Effective antibiotics in combination against extreme drug-resistant Pseudomonas aeruginosa with decreased susceptibility to polymyxin B

Objective

Extreme drug-resistant Pseudomonas aeruginosa (XDR-PA) with decreased susceptibility to polymyxin B (PB) has emerged in Singapore, causing infections in immunocompromised hosts. Combination therapy may be the only viable therapeutic option until new antibiotics become available. The objective of this study is to assess the in vitro activity of various antibiotics against local XDR-PA isolates.

Methods

PA isolates from all public hospitals in Singapore were systematically collected between 2006 and 2007. MICs were determined according to CLSI guidelines. All XDR-PA isolates identified were genotyped using a PCR-based method. Time-kill studies (TKS) were performed with approximately 10⁵ CFU/ml at baseline using clinically achievable unbound concentrations of amikacin (A), levofloxacin (L), meropenem (M), rifampicin (R) and PB alone and in combination. Bactericidal activity (primary endpoint) was defined as a ≥3 log₁₀ CFU/ml decrease in the colony count from the initial inoculum at 24 hours.

Results

22 clinical XDR-PA isolates with PB MIC 2–16 µg/ml were collected. From clonal typing, 5 clonal groups were identified and nine isolates exhibited clonal diversity. In TKS, meropenem plus PB, amikacin plus meropenem, amikacin plus rifampicin, amikacin plus PB exhibited bactericidal activity in 8/22, 3/22, 1/22 and 6/22 isolates at 24 hours respectively. Against the remaining ten isolates where none of the dual-drug combination achieved bactericidal activity against, only the triple-antibiotic combinations of ARP and AMP achieved bactericidal activity against 7/10 and 6/10 isolates respectively.

Conclusion

Bactericidal activity with sustained killing effect of ≥99.9% is critical for eradicating XDR-PA infections, especially in immunocompromised hosts. These findings underscore the difficulty of developing combination therapeutic options against XDR-PA, demonstrating that at least 3 antibiotics are required in combination and that efficacy is strain dependant.     

Evaluation of Trimethoprim/Sulfamethoxazole (SXT), Minocycline,Tigecycline, Moxifloxacin,and Ceftazidime Alone and in Combinations for SXT-Susceptible and SXT-Resistant Stenotrophomonas maltophilia by In Vitro Time-Kill Experiments

Background

The optimal therapy for infections caused by Stenotrophomonas maltophilia (S. maltophilia) has not yet been established. The objective of our study was to evaluate the efficacy of trimethoprim/sulfamethoxazole (SXT), minocycline, tigecycline, moxifloxacin, levofloxacin, ticarcillin-clavulanate, polymyxin E, chloramphenicol, and ceftazidime against clinical isolated S. maltophilia strains by susceptibility testing and carried out time-kill experiments in potential antimicrobials.

Methods

The agar dilution method was used to test susceptibility of nine candidate antimicrobials, and time-killing experiments were carried out to evaluate the efficacy of SXT, minocycline, tigecycline, moxifloxacin, levofloxacin, and ceftazidime both alone and in combinations at clinically relevant antimicrobial concentrations.

Results

The susceptibility to SXT, minocycline, tigecycline, moxifloxacin, levofloxacin, ticarcillin-clavulanate, chloramphenicol, polymyxin E, and ceftazidime were 93.8%, 95.0%, 83.8%, 80.0%, 76.3%, 76.3%, 37.5%, 22.5%, and 20.0% against 80 clinical consecutively isolated strains, respectively. Minocycline and tigecycline showed consistent active against 22 SXT-resistant strains. However, resistance rates were high in the remaining antimicrobial agents against SXT-resistant strains. In time-kill experiments, there were no synergisms in most drug combinations in time-kill experiments. SXT plus moxifloxacin displayed synergism when strains with low moxifloxacin MICs. Moxifloxacin plus Minocycline and moxifloxacin plus tigecycline displayed synergism in few strains. No antagonisms were found in these combinations. Overall, compared with single drug, the drug combinations demonstrated lower bacterial concentrations. Some combinations showed bactericidal activity.

