International Journal of Microbiology

Antioxidant Functions of Nitric Oxide Synthase in a Methicillin Sensitive Staphylococcus aureus

Manisha Vaish and Vineet K. Singh — Thu, 04 Apr 2013 11:33:22 +0000

Nitric oxide and its derivative peroxynitrites are generated by host defense system to control bacterial infection. However certain Gram positive bacteria including Staphylococcus aureus possess a gene encoding nitric oxide synthase (SaNOS) in their chromosome. In this study it was determined that under normal growth conditions, expression of SaNOS was highest during early exponential phase of the bacterial growth. In oxidative stress studies, deletion of SaNOS led to increased susceptibility of the mutant cells compared to wild-type S. aureus. While inhibition of SaNOS activity by the addition of L-NAME increased sensitivity of the wild-type S. aureus to oxidative stress, the addition of a nitric oxide donor, sodium nitroprusside, restored oxidative stress tolerance of the SaNOS mutant. The SaNOS mutant also showed reduced survival after phagocytosis by PMN cells with respect to wild-type S. aureus.

Viability Reagent, PrestoBlue, in Comparison with Other Available Reagents, Utilized in Cytotoxicity and Antimicrobial Assays

Thu, 04 Apr 2013 09:19:19 +0000

This study compared different commercially available viability reagents. The growth indicator reagents include p-iodonitrotetrazolium violet (INT), PrestoBlue, and Alamar Blue which were used for antimicrobial analysis against Streptococcus mutans, Prevotella intermedia, Propionibacterium acnes, and Mycobacterium tuberculosis. PrestoBlue and Alamar Blue are resazurin based reagents that resulted in a quick and easily distinguishable colour change that allowed for visual readings. INT and Sodium 3′-[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic acid hydrate (XTT) are tetrazolium based reagents which are converted to a formazan dye in the presence of metabolically active mitochondria enzyme. For cell viability analysis, reagents XTT and PrestoBlue were compared. PrestoBlue was able to clearly indicate the minimum inhibitory concentration (MIC) of various positive drug controls on various microbial strains. PrestoBlue was also a good indicator of the 50% inhibitory concentration (IC50) of positive drug controls on various cell lines.

Development of a Cell-Based Functional Assay for the Detection of Clostridium botulinum Neurotoxin Types A and E

Uma Basavanna, Tim Muruvanda, Eric W. Brown, and Shashi K. Sharma — Thu, 07 Mar 2013 10:43:21 +0000

The standard procedure for definitive detection of BoNT-producing Clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (MBA). The mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. Cell-based assays provide an alternative to the MBA in screening for BoNT-producing Clostridia. Here, we describe a cell-based assay utilizing a fluorescence reporter construct expressed in a neuronal cell model to study toxin activity in situ. Our data indicates that the assay can detect as little as 100 pM BoNT/A activity within living cells, and the assay is currently being evaluated for the analysis of BoNT in food matrices. Among available in vitro assays, we believe that cell-based assays are widely applicable in high-throughput screenings and have the potential to at least reduce and refine animal assays if not replace it.

Point Mutations in the folP Gene Partly Explain Sulfonamide Resistance of Streptococcus mutans

W. Buwembo, S. Aery, C. M. Rwenyonyi, G. Swedberg, and F. Kironde — Mon, 25 Feb 2013 08:19:15 +0000

Cotrimoxazole inhibits dhfr and dhps and reportedly selects for drug resistance in pathogens. Here, Streptococcus mutans isolates were obtained from saliva of HIV/AIDS patients taking cotrimoxazole prophylaxis in Uganda. The isolates were tested for resistance to cotrimoxazole and their folP DNA (which encodes sulfonamide-targeted enzyme dhps) cloned in pUC19. A set of recombinant plasmids carrying different point mutations in cloned folP were separately transformed into folP-deficient Escherichia coli. Using sulfonamide-containing media, we assessed the growth of folP-deficient bacteria harbouring plasmids with differing folP point mutations. Interestingly, cloned folP with three mutations (A37V, N172D, R193Q) derived from Streptococcus mutans 8 conferred substantial resistance against sulfonamide to folP-deficient bacteria. Indeed, change of any of the three residues (A37V, N172D, and R193Q) in plasmid-encoded folP diminished the bacterial resistance to sulfonamide while removal of all three mutations abolished the resistance. In contrast, plasmids carrying four other mutations (A46V, E80K, Q122H, and S146G) in folP did not similarly confer any sulfonamide resistance to folP-knockout bacteria. Nevertheless, sulfonamide resistance (MIC = 50 μM) of folP-knockout bacteria transformed with plasmid-encoded folP was much less than the resistance (MIC = 4 mM) expressed by chromosomally-encoded folP. Therefore, folP point mutations only partially explain bacterial resistance to sulfonamide.

