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Colistin is considered as one of the last-resort antibiotics and reliable antimicrobial susceptibility testing is therefore crucial. The reference standard for AST according to EUCAST and CLSI is broth microdilution (BMD). However, BMD is labor intensive to perform. Commercial antimicrobial susceptibility tests derived from BMD method are available. We investigated the performance of four different commercial tests: Sensititre™, SensiTest™ Colistin, Micronaut MIC Strip Colistin and UMIC Colistin using 70 clinical isolates (half of them was deemed by VITEK2 as resistant), including isolates from cystic fibrosis patients and mcr-1 bearing isolates. We used two reference standards: BMD and composite MIC as determined by all four tests. Sensititre™ had essential agreement (EA, defined as minimum inhibitory concentration within ± 1 dilution) of 87% and 89% compared to BMD and composite reference standard, respectively. For SensiTest™, the EA’s were 93% and 90%. For UMIC, 87% and 90%, and for Micronaut, 83% and 84%. All four tests demonstrated categorical agreement (CA) above 90%. CA for SensiTest™ and Micronaut was both 96%, UMIC 94%, and Sensititre™ 93%. All tests were reproducible as tested in two quality control isolates. In conclusion, in clinical isolates from a large referral center, the four commercial tests for determination of colistin minimum inhibitory concentrations showed acceptable performance.

OBJECTIVE: A prospective observational study was performed to assess the epidemiology and clinical impact of carbapenem-non-susceptible Klebsiella pneumoniae (CNKP) in intensive care units (ICUs) of the national referral hospital in Jakarta, Indonesia. MATERIALS/METHODS: Adult patients consecutively hospitalized for > 48 h in two ICUs of the national referral hospital were included from April until October 2013 and from April until August 2014. K. pneumoniae from clinical cultures and standardized screening of rectum and throat on admission, discharge and weekly if hospitalized > 7 days were collected. Environmental niches and healthcare workers (HCWs) were also screened. Susceptibility was determined phenotypically and the presence of carbapenemase genes by PCR. Raman spectroscopy as well as multiple-locus variable number tandem repeat analysis (MLVA) were used for typing. RESULTS: Twenty-two out of 412 (5.3%) patients carried CNKP on admission and 37/390 (9.5%) acquired CNKP during ICU stay. The acquisition rate was 24.7/1000 patient-days at risk. One out of 31 (3.2%) environmental isolates was a CNKP. None of the HCWs carried CNKP. Acquisition of CNKP was associated with longer ICU stay (adjusted Hazard Ratio: 2.32 [CI99: 1.35-3.68]). ICU survival was lower among patients with CNKP compared to patients with carbapenem-susceptible K. pneumoniae (aHR 2.57, p = 0.005). Ninety-six of the 100 (96%) CNKP isolates carried a carbapenemase gene, predominantly blaNDM. Raman typing revealed three major clusters among 48 Raman types identified, whereas MLVA distinguished six major clusters among a total of 30 different genotypes. CONCLUSIONS: NDM-producing CNKP are introduced into these ICUs and some strains expand clonally among patients and the environment, resulting in endemic CNKP. CNKP acquisition was associated with prolonged ICU stay and may affect ICU survival. TRIAL REGISTRATION: The study was registered at Netherlands Trial Register http://www.trialregister.nl. Can

BACKGROUND: Neonatal Staphylococcus aureus (S. aureus) bacteremia is an important cause of morbidity and mortality. In this study, we examined whether methicillin-susceptible S. aureus (MSSA) transmission and genetic makeup contribute to the occurrence of neonatal S. aureus bacteremia. METHODS: A retrospective, single-centre study was performed. All patients were included who suffered from S. aureus bacteremia in the neonatal intensive care unit (NICU), Erasmus MC-Sophia, Rotterdam, the Netherlands, between January 2011 and November 2017. Whole-genome sequencing (WGS) was used to characterize the S. aureus isolates, as was also done in comparison to reference genomes. Transmission was considered likely in case of genetically indistinguishable S. aureus isolates. RESULTS: Excluding coagulase-negative staphylococci (CoNS), S. aureus was the most common cause of neonatal bacteremia. Twelve percent (n = 112) of all 926 positive blood cultures from neonates grew S. aureus. Based on core genome multilocus sequence typing (cgMLST), 12 clusters of genetically indistinguishable MSSA isolates were found, containing 33 isolates in total (2-4 isolates per cluster). In seven of these clusters, at least two of the identified MSSA isolates were collected within a time period of one month. Six virulence genes were present in 98-100% of all MSSA isolates. In comparison to S. aureus reference genomes, toxin genes encoding staphylococcal enterotoxin A (sea) and toxic shock syndrome toxin 1 (tsst-1) were present more often in the genomes of bacteremia isolates. CONCLUSION: Transmission of MSSA is a contributing factor to the occurrence of S. aureus bacteremia in neonates. Sea and tsst-1 might play a role in neonatal S. aureus bacteremia.

