Jundishapur Journal of Microbiology
Volume:9 Issue: 10, Oct 2016

Vulvovaginal candidiasis is defined as vulvovaginitis associated with vaginal carriage of Candida spp. and is a common problem with a high rate of morbidity.
To investigate the distribution of Candida spp. and evaluate the corresponding antifungal susceptibility in women with genital tract infection in Chongqing, southwestern China.
Patients and Samples (n = 2.129) were obtained from female patients with symptoms of genital tract infection. Candida spp. were isolated from the specimens and were identified using a coloration medium and the VITEK 2 Compact automatic microbial identification system. Antifungal susceptibility testing was performed using the ATB FUNGUS drug susceptibility testing system.
From 2,129 samples, 478 (22.45%) isolates of Candida were isolated, of which 395 (82.64%) were Candida albicans, 39 (8.16%) were C. glabrata, 21 (4.39%) were C. tropicalis, 9 (1.88%) were C. parapsilosis, and 14 (2.93%) were other Candida spp. The resistance of C. albicans, C. glabrata, and C. tropicalis to 5 antifungal drugs (amphotericin B, voriconazole, fluconazole, 5-fluorocytosine, and itraconazole) ranged from 0.5% to 6.4%, 0% to 7.7%, and 0% to 9.6%, respectively.
Candida albicans was the major pathogen associated with candidiasis of the female genital tract in patients in Chongqing. The results of the antifungal sensitivity of the isolates suggest that it is important for clinicians to administer appropriate antifungals for the treatment of Candida spp. infections.

One-third of the world’s population is infected with Mycobacterium tuberculosis. Investigation of Toll-like receptors (TLRs) has revealed new information regarding the immunopathogenesis of this disease. Toll-like receptors can recognize various ligands with a lipoprotein structure in the bacilli. Toll-like receptor 2 and TLR-4 have been identified in association with tuberculosis infection.
The aim of our study was to investigate the relationship between TLR polymorphism and infection progress.
Twenty-nine patients with a radiologically, microbiologically, and clinically proven active tuberculosis diagnosis were included in this 25-month study. Toll-like receptor 2 and TLR-4 polymorphisms and allele distributions were compared between these 29 patients and 100 healthy control subjects. Peripheral blood samples were taken from all patients. Genotyping of TLR-2, TLR-4, and macrophage migration inhibitory factor was performed. The extraction step was completed with a Qiagen mini blood purification system kit (Qiagen, Ontario, Canada) using a peripheral blood sample. The genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism.
In total, 19 of the 29 patients with tuberculosis infection had a TLR-2 polymorphism, and 20 of the 100 healthy subjects had a TLR-2 polymorphism (P Toll-like receptor 2 polymorphism is a risk factor for tuberculosis infection. The limiting factor in this study was the lack of investigation of the interferon-γ and tumor necrosis factor-α levels, which are important in the development of infection. Detection of lower levels of these cytokines in bronchoalveolar lavage specimens, especially among patients with TLR-2 defects, will provide new data that may support the results of this study.

Accurate and rapid detection of drug-resistant Mycobacterium tuberculosis is fundamental for the successful treatment of tuberculosis (TB).
The aim of this study was to determine the frequency of common mutations leading to isoniazid (INH) and rifampicin (RMP) resistance.
Patients and In a cross-sectional study carried out in 2014, 90 patients with M. tuberculosis from five border provinces of Iran were selected. After a full clinical history and physical evaluation, real-time polymerase chain reaction (PCR) technique was performed for the detection of mutations in the patients’ katG and rpoB genes. The results were compared with results of a standard proportion method as well as a multiplex allele-specific PCR (MAS-PCR).
A total of 23 mutations were found in isolates among which, codon katG 315, rpoB P1 (511 - 519 sequence) and rpoB P2 (524-533 sequence) were responsible for seven, nine and seven cases, respectively. The mean (standard deviation (SD)) of melting temperature (Tm) in katG 315 codon, rpoB P1 and P2 sequences in susceptible and mutant isolates was as follows: katG 85.4°C (0.18) and 87.54°C (0.62); rpoΒ P1 84.6°C (0.61) and 82.9°C (0.38); rpoΒ P2 83.4°C (0.18) and 85.3°C (0.19), respectively. In comparison to the standard proportion test, the sensitivity of real-time PCR in detecting INH- and RMP-resistant mutations was 75% and 83.3%, respectively. In comparison to the MAS-PCR test, 100% of katG 315 mutations and 80% of rpoB mutations were determined. Overall, 10% of the patients were diagnosed with a recurrence of TB. Age and previous history of TB treatment increased mutation odds in rpoB sequences (P = 0.046, P = 0.036, respectively).
Detection of drug resistance associated with mutations through real-time PCR by melting analysis technique showed a high differentiating power. This technique had high concordance with the standard proportion test and MAS-PCR results.

