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Background Aeromonas hydrophila is a gram-negative facultative anaerobic coccobacillus, which is an environmental opportunistic pathogen. A. hydrophila are involved in several infectious diseases such as gastroenteritis, septicemia and wound infections. However, gastroenteritis caused by Aeromonas spp. are rare and the clinical relevance of Aeromonas species in stool specimens is still under debate. Case presentation Our case concerns a 32-year-old woman who presented at hospital with a worsening watery diarrhea and fever requiring intensive care. A cholera-like illness was diagnosed. The patient had a past history of an anti-Hu syndrome with a myenteric ganglionitis. A molecular multiplex RT-PCR (QIAstat-Dx Gastrointestinal Panel, QIAGEN) covering a broad spectrum of diverse gastrointestinal pathogens performed directly from the stool was negative but the stool culture revealed growth of A. hydrophila. Further investigations of the A. hydrophila strain in cell cultures revealed the presence of a cytotoxic enterotoxin. Conclusions Although A. hydrophila rarely causes gastroenteritis, Aeromonas spp. should be considered as a causative agent of severe gastroenteritis with a cholera-like presentation. This case highlights the need to perform culture methods from stool samples when PCR-based methods are negative and gastrointestinal infection is suspected.

Antibiotic tolerant Staphylococcus aureus pose a great challenge to clinicians as well as to microbiological laboratories and are one reason for treatment failure. Antibiotic tolerant strains survive transient antibiotic exposure despite being fully susceptible in vitro. Thus, fast and reliable methods to detect tolerance in the routine microbiology laboratory are urgently required.We therefore evaluated the feasibility of the replica plating tolerance isolation system (REPTIS) to detect antibiotic tolerance in S. aureus isolates derived directly from patients suffering from different types of infections and investigated possible connections to clinical presentations and patient characteristics.One hundred twenty-five S. aureus isolates were included. Replica plating of the original resistance testing plate was used to assess regrowth in the zones of inhibition, indicating antibiotic tolerance. Bacterial regrowth was assessed after 24 and 48 hours of incubation and an overall regrowth score (ORS) was assigned. Regrowth scores were compared to the clinical presentation.Bacterial regrowth was high for most antibiotics targeting protein synthesis and relatively low for antibiotics targeting other cellular functions such as DNA-replication, transcription and cell wall synthesis, with the exception of rifampicin. Isolates with a blaZ penicillinase had lower regrowth in penicillin and ampicillin. Low ORSs were more prevalent among isolates recovered from patients with immunosuppression or methicillin-resistant S. aureus (MRSA) isolates.In conclusion, REPTIS is useful to detect antibiotic tolerance in clinical microbiological routine diagnostics. Rapid detection of antibiotic tolerance offers a new diagnostic readout that might allow more tailored treatments in the future.

Helicobacter pylori is a major human pathogen that causes a wide range of gastrointestinal pathology. Progression of H. pylori induced gastritis to more severe disease has been found to highly correlate with the array of virulence factors expressed by the pathogen. The objective of this study was twofold: first, to characterize the genetic diversity of H. pylori strains isolated from 41 non-atrophic gastritis patients in Switzerland, an issue that has not been investigated to date. And second, to assess the prevalence and sequence variation of H. pylori virulence factors (cagA, vacA, iceA and dupA) and genes encoding outer membrane proteins (OMPs, babA, babB, sabA, sabB, hopZ, hopQ and oipA) by whole genome sequencing (WGS) using an Illumina MiSeq platform. WGS identified high genetic diversity in the analyzed H. pylori strains. Most H. pylori isolates were assigned to hpEurope (95.0%, 39/41), and the remaining ones (5.0%, 2/41) to hpEastAsia, subpopulation hspEAsia. Analysis of virulence factors revealed that 43.9% of the strains were cagA-positive, and the vacA s1 allele was detected in 56.0% of the isolates. The presence of cagA was found to be significantly associated (P &lt, 0.001) with the presence of vacA s1, babA2 and hopQ allele 1 as well as expression of oipA. Moreover, we found an association between the grade of gastritis and H. pylori abundance in the gastric mucosa, respectively and the presence of cagA, vacA s1 and hopQ allele 1. Among our 41 gastritis patients, we identified seven patients infected with H. pylori strains that carried a specific combination of virulence factors (i.e cagA, vacA s1 allele and babA2 allele), recently implicated in the development of more severe gastrointestinal pathology, like peptic ulcer disease and even gastric cancer. To this end, WGS can be employed for rapid and detailed characterization of virulence determinants in H. pylori, providing valuable insights into the pathogenic capacity of the bacterium. This could ultimately lead to a higher level of personalized treatment and management of patients suffering from H. pylori associated infections.

Matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS) has entered clinical laboratories, facilitating identification of bacteria. Here, we evaluated the MALDI Biotyper (Bruker Daltonics) for the identification of fastidious Gram-negative rods (GNR). Three sample preparation methods, direct colony transfer, direct transfer plus on-target formic acid preparation, and ethanol-formic acid extraction, were analyzed for 151 clinical isolates. Direct colony transfer applied with the manufacturer's interpretation criteria resulted in overall species and genus identification rates of 43.0% and 32.5%, respectively; 23.2% of the isolates were not identified, and two misidentifications (1.3%) were observed. The species identification rates increased to 46.4% and 53.7% for direct transfer plus formic acid preparation and ethanol-formic acid extraction, respectively. In addition, we evaluated score value cutoff alterations. The identification rates hardly increased by reducing the genus cutoff, while reducing the 2.0 species cutoff to 1.9 and to 1.8 increased the identification rates to up to 66.2% without increasing the rate of misidentifications. This study shows that fastidious GNR can reliably be identified using the MALDI Biotyper. However, the identification rates do not reach those of nonfastidious GNR such as theEnterobacteriaceae. In addition, two approaches optimizing the identification of fastidious GNR by the MALDI Biotyper were demonstrated: formic acid-based on-target sample treatment and reductions in cutoff scores to increase the species identification rates.

Corynebacterium spp. are rarely considered pathogens, but data on Corynebacterium spp. as a cause of orthopedic infections are sparse. Therefore, we asked how often Corynebacterium spp. caused an infection in a defined cohort of orthopedic patients with a positive culture. In addition, we aimed to determine the species variety and the susceptibility of isolated strains to define potential treatment strategies. We retrospectively assessed all bone and joint samples that were collected between 2006 and 2015 from an orthopedic ward and that were positive for Corynebacterium spp. by culture. The isolates were considered relevant to an infection if the same Corynebacterium sp. was present in at least two samples. We found 97 orthopedic cases with isolation of Corynebacterium spp. (128 positive samples). These were mainly Corynebacterium tuberculostearicum (n = 26), Corynebacterium amycolatum (n = 17), Corynebacterium striatum (n = 13), and Corynebacterium afermentans (n = 11). Compared to the species found in a cohort of patients with positive blood cultures hospitalized in nonorthopedic wards, we found significantly more C. striatum- and C. tuberculostearicum-positive cases but no C. jeikeium-positive cases in our orthopedic cohort. Only 16 out of 66 cases (24.2%) with an available diagnostic set of at least two samples had an infection. Antibiotic susceptibility testing (AST) showed various susceptibility results for all antibiotics except vancomycin and linezolid, to which 100% of the isolates were susceptible. The rates of susceptibility of corynebacteria isolated from orthopedic samples and of isolates from blood cultures were comparable. In conclusion, our study results confirmed that a Corynebacterium sp. is most often isolated as a contaminant in a cohort of orthopedic patients. AST is necessary to define the optimal treatment in orthopedic infections.

