Use of other antimicrobial drugs is associated with trimethoprim resistance in patients with urinary tract infections caused by E. coli.

In recent years, high frequencies of trimethoprim resistance in urinary tract infections (UTIs) caused by E. coli are have been reported. Co-resistance to other antimicrobial drugs may play a role in this increase. Therefore, we investigated whether previous use of other antimicrobial drugs was associated with trimethoprim resistance. We conducted a nested case-control study with urinary cultures with E. coli from participants of the Rotterdam Study sent in by general practitioners to the regional laboratory between 1 January 2000 and 1 April 2016. Multivariable logistic regression analysis was performed to study the association between prior prescriptions of several antimicrobial drug groups and trimethoprim resistance using individual participant data. Urinary cultures of 1264 individuals with a UTI caused by E. coli were included. When adjusted for previous other antimicrobial drug use, a history of > 3 prescriptions of extended-spectrum penicillins (OR 1.68; 95% CI 1.10-2.55) was significantly associated with trimethoprim resistance of E. coli as was the use of > 3 prescriptions of sulfonamides and trimethoprim (OR 2.22; 95% CI 1.51-3.26). The use of > 3 prescriptions of nitrofuran derivatives was associated with a lower frequency of trimethoprim resistance (OR 0.60; 95% CI 0.39-0.92), after adjustment for other antimicrobial drug prescriptions. We found that previous use of extended-spectrum penicillins is associated with trimethoprim resistance. On the contrary, previous nitrofurantoin use was associated with a lower frequency of trimethoprim resistance. Especially in individuals with recurrent UTI, co-resistance should be taken into account and susceptibility testing before starting trimethoprim should be considered.

Direct detection of extended-spectrum beta-lactamases (CTX-M) from blood cultures by LC-MS/MS bottom-up proteomics.

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.

Evaluation of the characteristics of leucyl-tRNA synthetase (LeuRS) inhibitor AN3365 in combination with different antibiotic classes.

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.

Decreased ciprofloxacin susceptibility in Salmonella Typhi and Paratyphi infections in ill-returned travellers: the impact on clinical outcome and future treatment options.

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 (ƒAUC(0-24)/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 ƒAUC(0-24)/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 ƒAUC0-24/MIC for levofloxacin was 60.5 ± 28.7 and for gatifloxacin, it was 97.9 ± 28.0. Increasing the dose to an adequate ƒAUC(0-24)/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 ƒAUC(0-24)/MIC ratio could be achieved by increasing the dose of ciprofloxacin or by the use of alternative fluoroquinolones.

Prevalence of and risk factors for extended-spectrum beta-lactamase genes carriership in a population-based cohort of middle-aged and elderly.

INTRODUCTION: Increasing resistance to beta-lactam antibiotics is an alarming development worldwide. Fecal carriership of TEM, SHV, CTX-M and CMY was studied in a community-dwelling population of middle-aged and elderly individuals. PATIENTS AND METHODS: Feces was obtained from individuals of the Rotterdam Study. Carriership of the TEM, SHV, CTX-M and CMY genes was determined using real-time polymerase chain reaction (qPCR). Possible associations were investigated between carriership of these genes and several risk factors, such as the use of antimicrobial drugs, diabetes mellitus, protein pump inhibitor (PPI) use, travelling, the composition of the gut microbiota, and intake of certain foods. RESULTS: The most prevalent gene was TEM (53.0%), followed by SHV (18.4%), CTX-M (5.4%) and CMY (3.6%). Use of penicillins with extended spectrum was associated with TEM carriership, whereas use of macrolides and lincosamides was associated with TEM and SHV carriership. Interestingly, use of PPIs was associated with a higher prevalence of carriership of TEM, SHV and CMY (TEM: odds ratio [OR] 1.34; 95% confidence interval [CI] 1.05-1.77; SHV: OR 2.17; 95%CI 1.55-2.87; CMY: OR 2.26; 95%CI 1.23-4.11). Furthermore, associations were found between the richness and composition of the gut microbiota and TEM and SHV carriership. CONCLUSIONS: The prevalence of carriership of TEM was substantial, but the prevalence of carriership of the extended-spectrum ß-lactamase gene, CTX-M and the AmpC ß-lactamase gene, CMY was relatively low in this community-dwelling, population-based cohort. The composition of the microbiota might play a role in the retention of resistance genes, but future studies are necessary to further elucidate this relationship.

