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Pathogens carried by ticks pose a threat to both human and animal health across the world. Typically associated with rural landscapes, ticks appear to adapt well to life in urban recreational areas. Although Dermacentor reticulatus is commonly found across Europe, data on the prevalence of pathogens in this tick species, in an urban environment, are very limited. PCR was used to examine 368 D. reticulatus individuals collected in the Zwierzyniecki Forest Nature Reserve in Białystok, Poland. In total, 10.3% of ticks were infected, with Babesia spp. (9.2%), Anaplasma phagocytophilum (0.8%) and Borrelia burgdorferi sensu lato (0.3%). Rickettsia spp., Bartonella spp., and Coxiella burnetii were not detected. Sequence analysis for Babesia-positive samples identified 79.4% of them as Babesia canis, 8.8% as Babesia microti, 5.9% as Babesia spp., 2.9% as Babesia venatorum, and 2.9% as Babesia vogeli. Results obtained in this study indicate that D. reticulatus ticks found within the urban premises of the study area are infected with at least three pathogens and therefore are an important factor in public health risk for tick-borne diseases.

The aim of this study was to investigate the synergy between ceftazidime-avibactam, ertapenem, fosfomycin, and tigecycline against carbapenemase-producing Klebsiella pneumoniae using the E test MIC:MIC (minimum inhibitory concentration) ratio synergy method. The results were interpreted using fractional inhibitory concentration index (FICI) to describe the effects of antimicrobial combinations in vitro. To assess the clinical significance of each antibiotic combination, the susceptible breakpoint index (SBPI) was calculated for each combination, and within each strain. The FICI method revealed that the most synergistic combinations against carbapenemase-producing K. pneumoniae were ceftazidime-avibactam with ertapenem and ceftazidime-avibactam with fosfomycin. This effect was demonstrated in 47% (9/19) of all tested clinical K. pneumoniae isolates. Considering the effects of all drug combinations in K. pneumoniae harboring blaKPC, blaNDM, and blaOXA-48 genes, we observed that the combination of ceftazidime-avibactam with fosfomycin was the most synergistic in New Delhi metallo-β-lactamase (NDM)-producing K. pneumoniae, and the combination of ceftazidime-avibactam with ertapenem was the most synergistic in K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae. In addition, all tested combinations were synergistic against oxacillinase (OXA)-48-producing K. pneumoniae, except the combination of ceftazidime-avibactam with tigecycline. The SBPI index showed that ceftazidime-avibactam in combination with fosfomycin reduced the MIC to less than the susceptibility breakpoint among all tested carbapenemase-producing K. pneumoniae. Moreover, the combinations of ceftazidime-avibactam with ertapenem, and ceftazidime-avibactam with tigecycline were able to reduce the MIC to less than the susceptibility breakpoint in all KPC- and OXA-48-producing K. pneumoniae.

Simple Summary Although the biological function of lncRNAs has not been fully elucidated, we know that the aberrant expression of lncRNAs can drive the cancer phenotype. Therefore, a growing area of research is focusing on lncRNAs as putative diagnostic biomarkers and therapeutic targets. The aim of the study was the appraisal of the diagnostic value of 14 differentially expressed lncRNA in the early stages of NSCLC. We established two classifiers. The first recognized cancerous from noncancerous tissues, the second successfully discriminated NSCLC subtypes (LUAD vs. LUSC). Our results indicate that the panel of 14 lncRNAs can be a promising tool to support a routine histopathological diagnosis of NSCLC. Abstract LncRNAs have arisen as new players in the world of non-coding RNA. Disrupted expression of these molecules can be tightly linked to the onset, promotion and progression of cancer. The present study estimated the usefulness of 14 lncRNAs (HAGLR, ADAMTS9-AS2, LINC00261, MCM3AP-AS1, TP53TG1, C14orf132, LINC00968, LINC00312, TP73-AS1, LOC344887, LINC00673, SOX2-OT, AFAP1-AS1, LOC730101) for early detection of non-small-cell lung cancer (NSCLC). The total RNA was isolated from paired fresh-frozen cancerous and noncancerous lung tissue from 92 NSCLC patients diagnosed with either adenocarcinoma (LUAD) or lung squamous cell carcinoma (LUSC). The expression level of lncRNAs was evaluated by a quantitative real-time PCR (qPCR). Based on Ct and delta Ct values, logistic regression and gradient boosting decision tree classifiers were built. The latter is a novel, advanced machine learning algorithm with great potential in medical science. The established predictive models showed that a set of 14 lncRNAs accurately discriminates cancerous from noncancerous lung tissues (AUC value of 0.98 ± 0.01) and NSCLC subtypes (AUC value of 0.84 ± 0.09), although the expression of a few molecules was statistically insignificant (SOX2-OT, AFAP1-AS1 and LOC730101 for tumor vs. normal tissue; and TP53TG1, C14orf132, LINC00968 and LOC730101 for LUAD vs. LUSC). However for subtypes discrimination, the simplified logistic regression model based on the four variables (delta Ct AFAP1-AS1, Ct SOX2-OT, Ct LINC00261, and delta Ct LINC00673) had even stronger diagnostic potential than the original one (AUC value of 0.88 ± 0.07). Our results demonstrate that the 14 lncRNA signature can be an auxiliary tool to endorse and complement the histological diagnosis of non-small-cell lung cancer.