Irrigation water is a major source of fresh produce contamination with undesired microorganisms including antibiotic-resistant bacteria (ARB), and contaminated fresh produce can transfer ARB to the consumer especially when consumed raw. Nevertheless, no legal guidelines exist so far regulating quality of irrigation water with respect to ARB. We therefore examined irrigation water from major vegetable growing areas for occurrence of antibiotic-resistant indicator bacteria Escherichia coli and Enterococcus spp., including extended-spectrum β-lactamase (ESBL)-producing E. coli and vancomycin-resistant Enterococcus spp. Occurrence of ARB strains was compared to total numbers of the respective species. We categorized water samples according to total numbers and found that categories with higher total E. coli or Enterococcus spp. numbers generally had an increased proportion of respective ARB-positive samples. We further detected high prevalence of ESBL-producing E. coli with eight positive samples of thirty-six (22%), while two presumptive vancomycin-resistant Enterococcus spp. were vancomycin-susceptible in confirmatory tests. In disk diffusion assays all ESBL-producing E. coli were multidrug-resistant (n = 21) and whole-genome sequencing of selected strains revealed a multitude of transmissible resistance genes (ARG), with bla_CTX-M-1 (4 of 11) and bla_CTX-M-15 (3 of 11) as the most frequent ESBL genes. Overall, the increased occurrence of indicator ARB with increased total indicator bacteria suggests that the latter might be a suitable estimate for presence of respective ARB strains. Finally, the high prevalence of ESBL-producing E. coli with transmissible ARG emphasizes the need to establish legal critical values and monitoring guidelines for ARB in irrigation water. ISSN:1932-6203

Background. Propionibacteria are important members of the human skin microbiota, but are also opportunistic pathogens associated with periprosthetic joint infections (PJI). While the role of Propionibacterium acnes in PJI has been widely described, insight into the capacity of Propionibacterium avidum to cause PJI is limited. Methods. An unusual cluster of four hip PJIs caused by P. avidum in one orthopedic center in 2015 prompted us to retrospectively identify and analyze clinical data related to previous P. avidum PJI cases (1997-2015). We also characterized the hemolytic and biofilm-producing capacity of our four clinical P. avidum strains isolated in 2015, and investigated their phylogenetic relationships by whole genome sequencing. Results. We retrospectively identified 13 P. avidum PJIs, with the majority being hip-related infections (n=11). Preoperative synovial fluid cultures were P. avidum positive in 63.6% of cases. Six out of 12 patients (50%) with available case histories were treated with an exchange of the prosthesis. In all but one of the six patients treated with debridement-retention of the prosthesis, treatment failed thus requiring a two-stage revision. The isolated P. avidum strains showed a more pronounced hemolytic activity, but a similar biofilm-forming ability when compared to P. acnes. Whole genome sequencing identified two phylogenetic clusters highly related to P. avidum PJI strains isolated in Sweden. Conclusions. We describe the largest series of P. avidum PJI predominantly located in the hip. Phylogenetic similarity of our P. avidum strains to PJI strains isolated elsewhere suggests these invasive lineages may be common.

Background. Chronic granulomatous disease (CGD) is a rare inherited disease of the phagocyte NADPH oxidase system that causes defective production of toxic oxygen metabolites, impaired bacterial and fungal killing, and recurrent life-threatening infections, mostly by catalase-producing organisms. We report for the first time, to our knowledge, chronic infections with Actinomyces species in 10 patients with CGD. Actinomycosis is a chronic granulomatous condition that commonly manifests as cervicofacial, pulmonary, or abdominal disease, caused by slowly progressive infection with oral and gastrointestinal commensal Actinomyces species. Treatment of actinomycosis is usually simple in immunocompetent individuals, requiring long-term, high-dose intravenous penicillin, but is more complicated in those with CGD because of delayed diagnosis and an increased risk of chronic invasive or debilitating disease. Methods. Actinomyces was identified by culture, staining, 16S ribosomal DNA polymerase chain reaction, and/ or a complement fixation test in 10 patients with CGD. Results. All 10 patients presented with a history of fever and elevated inflammatory signs without evident focus. Diagnosis was delayed and clinical course severe and protracted despite high-dose intravenous antibiotic therapy and/or surgery. These results suggest an unrecognized and unanticipated susceptibility to weakly pathogenic Actinomyces species in patients with CGD because these are catalase-negative organisms previously thought to be nonpathogenic in CGD. Conclusions. Actinomycosis should be vigorously sought and promptly treated in patients with CGD presenting with uncommon and prolonged clinical signs of infection. Actinomycosis is a catalase-negative infection important to consider in CGD.

Objectives Disc diffusion is a cost-efficient, low-complexity, reliable method for detection of blaZ -mediated benzylpenicillin resistance in Staphylococcus aureus if the zone edge is inspected. EUCAST breakpoints cannot fully separate β-lactamase-positive from β-lactamase-negative strains, and EUCAST recommends the zone edge test. Literature on nitrocefin-based testing and the zone edge test is scarce with wide variations in reported assay performance. Methods This study compared two different nitrocefin-based commercial and in-house tests and the EUCAST-based zone edge test for penicillinase detection in S. aureus applying a PCR-based gold standard. Results In total, 215 non-duplicate clinical S. aureus isolates were included in the study, of which 127 (59.1%) did not harbour a blaZ gene, whereas 88 (40.9%) were blaZ positive. This study showed that for blaZ detection the zone edge test is more sensitive (96.6%) than nitrocefin tests independent of using nitrocefin discs (87.5% sensitivity) or solution (89.8% sensitivity), and that the significant inter-person variations of the zone edge test are probably related to the training level of the individual investigators (individual sensitivity ranging from 68.2% to 96.6%, specificity ranging from 89.8% to 100%). Conclusions In addition to continued and strict training of investigators, we propose mandatory checking of benzylpenicillin zone edges, particularly in an investigation zone from 26 to 30 mm, which can result in improved specificity/positive predictive value of the zone edge test (from 98.4% to 100%) but retains the high sensitivity/negative predictive value of the method.

Toxigenic Corynebacterium diphtheriae is an important and potentially fatal threat to patients and public health. During the current dramatic influx of refugees into Europe, our objective was to use whole genome sequencing for the characterization of a suspected outbreak of C. diphtheriae wound infections among refugees. After conventional culture, we identified C. diphtheriae using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and investigated toxigenicity by PCR. Whole genome sequencing was performed on a MiSeq Illumina with >70×coverage, 2×250 bp read length, and mapping against a reference genome. Twenty cases of cutaneous C. diphtheriae in refugees from East African countries and Syria identified between April and August 2015 were included. Patients presented with wound infections shortly after arrival in Switzerland and Germany. Toxin production was detected in 9/20 (45%) isolates. Whole genome sequencing-based typing revealed relatedness between isolates using neighbour-joining algorithms. We detected three separate clusters among epidemiologically related refugees. Although the isolates within a cluster showed strong relatedness, isolates differed by >50 nucleotide polymorphisms. Toxigenic C. diphtheriae associated wound infections are currently observed more frequently in Europe, due to refugees travelling under poor hygienic conditions. Close genetic relatedness of C. diphtheriae isolates from 20 refugees with wound infections indicates likely transmission between patients. However, the diversity within each cluster and phylogenetic time-tree analysis suggest that transmissions happened several months ago, most likely outside Europe. Whole genome sequencing offers the potential to describe outbreaks at very high resolution and is a helpful tool in infection tracking and identification of transmission routes.

