Fingerprinting bacterial strains using ion mobility spectrometry

The detection of illicit explosives in a large room, container, or cargo hold is problematic due to the current limitations in sampling and detection of explosives and to the size and time constraints of the search. Solid Phase MicroExtraction (SPME) can be used to rapidly extract volatile and semi-volatile compounds from the headspace of an explosive and ion mobility spectrometry (IMS) is a rapid presumptive organic detection technique that has already found widespread use in the detection of hidden explosives. SPME has recently been coupled to IMS as a sample pre-concentration device in order to improve the detection of explosives concealed in open areas. Detection limits have been determined for the following taggants: 2-nitrotoluene (2-NT), 4-nitrotoluene (4-NT), 2,3-dimethyl-2,3-dinitro butane (DMNB) and the following volatile explosive compounds: 2,4-dinitrotoluene, (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT), and 2,4,6-trinitrotoluene (2,4,6-TNT). Nitrocellulose (NC) is also capable of being detected by the SPME-IMS system on a reliable basis. Results from these experiments point towards the usefulness of this technique as a potential screening tool for explosive compounds. Mass transport experiments are being conducted to determine the compound concentration in a flow of air for detection to occur. Further work will also be conducted using explosive odor signature compounds as potential illicit explosive detection compounds.

Bacterial identification is vital for healthcare and environmental quality control. Traditional bacterial identification methods require extensive sample preparation, including cultivation, staining, and microscopy, making them time-consuming and labor-intensive. This study proposes the application of Laser Speckle Contrast Imaging (LSCI) as a novel approach to capture variations in speckle patterns between the start and end of the lag caused by changes in the shape and arrangement of bacterial cells during cell division in liquid cultures at lower cell concentrations, such as in the lag phase. Our approach offers an efficient alternative to traditional methods of bacterial species identification demonstrated with Escherichia coli and Micrococcus luteus pairs. Also, the differentiation of strains ( E. coli ATCC25922 and DH5α) is carried out based on the percentage change in speckle contrast between the end of lag and mid-log phase of their growth curve.

Introducton: The Ion mobility spectrometry (IMS) is a highly sensitive method for detection of volatile organic compounds. There is no need for pre-analytical procedures in humidified air samples, i.e. exhaled air. The aim of the study was to prove the specificity of differentiation of different strains and subtypes of mycobacteria. Methods: Headspaces of agar and mycobacterial cultures were analysed. For comparison to MAP were used: mycobacterium avium avium, mycobacterium avium hominisuis, mycobacterium smegmatis, mycobacterium fortuitum. Using clusteranalysis and support vector machine the peaks were identified and compared to each other. The sensitivity and specificity was evaluated by Leave-One-Out cross validation discriminant analysis method. Results: 102 different clusters were detected. Many clusters represent the different relationship of different bacteria, domonstrating that for identification of specific bacteria a combination of clusters will be needed. Using more peaks the specificity for the identification of a certain germ will increase. Fig. 1: Example Boxplots of Cluster 39 and 48. Summary: Different bacteria species were possible to differentiate with 96% sensitivity and 84% specificity. The VOC-based detection of bacterial growth is highly specific and will be useful also for prevention of the risk of infection among staff.

Twelve bacterial species including Streptococcus uberis, S. parauberis, S. agalactiae, S. dysgalactiae, S. bovis, S. mitis, S. salivarius, S. saccharolyticus, Enterococcus faecium, E. faecalis, E. avium, and Aerococcus viridans were examined for their 16S ribosomal DNA fingerprint patterns. Oligonucleotide primers complementary to 16S rRNA genes were used to amplify by the polymerase chain reaction 16S ribosomal gene fragments from genomic DNAs. The molecular sizes of the amplified 16S ribosomal DNA (rDNA) fragments from the 12 species examined ranged from 1,400 to 1,500 bp. Restriction fragment length polymorphism analysis of 16S rDNA was performed with 11 different restriction endonucleases. All 12 species examined could be differentiated on the basis of characteristic 16S rDNA fingerprint patterns by using the restriction endonucleases HhaI, RsaI, and MspI. A scheme for the differentiation of the 12 species is presented. Eleven isolates representing 11 species were obtained from cows with intramammary infections and were examined by 16S rDNA fingerprinting. All 11 species isolated from cows were differentiated by using HhaI, RsaI, and MspI restriction endonucleases. The results of this study demonstrate the potential application of 16S rDNA fingerprinting for the identification and differentiation of bacterial species.

