Conventional Culture Methods Research Articles

A newly developed paper embedded microchip based on LAMP for rapid multiple detections of foodborne pathogens

BackgroundMicrofluidic chip detection technology is considered a potent tool for many bioanalytic applications. Rapid detection of foodborne pathogens in the early stages is imperative to prevent the outbreak of foodborne diseases, known as a severe threat to human health. Conventional bacterial culture methods for detecting foodborne pathogens are time-consuming, laborious, and lacking in pathogen diagnosis. To overcome this problem, we have created an embedded paper-based microchip based on isothermal loop amplification (LAMP), which can rapidly and sensitively detect foodborne pathogens.ResultsWe embed paper impregnated with LAMP reagent and specific primers in multiple reaction chambers of the microchip. The solution containing the target pathogen was injected into the center chamber and uniformly distributed into the reaction chamber by centrifugal force. The purified DNA of Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus, and Vibrio parahaemolyticus has been successfully amplified and directly detected on the microchip. The E. coli O157:H7 DNA was identified as low as 0.0134 ng μL− 1. Besides, the potential of this microchip in point-of-care testing was further tested by combining the on-chip sample purification module and using milk spiked with Salmonella spp.. The pyrolyzed milk sample was filtered through a polydopamine-coated paper embedded in the inside of the sample chamber. It was transported to the reaction chamber by centrifugal force for LAMP amplification. Then direct chip detection was performed in the reaction chamber embedded with calcein-soaked paper. The detection limit of Salmonella spp. in the sample measured by the microchip was approximately 12 CFU mL− 1.ConclusionThe paper embedded LAMP microchip offers inexpensive, user-friendly, and highly selective pathogen detection capabilities. It is expected to have great potential as a quick, efficient, and cost-effective solution for future foodborne pathogen detection.

Performance of MALDI-TOF Mass Spectrometry in the Philippines.

Identification of the causative pathogen in infectious diseases is important for surveillance and to guide treatment. In low- and middle-income countries (LMIC), conventional culture and identification methods, including biochemical methods, are reference-standard. Biochemical methods can lack sensitivity and specificity and have slow turnaround times, causing delays in definitive therapy. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI–TOF MS) is a rapid and accurate diagnostic method. Most studies comparing MALDI–TOF MS and biochemical methods are from high-income countries, with few reports from LMIC with tropical climates. The aim of this study was to assess the performance of MALDI–TOF MS compared to conventional methods in the Philippines. Clinical bacterial or fungal isolates were identified by both MALDI–TOF MS and automated (VITEK2) or manual biochemical methods in the San Lazaro Hospital, Metro Manila, the Philippines. The concordance between MALDI–TOF MS and automated (VITEK2) or manual biochemical methods was analyzed at the species and genus levels. In total, 3530 bacterial or fungal isolates were analyzed. The concordance rate between MALDI–TOF MS and biochemical methods was 96.2% at the species level and 99.9% at the genus level. Twenty-three isolates could not be identified by MALDI–TOF MS. In this setting, MALDI–TOF MS was accurate compared with biochemical methods, at both the genus and the species level. Additionally, MALDI–TOF MS improved the turnaround time for results. These advantages could lead to improved infection management and infection control in low- and middle-income countries, even though the initial cost is high.

Rapid Detection of Urinary Ureaplasma urealyticum and Mycoplasma hominis Isolated from Pregnant Women and Their Antibiotic Susceptibility Profile

Abstract Background Urinary tract infection is responsible for serious complications in pregnant women. Among different uropathogens, Ureaplasma urealyticum and Mycoplasma hominis are implicated in several urogenital infections. These organisms are not easily diagnosed by routine cultures and cannot be treated by common safe antibiotics used in pregnancy. This is the first study that aimed to rapidly detect these organisms in “urine of pregnant” women and determine their antibiotic susceptibility. Methods A total of 1500 pregnant women were included, 750 symptomatic patients and 750 controls (apparently healthy). Urine samples were taken aseptically from all subjects and analyzed using the Mycoplasma System Plus (MSP) kit. The specimens were further subjected to multiplex polymerase chain reaction (PCR) for the molecular confirmation. Results Of 750 urine specimens of the patients group, 223 specimens showed sterile pyuria suggesting the presence of mycoplasmas, whereas sterile pyuria was found in only 65 specimens of the control group. Of the 223 specimens of the patients group, 32 (14.3%) had U. urealyticum by MSP kit, 21 (9.4%) had M. hominis, 14 (6.3%) showed mixed coinfection, and 156 (70%) were negative. Seven (10.8%) of 65 specimens of the control group detected U. urealyticum. Conclusions There was a good agreement between the MSP kit and the multiplex PCR. The MSP kit was an easy and rapid alternative to the complicated conventional culture method for the detection of urinary mycoplasmas. Doxycycline and minocycline had the highest sensitivity rates; however, clindamycin would be the first choice for empirical treatment of pregnant women.