Background: Urinary tract infections (UTIs) are among the most prevalent microbial diseases and their financial burden on society is substantial. The continuing increase of antibiotic resistance worldwide is alarming so that well-tolerated, highly effective therapeutic alternatives are urgently needed. Objective: To investigate the effect of bacteriophages on Escherichia coli and Klebsiella pneumoniae strains isolated from the urine of patients suffering from UTIs. Material and methods: Forty-one E. coli and 9 K. pneumoniae strains, isolated from the urine of patients suffering from UTIs, were tested in vitro for their susceptibility toward bacteriophages. The bacteriophages originated from either commercially available bacteriophage cocktails registered in Georgia or from the bacteriophage collection of the George Eliava Institute of Bacteriophage, Microbiology and Virology. In vitro screening of bacterial strains was performed by use of the spot-test method. The experiments were implemented three times by different groups of scientists. Results: The lytic activity of the commercial bacteriophage cocktails on the 41 E. coli strains varied between 66% (Pyo bacteriophage) and 93% (Enko bacteriophage). After bacteriophage adaptation of the Pyo bacteriophage cocktail, its lytic activity was increased from 66 to 93% and only one E. coli strain remained resistant. One bacteriophage of the Eliava collection could lyse all 9 K. pneumoniae strains. Conclusions: Based on the high lytic activity and the potential of resistance optimization by direct adaption of bacteriophages as reported in this study, and in view of the continuing increase of antibiotic resistance worldwide, bacteriophage therapy is a promising treatment option for UTIs highly warranting randomized controlled trials.

Study Type - Therapy (case series) Level of Evidence 4 What's known on the subject? and What does the study add? Several studies describe a reduction of symptomatic urinary tract infections in patients with neurogenic detrusor overactivity after intradetrusor injections of botulinum neurotoxin A (BoNT/A). It was, however, unclear if a direct antibacterial effect of BoNT/A plays a role in this clinical observation. This is the first study to investigate a potential antibacterial effect of two frequently used BoNT/A formulations (i.e. Botox® and Dysport®), providing evidence that BoNT/A does not exert an antibacterial effect on lower urinary tract pathogens. OBJECTIVE: • To determine a potential direct antimicrobial effect of botulinum neurotoxin type A (BoNT/A). MATERIALS AND METHODS: • A prospective study was carried out using onabotulinumtoxin A (Botox®) and abobotulinumtoxin A (Dypsort®) in agar diffusion and broth microdilution assays with various clinical urinary tract isolates (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa, Acinetobacter baumannii, Citrobacter freundii, Klebsiella oxytoca and Bacillus subtilis). • Inhibition zones (mm) of bacteria around a disc containing 20 µL saline with 4 IU of Botox® were measured in the agar diffusion assay. • Minimal inhibitory concentrations (MICs, IU/mL) of both toxins for all bacteria were determined in the broth microdilution assay after overnight incubation at 35 °C. RESULTS: • There was no inhibition zone in the agar diffusion assays with any bacterial strain. • The microdilution test using Botox® and Dysport® showed bacterial growth in all dilutions, i.e. MICs > 20 and >100 IU/mL for Botox® and Dysport®, respectively. CONCLUSIONS: • BoNT/A has no direct antimicrobial effect. • The reduced frequency of symptomatic urinary tract infections (sUTIs) in patients with neurogenic detrusor overactivity (NDO) after BoNT/A intradetrusor injections seems to be caused by different indirect mechanisms, which are still not completely understood.

This chapter covers bacterial genera which are taxonomically diverse and belong to the families Cardiobacteriaceae, Flavobacteriaceae, Fusobacteriaceae, Neisseriaceae, Pasteurellaceae, and Porphyromonadaceae, but common traits justify their discussion as a group. It discusses only species that can be isolated from humans. The Pasteurellaceae consist of several genera, of which four are known to contain human pathogens: Actinobacillus, Aggregatibacter, Haemophilus, and Pasteurella. Phenotypic identification of fastidious gram-negative rods presents several challenges. Triple sugar iron or Kligler's agar may not support the growth of fastidious genera (e.g., Eikenella). Detection of antibodies directed against any of the bacteria discussed in the chapter has been tried on a small scale only and does not seem to offer much value. With specimens normally colonized with aerobic and anaerobic bacteria, as well as with specimens from wounds, e.g., bite wounds, the significance of the bacteria discussed in the chapter depends on their predominance and the absence of other potentially pathogenic bacteria. If these conditions are met, identification to the species level is needed for adequate interpretation and reporting as infectious agents and for susceptibility testing. If none of these conditions is present, a repeat culture and close cooperation between the microbiology laboratory and the physician are necessary for interpretation, for identification to the species or genus level, and for susceptibility testing.

Ninety strains of a collection of well-identified clinical isolates of gram-negative nonfermentative rods collected over a period of 5 years were evaluated using the new colorimetric VITEK 2 card. The VITEK 2 colorimetric system identified 53 (59%) of the isolates to the species level and 9 (10%) to the genus level; 28 (31%) isolates were misidentified. An algorithm combining the colorimetric VITEK 2 card and 16S rRNA gene sequencing for adequate identification of gram-negative nonfermentative rods was developed. According to this algorithm, any identification by the colorimetric VITEK 2 card other than Achromobacter xylosoxidans , Acinetobacter sp., Burkholderia cepacia complex, Pseudomonas aeruginosa , and Stenotrophomonas maltophilia should be subjected to 16S rRNA gene sequencing when accurate identification of nonfermentative rods is of concern.

The bacterial genera covered in this chapter are taxonomically diverse—they belong to the families Cardiobacteriaceae, Flavobacteriaceae, Leptotrichiaceae, Neisseriaceae, Pasteurellaceae, and Porphyromonadaceae—but common traits justify their discussion as a group. Most bacteria in this group are part of the microbiota of the nasopharynx and/or the oral cavity of animals and/or humans and are parasitic, with the only environmental genus being Chromobacterium. Transmission from animals occurs by contact (e.g., bites and licking of wounds), from humans to humans by droplets (e.g., directly with Kingella spp. or via human bites with Eikenella corrodens). The epidemiology and clinical significance of the genera Actinobacillus, Aggregatibacter, and Pasteurella, of the family Pasteurellaceae; the genera Cardiobacterium, Eikenella, and Kingella, belonging to the HACEK group; and other less frequently encountered genera are discussed separately. The identification of most genera is difficult by phenotypic methods. Direct microscopy may identify the corresponding bacteria; e.g., the typical morphology of spindle-shaped cells in the Gram stain and the anamnestic history of a dog bite allow the presumptive identification of Capnocytophaga canimorsus. Phenotypic key reactions as well as commercially available systems allow identification of the frequently encountered human isolates. If accurate identification of uncommon isolates is of concern, then molecular methods, e.g., 16S rRNA gene analysis, are necessary. The application of matrix-assisted laser desorption ionization–time-of-flight mass spectrometry shows promising results and is discussed here. Finally, susceptibility testing according to CLSI and EUCAST is described, especially for Pasteurella multocida.

Background Methicillin-resistant Staphylococcus aureus (MRSA) is a global epidemic threat. The aim of this study was to determine which globally known MRSA lineages are currently present at our tertiary care hospital in Switzerland, a hospital with low MRSA prevalence. In light of the increasing prevalence of multi drug resistance including vancomycin resistance we also assessed antibiotic susceptibilities. Methods The 146 MRSA strains collected over two years (March 2012 until February 2014) at the University Hospital Zurich, Switzerland, were analyzed by PFGE analysis of SmaI digests in combination with spa-typing. In addition, representative isolates were analyzed by multi locus sequence typing (MLST). Susceptibilities to eight antibiotics were assessed using the Kirby-Bauer disc diffusion method. Results Isolates showed resistance to erythromycin (48%), ciprofloxacin (43%), clindamycin (31%), tetracycline (22%), and gentamicin (16%). All isolates were susceptible to vancomycin, 95% were susceptible to sulfamethoxazole/trimethoprim and rifampicin, respectively. PFGE analysis revealed 22 different patterns, with four major patterns that accounted for 53.4% of all MRSA isolates, and seven sporadic patterns. Spa typing revealed 50 different spa types with the predominant types being t008 (14%), t002 (10%), and t127 (9%). 82% of the MRSA isolates could be assigned to six clonal complexes (CCs) namely CC1 (10%), CC5 (23%), CC8 (18%), CC22 (17%), CC30 (11%), and CC45 (3%) based on spa-types, PFGE patterns, and MLST. Two isolates could not be typed by either PFGE analysis or spa-typing and three isolates had spa-types that have not yet been described. Conclusions The combination of the two typing methods was more discriminatory as compared to the use of a single method. Several of the lineages that are predominant in Europe are present in our hospital. Resistances to antibiotics have decreased in comparison to a study conducted between 2004 and 2006. Electronic supplementary material The online version of this article (doi:10.1186/s12941-015-0075-3) contains supplementary material, which is available to authorized users.