Quorum sensing is a microbial cell-to-cell communication process. Quorum sensing bacteria produce and release extracellular messenger molecules called autoinducers. Gram-positive and Gram-negative, homoserine lactones, and oligopeptides are autoinducers used to communicate and regulate gene expression.
The goal of this study was to assess the impact of subinhibitory concentrations of Ferula assa-foetida l oleo-gum resin and Carum copticum fruit on the expression of tst and hld genes of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains.
This analytical study was performed using standard strains of MRSA (ATCC 33591) and MSSA (ATCC 29213). Suspensions of MRSA and MSSA bacteria were incubated at 37°C for 7 and 16 hours in the presence of ethanol extracts from F. assa-foetida and C. copticum. The expression of the hld and tst genes was then assessed using the real-time PCR protocol and SYBR Green Master Mix. The data analysis was carried out using the 2-ΔΔCT method.
The hld gene expression (RNAIII) of MRSA after 7 and 16 hours of exposure to the sMIC of the F. assa-foetida extract showed a fold change of -1 and 0.08, respectively, in comparison with controls. After 7 and 16 hours of exposure to the sMIC of the C. copticum extract, the fold change was -0.23 and -0.27, respectively. After exposure to the sMIC of the C. copticum extract for 16 hours, the fold change in the expression of the tst (TSST-1) MSSA gene was 0.37 lower than that of the control sample.
The results indicate that sMICs of ethanol extracts from F. assa-foetida and C. copticum can be used to control the expression of virulence genes in pathogenic bacteria, such as MRSA and MSSA.

The resistance of aminoglycosides in strains that produce beta-lactamase can be developed through the multidrug resistant encoding genes carried by common plasmids. Recently, the association between 16S rRNA methyltransferase resistance and beta-lactamase enzymes carried by the same plasmids has drawn increased attention from researchers, particularly the association in aminoglycoside-resistant strains with a minimum inhibitory concentration (MIC) of ≥ 256 µg/mL.
We aimed to investigate the co-existence of 16S rRNA methyltransferase and beta-lactamase genes in multidrug resistant (MDR) Klebsiella pneumoniae strains isolated from clinical samples.
We determined the molecular mechanisms of aminoglycoside resistance and its relationship with resistance to carbapenem and beta-lactam group antibiotics in 40 extended-spectrum beta-lactamase (ESBL)-positive carbapenem- and aminoglycoside-resistant K. pneumoniae strains. Multidrug resistant K. pneumoniae was isolated from various clinical samples in the faculty of medicine of Cukurova University, Turkey. First, the resistance of aminoglycoside and beta-lactam antibiotics was phenotypically investigated using the Kirby-Bauer disk diffusion test, double disk synergy test, and modified Hodge test. The MIC values of aminoglycoside were determined using the agar dilution method. Polymerase chain reaction was performed to detect the carbapenemases, ESBL, and 16S rRNA methyltransferase genes. The results were confirmed by a sequence analysis.
Twenty K. pneumoniae strains showed resistance to amikacin, and 40 were resistant to gentamicin. The MIC value was found to be > 512 µg/mL in five amikacin-resistant strains and > 128 µg/mL in 10 gentamicin-resistant isolates. The rmtC gene, a type of 16S rRNA methyltransferase, was amplified in four isolates (MIC amikacin: > 512 µg/mL, gentamicin: > 128 µg/mL). Of these four isolates, three had the blaNDM-1 gene and all contained at least one ESBL gene.
This study demonstrated the co-existence of rmtC and blaNDM-1 genes for the first time in Turkey. The spread of this resistant type should be monitored and limited through molecular surveillance.

