideline for phenotypic detection of extended spectrum β-lactamases (ESBLs) in Enterobacteriaceae, recommending ESBL confirmation with Etest or combination disc for isolates with a positive ESBL screen test (i.e. cefotaxime and/or ceftazidime MIC >1 mg/L or an automated system ESBL warning). Twenty laboratories submitted 443 Enterobacteriaceae with a positive ESBL screen test and their confirmation test result (74%Escherichia coli, 12%Enterobacter cloacae, 8%Klebsiella pneumoniae, 3%Proteus mirabilis, 2%Klebsiella oxytoca). Presence of ESBL genes was used as reference test. Accuracy of local phenotypic ESBL detection was 88%. The positive predictive value (PPV) of local screen tests was 70%, and differed per method (Vitek-2: 69%, Phoenix: 68%, disc diffusion: 92%), and species (95%K. pneumoniae-27%K. oxytoca). A low PPV (3%) was observed for isolates with automated system alarm but third-generation cephalosporin MICs <2 mg/L. Local ESBL confirmation had a PPV and negative predictive value (NPV) of 93% and 90%, respectively. Compared with centrally performed confirmation tests, 7% of local tests were misinterpreted. Combination disc was more specific than Etest (91% versus 61%). Confirmation tests were not reliable for P. mirabilis and K. oxytoca (PPV 33% and 38%, respectively, although NPVs were 100%). In conclusion, performance of Etests could be enhanced by education of technicians to improve their interpretation, by genotypic ESBL confirmation of P. mirabilis and K. oxytoca isolates with positive phenotypic ESBL confirmation, and by interpreting isolates with a positive ESBL alarm but an MIC <2 mg/L for cefotaxime and ceftazidime as ESBL-negative., (© 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.)

Purpose: It is widely assumed that closed suction systems (CSSs), as compared with open suction systems (OSSs), better guarantee optimal oxygenation with less disturbance of physiologic parameters in mechanically ventilated intensive care patients. We, therefore, quantified changes in heart rate (HR), mean arterial pressure (MAP), and peripheral oxygen saturation (Spo(2)) in patients undergoing endotracheal suctioning (ES) with CSS and OSS., Materials and Methods: We performed a prospective observational study nested within a crossover trial in 4 intensive care units between January 2007 and February 2008. Per unit, 50 ES procedures were selected at random, and HR, MAP, and Spo(2) were measured before and after ES., Results: In total, 197 complete ES procedures (103 OSS and 94 CSS) were monitored. Mean HR, MAP, and Spo(2) changed directly after ES and returned to baseline after 5 minutes. Changes in HR and MAP were comparable after using CSS and OSS, whereas in Spo(2), slightly better values were monitored 3 and 5 minutes after OSS, these differences being rather small (0.3%-0.7%) and clinically not relevant., Conclusions: Changes in HR, MAP, and Spo(2) were comparable and mild during and after CSS and OSS. Both systems can be considered equally safe., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The Check-MDR CT102 DNA microarray enables detection of the most prevalent carbapenemases (NDM, VIM, KPC, OXA-48 and IMP) and extended-spectrum β-lactamase (ESBL) gene families (SHV, TEM and CTX-M). The test performance of this microarray was evaluated with 95 Enterobacteriaceae isolates suspected of being carbapenemase producers, i.e. with meropenem MICs ≥0.5 mg l(-1). The collection of isolates contained 70 carbapenemase-producing isolates, including 37 bla(KPC)-, 20 bla(VIM)-, five bla(OXA-48)-, four bla(KPC)/bla(VIM)- and four bla(NDM)-positive isolates; and 25 carbapenemase-gene-negative isolates. ESBLs were produced by 51 of the isolates. PCR and sequencing of β-lactamase genes was used as reference test. For detection of carbapenemases, the sensitivity of the microarray was 97% (68/70), with 100% specificity. The two negative isolates tested positive when the microarray test was repeated; these isolates were an OXA-48- and a KPC-producing isolate. For ESBL detection, the sensitivity was 100% (51/51) and the specificity was 98% (43/44), although 20% of the SHV-12 ESBLs were categorized as SHV-2-like ESBLs. In conclusion, the CDT102 microarray is a rapid and accurate tool for the detection of carbapenemase and ESBL genes, although the array seems less suitable for epidemiology of ESBL genes.

