Bram M. W. Diederen

Methicillin- Resistant Staphylococcus aureus in Meat Products, the Netherlands

A new methicillin-resistant Staphylococcus aureus (MRSA) clone related to pig and cattle farming was detected in the Netherlands. We investigated the extent of S. aureus presence in meat and found 36 S. aureus strains in 79 samples. Two strains were MRSA; 1 was multilocus sequence type 398, the clone related to farming.

Comparative Evaluation of the VITEK 2, Disk Diffusion, Etest, Broth Microdilution, and Agar Dilution Susceptibility Testing Methods for Colistin in Clinical Isolates, Including Heteroresistant Enterobacter cloacae and Acinetobacter baumannii Strains

ABSTRACT Increasing antibiotic resistance in gram-negative bacteria has recently renewed interest in colistin as a therapeutic option. The increasing use of colistin necessitates the availability of rapid and reliable methods for colistin susceptibility testing. We compared seven methods of colistin susceptibility testing (disk diffusion, agar dilution on Mueller-Hinton [MH] and Isosensitest agar, Etest on MH and Isosensitest agar, broth microdilution, and VITEK 2) on 102 clinical isolates collected from patient materials during a selective digestive decontamination or selective oral decontamination trial in an intensive-care unit. Disk diffusion is an unreliable method to measure susceptibility to colistin. High error rates and low levels of reproducibility were observed in the disk diffusion test. The colistin Etest, agar dilution, and the VITEK 2 showed a high level of agreement with the broth microdilution reference method. Heteroresistance for colistin was observed in six Enterobacter cloacae isolates and in one Acinetobacter baumannii isolate. This is the first report of heteroresistance to colistin in E. cloacae isolates. Resistance to colistin in these isolates seemed to be induced upon exposure to colistin rather than being caused by stable mutations. Heteroresistant isolates could be detected in the broth microdilution, agar dilution, Etest, or disk diffusion test. The VITEK 2 displayed low sensitivity in the detection of heteroresistant subpopulations of E. cloacae. The VITEK 2 colistin susceptibility test can therefore be considered to be a reliable tool to determine susceptibility to colistin in isolates of genera that are known not to exhibit resistant subpopulations. In isolates of genera known to (occasionally) exhibit heteroresistance, an alternative susceptibility testing method capable of detecting heteroresistance should be used.

Performance of CHROMagar MRSA Medium for Detection of Methicillin-Resistant Staphylococcus aureus

ABSTRACT CHROMagar MRSA was evaluated for its ability to identify methicillin-resistant Staphylococcus aureus (MRSA). A well-defined collection consisting of 216 MRSA strains and 241 methicillin-susceptible Staphylococcus aureus isolates was used. The sensitivity of CHROMagar MRSA after 24 h of incubation was 95.4%, increasing to 100% after 48 h. The specificity was already 100% after 24 h.

Utility of Real-Time PCR for Diagnosis of Legionnaires’ Disease in Routine Clinical Practice

ABSTRACT The main aim of our study was to determine the added value of PCR for the diagnosis of Legionnaires’ disease (LD) in routine clinical practice. The specimens were samples submitted for routine diagnosis of pneumonia from December 2002 to November 2005. Patients were evaluated if, in addition to PCR, the results of at least one of the following diagnostic tests were available: (i) culture for Legionella spp. on buffered charcoal yeast extract agar or (ii) detection of Legionella pneumophila antigen in urine specimens. Of the 151 evaluated patients, 37 (25%) fulfilled the European Working Group on Legionella Infections criteria for a confirmed case of LD (the “gold standard”). An estimated sensitivity, specificity, and overall percent agreement of 86% (32 of 37; 95% confidence interval [CI] = 72 to 95%), 95% (107 of 112; 95% CI = 90 to 98%), and 93% (139 of 149), respectively, were found for 16S rRNA-based PCR, and corresponding values of 92% (34 of 37; 95% CI = 78 to 98%), 98% (110 of 112; 95% CI = 93 to 100%), and 97% (144 of 149), respectively, were found for the mip gene-based PCR. A total of 35 patients were diagnosed by using the urinary antigen test, and 34 were diagnosed by the 16S rRNA-based PCR. With the mip gene PCR one more case of LD (n = 36; not significant) was detected. By combining urinary antigen test and the mip gene PCR, LD was diagnosed in an additional 4 (11%) patients versus the use of the urinary antigen test alone. The addition of a L. pneumophila-specific mip gene PCR to a urinary antigen test is useful in patients with suspected LD who produce sputum and might allow the early detection of a significant number of additional patients.

