Modeling of Urinary Microbiota Associated With Cystitis.

A decade ago, when the Human Microbiome Project was starting, urinary tract (UT) was not included because the bladder and urine were considered to be sterile. Today, we are presented with evidence that healthy UT possesses native microbiota and any major event disrupting its "equilibrium" can impact the host also. This dysbiosis often leads to cystitis symptoms, which is the most frequent lower UT complaint, especially among women. Cystitis is one of the most common causes of antimicrobial drugs prescriptions in primary and secondary care and an important contributor to the problem of antimicrobial resistance. Despite this fact, we still have trouble distinguishing whether the primary cause of majority of cystitis cases is a single pathogen overgrowth, or a systemic disorder affecting entire UT microbiota. There are relatively few studies monitoring changes and dynamics of UT microbiota in cystitis patients, making this field of research still an unknown. In this study variations to the UT microbiota of cystitis patients were identified and microbial dynamics has been modeled. The microbial genetic profile of urine samples from 28 patients was analyzed by 16S rDNA Illumina sequencing and bioinformatics analysis. One patient with bacterial cystitis symptoms was prescribed therapy based on national guideline recommendations on antibacterial treatment of urinary tract infections (UTI) and UT microbiota change was monitored by 16S rDNA sequencing on 24 h basis during the entire therapy duration. The results of sequencing implied that a particular class of bacteria is associated with majority of cystitis cases in this study. The contributing role of this class of bacteria - Gammaproteobacteria, was further predicted by generalized Lotka-Volterra modeling (gLVM). Longitudinal microbiota insight obtained from a single patient under prescribed antimicrobial therapy revealed rapid and extensive changes in microbial composition and emphasized the need for current guidelines revision in regards to therapy duration. Models based on gLVM indicated protective role of two taxonomic classes of bacteria, Actinobacteria and Bacteroidia class, which appear to actively suppress pathogen overgrowth.

Identification of pathogens from native urine samples by MALDI-TOF/TOF tandem mass spectrometry.

BACKGROUND: Reliable high-throughput microbial pathogen identification in human urine samples is crucial for patients with cystitis symptoms. Currently employed methods are time-consuming and could lead to unnecessary or inadequate antibiotic treatment. Purpose of this study was to assess the potential of mass spectrometry for uropathogen identification from a native urine sample. METHODS: In total, 16 urine samples having more than 105 CFU/mL were collected from clinical outpatients. These samples were analysed using standard urine culture methods, followed by 16S rRNA gene sequencing serving as control and here described culture-independent MALDI-TOF/TOF MS method being tested. RESULTS: Here we present advantages and disadvantages of bottom-up proteomics, using MALDI-TOF/TOF tandem mass spectrometry, for culture-independent identification of uropathogens (e.g. directly from urine samples). The direct approach provided reliable identification of bacteria at the genus level in monobacterial samples. Taxonomic identifications obtained by proteomics were compared both to standard urine culture test used in clinics and genomic test based on 16S rRNA sequencing. CONCLUSIONS: Our findings indicate that mass spectrometry has great potential as a reliable high-throughput tool for microbial pathogen identification in human urine samples. In this case, the MALDI-TOF/TOF, was used as an analytical tool for the determination of bacteria in urine samples, and the results obtained emphasize high importance of storage conditions and sample preparation method impacting reliability of MS2 data analysis. The proposed method is simple enough to be utilized in existing clinical settings and is highly suitable for suspected single organism infectious etiologies. Further research is required in order to identify pathogens in polymicrobial urine samples.

Infection as a predictor of mortality in decompensated liver cirrhosis: exploring the relationship to severity of liver failure.