Over a period of 26 months, we have evaluated in a prospective fashion the use of 16S rRNA gene sequencing as a means of identifying clinically relevant isolates of nonfermenting gram-negative bacilli (non- Pseudomonas aeruginosa ) in the microbiology laboratory. The study was designed to compare phenotypic with molecular identification. Results of molecular analyses were compared with two commercially available identification systems (API 20 NE, VITEK 2 fluorescent card; bioMérieux, Marcy l'Etoile, France). By 16S rRNA gene sequence analyses, 92% of the isolates were assigned to species level and 8% to genus level. Using API 20 NE, 54% of the isolates were assigned to species and 7% to genus level, and 39% of the isolates could not be discriminated at any taxonomic level. The respective numbers for VITEK 2 were 53%, 1%, and 46%, respectively. Fifteen percent and 43% of the isolates corresponded to species not included in the API 20 NE and VITEK 2 databases, respectively. We conclude that 16S rRNA gene sequencing is an effective means for the identification of clinically relevant nonfermenting gram-negative bacilli. Based on our experience, we propose an algorithm for proper identification of nonfermenting gram-negative bacilli in the diagnostic laboratory.

Over a period of 18 months we have evaluated the use of 16S ribosomal DNA (rDNA) sequence analysis as a means of identifying aerobic catalase-negative gram-positive cocci in the clinical laboratory. A total of 171 clinically relevant strains were studied. The results of molecular analyses were compared with those obtained with a commercially available phenotypic identification system (API 20 Strep system; bioMérieux sa, Marcy l'Etoile, France). Phenotypic characterization identified 67 (39%) isolates to the species level and 32 (19%) to the genus level. Seventy-two (42%) isolates could not be discriminated at any taxonomic level. In comparison, 16S rDNA sequencing identified 138 (81%) isolates to the species level and 33 (19%) to the genus level. For 42 of 67 isolates assigned to a species with the API 20 Strep system, molecular analyses yielded discrepant results. Upon further analysis it was concluded that among the 42 isolates with discrepant results, 16S rDNA sequencing was correct for 32 isolates, the phenotypic identification was correct for 2 isolates, and the results for 8 isolates remained unresolved. We conclude that 16S rDNA sequencing is an effective means for the identification of aerobic catalase-negative gram-positive cocci. With the exception of Streptococcus pneumoniae and beta-hemolytic streptococci, we propose the use of 16S rDNA sequence analysis if adequate species identification is of concern.

An unknown, strictly anaerobic, Gram-positive, rod-shaped bacterium (strain MOL361T) was isolated from a blood culture of a febrile patient with acute appendicitis and characterized using phenotypic and molecular methods. Fatty acid analysis and biochemical examination indicated that the isolate most closely resembles members of the Gram-positive bacteria with low DNA G+C content. 16S rDNA sequencing revealed a relatively high overall similarity (97%) to an uncultured bacterium, but these two strains both exhibit low (

The activities of BAL9141 (formerly Ro 63-9141), a novel pyrrolidinone-3-ylidenemethyl cephalosporin, against 244 strains of gram-negative nonfermenters were evaluated. The overall MIC at which 50% of isolates are inhibited (MIC 50 ) and the overall MIC 90 were 2 and 64 μg/ml, respectively, which are similar to those of imipenem, lower than those of the other cephalosporins tested, amoxicillin, and the ticarcillin-clavulanic acid combination, and much higher than those of ciprofloxacin. BAL9141 shows species-dependent activity in vitro against a variety of gram-negative nonfermentative pathogens.

Background Clostridium difficile infection (CDI) remains a major health problem worldwide. Antibiotic use, in general, and clindamycin and ciprofloxacin, in particular, have been implicated in the pathogenesis of CDI. Here, we hypothesized that antibiotics that are highly active in vitro against C. difficile are less frequently associated with CDI than others. The primary goals of our study were to determine if antibiotic susceptibility and CDI are associated and whether the antimicrobial susceptibility of C. difficile changed over the years. Methods and results We examined a large panel of C. difficile strains collected in 2006-2008 at the University Hospital of Zurich. We found that the antimicrobial susceptibilities to amoxicillin/clavulanate, piperacillin/tazobactam, meropenem, clindamycin, ciprofloxacin, ceftriaxone, metronidazole and vancomycin were similar to those reported in the literature and that they are similar to those reported in other populations over the last two decades. Antibiotic activity did not prevent CDI. For example, thre use of meropenem, which is highly active against all strains tested, was a clear risk factor for CDI. Most of the antibiotics tested also showed a higher minimum inhibitory concentration distribution than that of EUCAST. All strains were susceptible to metronidazole. One strain was resistant to vancomycin. Conclusions Antibiotic susceptibilities of the collection of C. difficile from the University Hospital of Zurich are similar to those reported by others since the 1980. Patients treated with carbapenems and cephalosporins had the highest risk of developing CDI irrespective of the antimicrobial activity of carbapenems., BMC Infectious Diseases, 14, ISSN:1471-2334

Background Many bacteria causing systemic invasive infections originate from the oral cavity by entering the bloodstream. Recently, a novel pathogenic bacterium, Streptococcus tigurinus, was identified as causative agent of infective endocarditis, spondylodiscitis and meningitis. In this study, we sought to determine the prevalence of S. tigurinus in the human oral microbial flora and analyzed its association with periodontal disease or health. Results We developed a diagnostic highly sensitive and specific real-time TaqMan PCR assay for detection of S. tigurinus in clinical samples, based on the 16S rRNA gene. We analyzed saliva samples and subgingival plaque samples of a periodontally healthy control group (n = 26) and a periodontitis group (n = 25). Overall, S. tigurinus was detected in 27 (53%) out of 51 patients. There is no significant difference of the frequency of S. tigurinus detection by RT-PCR in the saliva and dental plaque samples in the two groups: in the control group, 14 (54%) out of 26 individuals had S. tigurinus either in the saliva samples and/or in the plaque samples; and in the periodontitis group, 13 (52%) out of 25 patients had S. tigurinus in the mouth samples, respectively (P = 0.895). The consumption of nicotine was no determining factor. Conclusion Although S. tigurinus was a frequently detected species of the human oral microbial flora, it was not associated with periodontal disease. Further investigations are required to determine whether S. tigurinus is a commensal or an opportunistic oral pathogen with a potential for development of invasive infections.

Ertapenem (MK-0826, L-749,345) is a 1-β-methyl carbapenem with a long serum half-life. Its in vitro activity was determined by broth microdilution against 3,478 bacteria from 12 centers in Europe and Australia, with imipenem, cefepime, ceftriaxone, and piperacillin-tazobactam used as comparators. Ertapenem was the most active agent tested against members of the family Enterobacteriaceae , with MICs at which 90% of isolates are inhibited (MIC 90 s) of ≤1 μg/ml for all species. Ertapenem also was more active than imipenem against fastidious gram-negative bacteria and Moraxella spp.; on the other hand, ertapenem was slightly less active than imipenem against streptococci, methicillin-susceptible staphylococci, and anaerobes, but its MIC 90 s for these groups remained ≤0.5 μg/ml. Acinetobacter spp. and Pseudomonas aeruginosa were also much less susceptible to ertapenem than imipenem, and most Enterococcus faecalis strains were resistant. Ertapenem resistance, based on a provisional NCCLS MIC breakpoint of ≥16 μg/ml, was seen in only 3 of 1,611 strains of the family Enterobacteriaceae tested, all of them Enterobacter aerogenes . Resistance was also seen in 2 of 135 anaerobes, comprising 1 Bacteroides fragilis strain and 1 Clostridium difficile strain. Ertapenem breakpoints for streptococci have not been established, but an unofficial susceptibility breakpoint of ≤2 μg/ml was adopted for clinical trials to generate corresponding clinical response data for isolates for which MICs were as high as 2 μg/ml. Of 234 Streptococcus pneumoniae strains tested, 2 required ertapenem MICs of 2 μg/ml and one required an MIC of 4 μg/ml, among 67 non- Streptococcus pyogenes , non- Streptococcus pneumoniae streptococci, single isolates required ertapenem MICs of 2 and 16 μg/ml. These streptococci also had diminished susceptibilities to other β-lactams, including imipenem as well as ertapenem. The Etest and disk diffusion gave susceptibility test results in good agreement with those of the broth microdilution method for ertapenem.