Performance of MRSA ID, a New Chromogenic Medium for Detection of Methicillin-Resistant Staphylococcus aureus

ABSTRACT MRSA ID was evaluated for its ability to identify methicillin-resistant Staphylococcus aureus. A well-defined collection of staphylococci was used (n = 998). The sensitivity after 24 h was 96.4%, increasing to 98.8% after 48 h. The specificity was 98.2% after 24 h and decreased to 89.7% after 48 h.

Detection of respiratory viruses and Legionella spp. by real-time polymerase chain reaction in patients with community acquired pneumonia

We conducted a study on throat swabs obtained from a group of hospitalized patients with community acquired pneumonia (CAP). Throat swab specimens from 242 adults admitted to hospital with CAP were tested. In total, 1 or more aetiological agents were identified by real-time PCR in 55 (23%) patients. The most frequently detected pathogens were coronavirus (17%), parainfluenza virus (6%) and influenza virus (4%). Overall, viral pathogens were identified by conventional techniques in 7 (2%) patients, and real-time PCR in 50 (21%) patients (p<0.0001). The diagnostic yield increased from 137 cases (57% of patients using conventional microbiological assays) to 158 cases (65% of patients using real-time PCR assays and conventional microbiological assays; p=0.06). A significantly higher percentage of mortality was present in patients with a mixed bacterial and viral infection. L. pneumophila PCR was positive in only 3 out of 11 cases (27%) of Legionnaires’ disease (LD). This study demonstrates that real-time PCR can increase the number of microbiological detections of respiratory pathogens, mainly as a result of detection of respiratory viruses.

Wild-type MIC distribution and epidemiological cut-off values in clinical Legionella pneumophila serogroup 1 isolates.

OBJECTIVES The purpose of this study was to establish wild-type (WT) distributions and determine the epidemiological cut-off values (ECOFF) in clinical L. pneumophila serogroup 1 isolates for 10 antimicrobials commonly used for the treatment of Legionella infections using a method feasible in a routine clinical laboratory. METHODS MICs of 183 clinical L. pneumophila serogroup 1 isolates, collected as part of an outbreak detection program, were tested using E-test methodology on buffered charcoal yeast extract agar supplemented with α-ketoglutarate (BCYE-α). The MICs were read after 2 days of incubation at 35 °C with increased humidity and without CO(2). ECOFFs were determined according to EUCAST methodology and expressed as WT ≤ X mg/L. RESULTS All antimicrobials showed a WT distribution, although the width varied from 2 two-fold dilutions to 8 dilutions, depending on antibiotic class. The ECOFFs determined were 1.0 mg/L for ciprofloxacin, 0.50 mg/L for levofloxacin, 1.0 mg/L for moxifloxacin, 1.0 mg/L for erythromycin, 1.0 mg/L for azithromycin, 0.50 mg/L for clarithromycin, 1.0 mg/L for cefotaxime, 0.032 mg/L for rifampicin, 16 mg/L for tigecycline, and 8 mg/L for doxycycline. CONCLUSION All isolates were inhibited by low concentrations of the fluoroquinolones and macrolides tested, with somewhat higher MICs for the fluoroquinolones. Rifampicin was found to be the most active against L. pneumophila isolates in vitro. These data can be used as a reference for the detection of resistance in clinical L. pneumophila isolates and as a setting of clinical breakpoints.

In Vitro Activity of Daptomycin against Methicillin-Resistant Staphylococcus aureus, Including Heterogeneously Glycopeptide-Resistant Strains

ABSTRACT The purpose of the present study was to assess the in vitro activity of daptomycin against a well-defined collection of methicillin-resistant Staphylococcus aureus (MRSA) isolates (n = 98), including heterogeneously glycopeptide-resistant MRSA (hGISA) strains. Susceptibility testing was performed by using the Etest system. Daptomycin was potent against both glycopeptide-susceptible and hGISA strains.

