MALDI-TOF MS Research Articles

Analyzing noncovalent interactions between notoginseng saponins and lysozyme by deposition scanning intensity fading MALDI-TOF mass spectrometry.

Analysis of noncovalent interactions between natural products and proteins is important for rapid screening of active ingredients and understanding their pharmacological activities. In this work, the intensity fading MALDI-TOF mass spectrometry (IF-MALDI-MS) method with improved reproducibility was implemented to investigate the binding interactions between saponins from Panax notoginseng and lysozyme. The benchmark IF-MALDI-MS experiment was established using N,N',N″-triacetylchitotriose-lysozyme as a model system. The reproducibility of ion intensities in IF-MALDI-MS was improved by scanning the whole sample deposition with a focused laser beam. The relative standard deviation (RSD) of deposition scanning IF-MALDI-MS is 5.7%. Similar decay trends of the relative intensities of notoginseng saponins against increasing amounts of lysozyme were observed for all six notoginseng saponins. The half-maximal fading concentration (FC50) was calculated to quantitatively characterize the binding affinity of each ligand based on the decay curve. According to the FC50 values obtained, the binding affinities of the six notoginseng saponins were evaluated in the following order: notoginsenoside S > notoginsenoside Fc > ginsenoside Rb1 > ginsenoside Rd > notoginsenoside Ft1 > ginsenoside Rg1. The binding order was in accordance with molecular docking studies, which showed hydrogen bonding might play a key role in stabilizing the binding interaction. Our results demonstrated that deposition scanning IF-MALDI-MS can provide valuable information on the noncovalent interactions between ligands and proteins.

Utility of MALDI-TOF MS for determination of species identity and blood meal sources of primary malaria vectors on the Kenyan coast

Background Protein analysis using matrix-assisted laser desorption/ionisation time-of-flight mass-spectrometry (MALDI-TOF MS) represents a promising tool for entomological surveillance. In this study we tested the discriminative power of this tool for measuring species and blood meal source of main Afrotropical malaria vectors on the Kenyan coast. Methods Mosquito collections were conducted along the coastal region of Kenya. MALDI-TOF MS spectra were obtained from each individual mosquito’s cephalothorax as well as the abdomens of blood-engorged mosquitoes. The same mosquitoes were also processed using gold standard tests: polymerase chain reaction (PCR) for species identification and enzyme linked immunosorbent assay (ELISA) for blood meal source identification. Results Of the 2,332 mosquitoes subjected to MALDI-TOF MS, 85% (1,971/2,332) were considered for database creation and validation. There was an overall accuracy of 97.5% in the identification of members of the An. gambiae (An. gambiae, 100%; An. arabiensis, 91.9%; An. merus, 97.5%; and An. quadriannulatus, 90.2%) and An. funestus (An. funestus, 94.2%; An. rivulorum, 99.4%; and An. leesoni, 94.1%) complexes. Furthermore, MALDI-TOF MS also provided accurate (94.5% accuracy) identification of blood host sources across all mosquito species. Conclusions This study provides further evidence of the discriminative power of MALDI-TOF MS to identify sibling species and blood meal source of Afrotropical malaria vectors, further supporting its utility in entomological surveillance. The low cost per sample (<0.2USD) and high throughput nature of the method represents a cost-effective alternative to molecular methods and could enable programs to increase the number of samples analysed and therefore improve the data generated from surveillance activities.

Characterization of Leuconostoc lactis Bacteremia during a 2-year Study at a Tertiary Care Center in North India-An Observational Analysis.

