تقویت تولید فیتاز از یک سوش جدید پروبیوتیک Bacillus Subtilis P6

Enhancement of Phytase Production from a New Probiotic Strain Bacillus subtilis P6

Abstract

An integrated classical and statistical optimization approach involving the combination of Placket–Burman design (PBD) and Central Composite Design (CCD) was employed for increasing phytase yield. PBD was used to evaluate the effect of 9 variables related to phytase production from probiotic strain B. subtilis, and three statistically significant variables, namely, glucose, beef extract and potassium phosphate were selected for further optimization studies. The levels of five variables for maximum phytase production were determined by a CCD. The optimum values for the factors were determined via response surface methodology (RSM) as: 6.59 gl-1 of glucose, 6 gl-1 of beef extract and 2.75 gl-1 of potassium phosphate respectively. Phytase production improved from 2.74 EUml-1 to 46.76 EUml-1 indicating 17-fold increase in activity after optimization.

1 Introduction

The growing awareness of the diet and health has prompted an increasing demand of food products that can support health apart from providing basic nutrition (Haros et al., 2009). Since last six decades, a lot of antibiotics have been incorporated in animal feed to improve their growth, efficiency and to protect them from pathogenic microorganisms.

But, antibiotic resistance has become a major public health concern today (Sharma et al., 2014). The gastrointestinal microflora plays beneficial role in the health and nutrition of animals, and probiotics, live microorganisms help in the maintenance of gut microflora. Probiotics have been strongly recommended as alternatives to antibiotics for food animals (Reid and Friendship, 2002). Recently, a great deal of attention has been devoted to the genuine value of bacterial species as multifunctional probiotics, which secrete various extracellular enzymes for enhancing feed digestibility as well as many antimicrobial compounds for improving animal performance (Lee et al., 2012); among them phytase holds the key position. Phytate is a natural phosphate reservoir in plant based animal feed and acts as an anti-nutritional factor in gut of animals and humans by restricting absorption of proteins, carbohydrates, amino acids and metals viz. Zn2+, Fe3+, Ca2+ and Mg2+ . However, in order to meet the phosphorus requirement, diet of monogastric animals are supplemented with inorganic phosphorus which causes the excretion of large amount of phosphorus in the excreta resulting in environmental pollution and several human health problems (Jorquera et al., 2011). Phytase (myo-inositol hexakisphosphate phosphohydrolase, E.C. 3.1.3.8 or E.C. 3.1.3.26) are group of enzymes that hydrolyze phytate to inositol phosphate, myo-inositol and inorganic phosphate. Today, phytase extracted from fungi (Nautophos® , Allzyme® SSF, Finase® P/L) is commercialized as feed supplement; on the other hand several bacterial species have been used as probiotics.

But till date, phytase producing probiotic strains have not been commercialized. However, a probiotic strain with phytase activity can perform double function and enhance productivity of animals manifolds. Simultaneously, they can also reduce the serious environmental problems caused by undigested phytate, which is main byproduct of human and animal excreta. Hence, from last few years, this area has become a major public health concern and is drawing the interest of health and research professionals all around the world.

One of the major cornerstones in biotechnology today is the optimization of the cultivation conditions for enhancing the productivity. Screening and evaluation of nutritional requirements of microorganisms is an important step in any bioprocess development.

Optimization studies involving one factor-ata-time approach is tedious, tends to overlook the interaction among the factors and might lead to misinterpretation of results. In contrast, statistical strategies are preferred and more advantageous and mitigate the error in determining the effects of parameters in an economical manner (Sharma and Satyanarayana, 2006). The empirical technique is a traditional optimization method employing one-factor-at-a-time strategies which is simple, easy and explains the individual effect of different components. Unfortunately, it is tedious and fails to explain the interactions among the factors (Awad et al., 2011). Statistical optimization is a proven tool for overcoming the limitations of the “one-factor at a time‟ method. It is more efficient technique since it can provide statistical data with a relatively small number of experiments.

In our previous endeavor, we isolated phytase producing potential probiotic strain Bacillus subtilis P6 from exotic environment (Sharma and Trivedi, 2015). Considering the high potential of phytase for use as feed supplement and its future prospective in animal feed and human nutrition, the present appraisal therefore aims at achieving the multifold improvement in the phytase production through sequential classical and statistical optimization strategy from probiotic strain Bacillus subtilis P6 so as to verify whether it can become a new kind of feed additive for food and animal feeding in the future.

یک بررسی در مورد روش های تشخیصی بیماری تب مالت

A Review on Diagnostic Methods of Brucellosis

Abstract

We reviewed different diagnostic methods of brucellosis in different livestock and humans. Bacteriological diagnosis, immunohistochemistry (an alternative technique for direct diagnosis) and different molecular methods for Brucella species genotyping are among the direct methods for diagnosis of brucellosis discussed in this review. The well-established indirect methods for diagnosis of brucellosis, serological and brucellin allergic skin tests were also critically conferred. Finally, for effective control and prevention of brucellosis around the world, the direct diagnostic methods are advised in order to develop vaccine against the circulating Brucella strain in the specific country.