Epidemiology of Respiratory Pathogens Among Children Hospitalized for Pneumonia in Xiamen: A Retrospective Study.

ObjectivesTo investigate the etiology of common respiratory pathogens in children < 2 years of age hospitalized with pneumonia in Xiamen from 2014 to 2017.MethodsThe medical records of 5581 children with pneumonia were retrospectively reviewed. Direct immunofluorescent test was used for respiratory virus testing. Bacteria were detected by conventional culture method. The results of pathogen detection at admission were analyzed as well as the clinical outcomes of children.ResultsThe burden of hospitalized children with pneumonia was highest among infants < 6 months old (58.2%). Respiratory syncytial virus (RSV) was the most common respiratory virus (26.0%) followed by parainfluenza (4.8%) and adenovirus (3.2%). Haemophilus influenzae was the most common bacteria detected (16.6%) followed by Moraxella catarrhalis (13.4%), Staphylococcus aureus (13.0%), Streptococcus pneumoniae (12.3%), Escherichia coli (5.1%) and Klebsiella pneumoniae (4.8%). Notably, RSV and K. pneumoniae were detected more frequently in severe pneumonia (35.0% and 10.9%) versus mild pneumonia (25.6% and 4.6%), with higher rates of ICU admissions, longer hospital stays and higher hospital costs compared to those infected with other respiratory pathogens.ConclusionsAmong children < 2 years of age hospitalized with pneumonia in Xiamen, RSV was the most common respiratory virus, while H. influenzae and S. pneumoniae remained the predominant bacterial pathogens detected. Considering the low implementation rate of vaccines against pneumococcal and Hib pneumonia in China, there is an urgent need to increase both vaccination rates to reduce pneumococcal and Hib disease burden.

Study of listeriosis in spontaneous abortions during preganacy at tertiary care centre

Listeriosis is an emerging zoonotic disease. Listeria monocytogenes is an uncommon cause of illness in general population, however, in some high risk groups including neonates, pregnant women, elderly persons, immunosuppressed transplant recipients and others with impaired cell mediated immunity, it is important cause of life threatening bacteremia meningoencephalitis. Listeriosis is 18 times more common in pregnancy (12/100,000) than in the non-pregnant population (0.7/100,000) and 16-27% of all infections with Listeria occur in pregnant women. Isolation of bacteria by conventional methods of microscopy and culture on selective agar PALCUM agar after enrichment of samples by using UVM1, UVM2 .Speciation of listeria done by carbohydrate fermentation test followed by study of pathogenicity by hemolysis on sheep blood agar and CAMP test followed by antimicrobial susceptibility test done.Out of total 131 patients having history of Spontaneous abortion were screened for Listeria, in that 10(7.6%) were carrying Listeria out of which 3(2.29%) of them found to be positive for L. monocytogenes and 07(5.34%) were other Listeria spp. The prevalence of L. monocytogenes in present study was 03(2.29%) from spontaneous abortion cases.Based on the results of present study it is concluded that Listeria monocytogenes is responsible for spontaneous abortions in spontaneous abortion during pregnancy in humans.

Carriage, Risk Factors, and Antimicrobial Resistance Patterns of Salmonella Isolates from Raw Beef in Jimma, Southwestern Ethiopia.

PurposeConsumption of raw beef infected with multidrug-resistant Salmonella is pertinent to the world public health risk of antimicrobial resistance. Henceforth, this study aimed to investigate the carriage, antimicrobial resistance (AR) patterns, and the revealing risk factors of Salmonella-contaminating beef in abattoirs and butcher shops in Jimma town.MethodsA cross-sectional study was conducted to investigate the carriage and AR patterns and to reveal the risk factors of beef contaminated by Salmonella spp. Three hundred and forty-eight swab samples were collected from abattoirs (n=210) and butcher shops (n=138) and the conventional cultural methods were employed for identification of Salmonella. Isolates were subjected to 12 antimicrobials using the Kirby–Bauer disk diffusion method for AR patterns.ResultsThe occurrence of Salmonella isolates from the abattoir samples was 11.4%, whereas about 6.52% of isolates were recovered from butcher shops. Educational status (abattoir: odds ratio (OR)=8.40, confidence interval (CI)=1.186–59.493; butcher shops: OR=9.17, CI=1.15–73.239), job related training (abattoir: OR=5.50, CI=1.065–28.416), contamination risk perception (abattoir: OR=5.31, CI=1.256–22.489), neatness of knives (abattoir: OR=7.6, CI=0.892–65.376), source of contamination (abattoir: OR=8.44, CI=1.682–42.39), wearing of protective cloth (butcher shops: OR=8.44, CI=1.682–42.39), manner of hand washing (butcher shops: OR=7.25, CI=1.210–43.442), and money handling (butcher shops: OR= 9.69, CI=1.578–59.474) were among the potential risk factors significantly associated with Salmonella carcass contamination in the abattoir and butcher shops. Of the 33 Salmonella isolates, 14 (58.3%) and six (66.7%) of the abattoir and butcher shops isolates, correspondingly, were resistant to two or more antibiotics.ConclusionThe finding of this investigation exhibited extensive multidrug-resistant Salmonella isolates in the study setting. Hence, establishing standard meat safety requirements and provision of training for meat handlers and prudent use of antimicrobials are recommended.