Leuconostoc species are regarded as important causes for many infections in immunocompromised patients. In this study, we assessed the characteristics of Leuconostoc spp. causing bacteremia in patients at our center. This observational analysis was conducted in the microbiology laboratory of a tertiary care center in northern India from July 2021 to July 2023. Patients in whom blood culture bottles were positive for Leuconostoc lactis were included in the study. Culture isolates were identified by MALDI-ToF MS as L. lactis and tested for antibiotic sensitivity results by Kirby-Bauer disk diffusion method. Demographic and clinical details were collected and analyzed. During the study period, 6,742 blood culture bottles flagged positive. Among these, L. lactis was isolated from 14 (0.21%) patients. The median patient age was 34 years. The male-to-female ratio was 2.5:1. All the patients with L. lactis bacteremia had an underlying condition leading to immunosuppression (e.g., carcinoma and chronic kidney disease). All the patients with L. lactis bacteremia had an intravascular device present at the time of bacteremia. All isolates in the study were sensitive to doxycycline, high level gentamicin, minocycline, ampicillin-sulbactam, and linezolid. Mortality was attributed to bacteremia by L. lactis in five patients. Appropriate and timely identification of the Leuconostoc species is important for the clinician to tailor regimens for the patients.

Campylobacter occurrence and antimicrobial resistance profile in under five-year-old diarrheal children, backyard farm animals, and companion pets.

Campylobacteriosis disproportionately affects children under five in low-income countries. However, epidemiological and antimicrobial resistance (AMR) information at the children-animal interface is lacking. We hypothesized that Campylobacter is a major cause of enteritis in children in Ethiopia, and contact with animals is a potential source of transmission. The objective of the study was to determine Campylobacter occurrence and its AMR in children under five with diarrhea, backyard farm animals, and companion pets. Stool from 303 children and feces from 711 animals were sampled. Campylobacter was isolated through membrane filtration on modified charcoal cefoperazone deoxycholate agar plates under microaerobic incubation, and the technique showed to be feasible for use in regions lacking organized laboratories. Typical isolates were characterized with MALDI-TOF MS and multiplex PCR. Of 303 children, 20% (n = 59) were infected, with a higher proportion in the 6 to 11-month age group. Campylobacter occurred in 64% (n = 14) of dogs and 44% (n = 112) of poultry. Campylobacter jejuni was present in both a child and animal species in 15% (n = 23) of 149 households positive for Campylobacter. MICs using the gradient strip diffusion test of 128 isolates displayed resistance rates of 20% to ciprofloxacin and 11% to doxycycline. MICs of ciprofloxacin and doxycycline varied between C. coli and C. jejuni, with higher resistance in C. coli and poultry isolates. Campylobacter infection in children and its prevalent excretion from backyard poultry and dogs is a understudied concern. The co-occurrence of C. jejuni in animals and children suggest household-level transmission As resistance to ciprofloxacin and doxycycline was observed, therapy of severe campylobacteriosis should consider susceptibility testing. Findings from this study can support evidence-based diagnosis, antimicrobial treatment, and further investigations on the spread of AMR mechanisms for informed One Health intervention.

MALDI glycotyping of O-antigens from a single colony of gram-negative bacteria

Polypeptide-targeted MALDI-TOF MS for microbial species identification has revolutionized microbiology. However, no practical MALDI-TOF MS identification method for O-antigen polysaccharides, a major indicator for epidemiological classification within a species of gram-negative bacteria, is available. We describe a simple MALDI glycotyping method for O-antigens that simultaneously identifies the molecular mass of the repeating units and the monosaccharide composition of the O-antigen. We analyzed the Escherichia coli O1, O6, and O157-type strains. Conventional species identification based on polypeptide patterns and O-antigen polysaccharide typing can be performed in parallel from a single colony using our MALDI-TOF MS workflow. Moreover, subtyping within the same O-antigen and parallel colony-specific O-antigen determination from mixed strains, including the simultaneous identification of multiple strains-derived O-antigens within selected colony, were performed. In MALDI glycotyping of two Enterobacteriaceae strains, a Citrobacter freundii strain serologically cross-reactive with E. coli O157 gave a MALDI spectral pattern identical to E. coli O157. On the other hand, an Edwardsiella tarda strain with no reported O-antigen cross-reactivity gave a MALDI spectral pattern of unknown O-antigen repeating units. The method described in this study allows the parallel and rapid identification of microbial genera, species, and serotypes of surface polysaccharides using a single MALDI-TOF MS instrument.