1 Introduction

Brucellosis is an ancient disease that can possibly be traced back to the 5th plague of Egypt around 1600 BC. Recent examination of the ancient Egyptian bones, dating to around 750 BC, showed evidence of sacroilitis and other osteoarticular lesions, common complications of brucellosis [1]. David Bruce isolated Brucella melitensis (B. melitensis) (Micrococcus melitensis at that time) in 1887 from the spleen of a British soldier who died from a febrile illness (Malta fever) common among military personnel stationed on Malta. For almost 20 years aіer isolation of M. melitensis, Malta fever remained a mystery and was thought to be a vector-borne disease until Нemistocles Zammit accidentally demonstrated the zoonotic nature of the disease in 1905 by isolating B. melitensis from goat’s milk. It was believed that goats were not the source of infection since they did not become ill when inoculated with Brucella cultures. Нe discovery that healthy goats could be carriers of the disease has been termed one of the greatest advances ever made in the study of epidemiology [2,3].

Brucellosis is caused by Gram-negative coccobacilli of the genus Brucella [4,5]. In livestock, the disease results in significant economic losses due to reproductive impairment caused by abortion, stillbirth or weak calves and neonatal mortality, infertility [6]. In humans, Brucella spp. infection causes a febrile disease that may be associated with a broad spectrum of symptoms, and it may be fatal in some cases [5,7]. Currently, there are ten spp. described in the genus Brucella. Each one may infect diوٴerent host spp., but each Brucella spp. has a preference for its host spp., B. melitensis (sheep and goats), B. abortus (cattle), B. suis (pigs), B. ovis (rams), B. canis (dogs), B. microti (rodents-Microtus arvalis), B. neotomae (rodents – Neotoma lepida), B. pinnipedialis (pinnipeds), B. ceti (cetacea), and B. inopinata (originally isolated from a human patient, but its preferential host is not known) [8,9]. Нree of this Brucella spp. can be subdivided in biotypes [10,11].

Нerefore, three biotypes (1-3) have been identified in B. melitensis; eight biotypes (1-7,9) in B. abortus; and five biotypes (1-5) in B. suis [12]. All Brucella spp. are considered potentially pathogenic for humans, with the exceptions of B. neotomae, B. microti, and B. ovis [6,9].

A precise diagnosis of Brucella spp. infection is important for the control of the disease in animals and consequently in man. Clinical diagnosis is based usually on the history of reproductive failures in livestock, but it is a presumptive diagnosis [13] that must be confirmed by laboratory methods [13,14]. Нe “gold standard” in the diagnosis of brucellosis is bacterial isolation from blood or bone marrow specimens that requires long cultivation periods (4 to 7 days up to 40 days) and oіen the blood cultures are unsuccessful [15]. Serological tests, such as serum agglutination test (SAT), rose Bengal plate test (RBPT), complement fixation test (CFT), and enzyme-linked immunosorbent assay (ELISA) are still frequently used [5,16]. Since the routine identification and diوٴerentiation of brucellosis suspected specimens, based on culture isolation and phenotypic characterization, requires biosafety level-3 (BSL-3) protocols for the high risk of laboratoryacquired infections [17], molecular methods have been explored in order to overcome these diٹculties. Furthermore, the polymerase chain reaction (PCR)-based assays have shown a higher sensitivity with respect to the standard microbiological assay for the diagnosis of brucellosis [18].

Нerefore, the objectives of this paper are to review a diagnostic methods that are used for isolation, screening, monitoring or epidemiological surveillance and complementary or confirmatory for brucellosis in livestock and humans.

شناسایی تنوع باکتریهای واژینال و رابطه آن با واژینوز باکتریال تشخیص داده شده بالینی با دناتورینگ الکتروفورز ژل گرادیانی و تحلیل تطابق

Identification of vaginal bacteria diversity and it’s association with clinically diagnosed bacterial vaginosis by denaturing gradient gel electrophoresis and correspondence analysis

Abstract

Bacterial vaginosis (BV) is a common complex associated with numerous adverse health outcomes, affecting women of different ages throughout the world. The etiology of BV remains poorly understood due to the difficulty of establishing a molecular genetic criterion to recognize the vaginal microbiota of BV-positive women from that of the normal women. We used techniques of broad-range PCR-DGGE and gel imaging analysis system cooperated with 16S rRNA gene sequencing and statistical analysis to investigate the community structure of the healthy and BV-affected vaginal microbial ecosystems. The community of vaginal bacteria detected in subjects with BV was far more luxuriant and diverse than in subjects without BV. The mean number of microbial species in 128 BV-positive women was nearly two times greater than in 68 subjects without BV(4.05 ± 1.96 versus 2.59 ± 1.14). Our sequencing efforts yielded many novel phylotypes (198 of our sequences represented 59 species), including several novel BV-associated bacteria (BVAB) and many belonging to opportunistic infections, which remain inexplicable for their roles in determining the health condition of vaginal microflora. This study identifies Algoriphagus aquatilis, Atopobium vaginae, Burkholderia fungorum, Megasphaera genomosp species as indicators to BV and subjects with BV harbor particularly taxon-rich and diverse bacterial communities. Maybe Bifidobacterium, Staphylococcus or even more alien species are commensal creatures in normal vaginal microbiota.

1 Introduction

Bacterial vaginosis (BV) is a common vaginal disorder characterized by an alteration of the normal vaginal bacterial microbiota to a heterogeneous state containing a complex population of anaerobic and microaerophilic organisms. BV is associated with sexually transmitted infections and adverse pregnancy outcomes such as low birth weight of foetus, premature birth, neonatal death and secondary infection after delivery and is also the risk factor for premature rupture of fetal membranes (PROM) (Goldenberg et al., 2008). Antibiotics treatment of the condition used to failure due to the uncertain variation of BV-associated bacteria (BVAB) (Beigi et al., 2004; Ferris et al., 2004). Many BV associated bacteria are resistant to culture and (or) difficult to detect by traditional methods, which thwart the exploration of the pathogenesis of BV. So far, there is a lack of effective prevention and treatment measures.

