Molecular Characterization of Skin Microbiota Between Cancer Cachexia Patients and Healthy Volunteers

Systemic inflammation contributes to both the development of cancer and of cachexia. The microenvironment of bacterial habitats might be changed during the progression of cancer cachexia. The aim of this study was to quantitatively and qualitatively compare the composition of the skin microbiota between cancer cachexia patients and healthy volunteers. Cutaneous bacteria were swabbed at the axillary fossa of 70 cancer cachexia patients and 34 healthy individuals from China. Nested-PCR-denaturing gradient gel electrophoresis (PCR-DGGE) with primers specifically targeting V3 region and quantitative PCR (qPCR) for total bacteria, Corynebacterium spp., Staphylococcus spp., and Staphylococcus epidermidis were performed on all samples. Barcoded 454 pyrosequencing of the V3–V4 regions was performed on 30 randomly selected samples. By comparing diversity and richness indices, we found that the skin microbiome of cachectic cancer patients is less diverse than that of healthy participants, though these differences were not significant. The main microbes that reside on human skin were divided into four phyla: Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes. Staphylococcus spp. and Corynebacterium spp. were the dominant bacteria at the genus level. Significantly fewer Corynebacterium spp. had been observed in cachexia patients compared to healthy subjects. These results suggest that the presence of cancer and cachexia alters human skin bacterial communities. Understanding the changes in microbiota during cancer cachexia may lead to new insights into the syndrome.     

Novel methicillin-resistant coagulase-negative Staphylococcus clone isolated from patients with haematological diseases at the Blood Bank Centre of Amazon,Brazil

Methicillin-resistant Staphylococcus remains a severe public health problem worldwide. This research was intended to identify the presence of methicillin-resistant coagulase-negative staphylococci clones and their staphylococcal cassette chromosome mec (SCCmec)-type isolate from patients with haematologic diseases presenting bacterial infections who were treated at the Blood Bank of the state of Amazonas in Brazil. Phenotypic and genotypic tests, such as SCCmec types and multilocus sequence typing (MLST), were developed to detect and characterise methicillin-resistant isolates. A total of 26 Gram-positive bacteria were isolated, such as: Staphylococcus epidermidis (8/27), Staphylococcus intermedius (4/27) and Staphylococcus aureus (4/27). Ten methicillin-resistant staphylococcal isolates were identified. MLST revealed three different sequence types: S. aureus ST243, S. epidermidis ST2 and a new clone of S. epidermidis, ST365. These findings reinforce the potential of dissemination presented by multi-resistant Staphylococcus and they suggest the introduction of monitoring actions to reduce the spread of pathogenic clonal lineages of S. aureus and S. epidermidis to avoid hospital infections and mortality risks.     

Bacterial community involved in traditional fermented soybean paste dajiang made in northeast China

Dajiang is a traditional fermented food prepared from soybeans that is still popular in northeast China. Although recent studies have revealed that a variety of bacterial species contribute to the production of fermented soybean products, little is known about bacterial communities involved in the fermentation of dajiang made in northeast China. In this study, 14 samples of naturally fermented dajiang were analyzed by denaturing gradient gel electrophoresis (DGGE) to determine the diversity of the bacteria involved in fermentation. Our results indicate that lactic acid bacteria, including Lactobacillus plantarum, uncultured Leuconostoc mesenteroides, Leuconostoc gasicomitatum, Enterococcus faecium, and Tetragenococcus halophilus, were the predominant species. This is the first report of Enterococcus spp. and Leuconostoc spp. in the Chinese fermented soybean paste dajiang using DGGE. The presence of Bacillus spp. (including Bacillus firmus), Oceanobacillus spp., and Paenibacillus glycanilyticus in the dajiang samples may be due to their salt tolerance. Potentially pathogenic Alphaproteobacteria and Staphylococcus epidermidis strains were also detected in this study. Moreover, three uncultured bacterial clones were found in some samples and require further study. The results reveal a high level of bacterial diversity in dajiang.     