Against a variety of antimicrobial resistant pathogens, the scientists attempted substitution of antimicrobial medicine with various nanoparticles and plant-based antibacterial substances.
The aim of this study was to assess the antibacterial effects of silver nanoparticles solely and in combination with Zataria multiflora essential oil and methanolic extract on some photogenic bacteria.
Minimum inhibitory concentrations (MICs) and fractional inhibitory concentrations (FICs) of plant essential oil, methanolic extract, and silver nanoparticles against bacteria were evaluated using the broth microdilution method and check board microtiter assays.
The results of the experiment showed that the MIC and minimal bacterial concentration (MBC) values of Ag-NPs against all strains were in the range of 15.625 - 500 µg/mL, and values for the essential oil and plant extract were in the range of 1.56 - 100 mg/mL.
Silver nanoparticles were observed to have additive effects with essential oil against Staphylococcus epidermidis and S. aureus. The obtained results suggest the need for further investigations of the antibacterial effects of the combination of silver nanoparticles with other plant extracts and essential oils.

In recent years, antibiotic resistance has been indicated as a paramount threat to public health. The use of bacteriophages appears to be a safer alternative for the control of bacterial infections.
The present study aims to explore sewage water for the presence of indigenous bacteriophages, and to investigate their antibacterial potential against Methicillin-resistant Staphylococcus aureus (MRSA).
Bacterial isolates were first collected and identified from pus samples taken from the surgical and burn units using standard microbiological procedures. A cefoxitin disk screen test was then used and interpreted according to the clinical laboratory standards institute (CLSI) guidelines for the detection of MRSA. The sewage samples were processed and the phages enriched using S. aureus as a host organism. Turbid and clear plaques of different sizes were isolated using an overlay method, purified, and then enumerated by means of a dilution method.
The phages exhibited good lytic activity against MRSA when tested in-vitro, and the highest activity was attained within three to six hours of phage infection. The isolated phage pq/48 was also found efficient in decreasing the bacterial count during an in-vivo trial in rabbits. A protein analysis using SDS-PAGE revealed 10 proteins of between 20 kDa and 155 kDa in size.
The overall results indicated that bacteriophages isolated from sewage exhibited excellent lytic activity against MRSA strains. In conclusion, bacteriophages can be further characterized and appear to be a promising candidate for phage therapy against MRSA in the future.

Outer membrane protein D (PD) is a highly conserved and stable protein in the outer membrane of both encapsulated (typeable) and non-capsulated (non-typeable) strains of Haemophilus influenzae. As an immunogen, PD is a potential candidate vaccine against non-typeable H. influenzae (NTHi) strains.
The aim of this study was to determine the cytokine pattern and the opsonic antibody response in a BALB/c mouse model versus PD from NTHi as a vaccine candidate.
Protein D was formulated with Freund’s and outer membrane vesicle (OMV) adjuvants and injected into experimental mice. Sera from all groups were collected. The bioactivity of the anti-PD antibody was determined by opsonophagocytic killing test. To evaluate the cytokine responses, the spleens were assembled, suspension of splenocytes was recalled with antigen, and culture supernatants were analyzed by ELISA for IL-4, IL-10, and IFN-γ cytokines.
Anti-PD antibodies promoted phagocytosis of NTHi in both immunized mice groups (those administered PD Freund’s and those administered PD OMV adjuvants, 92.8% and 83.5%, respectively, compared to the control group). In addition, the concentrations of three cytokines were increased markedly in immunized mice.
We conclude that immunization with PD protects mice against NTHi. It is associated with improvements in both cellular and humoral immune responses and opsonic antibody activity.

