Antimicrobial susceptibility profile of clinically relevant Bacteroides, Phocaeicola, Parabacteroides and Prevotella species, isolated by eight laboratories in the Netherlands.

OBJECTIVES: Recently, reports on antimicrobial-resistant Bacteroides and Prevotella isolates have increased in the Netherlands. This urged the need for a surveillance study on the antimicrobial susceptibility profile of Bacteroides, Phocaeicola, Parabacteroides and Prevotella isolates consecutively isolated from human clinical specimens at eight different Dutch laboratories. METHODS: Each laboratory collected 20-25 Bacteroides (including Phocaeicola and Parabacteroides) and 10-15 Prevotella isolates for 3 months. At the national reference laboratory, the MICs of amoxicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, meropenem, imipenem, metronidazole, clindamycin, tetracycline and moxifloxacin were determined using agar dilution. Isolates with a high MIC of metronidazole or a carbapenem, or harbouring cfiA, were subjected to WGS. RESULTS: Bacteroides thetaiotaomicron/faecis isolates had the highest MIC90 values, whereas Bacteroides fragilis had the lowest MIC90 values for amoxicillin/clavulanic acid, piperacillin/tazobactam, meropenem, imipenem and moxifloxacin. The antimicrobial profiles of the different Prevotella species were similar, except for amoxicillin, for which the MIC50 ranged from 0.125 to 16 mg/L for Prevotella bivia and Prevotella buccae, respectively. Three isolates with high metronidazole MICs were sequenced, of which one Bacteroides thetaiotaomicron isolate harboured a plasmid-located nimE gene and a Prevotella melaninogenica isolate harboured a nimA gene chromosomally.Five Bacteroides isolates harboured a cfiA gene and three had an IS element upstream, resulting in high MICs of carbapenems. The other two isolates harboured no IS element upstream of the cfiA gene and had low MICs of carbapenems. CONCLUSIONS: Variations in resistance between species were observed. To combat emerging resistance in anaerobes, monitoring resistance and conducting surveillance are essential.

Urine flow cytometry can rule out urinary tract infection, but cannot identify bacterial morphologies correctly.

The diagnosis of urinary tract infection (UTI) by urine culture is a time-consuming and costly procedure. Usage of a screening method, to identify negative samples, would therefore affect time-to-diagnosis and laboratory cost positively. Urine flow cytometers are able to identify particles in urine. Together with the introduction of a cut-off value, which determines if a urine sample is subsequently cultured or not, the number of cultures can be reduced, while maintaining a low level of false negatives and a high negative predictive value. Recently, Sysmex developed additional software for their urine flow cytometers. Besides measuring the number of bacteria present in urine, information is given on bacterial morphology, which may guide the physician in the choice of antibiotic. In this study, we evaluated this software update. The UF1000i classifies bacteria into two categories: 'rods' and 'cocci/mixed'. Compared to the actual morphology of the bacterial pathogen found, the 'rods' category scores reasonably well with 91% chance of classifying rod-shaped bacteria correctly. The 'cocci/mixed' category underperforms, with only 29% of spherical-shaped bacteria (cocci) classified as such. In its current version, the bacterial morphology software does not classify bacteria, according to their morphology, well enough to be of clinical use in this study population.

An evaluation of three processing methods and the effect of reduced culture times for faster direct identification of pathogens from BacT/ALERT blood cultures by MALDI-TOF MS.

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method for the identification of bacteria from agar media. Direct identification from positive blood cultures should decrease the time to obtaining the result. In this study, three different processing methods for the rapid direct identification of bacteria from positive blood culture bottles were compared. In total, 101 positive aerobe BacT/ALERT bottles were included in this study. Aliquots from all bottles were used for three bacterial processing methods, i.e. the commercially available Bruker's MALDI Sepsityper kit, the commercially available Molzym's MolYsis Basic5 kit and a centrifugation/washing method. In addition, the best method was used to evaluate the possibility of MALDI application after a reduced incubation time of 7 h of Staphylococcus aureus- and Escherichia coli-spiked (1,000, 100 and 10 colony-forming units [CFU]) aerobe BacT/ALERT blood cultures. Sixty-six (65%), 51 (50.5%) and 79 (78%) bottles were identified correctly at the species level when the centrifugation/washing method, MolYsis Basic 5 and Sepsityper were used, respectively. Incorrect identification was obtained in 35 (35%), 50 (49.5%) and 22 (22%) bottles, respectively. Gram-positive cocci were correctly identified in 33/52 (64%) of the cases. However, Gram-negative rods showed a correct identification in 45/47 (96%) of all bottles when the Sepsityper kit was used. Seven hours of pre-incubation of S. aureus- and E. coli-spiked aerobe BacT/ALERT blood cultures never resulted in reliable identification with MALDI-TOF MS. Sepsityper is superior for the direct identification of microorganisms from aerobe BacT/ALERT bottles. Gram-negative pathogens show better results compared to Gram-positive bacteria. Reduced incubation followed by MALDI-TOF MS did not result in faster reliable identification.