Conclusions

In S. maltophilia infections, susceptibility testing suggests that minocycline and SXT may be considered first-line therapeutic choices while tigecycline, moxifloxacin, levofloxacin, and ticarcillin-clavulanate may serve as second-line choices. Ceftazidime, colistin, and chloramphenicol show poor active against S. maltophilia. However, monotherapy is inadequate in infection management, especially in case of immunocompromised patients. Combination therapy, especially SXT plus moxifloxacin, may benefit than monotherapy in inhibiting or killing S. maltophilia.     

High Isoniazid Resistance Rates in Rifampicin Susceptible Mycobacterium tuberculosis Pulmonary Isolates from Pakistan

Background

Rapid new diagnostic methods (including Xpert MTB/RIF assay) use rifampicin resistance as a surrogate marker for multidrug resistant tuberculosis. Patients infected with rifampicin susceptible strains are prescribed first line anti-tuberculosis therapy. The roll out of such methods raises a concern that strains with resistance to other first line anti-tuberculosis drugs including isoniazid will be missed and inappropriate treatment given. To evaluate implications of using such methods review of resistance data from high burden settings such as ours is essential.

Objective

To determine resistance to first line anti-tuberculosis drugs amongst rifampicin susceptible pulmonary Mycobacterium tuberculosis (MTB) isolates from Pakistan.

Materials and Methods

Data of pulmonary Mycobacterium tuberculosis strains isolated in Aga Khan University Hospital (AKUH) laboratory (2009–2011) was retrospectively analyzed. Antimicrobial susceptibility profile of rifampicin susceptible isolates was evaluated for resistance to isoniazid, pyrazinamide, ethambutol, and streptomycin.

Results

Pulmonary specimens submitted to AKUH from 2009 to 2011 yielded 7738 strains of Mycobacterium tuberculosis. These included 54% (n 4183) rifampicin susceptible and 46% (n: 3555) rifampicin resistant strains. Analysis of rifampicin susceptible strains showed resistance to at least one of the first line drugs in 27% (n:1133) of isolates. Overall isoniazid resistance was 15.5% (n: 649), with an isoniazid mono-resistance rate of 4% (n: 174). Combined resistance to isoniazid, pyrazinamide, and ethambutol was noted in 1% (n: 40), while resistance to isoniazid, pyrazinamide, ethambutol, and streptomycin was observed in 1.7% (n: 70) of strains.

Conclusions

Our data suggests that techniques (including Xpert MTB/RIF assay) relying on rifampicin susceptibility as an indicator for initiating first line therapy will not detect patients infected with MTB strains resistant to other first line drugs (including isoniazid). The roll out of these techniques must therefore be accompanied by strict monitoring ensuring early resistance detection to increase chances of improved patient outcomes.     

Diversity of Acinetobacter baumannii in Four French Military Hospitals, as Assessed by Multiple Locus Variable Number of Tandem Repeats Analysis

Background

Infections by A. calcoaceticus-A. baumannii (ACB) complex isolates represent a serious threat for wounded and burn patients. Three international multidrug-resistant (MDR) clones (EU clone I-III) are responsible for a large proportion of nosocomial infections with A. baumannii but other emerging strains with high epidemic potential also occur.

Methodology/Principal Findings

We automatized a Multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) protocol and used it to investigate the genetic diversity of 136 ACB isolates from four military hospitals and one childrens hospital. Acinetobacter sp other than baumannii isolates represented 22.6% (31/137) with a majority being A. pittii. The genotyping protocol designed for A.baumannii was also efficient to cluster A. pittii isolates. Fifty-five percent of A. baumannii isolates belonged to the two international clones I and II, and we identified new clones which members were found in the different hospitals. Analysis of two CRISPR-cas systems helped define two clonal complexes and provided phylogenetic information to help trace back their emergence.

Conclusions/Significance

The increasing occurrence of A. baumannii infections in the hospital calls for measures to rapidly characterize the isolates and identify emerging clones. The automatized MLVA protocol can be the instrument for such surveys. In addition, the investigation of CRISPR/cas systems may give important keys to understand the evolution of some highly successful clonal complexes.     