According to Nugent’s classification (Nugent et al., 1991), a score ranged from 7 to 10 is considered as BV, a clinical condition dominated by the morphological identification of Gram-negative and-positive bacteria, without evidence of Lactobacillus morphotypes. In contrast, the score from 0 to 3 is considered an undisturbed vaginal microflora dominated by the Lactobacillus genus, identified as the principal Gram-positive rods bacteria.

In general, race (nationality) (Ness et al., 2003; Uscher-Pines et al., 2009), behavioral habits (Bradshaw et al., 2013), personal hygiene status (Brotman et al., 2008), and so on have been considered as causes of BV. Although there is agreement in the literature that no single agent likely causes BV, there is no consensus about what constitutes a pathogenic bacterial community in this syndrome. Comprehensive cultivation-independent comparisons of the vaginal bacterial communities between subjects with and without clinically defined BV have been rare.

With the development and application of culture-independent nucleic acid amplification test (NAAT) in recent years, it is possible to explore the structure and dynamic shift of microbiota (Larin et al., 2007; Burton and Reid, 2002; Zozaya-Hinchliffe et al., 2010; Ravel et al., 2011). Molecular methods have identified in the vagina of healthy, nonpregnant women the Lactobacillus genus living with a spectrum of bacteria including Gardnerella, Atopobium, Eggerthella, Megasphaera, Leptotrichia, Prevotella, Enterococcus, Bifidobacterium, Staphylococcus, Corynebacterium, Streptococcus, Bacterioides, Mycoplasma, Escherichia, Peptostreptococcus, Ureaplasma, Veillonela, and Candida species (Zhou et al., 2004; Ling et al., 2013; Hyman et al., 2005; Vitali et al., 2007; Nam et al., 2007). However, at present the spectrum of bacterial species that are correspondent to clinical diagnosis, i.e. the Nugent’s score is not well defined.

In this study, we investigated the composition of BVAB in countryside women of reproductive age. Women with and without BV was diagnosed by Nugent’s criteria and recruited as the research subject and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and DNA sequencing techniques were used to analysis the genetic information of 16S rRNA genes (16S rDNA) of vaginal bacteria. The sequence information of 16S rDNA of every detected bacterium was identified with gene bank data. In addition, DGGE Gel Image System was used to calculate the relative abundance of each bacterial species to the whole microbial population of one sample (vaginal swab). Eventually, appropriate statistical method was used to analyze the relationship between BV and bacterial composition, as well as the relationship between BV and other risk factors.

ارزیابی تشخیصی سنجش PCR ریل تایم کمی multiplex برای واژینوز باکتریال

Diagnostic Evaluation of a Multiplex Quantitative Real-Time PCR Assay for Bacterial Vaginosis

Abstract

Background: Quantitative multiplex PCR assay for Bacterial Vaginosis (BV) based on the detection of the predominant contributory targets was evaluated against the conventional Nugent Score that is laborious and subjective due to morphological assessment bias of BV-associated bacteria.

Methods: 125 dual vaginal specimens were collected from patients aged ≥18 years at the time of presentation at the provider office to perform real time PCR and Nugent Testing. PCR assessment of BV was performed by quantitation of DNA amounts of Gardnerella vaginalis, Atopobium vaginae, Lactobacillus spp., and total amount of bacterial DNA using a multiplex RT-PCR kit (ATRiDA, Netherlands). Discordant results were resolved by the Amsel criteria or ancillary testing such as BD Affirm, when available.

Results: Nugent score classified 36.36% of the patients in BV and 15.45% in transitional BV categories. In contrast, the PCR method called 48.18% as BV and 12.72% as transitional BV or BV of unspecified origin categories. The overall concordance between the two methods was 81.81%. None of the BV positives by Nugent method were missed by the PCR. There were only 2 intermediates by Nugent that were called normal by PCR. PCR method was more sensitive than the Nugent and picked an additional 11% positives.

Conclusions: PCR based molecular BV diagnosis can standardize women health testing by removing the bias due to subjective interpretation of Nugent scoring. Our study shows that PCR method is more sensitive than conventional testing and may be a promising replacement for laborious Nugent scoring method in an era of shrinking microbiology expertise.

1 Introduction

Bacterial vaginosis (BV) is a complex polymicrobial syndrome characterized by alterations of the vaginal flora with acquisition of diverse communities of anaerobic and facultative bacteria and depletion of the usually dominant Lactobacillus flora. BV is a cause of malodorous vaginal discharge, vulvovaginal irritation, and/or dysuria. Recurrent and/or untreated infections are associated with several obstetric and gynecologic complications, including preterm rupture of membranes, chorioamnionitis, puerperal endometritis, pelvic inflammatory disease, urinary tract infection, postoperative cellulitis, cervical dysplasia, and human immunodeficiency virus acquisition. The current diagnostic gold standard Nugent Score is based on morphological assessment of BV associated bacteria. The technique is laborious and subjective as some morphologically similar non-contributory bacteria can skew the results. As a consequence, the use of molecular techniques to detect predominant contributory targets is desirable to make the BV diagnosis. Molecular methods that solely depend on the detection of a single target such as Gardnerella vaginalis have limited utility due to low sensitivity and specificity [1]. Quantitative ratios of specific anaerobic microflora such as G vaginalis and Atopobium vaginae in relation to Lactobacillus spp. are highly sensitive and specific for the diagnosis of BV and is associated with disease recurrence. Since A. vaginae have advanced resistance to metronidazole, its identification can play an important role in helping to change therapy decisions [2,3].