There is a global increase in infections caused by Enterobacteriaceae with plasmid-borne β-lactamases that confer resistance to third-generation cephalosporins. The epidemiology of these bacteria is not well understood, and was, therefore, investigated in a selection of 636 clinical Enterobacteriaceae with a minimal inhibitory concentration >1 mg/L for ceftazidime/ceftriaxone from a national survey (75% E. coli, 11% E. cloacae, 11% K. pneumoniae, 2% K. oxytoca, 2% P. mirabilis). Isolates were investigated for extended-spectrum β-lactamases (ESBLs) and ampC genes using microarray, PCR, gene sequencing and molecular straintyping (Diversilab and multi-locus sequence typing (MLST)). ESBL genes were demonstrated in 512 isolates (81%); of which 446 (87%) belonged to the CTX-M family. Among 314 randomly selected and sequenced isolates, bla(CTX-M-15) was most prevalent (n = 124, 39%), followed by bla(CTX-M-1) (n = 47, 15%), bla(CTX-M-14) (n = 15, 5%), bla(SHV-12) (n = 24, 8%) and bla(TEM-52) (n = 13, 4%). Among 181 isolates with MIC ≥16 mg/L for cefoxitin plasmid encoded AmpCs were detected in 32 and 27 were of the CMY-2 group. Among 102 E. coli isolates with MIC ≥16 mg/L for cefoxitin ampC promoter mutations were identified in 29 (28%). Based on Diversilab genotyping of 608 isolates (similarity cut-off >98%) discriminatory indices of bacteria with ESBL and/or ampC genes were 0.994, 0.985 and 0.994 for E. coli, K. pneumoniae and E. cloacae, respectively. Based on similarity cut-off >95% two large clusters of E. coli were apparent (of 43 and 30 isolates) and 21 of 21 that were typed by belonged to ST131 of which 13 contained bla(CTX-M-15). Our findings demonstrate that bla(CTX-M-15) is the most prevalent ESBL and we report a larger than previously reported prevalence of ampC genes among Enterobacteriaceae responsible for resistance to third-generation cephalosporins.

Objectives: We quantified the association between antibiotic exposure and acquisition of antibiotic resistance in Pseudomonas aeruginosa and Enterobacter species in intensive care unit patients., Design: Prospective cohort study., Setting and Patients: In 1,201 patients, respiratory tract colonization was determined through regular screening on admission, twice weekly, and on discharge. Primary outcome was the acquisition of antibiotic resistance in previous antibiotic sensitive P. aeruginosa and Enterobacter species, with acquisition attributable to cross-transmission excluded based on genotyping and epidemiologic linkage. Cox regression analysis, adjusted for covariates, was performed to calculate hazard ratios of patients exposed to antibiotics compared to patients not exposed to antibiotics., Measurements and Main Results: In total, 194 and 171 patients were colonized with P. aeruginosa and Enterobacter species, respectively. Two or more cultures per episode were available for 126 and 108 patients. For P. aeruginosa, ceftazidime exposure was associated with 6.3 acquired antibiotic resistance events per 100 days of exposure, whereas incidence rates were lower for ciprofloxacin, meropenem, and piperacillin-tazobactam. In multivariate analysis, meropenem, ciprofloxacin, and ceftazidime were significantly associated with risk of resistance development in P. aeruginosa (adjusted hazard ratio, 11.1; 95% confidence interval, 2.4-51.5 for meropenem; adjusted hazard ratio, 4.1; 95% confidence interval, 1.1-16.2 for ciprofloxacin; adjusted hazard ratio, 2.5; 95% confidence interval, 1.1-5.5 for ceftazidime). For Enterobacter, ceftriaxone and ciprofloxacin exposure were associated with most antibiotic resistance acquisitions. No significant associations were found in multivariate analysis., Conclusions: Meropenem exposure is associated with the highest risk of resistance development in P. aeruginosa. Increasing carbapenem use attributable to emergence of Gram-negative bacteria producing extended-spectrum β-lactamases will enhance antibiotic resistance in P. aeruginosa.