Comparison between conventional and recent culture methods for detection of fish mycobacteria

The present study aimed to isolate and identify mycobacteria from Nile tilapia and fresh water as well as ornamental fish and aquarium water in Sohag Assuit and Qena Governorates. Conventional and recent methods (MB-Redox system) were used for isolation and detection of mycobacteria with comparison between them regarding to the rate of isolation time for growth and growth character of colonies. Three isolates were isolated from fresh water and identified as unclassified atypical mycobacteria and four isolates were recovered from aquarium water and identified as M. fortuitum and M. marinum. Eleven isolates were isolated from goldfish and identified as M. marinum, M. fortuitum and unclassified atypical mycobacteria. The recovery rate of MB-Redox system for mycobacteria was 12%, while in case of L-J medium was 10.66%. The mean time for detection of M. marinum was 3-7 days and 7-10 days for MBRedox system and Lowenstein – Jensen medium, respectively. The mean time for detection of M. fortuitum was 1-3 days for MB-Redox system, while in case of L-J medium was 3-6 days. The mean time for detection of other unidentified atypical mycobacteria was 3-8 days for MB-Redox system, while in case of L-J medium was 3-14 days. The detection of mycobacterial growth on MB-Redox system at different times revealed that the color of MB- Redox tubes appear yellow at time of inoculation changed to pink color after 3 days. The colonies appear as red particles on the surface. After 15 days, the color of MB-Redox medium changed to yellow with red particles on the surface.

Novel saltatory rolling circle amplification assay for rapid and visual detection of Campylobacter jejuni in chicken meat

Campylobacter jejuni is a widespread zoonotic foodborne pathogen and one of the major concerns in the poultry-based food industry. There is a need for a rapid, robust and equipment free detection system for C. jejuni as the conventional culture method is labour and resource-intensive. In this study, we have developed a simple saltatory rolling circle amplification (SRCA) assay integrating a cost-effective three-step centrifugation-based DNA extraction method for rapid and visual detection of C. jejuni in chicken meat. The specificity of the SRCA assay was established by using 17 C. jejuni and 18 non-C. jejuni strains. SRCA, end-point and real-time PCR were able to detect 50 fg, 5 pg and 500 fg C. jejuni DNA/tube, respectively. In the artificial-spiking study conducted in C. jejuni-free chicken meat, a limit of detection (LoD) of 3 log CFU/g and 2 log CFU/g were attained in 120 min (without enrichment) and 14 h (with 12 h enrichment), respectively. The LoD of the SRCA assay was 10- and 100-fold better than the end-point and real-time PCR, respectively. Therefore, keeping the end-user of resource-limited food analysis laboratories in mind, we have successfully developed a SRCA assay for rapid and accurate detection of C. jejuni in chicken meat.

Rapid detection and enumeration of aerobic mesophiles in raw foods using dielectrophoresis

The concept of dielectrophoresis (DEP), which involves the movement of neutral particles by induced polarization in nonuniform electric fields, has been exploited in various biological applications. However, only a few studies have investigated the use of DEP for detecting and enumerating microorganisms in foodstuffs. Therefore, we aimed to evaluate the accuracy and efficiency of a DEP-based method for enumerating viable bacteria in three raw foods: freshly cut lettuce, chicken breast, and minced pork. The DEP separation of bacterial cells was conducted at 20 V of output voltage and 6000 to 9000 kHZ of frequency with sample conductivity of 30–70 μS/cm. The accuracy and validity of the DEP method for enumerating viable bacteria were compared with those of the conventional culture method; no significant variation was observed. We found a high correlation between the data obtained using DEP and the conventional aerobic plate count culture method, with a high coefficient of determination (R2 > 0.90) regardless of the food product; the difference in cell count data between both methods was within 1.0 log CFU/mL. Moreover, we evaluated the efficiency of the DEP method for enumerating bacterial cells in chicken breasts subjected to either freezing or heat treatment. After thermal treatment at 55 °C and 60 °C, the viable cell counts determined via the DEP method were found to be lower than those obtained using the conventional culture method, which implies that the DEP method may not be suitable for the direct detection of injured cells. In addition to its high accuracy and efficiency, the DEP method enables the determination of viable cell counts within 30 min, compared to 48 h required for the conventional culture method. In conclusion, the DEP method may be a potential alternative tool for rapid determination of viable bacteria in a variety of foodstuffs.