Fluorescence-Based Comparative Evaluation of Bactericidal Potency and Food Application Potential of Anti-listerial Bacteriocin Produced by Lactic Acid Bacteria Isolated from Indigenous Samples

The aim of the present study was to ascertain the potency of anti-listerial bacteriocin produced by lactic acid bacteria (LAB) isolated from indigenous samples of dahi, dried fish, and salt-fermented cucumber. A total of 231 LAB isolates were obtained from the samples, of which 51 isolates displayed anti-listerial activity. The anti-listerial LAB were identified by PCR as Lactobacillus sp., Pediococcus sp., Enterococcus sp., and Lactococcus sp. PCR also enabled the detection of Class IIa bacteriocin-encoding genes such as enterocin A, pediocin, and plantaricin A in some of the LAB isolates. The culture filtrate from anti-listerial LAB isolates demonstrated bacteriocin-like inhibitory substance (BLIS) against common Gram-positive pathogenic bacteria such as Staphylococcus aureus, Enterococcus faecalis, and Bacillus cereus, and partial characterization of BLIS confirmed the production of bacteriocin by the LAB isolates. Sensitive fluorescence-based assays employing specific probes indicated the comparative potencies of the bacteriocin and clearly revealed the membrane-targeted anti-listerial activity of the purified bacteriocin produced by selected LAB isolates. The food application potential of plantaricin A produced by a native isolate Lactobacillus plantarum CRA52 was evidenced as the bacteriocin suppressed the growth of Listeria monocytogenes Scott A inoculated in paneer samples that were stored at 8?°C for 5?days.     

Microbial Quality and Direct PCR Identification of Lactic Acid Bacteria and Nonpathogenic Staphylococci from Artisanal Low-Acid Sausages

Detection of six species of lactic acid bacteria and six species of gram-positive catalase-positive cocci from low-acid fermented sausages (fuets and chorizos) was assessed by species-specific PCR. Without enrichment, Lactobacillus sakei and Lactobacillus curvatus were detected in 11.8% of the samples, and Lactobacillus plantarum and Staphylococcus xylosus were detected in 17.6%. Enriched samples allowed the detection of L. sakei and S. xylosus in all of the samples (100%) and of Enterococcus faecium in 11.8% of the sausages. The percentages of L. curvatus, L. plantarum, Staphylococcus carnosus, and Staphylococcus epidermidis varied depending on the sausage type. L. curvatus was detected in 80% of fuets and in 57% of chorizos. L. plantarum was found in 50% of fuets and 100% of chorizos. S. epidermidis was detected in only 11.8% of fuets, and S. carnosus was detected in only 5.9% of chorizos. Lactococcus lactis, Staphylococcus warneri, and Staphylococcus simulans were not detected in any sausage type. From a microbiological point of view, 70.6% of the samples could be considered of high quality, as they had low counts of Enterobacteriaceae and did not contain any of the food-borne pathogens assayed.     

Recognition of (Sesc) for Easy Identification of Staphylococcus Epidermidis and Molecular and Phenotypic Study of Β-Lactam Resistance in Staphylococcus Epidermidis Isolates in Isfahan

Background:Not only is it crucial to rapidly detect Staphylococcus epidermidis (S. epidermidis) isolates from a broad range of bacteria, but recognizing resistance agents can greatly improve current diagnostic and therapeutic strategies.Methods:The current cross-sectional study investigated 120 clinical isolates from a nosocomial S. epidermidis infection. The isolates were identified using common biochemical tests, and specific S. epidermidis surface protein C (SesC) primers were used to confirm the presence of S. epidermidis. PCR and special primers were used to detect the β-lactamase gene (blaZ). Methicillin resistance was measured using the agar screening method and antibiotic susceptibility was measured by disk diffusion. Results:100 samples were characterized as S. epidermidis using a phenotypic and genotypic methods. From the 100 specimens examined, 80% contained blaZ. According to agar screening, 60% of isolates were methicillin-resistant. S. epidermidis isolates demonstrated the highest resistance to penicillin (93%) and the highest sensitivity to cefazolin (39%).Conclusion:The increased resistance to β-lactam antibiotics in S. epidermidis isolates is alarming, and certain precautions should be taken by healthcare systems to continuously monitor the antimicrobial pattern of S. epidermidis, so that an appropriate drug treatment can be established.Key Words: Antibiotic resistance, β-lactam, Staphylococcus epidermidis     

