DNA sequence conservation between the Bacillus anthracis pXO2 plasmid and genomic sequence from closely related bacteria

Background  

Complete sequencing and annotation of the 96.2 kb Bacillus anthracis plasmid, pXO2, predicted 85 open reading frames (ORFs). Bacillus cereus and Bacillus thuringiensis isolates that ranged in genomic similarity to B. anthracis, as determined by amplified fragment length polymorphism (AFLP) analysis, were examined by PCR for the presence of sequences similar to 47 pXO2 ORFs.     

Evaluation of different methods to discriminate Bacillus anthracis from other bacteria of the Bacillus cereus group

Aims:  To evaluate different methods that are useful for rapid and definitive discrimination of Bacillus anthracis from other bacteria of the Bacillus cereus group in environmental samples like letters claimed to contain anthrax spores.
Methods and Results:  Characterized strains and bacteria from environmental samples were analysed by microbiological and molecular methods (PCR and restriction analysis). Environmental isolates often shared several microbiological features with B. anthracis , e.g. lack of β -haemolysis and phospholipase C activity, and only the gamma phage assay was specific for B. anthracis . PCR assays targeting markers from the virulence plasmids exclusively detected B. anthracis , but other PCR targets were also detected in nonanthrax isolates. Additionally, the restriction pattern in an Alu I restriction analysis of the SG-749 fragment is not 100% specific. The loci used for multiple-locus variable-number tandem repeat analysis of B. anthracis are also present in other members of the B. cereus group, but amplicon sizes are usually different.
Conclusions:  Environmental samples often contain borderline isolates closely related to B. anthracis both on microbiological and genetic levels. Real-time PCR targeting plasmidal and chromosomal markers should be used for rapid and definitive exclusion of a virulent strain of B. anthracis in such samples.
Significance and Impact of the Study:  This study gives an overview of the current microbiological and molecular methods used for identification of B. anthracis and shows that most assays have limits when borderline isolates present in environmental samples are analysed.     

The current MLVA typing scheme for <Emphasis Type="Italic">Enterococcus faecium</Emphasis> is less discriminatory than MLST and PFGE for epidemic-virulent,hospital-adapted clonal types

Background  

MLVA (multiple-locus variable-number tandem repeat analysis) is a reliable typing technique introduced recently to differentiate also isolates of Enterococcus faecium. We used the established VNTR (variable number of tandem repeats) scheme to test its suitability to differentiate 58 E. faecium isolates representing mainly outbreaks and clusters of infections and colonizations among patients from 31 German hospitals. All isolates were vancomycin-resistant (vanA type). Typing results for MLVA are compared with results of macrorestriction analysis in PFGE (pulsed-field gel electrophoresis) and MLST (multi-locus sequence typing).     

Allele distribution and genetic diversity of VNTR loci in <Emphasis Type="Italic">Salmonella enterica</Emphasis>serotype Enteritidis isolates from different sources

Background  

Salmonella enterica serotype Enteritidis (S. Enteritidis) is a zoonotic pathogen, which can be found in many sources including animals and the environment. However, little is known about the molecular relatedness among S. Enteritidis isolates from different sources. We have applied multiple-locus variable number tandem repeat analysis (MLVA) to study the genetic diversity of S. Enteritidis isolates from human and non-human sources.     

Bacillus amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for the production of Bikalga,an alkaline fermented food

Aims

To identify and screen dominant Bacillus spp. strains isolated from Bikalga, fermented seeds of Hibiscus sabdariffa for their antimicrobial activities in brain heart infusion (BHI) medium and in a H. sabdariffa seed‐based medium. Further, to characterize the antimicrobial substances produced.

Methods and Results

The strains were identified by gyrB gene sequencing and phenotypic tests as B. amyloliquefaciens ssp. plantarum. Their antimicrobial activity was determined by the agar spot and well assay, being inhibitory to a wide range of Gram‐positive and Gram‐negative pathogenic bacteria and fungi. Antimicrobial activity against Bacillus cereus was produced in H. sabdariffa seed‐based medium. PCR results revealed that the isolates have potential for the lipopeptides iturin, fengycin, surfactin, the polyketides difficidin, macrolactin, bacillaene and the dipeptide bacilysin production. Ultra‐high‐performance liquid chromatography‐time of flight mass spectrometry analysis of antimicrobial substance produced in BHI broth allowed identification of iturin, fengycin and surfactin.