Since the diagnostic characteristics of the Check-KPC ESBL microarray as a confirmation test on isolates obtained in a routine clinical setting have not been determined, we evaluated the microarray in a random selection of 346 clinical isolates with a positive ESBL screen test (MIC >1 mg/L for cefotaxime or ceftazidime or an ESBL alarm from the Phoenix or Vitek-2 expert system) collected from 31 clinical microbiology laboratories in the Netherlands in 2009. Using sequencing as the reference method the sensitivity of the microarray was 97% (237/245), the specificity 98% (97/99), the positive predictive value 99% (237/239) and the negative predictive value 92% (97/105)., (© 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.)

Intestinal carriage of extended-spectrum beta-lactamase (ESBL) -producing bacteria in food-producing animals and contamination of retail meat may contribute to increased incidences of infections with ESBL-producing bacteria in humans. Therefore, distribution of ESBL genes, plasmids and strain genotypes in Escherichia coli obtained from poultry and retail chicken meat in the Netherlands was determined and defined as 'poultry-associated' (PA). Subsequently, the proportion of E. coli isolates with PA ESBL genes, plasmids and strains was quantified in a representative sample of clinical isolates. The E. coli were derived from 98 retail chicken meat samples, a prevalence survey among poultry, and 516 human clinical samples from 31 laboratories collected during a 3-month period in 2009. Isolates were analysed using an ESBL-specific microarray, sequencing of ESBL genes, PCR-based replicon typing of plasmids, plasmid multi-locus sequence typing (pMLST) and strain genotyping (MLST). Six ESBL genes were defined as PA (bla(CTX-M-1) , bla(CTX-M-2) , bla(SHV-2) , bla(SHV-12) , bla(TEM-20) , bla(TEM-52) ): 35% of the human isolates contained PA ESBL genes and 19% contained PA ESBL genes located on IncI1 plasmids that were genetically indistinguishable from those obtained from poultry (meat). Of these ESBL genes, 86% were bla(CTX-M-1) and bla(TEM-52) genes, which were also the predominant genes in poultry (78%) and retail chicken meat (75%). Of the retail meat samples, 94% contained ESBL-producing isolates of which 39% belonged to E. coli genotypes also present in human samples. These findings are suggestive for transmission of ESBL genes, plasmids and E. coli isolates from poultry to humans, most likely through the food chain., (2011 The Authors. Clinical Microbiology and Infection; 2011 European Society of Clinical Microbiology and Infectious Diseases.)

