To screen or not to screen medical students for carriage of multidrug-resistant pathogens?

BACKGROUND: The carriage of multidrug-resistant (MDR) pathogens in medical students has not been studied extensively, despite the fact that they are in contact with patients and exposed to a hospital environment. AIM: To investigate the intestinal and nasal carriage of MDR pathogens among medical students and its association with their lifestyle and demographic data. METHODS: In 2021, first- and final-year medical students were invited to the study. Two rectal swabs were used for detection of extended-spectrum ß-lactamase (ESBL)-producing, colistin-, tigecycline- or carbapenem-resistant Gram-negative bacteria and vancomycin-resistant enterococci. Nasal swab was used for Staphylococcus aureus culture. S. aureus isolates were characterized by spa typing; Gram-negative resistant isolates and meticillin-resistant S. aureus (MRSA) were subjected to whole-genome short and/or long sequencing. FINDINGS: From 178 students, 80 (44.9%) showed nasal carriage of S. aureus; two isolates were MRSA. In rectal swabs, seven ESBL-producing strains were detected. Sixteen students were colonized by colistin-resistant bacteria, three isolates carried the mcr-1 gene (1.7%). The mcr-9 (10.7%, 19/178) and mcr-10 (2.2%, 4/178) genes were detected by quantitative polymerase chain reaction, but only two colistin-susceptible mcr-10-positive isolates were cultured. The S. aureus nasal carriage was negatively associated with antibiotic and probiotic consumption. S. aureus and colistin-resistant bacteria were detected more frequently among students in contact with livestock. CONCLUSION: Medical students can be colonized by (multi)drug-resistant bacteria with no difference between first- and final-year students. The participation of students in self-screening increases their awareness of possible colonization by resistant strains and their potential transmission due to poor hand hygiene.

Antimicrobial Activity of the Most Common Antibiotic-Releasing Systems Employed in Current Orthopedic Surgery: in vitro Study.

PURPOSE OF THE STUDY Infections of joint replacements represent one of the most serious problems in contemporary orthopedics. The joint infections treatment is usually multimodal and involves various combinations of drug delivery and surgical procedures. The aim of this study was to evaluate and compare the bacteriostatic and bactericidal properties of the most common antibiotic carriers used in orthopedic surgery: bone cements mixed with antibiotic and porous calcium sulfate mixed with antibiotic. MATERIAL AND METHODS Three commercial bone cements (Palacos®, Palacos® R+G, Vancogenx®) and commercial porous sulfate (Stimulan®) were prepared with a known concentration of vancomycin (a glycopeptide antibiotic). Specifically, for the purpose of our study, the testing specimens were prepared to release 0, 1, 2, 4, 8, 16, 32, 64, 128, 256, and 512 mg of vancomycin into 1 liter of solution. The specimens with increasing amount of antibiotic were placed in a separate tubes containing 5 mL of Mueller-Hinton broth inoculated with a suspension (0.1 m, McFarland 1) of the reference strain CCM 4223 Staphylococcus aureus to evaluate their bacteriostatic properties (broth dilution method). After this initial incubation and evaluation of the broth dilution method, an inoculum from each tube was transferred onto blood agar plates. After another 24-hour incubation under the same conditions, we evaluated the bactericidal properties (agar plate method). As many as 132 of independent experiments were performed (4 specimens × 11 concentrations × 3 repetitions = 132). RESULTS The bacteriostatic properties of all investigated samples were excellent, perhaps with the exception of the first bone cement (Palacos®). The sample Palacos® started to exhibit bacteriostatic properties at concentrations ≥ 8 mg/mL, while all other samples (Palacos R+G®, Vancogenx®, and Stimulan®) were bacteriostatic in the whole concentration range starting from 1 mg/mL. The bacteriocidic properties did not show such clear trends, but correlated quite well with different properties of the investigated samples during mixing - the most homogeneous samples seemed to exhibit the best and the most reproducible results. DISCUSSION The reliable and reproducible comparison of ATB carriers is a difficult task. The situation is complicated by high numbers of local antibiotic carriers on the market, numerous antibiotics used, and differences in clinical trials at different laboratories. Simple in vitro testing of bacteriostatic and bacteriocidic properties represents a simple and efficient approach to the problem. CONCLUSIONS The study confirmed that the two most common commercial systems used in the orthopedic surgery (bone cements and porous calcium sulfate) prevent bacterial growth (bacteriostatic effect), but they may not be 100% efficient in complete elimination of bacteria (bacteriocidic effect). The scattered results in the case of bacteriocidic tests seemed to be connected with the homogeneity of ATB dispersion in the systems and with the lower reproducibility of the employed agar plate method. Key words: local release of antibiotics; bone cements; calcium sulfate; antimicrobial susceptibility.

