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Here, we assessed the extraction efficiency of a deployable bench-top nucleic acid extractor EZ1 in comparison to the column-based approach with complex sample matrices.A total of 48 EDTA blood samples and 81 stool samples were extracted by EZ1 automated extraction and the column-based QIAamp DNA Mini Kit. Blood sample extractions were assessed by two real-time malaria PCRs, while stool samples were analyzed by six multiplex real-time PCR assays targeting bacterial, viral, and parasitic stool pathogens. Inhibition control PCR testing was performed as well.In total, 147 concordant and 13 discordant pathogen-specific PCR results were obtained. The latter comprised 11 positive results after column-based extraction only and two positive results after EZ1 extraction only. EZ1 extraction showed a higher frequency of inhibition. This phenomenon was, however, inconsistent for the different PCR schemes. In case of concordant PCR results, relevant differences of cycle threshold numbers for the compared extraction schemes were not observed.Switches from well-established column-based extraction to extraction with the automated EZ1 system do not lead to a relevantly reduced yield of target DNA when complex sample matrices are used. If sample inhibition is observed, column-based extraction from another sample aliquot may be considered.

Open access

Human immunodeficiency virus-positive individuals are at increased risk of both asymptomatic and symptomatic urinary tract infections. The aim of this study was to determine the prevalence of asymptomatic bacteriuria (ASB) in HIV-positive individuals, its associated factors including any correlation with the CD4 count of the patient, and the antibiotic susceptibility pattern of the isolated organisms. Midstream urine and blood samples were collected from 242 consenting HIV-positive patients who were attending routine follow-up clinic during the six-month period of the study. Microscopy, culture, and antibiotic susceptibility testing of the samples were carried out following standard protocols, and CD4 counts were also determined. Fifty one (21.1%) of the 242 individuals had significant bacteriuria. The predominant organism was Klebsiella spp. (35%) followed by Escherichia coli (31%). Prevalence of bacteriuria was higher in the women. Low CD4 counts and young age were significantly associated with the presence of bacteriuria. ASB prevalence is high in this population and related to the CD4 count level.

Open access

Haemophilus influenzae is a key pathogen of upper respiratory tract infections. Its reliable discrimination from nonpathogenic Haemophilus spp. is necessary because merely colonizing bacteria are frequent at primarily unsterile sites. Due to close phylogenetic relationship, it is not easy to discriminate H. influenzae from the colonizer Haemophilus haemolyticus. The frequency of H. haemolyticus isolations depends on factors like sampling site, patient condition, and geographic region.Biochemical discrimination has been shown to be nonreliable. Multiplex PCR including marker genes like sodC, fucK, and hpd or sequencing of the 16S rRNA gene, the P6 gene, or multilocus-sequence-typing is more promising. For the diagnostic routine, such techniques are too expensive and laborious. If available, matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry is a routine-compatible option and should be used in the first line. However, the used database should contain well-defined reference spectra, and the spectral difference between H. influenzae and H. haemolyticus is small. Fluorescence in-situ hybridization is an option for less well-equipped laboratories, but the available protocol will not lead to conclusive results in all instances. It can be used as a second line approach. Occasional ambiguous results have to be resolved by alternative molecular methods like 16S rRNA gene sequencing.