Objective: Cross-transmission of Gram-negative bacteria increases the likelihood of acquisition of infections and emergence of antibiotic resistance in intensive care units. Respiratory tracts of mechanically ventilated patients are frequently colonized with Gram-negative bacteria and endotracheal suctioning may facilitate cross-transmission. It is unknown whether closed suction systems, as compared with open suction systems, prevent cross-transmission. The objective was to determine whether closed suction systems, as compared with open suction systems, reduce the incidence of cross-transmission of Gram-negative bacteria in intensive care units., Design: We performed a prospective crossover study in which both systems were tested unitwide in four intensive care units., Setting: Two intensive care units from a university hospital and two from a teaching hospital participated in the trial between January 2007 and February 2008., Patients: All patients admitted to the intensive care unit for >24 hrs were included., Intervention: Closed suction systems and open suction systems were used for all patients requiring mechanical ventilation during 6-month clusters with the order of systems randomized per intensive care unit., Measurements and Main Results: Acquisition and cross-transmission rates of selected Gram-negative bacteria were determined through extensive microbiological surveillance and genotyping. Among 1,110 patients (585 with closed suction systems and 525 with open suction systems), acquisition for selected Gram-negative bacteria was 35.5 and 32.5 per 1,000 patient-days at risk during closed suction period and open suction period, respectively (adjusted hazard ratio, 1.14; 95% confidence interval, 0.9-1.4). During closed suction period, adjusted hazard ratios for acquisition were 0.66 (95% confidence interval, 0.45-0.97) for Pseudomonas aeruginosa and 2.03 (95% confidence interval, 1.15-3.57) for Acinetobacter species; acquisition rates of other pathogens did not differ significantly. Adjusted hazard ratios for cross-transmission during closed suction period 0.9 (0.4-1.9) for P. aeruginosa, 6.7 (1.5-30.1) for Acinetobacter, and 0.3 (0.03-2.7) for Enterobacter species. Overall cross-transmission rates were 5.9 (closed suction systems) and 4.7 (open suction systems) per 1,000 patient-days at risk., Conclusion: Closed suction systems failed to reduce cross-transmission and acquisition rates of the most relevant Gram-negative bacteria in intensive care unit patients.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Worldwide, resistance of Gram-negative micro-organisms to third-generation cephalosporins and carbapenems owing to β-lactamases is an increasing problem. Although the CTX-M, TEM and SHV extended-spectrum β-lactamases (ESBLs) are most widely disseminated, other β-lactamase families have also recently emerged, such as plasmid-mediated AmpC β-lactamases and carbapenemases. Here we describe a new set of multiplex polymerase chain reactions (PCRs) with one amplification protocol enabling detection of 25 prevalent β-lactamase families, including ESBLs, carbapenemases, plasmid-mediated AmpC β-lactamases and OXA β-lactamases., (Copyright © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Many bacterial typing methods are specific for one species only, time-consuming, or poorly reproducible. DiversiLab (DL; bioMérieux) potentially overcomes these limitations. In this study, we evaluated the DL system for the identification of hospital outbreaks of a number bacterial species. Appropriately typed clinical isolates were tested with DL. DL typing agreed with pulsed-field gel electrophoresis (PFGE) for Acinetobacter (n = 26) and Stenotrophomonas maltophilia (n = 13) isolates. With two exceptions, DL typing of Klebsiella isolates (n = 23) also correlated with PFGE, and in addition, PFGE-nontypeable (PFGE-NT) isolates could be typed. Enterobacter (n = 28) results also correlated with PFGE results; also, PFGE-NT isolates could be clustered. In a larger study (n = 270), a cluster of 30 isolates was observed that could be subdivided by PFGE. The results for Escherichia coli (n = 38) correlated less well with an experimental multilocus variable number of tandem repeats analysis (MLVA) scheme. Pseudomonas aeruginosa (n = 52) showed only a limited number of amplification products for most isolates. When multiple Pseudomonas isolates were assigned to a single type in DL, all except one showed multiple multilocus sequence types. Methicillin-resistant Staphylococcus aureus generally also showed a limited number of amplification products. Isolates that belonged to different outbreaks by other typing methods, including PFGE, spa typing, and MLVA, were grouped together in a number of cases. For Enterococcus faecium, the limited variability of the amplification products obtained made interpretation difficult and correlation with MLVA and esp gene typing was poor. All of the results are reflected in Simpson's index of diversity and adjusted Rand's and Wallace's coefficients. DL is a useful tool to help identify hospital outbreaks of Acinetobacter spp., S. maltophilia, the Enterobacter cloacae complex, Klebsiella spp., and, to a somewhat lesser extent, E. coli. In our study, DL was inadequate for P. aeruginosa, E. faecium, and MRSA. However, it should be noted that for the identification of outbreaks, epidemiological data should be combined with typing results.