Bacteriophage-Derived Peptidase Eliminates and Prevents Staphylococcal Biofilms

Sun, 03 Feb 2013 09:09:07 +0000

New antibacterial agents are urgently needed for the elimination of biofilm-forming bacteria that are highly resistant to traditional antimicrobial agents. Proliferation of such bacteria can lead to significant economic losses in the agri-food sector. This study demonstrates the potential of the bacteriophage-derived peptidase, , as a biocidal agent for the rapid disruption of biofilm-forming staphylococci, commonly associated with bovine mastitis. Purified applied to biofilms of Staphylococcus aureus DPC5246 completely eliminated the staphylococcal biofilms within 4 h. In addition, was able to prevent biofilm formation by this strain. The lysin also reduced S. aureus in a skin decolonization model. Our data demonstrates the potential of as a biocidal agent for prevention and treatment of biofilm-associated staphylococcal infections or as a decontaminating agent in the food and healthcare sectors.

Methicillin-Resistant Staphylococcus aureus Carriage among Students at a Historically Black University: A Case Study

Hua Shen, Eyitayo Akoda, and Kunyan Zhang — Sun, 20 Jan 2013 15:44:19 +0000

Background. Black people in the USA is afflicted with a higher rate of methicillin-resistant Staphylococcus aureus (MRSA) infection. This study determined the prevalence of MRSA carriage among black college students at a university setting. Methods. Hand and nasal swabs were collected and screened for MRSA by mannitol fermentation, coagulase, and DNase activities and their resistance to oxacillin. MRSA isolates were analyzed for antimicrobial resistance pattern, genetic profile for staphylococcal cassette chromosome mec (SCCmec) type, pulsed-field type, multilocus sequence type (ST), and the presence of Panton-Valentine leukocidin (PVL) gene. Results. MRSA was isolated from 1 of the 312 (0.3%) hand swabs and 2 of the 310 (0.65%) nasal swabs, respectively. All isolates lack multidrug resistance and have type IV SCCmec, characteristic of community-associated MRSA. These isolates were a ST8-MRSA-IVa-PVL(+) (USA300 strain), a ST8-MRSA-IVb-PVL(−), and a new MLST, ST2562-MRSA-IV-PVL(−), identified in this study. These isolates were thus not transmitted among students. Conclusion. We found a low rate of MRSA carriage among students in a black university. Our finding highlights the need of future study which involves multiinstitutions and other ethnic group to assess the association of black race with MRSA carriage.

Effect of Citrus Byproducts on Survival of O157:H7 and Non-O157 Escherichia coli Serogroups within In Vitro Bovine Ruminal Microbial Fermentations

Thu, 17 Jan 2013 15:23:33 +0000

Citrus byproducts (CBPs) are utilized as a low cost nutritional supplement to the diets of cattle and have been suggested to inhibit the growth of both Escherichia coli O157:H7 and Salmonella. The objective of this study was to examine the effects in vitro that varying concentrations of CBP in the powdered or pelleted variety have on the survival of Shiga-toxin Escherichia coli (STEC) serotypes O26:H11, O103:H8, O111:H8, O145:H28, and O157:H7 in bovine ruminal microorganism media. The O26:H11, O111:H8, O145:H28, and O157:H7 serotypes did not exhibit a change in populations in media supplemented with CBP with either variety. The O103:H8 serotype displayed a general trend for an approximate reduction in 5% powdered CBP and 20% pelleted CBP over 6 h. There was a trend for reductions in populations of a variant form of O157:H7 mutated in the stx1 and stx2 genes in higher concentrations of CBP. These results suggest that variations exist in the survival of these serotypes of STEC within mixed ruminal microorganism fluid media when supplemented with CBP. Further research is needed to determine why CBPs affect STEC serotypes differently.