Open access

Sepsis represents a life-threatening infection requiring the immediate start of antibacterial treatment to reduce morbidity. Thus, laboratories use direct antimicrobial susceptibility testing (AST) to rapidly generate preliminary results from positive blood cultures. As the direct AST has not yet been published to be evaluated with EUCAST breakpoints, the purpose of the study was to investigate the reliability of the direct agar diffusion test to correctly produce AST results from positive monobacterial blood cultures compared with the VITEK2-based definitive AST, when current EUCAST breakpoints were used.A total of 428 isolates from unselected monobacterial routine blood cultures and 110 challenge strains were included. Direct agar diffusion-based and standard VITEK2-based AST of 2803 bacterium-drug combinations yielded a total clinical category agreement of 95.47% with 1.28% very major errors and 3.42% combined major and minor errors. On the species level, very major errors were observed in the species-drug combinations Enterococcus spp.-high-level gentamicin (10.87%) and Staphylococcus spp.-rifampicin (5%), only. No very major errors occurred with Enterobacteriaceae and Pseudomonas aeruginosa.In most species-drug combinations, the direct agar diffusion test using EUCAST breakpoints precisely predicted the result of the definitive antibiotic susceptibility test and, thus, it can be used to optimize empiric antibiotic therapy until definitive results are available.

Open access
European Journal of Microbiology and Immunology
Authors: Volker Micheel, Benedikt Hogan, Rivo Rakotoarivelo, Raphael Rakotozandrindrainy, Fetra Razafimanatsoa, Tsiriniaina Razafindrabe, Jean Rakotondrainiarivelo, Sabine Crusius, Sven Poppert, Norbert Schwarz, Jürgen May, Hagen Frickmann, and Ralf Hagen

This study assesses the nasal occurrence of β-lactamase-producing Enterobacteriaceae both in patients in a hospital department of infectious diseases at admission and in healthy Madagascan students and health care workers.Nasal swabs from 681 students, 824 health care workers, and 169 patients were obtained in Antananarivo, Madagascar, and transferred to Germany. Screening for β-lactamase (ESBL, ampC) producing Enterobacteriaceae was performed by cultural and molecular approaches, comprising Brilliance ESBL agar, E-testing, ABCD-testing, and commercial hyplex ESBL and SuperBug ID PCR.Regarding ESBL-positive strains and strains with resistance against at least three out of the four tested bactericidal antibiotic drugs, 0.3% (five out of 1541) of the students and health care workers group showed nasal colonization, whereas colonization was observed in 7.1% (12 out of 169) of the hospitalized patients at admission. No appreciably reduced detection rates after sample storage and intercontinental transport were observed.A considerable proportion of nasal colonization with cephalosporin-resistant Enterobacteriaceae was demonstrated in Madagascan hospital patients at admission, posing a risk of developing future endogenous infections. The nasal colonization of healthy individuals was negligible. Good storage and transport stability of Enterobacteriaceae will allow for future studies even in areas difficult to access.

Open access
European Journal of Microbiology and Immunology
Authors: Cosme Alvarado-Esquivel, Renata Vazquez-Morales, Edgar Colado-Romero, Ramiro Guzmán-Sánchez, Oliver Liesenfeld, and Jitender Dubey

We performed a cross-sectional study to determine the seroprevalence of Toxoplasma gondii infection in 308 domestic pigs slaughtered in La Paz, Baja California Sur State, Mexico using the modified agglutination test (MAT, cut off 1:25). Forty (13%) of the 308 pigs were seropositive with MAT titers of 1:25 in 16, 1:50 in 5, 1:100 in 4, 1:200 in 5, 1:400 in 3, 1:800 in 3, 1:1600 in 2, and 1:3200 in 2. Multivariate analysis of pigs’ characteristics showed that seropositivity to T. gondii was negatively associated with mixed breed (OR = 0.02; 95% CI: 0.003–0.26; P = 0.001). Other variables including sex, type of raising, and municipality did not show an association with T. gondii seropositivity by multivariate analysis. The frequency of high antibody titers (≥1:400) was significantly higher (P < 0.001) in Landrace pigs than mixed breed pigs. The seroprevalence of T. gondii infection in pigs for slaughter in Baja California Sur State is low compared with seroprevalences reported in pigs in other Mexican states. Landrace pigs demonstrated higher seroprevalence rates and antibody levels than mixed breed pigs. This is the first report of T. gondii infection in pigs raised in a desert climate.

