Conventional Microbiological Techniques Research Articles

Usefulness of point-of-care multiplex PCR to rapidly identify pathogens responsible for ventilator-associated pneumonia and their resistance to antibiotics: an observational study

BackgroundThe use of multiplex PCR to shorten time to identification of pathogens and their resistance mechanisms for patients with ventilator-associated pneumonia (VAP) is attractive, but poorly studied. The multiplex PCR–based Unyvero pneumonia cartridge assay can directly identify 20 bacteria and one fungus, amongst the most frequently causing VAP, and 19 of their resistance markers in clinical specimens (bronchoalveolar lavage or tracheal aspirate), with a turnaround time of 4–5 h. We performed this study to evaluate the concordance between the multiplex PCR–based Unyvero pneumonia cartridge assay and conventional microbiological techniques to identify pathogens and their resistance mechanisms in patients with VAP.MethodsAll patients suspected of having VAP (January 2016 to January 2019), who underwent fiberoptic bronchoscopy with bronchoalveolar lavage fluid (BALF) and whose BALF microscopy examination revealed intracellular bacteria, were included. BALF conventional cultures (gold standard), antimicrobial susceptibility testing and processing for the Unyvero pneumonia cartridge were done. Culture and Unyvero results were compared.ResultsCompared to cultures of the 93 samples processed for both techniques, Unyvero correctly identified pathogens in 68 (73%) proven VAP episodes, was discordant for 25 (27%), detected no pathogen in 11 and overdetected a not otherwise found pathogen in six. For the eight remaining discordant results, the pathogen responsible for VAP was not included in the Unyvero cartridge panel or it grew at a non-significant level in culture. Amongst the 31 (33%) resistance mechanism discordances observed, 22 were resistance detection failures and 24 concerned Pseudomonas aeruginosa.ConclusionsCompared to conventional microbiological cultures, the Unyvero pneumonia cartridge had poor diagnostic performance: it correctly identified pathogens and their resistance mechanisms in 73% and 67% of VAP cases, respectively. The lack of performance on the resistance mechanism was more pronounced when the pathogen detected was a Pseudomonas aeruginosa.

Identification of Two Pathogens in a Culture‐Negative Prosthetic Joint Infection Using Nanopore Sequencing

Periprosthetic joint infection (PJI) following total joint arthroplasty (TJA) is challenging to diagnose due to lower yield in pathogenic identification (~50%) using conventional microbiologic culture techniques. Here we present a case in which two pathogens were detected using nanopore sequencing from a 50 year‐old male with a suspected, yet culture‐negative, PJI. Three swabs were taken from multiple sites of drainage surrounding the suspected infected right knee joint following a routine arthroplasty (original TKA was done three years prior). DNA was extracted from the swabs using the MasterPure™ Gram Positive DNA Purification Kit (Lucigen). Sample libraries were prepared using the Rapid PCR Barcoding Kit (Oxford Nanopore Technologies) and sequenced on a v9.4.1 MinION flow cell. A total of 1,056,710 reads from all three swabs combined were aligned using a minimum Q score of 7.0 to the RefSeq database on the EPI2ME platform using the “What’s in my pot?” app. A total of 5,540 reads (0.52%) aligned to the bacteria Corynebacterium with the most abundant species being detected as Corynebacterium simulans. A total of 1,886 (0.18%) mapped to the fungi Malassezia globosa. Due to the patient having a visible arthrocutaneous fistula combined with visualized drainage from the joint using a methylene blue challenge test, the prosthetic knee was removed and an antibiotic spacer that contained 6 gm vancomycin, 7.2 gm tobramycin and 100 mg of amphotericin B was implanted. The patient also received a 6 week course of ceftriaxone infusions and has shown rapid clinical improvement. While our sequencing results were not used directly for patient care in this case, it demonstrates the promise that nanopore sequencing has for future use in infectious disease diagnostics.