The aim of the study was to evaluate the diagnostic value of a multiplex quantitative real-time PCR assay (AmpliSens® Florocenosis/ Bacterial vaginosis-FRT PCR kit, ATRiDA, Netherlands) for Bacterial Vaginosis. A combination of Amsel critera and Nugent Graded gram stain (used as a gold standard) was used as the reference method.

شناسایی و مشخصات فیلوژنتیکی واژینولیزین Gardnerella vaginalis در نمونه های برگرفته از زنان بلغاری مبتلا به واژینوز باکتریال

Detection And Phylogenetic Characterization Of Gardnerella Vaginalis Vaginolysin In Samples From Bulgarian Women With Bacterial Vaginosis

Abstract

Recently, the intensive use of more advanced methods, including molecular genetic analysis, has aided the studies on the etiological role of Gardnerella vaginalis as the major pathogen of bacterial vaginosis (BV). The objective of our study was to propose a polymerase chain reaction (PCR)-based method detecting the G. vaginalis vaginolysin gene (vly) and to perform a phylogenetic analysis in an attempt to find possible correlations between clinical manifestations and gene mutations. Vaginal samples were collected from 1145 women with symptoms for vaginitis and 378 asymptomatic ones of reproductive age. After PCR detection of G. vaginalis 289 DNA samples were tested for vly using primers targeted at amplification of the whole gene. The presence of vly was detected in all 289 samples with positive PCR for G. vaginalis. Sixteen vly-positive PCR products were sequenced. The phylogenetic analysis based on the reference nucleic acid sequences deposited in the NCBI GenBank, covering the whole G. vaginalis vly gene, placed the studied isolates into three main groups. In conclusion, our experiments confirmed the major G. vaginalis virulence factor to be vaginolysin. The multiplex PCR results are promising and further studies should be carried out to determine the sensitivity range of the method and its applicability to diagnostics. Our phylogenetic analysis results, where some samples were grouped together, might imply a possible correlation between the clinical signs, the tendency to chronification, the cytotoxicity of vaginolysin and the vly gene mutations.

1 Introduction

Gardnerella vaginalis is a small, non-motile, Gram-variable coccobacillus that can grow in microaerophilic conditions. In the years following the discovery taxonomic status of this microbe remained uncertain due to some specifics in its cell wall structure: the cell wall stains Gram-positively in the exponential growth phase but, as the culture ages, the peptidoglycan layer becomes thinner and gives Gram-negative staining [1 ]. Thus, this Gram-variable microorganism was initially classified in the Corynebacterium and Haemophilus genera, but was later moved to a separate genus under the name G. vaginalis [ 1 ]. This bacterial species was proved to be a causative agent of genital tract infections in studies with volunteers [2 ] and in animal models [3 ]. It was also detected in exfoliated vaginal epithelium, in the absence of an inflammatory infiltrate. In recent years, the intensive use of more advanced methods, including molecular genetic analysis, has aided the studies on the etiological role of G. vaginalis, identifying it to be the major pathogen (in terms of frequency and quantity) in women with bacterial vaginosis (BV) [4–6]. Several members of the genus Lactobacillus inhibit pathogenic bacteria in healthy women‘s vaginal ecosystem but some substances produced by G. vaginalis could provide a synergistic effect for the growth of the other pathogens as well as an inhibitory effect against lactobacilli [3 ]. BV is the most frequent vaginal infection among women in the reproductive age group, and surveys on different continents alarm that it is especially frequent in younger women, under 30 years of age [5,7].

Several attempts have been made to classify G. vaginalis strains based on the phenotype; however, this proved to be clinically irrelevant [8 ]. The major G. vaginalis virulence factor is an exotoxin with cytotoxic activity (including hemolytic activity) against human cells. The hemolysin triggers a local immune response and the synthesis of secretary immunoglobulin A (sIgA) can therefore be used as a diagnostic marker for BV. This toxic product is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins [9 ]. The G. vaginalis hemolysin, termed vaginolysin (VLY), shows selective toxicity and specifically targets host cells that carry complement regulatory molecule CD59. The cytotoxicity mechanism is based on VLY-mediated pore formation, with the proline residue in the undecapeptide of domain 4 of the toxin molecule playing the main role. Mutations in this region generate a VLY toxoid and could possibly aid in the development of a vaccine [9 ].

The aim of the present study was to propose a polymerase chain reaction (PCR)-based method for detection of the G. vaginalis vly gene and to perform a phylogenetic analysis in an attempt to find possible correlations between some clinical manifestations and certain gene mutations.

