Dimitra P. Houhoula

Moxifloxacin resistance is prevalent among Bacteroides and Prevotella species in Greece

OBJECTIVES Moxifloxacin is recommended in the empirical treatment of infections involving Gram-negative anaerobes. However, current European data regarding its activity against anaerobic pathogens are limited. In order to evaluate its potency, we comparatively studied the activity of moxifloxacin against recently isolated Gram-negative anaerobes. METHODS Four hundred and ninety-five Gram-negative anaerobic clinical isolates (296 Bacteroides fragilis group, 58 non-fragilis Bacteroides spp. and 141 Prevotella spp.) were prospectively recovered in six Greek hospitals. Moxifloxacin MICs were determined in comparison with those of penicillin, piperacillin/tazobactam, cefoxitin, imipenem, metronidazole and clindamycin. RESULTS Overall moxifloxacin MIC(50) and MIC(90) were 2 and 32 mg/L, respectively. Based on the current CLSI breakpoints (susceptible, < or =2 mg/L; resistant, > or =8 mg/L), almost half of the total isolates (49%) were non-susceptible to moxifloxacin (32% resistant; 17% intermediate). This was more evident among the non-fragilis Bacteroides species, where 47% of the isolates were resistant and 14% intermediate to moxifloxacin. Species variation was noticed, with the highest non-susceptible rates detected among Prevotella oralis (90%), Prevotella bivia (80%), Bacteroides thetaiotaomicron (75%), Bacteroides uniformis (70%) and Bacteroides capillosus (67%) species. Among the 19 (4%) isolates that were metronidazole non-susceptible (MIC > or = 16 mg/L), only 4 (21%) were additionally non-susceptible to moxifloxacin. CONCLUSIONS High resistance rates to moxifloxacin among Bacteroides and Prevotella spp. were recorded, exceeding those previously reported in Europe and contraindicating its use as monotherapy for infections involving Gram-negative anaerobes without prior microbiological confirmation. For empirical usage, moxifloxacin should be combined with metronidazole in order to cover for these pathogens.

Two Cases of Infections Due to Multidrug-Resistant Bacteroides fragilis Group Strains

ABSTRACT Bacteroides fragilis group strains are still considered susceptible to most antimicrobial agents used for the treatment of infections caused by anaerobic organisms. We describe two cases of infections due to isolates simultaneously resistant to clindamycin, tetracycline, cefoxitin, piperacillin-tazobactam, and imipenem and, in one of the two cases, to metronidazole. Such infections, although still rare, do exist and tend to complicate treatment.

Ruling out Bacillus anthracis.

Optimization of methods for ruling out Bacillus anthracis leads to increased yields, faster turnaround times, and a lighter workload. We used 72 environmental non–B. anthracis bacilli to validate methods for ruling out B. anthracis. Most effective were horse blood agar, motility testing after a 2-h incubation in trypticase soy broth, and screening with a B. anthracis–selective agar.

Detection of Bacillus Galmette-Guérin (Mycobacterium bovis BCG) DNA in Urine and Blood Specimens after Intravesical Immunotherapy for Bladder Carcinoma

ABSTRACT A real-time PCR targeting IS6110 was employed for the detection of Mycobacterium tuberculosis DNA in specimens collected from 10 patients treated with intravesical M. bovis bacillus Galmette-Guérin (BCG) immunotherapy for bladder malignancy. BCG DNA was detected in all urine specimens taken 24 h after the instillations, as well as in 24% of the specimens collected 7 days after the instillations; it was also detected in a single specimen taken 6 weeks after the last instillation. BCG DNA was detected in 8.3% of the blood specimens taken 1 day after instillation, and its amplification was associated with cases of self-limiting fever. These findings give indications that this real-time PCR is helpful to recognize BCG bacteremic cases, which may lead to mycobacterial infection.