Molecular Characterization of Bacillus-Genus Bacteria with Fibrinolytic Potential Isolated from Squashes «NTETE» in Brazzaville in the Republic of Congo

Thromboses appearing in the blood and causing cardiovascular disease by the presence of fibrin remain a problem of concern worldwide. The fibrinolytic enzymes developed by bacteria of the Bacillus-genus are today an interesting and promising alternative to chemicals with multiple consequences. It is in this perspective that three (3) samples of cooked squash wrapped in sheets and consumed in Brazzaville and collected and in three Brazzaville markets were explored. The estimated count of the total flora of each sample for each of the three markets, namely Total, Moukondo and Tsiémé was carried out on PCA and represents respectively (5.2 ± 0.12) 107 CFU / g, (1.73 ± 0 , 16) 107 CFU / g, and (9.43 ± 1.06) 107 CFU / g while bacteria of the genus Bacillus are estimated in Mossel respectively at (3.5 ± 1.16) 106 CFU / g, (4, 01 ± 0.85) 106 CFU / g (8.96 ± 0.60) 106 CFU / g. Sixty-six isolates of bacteria of the genus Bacillus isolated from squash by conventional microbiology techniques have been phenotypically characterized. The morphological types characterized are essentially the bacillary form and the spherical form. The ability to produce fibrinolytic enzymes correlated with growth was assessed. The growth in terms of optical density varies from 0.800 to 0.97 and the enzymatic production in all the isolates tested varies from 12 to 21 mm. After DNA extraction from 36 isolates, PCR amplification of the rR16S gene revealed fragments of approximately 1500bp by electrophoresis on Agarose Gel. The sequencing of thirty-four (34) fragments made it possible to obtain fifteen (15) sequences having a strong similarity between them and also with the homologs of the databases (97% to 100%) and therefore the molecular identification of: Bacillus sp 40%, Bacillus amyloliquefaciens 6.66%, Bacillus subtilis 33.33%, Bacillus Pumilus 6.66%, Bacillus megaterum 6.66%, Bacillus velezensis 6.66%. Five (5) of these sequences have been submitted to GenBank and the accession numbers are successively: MK193815.1 (Bacillus subtilis strain ASM1), MK207434.1 (Bacillus subtilis strain ASM3), MK207435.1 (Bacillus pumilus strain ASM5) , MK207436.1 (Bacillus subtilis strain ASM4), MK207437.1 (Bacillus megaterium strain ASM2).The multiple alignment of sequences obtained shows a high conservation of this gene in bacteria of the genus Bacillus. The phylogenetic classification clearly shows this monophyletic class of bacteria of the genus Bacillus with very short distances(less than 3%).

Identification of Non-fermenters by Matrix Assisted Laser Desorption/Ionisation- Time of Flight Mass Spectrometry: Case Series of Achromobacter Infections

Achromobacter is a non-fermenting Gram Negative Bacillus (GNB) belonging to the family Alcaligenaceae. It is emerging as a pathogen, causing myriad of infections in different patient groups. Often wrongly identified by conventional microbiological techniques, Achromobacter when recovered from clinical specimens have often been discarded as contaminants. Accurate species identification of the currently known 21 species of Achromobacter, is difficult, labour-intensive and time consuming. Automation has made it possible to reliably and correctly identify Achromobacterspecies and other non-fermenters. Matrix Assisted Laser Desorption Ionisation-Time Of Flight-Mass Spectrometry (MALDI-TOF-MS) is a boon as it rapidly and accurately identifies Achromobacter species in clinical specimens. Three cases of Achromobacter infection, highlighting the pathogenic potential of the bacteria and the role of automation in its identification are presented with due consent from relatives or patient themselves. This case series highlights that Achromobacterwhen recovered from clinical specimens should not be discarded as contaminants especially if isolated repeatedly. Case one was a patient who had developed pyelonephritis and sepsis, associated with bacteraemia and ended fatally, Achromobacter was recovered on two occasions from the blood culture. Case two was a patient who was diagnosed of acute pancreatitis with an infected peripancreatic collection who recovered with prolonged therapy, Achromobacter was repeatedly isolated from the pus draining from the peripancreatic collection. Case three was a patient with an infected malignant ulcer with purulent discharge from the swelling. In all three cases Achromobacter was isolated and identified by MALDI-TOF-MS.

