Long-Term Prevention of Arthroplasty Infections via Incorporation of Activated AgNbO3 Nanoparticles in PMMA Bone Cement.

We investigated the possibility of loading PMMA bone cement with antimicrobial nanostructured AgNbO3 particles to counter biofilm formation at the cement-tissue interface. We found that a formulation containing (1-4)% AgNbO3 showed high antibacterial activity against Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa while not showing any toxicity against THP1 human cell lines. In addition, loading the particles did not impact the mechanical properties of the cement. The results thus obtained illustrate the potential of the approach to replace the current technique of mixing cement with conventional antibiotics, which is associated with shortcomings such as efficacy loss from antibiotic depletion.

Increased copy number of the target gene squalene monooxygenase as the main resistance mechanism to terbinafine in Leishmania infantum.

We use here two genomic screens in an attempt to understand the mode of action and resistance mechanism of terbinafine, an antifungal contemplated as a potential drug against the parasite Leishmania. One screen consisted in in vitro drug evolution where 5 independent mutants were selected step-by-step for terbinafine resistance. Sequencing of the genome of the 5 mutants revealed no single nucleotide polymorphisms related to the resistance phenotype. However, the ERG1 gene was found amplified as part of a linear amplicon, and transfection of ERG1 fully recapitulated the terbinafine resistance phenotype of the mutants. The second screen, Cos-seq, consisted in selecting a gene overexpression library with terbinafine followed by the sequencing of the enriched cosmids. This screen identified two cosmids derived from loci on chromosomes 13 and 29 encoding the squalene monooxygenase (ERG1) and the C8 sterol isomerase (ERG2), respectively. Transfection of the ERG1-cosmid, but not the ERG2-cosmid, produced resistance to terbinafine. Our screens suggest that ERG1 is the main, if not only, target for terbinafine in Leishmania and amplification of its gene is the main resistance mechanism.

Multidrug-resistant Salmonella strains from food animals as a potential source for human infection in Iran.

This study aimed to determine the frequency and characterization of Salmonella isolates from food-producing animals and human diarrheal samples in Shiraz, Iran. Overall, 105 Salmonella isolates were obtained from chicken meat (70/100, 70.0%), beef (19/100, 19.0%), lamb (11/100, 11.0%), and human stool (5/295, 1.7%). S. Infantis (40.9%), S. Enteritidis (29.5%), and S. Paratyphi B (8.6%) were the most prevalent serotypes. Totally 59.1% of the isolates were multidrug-resistant. High resistance to nalidixic acid (67.6%), tetracycline (62.9%), and trimethoprim-sulfamethoxazole (42.3%) were observed. Mutations in the gyrA and parC genes were detected in nalidixic acid-resistant isolates from chicken meat and human stool. No qnrA, qnrB or qnrS genes were detected. The blaTEM and blaCMY-2 genes were detected in ß-lactam-resistant isolates from beef and lamb. Despite the high genetic diversity of PFGE patterns, some isolates from different sources and times showed identical PFGE patterns suggesting specific Salmonella species circulate between various sources over time.

Prevalence, species diversity, and antimicrobial susceptibility of Campylobacter strains in patients with diarrhea and poultry meat samples: one-year prospective study.

Background and Objectives: Source tracking of antimicrobial resistance in Campylobacter is useful for control measures. In this study, Campylobacter-associated diarrhea and homology in antimicrobial resistance of humans and poultry meat isolates were investigated. Materials and Methods: A total of 400 stools of patients and 100 poultry meat samples were analyzed. Susceptibility of the isolates was detected by disk diffusion, Etest, and agar dilution methods. Mismatch amplification mutation assay was used for the detection of mutations in the gyrA quinolone resistance determining region (QRDR). Results: Campylobacter spp., including C. jejuni, C. coli, and C. lari, were detected in 35% of the chicken meat and 6.75% of the stool samples, respectively. The QRDR mutation was detected in most of the stool and chicken meat samples. Although the frequency of resistance to tetracycline (53.5% and 62.8%), erythromycin (39.2% and 37.1%), and gentamicin (32.1% and 31.4%) was relatively similar, higher frequency of resistance to ciprofloxacin (51.4% vs 28.6%) and nalidixic acid (42.15% vs 28.6%) among the chicken meat, and ampicillin (50% and 17.1%) among the human stool was detected. Conclusion: High percentage of poultry meat samples is contaminated with different Campylobacter species, which shows homology with the patients' isolates in Tehran.

Well-Tolerated Amphotericin B Derivatives That Effectively Treat Visceral Leishmaniasis.