Molecular Characterization of Invasive Meningococcal Isolates from Countries in the African Meningitis Belt before Introduction of a Serogroup A Conjugate Vaccine

Background

The serogroup A conjugate meningococcal vaccine, MenAfriVac, was introduced in mass vaccination campaigns in December 2010 in Burkina Faso, Mali and Niger. In the coming years, vaccination will be extended to other African countries at risk of epidemics. To document the molecular characteristics of disease-causing meningococcal strains circulating in the meningitis belt of Africa before vaccine introduction, the World Health Organization Collaborating Centers on Meningococci in Europe and United States established a common strain collection of 773 isolates from cases of invasive meningococcal disease collected between 2004 and 2010 from 13 sub-Saharan countries.

Methodology

All isolates were characterized by multilocus sequence typing, and 487 (62%) were also analyzed for genetic variation in the surface antigens PorA and FetA. Antibiotic susceptibility was tested for part of the collection.

Principal Findings

Only 19 sequence types (STs) belonging to 6 clonal complexes were revealed. ST-5 clonal complex dominated with 578 (74.8%) isolates. All ST-5 complex isolates were remarkably homogeneous in their PorA (P1.20,9) and FetA (F3-1) and characterized the serogroup A strains which have been responsible for most epidemics during this time period. Sixty-eight (8.8%) of the 773 isolates belonged to the ST-11 clonal complex which was mainly represented by serogroup W135, while an additional 38 (4.9%) W135 isolates belonged to the ST-175 complex. Forty-eight (6.2%) serogroup X isolates from West Africa belonged to the ST-181 complex, while serogroup X cases in Kenya and Uganda were caused by an unrelated clone, ST-5403. Serogroup X, ST-181, emerged in Burkina Faso before vaccine introduction.

Conclusions

In the seven years preceding introduction of a new serogroup A conjugate vaccine, serogroup A of the ST-5 clonal complex was identified as the predominant disease-causing strain.     

Molecular epidemiology of Neisseria meningitidis serogroup B in Brazil

Background

Neisseria meningitidis serogroup B has been predominant in Brazil, but no broadly effective vaccine is available to prevent endemic meningococcal disease. To understand genetic diversity among serogroup B strains in Brazil, we selected a nationally representative sample of clinical disease isolates from 2004, and a temporally representative sample for the state of São Paulo (1988–2006) for study (n = 372).

Methods

We performed multi-locus sequence typing (MLST) and sequence analysis of five outer membrane protein (OMP) genes, including novel vaccine targets fHbp and nadA.

Results

In 2004, strain B:4:P1.15,19 clonal complex ST-32/ET-5 (cc32) predominated throughout Brazil; regional variation in MLST sequence type (ST), fetA, and porB was significant but diversity was limited for nadA and fHbp. Between 1988 and 1996, the São Paulo isolates shifted from clonal complex ST-41/44/Lineage 3 (cc41/44) to cc32. OMP variation was associated with but not predicted by cc or ST. Overall, fHbp variant 1/subfamily B was present in 80% of isolates and showed little diversity. The majority of nadA were similar to reference allele 1.

Conclusions

A predominant serogroup B lineage has circulated in Brazil for over a decade with significant regional and temporal diversity in ST, fetA, and porB, but not in nadA and fHbp.     

Estimated Secondary Structure Propensities within V1/V2 Region of HIV gp120 Are an Important Global Antibody Neutralization Sensitivity Determinant

Background

Neutralization sensitivity of HIV-1 virus to antibodies and anti-sera varies greatly between the isolates. Significant role of V1/V2 domain as a global neutralization sensitivity regulator has been suggested. Recent X-ray structures revealed presence of well-defined tertiary structure within this domain but also demonstrated partial disorder and conformational heterogeneity.

Methods

Correlations of neutralization sensitivity with the conformational propensities for beta-strand and alpha-helix formation over the entire folded V1/V2 domain as well as within sliding 5-residue window were investigated. Analysis was based on a set of neutralization data for 106 HIV isolates for which consistent neutralization sensitivity measurements against multiple pools of human immune sera have been previously reported.