Bioprospección de bacterias ácido lácticas productoras de biofloculante aisladas de jugo de caña residual

The objective was to isolate bioflocculant-producing lactic acid bacteria (LAB) from residual sugarcane juice. For this reason, sugarcane juice samples were obtained from 15 randomly sampled residual sugarcane stalk samples. The isolation of LAB was then carried out by means of conventional microbiology techniques, using Agar Mayeux, Sandine and Elliker (MSE) culture medium at pH 7.2 and an incubation time of 30°C for 48 hours. Subsequently, pure cultures were made from the characteristic colonies of Leuconostoc mesenteroides (rubbery, viscous, translucent and creamy colonies) for their biochemical identification according to Bergey’s Manual of Bacteriological Determination. The identification and selection of L. mesenteroides subsp. mesenteroides was carried out according to the Kappa coefficient statistical method, with the purpose of using it in the production of dextran (bioflocculant) in an aerated-agitated bioreactor (Aplikon brand). The purity of the dextran was determined using the FT-IR technique, which was compared with the spectrum of pure dextran produced by the strain NRRL P-640. Four strains of Leuconostoc mesenteroides, LM (01-04) were isolated, of which the strain LM03 was identified as L. meenteroides subsp. mesenteroides. The dextran values produced by LM03 were 26.87 g/L at 80 hours (maximum concentration) and 2.61 g/L at 4 hours (minimum concentration). The dextran produced by LM03 is pure according to FT-IR analysis. In conclusion, it was possible to isolate BAL L. mesenteroides subsp. mesenteroides (strain LM03), which had the capacity to produce dextran, which can be used as a bioflocculant with different biotechnological and industrial uses.

Evaluation by Flow Cytometry of Escherichia coli Viability in Lettuce after Disinfection.

Foodborne outbreaks due to the consumption of ready-to-eat vegetables have increased worldwide, with Escherichia coli (E. coli) being one of the main sources responsible. Viable but nonculturable bacteria (VBNC) retain virulence even after some disinfection procedures and constitute a huge problem to public health due to their non-detectability through conventional microbiological techniques. Flow cytometry (FCM) is a promising tool in food microbiology as it enables the distinction of the different physiological states of bacteria after disinfection procedures within a short time. In this study, samples of lettuce inoculated with E. coli were subject to disinfection with sodium hypochlorite at free chlorine concentrations of 5, 10, 25, 50, and 100 mg·L−1 or with 35% peracetic acid at concentrations of 5, 10, 25, and 50 mg·L−1. The efficiency of these disinfectants on the viability of E. coli in lettuce was evaluated by flow cytometry with LIVE/DEAD stains. Results from this study suggest that FCM can effectively monitor cell viability. However, peracetic acid is more effective than sodium hypochlorite as, at half the concentration, it is enough to kill 100% of bacteria and always induces a lower percentage of VBNC. Finally, we can conclude that the recommended levels of chemical disinfectants for fresh fruit and vegetables are adequate when applied in lettuce. More importantly, it is possible to ensure that all cells of E. coli are dead and that there are no VBNC cells even with lower concentrations of those chemicals. These results can serve as guidance for lettuce disinfection, improving quality and the safety of consumption.

Improving the diagnosis of tuberculous meningitis: good, but not good enough

Tuberculosis (TB) is the leading infectious cause of death globally. Meningitis occurs in 1–2% of TB cases (more in HIV-endemic settings), but kills or disables up to 50% or more of those affected [1]. Tuberculous meningitis (TBM) is notoriously difficult to diagnose with conventional microbiological techniques because of the scarcity of bacilli and Mycobacterium tuberculosis DNA.