Antimicrobial efficacy and activity perseverance in arthroplasty of calcium sulfate beads containing vancomycin prepared ahead of time and stored in ready-to-use formula.

The use of local therapy with antibiotics in a suitable carrier is essential in the treatment and prevention of infections in orthopedic surgery and traumatology. In our orthopedic surgery department, a synthetic calcium sulfate hemihydrate (CaSO4·½H2O) is used as an antibiotic carrier, enabling the application of most types of intravenous antibiotics in the form of powder and liquid. This type of carrier with antibiotics is prepared in the theater during the procedure. During a surgical procedure, a small dead space is created (hand and foot area), which must be filled with an antibiotic carrier, and the situations arise where a large amount of the carrier is not used and thrown away. Therefore, we verified the efficacy of vancomycin in the pre-prepared carrier by an orientation microbiological method and by measuring the concentrations of the vancomycin released in active form and its two crystalline degradation products. Based on the agar diffusion test, we did not measure any difference in the effectiveness of the antibiotic in the carrier after its 12-day storage. Although vancomycin concentrations decreased by approximately 32% at the end of 12 days of storage, the concentrations of the released active form of vancomycin are many times higher than the minimum inhibitory concentrations for resistant strains of Staphylococcus aureus. Thus, the calcium sulfate carrier with vancomycin can be prepared several days in advance before its application, certainly up to 12 days.

[Revision of Infected Total Knee Arthroplasties with DAIR Method - Factors Affecting the Two-Year Survival Rate]. / Revize infektu totální náhrady kolenního kloubu metodou DAIR ­ faktory ovlivnující dvouleté prezití.

PURPOSE OF THE STUDY Periprosthetic joint infections in total knee arthroplasty (TKA) represent one of the most limiting factors of implantation. Frequency of this complication is up to 2.5% in primary implantation. Revision TKA with the use of DAIR (Debridement, Antibiotics and Implant Retention) procedure is a widely accepted method in treating infection, but the indication criteria have not been clearly defined as yet. The lack of uniformity prevails also with respect to the surgical technique and the importance of respective techniques for successful treatment. The purpose of this study was to evaluate the factors affecting the twoyear survival of TKA after treating the infection by DAIR. MATERIAL AND METHODS We conducted a monocentric retrospective analysis involving 52 cases of infected TKA managed with DAIR in the period between 2007 and 2016. The evaluation took into account such factors as the sex, age, history of revision surgery for aseptic or septic reasons, and pathogens. The patients were divided into groups based on the McPherson criteria. As to the procedure, we monitored the effect of administered antibiotics, time interval between the manifestation of symptoms of TKA infection and surgery, exchange of modular parts, and use of pulse lavage, continual lavage, local antibiotic carrier, or combination of these techniques. Treatment failure was defined as persistent infection and transition to chronic suppressive antibiotic therapy or need for revision surgery of the respective joint due to recurrent infection of TKA, or death directly associated with the treatment of infected TKA in the follow-up period of 2 years after DAIR. The R software (Team Development Core, 2017) was used to carry out the statistical analysis. The target variable was the failure at two years after surgery. The Generalized Linear Model (GLM) was used for the binary dependent variable - the socalled logistic model with a logit link function. RESULTS 32 of 52 patients (61.5%) were successfully treated, of whom 18 women (62.1%) and 14 men (60.9%). The effect of causative agent, administered antibiotics, polyethylene insert exchange, McPherson score or history of revision surgery of the respective joint for aseptic reasons was not confirmed. The history of revision surgery for infection of the affected joint had a strong negative impact on treatment success, 10 of 13 (76.9%) implants failed as against 10 of 39 (25.6%) implants with negative history of infection. The mean time from surgery to the manifestation of infection was 5.9 weeks (0.5-47.5). When surgery was performed within 2 weeks from the manifestation of infection, 1 of 15 (6.7%) cases failed. In case of a later surgery, 19 of 37 (51.4%) cases failed. As concerns the used surgical technique, 60% (9/15) failure was reported in case of the combination of pulse lavage and continual lavage, 36.4% (4/11) in case of the combination of pulse lavage and local antibiotic carrier, 25% (4/16) in case of separate continual lavage, and 66.7% (2/3) in case of continual lavage with local antibiotic carrier. DISCUSSION The importance of individual factors in revision surgery of periprosthetic joint infections of TKA remains unclear. The world literature indicates as a major negative effect the time factor, the positive history of infection of the affected implant, or other previous revision surgery for aseptic reasons. Ambiguous results are achieved in assessing the effect of the pathogen, administered antibiotics or presence of fistula, the statistical significance of which has not been confirmed in our study. Questionable is also the importance of individual surgical techniques. CONCLUSIONS DAIR is a suitable method in treating infections of stable TKA without the history of revision surgery for infection. The surgery should be performed within 2 weeks from the manifestation of symptoms. Key words: debridement, antibiotics, infection, implant retention, total knee arthroplasty.