Background: At present, phenotypic growth inhibition techniques are used in routine diagnostic microbiology to determine antimicrobial resistance of bacteria. Molecular techniques such as PCR are often used for confirmation but are indirect as they detect particular resistance genes. A direct technique would be able to detect the proteins of the resistance mechanism itself. In the present study targeted high resolution mass spectrometry assay was developed for the simultaneous detection of KPC, OXA-48-like, NDM, and VIM carbapenemases. Methods: Carbapenemase specific target peptides were defined by comparing available sequences in GenBank. Selected peptide sequences were validated using 62 Klebsiella pneumoniae and Escherichia coli isolates containing: 16 KPC, 21 OXA-48-like, 16 NDM, 13 VIM genes, and 21 carbapenemase negative isolates. Results: For each carbapenemase, two candidate peptides were validated. Method validation was performed in a blinded manner for all 83 isolates. All carbapenemases were detected. The majority was detected by both target peptides. All target peptides were 100% specific in the tested isolates and no peptide carry-over was detected. Conclusion: The applied targeted bottom-up mass spectrometry technique is able to accurately detect the four most prevalent carbapenemases in a single analysis.

Background: Carbapenem-nonsusceptible A. baumannii-calcoaceticus complex have emerged worldwide, but the epidemiology in Indonesian hospitals has not been studied. Methods: A prospective observational study was performed on the intensive care units (ICUs) of the national referral hospital in Jakarta-Indonesia, in 2013 and 2014. All consecutive adult patients admitted and hospitalized for >48 h in ICUs

Background: Antimicrobial resistance to ciprofloxacin is rising worldwide, especially in bacteria causing urinary tract infections (UTIs). Prudent use of current antibiotic drugs is therefore necessary. Objectives: We analysed (modifiable) risk factors for ciprofloxacin-resistant Escherichia coli. Methods: Urinary cultures of UTIs caused by E. coli were collected from participants in the Rotterdam Study, a prospective cohort study in an elderly population, and analysed for susceptibility to ciprofloxacin. Multivariate logistic regression was performed to investigate several possible risk factors for resistance. Results: Ciprofloxacin resistance in 1080 E. coli isolates was 10.2%. Multivariate analysis showed that higher age (OR 1.03; 95% CI 1.00-1.05) and use of two (OR 5.89; 95% CI 3.45-10.03) and three or more (OR 3.38; 95% CI 1.92-5.97) prescriptions of fluoroquinolones were associated with ciprofloxacin resistance, while no association between fluoroquinolone use more than 1 year before culture and ciprofloxacin resistance could be demonstrated. Furthermore, a high intake of pork (OR 3.68; 95% CI 1.36-9.99) and chicken (OR 2.72; 95% CI 1.08-6.85) and concomitant prescription of calcium supplements (OR 2.51; 95% CI 1.20-5.22) and proton pump inhibitors (OR 2.04; 95% CI 1.18-3.51) were associated with ciprofloxacin resistance. Conclusions: Ciprofloxacin resistance in community-acquired UTI was associated with a high intake of pork and chicken and with concomitant prescription of calcium supplements and proton pump inhibitors. Modification of antibiotic use in animals as well as temporarily stopping the prescription of concomitant calcium and proton pump inhibitors need further evaluation as strategies to prevent ciprofloxacin resistance.

