Antibiotic resistance, virulence genes, and phylogenetic groups of bacteria isolated from wild passerine birds in Iran.

Wild passerine birds may serve as environmental reservoirs and as vectors for the long-distance dispersal of microorganisms and resistance determinants. However, there is no much knowledge on pathogenic bacteria in wild birds in Iran. The present study aimed to analyze antibiotic resistance in wild passerine birds collected from the northeast region of Iran as the rich breeding bird fauna with a special focus on Escherichia coli virulence, integron, and phylogenetic groups. A total of 326 isolates were collected and identified from the cloaca of wild birds using a swab. The results showed a high percentage of resistance to tetracycline (45.8%) and ampicillin (26.7%). The resistance genes, tet(A), tet(B), tet(M), and tet(L) were detected in tetracycline-resistant isolates, while the blaTEM gene was the most prevalent in ampicillin-resistant isolates (38.6%). Out of the 129 E. coli isolates examined, 99 isolates were found to have virulence gene, with the highest prevalence of the fimbriae (fimH) gene (22.4%). Additionally, the E. coli strains were most often classified into phylogenetic groups B1 (48.8%) followed by B2 (19.3%). Also, the highest average frequency of class 1 integron was detected among our isolates. Results indicated that wild birds are reservoirs of multidrug resistance and virulence genes that may have the potential to be transferred to other organisms, including humans.

Encapsulation of Lactiplantibacillus plantarum probiotics through cross-linked chitosan and casein for improving their viability, antioxidant and detoxification.

In the present study, encapsulation of Lactiplantibacillus plantarum (L.p) was performed using chitosan and casein through calcium phosphate intercrossing. Chitosan and casein both considered as non-toxic and biocompatible food derived components with intrinsic antioxidant properties. Layer by layer strategy was performed for deposition of modified cross-linked chitosan along with casein as the novel protective layers on the surface of probiotics. After confirmation of successful encapsulation, the viability and antioxidant activity of encapsulated L.p was evaluated. The results showed enhanced survival and antioxidant activity of encapsulated L.p compared to free bacteria in simulated digestive conditions. The survival of free and encapsulated L.p was respectively 1.38 ±â€¯0.29 log cfu/ml and 6.99 ±â€¯0.12 log cfu/ml in SGF and 8.54 ±â€¯0.05 log cfu/ml and 7.25 ±â€¯0.23 log cfu/ml in SIJ after 2 h of incubation. HPLC analysis was also used to investigate the detoxification activity of probiotics toward Aflatoxin M1 and obtained results showed encapsulated bacteria could significantly reduce aflatoxin M1 (68.44 ±â€¯0.5 %) compared to free bacteria (43.76 ±â€¯0.54 %). The results of this research suggest that the chitosan/casein mediated encapsulation of L.p with layer-by-layer technology is an effective method to improve the survival and antioxidant properties of probiotics with enhanced detoxification of AFM1.

Role of host immunity and HBx among inactive chronic hepatitis B patients in a highly endemic region.

The hepatitis B virus (HBV) infection has a wide range, from fulminant hepatitis to inactive chronic hepatitis B (ICB) infection. The present study evaluated critical factors in the outcomes of HBV infection in a highly endemic region of Iran (approximately 12% HBV positive). The expression of seven genes involved in host immunity (Foxp3, T-bet, ROR-γt, AKT, CREB, IL-28/or IFN-λ2, and IL-28R) and HBx for viral activities were evaluated using real-time PCR, TaqMan method. A total of 58 subjects were randomly chosen, including 28 ICB and 30 healthy controls (HCs) from the Esfandiar district, South Khorasan province, Iran. The expression index of Foxp3 and ROR-γt was moderately up-regulated in ICBs but did not statistically significant. T-bet expression in ICB patients was significantly higher than in HCs (p = 0.004). Furthermore, evaluating two signalling pathways in Th activation and cell survival showed that the CREB pathway was significantly up-regulated in ICB patients compared to HCs (p = 0.006), but the AKT did not differ. In innate immune responses, the IL-28/or IFN-λ2 expression in ICB patients was significantly higher than in the HCs (p = 0.02). Surprisingly, only one ICB patient disclosed HBx expression, which shows deficient virus activity in these patients. The ICB condition seems to result from host immune pressure on HBV activities, up-regulation of T-bet and IFN-λ. The high expression of CREB may prevent Kupffer's pro-inflammatory reactions in the liver. Whereas the absence of HBx expression in ICB patients and, consequently, the inactivity of HBV may also confirm such immune pressure.