Bacteriophages and Their Role in Food Safety

Sanna M. Sillankorva, Hugo Oliveira, and Joana Azeredo — Tue, 18 Dec 2012 08:24:24 +0000

The interest for natural antimicrobial compounds has increased due to alterations in consumer positions towards the use of chemical preservatives in foodstuff and food processing surfaces. Bacteriophages fit in the class of natural antimicrobial and their effectiveness in controlling bacterial pathogens in agro-food industry has led to the development of different phage products already approved by USFDA and USDA. The majority of these products are to be used in farm animals or animal products such as carcasses, meats and also in agricultural and horticultural products. Treatment with specific phages in the food industry can prevent the decay of products and the spread of bacterial diseases and ultimately promote safe environments in animal and plant food production, processing, and handling. This is an overview of recent work carried out with phages as tools to promote food safety, starting with a general introduction describing the prevalence of foodborne pathogens and bacteriophages and a more detailed discussion on the use of phage therapy to prevent and treat experimentally induced infections of animals against the most common foodborne pathogens, the use of phages as biocontrol agents in foods, and also their use as biosanitizers of food contact surfaces.

Use of E-Beam for Shelf-Life Extension and Sanitizing of Marinated Pork Loin

Wed, 21 Nov 2012 10:55:35 +0000

The effectiveness of E-beam radiation to extend the shelf-life of marinated pork loin slices stored at 4 and 8°C (temperature abuse) has been studied. The shelf-life was extended from 7 to 16 and >20 days after the application of 1 and 2 kGy, respectively. In the event of a temperature abuse occuring during the product distribution (e.g., increase to 8°C), the shelf-life would be extended from 5 to 10 and 16 days, respectively, when applying the doses mentioned previously. From a public health point of view, the irradiation of marinated pork loin may be marketable for a longer period of time of up to two weeks, and guarantees a practically Salmonella and Listeria-free product. Minor changes are produced by the E-beam treatment in the main sensory and rheological characteristics. The odor was the most affected feature, but the off-odors diminished with increased storage. In any case, testers judged the samples to be adequate for marketing.

Diversity of Antimicrobial Resistance and Virulence Determinants in Pseudomonas aeruginosa Associated with Fresh Vegetables

Kashina Allydice-Francis and Paul D. Brown — Tue, 20 Nov 2012 15:12:12 +0000

With the increased focus on healthy eating and consuming raw vegetables, this study assessed the extent of contamination of fresh vegetables by Pseudomonas aeruginosa in Jamaica and examined the antibiotic susceptibility profiles and the presence of various virulence associated determinants of P. aeruginosa. Analyses indicated that vegetables from retail markets and supermarkets were widely contaminated by P. aeruginosa; produce from markets were more frequently contaminated, but the difference was not significant. Lettuce and carrots were the most frequently contaminated vegetables, while tomatoes were the least. Pigment production (Pyoverdine, pyocyanin, pyomelanin and pyorubin), fluorescein and alginate were common in these isolates. Imipenem, gentamicin and ciprofloxacin were the most inhibitory antimicrobial agents. However, isolates were resistant or showed reduced susceptibility to ampicillin, chloramphenicol, sulphamethoxazole/trimethoprim and aztreonam, and up to 35% of the isolates were resistant to four antimicrobial agents. As many as 30% of the isolates were positive for the fpv1 gene, and 13% had multiple genes. Sixty-four percent of the isolates harboured an exoenzyme gene (exoS, exoT, exoU or exoY), and multiple exo genes were common. We conclude that P. aeruginosa is a major contaminant of fresh vegetables, which might be a source of infection for susceptible persons within the community.

Prevalence and Antimicrobial Resistance of Thermophilic Campylobacter Isolated from Chicken in Côte d’Ivoire

Tue, 23 Oct 2012 13:09:25 +0000

Thermophilic Campylobacters are major causes of gastroenteritis in human. The main risk factor of infection is consumption of contaminated or by cross-contaminated poultry meat. In Côte d’Ivoire, gastroenteritis is usually observed but no case of human campylobacteriosis has been formally reported to date. The aims of this study were to determine prevalence and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from chickens ceaca in commercial slaughter in Abidjan. Between May and November 2009, one hundred and nineteen (119) chicken caeca samples were collected and analyzed by passive filtration method followed by molecular identification (PCR). From these 119 samples, 76 (63.8%) were positive to Campylobacter tests. Among the positive colonies, 51.3% were C. jejuni and 48.7% were C. coli. Of the 39 C. jejuni isolates, 79.5%, 38.5%, 17.9%, 10.3%, and 7.7% were, respectively, resistant, to nalidixic acid, ciprofloxacin, amoxicillin, erythromycin, and gentamicin. Among the 37 isolates of C. coli, 78.4%, 43.2%, 13.5%, 8.1%, and 0% were resistant, respectively, to the same antibiotics. In conclusion, we reported in this study the presence of high Campylobacter contamination of the studied chickens. Molecular identification of the bacteria was performed and determination of high resistance to antimicrobials of the fluoroquinolone family was revealed.