SET-M33 is a multimeric antimicrobial peptide active against Gram-negative bacteria _in vitro_ and _in vivo_. Insights into its killing mechanism could elucidate correlations with selectivity. SET-M33 showed concentration-dependent bactericidal activity against colistin-susceptible and resistant isolates of _P. aeruginosa_ and _K. pneumoniae_. Scanning and transmission microscopy studies showed that SET-M33 generated cell blisters, blebs, membrane stacks and deep craters in _K.

Rapid bacterial species identification and antibiotic susceptibility testing in positive blood cultures have an important impact on the antibiotic treatment for patients. To identify extended-spectrum beta-lactamases (ESBL) directly in positive blood culture bottles, we developed a workflow of saponin extraction followed by a bottom-up proteomics approach using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The workflow was applied to positive blood cultures with Escherichia coli and Klebsiella pneumoniae collected prospectively in two academic hospitals over a 4-month period. Of 170 positive blood cultures, 22 (12.9%) contained ESBL-positive isolates based on standard susceptibility testing. Proteomic analysis identified CTX-M ESBLs in 95% of these isolates directly in positive blood cultures, whereas no false positives were found in the non-ESBL producing positive blood cultures. The results were confirmed by molecular characterisation of beta-lactamase genes. Based on this proof-of-concept study, we conclude that LC-MS/MS-based protein analysis can directly identify extended-spectrum beta lactamases in E. coli and K. pneumoniae positive blood cultures, and could be further developed for application in routine diagnostics.

Aminoacyl tRNA synthetases are enzymes involved in the key process of coupling an amino acid to its cognate tRNA. AN3365 is a novel antibiotic that specifically targets leucyl-tRNA synthetase, whose development was halted after evaluation in phase II clinical trials owing to the rapid selection of resistance. In an attempt to bring AN3365 back into the developmental pipeline we have evaluated the efficacy of AN3365 in combination with different classes of antibiotic and characterized its mechanism of action. Although we detect no synergy or antagonism in combination with a range of antibiotic classes, a combination of AN3365 with colistin reduces the accumulation of AN3365-resistant and colistin resistance mutations. We also demonstrate that treatment with AN3365 results in the dramatic accumulation of the alarmone (p)ppGpp, the effector of the stringent response—a key player in antibiotic tolerance.

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Background: Infections with multidrug-resistant (MDR) microorganisms are an increasing threat to hospitalized patients. Although rapid typing of MDR microorganisms is required to apply targeted prevention measures, technical barriers often prevent this. We aimed to assess whether extended-spectrum beta-lactamase (ESBL)-producing Klebsiella species are transmitted between patients and whether routine, rapid typing is needed. Methods: For 43 months, the clonality of all ESBL-producing Klebsiella isolates from patients admitted to Erasmus MC University Medical Center in Rotterdam, the Netherlands was assessed with Raman spectroscopy. A cluster was defined as n ≥2 patients who had identical isolates. Primary patients were the first patients in each cluster. Secondary patients were those identified with an isolate clonally related to the isolate of the primary patient. Results: Isolates from 132 patients were analyzed. We identified 17 clusters, with 17 primary and 56 secondary patients. Fifty-nine patients had a unique isolate. Patients (n = 15) in four out of the 17 clusters were epidemiologically related. Ten of these 15 patients developed an infection. Conclusions: Clonal outbreaks of ESBL-producing Klebsiella species were detected in our hospital. Theoretically, after Raman spectroscopy had detected a cluster of n ≥2, six infections in secondary patients could have been prevented. These findings demonstrate that spread of ESBLproducing Klebsiella species occurs, even in a non-outbreak setting, and underscore the need for routine rapid typing of these MDR bacteria.

Antimicrobial susceptibility was analyzed for 354 typhoidal Salmonella isolates collected during 1999-2012 in the Netherlands. In 16.1% of all isolates and in 23.8% of all isolates that showed increased MICs for ciprofloxacin, the MIC for azithromycin was i