Updated Czech guidelines for the laboratory diagnosis of Clostridium difficile infections.

Clostridium difficile, a causative agent of intestinal infections (CDI) of varying severity, is an important nosocomial pathogen. Microbiological diagnosis, including an appropriate test algorithm and the corresponding interpretation of the results, is crucial for CDI confirmation. This update is based on the European guidance document for CDI laboratory diagnosis, taking into account the current CDI epidemiology and laboratory diagnostic approaches in the Czech Republic. Any diarrhoeal patient should be tested for CDI. The rectal swabs can only be used for testing in patients with paralytic ileus. Currently, a two-step test algorithm is recommended for CDI diagnosis. Due to a low positive predictive value, a single commercial test is not recommended as a stand-alone test for diagnosing CDI. Samples with a positive screening test (glutamate dehydrogenase or toxigenic strain nucleic acid) and a subsequent negative EIA (enzyme immunoassay) test for the presence of free toxins are diagnostically inconclusive. An option is to use a third confirmatory test; however, the current clinical status of the patient along with other laboratory findings should be considered in order to differentiate between ongoing CDI, carriage of a toxigenic strain of C. difficile, and other causes of diarrhoea. In general, when implementing a new diagnostic test, its sensitivity and specificity should be compared against the reference method. Diagnostic tests should refer to the data from published comparative studies and should not rely solely on information provided by the manufacturer. Currently, there is no commercial test available for detection of free C. difficile toxins in stool samples with 100 % sensitivity. Moreover, the pre-analytical conditions (storage and transport temperature of stool samples) and/or the initiation of an empirical therapy prior to the sampling may decrease the sensitivity of the assay.

[Endoprosthesis Infections - Guidelines for Antibiotic Therapy Common Guidelines of the Czech Society for Orthopaedics and Traumatology and the Society for Infectious Diseases of the Czech Medical Association of J. E. Purkyne]. / Infekce endoprotéz ­ doporucení antibiotické lécby Spolecné doporucení Ceské spolecnosti pro ortopedii a traumatologii a Spolecnosti infekcního lékarství Ceské lékarské spolecnosti J. E. Purkyne.

PURPOSE OF THE STUDY This study aims to articulate regional guidelines for curative and suppressive antibiotic therapy of total joint replacement infections. MATERIAL AND METHODS When developing the standard, used as source materials were the published foreign guidelines for antibiotic therapy of prosthetic joint infections, the analysis of resistance of bacterial strains conducted in the Hospital in Ceské Budejovice, a.s. and the assessment of strain resistance for the Czech Republic published by the European Antimicrobial Resistance Surveillance Network (EARS-Net). Considered was also the availability of individual antibiotics in the Czech Republic and restricted prescription according to the Summary of Product Characteristics as specified in the State Institute for Drug Control marketing authorisation. The expert group composed of orthopaedists, microbiologists and infectious disease specialists elaborated the basic antibiotic guideline for choosing an appropriate antibiotic/antifungal drug based on the usual susceptibility, its dose and dosage interval for initial and continuation therapy. The comments of individual specialists were gradually incorporated therein and in case of doubts majority rule was applied. The drafted document was sent for peer reviews to clinical orthopaedic, infectious disease and microbiological centres, whose comments were also incorporated and the finalised document was submitted for evaluation to specialised medical societies. RESULTS The outcome is the submitted guideline for antibiotic curative and suppressive therapy suitable for managing the prosthetic joint infections, which was approved by the committee of the Czech Society for Orthopaedics and Traumatology andthe Society for Infectious Diseases of the Czech Medical Association of J. E. Purkyne. DISCUSION Curative therapy of total joint replacement infections consists primarily in surgical treatment and has to be accompanied by adequate antibiotic therapy administered initially intravenously and later orally over a sufficient period of time. Bearing in mind the wide spectrum of pathogens that can cause infections of a joint replacement and their capacity to form a biofilm on foreign materials, the correct choice of an antibiotic, its dose and dosage interval are essential for successful treatment. Such standard should respect regional availability of antibiotics, regional pathogen resistance/susceptibility and ensure the achievement of sufficiently high concentrations at the requested location including anti-biofilm activity. CONCLUSIONS The submitted guideline is not the only treatment option for joint total replacement infections, but it makes the decisionmaking easier when treating these complications in the form of infections. The final choice of an antibiotic, its dose and duration of therapy shall be based on a critical assessment of results of microbiological (blood culture and molecular genetic) tests and reflect the patient s clinical condition. Since these are multidisciplinary issues, we consider useful for this guideline to be commented upon and approved by the committee of both the Society for Orthopaedics and Infectious Diseases so that it can become the starting point for treatment. Key words: total joint replacement infection, TEP, ATB, antibiotic therapy, consensus meeting, guideline.