This study explored the effects of water content and temperature on the mobility of exchangeable cations (termed the surface ionic mobility and hereafter ionic mobility) in the hydration layers of bentonite clay. The ionic mobility directly governs the surface conductivity of the clay. The investigation was done by measuring the bulk electrical conductivity (EC) of four sand–bentonite mixtures of different proportions for a wide range of water contents under constant temperature, and three bentonite samples at different water contents over 5–90°C. The ionic mobility was determined from the surface conductivity at the mean ionic strength of the hydration layers. The ionic mobility in the sand–bentonite samples increased with an increase in hydration layer thickness. For a given thickness of the hydration layer, the greater the bentonite content of a sample, the smaller was the ionic mobility. The ionic mobility in the bentonite samples at different water contents also increased, at reduced rates, with a rise in temperature. Consequently, the surface conductivity of the samples increased non-uniformly, at two different rates, with an increase in temperature. The increasing rate of this conductivity depended on temperature; over the low temperature range which depended on the water content, the rate was 0.013 dS/m.K, and over higher temperature range, the rate decreased to 0.008 dS/m.K. The commonly used temperature correction factor, 0.019 dS/m.K, for EC therefore did not hold true for the bentonite samples.

Diabetic foot is a major problem lead to a serious complications. Immune impairment in this group of patients predispose to bacterial infections. Different bacterial species were identified from samples taken from diabetic foot including gram positive and gram negative species. Antimicrobial considered important part of treatment regime and selection of effective antimicrobial will lead to better recovery results. This study was aimed to identify bacterial species infected diabetic foot and their antimicrobial patterns. Sterile cotton swab applied on the lesions and transferred to the laboratory immediately. Standard microbiology techniques were used to identify bacterial species whereas different media were used; MacConkey agar, Chocolate agar and blood agar. Multiple biochemical API 20 E test was used to identify gram negative bacteria. Catalase and coagulase test also used for gram positive collection identification. Disc diffusion method used to evaluate the antimicrobial susceptibility of collected identified isolates. Polymicrobial infections were 47.5% (19/40). Dominant combination of identified bacterial species was gram positive and gram negative group 15 (78.9). The highest prevalent bacterial species among gram positive isolates was S. aureus 31.7% (20/63) followed by coagulase negative staphylococci (CNS)15.9% (10/63) while Pseudomonas aeruginosa 17.5% (11/63) was the most prevalent among gram negative bacterial species. Methicillin resistant isolates was detected in 55% (n=11/20) of identified S. aureus collection. Antimicrobial resistance, Amikacin showed the highest effect against gram negative bacteria and ciprofloxacin against gram positive bacteria. High prevalence of polymicrobial infection in diabetic foot increase the concern of serious complications. S. aureus and CNS infection occurrence was high and their resistance to methicillin was a major concern. Most combination of polymicrobial infection consisted from gram positive and negative bacterial species which require antimicrobials effective against both groups.

Bacteria aggregate through various intercellular interactions to build biofilms, but the effect of environmental changes on them remains largely unexplored. Here, by using an experimental device that overcomes past difficulties, we observed the collective response of Escherichia coli aggregates to dynamic changes in the growth conditions. We discovered that nutrient starvation caused bacterial cells to arrange themselves into bundle-shaped clusters, developing a structure akin to that of smectic liquid crystals. The degree of the smectic-like bundle order was evaluated by a deep learning approach. Our experiments suggest that both the depletion attraction by extracellular polymeric substances and the growth arrest are essential for the bundle formation. Since these effects of nutrient starvation at the single-cell level are common to many bacterial species, bundle formation might also be a common collective behavior that bacterial cells may exhibit under harsh environments.

Fabric phase sorptive extraction (FPSE), a recently introduced novel sample preparation technology, has been evaluated for the extraction of benzodiazepines from human blood serum. FPSE utilizes a flexible fabric surface as the substrate platform for creating sol-gel hybrid organic-inorganic sorbent coatings. FPSE media can be introduced directly into the sample containing the target analyte(s), requiring no need for prior sample pretreatment or clean-up. Benzodiazepines were selected as model analytes because they represent one of the most widely used therapeutic drugs in psychiatry and are also amongst the most frequently encountered drugs in forensic toxicology. The chromatographic separation of target analytes was performed on a LiChroCART-LiChrospher®100 RP-18e (5 µm, 250 × 4 mm) analytical column, operated at room temperature. Ternary gradient elution was applied with a mobile phase that consisted of acetonitrile, methanol and ammonium acetate (0.05 M), which was delivered at a flow rate of 1.0 mL/min. Diode array detection was performed with monitoring at 240 nm. FPSE was performed using cellulose fabric extraction media coated with sol-gel poly(ethylene glycol) (sol-gel PEG). Absolute recovery values in the equilibrium state for the examined benzodiazepines were found to be 27% for bromazepam, 63% for lorazepam, 42 % for diazepam and 39% for alprazolam. Copyright © 2015 John Wiley & Sons, Ltd.