Results

Significant correlation between beta-sheet formation propensity of the folded segments of V1/V2 domain and neutralization sensitivity was observed. Strongest correlation peaks localized to the beta-strands B and C. Correlation persisted when subsets of HIV isolates belonging to clades B, C and circulating recombinant form BC where analyzed individually or in combinations.

Conclusions

Observed correlations suggest that stability of the beta-sheet structure and/or degree of structural disorder in the V1/V2 domain is an important determinant of the global neutralization sensitivity of HIV-1 virus. While specific mechanism is to yet to be investigated, plausible hypothesis is that less ordered V1/V2s may have stronger masking effect on various neutralizing epitopes, perhaps effectively occupying larger volume and thereby occluding antibody access.     

Complement Receptor 1 Variants Confer Protection from Severe Malaria in Odisha,India

Background

In Plasmodium falciparum infection, complement receptor-1 (CR1) on erythrocyte’s surface and ABO blood group play important roles in formation of rosettes which are presumed to be contributory in the pathogenesis of severe malaria. Although several studies have attempted to determine the association of CR1 polymorphisms with severe malaria, observations remain inconsistent. Therefore, a case control study and meta-analysis was performed to address this issue.

Methods

Common CR1 polymorphisms (intron 27 and exon 22) and blood group were typed in 353 cases of severe malaria (SM) [97 cerebral malaria (CM), 129 multi-organ dysfunction (MOD), 127 non-cerebral severe malaria (NCSM)], 141 un-complicated malaria and 100 healthy controls from an endemic region of Odisha, India. Relevant publications for meta-analysis were searched from the database.

Results

The homozygous polymorphisms of CR1 intron 27 and exon 22 (TT and GG) and alleles (T and G) that are associated with low expression of CR1 on red blood cells, conferred significant protection against CM, MOD and malaria deaths. Combined analysis showed significant association of blood group B/intron 27-AA/exon 22-AA with susceptibility to SM (CM and MOD). Meta-analysis revealed that the CR1 exon 22 low expression polymorphism is significantly associated with protection against severe malaria.

Conclusions

The results of the present study demonstrate that common CR1 variants significantly protect against severe malaria in an endemic area.     

The Changing Epidemiology of Meningococcal Disease in Quebec,Canada, 1991–2011: Potential Implications of Emergence of New Strains

Background

In order to inform meningococcal disease prevention strategies, we analysed the epidemiology of invasive meningococcal disease (IMD) in the province of Quebec, Canada, 10 years before and 10 years after the introduction of serogroup C conjugate vaccination.

Methodology

IMD cases reported to the provincial notifiable disease registry in 1991–2011 and isolates submitted for laboratory surveillance in 1997–2011 were analysed. Serogrouping, PCR testing and assignment of isolates to sequence types (ST) by using multilocus sequence typing (MLST) were performed.

Results

Yearly overall IMD incidence rates ranged from 2.2–2.3/100,000 in 1991–1992 to 0.49/100,000 in 1999–2000, increasing to 1.04/100,000 in 2011. Among the 945 IMD cases identified by laboratory surveillance in 1997–2011, 68%, 20%, 8%, and 3% were due to serogroups B, C, Y, and W135, respectively. Serogroup C IMD almost disappeared following the implementation of universal childhood immunization with monovalent C conjugate vaccines in 2002. Serogroup B has been responsible for 88% of all IMD cases and 61% of all IMD deaths over the last 3 years. The number and proportion of ST-269 clonal complex has been steadily increasing among the identified clonal complexes of serogroup B IMD since its first identification in 2003, representing 65% of serogroup B IMD in 2011. This clonal complex was first introduced in adolescent and young adults, then spread to other age groups.

Conclusion

Important changes in the epidemiology of IMD have been observed in Quebec during the last two decades. Serogroup C has been virtually eliminated. In recent years, most cases have been caused by the serogroup B ST-269 clonal complex. Although overall burden of IMD is low, the use of a vaccine with potential broad-spectrum coverage could further reduce the burden of disease. Acceptability, feasibility and cost-effectiveness studies coupled with ongoing clinical and molecular surveillance are necessary in guiding public policy decisions.     