Item does not contain fulltext, The emergence of decreased ciprofloxacin susceptibility (DCS) in Salmonella enterica serovar Typhi and serovar Paratyphi A, B or C limits treatment options. We studied the impact of DCS isolates on the fate of travellers returning with enteric fever and possible alternative treatment options. We evaluated the clinical features, susceptibility data and efficacy of empirical treatment in patients with positive blood cultures of a DCS isolate compared to patients infected with a ciprofloxacin-susceptible (CS) isolate in the period from January 2002 to August 2008. In addition, the pharmacokinetic and pharmacodynamic parameters of ciprofloxacin, levofloxacin and gatifloxacin were determined to assess if increasing the dose would result in adequate unbound fraction of the drug 24-h area under the concentration-time curve/minimum inhibitory concentration (fAUC0-24/MIC) ratio. Patients with DCS more often returned from the Indian subcontinent and had a longer fever clearance time and length of hospital stay compared to patients in whom the initial empirical therapy was adequate. The mean fAUC0-24/MIC was 41.3 +/- 18.8 in the patients with DCS and 585.4 +/- 219 in patients with a CS isolate. For DCS isolates, the mean fAUC0-24/MIC for levofloxacin was 60.5 +/- 28.7 and for gatifloxacin, it was 97.9 +/- 28.0. Increasing the dose to an adequate fAUC0-24/MIC ratio will lead to conceivably toxic drug levels in 50 % of the patients treated with ciprofloxacin. Emerging DCS isolates has led to the failure of empirical treatment in ill-returned travellers. We demonstrated that, in some cases, an adequate fAUC0-24/MIC ratio could be achieved by increasing the dose of ciprofloxacin or by the use of alternative fluoroquinolones.

Item does not contain fulltext, Coagulase-negative staphylococci (CoNS) isolated in neonatal late-onset sepsis are often antibiotic resistant. We analyzed CoNS from skin and feces of neonates during hospitalization. Antibiotic resistance of skin isolates increased during hospitalization, especially in Staphylococcus haemolyticus. Staphylococcus warneri showed low antibiotic resistance. Our data suggest that different CoNS species may play distinct roles in colonization.

Item does not contain fulltext, The efficacies of tigecycline and ceftazidime against fatal pneumonia in rats caused by an extended-spectrum beta-lactamase (ESBL)-positive Klebsiella pneumoniae strain or its wild-type (WT) progenitor were compared. Ceftazidime at 12.5 or 50 mg/kg of body weight twice daily (b.i.d.) was effective (50% or 100% rat survival) in pneumonia caused by the WT isolate but unsuccessful (100% rat mortality) in pneumonia caused by the ESBL-positive variant. In contrast, tigecycline at 6.25, 12.5, or 25 mg/kg b.i.d. showed dosage-dependent efficacy up to 100% rat survival irrespective of the ESBL character of the infecting organism.

A liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS) Escherichia coli strain, but during admission, a carbapenem-resistant (CR) E. coli strain was isolated. Analysis of the outer membrane protein profiles showed that both CS and CR E. coli lacked the porins OmpF and OmpC. Furthermore, PCR and sequence analysis revealed that both CS and CR E. coli possessed blaCTX-M-15 and blaOXA-1. The CR E. coli strain additionally harbored blaCMY-2 and demonstrated a>15-fold increase in-lactamase activity against nitrocefin, but no hydrolysis of meropenem was detected. However, nitrocefin hydrolysis appeared strongly inhibited by meropenem. Furthermore, the CMY-2 enzyme demonstrated lower electrophoretic mobility after its incubation either in vitro or in vivo with meropenem, indicative of its covalent modification with meropenem. The presence of the acyl-enzyme complex was confirmed by mass spectrometry. By transformation of the CMY-2-encoding plasmid into various E. coli strains, it was established that both porin deficiency and high-level expression of the enzyme were needed to confer meropenem resistance. In conclusion, carbapenem resistance emerged by a combination of elevated-lactamase production and lack of porin expression. Due to the reduced outer membrane permeability, only small amounts of meropenem can enter the periplasm, where they are trapped but not degraded by the large amount of the-lactamase. This study, therefore, provides evidence that the mechanism of trapping by CMY-2-lactamase plays a role in carbapenem resistance. Copyright

Coagulase-negative staphylococci (CoNS) isolated in neonatal late-onset sepsis are often antibiotic resistant. We analyzed CoNS from skin and feces of neonates during hospitalization. Antibiotic resistance of skin isolates increased during hospitalization, especially in Staphylococcus haemolyticus. Staphylococcus warneri showed low antibiotic resistance. Our data suggest that different CoNS species may play distinct roles in colonization. Copyright