BACKGROUND: Infections are common in patients with liver cirrhosis and increase mortality. We explored the relationship between infection and liver dysfunction in their effects on mortality. METHODS: Single-center data on decompensated liver cirrhosis patients hospitalized between March 2014 and December 2017 (index period) were reviewed until death, liver transplantation or 31 December 2018. Infections were classified as community-acquired infection (CAi) or hospital/healthcare associated infection (HCAi). Child-Pugh, model for the end-stage liver disease (MELD) and chronic liver failure-organ failure (CLiF-OF) scores indicated liver (dys)function. RESULTS: We enrolled 155 patients (85% alcoholic liver disease), 65 without infection at first hospitalization, 48 with CAi and 42 with HCAi. Multidrug resistant agents were confirmed in 2/48 (4.2%) CAi and 10/42 (23.8%) HCAi patients. At first hospitalization, infection was independently associated with worse liver dysfunction and vice versa, and with higher 30-day mortality [odds ratio (OR) = 2.73, 95% confidence interval (CI) 1.07-6.94]. The association was reduced with adjustment for MELD/CLiF-OF scores, but mediation analysis detected an indirect (via liver dysfunction) association. Twenty-eight patients were repeatedly hospitalized, 11 with new HCAi. HCAi was independently associated with twice higher risk of medium-term mortality and added an additional risk to any level of liver dysfunction, considering all or patients who survived the first 30 days. In those repeatedly hospitalized, HCAi appeared independently associated with a higher probability of infection and higher MELD scores at subsequent hospitalizations. CONCLUSION: Infection (particularly HCAi) adds mortality risk to any level of liver dysfunction in decompensated liver cirrhosis patients. Mechanisms of long(er)-term effects (in acute episode survivors) seemingly include enhanced deterioration of liver function.

Characterization of Isolates Associated with Emergence of OXA-48-Producing Klebsiella pneumoniae in Croatia.

Here, we report a retrospective study conducted to elucidate emergence, epidemiology, and molecular mechanisms of resistance underlying the early spread of OXA-48 carbapenemase-producing Enterobacteriaceae in Croatia. Retrospective screening for OXA-48 producers was performed on a collection of 296 nonrepetitive, carbapenem-nonsusceptible enterobacterial isolates collected from January 2011 to December 2012 from 40 participating centers in Croatia. Antimicrobial susceptibility profiles and production of carbapenemases were assessed phenotypically. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing were used for epidemiological analysis. Resistance genes were characterized by polymerase chain reaction (PCR) and sequencing. Plasmid localization of blaOXA-48 in isolates and transconjugants was investigated by S1-PFGE and Southern hybridization. PCR mapping was used for identification of genetic platform surrounding blaOXA-48. Out of 296 carbapenem-nonsusceptible isolates, blaOXA-48 gene was detected in 12 Klebsiella pneumoniae isolates. All OXA-48-producing isolates showed varying resistance to carbapenems and 11 were multidrug resistant. All coproduced additional beta-lactamases, including CTX-M-15, which was detected in eight isolates. Isolates were delineated in five clonal types by PFGE corresponding to five sequence types (STs) assigned ST15, ST16, ST37, ST528, and ST1418. All OXA-48 isolates conjugated successfully and other resistance determinants were not cotransferred. blaOXA-48 was carried on a ∼60 kb IncL/M plasmid and was detected within Tn1999.2 composite transposon. OXA-48, a class D carbapenemase, is emerging as a potentially significant contributor among carbapenem-resistant Enterobacteriaceae in Croatia, alongside class A and B carbapenemases. Polyclonal genetic background of K. pneumoniae isolates carrying ∼60 kb incL/M plasmid indicates that dissemination of the blaOXA48 gene is not driven exclusively by the spread of a single clone.

Deep brain stimulation for the early treatment of the minimally conscious state and vegetative state: experience in 14 patients.

OBJECTIVE An effective treatment of patients in a minimally conscious state (MCS) or vegetative state (VS) caused by hypoxic encephalopathy or traumatic brain injury (TBI) is not yet available. Deep brain stimulation (DBS) of the thalamic reticular nuclei has been attempted as a therapeutic procedure mainly in patients with TBI. The purpose of this study was to investigate the therapeutic use of DBS for patients in VS or MCS. METHODS Fourteen of 49 patients in VS or MCS qualified for inclusion in this study and underwent DBS. Of these 14 patients, 4 were in MCS and 10 were in VS. The etiology of VS or MCS was TBI in 4 cases and hypoxic encephalopathy due to cardiac arrest in 10. The selection criteria for DBS, evaluating the status of the cerebral cortex and thalamocortical reticular formation, included: neurological evaluation, electrophysiological evaluation, and the results of positron emission tomography (PET) and MRI examinations. The target for DBS was the centromedian-parafascicular (CM-pf) complex. The duration of follow-up ranged from 38 to 60 months. RESULTS Two MCS patients regained consciousness and regained their ability to walk, speak fluently, and live independently. One MCS patient reached the level of consciousness, but was still in a wheelchair at the time the article was written. One VS patient (who had suffered a cerebral ischemic lesion) improved to the level of consciousness and currently responds to simple commands. Three VS patients died of respiratory infection, sepsis, or cerebrovascular insult (1 of each). The other 7 patients remained without substantial improvement of consciousness. CONCLUSIONS Spontaneous recovery from MCS/VS to the level of consciousness with no or minimal need for assistance in everyday life is very rare. Therefore, if a patient in VS or MCS fulfills the selection criteria (presence of somatosensory evoked potentials from upper extremities, motor and brainstem auditory evoked potentials, with cerebral glucose metabolism affected not more than the level of hypometabolism, which is judged using PET), DBS could be a treatment option.