Exercise-based cardiac rehabilitation in patients with chronic heart failure: a Dutch practice guideline

Rationale

To improve the quality of exercise-based cardiac rehabilitation (CR) in patients with chronic heart failure (CHF) a practice guideline from the Dutch Royal Society for Physiotherapy (KNGF) has been developed.

Guideline development

A systematic literature search was performed to formulate conclusions on the efficacy of exercise-based intervention during all CR phases in patients with CHF. Evidence was graded (1–4) according the Dutch evidence-based guideline development criteria.

Clinical and research recommendations

Recommendations for exercise-based CR were formulated covering the following topics: mobilisation and treatment of pulmonary symptoms (if necessary) during the clinical phase, aerobic exercise, strength training (inspiratory muscle training and peripheral muscle training) and relaxation therapy during the outpatient CR phase, and adoption and monitoring training after outpatient CR.

Applicability and implementation issues

This guideline provides the physiotherapist with an evidence-based instrument to assist in clinical decision-making regarding patients with CHF. The implementation of the guideline in clinical practice needs further evaluation.

Conclusion

This guideline outlines best practice standards for physiotherapists concerning exercise-based CR in CHF patients. Research is needed on strategies to improve monitoring and follow-up of the maintenance of a physical active lifestyle after supervised CR.     

Activity and Interactions of Liposomal Antibiotics in Presence of Polyanions and Sputum of Patients with Cystic Fibrosis

Background

To compare the effectiveness of liposomal tobramycin or polymyxin B against Pseudomonas aeruginosa in the Cystic Fibrosis (CF) sputum and its inhibition by common polyanionic components such as DNA, F-actin, lipopolysaccharides (LPS), and lipoteichoic acid (LTA).

Methodology

Liposomal formulations were prepared from a mixture of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) or 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and Cholesterol (Chol), respectively. Stability of the formulations in different biological milieus and antibacterial activities compared to conventional forms in the presence of the aforementioned inhibitory factors or CF sputum were evaluated.

Results

The formulations were stable in all conditions tested with no significant differences compared to the controls. Inhibition of antibiotic formulations by DNA/F-actin and LPS/LTA was concentration dependent. DNA/F-actin (125 to 1000 mg/L) and LPS/LTA (1 to 1000 mg/L) inhibited conventional tobramycin bioactivity, whereas, liposome-entrapped tobramycin was inhibited at higher concentrations - DNA/F-actin (500 to 1000 mg/L) and LPS/LTA (100 to 1000 mg/L). Neither polymyxin B formulation was inactivated by DNA/F-actin, but LPS/LTA (1 to 1000 mg/L) inhibited the drug in conventional form completely and higher concentrations of the inhibitors (100 to 1000 mg/L) was required to inhibit the liposome-entrapped polymyxin B. Co-incubation with inhibitory factors (1000 mg/L) increased conventional (16-fold) and liposomal (4-fold) tobramycin minimum bactericidal concentrations (MBCs), while both polymyxin B formulations were inhibited 64-fold.

Conclusions

Liposome-entrapment reduced antibiotic inhibition up to 100-fold and the CFU of endogenous P. aeruginosa in sputum by 4-fold compared to the conventional antibiotic, suggesting their potential applications in CF lung infections.     

Primary Drug-Resistant Tuberculosis in Hanoi,Viet Nam: Present Status and Risk Factors

Introduction

Resistance of Mycobacterium tuberculosis (MTB) to anti-tuberculosis (TB) drugs presents a serious challenge to TB control worldwide. We investigated the status of drug resistance, including multidrug-resistant (MDR) TB, and possible risk factors among newly diagnosed TB patients in Hanoi, the capital of Viet Nam.

Methods

Clinical and epidemiological information was collected from 506 newly diagnosed patients with sputum smear- and culture-positive TB, and 489 (96.6%) MTB isolates were subjected to conventional drug susceptibility testing, spoligotyping, and 15-locus variable numbers of tandem repeats typing. Adjusted odds ratios (aORs) were calculated to analyze the risk factors for primary drug resistance.