Nasal carriage of methicillin-resistant and methicillin-sensitive strains of Staphylococcus sciuri in the Indonesian population.

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.

Differentiation between Streptococcus pneumoniae and other viridans group streptococci by matrix-assisted laser desorption/ionization time of flight mass spectrometry.

OBJECTIVES: Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is becoming the method of choice for bacterial identification. However, correct identification by MALDI-TOF of closely related microorganisms such as viridans streptococci is still cumbersome, especially in the identification of S. pneumoniae. By making use of additional spectra peaks for S. pneumoniae and other viridans group streptococci (VGS). We re-identified viridans streptococci that had been identified and characterized by molecular and phenotypic techniques by MALDI-TOF. METHODS: VGS isolates (n = 579), 496 S. pneumoniae and 83 non-S. pneumoniae were analysed using MALDI-TOF MS and the sensitivity and specificity of MALDI-TOF MS was assessed. Hereafter, mass spectra analysis was performed. Presumptive identification of proteins represented by discriminatory peaks was performed by molecular weight matching and the corresponding nucleotides sequences against different protein databases. RESULTS: Using the Bruker reference library, 495 of 496 S. pneumoniae isolates were identified as S. pneumoniae and one isolate was identified as non-S. pneumoniae. Of the 83 non-S. pneumoniae isolates, 37 were correctly identified as non-S. pneumoniae, and 46 isolates as S. pneumoniae. The sensitivity of the MALDI-TOF MS was 99.8% (95% confidence interval (CI) 98.9-100) and the specificity was 44.6% (95% CI 33.7-55.9). Eight spectra peaks were mostly present in one category (S. pneumoniae or other VGS) and absent in the other category and inversely. Two spectra peaks of these (m/z 3420 and 3436) were selected by logistic regression to generate three identification profiles. These profiles could differentiate between S. pneumoniae and other VGS with high sensitivity and specificity (99.4% and 98.8%, respectively). CONCLUSIONS: Spectral peaks analysis based identification is a powerful tool to differentiate S. pneumoniae from other VGS species with high specificity and sensitivity and is a useful method for pneumococcal identification in carriage studies. More research is needed to further confirm our findings. Extrapolation of these results to clinical strains need to be deeply investigated.

Needle-to-incubator transport time: logistic factors influencing transport time for blood culture specimens.

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 for blood cultures in a larger sample and identified predictors for transport times. A total of 4,322 blood cultures from 1,313 patients were included. The median transport time was 3.5 h, with 47% of cultures exceeding the recommended 4 h. Off-site location and type of clinical specialty were the most important predictors of long transport times. Cultures collected during weekend days or on wards at the largest distances from the laboratory were also associated with long transport times.

Immediate incubation of blood cultures outside routine laboratory hours of operation accelerates antibiotic switching.

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 median time from specimen collection until growth detection was reduced by 10.1 h in the intervention arm (P < 0.001). For 46 of 66 (70%) episodes in the intervention arm and for 51 of 85 (60%) episodes in the control arm, the antibiotic regimen was changed (not significant). The median time until the first change in the antibiotic regimen was 42.8 h in the intervention arm and 64.0 h in the control arm (P, 0.024). There was no difference in length of stay or hospital mortality. Immediate incubation of blood cultures outside laboratory hours reduces turnaround times and accelerates antibiotic switching.

Diet as a risk factor for antimicrobial resistance in community-acquired urinary tract infections in a middle-aged and elderly population: a case-control study.