Case Study of the Distribution of Mucosa-Associated Bifidobacterium Species, Lactobacillus Species, and Other Lactic Acid Bacteria in the Human Colon

The distribution of mucosa-associated bacteria, bifidobacteria and lactobacilli and closely related lactic acid bacteria, in biopsy samples from the ascending, transverse, and descending parts of the colon from four individuals was investigated by denaturing gradient gel electrophoresis (DGGE). Bifidobacterial genus-specific, Lactobacillus group-specific, and universal bacterial primers were used in a nested PCR approach to amplify a fragment of the 16S rRNA gene. DGGE profiles of the bifidobacterial community were relatively simple, with one or two amplicons detected at most sampling sites in the colon. DGGE profiles obtained with Lactobacillus group-specific primers were complex and varied with host and sampling site in the colon. The overall bacterial community varied with host but not sampling site.     

传统豆酱发酵过程中细菌多样性动态

细菌在豆酱发酵过程中起到非常重要的作用,并与豆酱的风味和质量密切相关,因此研究豆酱中细菌的多样性具有重要意义。以自然发酵的豆酱样品为研究对象,采用细菌16S rDNA的部分可变区的PCR-DGGE技术对自然发酵豆酱样品的细菌群落组成和优势菌群进行研究。结果表明,传统豆酱发酵过程细菌群体中既有原始种群的减少和增长,也有次级种群的增多和演变。在整个发酵过程中,初期和末期以不可培养细菌为主,初期细菌群体快速演替,细菌种群多样性指数在发酵42 d和56 d达到两次高峰。     

PCR detection of nitrite reductase genes (nirK and nirS) and use of active consortia of constructed ternary adherent staphylococcal cultures via mixture design for a denitrification process

In this study, three adherent Staphylococcus strains able to use nitrate as electron acceptor were isolated from textile wastewater activated sludge. Based on the biochemical profiles, bacterial strains were identified as Staphylococcus lentus, Staphylococcus warneri and Staphylococcus epidermidis the PCR amplification of the nir genes reveal that S. lentus and S. epidermidis were nirK positive and that S. epidermidis was nirS positive. The three strains were also icaA/icaD positive. To obtain the optimal formulation of pure cultures of the staphylococci, the influence of the different mixtures of organisms was studied using mixture design. The regression model of microorganism composition and main metabolites was established. The most predictable reduction of nitrate was 87.2 and 12% of COD consumption. The results suggested that the predictable production of nitrite would reach a minimum of 6.1 mg/l. Based on this, the response values that satisfied all expectations were optimized using MINITAB^® 14 analysis software. The most optimal proportion combination was 49.75% of S. lentus (curve value), 36.85% of S. warneri (curve value) and 13.39% S. epidermidis (curve value).     

Nested PCR-Denaturing Gradient Gel Electrophoresis Approach To Determine the Diversity of Sulfate-Reducing Bacteria in Complex Microbial Communities

Here, we describe a three-step nested-PCR-denaturing gradient gel electrophoresis (DGGE) strategy to detect sulfate-reducing bacteria (SRB) in complex microbial communities from industrial bioreactors. In the first step, the nearly complete 16S rRNA gene was amplified using bacterial primers. Subsequently, this product was used as a template in a second PCR with group-specific SRB primers. A third round of amplification was conducted to obtain fragments suitable for DGGE. The largest number of bands was observed in DGGE patterns of products obtained with primers specific for the Desulfovibrio-Desulfomicrobium group, indicating a large diversity of these SRBs. In addition, members of other phylogenetic SRB groups, i.e., Desulfotomaculum, Desulfobulbus, and Desulfococcus-Desulfonema-Desulfosarcina, were detected. Bands corresponding to Desulfobacterium and Desulfobacter were not detected in the bioreactor samples. Comparative sequence analysis of excised DGGE bands revealed the identity of the community members. The developed three-step PCR-DGGE strategy is a welcome tool for studying the diversity of sulfate-reducing bacteria.     