Antibiotic resistance, phylogenetic typing, and virulence genes profile analysis of uropathogenic Escherichia coli isolated from patients in southern Iraq.

Of the most common infectious diseases that occur mainly by uropathogenic Escherichia coli (UPEC) is urinary tract infections (UTIs). The purpose of this study was to investigate virulence factors, antibiotic resistance, and phylogenetic groups among UPEC strains isolated from patients with UTI in southern Iraq. A total of 100 UPEC isolates were collected from urine samples of UTI patients from various hospitals in southern Iraq, and confirmed by morphological and biochemical tests. Antimicrobial susceptibility testing on isolates was performed by disk diffusion method. Multiplex PCR techniques were used to evaluate the phylogenetic groups based on Clermont method and to detect the presence of six virulence factor genes. The majority of isolates belonged to the phylogenetic groups B2 (46%) and C (13%). The most prevalent virulence factors were fimH (96%), followed by aer (47%), papC (36%), cnf1 (17%), hly (15%), and afa (8%). Phenotypic testing showed that the isolates were most resistant to piperacillin, ticarcillin, amoxicillin/clavulanic acid (92%, 91%, and 88%, respectively) and most sensitive to amikacin and imipenem, respectively. The maximum antibiotic resistance and virulence factors were observed in the phylogenetic group B2. The results showed that the UPEC isolates had all six virulence factors with high frequency and the highest drug resistance. Besides, the results showed a direct relationship between virulence factors, gene diversity, phylogenetic background, and antimicrobial resistance in the UPEC isolates.

Antibacterial and Synergistic Effects of Herbal Extracts in Combination with Amikacin and Imipenem Against Multidrug-Resistant Isolates of Acinetobacter.

Acinetobacter species are defined as multidrug-resistant pathogens and the development of resistance against antimicrobials is a major problem in the treatment of infections caused by them. This study aimed to evaluate the antibacterial activity of aqueous and methanol extracts of Salvia chorassanica and Artemisia khorassanica on multidrug-resistant Acinetobacter isolates and also examining the interaction of the methanol extract of the plants with the combination of amikacin and imipenem. First, the presence of adeI and adeB genes in bacterial isolates was investigated. The aqueous and methanol extracts of the leaves of the plants were prepared by Maceration method. Minimum Inhibition Concentration (MIC) values were determined to evaluate the antibacterial activities of plant extracts and antibiotics. Combined effects of the antibiotics with plant extracts were performed using the checkerboard method. The accumulation assay was used to examine the inhibitory effects of plant extracts on the bacterial efflux pump. MIC results indicated that the methanol extracts were effective against Acinetobacter species. FICI values indicated that the combination of antibiotics with methanol plant extracts improves bacterial sensitivity to antibiotics. The extracts also exhibited efflux pump inhibitory activities. Consequently, combination of the plant extracts with antibiotics could enhance the antibiotic susceptibility of resistant pathogens.

Optical Properties of Methyl Orange-Doped Droplet and Photodynamic Therapy of Staphylococcus aureus.

Dye-doped droplets are known as mixtures of dyes with uniform solutions of water droplets in a continuous phase of oils with surfactants. To observe the relationship between water droplet structures and surfactant types on optical properties of dyes, a mixture of methyl orange (MO)-doped droplet prepared with benzane and hexane as oils and sodium bis(2-ethylhexyl) sulfosuccinate (AOT) as a surfactant was thus examined using Z-scan instrument, spectrophotometer, and fluorimeter in the present study. The findings revealed that nonlinear refractive (NLR) index, nonlinear absorption (NLA) coefficient, as well as fluorescence intensity of the MO had enhanced following a reduction in the droplet water content induced by changes in the non-centrosymmetric charge density distribution of this pH indicator. Moreover, the MO-doped droplet in a continuous phase of benzene investigated by 1H nuclear magnetic resonance (NMR) spectroscopy indicated that the MO had been located in the droplet in the vicinity of the hydrophilic part of the surfactant. Furthermore, the MO-doped droplets along with laser radiation were employed to perform antibacterial photodynamic therapy (APDT) of Staphylococcus aureus (S. aureus). It was ultimately concluded that the bacteria colony had also extremely diminished in the group treated by the MO-doped droplet.

