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

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.

Three Klebsiella pneumoniae lineages causing bloodstream infections variably dominated within a Greek hospital over a 15 year period.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a major clinical and public health threat. The rapid dissemination of this pathogen is driven by several successful clones worldwide. We aimed to investigate the CRKP clonal lineages, their antibiotic resistance determinants and their potential transmissions in a tertiary care hospital located in Athens, Greece. Between 2003 and 2018, 392 CRKP isolates from bloodstream infections were recovered from hospitalized patients. Whole genome sequencing (WGS) was performed on the Illumina platform to characterize 209 of these isolates. In total, 74 % (n=155) of 209 isolates belonged to three major clonal lineages: ST258 (n=108), ST147 (n=29) and ST11 (n=18). Acquired carbapenemase genes were the mechanisms of resistance in 205 isolates (bla KPC, n=123; bla VIM, n=56; bla NDM, n=20; bla OXA-48, n=6). Strong associations (P=0.0004) were observed between carbapenemase genes and clonal lineages. We first isolated bla VIM-1-carrying ST147 strains during the early sampling period in 2003, followed by the emergence of bla KPC-2-carrying ST258 in 2006 and bla NDM-1-carrying ST11 in 2013. Analysis of genetic distances between the isolates revealed six potential transmission events. When contextualizing the current collection with published data, ST147 reflected the global diversity, ST258 clustered with isolates representing the first introduction into Europe and ST11 formed a distinct geographically restricted lineage indicative of local spread. This study demonstrates the changing profile of bloodstream CRKP in a tertiary care hospital over a 15 year period and underlines the need for continued genomic surveys to develop strategies to contain further dissemination. This article contains data hosted by Microreact.

Epidemiological characteristics of infections caused by Bacteroides, Prevotella and Fusobacterium species: a prospective observational study.

In order to investigate differences among infections due to Gram-negative anaerobic bacteria (Bacteroides, Prevotella and Fusobacterium spp.), clinical, epidemiological, and microbiological data were collected and evaluated from 206 anaerobic infections. The most frequently isolated species was Bacteroides fragilis. The majority of the cases were intra-abdominal infections (49%) followed by skin and soft tissue infections (24.7%). Logistic regression analysis showed that Bacteroides spp. strains were more often isolated from intra-abdominal infections (p = 0.002), whereas Prevotella spp. were isolated more frequently from cases with shorter duration of hospitalization (p = 0.026), and less frequently from bloodstream infections (p = 0.049). In addition, Bacteroides spp. were associated with coinfection due to Enterobacteriaceae species (p = 0.007), whereas Prevotella spp. were associated with coinfection due to Staphylococcus spp. (p = 0.002). Patients with an infection due to B. fragilis, were more frequently admitted in a general surgical ward (p = 0.017), or have been treated with a 2nd generation cephalosporin before anaerobic infection onset (p = 0.05). Total mortality was 10.9% and was associated with bacteremia (p = 0.026), and hematological (p = 0.028), or solid organ malignancy (p = 0.007). Metronidazole resistance was detected only among Prevotella spp. (16.2%) and B. fragilis group (0.8%) isolates. In conclusion, this study indicated differences between infections due to the most frequently isolated Gram-negative anaerobic species, differences that may affect the design and implementation of empirical antimicrobial chemotherapy guidelines.

Failure of central venous catheter insertion and care bundles in a high central line-associated bloodstream infection rate, high bed occupancy hospital.

BACKGROUND: Our hospital has several characteristics different from the settings in which the central venous catheter (CVC) care bundle has been implemented so far, that is, care bundles or protocols are not systematically used, and the prevalence of central line-associated bloodstream infections (CLABSI) is high, as is bed occupancy rate. We examined the effectiveness of CVC care bundles. METHODS: Modified CVC bundles were implemented across all settings of our hospital. During both phases of the study, we collected data on CLABSI, and we monitored CVC insertion and management practices with direct observation audits. RESULTS: We have studied 913 CVC insertions (454 in PRE and 459 in POST) for 11,871 catheter-days. The incidence of CLABSI was 8.3 per 1,000 catheter-days PRE, and 7.6 per 1,000 catheter-days POST (incidence rate ratio, 0.92; 95% confidence interval, 0.60-1.40). Compliance with the CVC insertion bundle increased from 8.4%-74.3% (P < .0001). The CVC management bundle compliance also increased from 11.4%-57.7% (P < .0001). CONCLUSIONS: Despite improved compliance after the intervention, implementation of a modified CVC bundle failed to decrease CLABSI incidence. Higher bundle compliance rates may be necessary for a significant decrease in the incidence of CLABSI, along with the appropriate organizational culture and levels of staffing.