A multilocus sequence analysis (MLSA) was established and performed on the genus Thioclava, including 23 strains isolated from diverse marine environments, with the aim of better differentiation of strains and species within this genus. The study was based on sequences of 16S rRNA gene and five protein-coding housekeeping genes, gyrB, rpoD, dnaK, trpB, and recA. In contrast to 16S rRNA gene-based tree that was unable to separate some species within this genus, each tree based on a single housekeeping gene and MLSA had consistently defined seven clades, corresponding to the five established ones and two novel ones. The digital DNA-DNA hybridization and average nucleotide identity analyses based on genome sequences of the representative strains reconfirmed the validity of the MLSA analysis, and recommended a 97.3% MLSA similarity as the soft species threshold and nine species representing the five known and four putative novel species. Two of the four new species were identified as Thioclava sediminum sp. nov. (type strain TAW-CT134T = MCCC 1A10143T = LMG 29615T) and Thioclava marinus sp. nov. (type strain 11.10-0-13T = MCCC 1A03502T = LMG 29618T) by using a polyphasic taxonomic approach. Taken together, the newly established MLSA in this study first described the variability and phylogeny of the genus Thioclava which contributes to better understanding its ecology and evolution.

We have analyzed 11 strains and clones, representing five species (Penicillium janthinellum, P. citrioviridae, P. chrysogenum, Aspergillus niger, Trichoderma harzianum) and three genera of filamentous fungi, for the presence of hypervariable loci in their genomes by hybridization with simple repeat oligonucleotides and the DNA of phage M13. The oligonucleotide probes (CT)8, (GTG)5 and (GACA)4, as well as M13 DNA, are informative probes for fingerprinting in all genera and species tested. The probe (GATA)4 produced informative fingerprints only with the genomic DNA of A. niger. There was no similarity between the fingerprints originating from fungi of different genera and also little similarity between the fingerprints of different species belonging to the same genus. Fingerprints of strains of the same species differed only slightly from each other. Fingerprints of clones originating from one strain were identical. The results indicate that DNA fingerprinting is a powerful method to differentiate species and strains of filamentous fungi.

Background: The identification of bacterial species in fermented PDO (protected designation of origin) cheese is important since they contribute significantly to the final organoleptic properties, the ripening process, the shelf life, the safety and the overall quality of cheese. Methods: Ten commercial PDO feta cheeses from two geographic regions of Greece, Epirus and Thessaly, were analyzed by 16S metagenomic analysis. Results: The biodiversity of all the tested feta cheese samples consisted of five phyla, 17 families, 38 genera and 59 bacterial species. The dominant phylum identified was Firmicutes (49% of the species), followed by Proteobacteria (39% of the species), Bacteroidetes (7% of the species), Actinobacteria (4% of the species) and Tenericutes (1% of the species). Streptococcaceae and Lactobacillaceae were the most abundant families, in which starter cultures of lactic acid bacteria (LAB) belonged, but also 21 nonstarter lactic acid bacteria (NSLAB) were identified. Both geographical areas showed a distinctive microbiota fingerprint, which was ultimately overlapped by the application of starter cultures. In the rare biosphere of the feta cheese, Zobellella taiwanensis and Vibrio diazotrophicus, two Gram-negative bacteria which were not previously reported in dairy samples, were identified. Conclusions: The application of high-throughput DNA sequencing may provide a detailed microbial profile of commercial feta cheese produced with pasteurized milk.

The stable fly, Stomoxys calcitrans, has a great importance in medical and veterinary health due to its feeding and reproductive habits, which can disseminate some pathogenic agents among hosts even at long distances. This study aimed to describe, for the first time in Brazil, the bacterial microbiota in segments of the stable fly. Bacterial species were isolated from three different segments (cuticle, mouth parts and abdominal alimentary tract) of the stable fly. Twenty dairy farms were visited in four municipalities: Barra Mansa, Quatis, Resende and Rio Claro in the State of Rio de Janeiro in order to collect 20 flies in each site. Dissection of the flies and procedures of isolation and identification of bacterial species were performed. A total of 161 colonies of 33 distinct species were isolated, such as Escherichia coli, Staphylococcus aureus, and S. intermedius. Stomoxys calcitrans may harbor bacterial agents on their cuticle, mouth parts and abdominal alimentary tract and these bacteria may be pathogenic to their hosts.