Acceleration of the direct identification of Staphylococcus aureus versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods.

To accelerate differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CNS), this study aimed to compare six different DNA extraction methods from two commonly used blood culture materials, i.e. BACTEC and BacT/ALERT. Furthermore, we analysed the effect of reduced blood culture incubation for the detection of staphylococci directly from blood culture material. A real-time polymerase chain reaction (PCR) duplex assay was used to compare the six different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with methicillin-resistant S. aureus (MRSA). Bacterial DNA was isolated with automated extractor easyMAG (three protocols), automated extractor MagNA Pure LC (LC Microbiology Kit M(Grade)), a manual kit MolYsis Plus and a combination of MolYsis Plus and the easyMAG. The most optimal isolation method was used to evaluate reduced bacterial incubation times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the easyMAG resulted in the most sensitive detection of S. aureus, with a detection limit of 10 CFU/ml, in BacT/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S. aureus or CNS load of 1 CFU/ml blood can be detected after 5 h of incubation in BacT/ALERT 3D by combining the sensitive isolation method and the tuf LightCycler assay.

Capnocytophaga canimorsus infections in The Netherlands: a nationwide survey.

A retrospective nationwide survey on the occurrence of Capnocytophaga canimorsus and Capnocytopaga cynodegmi infections in The Netherlands over 3 years showed 32 cases, of which 31 were caused by C. canimorsus and one by an unspecified oxidase-positive Capnocytophaga strain. Twenty-eight patients had been diagnosed by blood culture, one by culture from both blood and cerebrospinal fluid (CSF), one by culture from a conjunctival swab, and two patients by 16S rRNA gene amplification by PCR directly from a blood or CSF specimen. The incidence rate was 0.67 infections per million population. Bacteraemia was found in 94% of the cases. The age range of patients was 38-80 years; 72% of them were male. Among 26 patients from whom clinical data were available, splenectomy was not reported, but alcoholism was reported in five. Nine patients (35%) had been admitted to the intensive-care unit, and three patients (13%) died. The mortality rate was much lower than observed in previous studies.

Decontamination of the digestive tract and oropharynx in ICU patients.

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

Psoas abscess: report of a series and review of the literature.

We describe a series of twelve patients with a psoas abscess seen in a three-year period in a university hospital and a large teaching hospital in the Netherlands. In our series, five of the 12 patients had a primary psoas abscess. The predisposing conditions were intravenous drug use, diabetes mellitus, prostate carcinoma and haematoma in the psoas muscle in a patient with haemophilia A. Seven of the 12 patients had a secondary psoas abscess. Five cases were due to vertebral osteomyelitis including two cases of tuberculosis. In the other two cases it was due to colitis and urinary tract infection. It is remarkable that in our series there was only one patient with a psoas abscess secondary to a disease of the digestive tract, while this is the most common cause of a secondary psoas abscess in the literature. There were two cases of tuberculosis which is an emerging disease again.

The DUEL study: a multi-center in vitro evaluation of linezolid compared with other antibiotics in the Netherlands.

OBJECTIVE: To evaluate bacterial susceptibility to linezolid in the Netherlands in comparison with other antibiotics. METHODS: Bacterial strains were isolated between September 1999 and January 2000 from patients presumed to require antibiotic treatment. The in vitro activity of 1226 strains from 34 participating laboratories was tested against linezolid, vancomycin, teicoplanin, oxacillin, penicillin, erythromycin, ampicillin and other antibiotics against enterococci, coagulase-negative staphylococci, Staphylococcus aureus and Streptococcus pneumoniae. Minimal inhibitory concentrations (MIC) were obtained with the E test on Mueller-Hinton agar: every laboratory included control strains. For vancomycin and teicoplanin only, brain-heart infusion agar and an inoculum of 2.0 McFarland was used for Staphylococcus aureus, coagulase-negative staphylococci and enterococci to support a better growth and clear recognition of hetero-resistant colonies. RESULTS: The values of MIC90 for linezolid were 1.5, 0.75, 0.75 and 1 mg/L for Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus pneumoniae and enterococci, respectively. Six enterococcal strains with decreased susceptibility against vancomycin or teicoplanin were identified as Enterococcus faecium, E. gallinarum and E. casseliflavus (two strains each) and they were found to harbor vanA, vanC1 and vanC2/3 genes, respectively. Nine per cent of Streptococcus pneumoniae (an increase from 1% 4 years ago) showed decreased susceptibility to erythromycin, of both the ermB and mefE type; there was no cross-resistance with linezolid. Twelve coagulase-negative staphylococcal strains were resistant to teicoplanin. CONCLUSION: Linezolid is a promising drug in the treatment of infections caused by Gram-positive cocci. Cross-resistance with other antibiotics tested was not found.

