Developing an algorithm for the diagnosis of abnormal vaginal discharge in a dutch clinical setting: a pilot study.

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.

The vaginal microbiota in the course of bacterial vaginosis treatment.

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.

Comparison of Amsel criteria, Nugent score, culture and two CE-IVD marked quantitative real-time PCRs with microbiota analysis for the diagnosis of bacterial vaginosis.

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.

Evaluation of a stepwise approach using microbiota analysis, species-specific qPCRs and culture for the diagnosis of lower respiratory tract infections.

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.

Immune status of health care workers to measles virus: evaluation of protective titers in four measles IgG EIAs.

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.

Impact of livestock-associated MRSA in a hospital setting.

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.

The role of systemic antibiotics in acquiring respiratory tract colonization with gram-negative bacteria in intensive care patients: a nested cohort study.

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.

Recommendations for the empirical treatment of complicated urinary tract infections using surveillance data on antimicrobial resistance in the Netherlands.

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.

Identical plasmid AmpC beta-lactamase genes and plasmid types in E. coli isolates from patients and poultry meat in the Netherlands.

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.

International multicenter evaluation of the DiversiLab bacterial typing system for Escherichia coli and Klebsiella spp.

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.

Consequences of switching from a fixed 2 : 1 ratio of amoxicillin/clavulanate (CLSI) to a fixed concentration of clavulanate (EUCAST) for susceptibility testing of Escherichia coli.

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.

Appropriateness of empirical treatment and outcome in bacteremia caused by extended-spectrum-ß-lactamase-producing bacteria.

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-ß-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 ß-lactam-ß-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.

Colistin resistance in gram-negative bacteria during prophylactic topical colistin use in intensive care units.

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.

Changes in heart rate, mean arterial pressure, and oxygen saturation after open and closed endotracheal suctioning: a prospective observational study.

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.

Detection of carbapenemase-producing Enterobacteriaceae with a commercial DNA microarray.

The Check-MDR CT102 DNA microarray enables detection of the most prevalent carbapenemases (NDM, VIM, KPC, OXA-48 and IMP) and extended-spectrum ß-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 ß-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.

Population distribution of Beta-lactamase conferring resistance to third-generation cephalosporins in human clinical Enterobacteriaceae in the Netherlands.

There is a global increase in infections caused by Enterobacteriaceae with plasmid-borne ß-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 ß-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.

Antibiotic exposure and resistance development in Pseudomonas aeruginosa and Enterobacter species in intensive care units.

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 ß-lactamases will enhance antibiotic resistance in P. aeruginosa.

Effect of open and closed endotracheal suctioning on cross-transmission with Gram-negative bacteria: a prospective crossover study.

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.

A set of multiplex PCRs for genotypic detection of extended-spectrum ß-lactamases, carbapenemases, plasmid-mediated AmpC ß-lactamases and OXA ß-lactamases.

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