Chemotherapy against the neglected tropical disease visceral leishmaniasis (VL) is suboptimal with only four licensed drugs. Amphotericin B (AmB), despite its toxicity, remained a second line drug for a long time. However, the demonstration that liposomal AmB is highly effective against VL propelled it, despite its cost, to a first line drug in many countries. While several ongoing efforts are aiming at finding cheaper and stable AmB-formulations, an alternative strategy is the development of less-toxic AmB derivatives. We show here that two less-toxic AmB derivatives with the carboxylate at position 16 of AmB derivatized to a methyl urea (AmB-MU) or amino urea (AmB-AU) are active in vitro against Leishmania donovani, both as free-living parasites as well as their intracellular form. Both less-toxic derivatives, similarly to AmB, target the ergosterol pathway of L. donovani. While the AmB-AU derivative showed female-specific liver toxicity in vivo, the AmB-MU derivative was well-tolerated and more effective than AmB against experimental VL. These studies are an important step for improving AmB-based therapy against a prevalent parasitic disease.

Diarrheagenic Escherichia coli and Shigella with High Rate of Extended-Spectrum Beta-Lactamase Production: Two Predominant Etiological Agents of Acute Diarrhea in Shiraz, Iran.

This study was conducted to find the etiology of acute diarrhea in Iranian children and determine the antimicrobial resistance patterns. The pathogenic bacteria were recovered from 110/269 (40.9%) diarrheal fecal samples with the following profiles: the most predominant pathogen was diarrheagenic Escherichia coli (DEC) (43.6%), comprising enteroaggregative E. coli (23.6%), enteropathogenic E. coli (10.9%), enteroinvasive E. coli (5.5%), and enterotoxigenic E. coli (3.6%); Shigella spp. (37.3%), Salmonella spp. (12.7%) and Campylobacter jejuni (6.4%) were ranked second and fourth in terms of prevalence, respectively. The rates of extended-spectrum beta-lactamase (ESBL) production were 66.7% and 53.7% in DEC and Shigella, respectively. Resistance to ampicillin (AMP) (95.1%), trimethoprim/sulfamethoxazole (SXT) (73.2%), azithromycin (ATH) (21.9%), and ciprofloxacin (CIP) (14.6%) was observed among Shigella isolates. Multidrug resistance phenotype was observed in 24.4% (10/41) of Shigella isolates, with the most common pattern of resistance to cefotaxime, ceftriaxone, ceftazidime, AMP, SXT, and ATH. This study indicates an alarming increase in the ESBL production of DEC and Shigella spp. and identifies them as the two most prevalent diarrhea-causing enteropathogens in the region. The results show that CIP could be an alternative to third-generation cephalosporins against these two pathogens. Therefore, it is proposed that further investigation be done in the pursuit of alternative antibiotics that are effective against the resistant cases. For instance, one study could look into the comparative clinical effectiveness of third-generation cephalosporins versus CIP, the latter not being presently the drug of choice for the treatment of acute diarrhea in children in Iran.

Genomic analyses of DNA transformation and penicillin resistance in Streptococcus pneumoniae clinical isolates.

Alterations in penicillin-binding proteins, the target enzymes for ß-lactam antibiotics, are recognized as primary penicillin resistance mechanisms in Streptococcus pneumoniae. Few studies have analyzed penicillin resistance at the genome scale, however, and we report the sequencing of S. pneumoniae R6 transformants generated while reconstructing the penicillin resistance phenotypes from three penicillin-resistant clinical isolates by serial genome transformation. The genome sequences of the three last-level transformants T2-18209, T5-1983, and T3-55938 revealed that 16.2 kb, 82.7 kb, and 137.2 kb of their genomes had been replaced with 5, 20, and 37 recombinant sequence segments derived from their respective parental clinical isolates, documenting the extent of DNA transformation between strains. A role in penicillin resistance was confirmed for some of the mutations identified in the transformants. Several multiple recombination events were also found to have happened at single loci coding for penicillin-binding proteins (PBPs) that increase resistance. Sequencing of the transformants with MICs for penicillin similar to those of the parent clinical strains confirmed the importance of mosaic PBP2x, -2b, and -1a as a driving force in penicillin resistance. A role in resistance for mosaic PBP2a was also observed for two of the resistant clinical isolates.

Genomic characterization of ciprofloxacin resistance in a laboratory-derived mutant and a clinical isolate of Streptococcus pneumoniae.