Adequate detection of carbapenemase-producing Enterobacteriaceae is crucial for infection control measures and appropriate choice of antimicrobial therapy. This guideline aims to improve the detection of carbapenemase-producing Enterobacteriaceae in the routine setting of clinical microbiology laboratories. Detection of carbapenemases in Enterobacteriaceae includes a screening step followed by a genotypic and optional phenotypic confirmatory step. For all Enterobacteriaceae, the meropenem screening breakpoint to detect carbapenemases is set at >or=0.5mg/L or a zone diameter of or=2mg/L or a zone diameter

Object: An important complication of external CSF drainage is bacterial meningitis or ventriculitis, resulting in increased morbidity, mortality, and health care costs. In 2003, a high rate (37%) of probable drain-related infections was identified at the authors' hospital. A multidisciplinary working group was installed to reduce this incidence to < 10% within 1.5 years., Methods: An intervention strategy based on 5 pillars (increased awareness, focused standard operating procedures, a diagnostic and therapeutic algorithm, timely administration of prophylaxis, and improvement of the drainage system) was designed and implemented from 2004 to 2006. During this period all patients with external CSF drainage were prospectively monitored., Results: Between 2004 and 2006, there were 467 patients in whom 579 drains (external ventricular and external lumbar) had been placed. The overall incidence of drain-related infections was 16.2% in 2004, 8.9% in 2005, and 11.3% in 2006. For external lumbar drains the number of infections per 100 drain days was 2.4 in 2004, 0.6 in 2005, and 0.8 in 2006. For external ventricular drains these rates were 1.7, 1.0, and 1.2, respectively. Meanwhile, the causative noncutaneous microorganisms, indicative for systemic-contamination during manipulation, decreased. By retrospective analysis, the proportion of patients with a probable drain-related infection decreased from 37% in 2003 to 9% in 2005 and 2006., Conclusions: The authors' multidisciplinary approach in which different preventive measures were combined was associated with a significant reduction in the incidence of drain-related secondary meningitis, and thus provides an important improvement of patient safety.

Horizontal gene transfer is a key step in the evolution of Enterobacteriaceae. By acquiring virulence determinants of foreign origin, commensals can evolve into pathogens. In Enterobacteriaceae, horizontal transfer of these virulence determinants is largely dependent on transfer by plasmids, phages, genomic islands (GIs) and genomic modules (GMs). The High Pathogenicity Island (HPI) is a GI encoding virulence genes that can be transferred between different Enterobacteriaceae. We investigated the HPI because it was present in an Enterobacter hormaechei outbreak strain (EHOS). Genome sequence analysis showed that the EHOS contained an integration site for mobile elements and harbored two GIs and three putative GMs, including a new variant of the HPI (HPI-ICEEh1). We demonstrate, for the first time, that combinatorial transfers of GIs and GMs between Enterobacter cloacae complex isolates must have occurred. Furthermore, the excision and circularization of several combinations of the GIs and GMs was demonstrated. Because of its flexibility, the multiple integration site of mobile DNA can be considered an integration hotspot (IHS) that increases the genomic plasticity of the bacterium. Multiple combinatorial transfers of diverse combinations of the HPI and other genomic elements among Enterobacteriaceae may accelerate the generation of new pathogenic strains.

This is the first report of 3 patients in whom carbapenemase-producing Klebsiella pneumoniae was identified in the Netherlands following foreign travel. They were a 55-year-old man who had undergone chemotherapy for lung cancer metastases, a 66-year-old woman and a 30-year-old man. The first patient was transferred from a Greek hospital; his isolate belonged to an epidemic clone (multilocus sequence type 258) with a KPC-2 carbapenemase gene. The patient died from pneumonia. The other two patients, who had been travelling around in India, were found to be colonised in the gasto-intestinal tract with different multiresistant K. pneumoniae isolates containing a New Delhi metallo-carbapenemase gene (NDM-1). The rapid emergence and dissemination of Enterobacteriaceae resistant to carbapenems such as imipenem and meropenem poses a considerable threat to clinical patient care and public health. Carbapenemase-producing strains are characterized by resistance to nearly all available beta-lactam antibiotics including cephalosporins and carbapenems. These strains are often also resistant to other classes of antibiotics. Invasive infections by these strains are associated with high morbidity and mortality rates. Adequate microbiological laboratory detection and infection control measures in hospital are pivotal to preventing dissemination in the Dutch healthcare setting.