Comparison of Penile Skin Swab with Intra-Urethral Swab and First Void Urine for Polymerase Chain Reaction-Based Diagnosis of Chlamydia trachomatis Urethritis in Male Patients

Chlamydia trachomatis is considered the most frequent cause of male non-gonococcal urethritis.1 Nevertheless, the pathogen may be also detected in urethral specimens of men suffering from gonococcal urethritis and in asymptomatic cases.2–4 Nucleic acid amplification techniques (NAATs) have currently become the diagnostic methods of choice and have increased the overall diagnostic yield. Preferred specimens are considered either the intraurethral swab (IUS) or more recently the first void urine (FVU), a noninvasive sampling method, which facilitates large epidemiologic studies.1,4,5 In addition, there are anecdotal reports comparing alternative noninvasive sampling methods, such as the glans-penis swab for supplementing molecular diagnosis.6 –9 In the current study we compared a noninvasive sampling method, the penile skin swab (PSS), with the IUS and FVU sampling procedures in the molecular diagnosis of male C. trachomatis urethritis. Between September 2005 and September 2006, a total of 210 male patients who were examined at the outpatient sexually transmitted diseases clinic of “Andreas Sygros” Hospital, Athens, Greece, for acute nongonococcal urethritis, were enrolled in the study. Exclusion criteria were urination during the previous 2 hours before examination, antibiotic treatment during the previous 3 weeks, or genital anatomical abnormalities (e.g., phimosis).10 The following specimens were taken by the clinician in the following order: first the PSS using the large swabs of the COBAS Amplicor swab specimen collection set, vigorously rolled over the glans and collar of the penis to ensure collection of a high number of epithelial cells; second the IUS using the thin swabs of the same set from the posterior 2 to 4 cm of the urethra; third the FVU catch (10–50 mL of urine).2 IUS and PSS samples were divided in 2 parts. The first part was processed immediately using the COBAS Amplicor CT assay (Amplicor PCR; Roche Molecular Systems, Branchburg, NJ), according to the manufacturer’s instructions. The polymerase chain reaction (PCR) amplification protocol targeted a 207 bp segment of the C. trachomatis conserved cryptic plasmid. The second half was put into 2-sucrose-phosphate transport medium, centrifuged at 4500 g for 30 minutes and stored at 70°C. FVU samples were also divided in half, the first part was processed immediately using the COBAS Amplicor CT assay and the second half was stored at 70°C. DNA extraction of the second half of the samples was performed using the NucleoSpin Tissue DNA extraction kit (Macherrey Nagel, Düren, Germany), according to the manufacturer’s instructions. A previously described nested-PCR (nPCR) protocol11 was used for amplification of a 1320 basepair segment of the omp1 gene.12 All specimens were tested neat and diluted 10 1 for inhibition detection. PCR products were separated in a 1.5% agarose gel (Ultra Pure Agarose, GIBCO, PAisley, Scotland), stained with 0.5 g/mL ethidium bromide, and documented under ultraviolet illumination. Positive patients were considered those who yielded a positive result of at least one of the IUS, FVU, or PSS specimens with both PCR assays, together with clinical and microbiologic indications of acute nongonococcal urethritis. The protocol of the study was reviewed and approved by the hospital’s Ethical Committee and a written consent was obtained from all patients. Supported by the Kapodistrias grant 70/3/8156 of the National and Kapodistrian University of Athens, Greece. Correspondence: Athanassios Tsakris, MD, PhD, MRCPath, Department of Microbiology, Medical School, National and Kapodistrian University of Athens, 75 Mikras Asias Street, 11527 Athens, Greece. E-mail: atsakris@med.uoa.gr. Received for publication January 21, 2008, and accepted May 27, 2008. From the *Department of Microbiology, Medical School, University of Athens, Athens, Greece; †Department of Microbiology, Andreas Sygros Hospital for Skin and Venereal Diseases, Athens, Greece; and ‡Department of Dermatology, Medical School, University of Athens, Andreas Sygros Hospital for Skin and Venereal Diseases, Athens, Greece Sexually Transmitted Diseases, December 2008, Vol. 35, No. 12, p.999–1001 DOI: 10.1097/OLQ.0b013e318182a586 Copyright

Molecular-beacon-based real-time PCR for detection and quantification of Mycobacterium tuberculosis DNA in clinical samples.

