Both biofilm cytotoxicity and monocytes' adhesion may be used as estimators of enterococcal virulence.

Our study aimed to identify markers of enterococci's virulence potential by evaluating the properties of strains of different sites of isolation. Enterococcal strains were isolated as commensals from faeces and as invasive strains from the urine and blood of patients from the University Clinical Centre, Gdansk, Poland. Changes in monocytes' susceptibility to the cytotoxic activity of isolates of different origins and their adherence to biofilm were evaluated using a flow cytometer. The bacterial protein profile was estimated by matrix assisted laser desorption ionization-time of flight mass spectrometer. The cytotoxicity of biofilm and monocytes' adherence to it were the most accurate factors in predicting the prevalence of the strain in the specific niche. Additionally, a bacterial protein with mass-to-charge ratio (m/z) 5000 was found to be responsible for the increased bacterial cytotoxicity, while monocytes' decreased adherence to biofilm was linked with the presence of proteins either with m/z 3330 or 2435. The results illustrate that monocytes' reaction when exposed to the bacterial biofilm can be used as an estimator of pathogens' virulence potential. The observed differences in monocytes' response are explainable by the bacterial proteins' profile. Additionally, the results indicate that the features of both bacteria and monocytes impact the outcome of the infection.

From the Friend to the Foe-Enterococcus faecalis Diverse Impact on the Human Immune System.

Enterococcus faecalis is a bacterium which accompanies us from the first days of our life. As a commensal it produces vitamins, metabolizes nutrients, and maintains intestinal pH. All of that happens in exchange for a niche to inhabit. It is not surprising then, that the bacterium was and is used as an element of many probiotics and its positive impact on the human immune system and the body in general is hard to ignore. This bacterium has also a dark side though. The plasticity and relative ease with which one acquires virulence traits, and the ability to hide from or even deceive and use the immune system to spread throughout the body make E. faecalis a more and more dangerous opponent. The statistics clearly show its increasing role, especially in the case of nosocomial infections. Here we present the summarization of current knowledge about E. faecalis, especially in the context of its relations with the human immune system.

Changes in the Protein Profile in Staphylococcal Strains from Patients Infected with the SARS-CoV-2 Virus.

Staphylococcus aureus strains are particularly often isolated from patients with SARS-CoV-2 infection. The aim of the current research was to determine whether the SARS-CoV-2 virus infection affects the protein profile of S. aureus. Bacteria were isolated from the forty swabs collected from the patients in the hospitals of the Pomeranian region. MALDI-TOF MS spectra were obtained using a Microflex LT instrument. Twenty-nine peaks were identified. The peak (2,430) is described here for the first time and was unique for the isolates from patients infected with the SARS-CoV-2 virus. These results support the hypothesis of bacterial adaptation to the conditions caused by viral infection.

Changes of urine isolates of Escherichia coli and Klebsiella pneumoniae biofilm affect monocytes' response.

The Gram negative rods as Escherichia coli and Klebsiella pneumoniae belong to the most common etiology agents of urinary tract infections. The aim of our study was to assess the diversity of biofilm formed in different urinary tract diseases and their impact on monocytes' adherence and activation. The bacteria were obtained from patients with different kidney problems. Some of the patients were after renal transplantation, some of them were not. Changes in the size and granularity of monocytes, as well as their adherence to biofilm, were assessed using FACSVerse flow cytometer after 1 h co-incubation of monocytes and bacterial biofilm in 37 °C. The obtained results were validated against monocytes incubated without bacteria. The isolates from patients with chronic kidney disease formed the most adherent biofilm regardless the presence or absence of inflammatory reaction. Adherence of monocytes also increased during therapy with immunosuppressive agents, but monocytes' response was different when cyclosporine or tacrolimus were used. Additionally the presence of inflammatory reaction in patients with kidney disease modified the monocytes response when the immunosuppressive drugs were used. Considering the obtained results, we conclude that the changes of monocytes' morphology in response to biofilm formed by Gram negative rods could become a tool to detect urinary tract infection, especially in those groups of patients, where the knowledge of ongoing inflammation is important and the standard tools fail to detect it.

Do the oral Staphylococcus aureus strains from denture wearers have a greater pathogenicity potential?