Results

Of 489 isolates, 298 (60.9%) were sensitive to all drugs tested. Resistance to isoniazid, rifampicin, streptomycin, ethambutol, and MDR accounted for 28.2%, 4.9%, 28.2%, 2.9%, and 4.5%, respectively. Of 24 isolates with rifampicin resistance, 22 (91.7%) were MDR and also resistant to streptomycin, except one case. Factors associated with isoniazid resistance included living in old urban areas, presence of the Beijing genotype, and clustered strains [aOR = 2.23, 95% confidence interval (CI) 1.15–4.35; 1.91, 1.18–3.10; and 1.69, 1.06–2.69, respectively). The Beijing genotype was also associated with streptomycin resistance (aOR = 2.10, 95% CI 1.29–3.40). Human immunodeficiency virus (HIV) coinfection was associated with rifampicin resistance and MDR (aOR = 5.42, 95% CI 2.07–14.14; 6.23, 2.34–16.58, respectively).

Conclusion

Isoniazid and streptomycin resistance was observed in more than a quarter of TB patients without treatment history in Hanoi. Transmission of isoniazid-resistant TB among younger people should be carefully monitored in urban areas, where Beijing strains and HIV coinfection are prevalent. Choosing an optimal treatment regimen on the basis of the results of drug susceptibility tests and monitoring of treatment adherence would minimize further development of drug resistance strains.     

Antibiotic Prescribing in DR Congo: A Knowledge,Attitude and Practice Survey among Medical Doctors and Students

Objectives

Antibiotic resistance (ABR) particularly hits resource poor countries, and is fuelled by irrational antibiotic (AB) prescribing. We surveyed knowledge, attitudes and practices of AB prescribing among medical students and doctors in Kisangani, DR Congo.

Methods

Self-administered questionnaires.

Results

A total of 184 questionnaires were completed (response rate 94.4%). Knowledge about AB was low (mean score 4.9/8 points), as was the estimation of local resistance rates of S. Typhi and Klebsiella spp.(correct by 42.5% and 6.9% of respondents respectively). ABR was recognized as a problem though less in their own practice (67.4%) than nation- or worldwide (92.9% and 85.5%, p<.0001). Confidence in AB prescribing was high (88.6%) and students consulted more frequently colleagues than medical doctors when prescribing (25.4% versus 11.6%, p  = 0.19). Sources of AB prescribing included pharmaceutical companies (73.9%), antibiotic guidelines (66.3%), university courses (63.6%), internet-sites (45.7%) and WHO guidelines (26.6%). Only 30.4% and 16.3% respondents perceived AB procured through the central procurement and local pharmacies as of good quality. Local AB guidelines and courses about AB prescribing are welcomed (73.4% and 98.8% respectively).

Conclusions

This data shows the need for interventions that support rational AB prescribing.     

Evaluation of Biochip System in Determining Isoniazid and Rifampicin Resistances of Mycobacterium Tuberculosis in Sputum Samples

Objective

To evaluate a biochip system in determining isoniazid and rifampicin resistances of Mycobacterium tuberculosis in sputum samples in a Chinese population.

Methods

We assembled 907 sputum smeared positive specimens of tuberculosis patients in total. Each sample would be separated into two parts for culture and biochip assay simultaneously. And those cultured positive and having full drug resistance results would be used as reference. The McNemar χ² test was adopted for evaluating the paired 2×2 table.

Results

Compared with drug sensitivity test, the agreement rates of the two methods in detecting rifampicin and isoniazid resistances were 93.37% and 94.49%, respectively. The sensitivity and specificity of biochip in detecting isoniazid were 74.31% and 96.92%, respectively. Meanwhile, the sensitivity and specificity for rifampicin were 79.76% and 96.53%, respectively. For multi-drug resistance, the sensitivity and specificity were 64.62% and 97.75%, respectively.

Conclusions

The biochip system is a rapid and accurate method for drug resistant tuberculosis diagnosis using sputum samples directly, especially for rifampicin resistance detection.     

Genetic Variation in Spatio-Temporal Confined USA300 Community-Associated MRSA Isolates: A Shift from Clonal Dispersion to Genetic Evolution?