A study was conducted to evaluate Sensititre® Automated Reading and Incubation System 2x System (ARIS), API® (API), and Bruker MALDI-TOF MS (MALDI) bacterial species identification systems using 132 diverse bacterial isolates from bovine milk samples and bulk tank milk received at the Penn State Animal Diagnostic Laboratory. The results were compared with 16S rRNA gene sequence analysis, which served as the reference method for species identification. The ARIS, API, and MALDI identified 0%, 40%, and 33.4% of species classified as Gram-positive rod isolates belonging to genera Arthrobacter, Bacillus, Brachybacterium, Brevibacterium, and Corynebacterium, respectively. It was observed that 76.5%, 93.9%, and 96.9% of catalase-negative, Gram-positive cocci (n = 33; Aerococcus, Enterococcus, Lactococcus, Streptococcus) were correctly identified to the species level by ARIS, API, and MALDI, respectively, while 33.4%, 84.5%, and 97.7% of catalase-positive, Gram-positive cocci (n = 45; Kocuria, Staphylococcus) were correctly identified to their species by ARIS, API, and MALDI, respectively. A total of 48 isolates (Acinetobacter, Citrobacter, Enterobacter, Escherichia, Klebsiella, Pantoea, Pasteurella, Providencia, Pseduomonas, Serratia) of Gram-negative bacteria were examined, of which 85.4%, 93.7%, and 95.8% of the isolates were correctly identified to the species level by ARIS, API, and MALDI, respectively. In our laboratory, the MALDI had the least costs associated with consumables and reagents compared to ARIS, API, and 16S rRNA identification methods. Identification of bacterial species was accomplished in <2 h using MALDI and 24 h for ARIS, API, and 16S rRNA identification systems.

Canine pyometra is a common and potentially life-threatening reproductive disorder in intact female dogs. This prospective study aimed to (1) investigate the bacterial spectrum and antimicrobial susceptibilities of bacterial isolates from the uterus and urine of dogs with pyometra, (2) assess the clonal relatedness and virulence factors of Escherichia coli isolates from individual dogs, and (3) determine the occurrence of concurrent and persistent bacteriuria or clinical urinary tract infections. Bacterial isolates from 208 uterine and 203 urine specimens collected during pyometra surgery were analyzed. Additionally, follow-up urine specimens were collected from 56 dogs with perioperative bacteriuria. Bacterial growth was detected in 87 % (180/208) of uterine specimens, while concurrent bacteriuria was observed in 33 % (67/203) of cases. In one-third (18/56) of these dogs the bacteriuria persisted, being primarily (15/18) asymptomatic. E. coli was the most common isolate in both uterine (71 %) and urine (81 %) specimens. Notably, altogether 32 distinct bacterial species were identified, with mixed growth in 15 % of the specimens. The vast majority of isolates were largely susceptible to tested antimicrobials. Identification of bacterial species was performed using MALDI-ToF MS, and antimicrobial susceptibility was assessed by disk diffusion. Whole-genome sequencing of 45 E. coli strains from fifteen dogs indicated high genetic similarities within individual dogs, supporting a clonal relationship. In conclusion, canine uteri with pyometra contained a plethora of bacterial species, predominantly E. coli, and antimicrobial resistance was rare. Concurrent and persistent E. coli bacteriuria was commonly caused by the same clone as found in the uterus.

Cell phones are the most common gadgets used in today’s world. Studies from different countries demonstrated that these electronic gadgets can carry different bacterial species, including potential pathogenic strains. These gadgets are in the hands of every health care professionals (HCPs) and can even be found in the operation theatres (OT). Moreover, healthcare workers tend to neglect the potential infectious threat that these gadgets carry. Hence, we investigated the microbial profile present on cell phones used by HCPs in United Arab Emirates (UAE). Standard microbiological procedures were used for collection and identification of bacterial species. Disk diffusion assay and crystal violet dye binding spectrophotometric assay were used for determining antibiogram and biofilm forming potential, respectively. Staphylococcus epidermidis was present in 64.1% of the samples, followed by Micrococci (22.3%), Streptococcus viridians (2.2%), Diphtheroids (2.9%), Bacillus spp. (5.2%), Enterobacter spp. (1.4%) and Pseudomonas spp. (1.4%). The tested strains exhibited varied degree of antibiotic resistance and biofilm producing potential. Our data highlights that mobile phones may just not be carriers for normal skin flora but also for antibiotic resistant, biofilm forming potential opportunistic pathogens and thus may serve as transmission vectors for bacteria. This is the first report from UAE analyzing carriage of bacteria in mobile gadgets used by HCPs. Furthermore, this would help to raise awareness among the HCPs about these electronic gadgets serving as a vehicle of transmission of bacterial pathogen.