Metal binding feature of copper‒induced metallothionein from freshwater crab Sinopotamon henanense reveals its Cu‒thionein character

Sinopotamon Henanense expresses two metal‒induced metallothioneins (MTs), Cd‒induced MT and Cu‒induced MT (ShCuMT). The Cd‒induced MT has been characterized as a Cd‒thiolate MT. However, it is unknown whether ShCuMT is a Cu‒thiolate MT. In the present study, ShCuMT was expressed heterologously in Escherichia coli and purified by Ni‒NTA column and superdex‒75 column. And its metal‒binding feature was evaluated by DTNB reaction, circular dichroism spectroscopy (CD), isothermal microtitration (ITC), electrospray flight mass spectrometry (ESI‒TOF‒MS), and matrix‒assisted laser desorption ionization flight mass spectrometry (MALDI‒TOF‒MS). Bioinformatics analysis demonstrated that ShCuMT possessed the cysteine‒triplet motif of a Cu‒specific MT. Expression and purification of ShCuMT illustrated that SUMO tag used as the production system for ShCuMT resulted in a high production yield. The stability order of ShCuMT binding metal ions were Cu (Ⅰ) > Cd (Ⅱ) > Zn (Ⅱ). The CD spectrum indicated that ShCuMT binding with Cu (I) exhibited a compact thiol metal clusters structure. Besides, there emerged no a visible nickel‒thiol absorption after Ni‒NTA column affinity chromatography. The ITC results implied that Cu‒ShCuMT possessed the optimal thermodynamic conformation and the highest stoichiometric number of Cu (Ⅰ). Overall, the results suggested that SUMO fusion system is a robust and inexpensive approach for ShCuMT expression and Ni‒NTA column had no influence on metal binding of ShCuMT and Cu(Ⅰ) was considered its cognate metal ion, and ShCuMT possessed canonical Cu‒thiolate characteristics. The metal binding feature of ShCuMT reported here contributes to elucidating the structure‒function relationship of ShCuMT in S. Henanense.

In vitro assessment of newer colistin-sparing antimicrobial agents for clinical isolates of carbapenem-resistant organisms

ObjectivesCarbapenem-resistant organisms (CROs) are a significant public health threat globally, particularly in countries like India with high antibiotic resistance rates. The current study investigates the prevalence of CROs, detects resistance mechanisms using phenotypic methods and assesses the efficacy of newer antibiotics against CROs. MethodsA prospective study conducted at a tertiary care hospital in India during 2021–23. Clinical specimens were processed and bacterial identification was performed using MALDI-TOF mass spectrometry followed by antimicrobial susceptibility testing using CLSI guidelines against a plethora of newer antibiotics for CROs. Carbapenemase production was detected using phenotypic methods, and the presence of the mcr-1 gene was assessed by real-time PCR. ResultsDuring the study period, predominantly (70 %) Gram-negative bacteria were isolated; amongst which 5692 strains were carbapenem-resistant, wherein resistance to different carbapenems ranged from 34.1% to 79 %. Majority of the carbapenemase producers were metallo-β-lactamases (MBL) producers (75 %). Colistin resistance was noted in 5.4 % of selected carbapenem-resistant isolates. Among newer antibiotics, cefiderocol demonstrated the lowest resistance rates (0–14 %), while resistance to newer β-lactam/β-lactamase inhibitor combinations was very high in carbapenem-resistant isolates. Fosfomycin, minocycline and tigecycline, each showing variable efficacy depending on the site of infection. Moreover, newer β-lactam/β-lactamase inhibitor combinations offer restricted benefits due to widespread prevalence of MBL in the region. ConclusionThe escalating prevalence of CROs in India underscores the urgency for alternative treatment options beyond colistin. Hence, highlighting the critical importance of developing effective strategies to combat carbapenem resistance.