Emergence of rifampin-resistant Streptococcus pneumoniae as a result of antimicrobial therapy for penicillin-resistant strains.

A multidrug-resistant strain of Streptococcus pneumoniae was isolated in The Netherlands during a nosocomial outbreak among 36 patients who mainly had chronic obstructive pulmonary disease. After the commencement of barrier nursing and short-term ceftriaxone-rifampin eradication therapy, the epidemic ceased. However, eradication therapy failed in 3 patients, and follow-up investigation of these patients showed the emergence of rifampin-resistant isolates.

Asymptomatic bacteriuria may be considered a complication in women with diabetes. Diabetes Mellitus Women Asymptomatic Bacteriuria Utrecht Study Group.

OBJECTIVE: To study the prevalence of and risk factors for asymptomatic bacteriuria (ASB) in women with and without diabetes. RESEARCH DESIGN AND METHODS: A total of 636 nonpregnant women with diabetes (type 1 and type 2) who were 18-75 years of age and had no abnormalities of the urinary tract, and 153 women without diabetes who were visiting the eye and trauma outpatient clinic (control subjects) were included. We defined ASB as the presence of at least 10(5) colony-forming units/ml of 1 or 2 bacterial species in a culture of clean-voided midstream urine from an individual without symptoms of a urinary tract infection (UTI). RESULTS: The prevalence of ASB was 26% in the diabetic women and 6% in the control subjects (P < 0.001). The prevalence of ASB in women with type 1 diabetes was 21%. Risk factors for ASB in type 1 diabetic women included a longer duration of diabetes, peripheral neuropathy, and macroalbuminuria. The prevalence of ASB was 29% in women with type 2 diabetes. Risk factors for ASB in type 2 diabetic women included age, macroalbuminuria, a lower BMI, and a UTI during the previous year. No association was evident between current HbA1c level and the presence of ASB. CONCLUSIONS: The prevalence of ASB is increased in women with diabetes and might be added to the list of diabetic complications in these women.

HIV-infected patient with a Rhodococcus equi pneumonia.

The case history of a HIV patient with a pulmonary infect of Rhodococcus equi is presented. He recovered after prolonged treatment with antibiotics and lobectomy. The Rhodococcus equi infection was the presenting symptom of his impaired immune status caused by HIV infection.

Evaluation of an in-house polymerase chain reaction for detection of Neisseria gonorrhoeae in urogenital samples.

AIM: To develop and evaluate a one day in-house polymerase chain reaction (PCR) assay for the detection of Neisseria gonorrhoeae DNA in urogenital samples. METHODS: 429 urogenital specimens were tested for the presence of N gonorrhoeae by in-house PCR and by Gen-Probe. The PCR assay amplifies target sequences within the N gonorrhoeae cppB gene on the 4.2 kb cryptic plasmid, after which amplicons are detected by a streptavidinbiotin based enzyme immunoassay using an internal probe. Discordant specimens were further evaluated by repeating the PCR and the Gen-Probe assay, and by an additional PCR using another set of 16S primers followed by radioactive detection of amplicons on a Southern blot. RESULTS: Of the 429 samples tested, 15 were found positive by in-house PCR, eight of which were confirmed by Gen-Probe. Of the seven discrepant samples, five were confirmed by 16S PCR and are also considered true positive. The remaining two samples were positive in the in-house PCR only, and are considered false positive. After resolution of discrepant samples, the sensitivities of the N gonorrhoeae assays were 100% and 61.5% for the in-house PCR and Gen-Probe, respectively, while specificities were comparable at 99.5% and 100%. CONCLUSIONS: The in-house PCR for the detection of N gonorrhoeae DNA is at least comparable to Gen-Probe in performance. An extended evaluation period should elucidate if the additional five GO-PCR positive specimens, confirmed by 16S PCR, are caused by persistence of DNA or whether the method is indeed more sensitive.

Hospital-related outbreak of infection with multidrug-resistant Streptococcus pneumoniae in the Netherlands.