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.

Colistin resistance in carbapenemase-producing Klebsiella pneumoniae bloodstream isolates: Evolution over 15 years and temporal association with colistin use by time series analysis.

Colistin is often the only available treatment option against infections caused by carbapenemase-producing Klebsiella pneumoniae (CP-Kp). In this study, the evolution of colistin resistance among CP-Kp and its relationship with colistin use in a tertiary-care hospital in Athens, Greece, was investigated. All CP-Kp blood isolates recovered between January 2002 and June 2016 were tested for susceptibility to colistin by agar dilution and broth microdilution methods. Data on colistin use were collected from the pharmacy database. Time series of colistin use and resistance were analysed using the Box and Jenkins method. A transfer function model was built to quantify the dynamic relationship between colistin use and resistance. Overall, 313 CP-Kp isolates were identified. The percentage colistin resistance increased from 0% in 2002 to 26.9% in 2016 (R2 = 0.5, P < 0.01). A temporal association between colistin use and resistance was observed; an increase in colistin use by 1 DDD/100 patient-days led to a 0.05 increase in the incidence rate of colistin resistance. The time lag between the effect of colistin use on subsequent variations in colistin resistance was 3 months. Colistin use and prior levels of colistin resistance could explain 69% of colistin resistance; in the remaining 31%, other factors might have played a role. The results presented here demonstrate a significant temporal association between colistin use and colistin resistance. These findings have important implications in implementing strategies to contain colistin resistance.

Differences in the evolution of imipenem susceptibility among Klebsiella pneumoniae and Escherichia coli isolates during a 6-year period in a tertiary care hospital.

The evolution of imipenem disk-diffusion susceptibility results of 2652 strains of Klebsiella pneumoniae and 7596 Escherichia coli isolated during the period 2000-2005 were analysed. Screening for production of metallo-beta-lactamases was performed using the EDTA-synergy method. The percentage rate of K. pneumoniae isolates having a zone diameter < or =25 mm increased from 20% in 2000 to 41% in 2005, whereas the respective rate of isolates having a zone diameter > or =30 mm decreased from 48 to 23%. These changes were more evident during 2000-2002, followed in 2003 by the isolation of the first imipenem-resistant strains. Regarding E. coli, a similar decrease was observed (the rates of isolates having a zone diameter < or =25 mm and > or =30 mm changed from 7% and 68% in 2000, to 32% and 36% in 2005, respectively) following the respective changes of K. pneumoniae. A total of 20 K. pneumoniae strains, but no E. coli, were confirmed as metallo-beta-lactamase producers. In conclusion, a decrease of the imipenem susceptibility prior to the isolation of the first resistant strains in a tertiary care hospital was detected, as well as differences in this decrease between the two species. These findings indicate that monitoring of the evolution of imipenem susceptibility in real-time may help in unveiling forthcoming resistance and in implementing the appropriate diagnostic techniques.

In vitro activities of tigecycline against recently isolated Gram-negative anaerobic bacteria in Greece, including metronidazole-resistant strains.

The in vitro activity of tigecycline was compared with those of benzylpenicillin, piperacillin + tazobactam, cefoxitin, imipenem, metronidazole, clindamycin, and tetracycline against 249 Gram-negative anaerobic bacteria (158 Bacteroides fragilis group, 27 non-fragilis Bacteroides spp., 44 Prevotella spp., and 20 miscellaneous), recently isolated from 8 general hospitals in Athens, Greece. Overall tigecycline MIC(50) and MIC(90) were 0.25 and 2 mg/L, respectively, whereas B. fragilis group MIC(50) and MIC(90) were 0.5 and 4 mg/L, respectively. In total, 93% of the isolates were susceptible to tigecycline (MIC /= 32 mg/L) was detected. In addition, tigecycline exhibited good activity against metronidazole- and tetracycline-resistant isolates (MIC(90), 0.5 and 8 mg/L, respectively). In summary, tigecycline exhibits good in vitro activity against Gram-negative anaerobic bacteria isolated in Greece, as well as stability to the most common occurring resistance mechanisms, attributes that make this parenteral agent an attractive alternative for use against infections involving these microorganisms.

Nationwide surveillance of resistance rates of Staphylococcus aureus clinical isolates from Greek hospitals, 2012-2013.