حساسیت ضد میکروبی و واژینولیزین در Gardnerella vaginalis از زنان سالم و مبتلا به واژینوز باکتریال

Antimicrobial susceptibility and vaginolysin in Gardnerella vaginalis from healthy and bacterial vaginosis diagnosed women

Abstract

Introduction: Bacterial vaginosis (BV) is a syndrome related to Gardnerella vaginalis and is characterized by an imbalance in the vaginal microbiota. This work focused on the evaluation of antimicrobial susceptibility patterns and the occurrence of the vaginolysin (vly) gene in G. vaginalis isolated from BV and non-BV patients. Methodology: The vaginal secretions were collected randomly and processed for G. vaginalis isolation. The isolates were presumptively identified by β-hemolysis and oxidase and catalase tests. Polymerase chain reaction (PCR) was performed to confirm bacterial identity and to detect the vly gene. Antimicrobial susceptibility patterns were determined. Results: Of 89 patients, G. vaginalis was isolated from 42 (37 BV and 5 non-BV), and 204 isolates were selected (179 from BV and 25 nonBV). The vly gene was detected in all G. vaginalis isolated from non-BV women and in 98.3% of the bacteria from BV patients. High resistance was observed for ampicillin (54.4%), metronidazole (59.8%), tinidazole (60.3%) and secnidazole (71.6%). Conclusions: Further studies are needed to better address the role of G. vaginalis and the vly gene in BV pathogenesis.

1 Introduction

Vaginal microbiota is defined as a diverse group of microorganisms that colonises the vagina without causing disease, considering the host’s regular homeostatic conditions [1]. Bacterial vaginosis (BV) is a polymicrobial syndrome mainly related to Gardnerella vaginalis, characterized by an imbalance in the healthy vaginal microbiota with an increase in anaerobic bacteria, particularly those producing H2O2, which leads to the onset of fetid vaginal discharge [2]. The prevalence of BV is difficult to determine, since a large proportion of infected women are asymptomatic and do not seek medical care, and are not therefore included in the studies. BV is the most common cause of vaginal discharge in women of reproductive age and is more common in black women than white, in those women with intrauterine devices (IUDs), in smokers, in women with multiple sexual partners, and in patients using antibiotics [3]. Anaerobic microorganisms associated with BV mainly include G. vaginalis, Ureaplasma urealyticum, M. hominis, Mobiluncus spp., Bacteroides spp., and Prevotella spp. The microorganisms involved and their products differ significantly between women with BV, and the risk of upper genital tract infections also probably varies between individuals [4]. The isolation of G. vaginalis may not be used for BV diagnosis because it is part of the vaginal microbiota of more than 50% of healthy women. A high concentration of G. vaginalis is often associated with the presence of BV [1].

As the clinical diagnosis of BV has been shown to be vulnerable, different methods have been proposed, such as those of Amsel and Nugent, which include clinical and laboratory observations [5]. Amsel criteria is used as the main clinical approach to BV diagnosis and requires three of the following four criteria: vaginal pH higher than 4.5; presence of adherent white vaginal discharge; finding of vaginal epithelial cells covered with adherent bacteria, the clue cells; and release of volatile amines following the addition of potassium hydroxide to a small amount of vaginal fluid [5,6]. As an alternative to the clinical diagnosis based on Amsel criteria, the Nugent score has been proposed to evaluate the vaginal microbiota through examination of vaginal smears under a microscope [7,8].

اهمیت تشخیص آزمایشگاهی در درمان بیماری تب مالت

Implications of laboratory diagnosis on brucellosis therapy

Brucellosis is a worldwide zoonosis with a huge economic impact on animal husbandry and public health. The diagnosis of human brucellosis can be protracted because the disease primarily presents as fever of unknown origin with unspecific clinical signs and symptoms. The isolation rate of the fastidious etiologic agent from blood cultures is low, and therefore laboratory diagnosis is mainly based on serologic and molecular testing. However, seronegative brucellosis patients have been described, and antibody titers of diagnostic significance are difficult to define. Whether the molecular detection of Brucella DNA in clinical samples should be followed by long-term antibiotic treatment or not is also a matter of debate. The aim of this article is to review and discuss the implications of laboratory test results in the diagnosis of human brucellosis on disease therapy.

Brucellosis is one of the world’s most widespread bacterial zoonoses, leading to tremendous economic losses in endemic regions and serious complaints in affected patients. The infection may be transmitted by direct animal contact, but is usually acquired through the consumption of contaminated food products of animal origin, mainly via unpasteurized goat’s milk and cheese. Furthermore, brucellosis is the most common bacterial laboratory-acquired infection worldwide [1]. Although many national and international programs have been established to eradicate the pathogen and control its spreading in animal husbandry, brucellosis is still a re-emerging disease. The surveillance of animal brucellosis is difficult due to bacterial persistence in wildlife and environmental reservoirs, with consecutive spill-over to domestic animals [2].

Brucellosis is caused by members of the genus Brucella (B.), which are gram-negative, facultative intracellular coccobacilli that were historically differentiated by their preferred animal host, varying pathogenicity and a few selected phenotypic traits. The genus comprises six classical species: B. melitensis bv 1–3 (primarily isolated from sheep and goats); B. abortus bv 1–6 and 9 (from cattle and other Bovidae); B. suis bv 1–3 (from pigs), bv 4 (from reindeer), bv 5 (from small rodents); B. canis (from dogs); B. ovis (from sheep); and B. neotomae (from desert wood rats). Recently, two novel species of marine origin, B. pinnipedialis (isolated from seals) and B. ceti (from dolphins and whales) [3], B. microti isolated from the common vole (Microtus arvalis) [4], red foxes (Vulpes vulpes) [5] and from soil [6], and B. inopinata isolated from a breast implant wound of a 71-year-old female patient [7] have been described. In the past, a lot of atypical Brucella strains arose. These could represent novel species or lineages of already described species, for example various Brucella strains originating from wild native rodent species in North Queensland, Australia [8], a novel Brucella isolate in association with two cases of stillbirth in nonhuman primates [9], and a B. inopinata-like strain (BO2), which was isolated from a lung biopsy of a 52-year-old Australian patient suffering from chronic destructive pneumonia [10].