Determination of imipenem efflux-mediated resistance in Acinetobacter spp., using an efflux pump inhibitor.

BACKGROUND AND OBJECTIVES: In recent years, reports of Acinetobacter strains resistant to all known antibiotics have caused a great concern in medical communities. Overexpression of efflux pumps is one of the major causes of resistance in bacteria. The aim of this study was to investigate the role of efflux pumps in conferring resistance to imipenem in clinically important Acinetobacter spp; Acinetobacter baumannii and Acinetobacter lwoffii. MATERIALS AND METHODS: A total number of 46 clinical Acinetobacter isolates, including 33 A. baumannii and 13 A. lwoffii isolates, previously collected from Shahid Kamyab and Ghaem hospitals of Mashhad, Iran were used in this study. Imipenem susceptibility testing was carried out by the disc diffusion method. Imipenem minimum inhibitory concentration (MIC) for resistant Acinetobacter isolates were determined both in the presence and absence of the efflux pumps inhibitor, carbonyl cyanide 3-chlorophenylhydrazone (CCCP). RESULTS: Resistance to imipenem was observed in 38 isolates including 30 A. baumannii and 8 A. lwoffii isolates. Experiments in the presence of CCCP showed a 2 to 16384 fold reduction in imipenem MICs in 14 A. baumannii and 2 A. lwoffii isolates. CONCLUSION: The results obtained showed high levels of resistance to imipenem and contribution of efflux pumps in conferring resistance in both Acinetobacter species in this study. Moreover, imipenem efflux mediated resistance highlights the importance of this mechanism not only in A. baumannii but also in non-baumannii Acinetobacter Spp. which have been neglected in antibiotic resistance studies.

Thiol-Capped Gold Nanoparticle Biosensors for Rapid and Sensitive Visual Colorimetric Detection of Klebsiella pneumoniae.

In the last few years, gold nanoparticle biosensors have been developed for rapid, precise, easy and inexpensive with high specificity and sensitivity detection of human, plant and animal pathogens. Klebsiella pneumoniae serotype K2 is one of the common gram-negative pathogens with high prevalence. Therefore, it is essential to provide the effective and exclusive method to detect the bacteria. Klebsiella pneumoniae serotype K2 strain ATCC9997 genomic DNA was applied to establish the detection protocol either with thiol-capped oligonucleotide probes and gold nanoparticles or polymerase chain reaction based on K2A gene sequence. In the presence of the genomic DNA and oligonucleotide probes, a change in the color of gold nanoparticles and maximum changes in wavelength at 550-650 nm was achieved. In addition, the result showed specificity of 15 × 105 CFU/mL and 9 pg/µL by gold nanoparticles probes. The lower limit of detection obtained by PCR method was 1 pg/µL. Moreover, results demonstrated a great specificity of the designed primers and probes for colorimetric detection assay and PCR. Colorimetric detection using gold nanoparticle probe with advantages such as the lower time required for detection and no need for expensive detection instrumentation compared to the biochemical and molecular methods could be introduced for rapid, accurate detection of the bacteria.

A rapid method for separating and concentration of food-borne pathogens using elution from ready-to-eat vegetables.

BACKGROUND AND OBJECTIVES: Traditional culture methods for detection of food-borne pathogens, a major public health problem, are simple, easily adaptable and very practical, but they can be laborious and time consuming. In this study, we eliminated culturing steps by developing a new separation method and therefore, decreased the detection time of food-borne pathogens (Salmonella enterica serovar Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes) to a few hours. MATERIALS AND METHODS: We used alkaline water and different alkaline buffers to elute bacteria from the lettuce surface as a model for ready-to-eat vegetables. Buffers used were as follows: 1) 0.05 M glycine; 2) 0.05 M glycine -100 mM Tris base -1% (w/v) beef extract; 3) buffer peptone water; 4) buffer phosphate saline. Buffers were adjusted to pH of 9, 9.5 and 10. In order to elute the bacteria, the lettuce pieces were suspended into buffers and shacked for 30, 45 and 60 min. Moreover, a multiplex PCR method for the simultaneous detection of food-borne pathogens was performed. RESULTS: The results showed that buffer peptone water at pH 9.5 for 45 min have high ability to elute bacteria from the lettuce surface and the bacteria can be detected using multiplex PCR. CONCLUSION: We developed a new rapid and efficient method for simultaneous separation of food-borne pathogens. This method eliminates culturing stages and permits the detection and identification of target pathogens in a few hours.