Ecological Succession and Viability of Human-Associated Microbiota on Restroom Surfaces

Human-associated bacteria dominate the built environment (BE). Following decontamination of floors, toilet seats, and soap dispensers in four public restrooms, in situ bacterial communities were characterized hourly, daily, and weekly to determine their successional ecology. The viability of cultivable bacteria, following the removal of dispersal agents (humans), was also assessed hourly. A late-successional community developed within 5 to 8 h on restroom floors and showed remarkable stability over weeks to months. Despite late-successional dominance by skin- and outdoor-associated bacteria, the most ubiquitous organisms were predominantly gut-associated taxa, which persisted following exclusion of humans. Staphylococcus represented the majority of the cultivable community, even after several hours of human exclusion. Methicillin-resistant Staphylococcus aureus (MRSA)-associated virulence genes were found on floors but were not present in assembled Staphylococcus pan-genomes. Viral abundances, which were predominantly enterophages, human papilloma virus, and herpesviruses, were significantly correlated with bacterial abundances and showed an unexpectedly low virus-to-bacterium ratio in surface-associated samples, suggesting that bacterial hosts are mostly dormant on BE surfaces.     

A novel application of Fourier-transformed infrared spectroscopy: classification of slime from staphylococci

Abstract

It has been proposed that the virulence of nosocomial Staphylococcus infections associated with indwelling medical devices is related to the ability of the bacterium to colonise these materials by forming a biofilm composed of multilayered cell clusters embedded in a slime matrix. However, the pathogenic role of exopolysaccharide biofilms is not fully understood. A new method was sought for differentiating the structure of slime from two closely related bacterial strains, Staphylococcus aureus and Staphylococcus epidermidis. Using PCR it was confirmed that these strains were positive for the icaA and icaD genes and the complete ica operon (2.7 kb). Monosaccharide analysis by thin-layer chromatography revealed an identical profile for both strains, with xylose and glucose present among the four visible bands. Using Fourier-transformed infrared spectroscopy and hierarchical cluster analysis, three of four S. aureus samples (75%), and four of five S. epidermidis samples were grouped according to species. A novel FTIR approach in classifying slime produced by S. aureus and S. epidermidis is reported.     

Antibiotic-resistant Staphylococcus epidermidis isolated from patients and healthy students comparing with antibiotic-resistant bacteria isolated from pasteurized milk

Antibiotic-resistant Staphylococci are a global issue affecting humans, animals, and numerous natural environments. Antibiotic-resistant Staphylococcus epidermidis is an opportunistic pathogen frequently isolated from patients and healthy individuals. This study aimed to examine the antibiotic resistance of S. epidermidis isolated from patients, healthy students and compare the results with antibiotic-resistant bacteria isolated from pasteurized milk. Clinical strain isolation was performed in several hospitals in the Riyadh. Skin swabs from 100 healthy undergraduate candidate students were obtained at King Saud University. The pasteurized milk samples were obtained from local market (company, X). After isolation, identification and susceptibility tests were performed using an automated system. A multiplex tuf gene-based PCR assay was used to confirm identification. Biofilm production and biofilm-related gene expression were studied. S. epidermidis represented 17% of clinical bacterial isolates, and 1.7% of isolates obtained from healthy students were multiantibiotic-resistant. All patient strains were teicoplanin- and vancomycin-susceptible, while all student strains were gentamicin-, levofloxacin-, moxifloxacin-, and trimethoprim/sulfamethoxazole-susceptible. All the bacteria isolated from pasteurized milk were benzylpenicillin and oxacillin-resistant strains. Of the S. epidermidis strains, 91% could produce biofilms, and mecA, icaADBR, ica-ADB, ica-AD, ica-A only, and ica-C only were expressed in 83, 17.1, 25.7, 37.1, 20, and 0% of the strains, respectively. This work demonstrates that S. epidermidis can be accurately identified using a multiplex tuf-based assay, and that multiantibiotic-resistant S. epidermidis strains are widespread amongst patients and healthy students.     