Objectives The growing incidence of multidrug-resistant (MDR) bacteria is an emerging challenge in modern medicine. The utility of carbapenems, which are considered ‘last-line' agents, is being diminished by the growing incidence of various resistance mechanisms in Gram-negative bacteria. A molecular investigation was performed of an MDR carbapenem-resistant Citrobacter freundii of sequence type 8 (ST8) isolated from a hematology patient with acute myeloid leukemia. Methods Multilocus sequence typing and analysis of the nucleotide sequence of the class I integron were performed using PCR and Sanger sequencing. Transformation of the resistance plasmid isolated following the alkaline lysis method was performed using chemically competent E. coli TOP10. Results Molecular analysis of the carbapenem-resistant C. freundii revealed the presence of the VIM-4 isoenzyme located on the ∼55-kb transferable resistance plasmid. Interestingly, the bla VIM-4 gene was inserted into an unusual gene cassette containing a 169-bp direct repeat of the 3′ segment of the bla VIM-4 gene. Conclusions All unusual gene cassettes containing VIM-DR (direct repeat) described thus far have been harbored by non-fermenters, i.e., Acinetobacter and Pseudomonas, underscoring the importance of resistance determinant mobility, which may go even beyond genus, family, and order boundaries. Great efforts need to be taken to explore pathways of resistance to ‘last-resort' antimicrobials, especially among clinically relevant pathogens.

Purpose: Enterococcus faecium strains have been reported worldwide as etiologic factors of many nosocomial infections, which are difficult to manage because of the constantly increasing resistance of these microorganisms to antibiotics and the ability to form biofilm. The aim of this study was to analyze the ability to produce a biofilm in E. faecium strains, depending on the patient’s clinical material. Materials and methods: Sixty-six E. faecium strains were investigated. Identification and susceptibility testing were conducted by the VITEK2 system. The ability to form biofilm was assessed by phenotypic methods. The presence of selected virulence genes was established by PCR followed by gel electrophoresis and sequencing. Results: Among the tested E. faecium isolates, 72.7% were biofilm-positive (BIO+) and 27.3% biofilm-negative (BIO-). Strains were collected mostly from rectal swabs (30.4%) and blood (18.3%). BIO+ strains from infections constituted 31.8% (52.4% isolated from blood) and from colonization 40.9% (48.2% from rectal swabs). 91.7% of the Blood Group strains and 68.5% of the Other Group strains produced biofilm. Strains from the Colonization Group produced biofilm in a proportion similar to the Infection Group (about 75%). There were no statistically significant differences in virulence and resistance, except for vancomycin (more resistant BIO+ Other than the BIO+ Blood Group, and more resistant BIO+ Colonization than BIO+ Infection Group) and teicoplanin (more resistant BIO+ Colonization than the BIO+ Infection Group). Conclusion: The majority of E. faecium isolates carries high levels of resistance to many antimicrobials, is well equipped with virulence genes, and possesses the ability to form biofilm.

Background The growing incidence of MDR Gram-negative bacteria is a rapidly emerging challenge in modern medicine. Objectives We sought to establish the role of intrinsic drug-resistance regulators in combination with specific genetic mutations in 11 Enterobacter cloacae isolates obtained from a single patient within a 7 week period. Methods The molecular characterization of eight carbapenem-resistant and three carbapenem-susceptible E. cloacae ST89 isolates included expression-level analysis and WGS. Quantitative PCR included: (i) chromosomal cephalosporinase gene (ampC); (ii) membrane permeability factor genes, e.g. ompF, ompC, acrA, acrB and tolC; and (iii) intrinsic regulatory genes, e.g. ramA, ampR, rob, marA and soxS, which confer reductions in antibiotic susceptibility. Results In this study we describe the influence of the alterations in membrane permeability (ompF and ompC levels), intrinsic regulatory genes (ramA, marA, soxS) and intrinsic chromosomal cephalosporinase AmpC on reductions in carbapenem susceptibility of E. cloacae clinical isolates. Interestingly, only the first isolate possessed the acquired VIM-4 carbapenemase, which has been lost in subsequent isolates. The remaining XDR E. cloacae ST89 isolates presented complex carbapenem-resistance pathways, which included perturbations in permeability of bacterial membranes mediated by overexpression of ramA, encoding an AraC/XylS global regulator. Moreover, susceptible isolates differed significantly from other isolates in terms of marA down-regulation and soxS up-regulation. Conclusions Molecular mechanisms of resistance among carbapenem-resistant E. cloacae included production of acquired VIM-4 carbapenemase, significant alterations in membrane permeability due to increased expression of ramA, encoding an AraC/XylS global regulator, and the overproduction of chromosomal AmpC cephalosporinase.