The broad-spectrum fluoroquinolone ciprofloxacin is a bactericidal antibiotic targeting DNA topoisomerase IV and DNA gyrase encoded by the parC and gyrA genes. Resistance to ciprofloxacin in Streptococcus pneumoniae mainly occurs through the acquisition of mutations in the quinolone resistance-determining region (QRDR) of the ParC and GyrA targets. A role in low-level ciprofloxacin resistance has also been attributed to efflux systems. To look into ciprofloxacin resistance at a genome-wide scale and to discover additional mutations implicated in resistance, we performed whole-genome sequencing of an S. pneumoniae isolate selected for resistance to ciprofloxacin in vitro (128 µg/ml) and of a clinical isolate displaying low-level ciprofloxacin resistance (2 µg/ml). Gene disruption and DNA transformation experiments with PCR fragments harboring the mutations identified in the in vitro S. pneumoniae mutant revealed that resistance is mainly due to QRDR mutations in parC and gyrA and to the overexpression of the ABC transporters PatA and PatB. In contrast, no QRDR mutations were identified in the genome of the S. pneumoniae clinical isolate with low-level resistance to ciprofloxacin. Assays performed in the presence of the efflux pump inhibitor reserpine suggested that resistance is likely mediated by efflux. Interestingly, the genome sequence of this clinical isolate also revealed mutations in the coding region of patA and patB that we implicated in resistance. Finally, a mutation in the NAD(P)H-dependent glycerol-3-phosphate dehydrogenase identified in the S. pneumoniae clinical strain was shown to protect against ciprofloxacin-mediated reactive oxygen species.

Genomic analysis and reconstruction of cefotaxime resistance in Streptococcus pneumoniae.

OBJECTIVES: To identify non-penicillin-binding protein (PBP) mutations contributing to resistance to the third-generation cephalosporin cefotaxime in Streptococcus pneumoniae at the genome-wide scale. METHODS: The genomes of two in vitro S. pneumoniae cefotaxime-resistant isolates and of two transformants serially transformed with the genomic DNA of cefotaxime-resistant mutants were determined by next-generation sequencing. A role in cefotaxime resistance for the mutations identified was confirmed by reconstructing resistance in a cefotaxime-susceptible background. RESULTS: Analysis of the genome assemblies revealed mutations in genes coding for the PBPs 2x, 2a and 3, of which pbp2x was the only mutated gene common to all mutants. The transformation of altered PBP alleles into S. pneumoniae R6 confirmed the role of PBP mutations in cefotaxime resistance, but these were not sufficient to fully explain the levels of resistance. Thirty-one additional genes were found to be mutated in at least one of the four sequenced genomes. Non-PBP resistance determinants appeared to be mostly lineage specific. Mutations in spr1333, spr0981, spr1704 and spr1098, encoding a peptidoglycan N-acetylglucosamine deacetylase, a glycosyltransferase, an ABC transporter and a sortase, respectively, were implicated in resistance by transformation experiments and allowed the reconstruction of the full level of resistance observed in the parent resistant strains. CONCLUSIONS: This whole-genome analysis coupled to functional studies has allowed the discovery of both known and novel cefotaxime resistance genes in S. pneumoniae.

T Cell Tolerance Following Bacterial Glutamic Acid Decarboxylase (GAD) Feeding in Streptozotocin-Induced Diabetes.

BACKGROUND: Autoimmune type 1 diabetes mellitus is caused by the T-cell mediated immune destruction of the insulin-producing beta-cell in pancreatic islets of Langerhans. The specificity of the auto-antibodies and of the auto-reactive T cells has been investigated, in which several auto-antigens were proposed. OBJECTIVE: The purpose of this study was to determine whether glutamic acid decarboxylase (GAD) feeding would induce oral tolerance of either the T cell or B cell compartment in streptozotocin (STZ) diabetic rats. METHODS: Experimental rats were fed 2 mg/kg of GAD (extracted from Escherichia coli) 14 days before being given intra-peritoneal injections of streptozotocin (STZ, 30 mg/kg body weight for 5 consecutive days). Of the two control groups, one was the diabetic control, which underwent STZ injections without being given the GAD; and the second was the normal control group. Systemic responses were compared between the three groups. T cell responses were assessed by a proliferation assay of spleen cells, and those of the B cell, by enzyme-linked immunosorbent assay ELISA for anti-GAD specific Abs in serum. RESULTS: Compared with the diabetic control group, a significant reduction was observed only in the proliferative response of spleen cells, but not in the level of anti-GAD antibody. CONCLUSION: We concluded that GAD feeding induces systemic T cell tolerance in STZ-induced diabetes.