Toxoplasma gondii is one of the most common causes of latent infections in humans worldwide. Detecting anti-Toxoplasma antibodies in serum using serological tests is a common method to diagnose toxoplasmosis.
In the present study, an indigenous ELISA kit was prepared using tachyzoites from the RH strain of T. gondii, and its sensitivity and specificity were compared with those of commercial kits.
To produce antigens, 0.02 mL of locally isolated T. gondii RH strain parasites along with 109 tachyzoites were injected into the peritoneal cavities of 50 laboratory mice (BALB/C). Parasites were collected after 4 days. After filtering and washing, the concentration of protein in sonicated tachyzoites was calculated using the Lowry protein assay. The dilution of antigen, serum and alkaline phosphatase conjugate was assessed in designing an indigenous ELISA method; then ELISA was performed based on these dilutions, and its sensitivity was determined using 200 serum samples. In addition, the specificity of the assay was evaluated using 40 serum samples from patients with tuberculosis, leukemia or hydatid cyst.
Indigenous ELISA was used to examine 100 serum samples containing anti-T. gondii IgG, with a sensitivity of 98% (commercial kits: 100%). Another 100 serum samples containing anti-T. gondii IgM were also tested, with a sensitivity of 99% (commercial kits: 100%). When 40 serum samples from patients with leukemia, hydatid cyst or tuberculosis were examined using anti-T. gondii IgG, the specificity was 100%, identical to commercial kits. However, the specificity of a similar test with anti-T. gondii IgM was just 28.6% for serum samples from leukemia patients, 21.4% for hydatid cyst and 16.7% for tuberculosis.
We found that purified locally isolated soluble crude antigens of the RH strain of T. gondii from the peritoneal cavity of mice may be one of the most promising antigens for detection of human toxoplasmosis in routine screening.

Antibiotic resistance among Staphylococcus aureus is of great concern worldwide. This resistance is further complicated by the ability of S. aureus to confer cross-resistance to other antibiotics due to the presence of resistance genes, such as erythromycin resistance methylase (erm) genes, which render the bacterium resistant to macrolide-lincosamide-streptogramin B (MLSB) antibiotics. Resistance to these antibiotics can lead to therapeutic failure, resulting in significant morbidity and mortality in patients with S. aureus infections.
This study was performed to examine the distribution of MLSB-resistant strains of methicillin-susceptible S. aureus (MSSA), which were obtained from hospitalized patients and normal healthy individuals (carriers) using phenotypic methods, such as the double-disk diffusion (D-test) and the genotypic method by polymerase chain reaction (PCR).
A total of 183 nonduplicative MSSA isolates obtained from hospitalized patients (133) and carriers (50) in our previous studies were randomly selected for the D-test. The guidelines of the Clinical and Laboratory Standards Institute (CLSI) were used for the interpretation of the results of this test. The detection of ermA, ermB, ermC and msrA genes by PCR was performed for isolates that had positive D-test results and that were resistant to erythromycin.
Of the 183 MSSA isolates, 97.2% and 98.4% were highly susceptible to erythromycin and clindamycin, respectively. MSLB resistance was detected in four isolates (2.2%). Of the 133 MSSA isolated from hospitalized patients, only 3.0% (4/133) and 2.3% (3/133) exhibited resistance to erythromycin and clindamycin, respectively. With regard to the MLSB resistance phenotypes, only 1.6% and 0.6% exhibited inducible MLSB (iMLSB) and MS phenotypes, respectively. The ermC gene was detected in all three iMLSB phenotypes, and the msrA gene was detected in the MS phenotype. Surprisingly, all MSSA isolates (100%) from carriers exhibited extremely high susceptibility to both antibiotics.
The prevalence rates of iMLSB MSSA isolates vary according to geographical locations and the local antibiotic policy. The low prevalence rate of iMLSB MSSA isolates could probably be related to the judicious use of antibiotics for treating S. aureus infections in our studied population. Nonetheless, continuous antibiotic surveillance is still necessary to control any emergence of resistance isolates so that targeted therapy and effective control can be implemented accordingly.