Use of Self-Organizing Map to Analyze Images of Fungi Colonies Grown from Triticum aestivum Seeds Disinfected by Ozone Treatment

Thierry Marique, Olivier Allard, and Martin Spanoghe — Tue, 16 Oct 2012 10:11:47 +0000

We submitted to ozone treatment Triticum aestivum (common wheat) seeds severely contaminated by fungi. Fungi colonies developed when seeds were placed over malt agar medium in Petri dishes; Fusarium sp. and Alternaria sp. were identified. However, conventional colonies counting did not allow a clear assessment of the effect of ozone disinfection. We thus used self-organizing maps (SOMs) to perform an image analysis of colonies surface area that clearly showed a significant disinfection effect on Fusarium sp.

Diversity across Seasons of Culturable Pseudomonas from a Desiccation Lagoon in Cuatro Cienegas, Mexico

Tue, 09 Oct 2012 16:15:34 +0000

Cuatro Cienegas basin (CCB) is a biodiversity reservoir within the Chihuahuan desert that includes several water systems subject to marked seasonality. While several studies have focused on biodiversity inventories, this is the first study that describes seasonal changes in diversity within the basin. We sampled Pseudomonas populations from a seasonally variable water system at four different sampling dates (August 2003, January 2004, January 2005, and August 2005). A total of 70 Pseudomonas isolates across seasons were obtained, genotyped by fingerprinting (BOX-PCR), and taxonomically characterized by 16S rDNA sequencing. We found 35 unique genotypes, and two numerically dominant lineages (16S rDNA sequences) that made up 64% of the sample: P. cuatrocienegasensis and P. otitidis. We did not recover genotypes across seasons, but lineages reoccurred across seasons; P. cuatrocienegasensis was isolated exclusively in winter, while P. otitidis was only recovered in summer. We statistically show that taxonomic identity of isolates is not independent of the sampling season, and that winter and summer populations are different. In addition to the genetic description of populations, we show exploratory measures of growth rates at different temperatures, suggesting physiological differences between populations. Altogether, the results indicate seasonal changes in diversity of free-living aquatic Pseudomonas populations from CCB.

Microbial Translocation and Infectious Diseases: What Is the Link?

Thu, 04 Oct 2012 15:40:36 +0000

Polymorphic Amplified Typing Sequences and Pulsed-Field Gel Electrophoresis Yield Comparable Results in the Strain Typing of a Diverse Set of Bovine Escherichia coli O157:H7 Isolates

Sun, 23 Sep 2012 08:16:04 +0000

Polymorphic amplified typing sequences (PATS), a PCR-based Escherichia coli O157:H7 (O157) strain typing system, targets insertions-deletions and single nucleotide polymorphisms at XbaI and AvrII restriction enzyme sites, respectively, and the virulence genes (stx1, stx2, eae, hlyA) in the O157 genome. In this study, the ability of PATS to discriminate O157 isolates associated with cattle was evaluated. An in-depth comparison of 25 bovine O157 isolates, from different geographic locations across Northwest United States, showed that about 85% of these isolates shared the same dendogram clade by PATS and pulsed-field gel electrophoresis (PFGE), irrespective of the restriction enzyme sites targeted. The Pearson’s correlation coefficient, r, calculated at about 0.4, 0.3, and 0.4 for XbaI-based, AvrII-based and combined-enzymes PATS and PFGE similarities, respectively, indicating that these profiles shared a good but not high correlation, an expected inference given that the two techniques discriminate differently. Isolates that grouped differently were better matched to their locations using PATS. Overall, PATS discriminated the bovine O157 isolates without interpretive biases or sophisticated analytical software, and effectively complemented while not duplicating PFGE. With its quick turnaround time, PATS has excellent potential as a convenient tool for early epidemiological or food safety investigations, enabling rapid notification/implementation of quarantine measures.