In order to assess the performance of two detection methods, a set of 93 recent clinical isolates of Staphylococcus aureus, including a large number of strains that demonstrated low-level methicillin-resistance were evaluated using the MRSA-Screen (Denka Seiken, Japan), a commercial latex agglutination test to detect penicillin-binding protein 2' (PBP2'), and a polymerase chain reaction assay using the LightCycler Instrument (Roche Diagnostics, Switzerland). The results show that the latex agglutination test is highly sensitive if performed after induction by cefoxitin. Inconclusive results can be rapidly confirmed on the same day by real-time polymerase chain reaction used to detect mecA and femA genes.

Summary The Vitek AutoMicrobic system in combination with the Gram-positive susceptibility card detects β-lactamase in staphylococci by utilizing penicillin as the substrate coupled with oxacillin as an inducer. The β-lactamase activity of 21 clinical isolates and two reference strains of Staphylococcus lugdunensis was determined with this automated system and compared with a liquid nitrocefin assay after induction with oxacillin. Eight (38 %) clinical isolates and the reference strain ATCC 49 576 of S. lugdunensis showed production of β-lactamase in both tests. Thirteen (62 %) clinical isolates and the type strain ATCC 43 809 were nitrocefin-negative. The Vitek AutoMicrobic system reported false-positive s-lactamase results for 9 of those 13 isolates and for the type strain of S. lugdunensis . Results for disk diffusion (ampicillin) were concordant with the nitrocefin assay. With one exception, the MICs for penicillin of the nitrocefin-negative strains were in the equivocal range of 0.06–0.12 mg/1 according to NCCLS. However, none of the nitrocefin-negative and Vitek-positive strains revealed any of the known staphylococcal genes for β-lactamase as investigated by Southern hybridization, supporting the fact that false-positive β-lactamase results may occur in the Vitek AutoMicrobic system. We conclude from our data that it may be justified to include S. lugdunensis in the quality control of Vitek cards containing s-lactamase tests.

The diagnostic value of IgM to Bartonella henselae was evaluated in 20 children with cat-scratch disease (CSD) and controls consisting of 20 blood donors and 20 children with enlarged lymph nodes without CSD by two indirect immunofluorescence assays (IFA). One was based on B. henselae cocultivated with Vero cells (host cell-associated IFA), and the other on B. henselae grown on agar (host cell-free IFA). With the host cell-associated IFA, 18 of 20 children with CSD revealed IgM, whereas only 14 did so with the host cell-free IFA. Sera of two blood donors as well as sera from three children with enlarged lymph nodes without CSD showed also positive IgM to cell-associated B. henselae. This study reveals that the IFA applied had sensitivities of 70 – 90% and specificities of 87.5 – 100% for detecting IgM to B. henselae. Additionally, 20 patients with IgM to Epstein-Barr virus (EBV) capsid antigen were tested for IgM to B. henselae. Sera of 16 and 9 of these patients revealed IgM to B. henselae with the host cell-associated and the host cell-free IFA, respectively. Using Western blot these sera were demonstrated to react against linearized proteins of Vero cells and of B. henselae. Thus, since acute EBV infection may substantially reduce the specificity of B. henselae-specific IgM tests, we conclude that diagnosis of CSD should be confirmed by a significant IgG titer to B. henselae or by detection of this pathogen.

Staphaurex Plus® (Murex Diagnostics), a rapid agglutination test for detecting Staphylococcus aureus , was combined with the free coagulase test to search for the coagulase-negative species Staphylococcus lugdunensis and for S. aureus . Staphaurex Plus® detected 29 S. lugdunensis and 1602 S. aureus in our routine laboratory over 6 months. Furthermore, 10 out of 902 coagulase-negative staphylococci other than S. lugdunensis reacted with Staphaurex Plus®. The sensitivity and specificity for detecting S. aureus were 99.7% and 95.7%, respectively. Clumping factor-positive S. lugdunensis can bind fibrinogen coated on the latex particles of Staphaurex Plus®. If this agglutination was considered true positive, the specificity for S. aureus was 98.9%. We conclude that Staphaurex Plus® is a highly sensitive and specific agglutination test for S. aureus and simultaneously detects clumping factor-positive S. lugdunensis .

Broad-range PCR amplification of part of the 16S rRNA gene followed by single-strand sequencing was applied to samples of 18 resected heart valves from patients with infective endocarditis. The PCR results were compared with those of cultures of valves and with those of previous blood cultures. For two patients there was agreement with the cultures of the valves; for nine patients there was agreement with the previous blood cultures, which were positive, while the cultures of the valves were negative; a Streptococcus sp. and Tropheryma whippelii each were found in one patient with negative cultures (valve and blood); for two patients the cultures of the valves as well as the PCR results were negative but the blood cultures were positive; for one patient amplification was inhibited; and for two patients the PCR results were positive but the amplicons could not be sequenced. It is concluded that broad-range PCR is a promising tool for patients with culture-negative endocarditis and allows the detection of rare, noncultivable organisms.

Objective We studied whether the change in antibiotic susceptibility testing (AST) guidelines from CLSI to EUCAST influenced cumulative antibiograms in a tertiary care hospital in Switzerland. Methods Antibiotic susceptibilities of non-duplicate isolates collected within a one-year period before (period A) and after (period B) changing AST interpretation from CLSI 2009 to EUCAST 1.3 (2011) guidelines were analysed. In addition, period B isolates were reinterpreted according to the CLSI 2009, CLSI 2013 and EUCAST 3.1 (2013) guidelines. Results The majority of species/drug combinations showed no differences in susceptibility rates comparing periods A and B. However, in some gram-negative bacilli, decreased susceptibility rates were observed when comparing CLSI 2009 with EUCAST 1.3 within period B: Escherichia coli / cefepime, 95.8% (CLSI 2009) vs. 93.1% (EUCAST 1.3), P=0.005; Enterobacter cloacae / cefepime, 97.0 (CLSI 2009) vs. 90.5% (EUCAST 1.3), P=0.012; Pseudomonas aeruginosa / meropenem, 88.1% (CLSI 2009) vs. 78.3% (EUCAST 1.3), P=0.002. These differences were still evident when comparing susceptibility rates according to the CLSI 2013 guideline with EUCAST 3.1 guideline. For P. aeruginosa and imipenem, a trend towards a lower antibiotic susceptibility rate in ICUs compared to general wards turned into a significant difference after the change to EUCAST: 87.9% vs. 79.8%, P=0.08 (CLSI 2009) and 86.3% vs. 76.8%, P=0.048 (EUCAST 1.3). Conclusions The change of AST guidelines from CLSI to EUCAST led to a clinically relevant decrease of susceptibility rates in cumulative antibiograms for defined species/drug combinations, particularly in those with considerable differences in clinical susceptibility breakpoints between the two guidelines.