Purpose To evaluate the in vitro efficacy of several anti-staphylococcal agents against a nationwide collection of contemporary Staphylococcus aureus clinical isolates from several healthcare centres in Greece. Methods Thirty hospitals throughout Greece (18 in Attica) provided all clinical isolates of S.aureus from April 2012 to May 2013 to a central lab to be re-submitted to susceptibility testing. The MICs were evaluated by Vitek® 2 with the exception of ceftaroline (OXOID M.I.C. Evaluator™). Vancomycin and daptomycin MICs were also evaluated by Etest®. Heterogeneously vancomycin-intermediate strains (hVISA) were detected by the Etest® GRD. VISA phenotype was confirmed by PAP-AUC. Results A total of 1005 isolates (39% MRSA) were studied. Susceptibility rates were: erythromycin 66.5%, clindamycin 79.2%, SXT 98.9%, rifampicin 97.3%, fusidic acid 67%, moxifloxacin 78.8%, vancomycin 99.9%, ceftaroline 92.9% and linezolid, tigecycline and daptomycin 100%. For mupirocin, high level resistance could be excluded for 98.9% of isolates. Vancomycin Etest® MIC50/90 were 1.5/1.5 mg/L, 58.5% of isolates exhibited a MIC > 1 and 8.7% a MIC of 2 mg/L, while Vitek® MIC50/90 were 1/1 and 3.1% showed MIC > 1 mg/L. One VISA strain was detected. Among the selected 175 isolates that were screened for hVISA phenotype, six (3.4%) were positive. In 315 bloodstream isolates, 64.1% had a vancomycin Etest® MIC > 1 mg/L. Conclusions This multi-centre surveillance study revealed that a significant percentage of contemporary S.aureus isolates from Greek patients have a vancomycin MIC (> 1 mg/L) that may compromise the clinical efficacy of the drug for the treatment of serious infections. The in vitro activity of SXT, rifampicin, mupirocin, linezolid, tigecycline, daptomycin and ceftaroline remains excellent.

Sonication assisted microbiological diagnosis of implant-related infection caused by Prevotella disiens and Staphylococcus epidermidis in a patient with cranioplasty.

BACKGROUND: Infections present a major complication of cranioplasty procedures and in many cases removal of the implant material becomes a necessity. Sonication of the artificial implant material has been used during the last years, in order to facilitate better diagnosis of these infections, nevertheless its use in cranial implant infections is still limited. CASE PRESENTATION: A case of a 63-year-old Caucasian male patient who underwent a decompressive craniectomy, due to intracranial hemorrhage, and a consequent cranioplasty using an autogenic bone flap fixed by titanium clamps, is reported. After three unsuccessful cranioplasty efforts to repair a persistent skin defect, removing the bone flap and the titanium clamps was a necessity. Tissue and bone cultures were unable to reveal any microorganism whilst sonication of the removed titanium clamps and consequent culture of the resulting sonication liquid yielded Prevotella disiens and Staphylococcus epidermidis. The patient was treated with daptomycin and metronidazole until discharge and the skin defect was successfully repaired. CONCLUSION: The present case report indicates that the use of the sonication procedure assisted the microbiological diagnosis. This is the first known neurosurgical case of the implementation of the sonication procedure.

Changes in the epidemiology of methicillin-resistant Staphylococcus aureus in a Greek tertiary care hospital, over an 8-year-period.

A total of 1019 non-replicate, consecutively isolated methicillin-resistant Staphylococcus aureus (MRSA) strains were collected from in-patients of a tertiary care general hospital in Athens, Greece, during the period 1994-2001. The susceptibility, resistance phenotypes and the dissemination of these isolates among hospital wards were studied. Total MRSA and gentamicin-resistant MRSA, as a proportion of the S. aureus isolates, increased from 33 and 9% in 1994 to 50.1 and 33.3% in 2001, respectively. Three main multi-resistant phenotypes predominated, representing 50.9% of the total MRSA isolates in 2001. MRSA strains susceptible to all antibiotics tested decreased to 1.9% in 1997 and again increased to 13.5% in 2001. A gradual decrease in the susceptibility of vancomycin during the 8-year-period was detected, but no vancomycin resistant S. aureus strains were isolated.

Detection of metallo-ß-lactamase genes in clinical specimens by a commercial multiplex PCR system.

hyplex®-MBL ID Multiplex PCR-ELISA, a novel method for identifying metallo-ß-lactamase genes directly in clinical specimens, was evaluated using a consecutive collection of 326 samples from three hospitals in Greece characterized by high prevalence of VIM producers. The method exhibited high sensitivity (98.0%) and specificity (98.6%) and was proven reliable in detecting bla(VIM) genes in blood, urine, pus, and sputum samples that, as confirmed by conventional methods, contained various VIM-producing species. Future multicenter studies should be considered for the thorough evaluation of this method and its potential diagnostic utility.

Two cases of infections due to multidrug-resistant Bacteroides fragilis group strains.

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.