Physicians’ awareness of the infection is very poor in many countries, and most cases correctly identified are clinically advanced. Because of its protean clinical manifestations, human brucellosis can be easily confused with other infectious and noninfectious diseases, leading to diagnostic delays and late onset of therapy. The isolation of the fastidious organisms is often unsuccessful or takes a long time, which is why the presumptive clinical diagnosis is usually confirmed by serologic tests. However, seronegative cases, cross-reactivity of anti-Brucella antibodies with many other clinically relevant bacteria, poorly defined cutoffs in serologic test systems, and so on can make the interpretation of the titers measured very difficult. Alternatively, molecular techniques can be used for the laboratory diagnosis of human brucellosis, but the detection of Brucella DNA does not prove an active infection with viable bacteria, and therefore does not effectively support therapeutic decision making.

The scope of this article is to review up-to-date laboratory techniques in the diagnosis of human brucellosis and to discuss if positive serologic or molecular tests should result in long-term antibiotic therapy.

شناسایی، کمیت سنجی و تعیین نوع واژینوز باکتریال در نمونه های واژنی بالینی غیرکشت شده توسط PCR کمّی

Identification, quantification and subtyping of Gardnerella vaginalis in noncultured clinical vaginal samples by quantitative PCR

Abstract

Gardnerella vaginalis is an important component of the human vaginal microflora. It is proposed to play a key role in the pathogenesis of bacterial vaginosis (BV), the most common vaginal condition. Here we describe the development, validation and comparative analysis of a novel molecular approach capable of G. vaginalis identification, quantification and subtyping in noncultured vaginal specimens. Using two quantitative PCR (qPCR) assays, we analysed G. vaginalis bacterial loads and clade distribution in 60 clinical vaginal-swab samples. A very high pathogen prevalence was revealed by species-specific qPCR not only among BV patients (100 %), but also in healthy women (97 %), although the G. vaginalis concentration was significantly lower in non-BV samples. G. vaginalis clades identified in vaginal specimens by subtyping multiplex qPCR, which targets four clade-specific genetic markers, had frequencies of 53 % for clade 1, 25 % for clade 2, 32 % for clade 3 and 83 % for clade 4. Multiple clades were found in 70 % of samples. Single G. vaginalis clades were represented by clade 1 and clade 4 in 28 % of specimens. A positive association with BV was shown for clade 1 and clade 3, while clade 2 was positively associated with intermediate vaginal microflora, but not with BV. Clade 4 demonstrated no correlation with the disorder. The presence of multiple clades had a high positive association with BV, whereas G. vaginalis identified as a single clade was negatively linked with the condition. Polyclonal G. vaginalis infection may be a risk factor for BV.

1 Introduction

Gardnerella vaginalis is a facultatively anaerobic, catalaseand oxidase-negative bacterium. G. vaginalis cells are pleomorphic, Gram-negative to Gram-variable, nonencapsulated and non-motile rods with a mean size of 0.5 to 1.5 mm (Greenwood & Pickett, 1980). Initially named Haemophilus vaginalis by discoverers, the microorganism was later referred to as Corynebacterium vaginale and taxonomically assigned as G. vaginalis (Gardner & Dukes, 1955; Piot et al., 1980). Currently it is the only species within the genus Gardnerella (List of Prokaryotic Names with Standing in Nomenclature – www.bacterio. net). G. vaginalis is mainly considered a part of the lower female genital tract microflora (Catlin, 1992). It can also be routinely isolated from the male urogenital tract as was reported in the first publication on this micro-organism and a number of other studies (Briselden & Hillier, 1990; Eren et al., 2011; Leopold, 1953; Swidsinski et al., 2010). The presence of G. vaginalis in the oral cavity and anal samples has also been described (El Aila et al., 2011; Holst, 1990; Marrazzo et al., 2012). Along with disorders in the urinary and genital tracts, G. vaginalis has been identified as a causative agent of bacteraemia, septicaemia with infective endocarditis, vertebral osteomyelitis, acute hip arthritis and retinal vasculitis (Graham et al., 2009; Lagace´- Wiens et al., 2008; Neri et al., 2009; Sivadon-Tardy et al., 2009; Yoon et al., 2010).