Conclusions

The Bacillus amyloliquefaciens ssp. plantarum exhibited broad‐spectrum antifungal and antibacterial properties. They produced several lipopeptide antibiotics and showed good potential for biological control of Bikalga.

Significance and Impact of the Study

Pathogenic bacteria often occur in spontaneous food fermentations. This is the first report to identify indigenous B. amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for safeguarding Bikalga.     

Molecular characterization of a lipase-producing <Emphasis Type="Italic">Bacillus pumilus</Emphasis> strain (NMSN-1d) utilizing colloidal water-dispersible polyurethane

Bacillus pumilus strain NMSN-1d isolated from polyurethane-contaminated water was found to grow in high salt concentration (NaCl 10%, w/v) and degrade Impranil-DLN, water-dispersible polyurethane. The genetic relatedness of the isolate has been established by standard molecular biological techniques and the enzyme(s) involved in polyurethane degradation were also studied. A total of nine bacterial strains were isolated from polyurethane-polluted sites and characterized by conventional, microbiological and biochemical methods. These isolates were subjected to 16S ribosomal RNA gene amplification by PCR using specific primers. The genetic relatedness of the isolates was also ascertained by ribotyping and BLAST analysis of the 16S ribosomal RNA gene sequences. The bacterial isolates were grown in yeast extract-salts minimal broth medium supplemented with water-dispersible polyurethane (Impranil DLN) as a sole source of carbon. The promising isolate utilizing polyurethane and producing lipase was identified as Bacillus pumilus NMSN-1d. The polyurethane degradation has been studied in polyurethane-Rhodamine-B and Luria-Bertani-polyurethane plate assays. The activity of hydrolytic enzymes such as lipase and esterase was confirmed on 2xYT-olive oil and tributyrin-Tween 20 plate assay. The newly isolated Bacillus pumilus appears promising in the management of polyurethane waste and in production of industrially important enzymes.     

A <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> isolation method utilizing a novel stain,low selection and high throughput produced atypical results

Background  

Bacillus thuringiensis is a bacterium known for producing protein crystals with insecticidal properties. These toxins are widely sought after for controlling agricultural pests due to both their specificity and their applicability in transgenic plants. There is great interest in isolating strains with improved or novel toxin characteristics, however isolating B. thuringiensis from the environment is time consuming and yields relatively few isolates of interest. New approaches to B. thuringiensis isolation have been, and continue to be sought. In this report, candidate B. thuringiensis isolates were recovered from environmental samples using a combination of a novel stain, high throughput and reduced selection. Isolates were further characterized by SDS-PAGE, light microscopy, PCR, probe hybridization, and with selected isolates, DNA sequencing, bioassay or Electron Microscopy.     

Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples

Aims

Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10‐fold serial dilutions of Bacillus anthracis spores using quantitative real‐time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 10¹ and 1·3 × 10² CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS).

Methods and Results

The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 ± 14, 68 ± 8, 66 ± 4 and 52 ± 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors.

Conclusions

Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit.

Significance and Impact of the Study

The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples.     

Test method development to evaluate hot,humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and B. thuringiensis Al Hakam spores

Aims

To develop test methods and evaluate the survival of Bacillus anthracis ?Sterne and Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air.

Methods and Results

Spores (>7 logs) of both strains were dried on six different test materials. Response surface methodology was employed to identify the limits of spore survival at optimal test combinations of temperature (60, 68, 77°C), relative humidity (60, 75, 90%) and time (1, 4, 7 days). No spores survived the harshest test run (77°C, 90% r.h., 7 days), while > 6·5 logs of spores survived the mildest test run (60°C, 60% r.h., 1 day). Spores of both strains inoculated on nylon webbing and polypropylene had greater survival rates at 68°C, 75% r.h., 4 days than spores on other materials. Electron microscopy showed no obvious physical damage to spores using hot, humid air, which contrasted with pH‐adjusted bleach decontamination.