Aim. Today, infections caused by vancomycin-resistant Enterococcus faecium (VRE) are a major problem in the healthcare system. The aim of this study was to compare the antibiotic resistance and virulence traits between vancomycin-susceptible E. faecium (VSE) and VRE clinical isolates.Material and Methods. Studies were performed on 66 E. faecium (32 VRE and 34 VSE) strains. Susceptibility testing and identification were performed, and strains were examined for ß-lactamase, hemolysin and biofilm production. Isolates were tested for the presence of 5 van genes, 8 virulence genes and 6 aminoglycoside-modifying enzyme (AME) genes. Obtained amplicons were subjected to electrophoretical separation and DNA sequencing.Results. Among 32 VRE isolates, 28 were found to have the VanA phenotype, and 4 the VanB. The most frequent resistance and virulence profile among VRE strains was resistance to ampicillin, imipenem, gentamicin, streptomycin, teicoplanin, and vancomycin with enterococcal surface protein (esp), endocarditis antigen (efaA), collagen adhesin (acm), and hialuronidase (hyl) genes; among VSE: resistance to ampicillin, imipenem, gentamicin, streptomycin with esp, efaA, acm, and hyl genes. Conclusions. Our findings prove that both VRE and VSE strains were well equipped with virulence and resistance genes, although VRE strains were characterized by a greater variety and a higher number of these genes. However, statistical analysis revealed no significant differences between VSE and VRE strains (p > 0.05). Nevertheless, our results suggest that VRE strains may slowly acquire and incorporate resistance and virulence genes, due to their ability to survive in a hospital environment for a long time.

Klebsiella pneumoniae infections have always been an important problem in public health, but today, the increasing resistance of these bacteria to antibiotics due to β-lactamases production has renewed interest in K. pneumoniae infections. The aim of the study was to present a case of a neurosurgical patient with multidrug-resistant K. pneumoniae ST11 infection after craniectomy. Four K. pneumoniae isolates from various clinical materials of the patient undergone identification and susceptibility testing with the Vitek2 system. Tests for β-lactamases production were performed according to EUCAST guidelines. Strains were analyzed for bla genes responsible for β-lactamase production (blaTEM, blaSHV, blaCTX-M, blaVIM, blaIMP, blaNDM, blaKPC, blaOXA-48) using PCR. Moreover, the genetic relatedness of these isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All tested strain presented multidrug resistance. The highest susceptibility was observed for imipenem, meropenem, and ertapenem. The strain isolated from the nervous system was ESBL-positive with blaSHV-11, blaTEM-1, and blaCTX-M-15 genes. Additionally, the strain from urine was blaKPC-3-positive. Molecular typing revealed that all strains belonged to the same clone and identified two PFGE profiles. The analysis of MLST allelic profile showed that tested K. pneumoniae strains belonged to ST11. Identification of ST11 K. pneumoniae as etiological factor of infection unfavorably impacts on prognosis among neurosurgical patient after craniectomy.

The present study was conducted to investigate the prevalence of genes encoding resistance to aminoglycosides and fluoroquinolones among twenty-five Pseudomonas aeruginosa isolated between 2002 and 2009. In PCR, following genes were detected: ant(2")-Ia in 9 (36.0%), aac(6')-Ib in 7 (28.0%), qnrB in 5 (20.0%), aph(3")-Ib in 2 (8.0%) of isolates.

The combination of an antifungal agent and drug carrier with adjunctive antimicrobial properties represents novel strategy of complex therapy in pharmaceutical technology. The goal of this study was to investigate the unmodified and ion cross-linked chitosan’s influence on anti-Candida activity of clotrimazole used as a model drug in hydrogels. It was particularly crucial to explore whether the chitosans’ structure modification by β-glycerophosphate altered its antifungal properties. Antifungal studies (performed by plate diffusion method according to CLSI reference protocol) revealed that hydrogels obtained with chitosan/β-glycerophosphate displayed lower anti-Candida effect, probably as a result of weakened polycationic properties of chitosan in the presence of ion cross-linker. Designed chitosan hydrogels with clotrimazole were found to be more efficient against tested Candida strains and showed more favorable drug release profile compared to commercially available product. These observations indicate that novel chitosan formulations may be considered as promising semi-solid delivery system of clotrimazole.