Rapid detection of common variants and deletions of CYP21A2 using MALDI-TOF MS

Newborn screening (NBS) for congenital adrenal hyperplasia (CAH) based on hormonal testing is successfully implemented in many countries. However, this method cannot detect non-classic CAH and has high false positive rates. We have developed a novel MALDI-TOF MS assay that can identify common variants and deletions of CYP21A2 in the Chinese population. Thirty-seven clinical patients with CAH confirmed by Sanger sequencing and MLPA analysis were detected by MALDI-TOF MS assay. Two CYP21A2 variants were detected in 30 patients and one CYP21A2 variant was detected in 7 patients. The MALDI-TOF MS assay detected 67 mutant alleles in 37 patients with a detection rate of 90.5%. Sanger sequencing revealed that three variants in seven patients were not included in the designed panel. Eleven distinct CYP21A2 variants were identified, including five missense variants, two nonsense variants, two large gene deletions, one splice variant, and one frameshift variant. The most frequent variant was c.293-13C > G (37.84%), followed by c.518T > A (21.62%) and exon 1–7 deletion (17.57%).The high-throughput MALDI-TOF MS assay that can simultaneously detect common variants and deletions of CYP21A2. This assay can be used for population-based genetic screening and rapid detection of suspected patients, and is expected to be a valuable complement to biochemical-based testing for the detection of CAH.

Identification of Enterococcus spp. from food sources by matrix-assisted laser desorption ionization-time of flight mass spectrometry and characterization of virulence factors, antibiotic resistance, and biofilm formation

The continuous detection of multi-drug-resistant enterococci in food source environments has aroused widespread concern. In this study, 198 samples from chicken products, animal feces, raw milk, and vegetables were collected in Japan and Egypt to investigate the prevalence of enterococci and virulence characterization. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed for species identification and taxonomic analysis of the isolates. The results showed that the rates of most virulence genes (efaA, gelE, asa1, ace, and hyl) in the Japanese isolates were slightly higher than those in the Egyptian isolates. The rate of efaA was the highest (94.9 %) among seven virulence genes detected, but the cylA gene was not detected in all isolates, which was in accordance with γ-type hemolysis phenotype. In Enterococcus faecalis, the rate of kanamycin-resistant strains was the highest (84.75 %) among the antibiotics tested. Moreover, 78 % of E. faecalis strains exhibited multi-drug resistance. Four moderately vancomycin-resistant strains were found in Egyptian isolates, but none were found in Japanese isolates. MALDI-TOF MS analysis correctly identified 98.5 % (68/69) of the Enterococcus isolates. In the principal component analysis dendrogram, strains isolated from the same region with the same virulence characteristics and similar biofilm-forming abilities were characterized by clustered distribution in different clusters. This finding highlights the potential of MALDI-TOF MS for classifying E. faecalis strains from food sources.

Biofilm Eradication and Inhibition of Methicillin-resistant Staphylococcus Clinical Isolates by Curcumin-Chitosan Magnetic Nanoparticles.

Biofilm-producing methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative Staphylococci (MR-CoNS) are a clinical challenge for the treatment of healthcare-associated infections. As alternative antimicrobial options are needed, we aimed to determine the effect of curcumin-chitosan magnetic nanoparticles on the biofilm of staphylococcal clinical isolates. MRSA and CoNS clinical isolates were identified by MALDI-TOF mass spectrometry. Antimicrobial susceptibility testing was performed by broth microdilution. Nanoparticles were synthesized by co-precipitation of magnetic nanoparticles (MNP) and encapsulation by ionotropic gelation of curcumin (Cur) and chitosan (Chi). Biofilm inhibition and eradication by nanoparticles with and without the addition of oxacillin was assessed on staphylococcal strains. Cur-Chi-MNP showed antimicrobial activity on planktonic cells of MRSA and MR-CoNS strains and inhibited biofilm of MRSA. The addition of OXA to Cur-Chi-MNP increased biofilm inhibition and eradication activity against all Staphylococci strains (p=0.0007); higher biofilm activity was observed in early biofilm stages. Cur-Chi-MNP showed antimicrobial and biofilm inhibition activity against S. aureus. The addition of OXA increased biofilm inhibition and eradication activity against all Staphylococci strains. A combination treatment of Cur-Chi-MNP and OXA could be potentially used to treat staphylococcal biofilm-associated infections in its early stages before the establishment of biofilm bacterial cells.