A rare case of necrotizing fasciitis caused by Vibrio cholerae O8 in an immunocompetent patient.

We report a case of necrotizing fasciitis of the leg caused by Vibrio cholerae O8 in a 63-year-old immunocompetent man after he had been fishing in a lake on a Croatian island. The strain was cytotoxic, invasive and adhesive and contained a fragment of the gene for El Tor-like hemolysin (El Tor hlyA). After surgical and antibiotic treatment, the patient fully recovered.

[IMPACT OF BIOFILM ON HEALING AND A METHOD FOR IDENTIFYING IT IN THE WOUND]. / UTJECAJ BIOFILMA NA CIJELJENJE RANE I POSTUPAK ZA IDENTIFIKACIJU BIOFILMA U RANI.

The skin microbiome is the aggregate of microorganisms that reside on the surface and in deep layers of the skin. Skin is colonized by bacteria, fungi, viruses and mites, maintaining a balance. Disruption in the ecosystem results in skin infections. Chronic wounds in diabetics, elderly and immobile individuals are at risk of skin organisms to invade and become pathogenic upon breach of the skin barrier. The bacteria of the skin microbiome may contribute to delayed healing and persistent inflammation. Staphylococcus epidermidis is an invasive skin organism that causes infection, i.e. hospital acquired infection (HAl) on medical devices and form biofilm. At the most basic level, biofilm can be described as bacteria embedded in a thick, slimy barrier of sugars and proteins. The biofilm barrier protects the microorganisms from external threats. Biofilms provide a reservoir of potentially infectious microorganisms that are resistant to antimicrobial agents, and their importance in the failure of medical devices and chronic inflammatory condition is increasingly being recognized. Particular research interest exists in the association of biofilms with wound infection and non-healing, i.e. chronic wounds. There is now strong evidence that biofilm is present in the majority of chronic wounds. Specialized microscopic techniques used since 2008 have allowed several research groups to demonstrate that 60% to 90% of chronic wounds have biofilm versus only 6% of acute wounds. While many studies confirm that chronic wounds often contain a polymicrobial flora, controversy remains with regard to whether these organisms directly contribute to non-healing. It seems most likely that individual bacteria themselves are not directly responsible for non-healing wounds. Rather, there is direct correlation between the presence of four or more distinct bacterial species in a wound and non-healing, suggesting that mixed microbial populations are the cause of pathology. The most reliable method to confirm the presence of a biofilm is specialized microscopy, e.g., bright-field, fluorescence in situ hybridization (FISH), and environmental scanning electron microscope (ESEM). Surface wound cultures underestimate total wound microbiota--misleading. Histological staining of deep debrided tissue shows evidence of biofilms. Now it is recognized that the majority of microbial species in chronic wounds are anaerobic bacteria (deep swabbing techniques yield similar findings to biopsies) if samples are processed within two hours. Traditional (wound culture method) cultures have limitations because all microbes (the organisms within the biofilm) cannot be isolated in culture or cannot be detected. Therefore, the most effective methods are molecular

Consensus statement: patient safety, healthcare-associated infections and hospital environmental surfaces.

Healthcare-associated infections have serious implications for both patients and hospitals. Environmental surface contamination is the key to transmission of nosocomial pathogens. Routine manual cleaning and disinfection eliminates visible soil and reduces environmental bioburden and risk of transmission, but may not address some surface contamination. Automated area decontamination technologies achieve more consistent and pervasive disinfection than manual methods, but it is challenging to demonstrate their efficacy within a randomized trial of the multiple interventions required to reduce healthcare-associated infection rates. Until data from multicenter observational studies are available, automated area decontamination technologies should be an adjunct to manual cleaning and disinfection within a total, multi-layered system and risk-based approach designed to control environmental pathogens and promote patient safety.