Evaluation of Two New Immunochromatographic Assays (Rapid U Legionella Antigen Test and SD Bioline Legionella Antigen Test) for Detection of Legionella pneumophila Serogroup 1 Antigen in Urine

ABSTRACT We evaluated two new immunochromatographic assays for their abilities to detect Legionella pneumophila serogroup 1 antigen in urine. The results were compared with those obtained by the Binax NOW urinary antigen test. The sensitivities and specificities were estimated to be 71.2% and 96.6%, respectively, for the Rapid U test; 31.5% and 98.9%, respectively, for the SD Bioline test; and 91.8% and 100%, respectively, for the Binax NOW test.

Isolation of ciprofloxacin-resistant Legionella pneumophila in a patient with severe pneumonia

Sir, Legionella species are responsible for 1%– 5% of cases of community-acquired pneumonia. Legionella pneumophila serogroup 1 (SG1) accounts for .90% of Legionnaires’ disease (LD) in North America and Europe and is the cause of significant mortality. The mortality rate among patients with L. pneumophila infections continues to be high, up to 26%. The antimicrobial agents most commonly used for treatment of LD are fluoroquinolones (e.g. ciprofloxacin or levofloxacin) and macrolides. In recent studies, we established wild-type distributions and determined the epidemiological cut-off values (ECOFFs) in clinical L. pneumophila SG1 isolates for 10 antimicrobials commonly used for the treatment of Legionella infections. A patient sought care at his general practitioner after several days of falling and body pains. On examination, the patient appeared ill and was sent to the emergency department of a nearby hospital. The initial chest radiograph demonstrated an infiltrate of the left lower lung field. The patient was admitted to the intensive care unit. Blood cultures were taken and antibiotic treatment was started with cefazolin and gentamicin. Urine was examined for the presence of Legionella antigens and when this test was reported positive, treatment was switched to 400 mg of ciprofloxacin intravenously twice daily. After initial improvement the clinical condition of the patient deteriorated, leading to intubation and mechanical ventilation. A new chest radiograph revealed a diffuse interstitial pneumonia. Bronchoalveolar lavage (BAL) was performed 4 days after treatment with ciprofloxacin was started and the patient slowly recovered. Eventually, culture of the BAL grew L. pneumophila SG1 after 4 days of ciprofloxacin treatment. After 10 days, the patient could be transferred to the ward. Therapy was then switched to 500 mg of clarithromycin orally twice daily. The patient’s further recovery was uncomplicated. The L. pneumophila SG1 strain was sent, as part of a national Legionella outbreak detection programme, to the reference laboratory for Legionella in Haarlem, The Netherlands. Susceptibility testing for ciprofloxacin was performed with Etest and an MIC value of ciprofloxacin of 2 mg/L was found. This value is outside the wild-type distribution range ECOFF1⁄41 mg/L as previously described and therefore potentially resistant. For sequencing of gyrA and gyrB (DNA gyrase) and parC and parE (topoisomerase IV) genes, extraction of L. pneumophila DNA was performed. The DNA extraction was performed by use of Qiagen’s BioRobot EZ1 (Hilden, Germany) according to the manufacturer’s instructions. The sequencing reaction was performed twice by using primer systems previously described for the L. pneumophila SG1 strain Paris. A comparative analysis of the obtained sequences was done using the published L. pneumophila genomes and data from the literature describing mutations in the quinolone resistance-determining region (QRDR) of type II topoisomerase of L. pneumophila by using DNAStar (WI, USA) software and the NCBI database. For control experiments, the wild-type strain MTZ OLDA and a spontaneous quinolone-resistant mutant of this strain were used. MTZ OLDA is an environmental isolate (L. pneumophila SG1), isolated from the water supply of a large building. A point mutation in the QRDR of the gyrA gene was identified and this mutation led to an amino acid exchange at position 83 (Escherichia coli numbering system). The result of this amino acid exchange is a change in ciprofloxacin susceptibility. Mutation at the same position (amino acid 83) has also been reported for other spontaneous quinolone-resistant mutants (Table 1). It is known that, in general, pathogens can become resistant during the course of a patient’s therapy and also induction of resistance upon exposure to antibiotics has been described. The origin of resistance in the clinical isolate is as yet unclear. There are two possibilities. The first is that the patient contracted an L. pneumophila SG1 strain with this point mutation from the environment. Alternatively, the mutation occurred during the Research letters