Lipopolysaccharide-Induced Cellular Activation May Participate in the Immunopathogenesis of Visceral Leishmaniasis Alone or in HIV Coinfection

Joanna Reis Santos-Oliveira and Alda Maria Da-Cruz — Wed, 22 Aug 2012 09:16:24 +0000

Visceral Leishmaniasis (VL) is an infectious disease which constitutes a serious public health problem, integrating the list of neglected tropical diseases. The disease is characterized by a Leishmania-specific immune suppression T-cell depletion and a decrease of other hematopoietic cells. In parallel, an immunostimulatory response also occurs, represented by polyclonal B lymphocytes, T-cell activation, and systemic proinflammatory responses. Parasite antigens were believed to mediate both suppression and activation mechanisms, but these concepts are constantly being revised. Similar to reports on HIV/AIDS, we have proposed that gut parasitation by amastigotes and lymphocyte depletion could also affect gut-associated lymphoid tissue, leading to mucosal barrier breach and predisposing to microbial translocation. An increment of plasmatic lipopolysaccharide (LPS) levels observed in Brazilian VL patients was implicated in the reduced blood CD4+ and CD8+ T cell counts, systemic T-cell activation, pro-inflammatory cytokines and MIF plasma levels, suggesting that a bacterial molecule not associated with Leishmania infection can exert deleterious effects on immune system. Recent results also pointed that the proinflammatory response was potentiated in VL/HIV-AIDS coinfected patients. The LPS-mediated cell activation adds another concept to the immunopathogenesis of VL and can bring a rational for new therapeutic interventions that could ameliorate the management of these patients.

Differential Adaptations of Methicillin-Resistant Staphylococcus aureus to Serial In Vitro Passage in Daptomycin: Evolution of Daptomycin Resistance and Role of Membrane Carotenoid Content and Fluidity

Nagendra N. Mishra, Aileen Rubio, Cynthia C. Nast, and Arnold S. Bayer — Thu, 16 Aug 2012 09:10:49 +0000

Previous studies showed serial 20 d in vitro passage of MRSA strain MW2 in sublethal daptomycin (DAP) resulted in diverse perturbations in both cell membrane (CM) and cell wall (CW) characteristics, including increased CM rigidity; increased CW thickness; “gain-in-function” single nucleotide polymorphisms (SNPs) in the mprF locus (i.e., increased synthesis and translocation of lysyl-phosphatidylglycerol (L-PG)); progressive accumulation of SNPs in yyc and rpo locus genes; reduced carotenoid production; cross-resistance to innate host defense peptides. The current study was designed to characterize the reproducibility of these phenotypic and genotypic modifications following in vitro serial passages of the same parental strain. After a second 20d serial in vitro passage of parental MW2, emergence of DAP-R was associated with evolution of several phenotypes closely mirroring previous passage outcomes. However, in contrast to the initial serial passage strain set, we observed (i) only modest increase in L-PG synthesis and no increase in L-PG outer CM translocation; (ii) significantly increased carotenoid synthesis (𝑃<0.05); (iii) a different order of SNP accumulations (𝑚𝑝𝑟𝐹≫𝑟𝑝𝑜𝐵≫𝑦𝑦𝑐𝐺); (iv) a different cadre and locations of such SNPs. Thus, MRSA strains are not “pre-programmed” to phenotypically and/or genotypically adapt in an identical manner during induction of DAP resistance.

Gut Microbial Translocation in Critically Ill Children and Effects of Supplementation with Pre- and Pro Biotics

Wed, 15 Aug 2012 13:02:07 +0000

Bacterial translocation as a direct cause of sepsis is an attractive hypothesis that presupposes that in specific situations bacteria cross the intestinal barrier, enter the systemic circulation, and cause a systemic inflammatory response syndrome. Critically ill children are at increased risk for bacterial translocation, particularly in the early postnatal age. Predisposing factors include intestinal obstruction, obstructive jaundice, intra-abdominal hypertension, intestinal ischemia/reperfusion injury and secondary ileus, and immaturity of the intestinal barrier per se. Despite good evidence from experimental studies to support the theory of bacterial translocation as a cause of sepsis, there is little evidence in human studies to confirm that translocation is directly correlated to bloodstream infections in critically ill children. This paper provides an overview of the gut microflora and its significance, a focus on the mechanisms employed by bacteria to gain access to the systemic circulation, and how critical illness creates a hostile environment in the gut and alters the microflora favoring the growth of pathogens that promote bacterial translocation. It also covers treatment with pre- and pro biotics during critical illness to restore the balance of microbial communities in a beneficial way with positive effects on intestinal permeability and bacterial translocation.