Isolation, identification, and characterization of thermophilic lactic acid bacteria isolated from whey of Kars Kashar cheeses.

Kars Kashar cheese is an artisanal pasta-filata type cheese and geographically marked in Eastern Anatolia of Turkey. The aims of this research were to determine for the first time thermophilic lactic acid bacteria (LAB) of Kars Kashar cheese and characterize the technological properties of obtained isolates. In our research, a number of 15 samples of whey were collected from the different villages in Kars. These samples were incubated at 45 °C and used as the source material for isolating thermophilic LAB. A total of 250 colonies were isolated from thermophilic whey, and 217 of them were determined to be presumptive LAB based on their Gram staining and catalase test. A total of 170 isolates were characterized by their phenotypic properties and identified using the MALDI-TOF mass spectrometry method. Phenotypic identification of isolates displayed that Enterococcus and Lactobacillus were the predominant microbiota. According to MALDI-TOF MS identification, 89 isolates were identified as Enterococcus (52.35%), 57 isolates as Lactobacillus (33.53%), 23 isolates as Streptococcus (13.53%), and one isolate as Lactococcus (0.59%). All thermophilic LAB isolates were successfully identified to the species level and it has been observed that MALDI-TOF MS can be successfully used for the identification of selected LAB. The acidification and proteolytic activities of the isolated thermophilic LAB were examined, and the isolates designated for use as starter cultures were also genotypically defined.

The Assessment of Heat-treated Turkish Type Dry Fermented Sausage (Sucuk) with Some Mixed Probiotic Lactic Acid Bacteria

T his study aims to obtain a combination of probiotic strains resistant to stress conditions, which good effect the qualitative properties of heat-treated sausage. Lactic acid bacteria (LAB) strains were identified by matrix-assisted laser desorption-ionization-time of flight mass spectrometry (MALDI-TOF MS) after phenotypic evaluation and evaluated for probiotic potential including acid and bile salt tolerance, antibiotic resistance, and antibacterial activity. All tested Lactiplantibacillus plantarum strains showed high antibacterial activity. L. plantarum (PeE2) and additionally two Enterococcus strains with the best probiotic properties were selected among the strains. Except for the control group, 6 different sucuk doughs were prepared using them as mixed cultures. The ripened sucuk doughs (18 h, 4 ºC) were subjected to fast-drying (30 min, 60 ºC), heat-treated (64 ºC of inner temperature) and rapid-drying (10 min, 75 ºC) stages. The quality characteristics of samples were determined during storage (days 7, 14, and 21). Coliform bacteria growth ≤1 log cfu/g determined on the final day of storage. The lowest TBARS value was determined in L. plantarum +E. faecium (1:1 ratio) mixed culture. Regarding L* and a* color values, lower results were observed in the control group during storage. Using probiotic culture significantly affected textural values, taste and general acceptability (p<0.05) and contributed to the quality parameters. The result of the study may support the development of a new approach for using new probiotics in meat products.

Bacterial discrimination by Fourier transform infrared spectroscopy, MALDI-mass spectrometry and whole-genome sequencing.

Aim: Proof-of-concept study, highlighting the clinical diagnostic ability of FT-IR compared with MALDI-TOF MS, combined with WGS. Materials & methods: 104 pathogenic isolates of Neisseriameningitidis, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus were analyzed. Results: Overall prediction accuracy was 99.6% in FT-IR and 95.8% in MALDI-TOF-MS. Analysis of N. meningitidis serogroups was superior in FT-IR compared with MALDI-TOF-MS. Phylogenetic relationship of S. pyogenes was similar by FT-IR and WGS, but not S. aureus or S. pneumoniae. Clinical severity was associated with the zinc ABC transporter and DNA repair genes in S. pneumoniae and cell wall proteins (biofilm formation, antibiotic and complement permeability) in S. aureus via WGS. Conclusion: FT-IR warrants further clinical evaluation as a promising diagnostic tool.