Can sputum gram stain be used to predict lower respiratory tract infection and guide empiric antimicrobial treatment: Experience from a tertiary care hospital

BackgroundThe mortality associated with lower respiratory tract infection is high. Indiscriminate use of antimicrobials leads to alteration of respiratory tract flora and development of multi-drug resistance. Rapid diagnostic tests to confirm infection can guide the clinicians about antimicrobial treatment. So the present study was planned to evaluate the role of direct gram stain examination as a rapid and simple test to help clinicians for appropriate patient management. MethodsThe present study was conducted on 1000 respiratory specimens which were processed using conventional microbiological techniques. Gram stain smear and culture results were compared statistically to assess the sensitivity, specificity, positive and negative predictive value. The agreement between gram stain smear examination and culture was calculated using kappa statistics. ResultsPotential pathogens were obtained from 28 of 209 deeply coughed out sputum samples (13.3%) and from 19 of 315 saliva mixed sputum samples (6%). Out of 473 tracheal aspirates, 115 (24.3%) had potential pathogens. The sensitivity for predicting infection was higher for good quality sputum samples (54%) as compared to poor quality sputum samples (37%). The gram stain and culture of tracheal samples had a good agreement for predicting infection whereas there was only moderate agreement for sputum sample. ConclusionGram stain smear examination from respiratory samples can be used to guide empiric antibiotic therapy pending final culture sensitivity results if the attending physicians ensure appropriate sample collection and transport. In absence of these supportive measures smear examination should not be relied upon for empiric treatment.

An outbreak of Salmonella Saintpaul in a Scottish childcare facility: the influence of parental under-reporting

BackgroundSalmonella outbreaks in childcare facilities are relatively rare, most often occurring secondary to contaminated food products or poor infection control practices. We report an outbreak of Salmonella Saintpaul at a pre-school facility in Ayrshire, Scotland with atypical clinical and epidemiological features.MethodsFollowing notification of the initial two cases, the multi-disciplinary Incident Management Team initiated enhanced active case finding and two environmental inspections of the site, including food preparation areas. Parent and staff interviews were conducted by the Public Health department covering attendance, symptomatology and risk factors for all probable and confirmed cases. Microbiological testing of stool samples and the facility water tank was conducted. Whole Genome Sequencing (WGS) was performed for positive stool samples at the national reference laboratory. Infection control measures were introduced iteratively due to the atypical progression of the outbreak.ResultsThere were 15 confirmed cases and 3 children admitted to hospital during the outbreak. However, 35.7% of cases reported extremely mild symptoms. The attack rate was 15.2%, and age of affected children ranged from 18 to 58 months (mean 35 months). All cases were the same Multilocus Sequence Type (MLST50). Epidemiological investigation strongly suggested person-to-person spread within the facility. Existing infection control practices were found to be of a high standard, but introduction of additional evidence-based control measures was inadequate in halting transmission. Facility staff reported concerns about lack of parental disclosure of gastrointestinal symptoms, particularly where these were mild, with 50.0% of cases having attended while symptomatic against public health advice. Voluntary two-week closure of the facility was implemented to halt transmission, following which there were no new cases. WGS results were unavailable until after the decision was taken to close the facility.ConclusionsThis is the first reported instance of a Salmonella Saintpaul outbreak at a childcare facility, or where person-to-person transmission is indicated. Clinicians should consider the influence of parental under-reporting on gastrointestinal outbreaks in childcare settings, particularly where perceived severity is low and financial or social pressures to attend work may reduce compliance. WGS cannot yet replace conventional microbiological techniques during short, localised outbreaks due to delays receiving results.