1 november 2010, Contains fulltext : 89077.pdf (publisher's version ) (Open Access), Coagulase-negative staphylococci (CoNS) are a major cause of sepsis in neonatal intensive care units (NICU) worldwide. Infecting strains of these commensal bacteria may originate from NICU personnel. Therefore, we studied the characteristics of CoNS isolates from NICU personnel and compared them to those of isolates from the general population and from sepsis patients. Furthermore, we studied the epidemiological effect on CoNS carriage of NICU personnel after a period of absence. In our study, we isolated CoNS from the thumbs of NICU personnel every 2 weeks during the summer of 2005 and sampled personnel returning from vacation and a control group from the general population. Furthermore, we collected sepsis isolates from this period. Isolates were tested for antibiotic resistance, mecA and icaA carriage, biofilm production, and genetic relatedness. We found that mecA and icaA carriage as well as penicillin, oxacillin, and gentamicin resistance were significantly more prevalent in CoNS strains from NICU personnel than in community isolates. Similar trends were observed when postvacation strains were compared to prevacation strains. Furthermore, genetic analysis showed that 90% of the blood isolates were closely related to strains found on the hands of NICU personnel. Our findings revealed that CoNS carried by NICU personnel differ from those in the general population. Hospital strains are replaced by community CoNS after a period of absence. NICU personnel are a likely cause for the cross-contamination of virulent CoNS that originate from the NICU to patients.

Coagulase-negative staphylococci (CoNS) are a major cause of sepsis in neonatal intensive care units (NICU) worldwide. Infecting strains of these commensal bacteria may originate from NICU personnel. Therefore, we studied the characteristics of CoNS isolates from NICU personnel and compared them to those of isolates from the general population and from sepsis patients. Furthermore, we studied the epidemiological effect on CoNS carriage of NICU personnel after a period of absence. In our study, we isolated CoNS from the thumbs of NICU personnel every 2 weeks during the summer of 2005 and sampled personnel returning from vacation and a control group from the general population. Furthermore, we collected sepsis isolates from this period. Isolates were tested for antibiotic resistance, mecA and icaA carriage, biofilm production, and genetic relatedness. We found that mecA and icaA carriage as well as penicillin, oxacillin, and gentamicin resistance were significantly more prevalent in CoNS strains from NICU personnel than in community isolates. Similar trends were observed when postvacation strains were compared to prevacation strains. Furthermore, genetic analysis showed that 90% of the blood isolates were closely related to strains found on the hands of NICU personnel. Our findings revealed that CoNS carried by NICU personnel differ from those in the general population. Hospital strains are replaced by community CoNS after a period of absence. NICU personnel are a likely cause for the cross-contamination of virulent CoNS that originate from the NICU to patients.

Staphylococcus sciuri strains were unexpectedly cultured from healthy persons and patients from Indonesia during a population-based survey on nasal Staphylococcus aureus carriage. Fifty-one S. sciuri isolates were further characterized. The S. aureus mecA gene was detected by PCR in 22 isolates (43.1%), whereas S. sciuri mecA was found in 33 isolates (64.7%). The staphylococcal cassette chromosome mec (SCCmec) regions of S. aureus mecA-positive isolates contained elements of classical S. aureus SCCmec types II and/or III. Copyright

01 maart 2010, Item does not contain fulltext, BACKGROUND: No detailed reports regarding extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae are currently available from Indonesia, the fourth most populous country in the world. METHODS: A survey was carried out to investigate the molecular epidemiology and genetic characteristics of clinical ESBL-producing Escherichia coli and Klebsiella pneumoniae isolates originating from the Dr. Soetomo Academic Hospital in Surabaya, Indonesia, over a 4 month period (January to April 2005). ESBLs were characterized by isoelectric focusing and PCR assays. Clonality of the isolates was assessed by PFGE and repetitive-sequence-based PCR (rep-PCR). Phylogenetic grouping was performed among CTX-M-15-producing E. coli. RESULTS: In total, 73 consecutive non-duplicate ESBL-positive E. coli and 72 K. pneumoniae strains were isolated. The bla(CTX-M-15) gene was found to be highly prevalent (69/73 strains, 94.5%) among the 73 ESBL-positive E. coli isolates. The gene was detected in both clonal and non-clonal isolates, as defined by PFGE and rep-PCR. Sixteen CTX-M-15-positive E. coli could be assigned to a single rep-PCR type and phylogenetic group B2 and belonged to the well-known O25b-ST131 clone. Among the 72 ESBL-positive K. pneumoniae isolates, bla(CTX-M-15) was again the most prevalent ESBL (40/72, 55.6%). Several SHV-type enzymes were also frequently detected: SHV-5 (n = 28); SHV-12 (n = 13); and SHV-2 (n = 6). TEM-type ESBLs were not detected in any of the isolates. CONCLUSIONS: Indonesia is another developing country affected by the emergence and spread of bacterial strains harbouring ESBL genes, including the CTX-M-15-producing B2-E. coli O25b-ST131 clone.