Conclusions

Test methods were developed to show that hot, humid air effectively inactivates B. anthracis ?Sterne and B. thuringiensis Al Hakam spores with similar kinetics.

Significance and Impact of the Study

Hot, humid air is a potential alternative to conventional chemical decontamination.     

Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization

Background  

Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are closely related members of the B. cereus-group of bacilli. Suppressive subtractive hybridization (SSH) was used to identify specific chromosomal sequences unique to B. anthracis.     

Molecular epidemiology of clinically high-risk Pseudomonas aeruginosa strains: Practical overview

In recent years, numerous outbreaks of multidrug-resistant Pseudomonas aeruginosa have been reported across the world. Once an outbreak occurs, besides routinely testing isolates for susceptibility to antimicrobials, it is required to check their virulence genotypes and clonality profiles. Replacing pulsed-field gel electrophoresis DNA fingerprinting are faster, easier-to-use, and less expensive polymerase chain reaction (PCR)-based methods for characterizing hospital isolates. P. aeruginosa possesses a mosaic genome structure and a highly conserved core genome displaying low sequence diversity and a highly variable accessory genome that communicates with other Pseudomonas species via horizontal gene transfer. Multiple-locus variable-number tandem-repeat analysis and multilocus sequence typing methods allow for phylogenetic analysis of isolates by PCR amplification of target genes with the support of Internet-based services. The target genes located in the core genome regions usually contain low-frequency mutations, allowing the resulting phylogenetic trees to infer evolutionary processes. The multiplex PCR-based open reading frame typing (POT) method, integron PCR, and exoenzyme genotyping can determine a genotype by PCR amplifying a specific insertion gene in the accessory genome region using a single or a multiple primer set. Thus, analyzing P. aeruginosa isolates for their clonality, virulence factors, and resistance characteristics is achievable by combining the clonality evaluation of the core genome based on multiple-locus targeting methods with other methods that can identify specific virulence and antimicrobial genes. Software packages such as eBURST, R, and Dendroscope, which are powerful tools for phylogenetic analyses, enable researchers and clinicians to visualize clonality associations in clinical isolates.     

Cereulide synthetase gene cluster from emetic Bacillus cereus: Structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1

Background  

Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that this toxin is produced by a nonribosomal peptide synthetase (NRPS), but its exact genetic organization and biochemical synthesis is unknown.     

Detection of a highly prevalent and potentially virulent strain of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> from nosocomial infections in a medical center

Background  

We correlated genotypes, virulence factors and antimicrobial susceptibility patterns of nosocomially identified Pseudomonas aeruginosa isolates from clinical specimens to those of environmental isolates encountered in the same units of a medical center. Antibiotic susceptibility testing, RAPD analysis and detection of enzymatic activities of extracellular virulence factors, were done on these isolates.     

Genetic Diversity among Bacillus anthracis Soil Isolates at Fine Geographic Scales

Environmental samples were collected from carcass sites during and after anthrax outbreaks in 2000 and 2001 in the bison (Bison bison) population within Wood Buffalo National Park and the Hook Lake Region north of Wood Buffalo National Park. Bacillus anthracis spores were isolated from these samples and confirmed using phenotypic characterization and real-time PCR. Confirmed B. anthracis isolates were typed using multiple-locus variable-number tandem repeat analysis (MLVA15) and single-nucleotide-repeat analysis (SNRA). B. anthracis isolates split into two clades based on MLVA15, while SNRA allowed some isolates between carcass sites to be distinguished from each other. SNRA polymorphisms were also present within a single carcass site. Some isolates from different carcass sites having the same SNRA type had divergent MLVA types; this finding leads to questions about hierarchical typing methods and the robustness of the fine-scale typing of Bacillus anthracis.     

Susceptibility patterns and cross resistances of antibiotics against Pseudomonas aeruginosa in a teaching hospital of Turkey

Background  

Pseudomonas aeruginosa is the third most common pathogen responsible for nosocomial infections and the prevalence of multiple resistant isolates has been increasing. Ninety-nine clinical isolates were studied in order to assess the current levels of susceptibility and cross-resistances of widely used antipseudomonal antibiotics against P. aeruginosa and to determine some resistance mechanisms by phenotypic methods.     