Immunoproteomic analysis of Lawsonia intracellularis identifies candidate neutralizing antibody targets for use in subunit vaccine development

Lawsonia intracellularis is an obligate intracellular microorganism and the causative agent of porcine proliferative enteropathy. Due to its obligate intracellular nature, characterization of antigens and proteins involved in host-pathogen interaction and immune recognition have been difficult to achieve using conventional microbiological techniques. In this work, we used 2-dimensional gel electrophoresis coupled with Western-immunoblotting, mass spectrometry and bioinformatics to identify bacterial proteins that interact in vitro with pig intestinal cells (IPEC-1), have immunogenic properties and the potential to be used as subunit vaccine antigens. We detected eleven immunogenic bacterial proteins from which fliC (LI0710), LI1153 (annotated by NCBI as Putative protein N), and LI0649 (annotated as autotransporter) were predicted to be expressed on the outer membrane while LI0169 (oppA; annotated as ABC dipeptide transport system) was predicted to be periplasmic with a transmembrane domain forming a central pore through the plasma membrane. Genes coding for these four proteins were cloned and expressed in Escherichia coli and the corresponding recombinant proteins were purified using affinity chromatography. Porcine hyperimmune serum against whole Lawsonia lysate established that all four recombinant proteins were immunogenic. Further, rabbit hyperimmune sera generated against the vaccine strain of L. intracellularis and rabbit serum specific for each recombinant protein showed an inhibitory effect on the attachment and penetration of live, avirulent L. intracellularis, thus indicating that each protein is a potential neutralizing antibody target and a candidate for subunit vaccine formulation.

English

In this study the microbiota of Clarias gariepinus tissues harvested from e-waste-soil polluted vials were assessed. Soil samples contaminated with e-waste were analyzed using standard analytical protocol while the microbial study was obtained using standard conventional microbiological techniques. The results revealed that the soil was sandy-loamy and blackish in colour. High organic matter (17.60%) and organic carbon (10.17%) were obtained. Also, higher calcium (182.00 mg/kg) and phosphorus (146.65 mg/kg) contents compared to other mineral constituents were recorded while, the heavy metals ranged from 0.32- 64.90 mg/kg. Bacterial count ranged from 9.0 × 102 to 4.0 × 103 cfu/L while the fungal count from 4.0 × 102 to 2.3 ×103 sfu/L. The genera of bacteria isolate were identified as Staphylococcus, Proteus, Bacillus, Listeria, Salmonella, Enterobacter, Pseudomonas, Lactobacillus and Corynebacterium and fungal isolates were Penicilliun, Candida, Articulospora, Aspergillus, Rhizopus, Mucor, Zoopage, Varcosporium and Rhodotorula. Microbial species richness of fish tissues from polluted vials indicates a more poly-diverse microbial community compared to those from unpolluted vial. Differences were observed in the occurrence of fungi on the surfaces of catfish tissues in control vial compare to those on fishes from polluted vials. Hence, there is a need for proper water management for fish culturing and caution in the exploration of fishes from polluted natural waters for human consumption. Key words: Clarias gariepinus, e-waste, fish tissues, microbiota, pollution, microbial species richness. &nbsp

Neonatal sepsis due to ESBL producing Klebsiella

Neonatal sepsis due to ESBL producing Klebsiella - IJMR- Print ISSN No: - 2394-546X Online ISSN No:- 2394-5478 Article DOI No:- 10.18231/j.ijmr.2019.024, Indian Journal of Microbiology Research-Indian J Microbiol Res

Rapid Pathogen Identification With Direct Application of MALDI-TOF Mass Spectrometry on an Endophthalmitis Vitreous Sample Without Prior Culture