Bacteria belonging to theEnterobacteriaceaefamily that produce extended-spectrumβ-lactamase (ESBL) enzymes are important pathogens of infections. Increasing numbers of ESBL-producing bacterial strains exhibiting multidrug resistance have been observed. The aim of the study was to evaluate the prevalence ofblaCTX-M,blaSHV, andblaTEMgenes among ESBL-producingKlebsiella pneumoniae,Escherichia coli, andProteus mirabilisstrains and to examine susceptibility to antibiotics of tested strains. In our study, thirty-six of the tested strains exhibitedblaCTX-Mgenes(blaCTX-M-15,blaCTX-M-3,blaCTX-M-91, andblaCTX-M-89). Moreover, twelve ESBL-positive strains harboredblaSHVgenes(blaSHV-18,blaSHV-7,blaSHV-2, andblaSHV-5), and the presence of ablaTEMgene(blaTEM-1)in twenty-five ESBL-positive strains was revealed. AmongK. pneumoniaethe multiple ESBL genotype composed ofblaCTX-M-15, blaCTX-M-3, blaSHV-18, blaSHV-7, blaSHV-2, andblaSHV-5genes encoding particular ESBL variants was observed. Analysis of bacterial susceptibility to antibiotics revealed that, amongβ-lactam antibiotics, the most effective againstE. colistrains was meropenem (100%), whereasK. pneumoniaewere completely susceptible to ertapenem and meropenem (100%), andP. mirabilisstrains were susceptible to ertapenem (91.7%). Moreover, among non-β-lactam antibiotics, gentamicin showed the highest activity toE. coli(91.7%) and ciprofloxacin the highest toK. pneumoniae(83.3%).P. mirabilisrevealed the highest susceptibility to amikacin (66.7%).

Purpose Strains of Acinetobacter spp. are responsible for a considerable percentage of hospital infections. These pathogens have colonized hospital environment and developed resistance to many currently available antibiotics. The aim of this study was one year-long analysis of the occurrence of multiresistant strains of Acinetobacter spp. in population of patients hospitalized in ICU of ED and determination of their genetic similarity. Material/Methods Subject of research was the population of patients admitted to ED of University Hospital in Bialystok in the period from 01.08.2010 to 01.08.2011. In the analysed group of patients, infections were identified on the basis of the guidelines of CDC. Identification and drug susceptibility of strains was specified using the automatic methods with the analyzer Vitek 2XL. Genotyping using Rep-PCR method in DiversiLab system was performed on strains of Acinetobacter spp. to determinate their genetic similarity. Results During analyzed period 405 patients were hospitalized, from 14 of them multiresistant strains of Acinetobacter spp. were isolated. Conducted genetic research allowed to detect 5 clones. Rep-PCR method in DiversiLab system enabled to learn that different clone of multiresistant strain of Acinetobacter spp. is responsible for variable forms of infection. Conclusions Results of conducted research suggest that genotyping with rep-PCR method in DiversiLab system is useful tool in diagnostics of clones of multiresistant pathogens isolated from patients requiring intensive care, hospitalized in ED. Genotyping with rep-PCR method combined with epidemiological investigation enables to establish ways of spreading of multiresistant strains of Acinetobacter spp. in ED.

The growing incidence of multidrug-resistant (MDR) bacteria is an emerging challenge in modern medicine. The utility of carbapenems, considered "last-line" agents in therapy of infections caused by MDR pathogens, is being diminished by the growing incidence of various resistance mechanisms. Enterobacter cloacae have lately begun to emerge as an important pathogen prone to exhibiting multiple drug resistance. We aimed to investigate the molecular basis of carbapenem-resistance in 44 E. cloacae clinical strains resistant to at least one carbapenem, and 21 susceptible strains. Molecular investigation of 65 E. cloacae clinical strains was based on quantitative polymerase chain reaction (qPCR) allowing for amplification of ampC, ompF, and ompC transcripts, and analysis of nucleotide sequences of alleles included in MLST scheme. Co-operation of three distinct carbapenem resistance mechanisms has been reported-production of OXA-48 (5%), AmpC overproduction (97.7%), and alterations in outer membrane (OM) transcriptome balance. Carbapenem-resistant E. cloacae were characterized by (1.) downregulation of ompF gene (53.4%), which encodes protein with extensive transmembrane channels, and (2.) the polarization of OM transcriptome-balance (79.1%), which was sloped toward ompC gene, encoding proteins recently reported to possess restrictive transmembrane channels. Subpopulations of carbapenem-susceptible strains showed relatively high degrees of sequence diversity without predominant types. ST-89 clearly dominates among carbapenem-resistant strains (88.6%) suggesting clonal spread of resistant strains. The growing prevalence of pathogens resistant to all currently available antimicrobial agents heralds the potential risk of a future "post-antibiotic era." Great efforts need to be taken to explore the background of resistance to "last resort" antimicrobials.