Antimicrobial Activity of Xoconostle Pears (Opuntia matudae) against Escherichia coli O157:H7 in Laboratory Medium

Saeed A. Hayek and Salam A. Ibrahim — Wed, 15 Aug 2012 09:02:29 +0000

The objective of this study was to investigate the antimicrobial activity of xoconostle pears (Opuntia matudae) against Escherichia coli O157:H7. Xoconostle pears were sliced, blended, and centrifuged. The supernatant was then filtered using a 0.45 μm filter to obtain direct extract. Direct extract of xoconostle pears was tested against four strains of E. coli O157:H7 in brain heart infusion (BHI) laboratory medium using growth over time and agar well diffusion assays. Our results showed that direct extract of xoconostle pears had a significant (𝑃<0.05) inhibitory effect at 4, 6, and 8% (v/v) concentrations and complete inhibitory effect at 10% (v/v) during 8 h of incubation at 37°C. Minimum inhibitory volume (MIV) was 400 μL mL−1 (v/v) and minimum lethal volume (MLV) was 650 μL mL−1 (v/v). The inhibitory effect of xoconostle pears found to be concentration dependent and not strain dependent. Thus, xoconostle pears extract has the potential to inhibit the growth of E. coli O157:H7 and could provide a natural means of controlling pathogenic contamination, thereby mitigating food safety risks.

Advances in Bacteriophage-Mediated Control of Plant Pathogens

Rebekah A. Frampton, Andrew R. Pitman, and Peter C. Fineran — Mon, 13 Aug 2012 13:33:41 +0000

There is continuing pressure to maximise food production given a growing global human population. Bacterial pathogens that infect important agricultural plants (phytopathogens) can reduce plant growth and the subsequent crop yield. Currently, phytopathogens are controlled through management programmes, which can include the application of antibiotics and copper sprays. However, the emergence of resistant bacteria and the desire to reduce usage of toxic products that accumulate in the environment mean there is a need to develop alternative control agents. An attractive option is the use of specific bacteriophages (phages), viruses that specifically kill bacteria, providing a more targeted approach. Typically, phages that target the phytopathogen are isolated and characterised to determine that they have features required for biocontrol. In addition, suitable formulation and delivery to affected plants are necessary to ensure the phages survive in the environment and do not have a deleterious effect on the plant or target beneficial bacteria. Phages have been isolated for different phytopathogens and have been used successfully in a number of trials and commercially. In this paper, we address recent progress in phage-mediated control of plant pathogens and overcoming the challenges, including those posed by CRISPR/Cas and abortive infection resistance systems.

Aromatic Compound-Dependent Staphylococcus aureus Is Safe in a Nasal Colonization Leukopenic Murine Model

Sun, 05 Aug 2012 10:20:54 +0000

Staphylococcus aureus nasal carriage is a risk factor for individuals suffering from trauma, surgical procedures, invasive devices, and/or decreased immunity. Recently, we demonstrated that artificial nasal colonization with an attenuated S. aureus mutant reduced by bacterial interference with the colonization of pathogenic strains of S. aureus. This could be an optional tool to diminish the rate of S. aureus infections in hospitalized patients. The aim of this study was to construct a safe ΔaroA mutant of S. aureus and to discriminate it from nasal colonizing and osteomyelitis S. aureus isolates by SmaI pulsed-field gel electrophoresis (PFGE) typing. The ΔaroA mutant, named RD17, exhibited an LD50 (3.2 × 106 colony-forming unit (CFU)) significantly higher than that of the parental strain (2.2 × 103 CFU). The colony number of the RD17 mutants recovered from nares of leukopenic mice was similar to that observed in the animals of the control group. Therefore, the ΔaroA mutant was demonstrated to be safe due to maintaining low growth levels in the nares regardless of immune status of the animals. PFGE typing allowed the unequivocal identification of the S. aureus and differentiation of aroA mutants in nasal colonizing and osteomyelitis isolates. This information could be important to discriminate endogenous infections from laboratory strains of S. aureus.