Purpose: We report a case of a 72-year-old man with bleb-related endophthalmitis (BRE) whose vitreous samples were directly analyzed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to rapidly identify the causative organism, whereas the results from conventional microbiological techniques were negative. Methods: We analyzed BRE vitreous samples with MALDI-TOF MS (Vitek MS, bioMérieux) for rapid pathogen identification without prior culture. Samples were also analyzed with standard microbiological methods. Results: Within 1 hour of sample acquisition, MALDI-TOF MS identified Gemella sanguinis from the undiluted vitreous sample from vitrectomy without prior culture with a confidence value of 99.7%. Gram stain and cultures from aqueous and vitreous samples were negative for 28 days after acquisition. The patient’s right-eye vision improved from hand motion to 20/50 2 months later. Conclusions: Our findings suggest that the direct analysis of intraocular samples with MALDI-TOF MS could be a novel, promising adjuvant method of rapid endophthalmitis diagnosis.

Nuclear magnetic resonance biosensor for rapid detection of Vibrio parahaemolyticus

BackgroundVibrio parahaemolyticus is a Gram-negative bacterium widely distributed in marine environments and a well-recognized invertebrate pathogen frequently isolated from seafood. V. parahaemolyticus may also spread into humans, via contaminated, raw, or undercooked seafood, causing gastroenteritis and diarrhea. MethodsA Nuclear Magnetic Resonance (NMR)-based detection system was used to detect pathogenic levels of this microorganism (105 CFU/ml) with Molecular Mirroring using iron nanoparticles coated with target-specific biomarkers capable of binding to DNA of the target microorganism. The NMR system generates a signal (in milliseconds) by measuring NMR spin–spin relaxation time T2, which correlates with the amount of microorganism DNA. ResultsCompared with conventional microbiology techniques such as real-time PCR (qPCR), the NMR biosensor showed similar limits of detection (LOD) at different concentrations (105–108 CFU/ml) using two DNA extraction methods. In addition, the NMR biosensor system can detect a wide range of microorganism DNAs in different matrices within a short period of time. ConclusionNMR biosensor represents a potential tool for diagnostic and quality control to ensure microbial pathogens such as V. parahaemolyticus are not the cause of infection. The “hybrid” technology (NMR and nanoparticle application) opens a new platform for detecting other microbial pathogens that have impacted human health, animal health and food safety.

The mechanism of action of pepR, a viral-derived peptide, against Staphylococcus aureus biofilms.

ObjectivesTo investigate the mechanism of action at the molecular level of pepR, a multifunctional peptide derived from the Dengue virus capsid protein, against Staphylococcus aureus biofilms.MethodsBiofilm mass, metabolic activity and viability were quantified using conventional microbiology techniques, while fluorescence imaging methods, including a real-time calcein release assay, were employed to investigate the kinetics of pepR activity at different biofilm depths.ResultsUsing flow cytometry-based assays, we showed that pepR is able to prevent staphylococcal biofilm formation due to a fast killing of planktonic bacteria, which in turn resulted from a peptide-induced increase in the permeability of the bacterial membranes. The activity of pepR against pre-formed biofilms was evaluated through the application of a quantitative live/dead confocal laser scanning microscopy (CLSM) assay. The results show that the bactericidal activity of pepR on pre-formed biofilms is dose and depth dependent. A CLSM-based assay of calcein release from biofilm-embedded bacteria was further developed to indirectly assess the diffusion and membrane permeabilization properties of pepR throughout the biofilm. A slower diffusion and delayed activity of the peptide at deeper layers of the biofilm were quantified.ConclusionsOverall, our results show that the activity of pepR on pre-formed biofilms is controlled by its diffusion along the biofilm layers, an effect that can be counteracted by an additional administration of peptide. Our study sheds new light on the antibiofilm mechanism of action of antimicrobial peptides, particularly the importance of their diffusion properties through the biofilm matrix on their activity.