Purpose Over the past years, an increase in resistance to aminoglycosides has been observed among Enterobacteriaceae rods. This resistance development reduces therapeutic options for infections caused by multidrug-resistance organisms. Because of the changing epidemiology of extended-spectrum β-lactamases (ESBLs) and resistance to aminoglycosides, we investigated the prevalence of the aac(3)-Ia, aac(6′)-Ib, ant(4′)-IIa, ant(2”)-Ia, and aph(3”)-Ib genes encoding aminoglycoside-modifying enzymes (AMEs) in ESBL-producing Escherichia coli as well as ESBL-non-producing isolates. To understand bacterial resistance to aminoglycoside antibiotics, we estimated resistance phenotypes and the presence of genes responsible for this resistance. Materials and methods The study was conducted on 44 E.coli strains originated from patients hospitalized at University Hospital of Bialystok. MIC values were obtained for gentamicin, amikacin, netilmicin, and tobramycin. Isolates were tested for the presence of the aac(3)-Ia, aac(6′)-Ib, ant(4′)-IIa, ant(2”)-Ia, and aph(3”)-Ib genes with the use of the PCR technique. Results Resistance to aminoglycosides was found in 79.5% of the isolates. The highest percentages of resistance were observed for tobramycin (70,5%) and gentamicin (59%), followed by netilmicin (43.2%) and amikacin (11.4%). PCR assays revealed the presence of aac(6′)-Ib among 26 (59.2%) strains, aph(3”)-Ib among 16 (36.2%), aac(3)-Ia among 7 (15.9%), and ant(2”)-Ia among 2 (4.6%) strains. Conclusions The enzymatic resistance against aminoglycosides in northeastern Poland among clinical isolates of E. coli is predominantly caused by aac(6′)-Ib and aph(3”)-Ib. Amikacin may be used for therapy of infections caused by ESBL-producing E. coli, because of the low rates of resistance.

The genus Bidens L. (Asteraceae) refers to several species of plants used in traditional phytotherapeutic preparations. B. tripartita, also known as bur marigold, is the most familiar plant and has been known as a remedy for chronic dysentery. The hydrodistilled essential oil of the aerial parts of the Polish B. tripartita was analyzed using gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) techniques. To exclude any potential toxic effects of the oil on human dermal fibroblasts, the MTT test (methyl thiazolyl tetrazolium) and COMET assay (single-cell gel electrophoresis) were performed. Novel gel formulations as topical carriers for essential oil obtained from B. tripartita were developed and characterized. The bioadhesive properties of the designed preparations in the ex vivo model using the skin of hairless mice were also evaluated. The therapeutic efficacy of the topical formulations is influenced by active phytoconstituents and vehicle characteristics. The antifungal properties of the essential oil of B. tripartita were also tested against Candida species, and this oil appears to be a promising topical anticandidal agent.

More and more is known about the role of venous wall abnormalities and valvular incompetence in the development of chronic venous disorders (CVD). Unfortunately detailed mechanisms of CVD pathophysiology are not well understood. Recent studies focus on involvement of the inflammatory process in the structural remodeling of venous valves and venous wall. The aim of this study is to investigate and to document the memory T cells homeostasis in CVD patients. In this study we present lymphocytic changes in blood from varicose veins in terms of total CD4+ and CD8+ T cells and their particular subsets of memory T cells: TN, TCM and TEM. Results suggest that immunological memory may be involved in the CVD development.

T cell memory in comparison with B cell memory is not well understood. This review focuses on CD8+ and CD4+ memory T cells. In this article we try to define memory cells and also present models of memory T cells formation. We would also like to delineate their differentiation into distinct subsets. Long-lived memory T cells consist in two main subsets: TCM and TEM. Recent studies have shown that not all cells considered to be memory cells differentiate into TCM and TEM, but a small proportion of theses cells exhibit naive cells phenotype. Memory T cells constitute a heterogeneous population of cells. In this study we lay stress on characteristic of main memory T cells subsets and their alleged participation in immune response upon reexposure to the Ag.

Programmed death-1 (PD-1) is a critical mediator of virus-specific CD8+ T-cell exhaustion. Here, we examined the expression of PD-1 on simian immunodeficiency virus (SIV)-specific CD8+ T cells and its possible involvement in regulation of cytokine production, proliferation, and survival of these cells. The majority of SIV-specific CD8+ T cells expressed a PD-1high phenotype, independent of their differentiation status, in all tissues tested. PD-1 expression gradually declined on CD8+ T cells specific for SIV-derived epitopes that had undergone mutational escape, indicating that antigen-specific TCR stimulation is the primary determinant of PD-1 expression. SIV-specific PD-1highCD8+ T cells produced IFN-γ, TNF-α, and IL-2 under cognate peptide stimulation. While CD8+ T cells that proliferated in response to antigen had a PD-1high phenotype, it was determined that there was a reduced proliferative capacity of PD-1high compared with PD-1low SIV-specific CD8+ T cells. PD-1high SIV-specific CD8+ T cells were highly susceptible to cell death leading to loss of such cells after in vitro stimulation. Thus, PD-1 is a negative regulator of SIV-specific CD8+ T cells, operating predominantly through the induction of cell death. Manipulation of the interaction of PD-1 with its ligands could thus potentially restore the CD8+ T-cell responses in SIV infection.