Introduction

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are increasingly isolated, with USA300-0114 being the predominant clone in the USA. Comparative whole genome sequencing of USA300 isolates collected in 2002, 2003 and 2005 showed a limited number of single nucleotide polymorphisms and regions of difference. This suggests that USA300 has undergone rapid clonal expansion without great genomic diversification. However, whole genome comparison of CA-MRSA has been limited to isolates belonging to USA300. The aim of this study was to compare the genetic repertoire of different CA-MRSA clones with that of HA-MRSA from the USA and Europe through comparative genomic hybridization (CGH) to identify genetic clues that may explain the successful and rapid emergence of CA-MRSA.

Materials and Methods

Hierarchical clustering based on CGH of 48 MRSA isolates from the community and nosocomial infections from Europe and the USA revealed dispersed clustering of the 19 CA-MRSA isolates. This means that these 19 CA-MRSA isolates do not share a unique genetic make-up. Only the PVL genes were commonly present in all CA-MRSA isolates. However, 10 genes were variably present among 14 USA300 isolates. Most of these genes were present on mobile elements.

Conclusion

The genetic variation present among the 14 USA300 isolates is remarkable considering the fact that the isolates were recovered within one month and originated from a confined geographic area, suggesting continuous evolution of this clone.     

Construction of an expression plasmid (vector) encoding Brucella melitensis outer membrane protein,a candidate for DNA vaccine

Background:

DNA vaccination with plasmid encoding bacterial, viral, and parasitic immunogens has been shown to be an attractive method to induce efficient immune responses. Bacteria of the genus Brucella are facultative intracellular pathogens for which new and efficient vaccines are needed.

Methods:

To evaluate the use of a DNA immunization strategy for protection against brucellosis, a plasmid containing the DNA encoding the Brucella melitensis (B. melitensis) 31 kDa outer membrane protein, as a potent immunogenic target, was constructed.

Results:

The constructed plasmid, pcDNA3.1+omp31, was injected intramuscularly into mice and the expression of omp31 RNA was assessed by RT-PCR. The integrity of the pcDNA3.1+omp31 construct was confirmed with restriction analysis and sequencing. Omp31 mRNA expression was verified by RT-PCR.

Conclusion:

Our results indicate that the pcDNA3.1+omp31 eukaryotic expression vector expresses omp31 mRNA and could be useful as a vaccine candidate.Key Words: Brucella melitensis, omp31, DNA Vaccine, pcDNA3.1     

A Detailed Phylogenetic Analysis of FIV in the United States

Background

Feline immunodeficiency virus (FIV) is a lentivirus associated with AIDS-like illnesses in cats and has been used as a model for the study of human immunodeficiency virus (HIV). A feature of HIV and FIV infection is the continually increasing divergence among viral isolates between different individuals, as well as within the same individuals.

Methodology/Principal Findings

The goal of this study was to determine the phylogenetic patterns of viral isolates obtained within the United States (U.S.) by focusing on the variable, V3-V4, region of the FIV envelope gene.

Conclusions/Significance

Data indicate that FIV, from within the U.S., localize to four viral clades, A, B, C, and F. Also shown is the geographic isolation of strains where clade A and clade B are found predominately on the west coast; however, clade B is also found throughout the U.S. and represents the predominant clade. This study presents a complete and conclusive analysis of FIV isolates from within the U.S. and may be used as the essential basis for the development of an effective multi-clade vaccine.     

The Impact of spgM,rpfF, rmlA Gene Distribution on Biofilm Formation in Stenotrophomonas maltophilia

Background

Stenotrophomonas maltophilia is emerging as one of the most frequently found bacteria in chronic pulmonary infection. Biofilm is increasingly recognized as a contributing factor to disease pathogenesis. In the present study, a total of 37 isolates of S. maltophilia obtained from chronic pulmonary infection patients were evaluated to the relationship between biofilm production and the relative genes expression.

Methods

The clonal relatedness of isolates was determined by pulse-field gel electrophoresis. Biofilm formation assays were performed by crystal violet assay, and confirmed by Electron microscopy analysis and CLSM analysis. PCR was employed to learn gene distribution and expression.