A micro-Raman and chemometric study of urinary tract infection-causing bacterial pathogens in mixed cultures

Detection of urinary tract infection (UTI)-causing bacteria uses conventional time-consuming microbiological techniques. The current need is to use a fast and reliable method of bacterial identification. In order to unambiguously distinguish the UTI-causing five bacterial species used in the current study, micro-Raman spectra were obtained from a home-assembled micro-Raman system and analyzed by multivariate statistical techniques such as principal component analysis (PCA), partial least square-discriminate analysis (PLS-DA), and support vector machine (SVM). Also, the micro-Raman spectra recorded from samples containing two and three bacterial species were tested and validated against the aforementioned calibration models using PLS-DA and SVM. The prediction accuracies of up to 73 and 89% were achieved with PLS-DA and SVM, respectively. Taken together, the present study depicts the capturing of unique micro-Raman spectral features manifesting from the biochemical content of each bacterium. Also, micro-Raman spectroscopy combined with multivariate data analysis can therefore be a reliable and faster technique for the diagnosis of UTI-causing bacteria. Graphical Abstract.

Nasopharyngeal Carriage of Antimicrobial-Resistant Pneumococci in an Intensively Sampled South African Birth Cohort.

Introduction: Nasopharyngeal (NP) colonization by Streptococcus pneumoniae (pneumococcus) precedes the development of respiratory tract infection. Colonization by antimicrobial-resistant pneumococci, especially in infants, is a major public health concern. We longitudinally investigated antimicrobial-resistance amongst pneumococci colonizing the nasopharynx of South African infants immunized with the 13-valent pneumococcal conjugate vaccine (PCV13).Methods: NP swabs were collected every second week from birth through the first year of life from 137 infants. Pneumococci were identified and serotyped using conventional microbiological techniques, and their antibiotic susceptibility profiles determined by disk diffusion and E-test.Results: All infants were immunized with 3 doses of PCV13. 1520 pneumococci (760 non-repeat) isolates were recovered from 137 infants; including non-typeable (n = 99), PCV13 (n = 133) and non-PCV13 serotypes (n = 528). The prevalence of penicillin, erythromycin, and cotrimoxazole non-susceptibility was 19% (95% CI 17–22%) (3% fully resistant), 18% (95% CI 15–21%) (14% fully resistant), and 45% (95% CI 42–49%) (36% fully resistant), respectively. The predominant penicillin-non-susceptible serotypes included 19A, 19F, 15B/15C, 15A, and 21, while susceptible serotypes included 23A, 34, and 17A. Multidrug-resistance (MDR) was observed in 9% (95% CI 7–11%) of the isolates. PCV13 serotypes were more likely to be non-susceptible, compared to non-PCV13 serotypes, to penicillin (26% vs. 16%, p = 0.007), erythromycin (23% vs. 15%, p = 0.027) and cotrimoxazole (62% vs. 41%, p < 0.001). Non-susceptibility to penicillin, erythromycin, and cotrimoxazole remained relatively constant through the first year of life (X2 test for trend: p = 0.184, p = 0.171, and p = 0.572, respectively). Overall, penicillin or erythromycin-non-susceptible pneumococci were carried for a shorter duration than susceptible pneumococci [penicillin (mean days, 18 vs. 21, p = 0.013) and erythromycin (mean days, 18 vs. 21, p = 0.035)]. Within individual infants carrying the same serotype longitudinally, changes in antibiotic susceptibility were observed over time in 45% (61/137) of infants and these changes were predominantly for penicillin (76%, 79/104).Conclusion: Prevalence of NP carriage with antibiotic-non-susceptible pneumococci was relatively constant throughout the first year of life. PCV13 serotypes were more commonly non-susceptible to penicillin, erythromycin, and cotrimoxazole. Penicillin or erythromycin-non-susceptible pneumococci were carried for a shorter duration than penicillin or erythromycin-susceptible pneumococci.

Estimating Bacterial Concentrations in Fibrous Substrates Through a Combination of Scanning Electron Microscopy and ImageJ.