Due to the overuse of antibiotics in livestock as a growth-promoting agent, the emergence of multi-antibiotic resistant bacteria is becoming a concern.
In this study, we aimed to detect the presence and discover the molecular determinants of foodborne bacteria in retail sausages resistant towards the antibacterial agent amoxicillin-clavulanate.
Two grams of sausages were chopped into small pieces and transferred into sterile Luria-Bertani (LB) enrichment broths overnight before they were plated on MacConkey agar petri dishes. The bacteria isolated were then screened for amoxicillin-clavulanate resistance, and an antimicrobial susceptibility test of each isolate was performed by using the disc diffusion method. Double synergy and phenotypic tests were carried out to detect the presence of extended spectrum β-lactamase (ESBL). API 20E kit was used to identify the Enterobacteriaceae. All isolates were further examined by polymerase chain reaction (PCR) for resistant genes blaOXA-1, blaOXA-10, plasmid-mediated AmpC (blaCMY and blaDHA), and the chromosome-mediated AmpC, Sul1, blaTEM, and blaSHV genes.
A total of 18 amoxicillin-clavulanate resistant isolates were obtained from seven different types of retail sausages. Only half of them were identified as Enterobacteriaceae, but none were ESBL-producers. All the 18 isolated strains demonstrated resistance towards amoxicillin-clavulanate, penicillin and oxacillin (100%), cefotaxime (71.4%), cefpodoxime (66.7%), and ampicillin (83.3%). blaTEM was the most frequently detected β-lactamase gene. Both plasmid- and chromosomal-bound blaTEM genes were detected in all of the isolated Enterobacteriaceae. blaSHV and Sul1 accounted for 22.2% and 11.1% of the amoxicillin-clavulanate resistant isolates, respectively, whereas blaAMPC, blaCMY, blaDHA, blaOXA-1, and blaOXA-10 were not found in any of the isolates. The only one ESBL-producing bacteria detected in this study was Chryseobacterium meningosepticum, which harbored the blaTEM gene.
The multidrug resistant bacteria that carry antibiotic resistant genes from retail sausages may increase the risk of transmission to humans via the consumption of contaminated sausages. Stricter measures must be taken to address the use of antibiotics in animal agriculture and to consider their potential impact on human health..

Campylobacter jejuni is one of the major causes of infectious diarrhea worldwide. The distending cytolethal toxin (CDT) of Campylobacter spp. interferes with normal cell cycle progression. This toxic effect is considered a result of DNase activity that produces chromosomal DNA damage. To perform this event, the toxin must be endocytosed and translocated to the nucleus.
The aim of this study was to evaluate the role of the cytoskeleton in the translocation of CDT to the nucleus.
Campylobacter jejuni ATCC 33291 and seven isolates donated from Instituto de Biotecnologia were used in this study. The presence of CDT genes in C. jejuni strains was determined by PCR. To evaluate the effect of CDT, HeLa cells were treated with bacterial lysate, and the damage and morphological changes were analyzed by microscopy, immunofluorescence staining, and flow cytometry. To evaluate the role of the cytoskeleton, HeLa cells were treated with either latrunculin A or by nocodazole and analyzed by microscopy, flow cytometry, and immunoquantification (ELISA).
The results obtained showed that the eight strains of C. jejuni, including the reference strain, had the ability to produce the toxin. Usage of latrunculin A and nocodazole, two cytoskeletal inhibitors, blocked the toxic effect in cells treated with the toxin. This phenomenon was evident in flow cytometry analysis and immunoquantification of Cdc2-phosphorylated.
This work showed that the cytotoxic activity of the C. jejuni CDT is dependent on its endocytosis. The alteration in the microtubules and actin filaments caused a blockage transit of the toxin, preventing it from reaching the nucleus of the cell, as well as preventing DNA fragmentation and alteration of the cell cycle. The CDT toxin appears to be an important element for the pathogenesis of campylobacteriosis, since all clinical isolates showed the presence of cdtA, cdtB and cdtC genes.