Although real-time PCR (RT-PCR) has been extensively evaluated for diagnosis of Mycobacterium tuberculosis infections (5), data are limited on molecular-beacon (MB) applications. An MB-based RT-PCR protocol was designed and evaluated for direct M. tuberculosis detection and quantification in clinical specimens. A total of 1,019 samples (417 pulmonary and 602 extrapulmonary) were consecutively and prospectively collected for tuberculosis (TB) diagnosis. They were processed using standard methodology (4) and divided into two parts. The first half of each sample was used for acid-fast staining and culture, while the second half was stored at −70°C. TB diagnosis of patients followed previous definitions (11). After diagnosis elucidation, the second part of each specimen obtained from TB-positive patients, as well as an equal number of specimens obtained from TB-negative patients, was thawed and DNA was extracted using a QIAamp DNA Mini kit (Qiagen, Hilden, Germany). The RT-PCR targeted the IS6110 sequence, and the following primers, amplifying a 161-bp fragment, and MB were designed, using Beacon Designer 5.1 software (Premier Biosoft, Palo Alto, CA): TB2F (5′-GTCCACGCCGCCAACTACG-3′), TΒ2R (5′-GTTAGGTGCTGGTGGTCCGAAG-3′), and TB2B (6-carboxyfluorescein-5′-CGCGATCGCCACAGCCCGTCCCGCCGATGATCGCG-3′-benzoic acid succinimidyl ester). Conditions consisted of 2 min of denaturation at 95°C, 50 cycles of 45 s of denaturation at 93°C, 90 s of annealing at 60°C, and 2 min of extension at 72°C, and finally, 7 min of extension at 72°C. DNA extracted from the M. tuberculosis H37Rv strain (ATCC 25618) was used for the quantification standard curve. DNA from an M. avium and an M. chelonae clinical isolate, from an M. bovis BCG strain, and from Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, and Bacteroides fragilis ATCC 25285 strains was used for specificity confirmation. The median and mean of the quantified DNA and cycle threshold values were calculated using Instat 3.0 software (GraphPad Software, San Diego, CA 92130). A previously reported (8) single-step conventional PCR (C-PCR) was used for comparisons. With the RT-PCR protocol, a positive signal was detected using DNA only from the M. tuberculosis and the M. bovis strains. RT-PCR analytical sensitivity was 7 fg of DNA, corresponding to approximately 1.4 mycobacterial genomes. Overall, 32 patients were TB positive, and 40 specimens were obtained from those patients (Tables ​(Tables11 and ​and2).2). A total of 40 specimens (10 pulmonary and 30 extrapulmonary) were also obtained from 34 TB-negative patients. The two PCR assays showed equally high levels of sensitivity (Table ​(Table1),1), although C-PCR performed better among pulmonary specimens and RT-PCR performed better among extrapulmonary specimens. Combining the two assays yielded 100% sensitivity. After testing the samples diluted 10−1, we found three samples to be inhibitory (one with both protocols and two with PCR only). DNA quantification results from comparisons of the main specimen types are shown in Table ​Table2.2. The mean (± standard deviation) cycle threshold value for the RT-PCR was 33.5 (± 5.3; range, 19.3 to 40.7). TABLE 1. Sensitivities, specificities, and positive and negative predictive values of the two PCR assays TABLE 2. DNA quantification of the main specimen types by use of the RT-PCR protocol In the literature, TaqMan and linear fluorescence probes have mostly been applied for direct diagnosis of TB (1, 3, 5). MB technology has been evaluated for direct detection of M. avium (2, 6) as well as for detection of mutation-specific resistance in M. tuberculosis isolates (9, 10) and, in only one report, for direct TB diagnosis using pulmonary specimens (7). In the present study, a newly designed MB RT-PCR was developed and proved to be a promising tool, showing high sensitivity and specificity for a collection of pulmonary and extrapulmonary specimens, which in most cases (29 out of 40) were acid-fast staining and culture negative. Our report has also shown that pulmonary and pus specimens had the highest median DNA loads, in contrast to the results seen with lymph nodes. Although lymph node biopsy is commonly used for extrapulmonary TB diagnosis, our results indicated that a specimen from the primal focal point of the disease, if retrievable, may yield higher mycobacterial loads.