We used flow cytometry to compare the phagocytic activity of monocytes against Staphylococcus aureus strains (both biofilm and planktonic cells) isolated from denture wearers and non-wearers. Staphylococcal strains were cultured in Brain Heart Infusion broth in both planktonic and biofilm form and were stained with a fluorescent reporter (propidium iodide) and incubated with monocytes. The fluorescence of the monocytes containing phagocytized bacteria was determined by flow cytometry and normalized to that of the bacterial strains used in the experiment. Staphylococcal strains from denture wearers caused greater activation of monocytes but were less prone to phagocytosis. The percentage of monocytes containing bacterial cells after exposition to staphylococcal strains varied from 2.7% to 81.4% for planktonic cells. For biofilm-released cells, this value ranged from 0.6% to 36.2%. The effectiveness of phagocytosis, estimated based on an increase in monocyte fluorescence, amounted to 32.4 and 71 FL2 units for the biofilm and planktonic cells, respectively. The lesser efficiency of phagocytosis against biofilm S. aureus in denture wearers suggests that they might have been colonized with the strains which were less prone to eradication than those from non-wearers.

The high PMNs phagocytosis resistance of enterococcal isolates from RTx patients.

Infections caused by opportunistic pathogens such as enterococci remain difficult to manage, especially in immunocompromised patients. Because of infections' limited symptoms in such patients the additional problems are to find proper diagnostic criteria and the management of infection. Here we aimed to compare the resistance of commensal enterococcal strains and RTx patients' isolates, to PMNs phagocytosis. Thirty-six enterococcal urine and faecal isolates from RTx patients and 17 faecal isolates from healthy volunteers were cultured in planktonic and biofilm forms in 37°C or 42°C. Another tested variable was the addition of immunosuppressant to the culture media. Bacterial cells were stained with fluorescent reporter (CFDA, PI) and incubated with PMNs. Results of phagocytosis were estimated as a mean fluorescence intensity (MFI) of PMNs using flow cytometry. Commensal enterococci cultured in all abovementioned (37°C and 42°C/the addition of immunosuppressant) conditions were less resistant to phagocytosis compared to RTx isolates. Observed significant difference in phagocytosis resistance suggests that patients in immunosuppression are colonized with high risk strains which may lead to the development of infection.

Prophages in enterococcal isolates from renal transplant recipients: renal failure etiologies promote selection of strains.

Infections caused by commensal bacteria may be fatal for the patients under immunosuppressive therapy. This results also from difficulty in identification of high risk strains. Enterococcal infections are increasingly frequent but despite many studies on virulence traits, the difference between commensal and pathogenic strains remains unclear. Prophages are newly described as important elements in competition between strains during colonization, as well as pathogenicity of the strains. Here we evaluate a difference in presence of pp4, pp1, and pp7 prophages and ASA (aggregation substance) gene expression in enterococcal isolates from renal transplant recipients (RTx) with different etiology of the end-stage renal failure. Prophages sequence was screened by PCR in strains of Enterococcus faecalis isolated from urine and feces of 19 RTx hospitalized at Medical University of Gdansk and 18 healthy volunteers. FLOW-FISH method with use of linear locked nucleic acid (LNA) probe was used to assess the ASA gene expression. Additionally, ability of biofilm formation was screened by crystal violet staining method. Presence of prophages was more frequent in fecal isolates from immunocompromised patients than in isolates from healthy volunteers. Additionally, both composition of prophages and ASA gene expression were related to the etiology of renal disease.

Low metabolic activity of biofilm formed by Enterococcus faecalis isolated from healthy humans and wild mallards (Anas platyrhynchos).

It is widely known that Enterococcus faecalis virulence is related to its biofilm formation. Although Enterococci are common commensal organisms of the gastrointestinal tract, the difference between commensal and pathogen strains remain unclear. In this study, we compare the biochemical profile of the biofilms formed by two groups of medical and two groups of commensal strains. The medical strains were isolated as pathogens from infections of urinary tract and other infections (wounds, pus and bedsores), and the commensal strains were taken from faeces of healthy volunteers and faeces of wild mallards (Anas platyrhynchos) living in an urban environment. The properties of biofilms formed by medical and commensal strains differed significantly. Commensal strains showed lower metabolic activity and glucose uptake and higher biofilm biomass than the medical ones. Consistent with glucose uptake experiments, we found that the glucose dehydrogenase gene was more expressed in medical strains. These results indicate that higher metabolic activity and lower protein concentration of E. faecalis cells within biofilms are formed during infections.

Increased pheromone cCF10 expression in Enterococcus faecalis biofilm formed by isolates from renal transplant patients.