The use of paleo-imaging and microbiological testing in the analysis of antique cultural material: multislice tomography, and microbial analysis of the Trogir Cathedral cope hood depicting St. Martin and a beggar.

Paleoradiology is the study of biological and other materials from archeological settings through the use of various medical imaging techniques. Although it is most often used in the scientific study of ancient human remains, it can also be used to study metals, ceramics, paper, and clothes. The aim of this study was to test two paleoimaging techniques (MSCT and mammography) in the analysis of an important Croatian liturgical vestment: the hood of a bishop's cope from St. Lawrence's Treasury in Trogir depicting St. Martin and a beggar. To ensure a safe environment for scientists participating in the analysis, a preliminary microbiological analysis was performed, which contributed to the database of microbiological flora found on Croatian archeological remains and relics studied to date. Due to a great amount of metal filaments, the paleoradiological analysis did not produce satisfactory results. However, a digitally enhanced image clearly showed fine metal embroidery of the hood that was not so easily perceived by naked eye. This article argues in favor of expanding paleoradiological studies on materials other than human remains and also of publishing unsatisfactory results, as important lessons for future development of techniques and methods to analyze ancient remains and seek answers about human historical and cultural heritage.

Microbiological analysis of the mummy of St. Marcian.

The paper provides the results of the microbiological analysis of samples taken from the mummified remains of St. Marcian. Microorganisms found belong to bacteria and fungi of the genera Aspergillus, Bacillus, Penicillium, Sarcina, and Shewanella which are all apart of the common air, soil, and human microbial flora and therefore present no health hazard.

Nightmare complication after lumbar disc surgery: cranial nontraumatic acute epidural hematoma.

STUDY DESIGN: Case report. OBJECTIVE: The nontraumatic cranial epidural hematoma is an extremely rare clinical entity and it commonly occurs in the presence of infectious diseases as complication of sinusitis, coagulopathy, vascular malformation of the dura, hemorrhagic tumor, in patients with sickle cell disease, systemic lupus erythematosus, after open heart surgery, and in patients on hemodialysis. SUMMARY OF BACKGROUND DATA: We reported a case of spontaneous epidural hematoma that occurred after herniated disc surgery. There was no history of preceding head trauma and patient medical history was unremarkable. To date, no report is found of cranial epidural hematoma after lumbar discectomy. METHODS: A 58-year-old woman was operated because of a herniated disc at lumbar L3-L4 level and spinal stenosis. Bilateral L3-L4 interlaminectomies and L3-L4 discectomy were performed. Hemovac drain was placed in epidural space. Four hours after surgery, the patient developed left side weakness accompanied with nausea, vomiting, and severe headache. Urgent computed tomography of the head showed acute cranial epidural hematoma and the patient underwent emergent surgery. There was no evidence of trauma of the scalp or any other anatomical abnormality. Control computed tomography 14 hours after evacuation of the hematoma showed new extradural hematoma at the site of the previous one. Patient underwent second surgery. RESULTS: Nine days after operation of the spine, patient recovered completely with no neurological deficit, but the cerebrospinal fluid showed at the place of spinal wound. Patient was operated once more and a minor dural tear was revealed, which was repaired. The patient was discharged on ninth postoperative and she recovered completely. CONCLUSION: There have been described rare cases of spontaneous subdural, intracerebral, and intracerebellar hematoma caused by intracranial hypotension, but this is the first described case of spontaneous cranial epidural hematoma after lumbar disc surgery caused by intracranial hypotension.

Microbiological analysis of a mummy from the archeological museum in Zagreb.

In this paper we report the results of the microbiological analysis of the samples taken from the mummy from the collection of the Archaeological museum in Zagreb, Croatia. Samples were taken from specific places such as oral, orbital, abdominal cavity and bandages surrounding the mummy, and analyzed in Department of Microbiology and Hospital Infections in University Hospital "Dubrava" in Zagreb and in National Reference Laboratory for systemic mycoses of Croatian National Institute of Public Health in Zagreb. The analysis indicated that all of the found organisms were non-primary pathogenic and are not harmful for healthy humans. Isolated microorganisms mainly belonged to the group of saprophytic fungi as listed: Monilia spp., Penicillium spp., Alternaria spp., Aspergillus fumigatus, Aspergillus nidulans, Rhizopus spp. and Chrysosporium spp. and to the genus of saprophytic bacteria, Bacillus spp.