Conventional signal-based microanalytical techniques for estimating bacterial concentrations are often susceptible to false signals. A visual quantification, therefore, may compliment such techniques by providing additional information and support better management decisions in the event of outbreaks. Herein, we explore a method that combines electron microscopy (EM) and image-analysis techniques and allows both visualization and quantification of pathogenic bacteria adherent even to complex nonuniform substrates. Both the estimation and imaging parameters were optimized to reduce the estimation error ( E, %) to close to ±5%. The method was validated against conventional microbiological techniques such as the use of optical density, flow cytometry, and quantitative real-time PCR (qPCR). It could easily be tailored to estimate different species of pathogens, such as Escherichia coli O157, Listeria innocua, Staphylococcus aureus, Enterococcus faecalis, and Bacillus anthracis, on samples similar to those in real-time contamination scenarios. The present method is sensitive enough to detect ∼100 bacterial CFU/mL but has the potential to estimate even lower concentrations with increased imaging and computation times. Overall, this imaging-based method may greatly complement any signal-based pathogen-detection technique, especially in negating false signals, and therefore may significantly contribute to the field of analytical microbiology and biochemistry.

Food inspection of the maltese cheeselet using hyperspectral imaging. The ‘food inspection using hyperspectral imaging’ (FIHI) project is financed by the Malta Council for Science and Technology, for and on behalf of the Foundation for Science and Technology, through the FUSION: R&amp;I technology Development Programme

The island of Malta has a rich heritage, which is evident in the rustic appeal of this island. Ġbejna forms an integral part of the Maltese food heritage. This artisan product is made from sheep or goat milk curds and aged for several months to develop its distinctive taste. During the ageing process, the cheese can become spoiled by fungi and unsafe for human consumption. This is a significant public health risk and a financial liability for producers. Conventional microbiology techniques do not detect these slow-growing pigment-less fungi early, allowing occasional distribution of contaminated products. We propose the use of hyperspectral imaging to detect these fungi during the early stages of cheese production. In contrast with a typical digital camera, which compiles the light signal into three broad wavelength bands; red, green and blue, a hyperspectral camera records numerous narrow and contiguous wavelength bands reflected from an object. This produces a series of images, each corresponding to the reflected electromagnetic energy in the respective narrow band of wavelengths. This image series may, in turn, be used for early fungal detection and identification. To test this hypothesis, a model cheeselet was produced to conduct compatibility and stability studies, through measurements of colony forming units, water activity, moisture levels, pH, protein and sugar content. The ġbejna model was then challenged with fungal strains isolated from commercial ġbejna and imaged using a hyperspectral camera. An algorithm is under development to differentiate contaminated samples from uncontaminated samples using image analysis and multivariate statistics.

Methylglyoxal, the Major Antibacterial Factor in Manuka Honey: An Alternative to Preserve Natural Cosmetics?

Microbial safety is an essential prerequisite of cosmetics, and preservatives are required to prevent product spoilage and damage to consumers’ health. Consumer concern about the safety of some cosmetic ingredients and the increasing demand for more natural beauty products has driven cosmetic industries and formulators to find natural alternatives to replace synthetic preservatives currently used. In this study, methylglyoxal (MGO, the main factor responsible for the antimicrobial activity of manuka honey) was tested for antimicrobial activity against a panel of selected bacteria and mycetes by using conventional microbiological techniques (determination of M.I.C., time-kill assay), and its potential preservative in an O/W emulsion was investigated (challenge test). MGO showed a remarkable and fast antibacterial activity (M.I.C. values 0.150–0.310 mg/mL), while the inhibitory activity against fungi was less marked (M.I.C. values 1.25–10 mg/mL); chitosan has proven to be a synergist of antimicrobial effectiveness of MGO. Results of the challenge test showed that the addition of MGO to a cream formulation was efficient against microbial contamination. On the basis of our results, MGO appears to be a good candidate as a cosmetic preservative of natural origin; further studies are needed to confirm its applicability and its safety.