OBJECTIVES: There is an ongoing debate as to what extent antimicrobial resistance (AMR) can be transmitted from animals to humans via the consumption of animal products. Because epidemiological data on the role of diet in AMR in humans are lacking, we investigated this association between diet and AMR for different antimicrobial drugs in Escherichia coli (E. coli) in urinary tract infections (UTIs). METHODS: Susceptibility of E. coli in urinary cultures and information on diet (with food frequency questionnaires) were obtained from participants of the Rotterdam study, a population-based prospective cohort study. The association between intake of several food groups (meat, seafood, eggs, dairy products, crops) and resistance of E. coli to several antimicrobial drugs (amoxicillin, amoxicillin-clavulanic acid, trimethoprim, sulfamethoxazole-trimethoprim, first-generation cephalosporins, cefotaxime, nitrofurantoin, norfloxacin) was studied. RESULTS: Urinary cultures with E. coli were obtained from 612 individuals, of whom 481 (78.6%) were women. Resistance rates varied from 246/611 (40.3%) for amoxicillin and 167/612 (27.3%) for trimethoprim to only 29/612 (4.7%) for nitrofurantoin and 16/462 (3.5%) for cefotaxime. A higher intake of chicken was associated with cefotaxime resistance (OR 2.18; 95% CI 1.05-4.51 per tertile increase); a higher intake of pork was associated with norfloxacin resistance (OR 1.42; 95% CI 1.04-1.95 per quartile increase). In contrast, a higher intake of cheese was associated with lower AMR to amoxicillin (OR 0.84; 95% CI 0.72-0.99 per quartile increase) and amoxicillin-clavulanic acid (OR 0.67; 95% CI 0.53-0.86 per quartile increase). CONCLUSIONS: These findings support the hypothesis that diet may play a role in the AMR of E. coli in UTIs.

Rapid identification and antimicrobial susceptibility testing reduce antibiotic use and accelerate pathogen-directed antibiotic use.

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.

Unexpected mechanisms of resistance in Dutch Pseudomonas aeruginosa isolates collected during 14 years of surveillance.

Pseudomonas aeruginosa is one of the most important causes of infection in intensive care units (ICUs). It is intrinsically resistant to many antimicrobials and easily acquires additional resistance genes via horizontal gene transfer of mobile genetic elements. In this study, 1528 P. aeruginosa isolates obtained from a Dutch national surveillance programme between the years 1998-2011 were analysed for the presence of extended-spectrum ß-lactamase (ESBL) genes (blaCTX-M, blaSHV, blaTEM, blaBEL, blaPER, blaVEB and blaOXA-10) and metallo-ß-lactamase (MBL) genes (blaIMP, blaVIM and blaNDM). Of the ceftazidime-resistant isolates, 6.2% tested phenotypically positive for ESBL. Moreover, a Verona integron-encoded MBL (VIM) gene was found in 3.1% of isolates that were phenotypically resistant to imipenem and/or meropenem. Multilocus sequence typing (MLST) of ESBL-positive isolates indicated ST1216, ST111 and ST622, with all blaVIM-positive isolates belonging to the ST111 clone. Although the prevalence of ESBL and MBL phenotypes in this Dutch national surveillance collection of >1500 ICU P. aeruginosa isolates was very low, all VIM-producing isolates belonged to the high risk-associated, international, clonal complex CC111, and most ESBL-producing isolates belonged to clonal complexes known for their successful spread, e.g. CC111 and CC235. These data indicate that high-risk clones of P. aeruginosa were present in the Netherlands between 1998-2011 and probably spread unnoticed throughout Dutch hospitals.

[Antibiotic resistance: epidemiological developments and preventive measures]. / Antibioticumresistentie: epidemiologische ontwikkelingen en preventieve maatregelen.