Membrane organization and cell fusion during mating in fission yeast requires multipass membrane protein Prm1.

The involvement of Schizosaccharomyces pombe prm1(+) in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell-cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1(+) and the dni(+) genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell-cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell-cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion.

Regulation of cell wall synthesis by the clathrin light chain is essential for viability in Schizosaccharomyces pombe.

The regulation of cell wall synthesis by the clathrin light chain has been addressed. Schizosaccharomyces pombe clc1Δ mutant was inviable in the absence of osmotic stabilization; when grown in sorbitol-supplemented medium clc1Δ cells grew slowly, formed aggregates, and had strong defects in morphology. Additionally, clc1Δ cells exhibited an altered cell wall composition. A mutant that allowed modulating the amount of Clc1p was created to analyze in more detail the dependence of cell wall synthesis on clathrin. A 40% reduction in the amount of Clc1p did not affect acid phosphatase secretion and bulk lipid internalization. Under these conditions, ß(1,3)glucan synthase activity and cell wall synthesis were reduced. Also, the delivery of glucan synthases to the cell surface, and the secretion of the Eng1p glucanase were defective. These results suggest that the defects in the cell wall observed in the conditional mutant were due to a defective secretion of enzymes involved in the synthesis/remodelling of this structure, rather than to their endocytosis. Our results show that a reduction in the amount of clathrin that has minor effects on general vesicle trafficking has a strong impact on cell wall synthesis, and suggest that this is the reason for the lethality of clc1Δ cells in the absence of osmotic stabilization.

The integrity of the cytokinesis machinery under stress conditions requires the glucan synthase Bgs1p and its regulator Cfh3p.

In yeast, cytokinesis requires coordination between nuclear division, acto-myosin ring contraction, and septum synthesis. We studied the role of the Schizosaccharomyces pombe Bgs1p and Cfh3p proteins during cytokinesis under stress conditions. Cfh3p formed a ring in the septal area that contracted during mitosis; Cfh3p colocalized and co-immunoprecipitated with Cdc15p, showing that Cfh3p interacted with the contractile acto-myosin ring. In a wild-type strain, a significant number of contractile rings collapsed under stress conditions and this number increased dramatically in the cfh3Δ, bgs1cps1-191, and cfh3Δ bgs1/cps1-191. Our results show that after osmotic shock Cfh3p is essential for the stability of the (1,3) glucan synthase Bgs1p in the septal area, but not at the cell poles. Finally, cells adapted to stress; they repaired their contractile rings and re-localized Bgs1p to the cell surface some time after osmotic shock. A detailed analysis of the cytokinesis machinery in the presence of KCl revealed that the actomyosin ring collapsed before Bgs1p was internalized, and that it was repaired before Bgs1p re-localized to the cell surface. In the cfh3Δ, bgs1/cps1-191, and cfh3Δ bgs1/cps1-191 mutants, which have reduced glucan synthesis, the damage produced to the ring had stronger consequences, suggesting that an intact primary septum contributes to ring stability. The results show that the contractile actomyosin ring is very sensitive to stress, and that cells have efficient mechanisms to remedy the damage produced in this structure.

The FN3 and BRCT motifs in the exomer component Chs5p define a conserved module that is necessary and sufficient for its function.

Chs5p is a component of the exomer, a coat complex required to transport the chitin synthase Chs3p from the trans-Golgi network to the plasma membrane. The Chs5p N-terminal region exhibits fibronectin type III (FN3) and BRCT domains. FN3 domains are present in proteins that mediate adhesion processes, whereas BRCT domains are involved in DNA repair. Several fungi--including Schizosaccharomyces pombe, which has no detectable amounts of chitin--have proteins similar to Chs5p. Here we show that the FN3 and BRCT motifs in Chs5p behave as a module that is necessary and sufficient for Chs5p localization and for cargo delivery. The N-terminal regions of S. cerevisiae Chs5p and S. pombe Cfr1p are interchangeable in terms of Golgi localization, but not in terms of exomer assembly, showing that the conserved function of this module is protein retention in this organelle and that the interaction between the exomer components is organism-specific.