Increasing incidence of Clostridium difficile ribotype 001 associated with severe course of the infection and previous fluoroquinolone use in the Czech Republic, 2015.

The aim of the study was to provide an update on the epidemiology of C. difficile infection (CDI) in a representative number of hospitals within the Czech Republic in 2015. In 2015, twenty-eight Czech hospitals were invited to participate in a CDI study. Laboratories sent the first 20 consecutive C. difficile isolates for characterization by capillary-electrophoresis (CE) ribotyping and the presence of toxin genes and collected patient data on previous hospitalization, antibiotic treatment, the number of CDI episodes and the course of CDI. The mean incidence of CDI was 5.2 [95% CI 4.2-6.2] cases per 10,000 patient-bed days in 27 hospitals in 2015. Of 490 C. difficile isolates, the prevalent PCR ribotypes (RTs) were 001 (n = 164, 33.5%) and 176 (n = 125, 25.5%) followed by 014 (n = 37, 7.6%), 012 (n = 17, 3.5%), 020 (n = 16, 3.3%), 017 (n = 14, 2.9%) and 002 (n = 11, 2.2%). A severe course of CDI was reported in 104 cases (21.2%) with significant association with RT001 infection (p = 0.03). CDI recurrence was 10.8% (n = 53). The previous use of fluoroquinolones was associated with RTs 001 and 176 CDIs (p = 0.046 and p = 0.041). We observed a persistence of RTs 001 and 176 CDIs in the Czech Republic that was associated with the previous use of fluoroquinolones. This highlights the need for a reduction in fluoroquinolone use in Czech hospital settings.

[Clostridium difficile remains a medical challenge]. / Clostridium difficile stále aktuální.

Intestinal infections caused by the Clostridium difficile (CDI) bacterium currently represent a serious medical problem. They belong to the most frequent nosocomial infections and, in some countries, a community-acquired disease with a significantly increased incidence of community associated CDI is reported. The infection can manifest as mind diarrhea, but also as a life-threatening illness accompanied by paralytic ileus and painful distension of the colon, developing into secondary sepsis. Recurrent forms difficult to manage are a relatively common complication of the disease. Severity of infection may be influenced by the virulence of the causative strain. Severe course of the disease is associated with ribotypes 027, 078, 001. In the Czech Republic, ribotypes 001 and 176 have predominated over the last years. Laboratory diagnosis is based on the detection of C. difficile glutamate dehydrogenase and free clostridium toxins (A,B) in a diarrheal stool sample or culture of C. difficile in anaerobic conditions. Metronidazole, vancomycin and fidaxomicin are the drugs of choice in the treatment of aC. difficile with administration according to the actual treatment guidelines. Fecal bacteriotherapy is recommended in treatment and prevention of recurrent CDI. Surgery is indicated in progressive complicated forms when no response to medication is achieved and the patient is in a critical condition.Key words: Clostridium difficile intestinal infection epidemic ribotypes clostridium colitis treatment.

Molecular characterisation of Czech Clostridium difficile isolates collected in 2013-2015.

Clostridium difficile is a leading nosocomial pathogen and molecular typing is a crucial part of monitoring its occurrence and spread. Over a three-year period (2013-2015), clinical C. difficile isolates from 32 Czech hospitals were collected for molecular characterisation. Of 2201 C. difficile isolates, 177 (8%) were non-toxigenic, 2024 (92%) were toxigenic (tcdA and tcdB) and of these, 677 (33.5%) carried genes for binary toxin production (cdtA, cdtB). Capillary-electrophoresis (CE) ribotyping of the 2201 isolates yielded 166 different CE-ribotyping profiles, of which 53 were represented by at least two isolates for each profile. Of these, 29 CE-ribotyping patterns were common to the Leeds-Leiden C. difficile reference strain library and the WEBRIBO database (83.7% isolates), and 24 patterns were recognized only by the WEBRIBO database (11.2% isolates). Isolates belonging to these 53 CE-ribotyping profiles comprised 94.9% of all isolates. The ten most frequent CE-ribotyping profiles were 176 (n=588, 26.7%), 001 (n=456, 20.7%), 014 (n=176, 8%), 012 (n=127, 5.8%), 017 (n=85, 3.9%), 020 (n=68, 3.1%), 596 (n=55, 2.5%), 002-like (n=45, 2.1%), 010 (n=35, 1.6%) and 078 (n=34, 1.6%). Multi-locus sequence typing (MLST) of seven housekeeping genes performed in one isolate of each of 53 different CE-ribotyping profiles revealed 40 different sequence types (STs). We conclude that molecular characterisation of Czech C. difficile isolates revealed a high diversity of CE-ribotyping profiles; the prevailing RTs were 001 (20.7%) and 176 (027-like, 26.7%).