The maximum recommended transport time for blood cultures is 4 h [L. S. Garcia (ed.), 2007 Update: Clinical Microbiology Procedures Handbook, 2nd ed., 2007]. In a previous study, we found that the average transport time was 10 h. In this cohort study, we measured transport times fo

The aim of this prospective randomized controlled clinical trial was to assess the impact of immediate incubation of blood cultures delivered to the laboratory outside its hours of operation on turnaround times, antibiotic prescription practices, and patient outcomes. A continuously monitoring blood culture incubator was placed outside the laboratory, which was switched on (intervention arm) and off (control arm) in a randomized manner. Included were new bacteremia episodes of patients older than 18 years. During the 30-week study period, the first positive blood culture specimen of an episode had to be brought to the laboratory outside its hours of operation. The med

Introduction: Rapid bacterial identification and susceptibility tests can lead to earlier microbiological diagnosis and pathogen-directed, appropriate therapy. We studied whether accelerated diagnostics affected antibiotic use and patient outcomes. Patients and methods: A prospective randomized clinical trial was performed over a 2-year period. Inpatients were selected on the basis of a positive culture from normally sterile body fluids and randomly assigned to either a rapid intervention arm or the control arm. The intervention arm used the Vitek 2 automated identification and susceptibility testing device, combined with direct inoculation of blood cultures. In the control arm, the Vitek 1 system inoculated from subcultures was used. Follow-up was 4 weeks after randomization. Results: A total of 1498 patients were randomized: 746 in the intervention arm and 752 in the control arm. For susceptibility testing, the rapid arm was 22 h faster than the control arm, and for identification, it was 13 h faster (P < 0.0001). In the rapid arm, antibiotic use was 6 defined daily doses lower per patient than in the control arm (P = 0.012). Whereas antibiotics were switched more in the rapid group on the day of randomization (P = 0.006), in the control group they were switched more on day two (P = 0.02). Mortality rates did not differ significantly between the two groups (17.6% versus 15.2%). Conclusions: While rapid bacterial identification and susceptibility testing led to earlier changes and a significant reduction in antibiotic use, they did not reduce mortality.

The pharmacokinetic/pharmacodynamic (PK/PD) indices that define the therapeutic effect of the betalactam ceftazidime in a rat model of Klebsiella pneumoniae lung infection were investigated in relation to treatment duration and treatment endpoint. Treatment was started 24 h after infection with dosing regimens of 3.1 up to 1,600 mg/kg of body weight/day and dosing every 6, 12, or 24 h. When animals were treated for a relatively short period of 48 h, the duration of time that unbound

Contains fulltext : 32756.pdf (publisher's version ) (Open Access), A randomized double-blind trial with a 7-valent pneumococcal conjugate vaccine was conducted in The Netherlands among 383 children, aged 1 to 7 years, with a history of recurrent acute otitis media. No effect of vaccination on the pneumococcal colonization rate was found. However, a shift in serotype distribution was clearly observed (R. Veenhoven et al., Lancet 361:2189-2195, 2003). We investigated the molecular epidemiology of 921 pneumococcal isolates retrieved from both the pneumococcal vaccine (PV) and control vaccine (CV) groups during the vaccination study. Within individuals a high turnover rate of pneumococcal restriction fragment end labeling genotypes, which was unaffected by vaccination, was observed. Comparison of the genetic structures before and after completion of the vaccination scheme revealed that, despite a shift in serotypes, there was clustering of 70% of the pneumococcal populations. The remaining isolates (30%) were equally observed in the PV and CV groups. In addition, the degree of genetic clustering was unaffected by vaccination. However, within the population genetic structure, nonvaccine serotype clusters with the serotypes 11, 15, and 23B became predominant over vaccine-type clusters after vaccination. Finally, overall pneumococcal resistance was low (14%), and, albeit not significant, a reduction in pneumococcal resistance as a result of pneumococcal vaccination was observed. Molecular surveillance of colonization in Dutch children shows no effect of pneumococcal conjugate vaccination on the degree of genetic clustering and the genetic structure of the pneumococcal population. However, within the genetic pneumococcal population structure, a clear shift toward nonvaccine serotype clusters was observed.