Communities of arbuscular mycorrhizal fungi and bacteria in the rhizosphere of Caragana korshinkii and Hippophae rhamnoides in Zhifanggou watershed

The process of ecological restoration and reconstruction in Zhifanggou watershed forms a special ecosystem on the Loess Plateau. Little is known about the communities of arbuscular mycorrhizal fungi (AMF) and bacteria in this ecosystem. The aim of this study was to analyze the communities of AMF and bacteria, and their relationship in the rhizosphere of Caragana korshinkii and Hippophae rhamnoides in Zhifanggou watershed. Soil samples were collected from Zhifanggou watershed. The communities of AMF and bacteria were analyzed by using nested PCR of rDNA fragments and denaturing gradient gel electrophoresis (DGGE). Diversity analysis revealed that the bacterial Shannon diversity index was higher than that of AMF, and the AMF and bacterial Shannon diversity index in the rhizosphere of H. rhamnoides was higher than that of C. korshinkii. Principal component analysis (PCA) revealed that host plant had a significant influence on the bacterial community structure, but no strict specificity with AMF. Correlation analysis showed that the AMF communities had a significant positive correlation with the bacterial communities, and that indicated a significant effect of AMF on bacteria.     

Bacterial Community Structure in the Hyperarid Core of the Atacama Desert, Chile

Soils from the hyperarid Atacama Desert of northern Chile were sampled along an east-west elevational transect (23.75 to 24.70°S) through the driest sector to compare the relative structure of bacterial communities. Analysis of denaturing gradient gel electrophoresis (DGGE) profiles from each of the samples revealed that microbial communities from the extreme hyperarid core of the desert clustered separately from all of the remaining communities. Bands sequenced from DGGE profiles of two samples taken at a 22-month interval from this core region revealed the presence of similar populations dominated by bacteria from the Gemmatimonadetes and Planctomycetes phyla.     

Dandruff Is Associated with Disequilibrium in the Proportion of the Major Bacterial and Fungal Populations Colonizing the Scalp

The bacterial and fungal communities associated with dandruff were investigated using culture-independent methodologies in the French subjects. The major bacterial and fungal species inhabiting the scalp subject’s were identified by cloning and sequencing of the conserved ribosomal unit regions (16S for bacterial and 28S-ITS for fungal) and were further quantified by quantitative PCR. The two main bacterial species found on the scalp surface were Propionibacterium acnes and Staphylococcus epidermidis, while Malassezia restricta was the main fungal inhabitant. Dandruff was correlated with a higher incidence of M. restricta and S. epidermidis and a lower incidence of P. acnes compared to the control population (p<0.05). These results suggested for the first time using molecular methods, that dandruff is linked to the balance between bacteria and fungi of the host scalp surface.     

Electrical protein array chips for the detection of staphylococcal virulence factors

A new approach for the detection of virulence factors of Staphylococcus aureus and Staphylococcus epidermidis using an electrical protein array chip technology is presented. The procedure is based on an enzyme-linked sandwich immunoassay, which includes recognition and binding of virulence factors by specific capture and detection antibodies. Detection of antibody-bound virulence factors is achieved by measuring the electrical current generated by redox recycling of an enzymatically released substance. The current (measured in nanoampere) corresponds to the amount of the target molecule in the analyzed sample. The electrical protein chip allows for a fast detection of Staphylococcus enterotoxin B (SEB) of S. aureus and immunodominant antigen A homologue (IsaA homologue) of S. epidermidis in different liquid matrices. The S. aureus SEB virulence factor could be detected in minimal medium, milk, and urine in a concentration of 1 ng/ml within less than 23 min. Furthermore, a simultaneous detection of SEB of S. aureus and IsaA homologue of S. epidermidis in a single assay could be demonstrated.     