This study compared three sample preparation methods (direct transfer, the direct transfer-formic acid method with on-target formic acid treatment, and ethanol-formic acid extraction) for the identification of Gram-positive cocci with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). A total of 156 Gram-positive cocci representing the clinically most important genera, Aerococcus , Enterococcus , Staphylococcus , and Streptococcus , as well as more rare genera, such as Gemella and Granulicatella , were analyzed using a Bruker MALDI Biotyper. The rate of correct genus-level identifications was approximately 99% for all three sample preparation methods. The species identification rate was significantly higher for the direct transfer-formic acid method and ethanol-formic acid extraction (both 77.6%) than for direct transfer (64.1%). Using direct transfer-formic acid compared to direct transfer, the total time to result was increased by 22.6%, 16.4%, and 8.5% analyzing 12, 48, and 96 samples per run, respectively. In a subsequent prospective study, 1,619 clinical isolates of Gram-positive cocci were analyzed under routine conditions by MALDI-TOF MS, using the direct transfer-formic acid preparation, and by conventional biochemical methods. For 95.6% of the isolates, a congruence between conventional and MALDI-TOF MS identification was observed. Two major limitations were found using MALDI-TOF MS: the differentiation of members of the Streptococcus mitis group and the identification of Streptococcus dysgalactiae . The Bruker MALDI Biotyper system using the direct transfer-formic acid sample preparation method was shown to be a highly reliable tool for the identification of Gram-positive cocci. We here suggest a practical algorithm for the clinical laboratory combining MALDI-TOF MS with phenotypic and molecular methods.

Background Neisseria gonorrhoeae can rapidly develop resistance to antimicrobial agents. Over the last years, decreased gonococcal susceptibility to third-generation cephalosporins, especially cefixime, emerged worldwide. Therefore, current international guidelines recommend dual therapy for gonorrhoea with ceftriaxone plus either azithromycin or doxycycline. Gonococcal susceptibility data in Switzerland are sparse. Methods We investigated the prevalence of antibiotic susceptibility of N. gonorrhoeae in specimens collected between 1990 and 2012 at the University of Zurich, Switzerland. Minimum inhibitory concentrations (MICs) for cefixime, ceftriaxone, ciprofloxacin, and penicillin were determined by Etests. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were used to define reduced susceptibility. Results A total of 320 isolates were tested. Between 1990 and 2006 all tested samples were susceptible to both cephalosporins. Subsequently, the prevalence of elevated MICs for cefixime increased to 10.4% (2007/2008), 11.5% (2009/2010), and 11.4% (2011/2012); and for ceftriaxone to 2.4% (2007/2008), 4.7% (2009/2010), and 0% (2011/2012), respectively. The prevalence of resistance to ciprofloxacin (72.7%) and penicillin (22.7%) was high in 2011/2012. Conclusions Decreasing susceptibility of N. gonorrhoeae to third-generation cephalosporins in Switzerland supports treatment recommendations with ceftriaxone plus azithromycin or doxycycline. Health-care providers need to be aware of possible treatment failures with cephalosporins. Continued surveillance of gonococcal antimicrobial resistance is essential.

Commercial blood culture systems support the growth of fastidious bacteria in blood. However, normally sterile body fluids (NSBF) other than blood are frequently transported and cultivated in blood culture bottles. In a 2-year period only 9 isolates of Haemophilus influenzae and 5 isolates of H. parainfluenzae were cultivated out of 7196 NSBF in our routine laboratory. The growth of H. influenzae and H. parainfluenzae in blood culture bottles without blood was compared to growth in different supplemented liquid media. Inocula of 108 bacteria of 12 H. influenzae and 16 H. parainfluenzae strains were not detected in blood culture bottles ( BacT Alert and BacT Alert -FAN ) without addition of blood. Inocula of 102 to 106 bacteria of 2 H. influenzae and 4 H. parainfluenzae strains grew in BacT Alert bottles with blood only and in brain heart infusion (BHI) broth only when supplemented with X- and V-factor. Inocula of 108 bacteria of these strains did not survive in BHI-Liquoid bottles if blood was added after a delay of 24 h. We conclude if X- and/ or V-factor dependent organisms are expected from NSBF, transport and cultivation in blood culture bottles without blood cannot be recommended. Rapid transport to the laboratory and inoculation of both liquid and solid media containing X- and V-factor are crucial for optimal recovery of these fastidious organisms.

Our aim was to evaluate the clinical impact of routine amniotic fluid and neonatal surface swab microbiology at Caesarean section. Microbiology data from 1 537 neonates delivered by Caesarean section were analysed in the light of clinical outcome. 1 340 (87%) neonates had non-pathogenic bacteria or negative culture results from both amniotic fluid and surface swab samples. Of the 197 (13%) neonates with pathogenic bacteria, 22 (1.4%) were diagnosed with infection, but only in 6 (0.4%) were the bacteria presumed to be responsible for the infection. Amniotic fluid and surface swab culture had sensitivities of 54% and 35%, and positive predictive values of 14% and 17%, respectively, for detecting a neonate at risk of infection. Amniotic fluid and neonatal surface swab microbiology at Caesarean section contributes little if anything to postnatal management and can be safely dropped from operative routine.

Background: Extended-spectrum β-lactamases (ESBLs) are an increasing challenge in the treatment of urinary tract infections (UTIs), and also in the community. We aimed to investigate the characteristics of patients with UTIs due to ESBL-producing Escherichia coli and to assess the risk factors for ESBLs in community-acquired isolates. Methods: We performed a retrospective study from January 1, 2007 to December 31, 2009 at a tertiary care teaching hospital in Switzerland, comparing patients with community-acquired versus healthcare-associated UTIs due to ESBL-producing E. coli. Additionally, we investigated the antimicrobial susceptibility of these isolates. Results: A total of 123 patients were studied, of whom 79 (64%) had community-acquired and 44 (36%) had healthcare-associated UTIs. Community-acquired isolates were associated with acute uncomplicated UTIs (odds ratio [OR] 6.62, 95% confidence interval [CI] 1.83-36.5, P

Two mechanisms account for AmpC activity in Escherichia coli , namely, mutations in the ampC promoter and attenuator regions resulting in ampC overexpression and acquisition of plasmid-carried ampC genes. In this study, we analyzed 51 clinical E. coli isolates with reduced susceptibility to amoxicillin-clavulanic acid, piperacillin-tazobactam, or extended-spectrum cephalosporins for the presence of AmpC production. Three phenotypic AmpC confirmation assays (cefoxitin-cloxacillin disk diffusion test, cefoxitin-EDTA disk diffusion test, and AmpC Etest) were compared for the detection of AmpC activity. All 51 isolates were characterized genetically by mutational analysis of the chromosomal ampC promoter/attenuator region and by PCR detection of plasmid-carried ampC genes. Altogether, 21/51 (41%) E. coli isolates were considered true AmpC producers. AmpC activity due to chromosomal ampC promoter/attenuator mutations was found in 12/21 strains, and plasmid-carried ampC genes were detected in 8/21 isolates. One strain contained both ampC promoter mutations and a plasmid-carried ampC gene. All three phenotypic tests were able to detect the majority (>90%) of AmpC-positive strains correctly. Cefoxitin resistance was found to be a discriminative parameter, detecting 20/21 AmpC-producing strains. Susceptibility to extended-spectrum cephalosporins, e.g., ceftriaxone, ceftazidime, and cefotaxime, was found in 9 of the 21 AmpC-positive strains. Considering the elevated zone diameter breakpoints of the 2010 CLSI guidelines, 2/21 AmpC-positive strains were categorized as susceptible to extended-spectrum cephalosporins.

A case of laboratory-acquired infection with Escherichia coli O157:H7 is presented. Evidence of the identity of the infecting strain was provided by toxin type and plasmid profiles. Because no obvious technical errors in laboratory practices could be demonstrated we conclude that the infecting dose for E. coli O157:H7 may be small. The clinical course was uncomplicated; during reconvalescence, the patient's serum recognized a unique 87 kDa band on immunoblots of the infecting strain.