Enterobacteriaceae that contain the High Pathogenicity Island (HPI), which encodes the siderophore yersiniabactin, display increased virulence. This increased virulence may be explained by the increased iron scavenging of the bacteria, which would both enhance bacterial growth and limit the availability of iron to cells of the innate immune system, which require iron to catalyze the Haber-Weiss reaction that produces hydroxyl radicals. In this study, we show that yersiniabactin increases bacterial growth when iron-saturated lactoferrin is the main iron source. This suggests that yersiniabactin provides bacteria with additional iron from saturated lactoferrin during infection. Furthermore, the production of ROS by polymorphonuclear leukocytes, monocytes, and a mouse macrophage cell line is blocked by yersiniabactin, as yersiniabactin reduces iron availability to the cells. Importantly, iron functions as a catalyst during the Haber-Weiss reaction, which generates hydroxyl radicals. While the physiologic role of the Haber-Weiss reaction in the production of hydroxyl radicals has been controversial, the siderophores yersiniabactin, aerobactin, and deferoxamine and the iron-chelator deferiprone also reduce ROS production in activated innate immune cells. This suggests that this reaction takes place under physiological conditions. Of the tested iron chelators, yersiniabactin was the most effective in reducing the ROS production in the tested innate immune cells. The likely decreased bacterial killing by innate immune cells resulting from the reduced production of hydroxyl radicals may explain why the HPI-containing Enterobacteriaceae are more virulent. This model centered on the reduced killing capacity of innate immune cells, which is indirectly caused by yersiniabactin, is in agreement with the observation that the highly pathogenic group of Yersinia is more lethal than the weakly pathogenic and the non-pathogenic group.

Bacterial strains differ in their ability to cause hospital outbreaks. Using comparative genomic hybridization, Enterobacter cloacae complex isolates were studied to identify genetic markers specific for Enterobacter cloacae complex outbreak strains. No outbreak-specific genes were found that were common in all investigated outbreak strains. Therefore, the aim of our study was to identify specific genetic markers for an Enterobacter hormaechei outbreak strain (EHOS) that caused a nationwide outbreak in The Netherlands. Most EHOS isolates carried a large conjugative plasmid (pQC) containing genes encoding heavy-metal resistance, mobile elements, pili-associated proteins and exported proteins as well as multiple-resistance genes. Furthermore, the chromosomally encoded high-pathogenicity island (HPI) was highly associated with the EHOS strain. In addition, other DNA fragments were identified that were associated with virulence: three DNA fragments known to be located on a virulence plasmid (pLVPK), as well as phage- and plasmid-related sequences. Also, four DNA fragments encoding putative pili with the most homology to pili of Salmonella enterica were associated with the EHOS. Finally, four DNA fragments encoding putative outer-membrane proteins were negatively associated with the EHOS. In conclusion, resistance and putative virulence genes were identified in the EHOS that may have contributed to increased epidemicity. The high number of genes detected in the EHOS that were related to transferable elements reflects the genomic plasticity of the E. cloacae complex and may explain the emergence of the EHOS in the hospital environment.

Objectives: The aim of this study was to test the hypothesis that trimethoprim/sulfamethoxazole selects for integron-positive and multidrug-resistant Enterobacteriaceae in the intestinal flora., Methods: During 1 year of follow-up, antibiotic susceptibility and the presence of integrons were determined in faecal Enterobacteriaceae isolated from 99 children with chronic active otitis media, randomly assigned to treatment with trimethoprim/sulfamethoxazole or placebo (http://www.clinicaltrials.gov/; trial registration number NCT00189098)., Results: At 6 and 12 weeks of follow-up, 32 (91%) and 24 (67%) children in the trimethoprim/sulfamethoxazole group carried trimethoprim/sulfamethoxazole-resistant Enterobacteriaceae versus 10 (21%) and 8 (17%) children in the placebo group [rate differences (RDs): 70 (95% CI: 55; 85) and 50 (95% CI: 31; 69)], respectively. Multiresistance also increased during trimethoprim/sulfamethoxazole treatment. At 6 weeks of follow-up, the integron prevalence was 26 (79%) in the trimethoprim/sulfamethoxazole group and 10 (22%) in the placebo group [RD: 57 (95% CI: 39; 75)]. After 12 weeks the integron prevalence, and after 1 year the susceptibility levels, had returned to baseline values., Conclusions: Initially, trimethoprim/sulfamethoxazole usage was strongly associated with the appearance of integron-positive (multi)drug-resistant Enterobacteriaceae in the intestinal flora. After prolonged exposure to trimethoprim/sulfamethoxazole, however, this population of Enterobacteriaceae was substituted by a population with non-integron-associated resistance mechanisms. After trimethoprim/sulfamethoxazole was discontinued, susceptibility rates to all antibiotics returned to baseline levels.