Detection of KPC enzyme by MALDI-TOF MS from bacteria impregnated in filter paper

The main mechanism that causes resistance to carbapenem, one of the most potent antibiotic available, in Enterobacterales bacterial isolates, is due to Klebsiella pneumoniae carbapenemase (KPC) production by the bacterium. KPC is spread worldwide, requiring laboratories to be capable of identifying this enzyme, however some methods can be expensive for small laboratories, especially in developing countries. Therefore, the development of methods with low cost of reagents for the detection of KPC enzyme is necessary. The objective of this study was to evaluate the detection of KPC enzyme by MALDI-TOF MS from inactivated bacteria impregnated in filter paper. A total of 129 Enterobacterales isolates were impregnated in filter paper, and after 7 days at room temperature, they were subjected to a protein extraction protocol and spectra acquisition, in triplicates, by MALDI-TOF MS. The spectra were evaluated and KPC was identified according to the presence of a peak of 28,712.62 ± 27.80 m/z. Considering the presence of the KPC peak in at least one spectrum of the triplicates, this method presented 60.8% sensitivity and 96.4% specificity. However, considering the presence of KPC peak in at least two spectra of the triplicate, a specificity of 100% was achieved. The detection of KPC enzyme from inactivated bacteria impregnated in filter paper can be used as a method to confirm the presence of KPC, which could be very significant for small laboratories with limited resources.

Non-Flammable Epoxy Composition Based on Epoxy Resin DER-331 and 4-(β-Carboxyethenyl)phenoxy-phenoxycyclotriphosphazenes with Increased Adhesion to Metals

Functional cyclophosphazenes have proven to be effective modifiers of polymer materials, significantly improving their performance properties, such as adhesive characteristics, mechanical strength, thermal stability, fire resistance, etc. In this study, 4-(β-carboxyethenyl)phenoxy-phenoxycyclotriphosphazenes (CPPP) were obtained by the condensation of 4-formylphenoxy-phenoxycyclotriphosphazene with malonic acid. Its structure was studied using 31P, 1H, and 13C NMR spectroscopy and MALDI-TOF mass spectrometry, and the thermal properties were determined by DSC and TGA methods. Molecular modeling using the MM2 method showed that CPPPs are nanosized with diameters of spheres described around the molecules in the range of 1.34–1.93 nm, which allows them to be classified as nanosized structures. The epoxy resin DER-331 was cured with CPPP, and the conversion of epoxy groups was assessed using IR spectroscopy. Using optical interferometry, it was shown that CPPPs are well compatible with epoxy resin in the temperature range from 80 to 130 °C. It was established that the cured epoxy composition was fire resistant, as it successfully passed the UL-94 vertical combustion test due to the formation of porous coke during the combustion process and also had high heat resistance and thermal stability (decomposition onset temperature about 300 °C, glass transition temperature 230 °C). The composition has low water absorption, high resistance to fresh and salt water, fire resistance, and adhesive strength to steel and aluminum (11 ± 0.2 MPa), which makes it promising for use as an adhesive composition for gluing parts in the shipbuilding and automotive industries, the aviation industry, and radio electronics.

Genomic characterization of carbapenem and colistin-resistant Klebsiella pneumoniae isolates from humans and dogs.