European countries differ with regard to the occurrence of resistant micro-organisms. This indicates that the development of resistance can be influenced. Resistance levels are low in the Netherlands, but the resistance to various antibiotics is increasing. Factors that are known to contribute to antibiotic resistance are: the dosage and duration of antibiotic exposure and the type of antibiotic used in combination with a specific micro-organism. The selection pressure can be decreased by making use of antibiotics with a narrow spectrum. A new pharmacodynamic finding is that the dosage of antibiotics can be regulated in a way that minimises the selection of resistant clones. Implementation ofnational guidelines for the treatment of infections will contribute to more efficient use of antibiotics and the control of antibiotic resistance in the Netherlands.

Antimicrobial resistance trends in blood culture positive Salmonella Paratyphi A isolates from Pondicherry, India.

Enteric fever is a public health problem with the upsurge in the occurrence of Salmonella isolates that are resistant to ciprofloxacin. In this study, a total of 284 blood culture isolates of S. Paratyphi A were investigated. Of these isolates, 281 (98.9%) were nalidixic acid resistant. A high rate (6.3%) of high-level resistance (≥ 4 µg/mL) was found to ciprofloxacin. The isolates with ciprofloxacin minimum inhibitory concentrations (MICs) of ≥ 12 µg/mL had 4 mutations, 2 mutations within the quinolone resistance-determining region of gyrA and 2 mutations also in parC. According to the Clinical Laboratory Standards Institute 2012 MIC breakpoints, 75.0% of isolates were resistant to ciprofloxacin. Finally, 3 major pulsed-field gel electrophoresis patterns were observed among the S. Paratyphi A isolates. The spread of fluoroquinolone resistant S. Paratyphi A necessitates a change toward 'evidence-based' treatment for enteric fever. The research provides a perspective on the increasing prevalence of antimicrobial resistant S. Paratyphi A isolates in this region of India.

Monitoring persistence of coagulase-negative staphylococci in a hematology department using phenotypic and genotypic strategies.

OBJECTIVE: To determine persistence of coagulase-negative staphylococci (CNS) on a hematology-oncology ward and to determine the value of phenotypic and genotypic procedures for establishing clonality among CNS isolates. DESIGN: Strains of CNS isolated from bacteremic patients (n = 139) were typed by biochemical reactivity, antibiotic susceptibility, DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE), and arbitrary primed polymerase chain reaction (AP PCR). Coagulase-negative staphylococci were subgrouped in a random collection (n = 20) used for the evaluation of the typing procedures and a collection of 119 CNS isolates from hematologic patients displaying multiple bacteremic episodes. RESULTS: Analysis of the reference collection demonstrated the usefulness of the DNA typing procedures, indicating that AP PCR and PFGE can be used for epidemiologic typing of CNS in a concordant fashion. Certain strains appeared to be permanent colonizers of the hematology ward or ward-related personnel. In individual patients, persistent colonization by a single type was demonstrated. However, a number of patients also experienced bacteremic episodes caused by CNS belonging to different types. CONCLUSION: We conclude that monitoring of CNS infections on a hematology ward by various genotypic techniques provides insight into nosocomial epidemiology and elucidates the complexity of the infections taking place. DNA typing is preferred over phenotypic procedures and can identify persistent CNS strains in a given location.

Molecular epidemiology of drug-resistant pneumococci: toward an international approach.