Renal transplant recipients are at a high risk of developing infectious complications even caused by commensal bacteria. This is because of various physiological non-immunological, and immunological protective mechanisms are not fully efficient in RTx patients. Therefore, rapid and precise diagnostic tools are essential in this particular group of patients. We aimed to develop simple and sensitive protocol Flow-Fish for the study of gene expression in enterococci and to compare expression of genes involved in virulence regulation in biofilm and planktonic form of Enterococcus faecalis. Proper optimization of the method was demonstrated with analysis of dehydrogenase gene expression. According to expectation reduction of the dehydrogenase gene expression was observed in biofilm. Furthermore, expression of studied gene was higher in clinical than in commensal strains. We have also found that in contrast to dehydrogenase gene, pheromone cCF10 gene expression increasing then clinical strains formed biofilm.

Flow cytometry approach study of Enterococcus faecalis vancomycin resistance by detection of Vancomycin@FL binding to the bacterial cells.

We studied the usefulness of flow cytometry for detection of vancomycin resistance in Enterococcus faecalis by direct binding of commercially available fluorescent vancomycin to cells obtained from culture. The cells were stained with Vancomycin@FL, sonicated and additionally stained with propidium iodide (PI). Regarding to inductive mechanism of vanA-mediated vancomycin resistance, resistant reference strain was also pre-incubated with vancomycin. PI staining divided cells into two subpopulations. There were significantly lower mean FL1 fluorescence values and mean fluorescence per particle (FL1/FSC) in reference vancomycin-resistant strain than in reference and clinical strains sensitive to this antibiotic. Pre-incubation with vancomycin of vancomycin resistant enterococci strain modified Vancomycin@FL binding, however, cells remained easy to differ. We have demonstrated new, quick and sensitive method for detection of vancomycin resistant strains of E. faecalis. The study proved possibility of detection of vancomycin resistance caused by presence of vanA gene by staining cells with Vancomycin@FL. Flow cytometry approach study of E. faecalis vancomycin resistance by detection of Vancomycin@FL binding to the bacterial cells.

Heterogeneity of methicillin-resistant Staphylococcus aureus strains (MRSA) characterized by flow cytometry.

We used fluorescent penicillin Bocillin FL for characterization of control methicillin-resistant Staphylococcus aureus (MRSA) strains belonging to one of four heterogenic classes and comparing them with clinical MRSA isolates. Significant differences in percentage of fluorescent cells and reduction of Bocillin FL binding after incubation with methicillin between control strains from classes I and IV were observed, whereas the strains from classes II and III were differed after incubation with methicillin. According to this criteria, 55.8% of the clinical isolates population were similar to the strain of class IV or homogenic resistant, 11.8% was found as I, and 32.3% were categorized as class II or III. However, continuous diversity of measured features was also discussed.

[The prevalence of tetracycline resistance genes in clinical and commensal strains of Enterococcus faecalis isolated in Gdansk]. / Wystepowanie i zróznicowanie genów odpowiedzialnych za opornosc na tetracykline szczepów Enterococcus faecalis izolowanych w regionie gdanskim.

The common use of antibiotics is responsible for selecting of drug resistance not only in pathogenic, clinical bacteria but also in commensal, not pathogenic strains which could cause the rapid dissemination of the resistance to these antibacterial agents. However, information regarding the antibiotic resistance of commensal bacteria is very scarce, and the data is based mostly on phenotypical research. Therefore the use of genotyping methods for detection of tetracycline resistance genes, in commensal and medical isolates of bacteria, is essential, for understanding the spread of antibiotic resistance. In this study 24 commensal and 27 clinical isolates of Enterococcus faecalis has been screened by PCR methods for tet(M), tet(S) genes and Tn916 and Tn5397 transpozons. Subsequently, the tet(M) gene amplicones were sequenced and phylogenetic analysis was performed. We have found that the prevalence of tet(S) gene varied significantly between commensal and clinical strains. Moreover, the frequency of transpozons in clinical isolates was much higher comparing to strains isolated from healthy individuals. The phylogenetic analysis did not show significant differences between clinical and commensal strains but it could suggest that the genetic similarity between these two groups could be favourable factor for broad range spread of tet(M) gene.

Living donor liver graft salvage after rupture of hepatic artery pseudoaneurysm.