A randomized double-blind trial with a 7-valent pneumococcal conjugate vaccine was conducted in The Netherlands among 383 children, aged 1 to 7 years, with a history of recurrent acute otitis media. No effect of vaccination on the pneumococcal colonization rate was found. However, a shift in serotype distribution was clearly observed (R. Veenhoven et al., Lancet 361:2189-2195, 2003). We investigated the molecular epidemiology of 921 pneumococcal isolates retrieved from both the pneumococcal vaccine (PV) and control vaccine (CV) groups during the vaccination study. Within individuals a high turnover rate of pneumococcal restriction fragment end labeling genotypes, which was unaffected by vaccination, was observed. Comparison of the genetic structures before and after completion of the vaccination scheme revealed that, despite a shift in serotypes, there was clustering of 70% of the pneumococcal populations. The remaining isolates (30%) were equally observed in the PV and CV groups. In addition, the degree of genetic clustering was unaffected by vaccination. However, within the population genetic structure, nonvaccine serotype clusters with the serotypes 11, 15, and 23B became predominant over vaccine-type clusters after vaccination. Finally, overall pneumococcal resistance was low (14%), and, albeit not significant, a reduction in pneumococcal resistance as a result of pneumococcal vaccination was observed. Molecular surveillance of colonization in Dutch children shows no effect of pneumococcal conjugate vaccination on the degree of genetic clustering and the genetic structure of the pneumococcal population. However, within the genetic pneumococcal population structure, a clear shift toward nonvaccine serotype clusters was observed.

The performances of the BDProbeTec ET (Becton Dickinson) and COBAS AMPLICOR MTB (Roche) were retrospectively evaluated for detecting Mycobacterium tuberculosis complex in various respiratory specimens. The BACTEC and MGIT liquid culture system (Becton Dickinson) was used as a reference method. A total of 824 respiratory specimens, comprised of sputa, bronchoalveolar lavage fluid, and bronchial and tracheal aspirates from 580 patients, were evaluated. Out of 824 clinical specimens, 109 specimens from 43 patients were culture positive for M. tuberculosis. Of these 109 specimens, 67 were smear positive, 85 were positive by the COBAS AMPLICOR MTB test, and 94 were positive by the BDProbeTec ET. Of the 715 culture-negative specimens, 17 were positive by the auramine staining, 11 were positive by the COBAS AMPLICOR MTB test, and 12 were positive by the BDProbeTec ET. After discrepancy analysis and review of the patients' clinical data, 130 specimens from 50 patients were considered "true-positive" specimens. This resulted in the following sensitivities: microscopy, 61.5%; COBAS AMPLICOR MTB test, 78.0%; and BDProbeTec ET, 86.2%. The specificities of each system, based on the clinical diagnosis, were 99.7% for microscopy, 99.9% for the COBAS AMPLICOR MTB test, and 99.9% for the BDProbeTec ET. The data presented represent a considerable number of specimens evaluated with a considerable number of culture- and auramine-positive and culture-positive and auramine-negative results and therefore give a realistic view of how the data should be interpreted in a daily routine situation. Specifically, the data with regard to the culture-positive and auramine-negative specimens are useful, because in a routine situation, auramine-negative specimens are sometimes accepted, on clinical indications, to be analyzed by an amplification method.