Carbapenem and colistin-resistant Enterobacteriaceae, including Klebsiella pneumoniae, have become a growing global concern, posing a significant threat to public health. Currently, there is limited information about the genetic background of carbapenem and colistin-resistant K. pneumoniae isolates infecting humans and dogs in Thailand. This study aimed to characterize carbapenem and colistin-resistant genes in six resistant K. pneumoniae clinical isolates (three from humans and three from dogs) which differed in their pulse field gel electrophoresis profiles. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), antimicrobial susceptibility testing, and whole-genome sequencing were employed to identify and analyze the isolates. All six isolates were carbapenemase-producing K. pneumoniae isolates with chromosomally carried blaSHV, fosA, oqxA and oqxB genes, as well as nine to 21 virulence genes. The isolates belonged to five multilocus sequence types (STs): one isolate from a human and one from a dog belonged to ST16, with the other two human isolates being from ST340 and ST1269 and the other two dog isolates were ST147 and ST15. One human isolate and two dog isolates harbored the same blaOXA-232 gene on the ColKP3 plasmid, and one dog isolate carried the blaOXA-48 gene on the IncFII plasmid. Notably, one human isolate exhibited resistance to colistin mediated by the mcr-3.5 gene carried on the IncFII plasmid, which co-existed with resistance determinants to other antibiotics, including aminoglycosides and quinolones. In conclusion, this study provides a comprehensive characterization of both chromosome- and plasmid-mediated carbapenem and colistin resistance in a set of K. pneumoniae clinical isolates from unrelated humans and dogs in Thailand. The similarities and differences found contribute to our understanding of the potential widescale dissemination of these important resistance genes among clinical isolates from humans and animals, which in turn may contribute to outbreaks of emerging resistant clones in hospital settings.

Monitoring of enteropathogenic Gram-negative bacteria in wastewater treatment plants: a multimethod approach.

The wastewater treatment processes are associated with the emission of microbial aerosols, including enteropathogenic bacteria. Their presence in this work environment poses a real threat to the health of employees, both through the possibility of direct inhalation of the contaminated air and indirectly through the pollution of all types of surfaces with such bioaerosol particles. This study aimed to investigate the prevalence of enteropathogenic bacteria in the air, on surfaces, and in wastewater samples collected in four wastewater treatment plants (WWTPs). The effectiveness of conventional culture-biochemical, as well as spectrometric and molecular methods for the rapid detection of enteropathogenic bacteria at workstations related to particular stages of wastewater processing, was also evaluated. Bioaerosol, surface swab, and influent and effluent samples were collected from wastewater plants employing mechanical-biological treatment technologies. The air samples were collected using MAS-100 NT impactor placed at a height of 1.5m above the floor or ground, simulating aspiration from the human breathing zone. Surface samples were collected with sterile swabs from different surfaces (valves, handles, handrails, and coveyor belts) at workplaces. The raw influent and treated effluent wastewater samples were aseptically collected using sterile bottles. The identification of bacterial entheropathogens was simultaneously conducted using a culture-based method supplemented with biochemical (API) tests, mass-spectrometry (MALDI TOF MS), and molecular (multiplex real-time PCR) methods. This study confirmed the common presence of bacterial pathogens (including enteropathogenic and enterotoxigenic Escherichia coli, Salmonella spp., Campylobacter spp., and Yersinia enterocolitica) in all air, surface, and wastewater samples at studied workplaces. Higher concentrations of enteropathogenic bacteria were observed in the air and on surfaces at workplaces where treatment processes were not hermetized. The results of this study underline that identification of enteropathogenic bacteria in WWTPs is of great importance for the correct risk assessment at workplaces. From the analytical point of view, the control of enteropathogenic bacterial air and surface pollution using rapid multiplex-PCR method should be routinely performed as a part of hygienic quality assessment in WWTPs.