Comparison of Disk Diffusion and Etest Methods to Determine the Susceptibility of Staphylococcus aureus Circulating in Riyadh, Saudi Arabia to Fusidic Acid

Ali M. Somily, David R. Peaper, Elijah Paintsil, and Thomas S. Murray — Tue, 24 Jul 2012 09:05:10 +0000

Fusidic acid is a common therapy for staphylococcal infections in Saudi Arabia, but reports have suggested high rates of resistance among clinical isolates. Susceptibility testing of S. aureus to fusidic acid is further complicated by the lack of consensus on mean inhibitory concentrations (MIC) and disk diffusion cutoffs to determine resistance. The purpose of this study was to determine the correlation between disk diffusion and Etest determined MIC susceptibility results in clinical isolates of S. aureus from a large academic hospital in Riyadh, Saudi Arabia. Our data demonstrate excellent correlation between Etest determined MIC and disk diffusion susceptibility data, using either previously proposed zone sizes of ≥21 mm as susceptible and ≤18 mm as resistant or the EUCAST recommended zone size of ≤24 mm for resistance, in an area with relatively high rates of fusidic acid resistance.

Microbial Translocation in Chronic Liver Diseases

Tue, 17 Jul 2012 08:01:41 +0000

The intestinal microflora is not only involved in the digestion of nutrients, but also in local immunity, forming a barrier against pathogenic microorganisms. The derangement of the gut microflora may lead to microbial translocation, defined as the passage of viable microorganisms or bacterial products (i.e., LPS, lipopeptides) from the intestinal lumen to the mesenteric lymph nodes and other extraintestinal sites. The most recent evidence suggests that microbial translocation (MT) may occur not only in cirrhosis, but also in the early stage of several liver diseases, including alcoholic hepatopathy and nonalcoholic fatty liver disease. Different mechanisms, such as small intestinal bacterial overgrowth, increased permeability of intestinal mucosa, and impaired immunity, may favor MT. Furthermore, MT has been implicated in the pathogenesis of the complications of cirrhosis, which are a significant cause of morbidity and mortality in cirrhotic subjects. Therapeutic strategies aiming at modulating the gut microflora and reducing MT have focused on antibiotic-based options, such as selective intestinal decontamination, and nonantibiotic-based options, such as prokinetics and probiotics. In particular, probiotics may represent an attractive strategy, even though the promising results of experimental models and limited clinical studies need to be confirmed in larger randomized trials.

Plant Growth Promoting of Endophytic Sporosarcina aquimarina SjAM16103 Isolated from the Pneumatophores of Avicennia marina L.

S. Rylo Sona Janarthine and P. Eganathan — Tue, 03 Jul 2012 08:49:12 +0000

Endophytic Sporosarcina aquimarina SjAM16103 was isolated from the inner tissues of pneumatophores of mangrove plant Avicennia marina along with Bacillus sp. and Enterobacter sp. Endophytic S. aquimarina SjAM16103 was Gram variable, and motile bacterium measured 0.6–0.9 μm wide by 1.7–2.0 μm long and light orange-brown coloured in 3-day cultures on tryptone broth at 26°C. Nucleotide sequence of this strain has been deposited in the GenBank under accession number GU930359. This endophytic bacterium produced 2.37 μMol/mL of indole acetic acid and siderophore as it metabolites. This strain could solubilize phosphate molecules and fixes atmospheric nitrogen. Endophytic S. aquimarina SjAM16103 was inoculated into four different plants under in vitro method to analyse its growth-promoting activity and role inside the host plants. The growth of endophytic S. aquimarina SjAM16103 inoculated explants were highly significant than the uninoculated control explants. Root hairs and early root development were observed in the endophytic S. aquimarina SjAM16103 inoculated explants.

Antimicrobial Peptides as Therapeutic Agents

Mathew Upton, Paul Cotter, and John Tagg — Tue, 19 Jun 2012 10:22:30 +0000

Listeria monocytogenes in Ready-to-Eat Seafood and Potential Hazards for the Consumers

Sat, 16 Jun 2012 15:55:55 +0000

The risk of exposure to Listeria monocytogenes (L. monocytogenes) when consuming Ready-to-Eat (RTE) seafood was assessed in the Veneto Region (Italy). Thirty-eight samples were analyzed, each sample consisted of three subunits belonging to the same batches. The first of the three units was examined immediately, the second was stored at +4°C (for all of its shelf-life) and the third at +10°C (for the latter third of its shelf-life) before the analysis. Chemical-physical and microbiological parameters were tested simultaneously. Culture results showed the presence of viable L. monocytogenes in 9 (23,68%) of the 38 samples analysed, 3 (33,33%) of which with a concentration >100 cfu/g. PCR tests yielded 12 L. monocytogenes positive samples. Semipreserves with aw (water activity) and pH values that favour L. monocytogenes growth were the only ones to result positive to microbiological and PCR tests. Temperature proved to be an important factor as it limits the growth of L. monocytogenes, including products with potentially high competitive microbial charges. Four different serotypes were recovered and ribotyping has helped to highlight the genomic variability of L. monocytogenes strains in food. This supports the hypothesis that L. monocytogenes continues to evolve genetically to the detriment of phenotypic conservation.