Background: Selective digestive tract decontamination (SDD) and selective oropharyngeal decontamination (SOD) are infection-prevention measures used in the treatment of some patients in intensive care, but reported effects on patient outcome are conflicting., Methods: We evaluated the effectiveness of SDD and SOD in a crossover study using cluster randomization in 13 intensive care units (ICUs), all in The Netherlands. Patients with an expected duration of intubation of more than 48 hours or an expected ICU stay of more than 72 hours were eligible. In each ICU, three regimens (SDD, SOD, and standard care) were applied in random order over the course of 6 months. Mortality at day 28 was the primary end point. SDD consisted of 4 days of intravenous cefotaxime and topical application of tobramycin, colistin, and amphotericin B in the oropharynx and stomach. SOD consisted of oropharyngeal application only of the same antibiotics. Monthly point-prevalence studies were performed to analyze antibiotic resistance., Results: A total of 5939 patients were enrolled in the study, with 1990 assigned to standard care, 1904 to SOD, and 2045 to SDD; crude mortality in the groups at day 28 was 27.5%, 26.6%, and 26.9%, respectively. In a random-effects logistic-regression model with age, sex, Acute Physiology and Chronic Health Evaluation (APACHE II) score, intubation status, and medical specialty used as covariates, odds ratios for death at day 28 in the SOD and SDD groups, as compared with the standard-care group, were 0.86 (95% confidence interval [CI], 0.74 to 0.99) and 0.83 (95% CI, 0.72 to 0.97), respectively., Conclusions: In an ICU population in which the mortality rate associated with standard care was 27.5% at day 28, the rate was reduced by an estimated 3.5 percentage points with SDD and by 2.9 percentage points with SOD. (Controlled Clinical Trials number, ISRCTN35176830.), (2009 Massachusetts Medical Society)

Background: Isolates of the Enterobacter cloacae complex have been increasingly isolated as nosocomial pathogens, but phenotypic identification of the E. cloacae complex is unreliable and irreproducible. Identification of species based on currently available genotyping tools is already superior to phenotypic identification, but the taxonomy of isolates belonging to this complex is cumbersome., Methodology/principal Findings: This study shows that multilocus sequence analysis and comparative genomic hybridization based on a mixed genome array is a powerful method for studying species assignment within the E. cloacae complex. The E. cloacae complex is shown to be evolutionarily divided into two clades that are genetically distinct from each other. The younger first clade is genetically more homogenous, contains the Enterobacter hormaechei species and is the most frequently cultured Enterobacter species in hospitals. The second and older clade consists of several (sub)species that are genetically more heterogeneous. Genetic markers were identified that could discriminate between the two clades and cluster 1., Conclusions/significance: Based on genomic differences it is concluded that some previously defined (clonal and heterogenic) (sub)species of the E. cloacae complex have to be redefined because of disagreements with known or proposed nomenclature. However, further improved identification of the redefined species will be possible based on novel markers presented here.