An increase in the antibiotic resistance among Enterococcus faecium strains has been observed worldwide. Moreover, this bacteria has the ability to produce several virulence factors and to form biofilm that plays an important role in human infections. This study was designed to compare the antibiotic resistance and the prevalence of genes encoding surface protein (esp), aggregation substance (as), surface adhesin (efaA), collagen adhesin (ace), gelatinase (gelE), and hialuronidase (hyl) between biofilm-producing and non-producing E. faecium strains. Therefore, ninety E. faecium clinical isolates were tested for biofilm-forming ability, and then were assigned to two groups: biofilm-positive (BIO(+), n =70) and biofilm-negative (BIO(-), n = 20). Comparison of these groups showed that BIO(+) isolates were resistant to β-lactams, whereas 10% of BIO(-) strains were susceptible to ampicillin (statistically significant difference, p = 0.007) and 5% to imipenem. Linezolid and tigecycline were the only antibiotics active against all tested isolates. Analysis of the virulence factors revealed that ace, efaA, and gelE genes occurred more frequently in BIO(-) strains (ace in 50% BIO(+) vs. 75% BIO(-); efaA 44.3% vs. 85%; gelE 2.9% vs. 15%, respectively), while hyl gene appeared more frequently in BIO(+) isolates (87.1% BIO(+) vs. 65% BIO(-)). These differences were significant (p < 0.05). We concluded that BIO(+) strains were more resistant to antibiotics than BIO(-) strains, but interestingly, BIO(-) isolates were characterized by possession of higher virulence capabilities.

An increase in the antibiotic resistance among members of theEnterobacteriaceaefamily has been observed worldwide. Multidrug-resistant Gram-negative rods are increasingly reported. The treatment of infections caused byEscherichia coliand otherEnterobacteriaceaehas become an important clinical problem associated with reduced therapeutic possibilities. Antimicrobial carbapenems are considered the last line of defense against multidrug-resistant Gram-negative bacteria. Unfortunately, an increase of carbapenem resistance due to the production ofKlebsiella pneumoniaecarbapenemase (KPC) enzymes has been observed. In this study we describe the ability ofE. colito produce carbapenemase enzymes based on the results of the combination disc assay with boronic acid performed according to guidelines established by the European Community on Antimicrobial Susceptibility Testing (EUCAST) and the biochemical Carba NP test. Moreover, we evaluated the presence of genes responsible for the production of carbapenemases (blaKPC,blaVIM,blaIMP,blaOXA-48) and genes encoding otherβ-lactamases (blaSHV,blaTEM,blaCTX-M) amongE. coliisolate. The tested isolate ofE. colithat possessed theblaKPC-3andblaTEM-34genes was identified. The tested strain exhibited susceptibility to colistin (0.38 μg/mL) and tigecycline (1 μg/mL). This is the first detection ofblaKPC-3in anE. coliST479 in Poland.

An HIV-1 vaccine able to induce broad CD4+ and CD8+ T cell responses may provide long-term control of viral replication. In this study we directly assess the relative benefit of immunization with vaccines expressing three structural Ags (Gag, Pol, and Env), three early regulatory proteins (Rev, Tat, and Nef), or a complex vaccine expressing all six Ags. The simultaneous administration of all six Ags during vaccination resulted in Ag competition manifested by a relative reduction of CD8+ T cell and lymphoproliferative responses to individual Ags. Despite the Ag competition, vaccination with all six Ags resulted in a delay in the onset and a decrease in the extent of acute viremia after mucosal challenge exposure to highly pathogenic SIVmac251. Reduced levels of acute viremia correlated with lower post-set point viremia and long-term control of infection. In immunized animals, virus-specific CD4+ T cell and lymphoproliferative responses were preserved during acute viremia, and the maintenance of these responses predicted the long-term virological outcome. Taken together, these results suggest that the breadth of the immune response is probably more important than high frequency responses to a limited number of epitopes. These data provide the first clear evidence of the importance of nonstructural HIV Ags as components of an HIV-1 vaccine.

Plasma virus in human immunodeficiency virus type 1/simian immunodeficiency virus (HIV-1/SIV) infection most likely results from the combination of viruses produced in different tissues. As immunological pressure may be higher in effector sites than secondary lymphoid tissues, we investigated quantitative and qualitative changes in viral RNA in blood and tissues of 10 Mamu-A*01-positive SIV-infected macaques in parallel with the frequency of CD8+ T cells recognizing the dominant Gag181-189 CM9 epitope. The plasma virus level in these macaques directly correlated with the viral RNA levels in lymph nodes, spleen, lungs, colon, and jejunum. In contrast, the frequency of the Gag181-189 CM9 tetramer did not correlate with SIV RNA levels in any compartment. We investigated the presence of viral immune escape in RNA from several tissues. The complete substitution of wild-type genotype with viral immune-escape variant within the Gag181-189 CM9 epitope was associated with low tetramer response in all tissues and blood of two macaques. In one macaque, the replacement of wild type with an immune-escape mutant was asynchronous. While the mutant virus was prevalent in blood and effector tissues (lungs, jejunum, and colon), secondary lymphoid organs such as spleen and lymph nodes still retained 80% and 40%, respectively, of the wild-type virus. These results may imply that there are differences in the immunological pressure exerted by cytotoxic T lymphocytes (CTLs) in tissue compartments of SIVmac251-infected macaques.