Hepatic artery pseudoaneurysm (HAP) is an uncommon but life-threatening complication of liver transplantation (LTx). It is often associated with a local infection. Prompt diagnosis and intervention are necessary. We report the first occurrence of such complication in the setting of adult living donor liver transplant. A 48-year-old female with primary sclerosing cholangitis underwent living donor right lobe LTx. Her postoperative course was uneventful. A month later, she developed massive gastrointestinal bleeding, with negative endoscopy and angiography. She rebled 2 weeks later, and an HAP was shown on angiography. On exploration, she was found to have an HAP caused by bile leakage from an accessory bile duct and a dissection of the native artery, likely a result of the angiography. The liver was revascularized using a cadaveric iliac artery conduit between the donor hepatic artery and the aorta, and the hepaticojejunostomy was reconstructed. Biliary complications are the most frequent complications in living donor LTx. A clinically silent bile leak can cause an HAP, resulting in massive gastrointestinal bleeding. Surgical repair and biliary reconstruction can yield an excellent clinical result.

[Non-specific adherence of Enterococcus faecalis strains isolated from healthy voluntaries and infection of urinary tract]. / Róznice w nieswoistej adhezji szczepów Enterococcus faecalis izolowanych od nosicieli oraz z zakazen ukladu moczowo-plciowego.

Enterococcus faecalis is a component of human and animal gastrointestinal microflora. However, the adhesion is considered to be the key step in the pathogenesis of enterococcal infections and the first step of biofilm formation. We aimed to compare and evaluate adherence of strains considered to be commensal flora (isolated from healthy volunteers) and strains isolated as a pathogen from medical samples in Gdansk Region The additional aim of this study was to analyze influence of subinhibitory concentration of gentamycin and cAD1 pheromone. Comparison involved 20 strains isolated from healthy voluntaries, 23 strains isolated as etiological agent of urinary tract infection and 16 HLAR strains from other infections. Adherence ability was tasted by turbidymetric method at 550 nm, as o reduction of bacterial inoculum after incubation with hydroksyapatite. Results showed significant difference between commensal and virulent strains as well as between none-inducted and inducted by pheromone. In contrast there was no difference between inducted and non-inducted virulent strains as well as between inducted virulent and inducted not virulent strains. The result shows ability to differentiate this group by non-specific adherence.

A genetic association study of the functional A118G polymorphism of the human mu-opioid receptor gene in patients with acute and chronic pain.

In this prospective, observational study we explored whether A118G single nucleotide polymorphism in the human mu-opioid receptor (MOR) gene could explain the inter-individual differences in opioid analgesic requirements in patients with acute postoperative pain and chronic pain. The frequency of the wild-type A118 MOR (major) and variant G118 MOR (minor) alleles in the subjects with chronic, noncancer pain (n = 121) and opioid-naïve subjects with acute postoperative pain (n = 101), serving as the control group, were examined. The relationships among the A118G MOR genotype, opioid requirements, and the numerical pain score were analyzed in both groups. The frequency of the minor allele was significantly lower in the subjects with chronic pain when compared with the group with acute postoperative pain (0.079 versus 0.158; P = 0.009 by chi2 test). No statistically significant association was observed between the presence of A118G MOR polymorphism and the average postoperative pain score or the doses of morphine used in the immediate postoperative period. In the high-quartile, opioid utilization, chronic pain patients, the homozygotic carriers of the major allele required significantly higher opioid dose than did the carriers of the minor allele. The results indicate that although the presence of the minor allele does not appear to affect opioid analgesic use in acute postoperative pain, the minor allele is less common in chronic pain patients, especially in those requiring higher doses of opioid analgesics.

Minilaparoscopic appendectomy for acute appendicitis.

BACKGROUND: Minilaparoscopic appendectomy for appendicitis is not a well-established procedure. This approach provides less abdominal wall trauma, fewer complications, and excellent cosmetic results. Our aim was to show the feasibility and safety of the minilaparoscopic approach. METHODS: Minilaparoscopic appendectomy was performed in 37 patients. Two 2.2-mm trocars were used to manipulate a 2.2-mm, 0-degree laparoscope and for grasper access. A 5-mm trocar was used for the ultrasonic scalpel. RESULTS: No deaths occurred. In 3 patients (8%), appendectomy was aborted due to pathology of the ovary. Conversion to the open approach occurred in 2.7% of patients. The average operating time was 34 minutes (range, 15 to 80), and the median length of hospital stay was 1.2 day (range, 1 to 5). CONCLUSIONS: The minilaparoscopic approach a) has the same advantages as the conventional laparoscopic approach in terms of better diagnostic accuracy and safety; b) a low incidence of complications; and c) yields excellent cosmetic results.

Prevention of postoperative nausea and vomiting with granisetron and dolasetron in relation to CYP2D6 genotype.