Recurrent soft tissue abscesses of the jaw, wrist, and arm developed in a 73-year-old housewife with nephrotic syndrome and immunoglobulin A(κ) gammopathy of unknown etiology. Conventional cultures remained negative, despite visible gram-negative rods on microscopy. Broad-spectrum PCR revealed Legionella cincinnatiensis , which was confirmed by isolation of the organism on special Legionella medium. Infections due to Legionella species outside the lungs are rare. L. cincinnatiensis has been implicated in only four cases of clinical infection; these involved the lungs in three patients and the central nervous system in one patient. We conclude that broad-spectrum PCR can be a valuable tool for the evaluation of culture-negative infections with a high probability of bacterial origin and that Legionella might be an underdiagnosed cause of pyogenic soft tissue infection.

Background Our aim is to elucidate shifts in the bacterial spectrum colonising burn wounds and corresponding antibiotic susceptibilities during a 20-year study period. Methods Microbiological results from burn patients collected between 1986 and 2005 were analysed retrospectively. Results Staphylococcus aureus was isolated most frequently (20.8%), followed by Escherichia coli (13.9%), Pseudomonas aeruginosa (11.8%), coagulase-negative staphylococci (CNS) (10.9%), Enterococcus sp. (9.7%), Enterobacter cloacae (5.6%), Klebsiella pneumoniae (5%), Acinetobacter sp. (3.2%), Proteus mirabilis (2%) and Stenotrophomonas maltophilia (1.4%). Susceptibility of S. aureus to broad-spectrum substances such as ciprofloxacin or penicillinase-stable penicillins has waned, others such as cotrimoxazole or netilmicin remained effective. Not a single resistance against vancomycin was recorded. Increases in methicillin-resistant S. aureus (MRSA) were pronounced (3% in 1986–1997 (the first of the three study periods) to 16% in 1998–2001 and 13% in 2002–2005). Results for methicillin-resistant CNS (MRCNS) show an even greater increase. P. aeruginosa has shown increasing susceptibility against netilmicin (1986–1989: 84%, 2002–2005: 95%). Susceptibility of P. aeruginosa to ceftazidime has decreased markedly. S. maltophilia has shown clinically relevant susceptibility mainly against ciprofloxacin. Acinetobacter sp. have shown little susceptibility to most antibiotics. Imipenem or meropenem have been very reliable reserve antibiotics throughout the study period for the fermenting Enterobacteriaceae (E. coli, K. pneumoniae, E. cloacae and P. mirabilis), with susceptibilities of or near 100%. Conclusion In-depth knowledge of the bacteria causing infectious complications and of their antibiotic susceptibilities is a prerequisite for treating burn patients. Our study shows shifts in the microbial spectrum and their antibiogram, which mandate frequent reassessments.

Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae account for approximately 80% of the cases of acute bacterial meningitis [1]. Since the introduction of H. influenzae type b vaccination, however, H. influenzae meningitis has become rarely seen. Capnocytophaga canimorsus is an even less-frequent cause of bacterial meningitis, with only 19 cases having been published so far in the English-language literature [2]. However, since it is fashionable for humans to own animals as pets and for defense, the true incidence of this pathogen may be higher. Reported here is the case of a patient with bacteremia and meningitis after a dog bite, in which the etiologic diagnosis was difficult to establish. A previously healthy 54-year-old Caucasian man who regularly consumed alcohol (approximately 85 g ethanol/ day) was admitted to the emergency room with a 3-day history of increasing headache and chills, associated with vomiting, diarrhea, and arthralgia. He had been bitten on his left index finger by his pet dog 10 days prior to presentation. Physical examination showed a temperature of 38.6°C, blood pressure of 120/80 mmHg and pulse rate of 100 beats/min. He was alert and oriented. Flexion of the cervical spine was painful, but there were no focal neurological deficits. Laboratory findings revealed an elevated level of Creactive protein (193 mg/l), leukocytosis (12.9×10/l with 32% of neutrophils showing band forms), a decreased platelet count (56×10/l) and a normal international normalized ratio value (1.0). Lumbar puncture showed turbid cerebrospinal fluid (CSF) with 1001 leukocytes/μl (82% neutrophils), an elevated protein level (1.69 g/l) and low glucose concentration (1.8 mmol/l or 30% of the blood glucose concentration). Even though no microorganisms were seen in a Gram-stained specimen of CSF, the diagnosis of bacterial meningitis was made. Empirical antibiotic therapy with ceftriaxone and a 4-day course of intravenous dexamethasone were initiated. Gram-negative bacteria grew in both the CSF culture media and in an aerobic blood-culture flask, after 5 and 7 days, respectively. Further differentiation of the bacteria was not possible and the colonies were sent to the reference laboratory for further testing. After failing to subculture the colonies, identification was achieved using broad-range polymerase chain reaction (PCR) and analysis of 16S ribosomal DNA sequences of the colonies, as described previously [3]. The molecular identification revealed C. canimorsus (Fig. 1). Retrospective Gram stain of the colonies showed gram-negative rods with fusiform shapes suggestive of a Capnocytophaga sp. Our patient’s condition improved rapidly on ceftriaxone therapy, which was administered for 13 days, and he was discharged 1 day after treatment ended. C. canimorsus is a slow-growing, capnophilic, gramnegative rod that was first isolated in 1976. Its initial name DF-2 (dysgonic fermenter) [4] was changed to C. canimorsus, [5] since canimorsus is Latin for dog bite and the bacterium is part of the normal oral flora of dogs. In a previous study, Westwell et al. [6] were able to isolate C. canimorsus from the oral cavity of 24% of 180 dogs and 17% of cats. Most patients with C. canimorsus infection report an antecedent dog bite (54%), scratch (8.5%) or at least contact (27%) [7]. On average, 7 days pass between the time of the dog bite and hospitalization (range, 3–14 days) [2], and the clinical presentation spans from mild to severe disease. Host risk factors for a fulminant clinical course, i.e. meningitis or sepsis with shock and disseminated intravascular coagulation, include asplenia, alcoholism, and immunosuppression with glucocorticoid or chemotherapy [8]. However, in 37% of patients with C. canimorsus meningitis no such risk J. Gottwein . T. Herren (*) Department of Medicine, Limmattal Hospital, Urdorferstrasse 100, 8952 Schlieren, Switzerland e-mail: thomas.herren@spital-limmattal.ch Tel.: +41-44-7332843 Fax: +41-44-7332899

Background:: Successful treatment of allograft infections by the temporary implantation of an antibiotic-loaded polymethylmethacrylate cement spacer depends on the diffusion of antibiotics out of the cement and inhibition of bacterial growth in the surrounding tissue. We investigated with an in vitro model how long antibiotics are released by the cement and if gentamicin-resistant coagulase-negative staphylococci (CNS) are inhibited by vancomycin mixed with the gentamicin-loaded cement. Materials and Methods:: Four formulations of antibiotic-loaded cement disks, i.e. gentamicin, tobramycin, vancomycin and tobramycin combined with vancomycin, respectively, were used to test the inhibition of eight isolates of Staphylococcus epidermidis and two reference strains of Staphylococcus aureus by an agar diffusion test on Mueller-Hinton (MH) agar similar to the routine laboratory disk diffusion method. Moreover, cement spacer cylinders loaded with gentamicin alone or combined with vancomycin were submerged in MH agar for weeks and the capacity to inhibit five different isolates of S. epidermidis was measured. Results:: The size of the inhibition zones around the antibiotic-loaded cement disks correlated with the minimal inhibitory concentration (MIC) of the antibiotics against the tested strains. All five strains of S. epidermidis were inhibited by vancomycin-loaded cement spacers for at least 30 days. However, two gentamicin-resistant S. epidermidis strains with MICs of 4 mg/l and 16 mg/l could not be inhibited longer than 3 days by the gentamicin-loaded cement spacer. Conclusion:: The in vitro data suggest that antibiotic-loaded cement spacers inhibit susceptible bacteria for 4-6 weeks. The addition of vancomycin to commercial aminoglycoside-loaded cements might be helpful in allograft infections in tumor patients to inhibit a broad range of bacteria including gentamicin-resistant CNS very commonly found in such infections