IL-2, the first cytokine discovered with T cell growth factor activity, is now known to have pleiotropic effects on T cells. For example, it can promote growth, survival, and differentiation of Ag-selected cells, or facilitate Ag-induced cell death of T cells when Ag persists, and in vivo, it is thought to contribute to the regulation of the size of adaptive T cell response. IL-2 is deficient in HIV-1 infection and has been used in the management of HIV-1-infected individuals undergoing antiretroviral therapy. In this study, we investigated how continuous low-dose IL-2 affected the CD4+ and CD8+ T cell response induced by two inoculations of a canarypox recombinant SIV-based vaccine candidate in healthy macaques chronically infected with SIVmac251. These macaques had normal levels of CD4+ T cells at the beginning of antiretroviral therapy treatment. Vaccination in the presence of IL-2 significantly augmented Gag-specific CD8+ T cell responses, but actually reduced Gag-specific CD4+ T cell responses. Although IL-2 at low doses did not change the overall concentration of circulating CD4+ or CD8+ T cells, it expanded the frequency of CD4+CD25+ T cells. Depletion of the CD4+CD25+ T cells in vitro, however, did not result in a reconstitution of Gag-specific CD4+ responses or augmentation of SIV-specific CD8+ T cell responses. Thus, we conclude that the decrease in virus-specific CD4+ T cell response may be due to IL-2-promoted redistribution of cells from the circulation, or due to Ag-induced cell death, rather than suppression by a T regulatory population.

Infection with human immunodeficiency virus or simian immunodeficiency virus (SIV) induces virus-specific CD8+T cells that traffic to lymphoid and nonlymphoid tissues. In this study, we used Gag-specific tetramer staining to investigate the frequency of CD8+T cells in peripheral blood and the central nervous system of Mamu-A*01-positive SIV-infected rhesus macaques. Most of these infected macaques were vaccinated prior to SIVmac251 exposure. The frequency of Gag181-189CM9 tetramer-positive cells was consistently higher in the cerebrospinal fluid and the brain than in the blood of all animals studied and did not correlate with either plasma viremia or CD4+-T-cell level. Little or no infection in the brain was documented for most animals by nucleic acid sequence-based amplification or in situ hybridization. These data suggest that this Gag-specific response may contribute to the containment of viral replication in this locale.

We have modeled smallpox vaccination with Dryvax (Wyeth) in rhesus macaques that had depletion of CD4(+) T cells induced by infection with simian immunodeficiency virus or simian/human immunodeficiency virus. Smallpox vaccination induced significantly larger skin lesions in immunocompromised macaques than in healthy macaques. Unexpectedly, "progressive vaccinia" was infrequent. Vaccination of immunocompromised macaques with the genetically-engineered, replication-deficient poxvirus NYVAC, before or after retrovirus infection, was safe and lessened the severity of Dryvax-induced skin lesions. Neutralizing antibodies to vaccinia were induced by NYVAC, even in macaques with severe CD4(+) T cell depletion, and their titers inversely correlated with the time to complete resolution of the skin lesions. Together, these results provide the proof of concept, in macaque models that mirror human immunodeficiency virus type 1 infection, that a prime-boost approach with a highly attenuated poxvirus followed by Dryvax increases the safety of smallpox vaccination, and they highlight the importance of neutralizing antibodies in protection against virulent poxvirus.

A therapeutic vaccine for individuals infected with HIV-1 and treated with antiretroviral therapy (ART) should be able to replenish virus-specific CD4+ T-cells and broaden the virus-specific CD8+ T-cell response in order to maintain CD8+ T-cell function and minimize viral immune escape after ART cessation. Because a combination of DNA and recombinant poxvirus vaccine modalities induces high levels of virus-specific CD4+ T-cell response and broadens the cytolytic activity in naive macaques, we investigated whether the same results could be obtained in SIVmac251-infected macaques. The macaques studied here were long-term nonprogressors that naturally contained viremia but were nevertheless treated with a combination of antiviral drugs to assess more carefully the effect of vaccination in the context of ART. The combination of a DNA expressing the gag and pol genes (DNA-SIV-gp) of SIVmac239 followed by a recombinant fowlpox expressing the same SIVmac genes (FP-SIV-gp) was significantly more immunogenic than two immunizations of FP-SIV-gp in SIVmac251-infected macaques treated with ART. The DNA/FP combination significantly expanded and broadened Gag-specific T-cell responses measured by tetramer staining, ELISPOT, and intracellular cytokine staining and measurement of ex vivo cytolytic function. Importantly, the combination of these vaccine modalities also induced a sizeable expansion in most macaques of Gag-specific CD8-(CD4+) T-cells able to produce TNF-α. Hopefully, this modality of vaccine combination may be useful in the clinical management of HIV-1-infected individuals.