The effects of ceftizoxime (CZX), piperacillin (PIP), and PIP-tazobactam (PT) concentrations on the antibacterial activity and selection of resistant mutants of Bacteroides fragilis and Enterobacter cloacae were investigated in vitro in a mixed-culture anaerobic time-kill study and in vivo in a mixed-infection abscess model. Mixed cultures were incubated for 24 h with 0.125 to 512 micro g of CZX per ml or 0.125 to 2,048 micro g of PIP or PT per ml. Mice were treated every 2 h for 24 h with CZX at 6 to 1,536 mg/kg/day or with PIP or PT at 24 to 6,144 mg/kg/day starting 30 min before inoculation with different B. fragilis-E. cloacae combinations. There was a good correlation between the in vitro and in vivo activities of the antibiotics and their MICs obtained with high inocula (10(8) CFU/ml). The respective 50% effective doses (milligrams per kilogram per day) with B. fragilis and E. cloacae 22491 were 771 and 521 for CZX, 416 and 643 for PIP, and 85 and 554 for PT, and with the B. fragilis-E. cloacae 032349 combination, they were 81 and 21 for CZX and 77 and 766 for PT. Resistant mutants of E. cloacae 22491 were preferentially selected in vitro with 2 to 64 micro g of CZX per ml and in vivo with CZX at 12 to 384 mg/kg/day. There was no preferential selection of CZX-resistant B. fragilis or E. cloacae 032349. For CZX-resistant E. cloacae 22491, we found a 16- to 512-fold increase in the MIC of CZX and increased MICs of other expanded-spectrum cephalosporins, owing in part to the production of a stably derepressed cephalosporinase. In vitro and in vivo, PT did not select resistant mutants of E. cloacae and B. fragilis. Results demonstrate the adverse microbiological outcome of choosing an expanded-spectrum cephalosporin like CZX for empirical treatment of mixed infections involving a susceptible Enterobacter strain.

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To determine the efficacy of trovafloxacin as a possible treatment for intra-abdominal abscesses, we have developed an anaerobic time-kill technique using different inocula to study the in vitro killing of Bacteroides fragilis in pure culture or in mixed culture with either Escherichia coli or a vancomycin-resistant strain of Enterococcus faecium (VREF). With inocula of 5 x 10(5) CFU/ml and trovafloxacin concentrations of /=6.1 (log(10) CFU/ml) was attained with all pure and mixed cultures within 24 h. With inocula of 10(8) CFU/ml, a similar E(max) and a similar concentration to produce 50% of E(max) (EC(50)) for B. fragilis were found in both pure cultures and mixed cultures with E. coli. However, to produce a similar killing of B. fragilis in the mixed cultures with VREF, a 14-fold increase in the concentration of trovafloxacin was required. A vancomycin-susceptible strain of E. faecium and a trovafloxacin-resistant strain of E. coli were also found to confer a similar "protective" effect on B. fragilis against the activity of trovafloxacin. Using inocula of 10(9) CFU/ml, the activity of trovafloxacin was retained for E. coli and B. fragilis and was negligible against VREF. We conclude that this is a useful technique to study the anaerobic killing of mixed cultures in vitro and may be of value in predicting the killing of mixed infections in vivo. The importance of using mixed cultures and not pure cultures is clearly shown by the difference in the killing of B. fragilis in the mixed cultures tested. Trovafloxacin will probably be ineffective in the treatment of infections involving large numbers of enterococci. However, due to its ability to retain activity against large cultures of B. fragilis and E. coli, trovaflo

The pharmacodynamic and pharmacokinetic properties of trovafloxacin were studied in a standardized murine model of established subcutaneous abscesses. Daily dosing regimens of 37.5 to 300 mg/kg every 8 h (q8h) or every 24 h (q24h) were started 3 days after inoculation with mixtures containing either Bacteroides fragilis-Escherichia coli-autoclaved cecal contents (ACC) or B. fragilis-vancomycin-resistant Enterococcus faecium (VREF)-ACC. Treatment was continued for 3 or 5 days. The efficacy of treatment was determined by the decrease in abscess bacterial counts and abscess wei

We evaluated the accuracy of the VITEK 2 fully automated system to detect and identify glycopeptide-resistant enterococci (GRE) compared to a reference agar dilution method. The sensitivity of vancomycin susceptibility testing with VITEK 2 for the detection of vanA, vanB, and vanC1 strains was 100%. The sensitivity of vancomycin susceptibility testing of vanC2 strains was 77%. The sensitivity of teicoplanin susceptibility testing of vanA strains was 9