An international multicenter study was undertaken to investigate the epidemiological dynamics of penicillin-resistant pneumococci. We compared the molecular epidemiological characteristics of 205 penicillin-resistant isolates originating from The Netherlands, Thailand, United States, Spain, Greece, Poland, Cuba, Germany, Finland, United Kingdom, South Africa, Hungary, Portugal, Croatia, and the Czech Republic. Eighty-four distinct restriction fragment end labeling (RFEL) types were observed. Twenty-eight genetic types were shared by two or more strains. Five genetic clusters consisted of strains originating from different countries, illustrating dissemination of penicillin-resistant pneumococci among countries. The strains displaying the two predominant RFEL types corresponding with the pandemic clones 23F and 9V were found in 10 and 6 different countries, respectively. This clearly demonstrates the pandemic behavior of these two clones. Twelve out of the 28 genetic clusters contained two or more serotypes. This finding indicates frequent horizontal transfer of capsular genes. Within distinct RFEL types, identical penicillin binding protein (PBP) genotypes were often observed, suggesting a high frequency of horizontal transfer of penicillin resistance genes. The most predominant PBP type was found in 15 distinct RFEL types, comprised 44% of the entire collection, and was observed in 11 countries. The vast majority of the strains belonging to the pandemic clones 23F and 9V shared this predominant PBP type. We hypothesize that the clones 23F and 9V are responsible for the worldwide increase of penicillin-resistance, because they serve as a genetic reservoir for susceptible pneumococci to acquire penicillin resistance.

Investigation into the repeated recovery of coagulase-negative staphylococci from blood taken at the end of cardiopulmonary by-pass.

Forty-six strains of coagulase-negative staphylococci (CNS) were typed by biochemical reactivity, antibiotic susceptibility pattern, macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) and arbitrary primed polymerase chain reaction (AP PCR). Twenty-four strains were obtained in 1993 from blood cultures of as many patients coupled to a heart-lung machine during cardiac surgery. Since over 30% of the latter belonged to a single type, it was concluded that during the year of analysis a single clone of CNS persisted in this hospital setting. Subsequent epidemiological surveillance putatively identified four possible carriers among surgical personnel. For this reason, 22 strains collected from the hands and nose of two cardiac surgeons and two perfusionists were also tested; none were identical to the persistent clone. Thus either the operation equipment was colonized longitudinally or the causative CNS had disappeared from the suspect individuals' flora. Longitudinal monitoring of CNS infections by various techniques gives a valuable insight into nosocomial epidemiology and elucidates the complexity of the CNS colonization.

Towards a better diagnosis of throat infections (with group A beta-haemolytic streptococcus) in general practice.

BACKGROUND: Sore throat is a common complaint in general practice. However, management strategies are not very clear. A better diagnostic procedure is needed to prevent the overuse of antibiotics. AIM: To assess the diagnostic value of a rapid streptococcal antigen detection test in addition to four clinical features in patients with sore throat, using throat culture and antibody titres as reference tests. METHOD: Four clinical features [fever (history) > or = 38.0 degrees C, lack of cough, tonsillar exudate, and anterior cervical lymphadenopathy] were registered in 558 patients aged 4 to 60 years presenting with sore throat of no more than 14 days' duration. A rapid diagnostic test was performed, as well as a throat culture and antibody titres [fourfold increase in anti-streptolysin-O (ASO) and/or anti-deoxyribonuclease B (anti-DNAase B)] in patients aged 11 years and older. RESULTS: Throat cultures were positive for group A beta-haemolytic streptococcus (GABHS) in 33% of the patients. Rapid tests were positive in 24%. Compared with the throat culture, the sensitivity of the rapid test was 65%, the specificity 96%, the positive predictive value 88%, and the negative predictive value 85%. However, for patients with three or four clinical features, the sensitivity of the rapid test was considerably higher at 75%. Children (< or = 14 years) had a slightly raised specificity and raised positive predictive value and prevalence. With the antibody titres as a reference, the rapid test performed as well as the throat culture with regard to its predictive value. CONCLUSION: For the management of patients with sore throat in general practice, a rapid test may have an additional value, especially in patients with a high chance of having GABHS infection. However, as the sensitivity of the test studied is low, tests with a higher sensitivity are needed.

Whole-genome mapping for high-resolution genotyping of Pseudomonas aeruginosa.

A variety of molecular typing techniques have been developed to investigate the clonal relationship among bacterial isolates, including those associated with nosocomial infections. In this study, the authors evaluated whole-genome mapping as a tool to investigate the genetic relatedness between Pseudomonas aeruginosa isolates, including metallo beta-lactamase-positive outbreak isolates.