Different steps of sexual development are differentially regulated by the Sec8p and Exo70p exocyst subunits.

In this paper we show that in Schizosaccharomyces pombe, mating-specific cell adhesion is dependent on the exocyst subunit Sec8p, but independent of the exocyst subunit Exo70p. In the absence of Exo70p, the forespore membrane does not develop properly and the leading edge protein Meu14p is abnormally distributed. Additionally, the spindle pole body is aberrant in a significant number of exo70Delta asci. In both the sec8-1 and the exo70Delta mutants, the development of the spore cell wall is impaired. These results show that different steps of sexual development are differentially regulated by the exocyst and suggest the existence of exocyst subcomplexes with distinct roles in mating.

The fission yeast SEL1 domain protein Cfh3p: a novel regulator of the glucan synthase Bgs1p whose function is more relevant under stress conditions.

In Schizosaccharomyces pombe, Bgs1/Cps1p is a beta(1,3)-glucan synthase required for linear beta(1,3)-glucan synthesis and primary septum formation. Here, we have studied the regulation of Bgs1p by Cfh3/Chr4p, a member of a family of conserved adaptor proteins, which resembles the chitin synthase regulator Chs4p from Saccharomyces cerevisiae and Candida albicans. cfh3Delta cells showed a genetic interaction with cps1-191, and Cfh3p co-immunoprecipitated with Bgs1/Cps1p. In the absence of cfh3(+), cells were more sensitive to digestion by glucanases, and both Calcofluor staining and glucan synthesis were reduced. We found that in a wild-type strain, beta(1,3)-glucan synthesis was reduced under stress conditions. In the cfh3Delta, cps1-191, and cfh3Delta cps1-191 strains, beta(1,3)-glucan synthesis was further reduced, and growth was impaired under stress conditions, suggesting that Cfh3p and Bgs1p might play a role in ensuring growth in unfavorable environments. In a cfh3Delta mutant, Bgs1p was delocalized when the cells were distressed, but a blockade in endocytosis prevented this delocalization. Finally, we found that the SEL1 repeats are required for Cfh3p function. These results show that Cfh3p is a regulatory protein for Bgs1p and that its function is particularly necessary when the cells are undergoing stress.

The Schizosaccharomyces pombe Map4 adhesin is a glycoprotein that can be extracted from the cell wall with alkali but not with beta-glucanases and requires the C-terminal DIPSY domain for function.

SUMMARY: In fungi, cell adhesion is required for flocculation, mating and virulence, and it is mediated by covalently bound cell wall proteins termed adhesins. Map4, an adhesin required for mating in Schizosaccharomyces pombe, is N-glycosylated and O-glycosylated, and is an endogenous substrate for the mannosyl transferase Oma4p. Map4 has a modular structure with an N-terminal signal peptide, a serine and threonine (S/T)-rich domain that includes nine repeats of 36 amino acids (rich in serine and threonine residues, but lacking glutamines), and a C-terminal DIPSY domain with no glycosylphosphatidyl inositol (GPI)-anchor signal. Map4 can be extracted from cell walls with SDS/mercaptoethanol sample buffer or with mild alkali solutions. After extensive extraction with hot sample buffer, no more protein can be released by beta-glucanases or alkali. Additionally, none of the cysteine residues of the protein is required for its retention at the cell wall. These results show that Map4 is not directly bound to beta-glucans and point to the existence of alkali- and SDS/mercaptoethanol-sensitive linkages between cell wall proteins. The N-terminal S/T-rich regions are required for cell wall attachment, but the C-terminal DIPSY domain is required for agglutination and mating in liquid and solid media.

The fission yeast Map4 protein is a novel adhesin required for mating.

Cell adhesion is required for many cellular processes. In fungi, cell-cell contact during mating, flocculation or virulence is mediated by adhesins, which typically are glycosyl phosphatidyl inositol (GPI)-modified cell wall glycoproteins. Proteins with internal repeats (PIR) are surface proteins involved in the response to stress. In Schizosaccharomyces pombe no adhesins or PIR proteins have been described. Here we study the S. pombe Map4p, which defines a new class of surface protein that is not GPI-modified and has a serine/threonine rich domain and internal repeats that differ from those present in PIR proteins. Map4p is a mating type-specific adhesin required for mating in h(+) cells and enhances cell adhesion when overexpressed.