Open access

The qac genes of Staphylococcus species encode multidrug efflux pumps: membrane proteins that export toxic molecules and thus increase tolerance to a variety of compounds such as disinfecting agents, including quaternary ammonium compounds (for which they are named), intercalating dyes and some antibiotics. In Stapylococcus species, six different plasmid-encoded Qac efflux pumps have been described, and they belong to two major protein families. QacA and QacB are members of the Major Facilitator Superfamily, while QacC, QacG, QacH, and QacJ all belong to the Small Multidrug Resistance (SMR) family. Not all SMR proteins are called Qac and the reverse is also true, which has caused confusion in the literature and in gene annotations. The discovery of qac genes and their presence in various staphylococcal populations is briefly reviewed. A sequence comparison revealed that some of the PCR primers described in the literature for qac detection may miss particular qac genes due to lack of DNA conservation. Despite their resemblance in substrate specificity, the Qac proteins belonging to the two protein families have little in common. QacA and QacB are highly conserved in Staphylococcus species, while qacA was also detected in Enterococcus faecalis, suggesting that these plasmid-born genes have spread across bacterial genera. Nevertheless, these qacA and qacB genes are quite dissimilar to their closest homologues in other organisms. In contrast, SMR-type Qac proteins display considerable sequence variation, despite their short length, even within the Staphylococcus genus. Phylogenetic analysis of these genes identified similarity to a large number of other SMR members, found in staphylococci as well as in other genera. A number of phylogenetic trees of SMR Qac proteins are presented here, starting with genes present in S. aureus and S. epidermidis, and extending this to related genes found in other species of this genus, and finally to genes found in other genera.

Open access
European Journal of Microbiology and Immunology
Authors: Trudy M. Wassenaar, Anke Zschüttig, Claudia Beimfohr, Thomas Geske, Christian Auerbach, Helen Cook, Kurt Zimmermann, and Florian Gunzer

The probiotic product Symbioflor2 (DSM 17252) is a bacterial concentrate of six different Escherichia coli genotypes, whose complete genome sequences are compared here, between each other as well as to other E. coli genomes. The genome sequences of Symbioflor2 E. coli components contained a number of virulence-associated genes. Their presence seems to be in conflict with a recorded history of safe use, and with the observed low frequency of adverse effects over a period of more than 6 years. The genome sequences were used to identify unique sequences for each component, for which strain-specific hybridization probes were designed. A colonization study was conducted whereby five volunteers were exposed to an exceptionally high single dose. The results showed that the probiotic E. coli could be detected for 3 months or longer in their stools, and this was in particular the case for those components containing higher numbers of virulence-associated genes. Adverse effects from this long-term colonization were absent. Thus, the presence of the identified virulence genes does not result in a pathogenic phenotype in the genetic background of these probiotic E. coli.

Open access

The collection of epiphyllous bryophytes in the lowland rainforests of Phang-Nga province and in the neighbouring Phuket and Surat Thani provinces resulted in 54 liverwort and one moss species, of which 14 are new records for the bryoflora of Thailand. Epiphyllous bryophyte assemblages from nine localities are evaluated for species richness and beta diversity, as well as for their phytogeographical status.

Open access

With microbiome research being a fiercely contested playground in science, new data are being published at tremendous pace. The review at hand serves to critically revise four microbial metabolites widely applied in research: butyric acid, flagellin, lipoteichoic acid, and propionic acid. All four metabolites are physiologically present in healthy humans. Nevertheless, all four are likewise involved in pathologies ranging from cancer to mental retardation. Their inflammatory potential is equally friend and foe. The authors systematically analyze positive and negative attributes of the aforementioned substances, indicating chances and dangers with the use of pre- and probiotic therapeutics. Furthermore, the widespread actions of microbial metabolites on distinct organs and diseases are reconciled. Moreover, the review serves as critical discourse on scientific methods commonly employed in microbiome research and comparability as well as reproducibility issues arising thereof.

Open access