Since the moment of discovery G. vaginalis has been linked with bacterial vaginosis (BV), the most common vaginal condition. BV is characterized by the elevation of vaginal pH and clinical symptoms, such as malodorous vaginal discharge and the presence of clue cells visualized on wet mount, although a large percentage of women with BV can be asymptomatic (Amsel et al., 1983; Schwebke & Desmond, 2007). The disorder is associated with a dramatic change in vaginal microflora, when protective Lactobacillus species are depleted and replaced by fastidious anaerobic bacteria including Prevotella, Atopobium, Megasphaera, Sneathia and G. vaginalis (Eschenbach, 1993; Forsum et al., 2005; Nugent et al., 1991). Described as ‘a single etiological agent’ of BV by the discoverers, G. vaginalis, however, failed to fulfil Koch’s postulates for microbial infection in multiple subsequent studies (Catlin, 1992; Holst, 1990; Menard et al., 2008; Zozaya-Hinchliffe et al., 2010). The complex polymicrobial nature of BV and the presence of G. vaginalis in the vaginal milieu of healthy individuals argue against the definition of G. vaginalis as a sole disease-causative species. The micro-organism, nevertheless, remains a prime suspect in the pathogenesis of BV since it is present in 95–100 % of BV patients, although the association of G. vaginalis with Atopobium or Megasphaera has been shown to display better predictive value for this condition (Fredricks et al., 2009; Menard et al., 2008; Muzny & Schwebke, 2013; Schellenberg et al., 2011). It was proposed that G. vaginalis might serve as an initial colonizer critical for the development of BV (Patterson et al., 2010). Some studies suggest that G. vaginalis dominance in the vaginal microflora can represent a distinct transitional or intermediate state between healthy microflora occupied by lactobacilli and BV-type microflora populated with fastidious anaerobes (Schellenberg et al., 2011). After more than half a century the role of G. vaginalis in such an ‘enigmatic’ condition as BV remains obscure.

واژینوز باکتریال مرتبط با Gardnerella vaginalis در زنان بلغاری

Gardnerella vaginalis-associated bacterial vaginosis in Bulgarian women

Abstract

Background Bacterial vaginosis (BV) is the most common cause of vaginal discharge in women of reproductive age. The purpose of this study was to determine the frequency of BV in Bulgarian pregnant and nonpregnant women from several age ranges and to compare three different laboratory methods for Gardnerella vaginalis detection in patents suffering from BV.

Methods Between September 2011 and June 2012, 809 women of 16–40 years of age separated in two major groups: nonpregnant – 469 (355 with and 114 without symptoms) and pregnant – 340 (213 and 127 respectively) were enrolled for the study. The women underwent three different laboratory tests simultaneously: scoring of Gram staining of vaginal smear, culture, and polymerase chain reaction (PCR) assay for G. vaginalis.

Results The microscopic method detected high frequency of BV in symptomatic (57%) whereas only a minority of asymptomatic subjects (14%) were detected. G. vaginalis-associated BV was diagnosed in approximately equal proportions when evaluated with PCR and microscopic method for both pregnant and nonpregnant women. The comparative analysis of microscopic evaluation, culture and PCR assays demonstrated greater concurrence (about 90%) between Gram staining and PCR detection for BV, than both methods compared to culture. The combination of microscopy and PCR turned out to be very reliable and repeatable for detecting G. vaginalis-associated BV.

Conclusions This is the first comparative investigation on the epidemiology of G. vaginalis-associated BV in Bulgaria. The established highest frequency in the young Bulgarian women (21–30 years) is alarming and should be considered in prophylaxis and reproductive programmes.

1 Introduction

Bacterial vaginosis (BV) is the most common cause of unpleasant vaginal odor and discharge in women of reproductive age.1,2 It is induced by an imbalance in naturally occurring microflora. Any change in the resident flora including reduction of lactobacilli allows for different anaerobic bacteria to gain a foothold and multiply.3–7 Nevertheless the process is multifactorial and the initial mechanism of replacement of normal lactobacillary flora by opportunistic pathogens in vaginal ecosystem and the role of intrinsic host factors still remains unclear, requiring more research to be conducted.5–8 The essential participants in pathological polymicrobial associations, which could be used as markers for BV, are Gardnerella vaginalis (that grows under appropriate microaerophilic conditions) and anaerobic Atopobium vaginae.3–8 Other microorganisms involved in BV microbiota are very diverse and include anaerobes, such as Peptostreptococcus spp., Mobiluncus spp., Prevotella spp., Bacteroides spp., Fusobacterium spp., and facultative anaerobes.6–11 It is not clear yet if the BV is a sexually transmitted disease, but it is more common in promiscuous women with hazardous sexual behavior (with multiple and/or new sexual partners; or with female partners, sex during menses).12–18 BV can be an independent risk factor for acquisition of any other sexually transmitted infection.1,15,17 It has also been shown to be a cause for serious health problems as preterm birth, postpartum fever, development of endometritis, post-hysterectomy or postabortal sepsis, and pelvic inflammatory disease.19–21

The purpose of this study was to determine the frequency of BV and G. vaginalis-associated BV in Bulgarian pregnant and nonpregnant women from different age ranges and to compare three distinct laboratory methods for G. vaginalis detections in patients suffering from BV.

بینش هایی نسبت به سیستم CRISPR/Cas در گاردنلاواژینالیس

Insights into the CRISPR/Cas system of Gardnerella vaginalis

Abstract

Background: Gardnerella vaginalis is identified as the predominant colonist of the vaginal tracts of women diagnosed with bacterial vaginosis (BV). G. vaginalis can be isolated from healthy women, and an asymptomatic BV state is also recognised. The association of G. vaginalis with different clinical phenotypes could be explained by different cytotoxicity of the strains, presumably based on disparate gene content. The contribution of horizontal gene transfer to shaping the genomes of G. vaginalis is acknowledged. The CRISPR loci of the recently discovered CRISPR/Cas microbial defence system provide a historical view of the exposure of prokaryotes to a variety of foreign genetic elements.