A large outbreak with an aminoglycoside-resistant Enterobacter cloacae (AREC) clone occurred at the University Medical Center Utrecht beginning in 2001 and continued up through the time that this study was completed. This clone (genotype I) contains a conjugative R plasmid carrying the qnrA1, bla(CTX-M-9), and aadB genes, encoding resistance to quinolones, extended-spectrum beta-lactamases, and aminoglycosides, respectively. The aim of this study was to determine whether this clone was more transmissible than other AREC strains. Therefore, the dissemination of this genotype and of other E. cloacae strains was studied. In addition, infection control measures taken were evaluated. Pulsed-field gel electrophoresis analysis divided the 191 AREC strains into 42 different genotypes, of which 5 (12%) involved at least three patients. Aside from this outbreak (133 patients), only two other small outbreaks occurred, showing that the infection control measures were successful for all strains but one. Among 324 aminoglycoside-susceptible E. cloacae strains, 34/166 (20%) genotypes were identified from at least three patients, but only 4 involved small outbreaks. The outbreak strain was also detected in 11 of 15 other Dutch hospitals and caused outbreaks in at least 4. Evaluation of infection control measures showed that the outbreak strain disseminated throughout the hospital despite adequate implementation of internationally accepted guidelines on the control of multidrug-resistant Enterobacteriaceae (MRE). In conclusion, some MRE strains are better able to spread than others, and these strains may not be controlled by the current infection control guidelines. Strategies to identify such strains in an early phase and adapted guidelines for such "superbugs" are needed to prevent these clones from becoming endemic.

The 'Stichting Werkgroep Antibioticabeleid' (Dutch Working Party on Antibiotic Policy) has developed an electronic national antibiotic guide for the antibiotic treatment and prophylaxis of common infectious diseases in hospitals. This guide also contains information on the most important characteristics of antimicrobial drugs. Advice on antibiotic treatment is based on existing national evidence-based guidelines, where available. Where no guideline is available, the advice is based on an inventory of the antibiotic policies of the 12 Dutch centres with an infectious disease or medical microbiology training programme. The national antibiotic guide can be accessed through the SWAB website (www.swab.nl) and can also be downloaded on PDA/PocketPC, free of charge. Every hospital antibiotic formulary committee in the Netherlands will be offered the opportunity to edit The national version for local use.

Objectives: To determine the optimal approach for nasopharyngeal culture and to establish which approach children tolerate best., Design: Cross-sectional study., Setting: A pediatric otolaryngology department of a Dutch tertiary care hospital., Patients: A cohort of 42 children with chronic suppurative otitis media., Intervention: Paired nasopharyngeal samples were collected transorally and transnasally and cultured for potential aerobic pathogens., Main Outcome Measures: The isolation rate of both samples and the amount of discomfort measured by the visual analog scale., Results: Forty-six (87%) of 53 samples obtained transnasally were culture positive vs 40 (75%) of 53 samples obtained transorally (P = .20). Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus were found more frequently with the transnasal than with the transoral approach: 34% vs 13% (P = .003), 62% vs 51% (P = .20), 30% vs 19% (P = .15), and 21% vs 11% (P = .18), respectively. Mean (SD) visual analog scale scores were 5.3 (1.0) and 3.4 (1.7) (P<.001) for the transnasal and transoral approaches, respectively., Conclusions: Although the transoral approach is better tolerated in children, the isolation rate of the transnasal approach is higher, especially for S. pneumoniae. The transnasal sampling technique should therefore be the preferred approach for detection of potential pathogens in the nasopharynx in children.

An outbreak of Enterobacter cloacae infections with variable susceptibility to fluoroquinolones occurred in the University Medical Center Utrecht in the Netherlands in 2002. Our investigation showed that a qnrA1 gene was present in 78 (94%) of 83 outbreak isolates and that a qnrA1-encoding plasmid transferred to other strains of the same species and other species. The earliest isolate carrying this same plasmid was isolated in 1999. qnrA1 was located in a complex integron consisting of the intI1, aadB, qacEDelta1, sul1, orf513, qnrA1, ampR, qacEDelta1, and sul1 genes that were not described previously. On the same plasmid, 2 other class 1 integrons were present. One was a new integron associated with the bla(CTX-M-9) extended-spectrum beta-lactamase.