Identification of pathogenic bacteria in blood cultures: Comparison between conventional and PCR methods

Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Acinetobacter baumanii, and Klebsiella pneumoniae were found to be the most prevalent bacteremia-causing bacteria in a survey in a medical center. A PCR method for identification of these five most common pathogens in blood cultures was developed. A unique sequence was chosen for each pathogen and used for primer design. Sixty-one blood samples (from hospitalized patients) in which bacterial growth was detected were processed in parallel by conventional microbiological methods and by the PCR method. The results obtained by PCR were identical to those obtained by conventional methods in 93.4% of the cases. PCR failed to identify bacteria which were found conventionally in only 6.6% of the cases (mostly bacteria not included in the PCR cassette). Another group of eighty-eight blood samples from patients were processed immediately upon their arrival at the laboratory by taking aliquots for the PCR method. The blood sample bottles were processed in parallel by conventional methods. In 78.4% of the cases the results of both methods were identical. In 12.5% of the cases, PCR afforded identification of bacteria but conventional methods showed no bacteria in the sample. On the other hand, PCR afforded 9.1% negative results while conventional methods identified bacteria not included in the PCR cassette. It is concluded that the molecular method appears to be a specific and precise method for identifying pathogenic bacteria in blood samples.     

Antibiotic resistance profiles of coagulase-positive and coagulase-negative staphylococci from pit latrine fecal sludge in a peri-urban South African community

The aim of this study was to assess pit latrine samples from a peri-urban community in KwaZulu-Natal (South Africa) for the presence of multidrug-resistant (MDR) Staphylococcus spp. Standard procedures were used to isolate Staphylococcus spp. from pit latrine fecal sludge samples, with confirmation at genus level by polymerase chain reaction (PCR). Sixty-eight randomly selected pit latrine Staphylococcus spp. isolates were further characterized by using established disk diffusion procedures. An average Staphylococcus spp. count of 2.1?×?10⁵ CFU per g fecal material was established using two randomly selected pit latrine samples. Of the 68-selected Staphylococcus spp. pit latrine isolates, 49% were identified as coagulase positive, 51% as coagulase negative and 65% (12 coagulase positive, 32 coagulase negative isolates) were categorized as MDR. The majority (66/68) of Staphylococcus spp. isolates displayed resistance to fusidic acid while only 5/68 isolates displayed resistance to chloramphenicol. The pit latrine samples analyzed in this study are a source of MDR Staphylococcus spp., highlighting the need for proper hygiene and sanitation regimes in rural communities using these facilities.     

Changes in bacterial communities from swine feces during continuous culture with starch

Bacteria from swine feces were grown in continuous culture with starch as the sole carbohydrate in order to monitor changes during fermentation and to determine how similar fermenter communities were to each other. DNA extracted from fermenter samples was analyzed by denaturing gradient gel electrophoresis (DGGE). A significant decrease in diversity was observed, the Shannon–Weaver index dropped from 1.92 to 1.13 after 14 days of fermentation. Likewise, similarity of fermenter communities to those in the fecal inoculum also decreased over time. Both diversity and similarity to the inoculum decreased most rapidly in the first few days of fermentation, reflecting a period of adaptation. Sequencing of DGGE bands indicated that the same species were present in replicate fermenters. Most of these bacteria were placed in the Clostridium coccoides/Eubacterium rectale group (likely saccharolytic butyrate producers), a dominant bacterial group in the intestinal tract of pigs. DGGE proved useful to monitor swine fecal communities in vitro and indicated the selection and maintenance of native swine intestinal bacteria during continuous culture.