[ISKRA guidelines for antimicrobial prophylaxis in surgery--Croatian National Guidelines]. / Smjernice ISKRA za antimikrobnu profilaksu u kirurgiji--Hrvatske Nacionalne Smjernice.

Summary. The development of the Guidelines for perioperative prophylactic use of antimicrobial agents (further on Guidelines) was initiated by the Interdisciplinary Section for Antibiotic Resistance Control (ISKRA) of the Croatian Ministry of Health and Social Welfare in accordance with the principles of AGREE (Appraisal of Guidelines for Research and Evaluation) methodology which means that the guidelines are the result of a consensus between all involved professional societies. Guidelines were composed in order to improve antibiotic use in surgical professions. Data obtained from observational studies have shown that the use of antimicrobials in surgical professions is unsatisfactory, and since around 50% of all prescribed drugs in surgical professions refer to perioperative prophylaxis, such guidelines could significantly improve current negative trend and reduce the occurrence of infections in surgical patients as well as slow down the selection of resistant bacteria. In the introductory part of the guidelines, principles of perioperative prophylaxis are presented. The advantages and risks of prophylaxis are listed as well as factors that determine prophylaxis effectiveness. For easier orientation, surgical professions have been divided into basic surgical fields. In each field, the specificity of the field has been described followed by uniform structured tables and with every listed surgical procedure there is the most probable cause of infection, the drug of choice for prophylaxis, alternative drug, remark for particular surgical procedure and finally the grade of recommendation. The Guidelines do not cover perioperative prophylaxis in immunocompromised patients nor perioperative prophylaxis in children. The Guidelines do not cover all possible surgical interventions, but can be used as a basis for most surgical procedures performed in our hospitals. At the very end of these Guidelines, a comprehensive list of references enables all those interested to find further information and details about this topic. The revision of the Guidelines is planned in three years' time.

[Biofilm--still a problem?]. / Biofilm--jos uvijek problem?

Biofilm formation is a crucial step in the pathogenesis of many subacute and chronic bacterial infections, including foreign body-related infections. Biofilms are difficult to eradicate with conventional antimicrobial agents. Bacterial biofilms have several potential antimicrobial resistance mechanisms. Persister cells play a major role in the tolerance of biofilm bacteria to antimicrobial agents. The most common microorganisms responsible for about two thirds of infections with foreign material are biofilm-forming bacteria such as staphylococci (Staphylococcus aureus, S. epidermidis, S. lungdunensis), followed by Pseudomonas aeruginosa and Enterococcus spp, as well as fungal infections such as Candida sp. Preventing early colonisation is a good strategy for reducing morbidity and furthermore, reducing biofilm-associated infection reduces the need for antibiotic use.

[Antibiotics for pancreatitis--still controversial?]. / Antibiotici i pankreatitis--jos uvijek sporno?

Summary. Infection frequently complicates the course of severe acute pancreatitis and might manifest as infected pancreatic necrosis, pancreatic abscess or an infected peripancreatic fluid collection. Pancreatic necrosis occurs in roughly 21% of all cases of pancreatitis. In patients with necrosis involving more than one-half of the pancreas, the incidence of subsequent infection is as high as 40%-70%. More than 50% of these infection yield a polymicrobial isolate with predominance of enteric bacteria but recently, the microbiologic pattern has shifted toward more resistant gram-negative bacilli, gram-positive cocci and yeast, a reflection of exposure to broad-spectrum antimicrobial agents. Given the morbidity associated with infection, many commentators have advocated prophylactic antimicrobial therapy in patients with necrosis to the point that this measure has been incorporated into routine practice. However, there is controversy over the risks and potential benefit. Currently, advise against the routine use of prophylactic systemic antibiotics and antifungals (side-effect selection of resistant microbes and fungi). However, there may be some patients who benefit from prophylaxis, and additional studies and investigations are ongoing. Antibiotics should not be given early in the disease course because most symptoms are due to the inflammatory response, not an infectious etiology. Antibiotics are indicated when CT scans indicate a pancreatic phlegmon, empirically in the case of severe pancreatitis associated with septic shock, or with documented fine - needle aspiration biopsy identification of bacteria. Under those circumstances, antibiotic coverage is warranted to prevent systemic gram-negative sepsis. Infected pancreatic necrosis should be treated with carbapenems because they can effectively penetrate pancreatic tissue. Other conditions, such as biliary pancreatitis associated with cholangitis, mandate antibiotic coverage.