Background: : Antimicrobial resistance of Helicobacter pylori is the main reason for eradication failure. We have studied the feasibility of a commercial transport medium for cultural recovery and subsequent drug susceptibility testing. Patients and Methods: : From March to December 2000, 79 consecutive gastric biopsies, positive in a rapid urease test, were transferred into a commercial transport medium and sent within 24 hours from the district hospital to the microbiological laboratory for culture and susceptibility testing. A commercial agar plate and an in-house Wilkins-Chalgren agar plate were used for culture. Susceptibility data were compared with data collected from 1992 to 2003 in the University Hospital of Zurich. Results: : Cultural recovery and susceptibility testing of H. pylori was successful in 55 of 79 patients. In 17 cases cultural recovery failed because of technical problems (n = 14), long transport time (n = 1) and unknown reason (n = 2). Failure of susceptibility testing (n = 7) was mainly due to fungal overgrowth. Resistance to metronidazole and clarithromycin was found in 15 (27%) and in 12 patients (22%), respectively; resistance to amoxicillin was not observed. Five patients (9%) showed resistance both to metronidazole and to clarithromycin. Eradication therapy failed in all patients with macrolide resistance. Resistance rates were higher in females than in males; 30% vs 12% for clarithromycin and 33% vs 20% for metronidazole. Resistance to metronidazole was significantly lower in Swiss patients (15%) than in non-Swiss patients (39%). Conclusion: : Antimicrobial resistance data can reliably be obtained by sending the biopsy specimen in a commercial transport medium to a microbiological laboratory. This is especially important after eradication failure. Resistance to metronidazole and clarithromycin is highly prevalent and more common in women and non-Swiss patients

A Gram-positive, facultatively anaerobic, rod-shaped, endospore-forming bacterium (strain MOL722(T)) was characterized using phenotypic and molecular methods. Fatty acid analysis and biochemical examination indicated that the isolate belongs to the Gram-positive bacteria with low DNA G+C content, probably to the genus Paenibacillus. Direct sequencing of the 16S rRNA gene showed ambiguities, suggesting heterogeneity. Cloned 16S rDNA yielded seven different sequences varying at 15 positions, with one being an insertion. The isolate shares 98.5% sequence similarity with Paenibacillus sp. P15-9, but less than 94% similarity with other paenibacilli and bacilli. Phylogenetic analysis with different treeing methods revealed that strain MOL722(T), together with Paenibacillus sp. P15-9, forms a novel monophyletic clade within the genus Paenibacillus. Based on phenotypic and phylogenetic evidence, it is proposed that the unknown bacterium is classified as the novel species Paenibacillus turicensis sp. nov., the type strain of which is strain MOL722(T) (= DSM 14349(T) = NCCB 100011(T)).

A 71-year-old female patient was hospitalized with membranous laryngopharyngitis typical of classical diphtheria. A toxigenic strain of Corynebacterium ulcerans was isolated from the throat. The patient was treated for 6 days with amoxicillin-clavulanic acid and recovered without complications. This second reported case of diphtheric laryngopharyngitis caused by C. ulcerans in Switzerland is a reminder that C. ulcerans should be included as a possible agent in patients with classical diphtheria symptoms.

We investigated an outbreak of Serratia marcescens in the neonatal intensive care unit (NICU) of the University Hospital of Zurich. S. marcescens infection was detected in 4 children transferred from the NICU to the University Children's Hospital (Zurich). All isolates showed identical banding patterns by pulsed-field gel electrophoresis (PFGE). In a prevalence survey, 11 of 20 neonates were found to be colonized. S. marcescens was isolated from bottles of liquid theophylline. Despite replacement of these bottles, S. marcescens colonization was detected in additional patients. Prospective collection of stool and gastric aspirate specimens revealed that colonization occurred in some babies within 24 hours after delivery. These isolates showed a different genotype. Cultures of milk from used milk bottles yielded S. marcescens. These isolates showed a third genotype. The method of reprocessing bottles was changed to thermal disinfection. In follow-up prevalence studies, 0 of 29 neonates were found to be colonized by S. marcescens. In summary, 3 consecutive outbreaks caused by 3 genetically unrelated clones of S. marcescens could be documented. Contaminated milk could be identified as the source of at least the third outbreak.

In a recent article, Hussain et al. evaluated a commercial latex agglutination test (LA), the MRSA-Screen (Denka Seiken Co., Niigata, Japan), for rapid detection of penicillin-binding protein 2a (PBP2a) in mecA-positive and mecA-negative coagulase-negative staphylococci (CNS) (2). The sensitivity and specificity were 100 and 99.5% (2), respectively, although the MRSA-Screen was initially developed for rapid detection of PBP2a in methicillin-resistant Staphylococcus aureus (MRSA) (1). However, this was a retrospective study with selected CNS strains. Furthermore, they used oxacillin-induced colonies for the LA since without induction only 72 (57.6%) of 125 mecA-positive CNS gave a positive result and additionally required an extended reaction time, i.e., 3 to 15 min, for agglutination (2). We recently presented a retrospective evaluation of the MRSA-Screen with 60 mecA-positive and 60 mecA-negative CNS, mainly (72%) Staphylococcus epidermidis (R. Zbinden, E. Rundler, V. Kaspar, and B. Berger-Bachi, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. C-234, 1999). In contrast to the results of the study of Hussain et al., all mecA-positive CNS showed positive agglutination after 3 min without induction; one isolate, i.e., Staphylococcus lugdunensis, revealed a false-positive result. Thereafter, we tested prospectively 80 unselected freshly isolated CNS. They were subsequently verified for the presence of the mecA gene by PCR (unpublished data). Thirty-eight mecA-positive CNS, of which 34 were S. epidermidis, were analyzed by the MRSA-Screen. Thirty-two were clearly positive, 4 were weakly positive, and 2 were negative after 3 min. However, one of the latter became positive after longer reaction time, i.e., 13 min, and the second was positive after induction. Therefore, we prospectively tested 30 freshly isolated CNS by the MRSA-Screen from the blood agar and from the edge of the inhibition zone of the oxacillin disk from the routinely used Mueller-Hinton susceptibility plate (unpublished data). Only 1 of 10 oxacillin disk-resistant CNS showed stronger agglutination from the induced oxacillin inhibition edge. Since an agglutination time of 10 min instead of 3 min was proposed (4), we also observed agglutination after 10 min for the oxacillin disk-susceptible strains. However, one mecA-negative CNS out of 20 oxacillin disk-susceptible CNS revealed a false-positive result after 10 min. Even if our results did not reflect the same importance of induction before using MRSA-Screen as observed by Hussain et al., we agree that induction may be helpful in order to avoid false-positive results due to extended reaction time. The phenotypic detection of methicillin resistance in CNS is problematic, as Hussain et al. have shown in an earlier report (3). Staphylococcus cohnii, Staphylococcus saprophyticus, Staphylococcus warneri, Staphylococcus lugdunensis, and Staphylococcus xylosus showed 94.6% false-positive oxacillin resistance according to the new NCCLS breakpoints in the agar dilution test. However, the new NCCLS interpretative guidelines correctly classify the vast majority of clinically significant CNS, i.e., S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis (3). The authors have now demonstrated that the MRSA-Screen is effective in detecting PBP2a in CNS and is better than the conventional susceptibility tests in classifying mecA-negative CNS as oxacillin-susceptible (2). We propose larger prospective studies with consecutive freshly isolated CNS, including phenotypically oxacillin-susceptible, mecA-positive CNS to evaluate the MRSA-Screen in the routine laboratory. If the correlation of the MRSA-Screen and mecA positivity is over 99% in such prospective studies under routine laboratory conditions, the easy rapid MRSA-Screen might replace the mecA PCR for CNS with phenotypically unclear methicillin susceptibility. Ed. Note: The authors of the published article were given the opportunity and declined to respond.