We investigated the efficacy of granisetron and dolasetron in preventing postoperative nausea and vomiting. Because the metabolism of the various antiemetic 5-hydroxytryptamine type 3 (5-HT3) antagonists involves different isoforms of the hepatic cytochrome P450 system, we examined the relationship between the clinical efficacy of these drugs and polymorphic cytochrome P450 2D6 (CYP2D6) genotype. This prospective, randomized, double-blind study involved 150 adult patients with a moderate to high risk for postoperative nausea and vomiting. All subjects received dexamethasone at induction of anesthesia followed by either 12.5 mg of dolasetron or 1 mg of granisetron. We analyzed the number of complete responders (no vomiting or rescue medication) during the first 24 hours after surgery. CYP2D6 genotyping was performed using a TaqMan real-time polymerase chain reaction. A complete response was more frequent in the granisetron group (54.7%) compared with the dolasetron group (38.7%, P < 0.05). In subjects receiving dolasetron, carriers of the duplication of the CYP2D6 allele predicting ultrarapid metabolizer status had more frequent vomiting episodes (P < 0.05) than patients in the granisetron group. It is postulated that the difference in the antiemetic efficacy between two investigated 5-HT3 receptor antagonists may be associated with differences in the carrier status for the duplication of the CYP2D6 allele.

Effect of valdecoxib pretreatment on pain and secondary hyperalgesia: a randomized controlled trial in healthy volunteers [ISRCTN05282752, NCT00260325].

BACKGROUND: Induction of the COX-2 isoenzyme appears to play a major role in the genesis of central sensitization after nociceptive stimulation. This study aimed to investigate the efficacy of a single, oral dose of the specific COX-2 inhibitor-valdecoxib in attenuating the central sensitization - induced secondary hyperalgesia in a heat/capsaicin pain model in healthy volunteers. METHODS: The study was a randomized, double blind, placebo controlled, crossover, single dose efficacy trial using 20 healthy volunteers. Two hours following placebo or 40 mg, PO valdecoxib, participants underwent skin sensitization with heat/capsaicin, as well as supra-threshold pain and re-kindling measurements according to an established, validated pain model. Subjects rated pain intensity and unpleasantness on a visual analog scale and the area of secondary hyperalgesia was serially mapped. RESULTS: The area of secondary hyperalgesia produced after 40 mg of valdecoxib was no different than that after placebo. Furthermore, there were no significantly relevant differences when volunteers were treated with valdecoxib or placebo in relation to either cold- or hot pain threshold or the intensity of pain after supra-threshold, thermal pain stimulation. CONCLUSION: We demonstrated that a single, oral dose of valdecoxib when does not attenuate secondary hyperalgesia induced by heat/capsaicin in a cutaneous sensitization pain model in healthy volunteers.

Thymoglobulin induction protects liver allografts from ischemia/reperfusion injury.

BACKGROUND: Interventions that minimize hepatic ischemia/reperfusion injury (IRI) can expand the donor organ pool. Thymoglobulin (TG) induction therapy has been shown to ameliorate delayed graft function and possibly decrease IRI in cadaver renal transplants recipients. This controlled randomized trial was designated to assess the ability of TG to protect against IRI in liver transplant recipients. PATIENTS AND METHODS: Twenty-two cadaveric liver transplant recipients were randomized to receive either TG (1.5 mg/kg/dose) during the anhepatic period and QOD x2 doses or no TG. No differences in recipients' demographics were present and donor characteristics were similar in terms of age, cause of death, and cold ischemia time. Maintenance immunosupression consisted of Tacrolimus (or Cyclosporine) and steroids for both groups. Donor biopsies were obtained during organ procurement, cold storage and 1 h after re-vascularization. Post-operative liver function tests were monitored. Early graft function, length of stay, patient and graft survival rates, incidence of primary non-function and rate of rejection were assessed. RESULTS: Patient and graft survival at 3 months was 100%. There was no incidence of primary graft non-function and no need for re-transplantation. The incidence of acute rejection was similar between the two groups. Patients in the TG group had significant decreases in alanine aminotransferase test at day 1 compared to the control group (p = 0.02). There were also near significant decreases of total bilirubin at day 5 and shorter length of hospitalization. Liver biopsy (at procurement, when cold, and post-reperfusion) of TG group demonstrated a trend for increased central ballooning. CONCLUSION: The TG allowed for more compromised liver grafts to be transplanted with less clinical evidence of IRI and improved function. Further studies on the degree of apoptosis in the liver biopsy post-reperfusion are underway.