As most human immunodeficiency virus (HIV) infection occurs via mucosal surfaces, an important goal of vaccination may be the induction of virus-specific immune responses at mucosal sites to contain viral infection early on. Here we designed a study in macaques carrying the major histocompatibility complex class I Mamu-A∗01 molecule to assess the capacity of the highly attenuated poxvirus NYVAC/simian immunodeficiency virus (SIV) SIVgpevaccine candidate administered by the intranasal, intramuscular, or intrarectal route to induce mucosal immunity. All macaques, including one naive macaque, were exposed to SIVmac251by the intrarectal route and sacrificed 48 h after infection. The kinetics of immune response at various time points following immunization with NYVAC/SIVgpeand the anamnestic response to SIVmac251at 48 h after challenge were assessed in blood, in serial rectal and vaginal biopsy samples, and in tissues at euthanasia with an SIVmacGag-specific tetramer. In addition, at euthanasia, antigen-specific cells producing gamma interferon or tumor necrosis factor alpha from the jejunum lamina propria were quantified in all macaques. Surprisingly, antigen-specific CD8+T cells were found in the mucosal tissues of all immunized macaques regardless of whether the vaccine was administered by a mucosal route (intranasal or intrarectal) or systemically. In addition, following mucosal SIVmac251challenge, antigen-specific responses were mainly confined to mucosal tissues, again regardless of the route of immunization. We conclude that immunization with a live vector vaccine results in the appearance of CD8+T-cell responses at mucosal sites even when the vaccine is delivered by nonmucosal routes.

A cohort of rhesus macaques with long-standing SIVmac251 infection (≥5 mo) was treated with continuous antiretroviral therapy (ART). A group of eight macaques was vaccinated with or without simultaneous administration of low dose IL-2 with the highly attenuated poxvirus vector (NYVAC) vaccine candidate expressing the SIVmac structural gag-pol-env (gpe) genes and a novel chimeric fusion protein derived from the rev-tat-nef (rtn) regulatory genes. Control groups consisted of mock-vaccinated macaques or animals treated only with IL-2. Vaccination significantly expanded both virus-specific CD4+ and CD8+ T cell responses, and IL-2 further increased the vaccine-induced response to an immunodominant Gag epitope. Following antiretroviral treatment interruption, the viral set point was significantly lower in vaccinated than in control macaques for at least 4 consecutive mo, and viral containment was inversely correlated with vaccine-induced, virus-specific CD4+ and CD8+ T cell responses. These data provide the proof of concept that therapeutic vaccination before cessation of ART may be a feasible approach in the clinical management of HIV-1 infection.

Most vaccine modalities for human immunodeficiency virus type 1 (HIV-1) tested for immunogenicity and efficacy in the SIVmac (simian immunodeficiency virus) macaque model do not include the viral regulatory proteins. Because viral regulatory proteins are expressed early during the virus life cycle and represent an additional source of antigens, their inclusion as a vaccine component may increase the overall virus-specific immune response in vaccinees. However, at least two of the early proteins, Tat and Nef, may be immunosuppressive, limiting their usefulness as components of an SIV vaccine. We have constructed a polyvalent chimeric protein in which the open reading frames for Tat and Nef have been reassorted and the nuclear localization sequence for Tat and Rev and the myristoylation site for Nef have been removed. The resulting DNA plasmid (pDNA-SIV-Retanef) (pDNA-SIV-RTN) encodes a protein of 55 kDa (Retanef) that localizes at the steady state in the cytoplasma of transfected cells. Both the DNA-SIV-RTN and the highly attenuated recombinant poxvirus vector NYVAC-SIV-RTN were demonstrated to be immunogenic in SIVmac251-infected macaques treated with ART as well as in naive macaques. An equivalent strategy may be used for the generation of polyvalent antigens encoding the regulatory proteins in a HIV-1 vaccine candidate.

Most human immunodeficiency virus (HIV) type 1 infections occur by the mucosal route. Thus, it is important to assess the immune responses to HIV in the vaginal, cervical, and rectal compartments. Here we quantitated the virus-specific CD8+T-cell response and characterized the phenotype of lymphocytes in the genital tracts of naive macaques, macaques acutely or chronically infected with simian immunodeficiency virus SIVmac251, and macaques chronically infected with chimeric simian/human immunodeficiency virus SHIVKU2.Vaginal biopsy samples or samples obtained at the time of euthanasia were used in this analysis. The percentage of Gag-specific, tetramer-positive T cells was as high as 13 to 14% of the CD3+CD8+T-cell population in the vaginal and cervical laminae propriae of both SIVmac251and SHIVKU2chronically infected macaques. In most cases, the frequency of this response in the cervicovaginal compartment far exceeded the frequency in the blood or the draining iliac lymph node. Vaginal laminae propriae of naive macaques contained 55 to 65% CD3+CD8+cells and 28 to 34% CD3+CD4+cells, while the majority of intraepithelial cells were CD8+T cells (75 to 85%). For the same cells, the surface expression of CD62L was low whereas that of αEβ7 was high. No difference in the expression of CD45RA on CD8+T cells was observed in the chronic stage of SIVmac251infection. Although no decrease in the percentage of CD4+cells in the genital tract was observed within the first 12 days of infection, by 6 weeks from SIVmac251infection and thereafter the percentage of CD4+T cells was decreased in the laminae propriae of the vagina and cervix. Expression of CD45RA did not differ in naive and acutely SIVmac251infected macaques. Information on the quality and quantity of local immune responses may help in the design of vaccine strategies aimed at containing viral replication at the site of viral encounter.