[Diagnosis of infections caused by Clostridium difficile in the Czech Republic: availability, possibilities, and interpretation of laboratory results]. / Diagnostika infekcí vyvolaných Clostridium difficile v Ceské republice--dostupnost, moznosti, interpretace laboratorních nálezu.

OBJECTIVE: To assess the availability of the laboratory diagnosis of infections caused by C. difficile in the Czech Republic (CR), including the range of tests used, possible combinations, and adequate interpretation of model results. MATERIAL AND METHODS: Data were collected through a web questionnaire survey with the participation of representatives of 61 public and private microbiological laboratories. The questionnaire addressed the use of diagnostic test kits and culture media in the diagnosis of C. difficile infection (CDI). In addition, the respondents were asked to interpret a glutamate dehydrogenase (GDH) positive and, at the same time, toxin A/B negative result, without or with laboratory confirmation if available. RESULTS: In the CR, the most commonly used test in the diagnosis of CDI is the C. DIFF Quik Chek Complete® test (Alere) for the detection of GDH and A/B toxins, as reported by 50 (82%) laboratories. Anaerobic culture is performed by 43 (70.5%) laboratories, 21 (48.8%) of which use selective agar (Oxoid). Direct detection of DNA of toxigenic C. difficile is feasible in 17 (27.9%) laboratories, with most of them (15 laboratories) using the closed system Xpert® C. difficile (Cepheid). The diagnosis based only on the detection of GDH and A/B toxins is used by 13 (21.3%) laboratories. Two (3.3%) laboratories detect A/B toxins alone and three (4.9%) laboratories carry out the detection of A/B toxins followed by anaerobic culture. A three step scheme: detection of GDH and A/B toxins with subsequent anaerobic culture is used by 26 (42.6%) laboratories. The detection of GDH and A/B toxins along with a PCR assay are provided by three (4.9%) laboratories. A complete diagnostic scheme for CDI (detection of GDH and A/B toxins, direct detection of DNA, and aerobic culture) is feasible in 14 (23%) laboratories. CONCLUSION: This questionnaire survey study identified 24 different testing algorithms to be in use within the study period (April to July 2014) in the CR. Five (8.2%) laboratories have no highly sensitive screening test such as the detection of GDH or nucleic acid amplification test (NAAT) included in their testing algorithm. Thirteen (21.3%) laboratories perform the detection of GDH and A/B toxins but have no confirmation method to be used if only one test turns out positive. In the case of GDH positivity and A/B toxin negativity, the result should be provided with a supplementary comment on further possibilities for the laboratory detection of CDI and the claimed sensitivity of the test used. If no confirmation test is available, the result should be considered as epidemiologically and clinically significant, once other possible causes of diarrhoea are ruled out.

The emergence of Clostridium difficile PCR-ribotype 001 in Slovakia.

PURPOSE: The purpose of this study was to determine the incidence of Clostridium difficile infections (CDI) and to characterise the isolates in 14 departments of ten academic hospitals in Slovakia. METHODS: During a one-month study (September 2012) all unformed stool samples were investigated using a rapid test to detect the presence of GDH and toxins A/B. Positive samples were cultured anaerobically and C. difficile isolates were characterised by ribotyping, multiple-locus variable-number tandem repeats analysis, and gyrA, rpoB and ermB investigation. RESULTS: A total of 194 unformed stool samples were investigated and 38 (19.6 %) had a positive rapid test. Of 38 samples, 27 revealed a positive result for GDH and free toxins A/B in the stool, and 11 samples only for the presence of GDH. The mean CDI incidence in 2012 was 5.2 cases per 10,000 patient bed-days. Twenty C. difficile isolates were available for molecular analysis; seventeen belonged to PCR-ribotype 001 (85 %) whereas the remaining three isolates were identified as PCR-ribotypes 017, 078 and 449. MLVA of the PCR-ribotype 001 isolates identified two clonal complexes and a close genetic relatedness between isolates from six different hospitals. Molecular analysis of antibiotic-resistance determinants revealed the presence of ermB gene encoding resistance to the MLSB group of antibiotics in 90 % of isolates, and Thr82Ile amino acid substitution in the gyrA gene associated with resistance to fluoroquinolones in 85 % of isolates. CONCLUSIONS: We conclude that C. difficile PCR-ribotype 001 is the predominant PCR-ribotype in Slovakia with a strong potential for clonal spread and development of multidrug resistance.