A comparative study of commercially available, minimally invasive, sampling methods on Early Neolithic humeri analysed via palaeoproteomics

Due to methodological advances in the archaeological sciences, an increasing number of archaeological specimens undergo destructive sampling. However, the preservation of cultural heritage is a primary concern. This leads to a dilemma between accessing sample material and obtaining sufficient information for a meaningful analytical outcome. Ideally, sampling a specimen would preserve the object for further macro, micro, and molecular analyses. For palaeoproteomics, a number of minimally invasive sampling approaches have been proposed, representing different benefits and limitations. There have been studies comparing a selection of these protocols, however, these have focused on specimens from a homogenous preservation environment using Zooarchaeology by Mass Spectrometry (ZooMS). Here we expand on earlier work by extending the comparison to specimens from two highly different preservation environments through both ZooMS and liquid-chromatography tandem mass spectrometry (LC-MS/MS). We compare five sampling approaches and seven extraction protocols in total, on 10 Bos sp. humeri from the Early Neolithic site of La Draga, Spain, utilising MALDI-ToF MS and LC-MS/MS to generate proteomic output, while assessing protocol invasiveness using microscopy and 3D imaging. Five humeri originate from Sector A, which is mostly related to dry, terrestrial preservation conditions, while the other five humeri stem from Sector B, which is characterised by its phreatic/aquatic preservation conditions. We show that there is a significant difference in protein recovery and taxonomic specificity between the sampling techniques applied, as well as between burial conditions. Additionally, various surface modifications were observed depending on the specific sampling technique applied. It is therefore essential to assess protein preservation for each sedimentological context within an archaeological site before performing extensive sampling, as protein preservation can be highly inter- and intra-site-specific.

Correlation between small-cell lung cancer serum protein/peptides determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and chemotherapy efficacy

BackgroundCurrently, no effective measures are available to predict the curative efficacy of small-cell lung cancer (SCLC) chemotherapy. We expect to develop a method for effectively predicting the SCLC chemotherapy efficacy and prognosis in clinical practice in order to offer more pertinent therapeutic protocols for individual patients.MethodsWe adopted matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and ClinPro Tools system to detect serum samples from 154 SCLC patients with different curative efficacy of standard chemotherapy and analyze the different peptides/proteins of SCLC patients to discover predictive tumor markers related to chemotherapy efficacy. Ten peptide/protein peaks were significantly different in the two groups.ResultsA genetic algorithm model consisting of four peptides/proteins was developed from the training group to separate patients with different chemotherapy efficacies. Among them, three peptides/proteins (m/z 3323.35, 6649.03 and 6451.08) showed high expression in the disease progression group, whereas the peptide/protein at m/z 4283.18 was highly expressed in the disease response group. The classifier exhibited an accuracy of 91.4% (53/58) in the validation group. The survival analysis showed that the median progression-free survival (PFS) of 30 SCLC patients in disease response group was 9.0 months; in 28 cases in disease progression group, the median PFS was 3.0 months, a statistically significant difference (χ2 = 46.98, P < 0.001). The median overall survival (OS) of the two groups was 13.0 months and 7.0 months, a statistically significant difference (χ2 = 40.64, P < 0.001).ConclusionsThese peptides/proteins may be used as potential biological markers for prediction of the curative efficacy and prognosis for SCLC patients treated with standard regimen chemotherapy.

Isolation of a CTX-M-55 (ESBL)-Producing Escherichia coli Strain of the Global ST6448 Clone from a Captive Orangutan in the USA.

The purpose of this study was to determine if orangutans (Pongo spp.) living in captivity at a zoo in Wisconsin were colonized with antimicrobial-resistant bacteria and, if found, to identify underlying genetic mechanisms contributing to their resistant phenotypes. We hypothesize that since antimicrobial-resistant bacteria are so prevalent within humans, the animals could also be carriers of such strains given the daily contact between the animals and the zoo staff that care for them. To test this theory, fecal samples from two orangutans were examined for resistant bacteria by inoculation on HardyCHROM™ ESBL and HardyCHROM™ CRE agars. Isolates were identified using MALDI-TOF mass spectrometry and antimicrobial susceptibility testing was performed using a Microscan autoSCAN-4 System. An isolate was selected for additional characterization, including whole genome sequencing (WGS). Using the Type (Strain) Genome Server (TYGS) the bacterium was identified as Escherichia coli. The sequence type identified was (ST/phylogenetic group/β-lactamase): ST6448/B1/CTX-M-55.