Multidrug-resistant strains of Streptococcus pneumoniae were isolated over a two-year period (July 1995 until August 1997) from the sputum of 36 patients who were hospitalized in a Dutch medical centre. Nosocomial transmission was confirmed by typing of the bacterial isolates: all 36 multidrug-resistant isolates shared the same genotype, serotype, and displayed overlapping drug resistance profiles. Thirty-two of the 36 (89%) patients had chronic obstructive pulmonary disease (COPD). The outbreak was initiated by a 76-year old patient, who had been colonized with the same strain since 1993. Because staff screening of the hospital and pulmonary function department was negative, patient-to-patient spread was the most likely cause of this outbreak. The epidemic ceased following the commencement of barrier nursing, a treatment course of ceftriaxone, and a five-day rifampicin eradication therapy for the positive patients. The outbreak resulted from failure to recognize quickly the rapid transmission of this multidrug-resistant pneumococcal clone. We conclude that patients with COPD are at high risk of acquiring multidrug resistant pneumococci, and suggest that COPD patients who are colonized or infected with multidrug-resistant pneumococci should be isolated to prevent future transmission.

Prevalence of primary Helicobacter pylori resistance to metronidazole and clarithromycin in The Netherlands.

The minimum inhibitory concentrations of metronidazole and clarithromycin were determined for 780 Helicobacter pylori strains collected in 1994 and 1995 from three different regions in The Netherlands. The overall prevalence of primary metronidazole resistance was 17%, with resistance found more frequently in women (24%) than in men (13%). There was no significant difference between the levels of resistance in the three regions. Primary clarithromycin resistance was rare (1%) and relatively infrequent as compared to that found in other countries. Four of the six strains resistant to clarithromycin were also resistant to metronidazole.

Comparison of fluorescent BACTEC 9000 MB system, Septi-Chek AFB system, and Lowenstein-Jensen medium for detection of mycobacteria.

The newly developed fluorescent BACTEC 9000 MB system for automated culture of mycobacteria was compared with the Septi-Chek AFB system and Lowenstein-Jensen medium (LJ). A total of 2,005 clinical specimens were included in the study. Mycobacteria were isolated from 202 (10.1%) specimens, including 155 Mycobacterium tuberculosis complex isolates and 47 Mycobacteria other than M. tuberculosis isolates. Of 131 isolates detected by the BACTEC system, the Septi-Chek AFB system, or both, 120 (91.6%) were detected by the BACTEC system and 105 (80.2%) were detected by the Septi-Chek AFB system (P < 0.02). The recovery rate in the BACTEC system compared with that in the Septi-Chek AFB system was significantly higher for M. tuberculosis complex isolates (P < 0.005) and for isolates from acid-fast smear-negative specimens (P < 0.01). Of 148 isolates detected by the BACTEC system, LJ, or both, 142 (95.9%) were detected by the BACTEC system and 118 (79.9%) were detected by LJ (P < 0.001). The recovery rate in the BACTEC system compared with that on LJ was significantly higher for M. tuberculosis complex isolates (P < 0.001). The BACTEC system detected more mycobacteria from both smear-positive and smear-negative specimens than LJ. The mean times to detection of mycobacteria were 17.6 days for the BACTEC system, 26.0 days for the Septi-Chek AFB system, and 29.4 days for LJ. The BACTEC fluorescent 9000 MB system is a rapid, sensitive, and efficient method for the isolation of mycobacteria.

Comparison of three commercially available enzyme-linked immunosorbent assays and biopsy-dependent diagnosis for detecting Helicobacter pylori infection.

We evaluated the performance of three enzyme-linked immunosorbent assays (ELISAs) in detecting serum immunoglobulin G (IgG) and IgA antibodies to Helicobacter pylori; two were new ones from Pyloriset (Pyloriset EIA-G update and Pyloriset EIA-A update; Orion Diagnostica, Espoo, Finland), and the third was the Malakit EIA-G (Biolab, Limal, Belgium). Serum samples from 154 dyspeptic patients were collected. As a reference method, multiple biopsy specimens from different anatomical areas of the stomach were obtained by endoscopy and were analyzed by culture and/or histology and direct urease testing. Accordingly, 126 patients (82%) were found to be H. pylori positive and 28 patients (18%) were found to be H. pylori negative. To validate serology as a predictor of H. pylori infection, sensitivity, specificity, positive and negative predictive values, and accuracy of the assays were calculated against the H. pylori status as determined by the reference method. The corresponding data for the different ELISAs were 100%, 79%, 95%, 100%, and 96% for the Pyloriset ELA-G update, 81%, 89%, 97%, 52%, and 82% for the Pyloriset EIA-A update, and 87%, 86%, 96%, 60%, and 87% for the Malakit EIA-G, respectively. We conclude that the Pyloriset EIA-G update is a reliable and accurate test and that because of its 100% sensitivity, conjunctional IgA testing is not necessary. Its 100% negative predictive value makes it a very useful screening test. For purposes of excluding infection with H. pylori, the performance of the Malakit EIA-G is moderate but can be improved by conjunctional IgA testing. The Pyloriset EIA-A update can be useful as such a conjunctional test.