Impact of HMGB1/TLR Ligand Complexes on HIV-1 Replication: Possible Role for Flagellin during HIV-1 Infection

Wed, 06 Jun 2012 09:24:34 +0000

Objective. We hypothesized that HMGB1 in complex with bacterial components, such as flagellin, CpG-ODN, and LPS, promotes HIV-1 replication. Furthermore, we studied the levels of antiflagellin antibodies during HIV-1-infection. Methods. Chronically HIV-1-infected U1 cells were stimulated with necrotic extract/recombinant HMGB1 in complex with TLR ligands or alone. HIV-1 replication was estimated by p24 antigen in culture supernatants 48–72 hours after stimulation. The presence of systemic anti-flagellin IgG was determined in 51 HIV-1-infected patients and 19 controls by immunoblotting or in-house ELISA. Results. Flagellin, LPS, and CpG-ODN induced stronger HIV-1 replication when incubated together with necrotic extract or recombinant HMGB1 than activation by any of the compounds alone. Moreover, the stimulatory effect of necrotic extract was inhibited by depletion of HMGB1. Elevated levels of anti-flagellin antibodies were present in plasma from HIV-1-infected patients and significantly decreased during 2 years of antiretroviral therapy. Conclusions. Our findings implicate a possible role of HGMB1-bacterial complexes, as a consequence of microbial translocation and cell necrosis, for immune activation in HIV-1 pathogenesis. We propose that flagellin is an important microbial product, that modulates viral replication and induces adaptive immune responses in vivo.

Host-Fungal Interactions: Pathogenicity versus Immunity

Arianna Tavanti, Julian R. Naglik, and Nir Osherov — Sun, 27 May 2012 11:09:58 +0000

Comparative Genomic Analysis of Lactococcus garvieae Strains Isolated from Different Sources Reveals Candidate Virulence Genes

Eiji Miyauchi, Hidehiro Toh, Akiyo Nakano, Soichi Tanabe, and Hidetoshi Morita — Wed, 09 May 2012 12:15:01 +0000

Lactococcus garvieae is a major pathogen for fish. Two complete (ATCC 49156 and Lg2) and three draft (UNIUD074, 8831, and 21881) genome sequences of L. garvieae have recently been released. We here present the results of a comparative genomic analysis of these fish and human isolates of L. garvieae. The pangenome comprised 1,542 core and 1,378 dispensable genes. The sequenced L. garvieae strains shared most of the possible virulence genes, but the capsule gene cluster was found only in fish-pathogenic strain Lg2. The absence of the capsule gene cluster in other nonpathogenic strains isolated from mastitis and vegetable was also confirmed by PCR. The fish and human isolates of L. garvieae contained the specific two and four adhesin genes, respectively, indicating that these adhesion proteins may be involved in the host specificity differences of L. garvieae. The discoveries revealed by the pangenomic analysis may provide significant insights into the biology of L. garvieae.

Relationship between the Presence of the nalC Mutation and Multidrug Resistance in Pseudomonas aeruginosa

Mon, 07 May 2012 14:51:15 +0000

Objectives. The current study was conducted to determine the relationship between the presences of significant multidrug resistance in Pseudomonas aeruginosa (P. aeruginosa) having intact mexR genes (nalC) to different antibiotics. Methods. In order to identify nalC, fifty strains of P. aeruginosa were obtained. All isolates were found in urinary tract infections. They were evaluated against different antibiotics. The nalC mutant was identified by PCR. Results. The 50 clinical isolates of P. aeruginosa originated from two hospitals in Iran, in which 32 isolates were found in Milad hospital, and 18 isolates were collected in the Ilam Hospital. The results in Milad hospital of nalC revealed that all P. aeruginosa resistant to oxacillin showed the presence of nalC. In Ilam hospital only three (16.6%) isolates were resistant to oxacilin and aztreonam, and among these three isolates only one isolate revealed resistance to ceftazidime and amikacin. The resistant isolates showed the presence of both OXA-10 and nalC. Conclusion. Our results showed that the presence of nalC was observed among P. aeruginosa resistance to oxacilin. Thus, the finding suggested relationship between oxacilin resistance and presence of nalC and consequently overproduction of the MexABOprM efflux system.