[Diagnosis of Clostridium difficile infections: comparative study of two immuno enzyme assays with confirmation by PCR and culture followed by PCR ribotyping]. / Diagnostika Clostridium difficile infekcí - porovnávací studie dvou imunoenzymatických metod s konfirmací pomocí PCR a kultivace s následnou ribotypizací kmene*

STUDY OBJECTIVE: Comparison of two commercially avail-able tests for the detection of Clostridium difficile Glutamate Dehydrogenase (GDH) and toxins A and B for their sensitivity and specificity. MATERIAL AND METHODS: Eighty-six stool samples from patients hospitalised in the Motol University Hospital were analysed. GDH and toxins A and B were assayed in parallel by two tests: C. difficile Quik Chek Complete® (Techlab, USA) and Liaison® C. difficile GDH and Toxins AαB (DiaSorin, USA). From the stool samples, nucleic acids were also isolated using the UltraClean® Fecal DNA kit (MoBio Laboratories, USA). The commercially available C. difficile Elite MGB® kit (Nanogen, Italy) was used for the polymerase chain reaction (PCR). Anaerobic culture on C. difficile selective medium (Oxoid) was performed for all positive samples at least in one test. Pure isolates were characterized by PCR ribotyping. RESULTS: Thirty-six (42%) samples were GDH negative and toxin A/B negative by both tests. Twenty (23%) samples were GDH positive and toxin A/B positive by both tests. Nine (10%) samples were GDH positive and toxin negative by both tests, but were positive by PCR. Eleven (13%) samples that were GDH positive and toxin negative by both tests remained negative by PCR. Six (7%) samples only were GDH positive and toxin positive by the Liaison® test alone. Four (5%) samples were GDH-positive by theLiaison® test alone. Culture failure was observed in 11 (13%) samples, of which seven were positive by PCR. PCR was inhibited in five (6%) samples. The following toxigenic ribotypes: AI-3, 001, 002, 012,014, 017, 020, 049, 054, 078, 176, 203, and 413 and non-toxigenic ribotypes: AI-34, AI-61, 010, 485, 495, and 596 were identified. CONCLUSION: The Liaison® test had seven percent higher sensitivity for the detection of toxins A/B. The two-step protocol of the tests is also cost-saving. The savings can be used e.g. for incorporating the PCR techniques into the diagnostic algorithm of the laboratory.

[Antibiotic resistance in nontyphoidal salmonellae serovars in the Czech Republic]. / Antibiotická rezistence u netyfových sérovaru Salmonella spp. v Ceské republice.

STUDY AIM: To determine antibiotic resistance and incidence of multidrug resistance among Nontyphoidal salmonellae serovars isolated from humans. MATERIAL AND METHODS: Consecutive Salmonella isolates from patients, recovered in 48 microbiology laboratories in May 2012, were analyzed in the respective reference laboratories at the National Institute of Public Health. Strains were re-identified and differentiated into serovars. Their minimum inhibitory concentrations (MICs) to 11 antibiotics were determined by the microdilution method. RESULTS: Of 25 serovars identified among 637 strains of Salmonella enterica, the most frequent were Enteritidis (87.0 %), Typhimurium (4.9 %), and monophasic Typhimurium 4,[5],12:i:- (2.0 %) and Mbandaka (0.6 %); other serovars were rare. Altogether 558 strains (87.6 %) were susceptible to all antibiotics tested and the remaining 79 strains were resistant to one or more antibiotics. The prevalence rates of resistance to individual antibiotics among 637 study strains were as follows: ampicillin 8.5%, tetracycline 5.7%, sulfamethoxazole 5.2%, cipro-floxacin 3.8%, and chloramphenicol 2.5%. Resistance to gentamicin, trimethoprim, and third and fourth generation cephalosporins was rare ( 0.5%) and none of the study strains showed resistance to meropenem. Three producers of extended spectrum beta-lactamase were multidrug resistant and two of them recovered from twins exhibited a different pattern of resistance. Resistant strains were most often assigned to the following serovars: Enteritidis (49.4%), Typhimurium (26.6%), and monophasic Typhimurium (15.2%). While only 7% (39 of 554 strains) of Enteritidis strains were resistant, the serovars Typhimurium and its monophasic variant 4,[5],12:i:- showed high rates of resistance, i.e. 66.7 and 92.3%, respectively. Furthermore, resistance was revealed in all strains of the serovars Virchow (n = 3), Kentucky (n = 1), and Newport (n = 1), in two of three strains of the serovar Infantis, and in one of two strains of the serovar Stanley. All five blood isolates were assigned to the serovar Enteritidis and one of them showed resistance to ciprofloxacin. Of 79 resistant strains, 26.6% showed resistance to ampicillin only and 24.1% to ciprofloxacin only, with multidrug resistance, i.e. resistance to three or more antibiotics, confirmed in 43.0% of strains. CONCLUSION: Despite a relatively low prevalence of resistance to the antibiotics tested among 637 study strains, the following alarming findings were made: Detection of Salmonella enterica strains resistant to ciprofloxacin as the drug of choice or to higher generation cephalosporins and multidrug resistance revealed in two thirds of the strains of the serovar Typhimurium and in all but one strains of its monophasic variant 4,[5],12:i:-.