Comparative evaluation of conventional and real-time PCR assays for detecting Bacteroides fragilis in clinical samples

ABSTRACT A conventional PCR and a real-time PCR for detecting Bacteroides fragilis were evaluated against clinical specimens. Analytical sensitivities were 100 and 40 fg of DNA, respectively. Detection limits were 100 and 10 CFU/ml, respectively. A total of six culture-negative specimens were positive by PCR. Altering the gold standard from “positive culture” to “positive culture or both PCR assays positive” resulted in sensitivities of 91.7% and 100%, respectively, and in specificities of 100% and 98.6%, respectively.

High level of heterogeneity among Listeria monocytogenes isolates from clinical and food origin specimens in Greece.

In order to examine the genetic variation of clinical and food isolates of Listeria monocytogenes in Greece, a total of 61 L. monocytogenes non-duplicate isolates, recovered from clinical specimens (n=19) and food (n=42), were serotyped and genotyped using two different Random Amplification of Polymorphic DNA (RAPD) protocols and Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). Serotype group 4b, 4d, 4e prevailed (39.4%), among both clinical and food isolates, followed by serotype group 1/2a, 3a (23.0%), which nevertheless was detected only among food isolates. The most discriminatory typing protocol was MLVA, which grouped four isolates into two pairs, while the remaining isolates produced unique fingerprints. Similar results were obtained when taking into account the combination of the two RAPD protocols (Simpson index 0.999); six isolates were grouped into three pairs, two of which were the pairs that were identified also by MLVA. Single use of each RAPD protocol resulted in inferior discrimination (Simpson index 0.978 and 0.997, respectively). In conclusion, the two molecular procedures, MLVA, and the combined RAPD protocols, produced similar results, showing that L. monocytogenes isolates from clinical and food specimens were highly heterogenous and that clustering was very uncommon.

Food Safety and Label Claims for Hazelnut Allergy Traces: Evaluation of Two PCR Assays

Abstract Houhoula D.P., Lagou K., Varvaresou M., Giannakourou M., Bratakos S.M., Lougovois V., Tsaknis J., Koussissis S. (2015): Food safety and label claims for hazelnut allergy traces: evaluation of two PCR as-says . Czech J. Food Sci., 33: 410–415. The molecular techniques (C-PCR, RT-PCR) in the detection and quantification of allergic substances of hazelnut in various categories of food commodities, e.g. breakfast cereals, chocolates and biscuits, frequently involved in allergic outbreaks was implementated. For the detection of hazelnut a gene coding the major allergenic protein Cor a1 was selected. In some instances, the presence of hazelnuts is not declared on the label and the products may carry no warn -ing for potentially allergenic substances, usually referred to as “traces”. A total of 150 samples were collected from local supermarkets and analysed for the purpose of the study. From these, a total of 38 (25.3%) specimens contained hazelnut, 30 (20.0%) contained “traces” of hazelnut, 26 (17.3%) contained a label warning for the possible presence of “traces” of allergenic substances, and 56 (37.3%) specimens contained no food allergy labels. Among them, using the C-PCR, 36 (94.7%), 10 (33.3%), 5 (19.2%), and 5 (8.9%) specimens were detected as positive, respectively. Using the RT-PCR, 38 (100%), 15 (50%), 7 (26.9%) and 8 (14.3%) specimens were detected as positive, respectively. Finally, by combining both methods, 38 (100%), 17 (56.7%), 9 (34.6%), and 10 (17.9%) specimens were identified as positive, respectively.