Results: The CRISPR/Cas loci were analysed using available sequence data from three G. vaginalis complete genomes and 18 G. vaginalis draft genomes in the NCBI database, as well as PCR amplicons of the genomic DNA of 17 clinical isolates. The cas genes in the CRISPR/Cas loci of G. vaginalis belong to the E. coli subtype. Approximately 20% of the spacers had matches in the GenBank database. Sequence analysis of the CRISPR arrays revealed that nearly half of the spacers matched G. vaginalis chromosomal sequences. The spacers that matched G. vaginalis chromosomal sequences were determined to not be self-targeting and were presumably neither constituents of mobile-element-associated genes nor derived from plasmids/viruses. The protospacers targeted by these spacers displayed conserved protospacer-adjacent motifs.

Conclusions: The CRISPR/Cas system has been identified in about one half of the analysed G. vaginalis strains. Our analysis of CRISPR sequences did not reveal a potential link between their presence and the virulence of the G. vaginalis strains. Based on the origins of the spacers found in the G. vaginalis CRISPR arrays, we hypothesise that the transfer of genetic material among G. vaginalis strains could be regulated by the CRISPR/Cas mechanism. The present study is the first attempt to determine and analyse the CRISPR loci of bacteria isolated from the human vaginal tract.

1 Background

Gardnerella vaginalis, a facultatively anaerobic bacterium of the Bifidobacteriaceae family, is strongly associated with bacterial vaginosis (BV): a disease characterised by malodorous vaginal discharge [1-3]. Women with BV are at risk of poor reproductive health outcomes and the acquisition of some sexually transmitted diseases [2,4]. BV is defined as a shift in microbial species from hydrogen peroxide producing Lactobacillus to anaerobic bacteria including G. vaginalis, Atopobium vaginae, Prevotella, Peptostreptococcus, and Bacteroides spp. [5,6]. The gold standard for laboratory diagnosis of BV is the Gram stain, which is used to determine the relative concentrations of lactobacilli and the bacteria characteristic of BV [7]. The state of asymptomatic BV has also been recognised, although Gram stains revealed a decrease in lactobacilli and an increase in the abundance of anaerobes specific to BV [8]. The same G. vaginalis that is recovered as the prevailing inhabitant of the vaginal tracts of women diagnosed with BV is also found in BV-negative women, though at much lower numbers [5,9,10]. The issue of G. vaginalis commensalism is still unclear, as the vaginal bacterial community is dynamic and tends to change during the menstrual cycle to produce transient dominance of G. vaginalis in healthy women [11,12]. Using culture- independent techniques, it was demonstrated that the vaginal microbiota may differ among human populations: Hispanic and non-Hispanic black women have significantly more anaerobes and fewer lactobacilli than Asian and Caucasian women [12]. Thus, low counts of Lactobacillus do not necessarily indicate the BV state [6,13].

The association of G. vaginalis with different clinical phenotypes could be explained by different cytotoxicity of the strains, presumably based on disparities in their gene content. Until recently, surprisingly little has been known about the genetics of G. vaginalis. In 2010, the genomes of several G. vaginalis strains from the vaginas of BV and non-BV patients were sequenced, providing information about the bacterium and enabling comparative genomic analyses [14,15]. Attempts have also been made to expand the knowledge of the genotypic and phenotypic diversity of G. vaginalis strains in terms of virulence factors: particularly vaginolysin, sialidase, and biofilm-forming proteins [16-18]. The development of methods for the genotypic differentiation of G. vaginalis revealed that the genomes exhibit great variability. Therefore, some conventional methods, including pulse field gel electrophoresis, restriction fragment length polymorphism, classical ribotyping with Southern blot, and restriction enzyme analysis, are not applicable for typing this species [19-21]. The amplified ribosomal DNA restriction analysis method, while applicable to the genotypic differentiation of G. vaginalis, has been found to not be discriminatory enough for pathogenetic and epidemiological studies of G. vaginalis [17,18].

Recent data from G. vaginalis comparative genomic analyses have indicated that the bacterium possesses a small core genome, consisting of 746 genes, that accounts for only 27% of the pan-genome of the species [22]. The small number of unique genes (21) in the individual strains of G. vaginalis and the genomic plasticity among the strains suggest that horizontal gene transfer (HGT) is active; but there is frequent homologous recombination among G. vaginalis strains, as well as the intake of foreign DNA from other species [15,22].

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and their associated cas genes constitute a bacterial and archaeal defence mechanism against exogenous nucleic acids [23]. The majority of archaea and approximately half of bacterial genomes contain CRISPR loci [24]. CRISPR loci consist of unique sequences (spacers) that intercalate between short conserved repeat sequences. The spacer sequences often originate from invading viruses and plasmids [25,26]. The CRISPR/Cas defence mechanism relies on RNA interference that prevents bacteriophage infection and plasmid conjugation, thus restricting two routes of HGT [27]. Analyses of CRISPR sequences have been used in a variety of applications including strain genotyping and epidemiological study, detection of evolutionary events and bottlenecks, investigation of the history of virus exposure, and host population dynamics, providing insights into the dominant routes of HGT [28-32]. The current study targeted the detection and analysis of CRISPR loci in the genomes of 17 G. vaginalis strains isolated from the vaginal tracts of women diagnosed with BV [18], and also in the genomes of 21 G. vaginalis strains deposited in the NCBI genome database.

In the current study, we examined the origins of CRISPR spacers representing the immunological memory of G. vaginalis strains, and we hypothesised about the impact of CRISPR/Cas on the emergence of genetic variability of G. vaginalis strains. Also, we demonstrated the restricted distribution of the CRISPR loci among the G. vaginalis strains.