Bone grafts as vancomycin carriers in local therapy of resistant infections.

The level of an antibiotic capable of inhibiting the etiological agent at the site of infection is an essential prerequisite for successful antibiotic therapy. In some cases, locally applied antibiotics may compensate for limitations of systemic administration and shorten systemic therapy. We aimed at verifying to what extent vancomycin (Van) bound to ground bone grafts is usable in the treatment of serious infections. The levels of released Van significantly exceeded the Van minimum inhibitory concentration, which can suppress Van-sensitive staphylococci and Van intermediate Staphylococcus aureus, for the whole period of a 16-day measurement. Our results indicate that bone grafts can be used as Van carriers in therapy of osteomyelitis caused by Van-sensitive Staphylococcus strains.

Isolation of rat lung mast cells for purposes of one-week cultivation using novel Percoll variant Percoll PLUS.

Prolonged cultivation of separated rat lung mast cells (LMC) in vitro is necessary to better investigate a possible role of LMC in different stages of tissue remodeling induced by hypoxia. Rat lung mast cells (LMC) were separated using a protocol including an improved proteolytic extraction and two subsequent density gradient separations on Ficoll-Paque PLUS and a new generation of Percoll, i.e. Percoll PLUS. Instead of usual isotonic stock Percoll solution, an alternative "asymptotically isotonic" stock solution was more successful in our density separation of LMC on Percoll PLUS. Separated cells were cultivated for six days in media including stem cell factor, interleukins IL-3 and IL-6, and one of two alternative mixtures of antibiotics. These cultivations were performed without any contamination and with only rare changes in cell size and morphology. Model co-cultivation of two allogenic fractions of LMC often caused considerable rapid changes in cell morphology and size. In contrast to these observations no or rare morphological changes were found after cultivation under hypoxic conditions. In conclusions, we modified separation on Percoll PLUS to be widely used, altered LMC separation with respect to purposes of long-lasting cultivation and observed some model morphological changes of LMC.

[In vitro assessment of vancomycin released from bone grafts]. / Monitoring vankomycinu uvolnovaného z kostních stepu v pokuse in vitro.

PURPOSE OF THE STUDY: Infections of the musculoskeletal system present a serious problem in orthopaedic and trauma medicine because, typically, they are often recurrent and associated with the development of resistance to antibiotics. The aim of this study was to ascertain whether the local concentration of vancomycin released from cancellous bone grafts exceeded the minimum inhibitory concentration (MIC) for vancomycin-resistant Staphylococcus aureus (VRSA ≥ 16 mg/L) during a 16-day in vitro experiment. MATERIAL AND METHODS: Morselised grafts of spongy bone were selected as ideal local carriers of antibiotic. They were impregnated with vancomycin (Edicin®). Its concentration was assessed by Agilent 1200 high performance liquid chromatography coupled with a diode array detector (Agilent Technologies, USA). Morselised bone was impregnated with vancomycin at 0.1 g antibiotic per 10 g bone, and 20 samples each weighing 1 g were prepared. They were placed in test tubes with phosphate buffer at pH = 7.4 and maintained in a thermostat at 37°C. During the 16-day period, buffer samples were taken at intervals and examined for vancomycin concentration by the above-described method. RESULTS: During the whole experimental period, the level of released vancomycin was high above the MIC for VRSA. The maxi- mum average concentration was obtained between day 2 and day 4 and it reached 507.68 mg/L. At this interval the vancomycin level was stable, because there was no significant difference (p >.0.005) between the values of the 2nd and the 4th day. Then a gradual decrease in antibiotic levels was detected, with an average concentration of 332.29 mg/L recorded at 16 days. DISCUSSION: Recently, the occurrence of methicilin-resistant Staphylococcus aureus (MRSA) infections has been increasing as well as the risk of VRSA infections, and therefore our experiment was set up to assess the releasing properties of bone grafts impregnated with vancomycin The levels of released vancomycin were much higher than the MIC for VRSA for the whole period of measurement. This finding is different from the results of an in vitro study by Witso et al., in which the vancomycin level dropped below the MIC after 2 weeks. The decrease in vancomycin levels following its maximum values was greater than it had been expected although the samples were diluted only minimally..There are several explanations for this finding. However, from the clinical point of view it is important that, for a sufficiently long period, vancomycin is maintained at a level exceeding the MIC for VRSA. CONCLUSIONS: In an in vitro experiment under conditions simulating a human internal environment, the elution of antibiotic from vancomycin-impregnated cancellous bone grafts was investigated. The local vancomycin concentrations much exceeded the MIC for VRSA for more than 2 weeks. The highest levels, i.e. the total vancomycin amount, were recorded at 2 to 4 days after carrier application. Based on the experimental results, vancomycin-loaded bone grafts can be recommended for local treatment of the musculoskeletal system infected with antibiotic-sensitive staphylococci, MRSA strains or possibly also for VRSA infections.