Microbial sensor for drug susceptibility testing of Mycobacterium tuberculosis

Aims

Drug susceptibility testing (DST) of clinical isolates of Mycobacterium tuberculosis is critical in treating tuberculosis. We demonstrate the possibility of using a microbial sensor to perform DST of M. tuberculosis and shorten the time required for DST.

Methods and Results

The sensor is made of an oxygen electrode with M. tuberculosis cells attached to its surface. This sensor monitors the residual oxygen consumption of M. tuberculosis cells after treatment with anti‐TB drugs with glycerine as a carbon source. In principle, after drug pretreatment for 4–5 days, the response differences between the sensors made of drug‐sensitive isolates are distinguishable from the sensors made of drug‐resistant isolates. The susceptibility of the M. tuberculosis H37Ra strain, its mutants and 35 clinical isolates to six common anti‐TB drugs: rifampicin, isoniazid, streptomycin, ethambutol, levofloxacin and para‐aminosalicylic acid were tested using the proposed method. The results agreed well with the gold standard method (LJ) and were determined in significantly less time. The whole procedure takes approximately 11 days and therefore has the potential to inform clinical decisions.

Conclusions

To our knowledge, this is the first study that demonstrates the possible application of a dissolved oxygen electrode‐based microbial sensor in M. tuberculosis drug resistance testing. This study used the microbial sensor to perform DST of M. tuberculosis and shorten the time required for DST.

Significance and Impact of the Study

The overall detection result of the microbial sensor agreed well with that of the conventional LJ proportion method and takes less time than the existing phenotypic methods. In future studies, we will build an O₂ electrode array microbial sensor reactor to enable a high‐throughput drug resistance analysis.     

Genetic diversity of clinical isolates of <Emphasis Type="Italic">Bacillus cereus</Emphasis>using multilocus sequence typing

Background  

Bacillus cereus is most commonly associated with foodborne illness (diarrheal and emetic) but is also an opportunistic pathogen that can cause severe and fatal infections. Several multilocus sequence typing (MLST) schemes have recently been developed to genotype B. cereus and analysis has suggested a clonal or weakly clonal population structure for B. cereus and its close relatives B. anthracis and B. thuringiensis. In this study we used MLST to determine if B. cereus isolates associated with illnesses of varying severity (e.g., severe, systemic vs. gastrointestinal (GI) illness) were clonal or formed clonal complexes.     

The antimicrobial susceptibility of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> isolates using three different methods and their genetic relatedness

Background  

Stenotrophomonas maltophilia is inherently resistant to many antimicrobials. So far, antimicrobial susceptibility tests for S. maltophilia have not been fully standardized. The purpose of the study was to compare the susceptibility of S. maltophilia isolates against seven different antimicrobials using three different methods and to investigate their genetic relatedness.     

Validation of a same-day real-time PCR method for screening of meat and carcass swabs for Salmonella

Background  

One of the major sources of human Salmonella infections is meat. Therefore, efficient and rapid monitoring of Salmonella in the meat production chain is necessary. Validation of alternative methods is needed to prove that the performance is equal to established methods. Very few of the published PCR methods for Salmonella have been validated in collaborative studies. This study describes a validation including comparative and collaborative trials, based on the recommendations from the Nordic organization for validation of alternative microbiological methods (NordVal) of a same-day, non-commercial real-time PCR method for detection of Salmonella in meat and carcass swabs.     

Detection of <Emphasis Type="Italic">Francisella tularensis</Emphasis> in ticks and identification of their genotypes using multiple-locus variable-number tandem repeat analysis

Background  

Tularemia was reported in China over 50 years ago, however, many epidemical characteristics remain unclear. In the present study, the prevalence of Francisella tularensis in ticks was investigated during an epidemiological surveillance in China and then we measured their genetic diversity by conducting multiple-locus variable- number tandem repeat analysis (MLVA).