Results

Twenty-four pulsotypes were designated for 37 S. maltophilia isolates, and these 24 pulsotypes exhibited various levels of biofilm production, 8 strong biofilm-producing S. maltophilia strains with OD492 value above 0.6, 14 middle biofilm-producing strains with OD492 average value of 0.4 and 2 weak biofilm-producing strains with OD492 average value of 0.19. CLSM analysis showed that the isolates from the early stage of chronic infection enable to form more highly structured and multilayered biofim than those in the late stage. The prevalence of spgM, rmlA, and rpfF genes was 83.3%, 87.5%, and 50.0% in 24 S. maltophilia strains, respectively, and the presence of rmlA, spgM or rpfF had a close relationship with biofilm formation but did not significantly affect the mean amount of biofilm. Significant mutations of spgM and rmlA were found in both strong and weak biofilm-producing strains.

Conclusion

Mutations in spgM and rmlA may be relevant to biofilm formation in the clinical isolates of S. maltophilia.     

Trampling, defoliation and physiological integration affect growth, morphological and mechanical properties of a root-suckering clonal tree

Background and Aims

Grazing is a complex process involving the simultaneous occurrence of both trampling and defoliation. Clonal plants are a common feature of heavily grazed ecosystems where large herbivores inflict the simultaneous pressures of trampling and defoliation on the vegetation. We test the hypothesis that physiological integration (resource sharing between interconnected ramets) may help plants to deal with the interactive effects of trampling and defoliation.

Methods

In a field study, small and large ramets of the root-suckering clonal tree Populus simonii were subjected to two levels of trampling and defoliation, while connected or disconnected to other ramets. Plant responses were quantified via survival, growth, morphological and stem mechanical traits.

Key Results

Disconnection and trampling increased mortality, especially in small ramets. Trampling increased stem length, basal diameter, fibrous root mass, stem stiffness and resistance to deflection in connected ramets, but decreased them in disconnected ones. Trampling decreased vertical height more in disconnected than in connected ramets, and reduced stem mass in disconnected ramets but not in connected ramets. Defoliation reduced basal diameter, leaf mass, stem mass and leaf area ratio, but did not interact with trampling or disconnection.

Conclusions

Although clonal integration did not influence defoliation response, it did alleviate the effects of trampling. We suggest that by facilitating resource transport between ramets, clonal integration compensates for trampling-induced damage to fine roots.     

Long-Term Effect of Antibodies against Infused Alpha-Galactosidase A in Fabry Disease on Plasma and Urinary (lyso)Gb3 Reduction and Treatment Outcome

Introduction

Enzyme replacement therapy (ERT) with alpha-Galactosidase A (aGal A) may cause antibody (AB) formation against aGal A in males with Fabry disease (FD). Anti agalsidase ABs negatively influence globotriaosylceramide (Gb3) reduction. We investigated the impact of agalsidase AB on Gb3 and lysoGb3 and clinical outcome in Fabry patients on ERT.

Methods

Adult male and female patients on ERT for at least one year were included. Urinary Gb3 was measured by HPLC, plasma lysoGb3 by LC-ESI-MS/MS and AB with a neutralization assay.

Results

Of the 59 patients evaluable patients, 0/30 females and 17/29 males developed anti-agalsidase antibodies (AB+). Only 3/17 males had transient (low) titers (tolerized). All AB+ patients developed antibodies during the first year of treatment. Change of agalsidase preparation (or dose) did not induce antibody formation. AB+ males had significant less decline in plasma lysoGb3 compared to AB− males (p = 0.04). Urinary Gb3 levels decreased markedly in AB− but remained comparable to baseline in AB+ males (p<0.01). (Lyso)Gb3 reduction in plasma and urine on ERT was correlated with LVmass reduction in females and development white matter lesions and stroke.

Conclusion

In male patients antibodies against aGal A remained present up to 10 years of ERT. The presence of these antibodies is associated with a less robust decrease in plasma lysoGb3 and a profound negative effect on urinary Gb3 reduction, which may reflect worse treatment outcome.