Stenotrophomonas maltophilia: Significant contemporary hospital pathogen - review.

Stenotrophomonas maltophilia (Sm) plays an important role as an opportunistic pathogen in immunocompromised individuals. The growing detection rates of this bacterium in hospitalized patients are associated with the invasiveness of therapeutic and diagnostic procedures and the selection pressure of antibiotic therapy. A broad range of infections that can be caused by Sm is frequently bound to biofilm. The high level of intrinsic resistance to many unrelated antibiotics and increasing acquired resistance to the drug of choice, trimethoprim-sulfamethoxazole pose a threat for the near future when our treatment options may become depleted. Prevention of colonization and infection consists in consequent implementation of the rules governing nosocomial infection control, rational use of antibiotics including the optimization of selection and testing of antimicrobial agents suitable for the treatment of stenotrophomonad infections.

[Late hematogenous infection of prosthetic joint]. / Pozdní hematogenní infekce kloubních náhrad.

The importance of prevention in late hematogenous infection is well understood but, because responsibility lies with general practitioners and other specialists, the orthopedic surgeon is usually not much interested. In both our and other countries, discussions are taking place on whether and to what extent antibiotic prevention should be carried out. Antibiotic prophylaxis of hematogenous infection is not indicated for all patients with joint arthroplasty, but only for a limited, defined group of patients at high risk. In these, however, the present state of knowledge suggests that prevention is necessary. A preventive treatment of late hematogenous infection is used for a procedure or a disease associated with risks in all the patients involved within two years of prosthetic joint implantation and, after this period, only in immunosuppressed patients. Surgery on the urogenital tract associated with the risk of bacteremia includes prostate gland surgery, operations for urinary bladder tumors, nephrolithotomy, extracorporeal lithotripsy and prostate biopsy. Certain conditions, such as urinary catheter presence, intermittent catheterization, urethral stent presence, urine retention and a history of urinary tract infection or prostate inflammation, pose an increased risk of bacterial colonization for the urogenital system. Dental procedures associated with a risk of bacteremia include tooth extraction, surgery on the parodontium, surgical extraction of an impacted tooth, dental implant treatment, procedures in a tooth's apical region, initial application of an orthodontic apparatus, intraligamentous blocks and also cleaning teeth and implants expected to bleed. Gynecological surgery with a risk of bacteremia are abdominal, vaginal and laparoscopic hysterectomies, surgery for cancer contaminated with vaginal bacteria, reconstruction surgery, operations on the pelvic floor for defects associated with urinary incontinence and use of xenotransplants. In obstetrics, a cesarean section carries some risks. In general surgery, the preventive administration of antibiotics is indicated, apart from situations always requiring antibiotic therapy, also for advanced forms of acute appendicitis, perirectal abscess, invasive endoscopy procedures on the colon, soft tissue phlegmona or abscess, surgical treatment of venous ulceration and pressure sores, and limb amputation. When inserting any piercing in patients with joint replacement at risk, it is recommended to do it with antibiotic administration; also, it is necessary to responsibly treat any inflammatory complication. The system of prevention for the late hematogenous infections of prosthetic joints is not developed as thoroughly as, for instance, it is in cardiology for patients with valve reconstruction. Because of the reasons given above, it is advisable to set up unambiguous guidelines for the prevention of late hematogenous infection in patients with joint replacement.