Canine induced pluripotent stem cell maintenance under feeder-free and chemically-defined conditions.

Canine induced pluripotent stem cells (ciPSCs) provide a platform for regenerative veterinary medicine, disease modeling, and drug discovery. However, in the conventional method, ciPSCs are maintained using chemically-undefined media containing unknown animal components under on-murine embryonic fibroblast feeder conditions, which were reported to modify cell surface of iPSCs and increases the risk of immune rejection when the cells are transplanted into patients. Moreover, in the conventional method, ciPSCs are mechanically passaged, which requires much time and effort. Therefore, the large-scale expansion of ciPSCs is difficult, which should be resolved for using ciPSCs in clinical application and research. Here, it was shown that StemFit® AK02N and iMatrix-511 could maintain the pluripotency of ciPSCs using conventional culture method. Furthermore, it was demonstrated that the feeder-free and chemically-defined ciPSC culture systems using StemFit® AK02N and iMatrix-511 could stably maintain and allow the easy expansion of ciPSCs generated using N2B27 and StemFit® AK02N, without causing karyotype abnormalities. ciPSCs expressed several pluripotency markers and formed teratomas, including cells derived from three germ layers.

Three-dimensional spheroid culture induces apical-basal polarity and the original characteristics of immortalized human renal proximal tubule epithelial cells

The proximal tubules, which are part of the kidney, maintain blood homeostasis by absorbing amino acids, glucose, water, and ions such as sodium (Na), potassium, and bicarbonate. Proximal tubule dysfunction is associated with the pathogenesis of many kidney diseases. Renal proximal tubular epithelial cells (RPTECs) are responsible for the main functions of the proximal tubules. Therefore, in vitro experiments using RPTECs would greatly enhance our understanding of nephron physiology and pathobiology. It is preferable to use immortalized cell lines, such as human kidney-2 (HK-2) cells, because they are derived from humans and maintain growth indefinitely. However, tissue-specific RPTEC phenotypes, including apical-basal polarization, are frequently lost in conventional two-dimensional culture methods in part due to microenvironmental deficiencies. To overcome this limitation, we developed a three-dimensional (3D) spheroid culture method for HK-2 cells using an extracellular matrix. HK-2 spheroids in 3D culture formed a tubule-like architecture with cellular polarity and showed markedly restored Na transport function. 3D culture of HK-2 cells also increased expression of kidney development–related genes, including WNT9B. Models of human renal tubules using HK-2 spheroids will greatly improve our understanding of the physiology and pathobiology of the kidney.

Trichoderma Strains – Antagonists of Plant Pathogenic Micromycetes

Species of the genus Trichoderma are known as the producers of many biologically active substances, in particular, enzymes that have found their practical application in many industries. In addition, the active Trichoderma strains are used as biological control agents of plant pathogenic micromycetes. Trichoderma strains are able to exhibit antagonistic properties against plant pathogens very effectively due to their peculiarities: high growth rate; synthesis of chitinolytic enzymes and secondary metabolites with antifungal activity. Thus, highly active Trichoderma strains has been successfully used to control plant pathogenic isolates of Fusarium, Alternaria, Botrytis, Sclerotinia, Verticillium, Pythium and other genera. The aim of the study was to evaluate the antagonistic activity of 100 Trichoderma strains against eight test-cultures of plant pathogenic micromycetes of different species and to select the most active strains for further studies of their physiological properties. Methods. Cultures of the studied fungi were grown on potato-dextrose agar. The antagonistic activity of fungi of the genus Trichoderma against fungal plant pathogens was carried out using the conventional method of dual culture. The percentages of growth inhibition of plant pathogens were calculated, and on the basis of these values the antagonistic activity of strains were concluded. Microsoft Excel and Origin 8.0 (OriginLab) packages were used for statistical data processing. Results. According to the results of a study of the antagonistic activity of Trichoderma strains against plant pathogenic micromycetes in general, it was shown that the strains have a high level of antifungal activity. Thus, the most numerous group (38%) of the total studied Trichoderma strains were “highly active” ones that inhibited the growth of plant pathogens from 70 to 80%. The second largest group (27%) was “moderately active” strains with 60–70% inhibition of plant pathogenic test cultures. The smallest (6%) but the most active group consisted of the “most active” strains with an average value of the inhibition more than 80%. Thus, almost three quarters (71%) of the studied Trichoderma strains showed a high level of antagonistic activity against plant pathogens with the inhibition more than 60%. In addition, only 17% of Trichoderma strains were “inactive” and 12% of them showed insufficient activity with growth inhibition of plant pathogens less than 50%. Notably, 35% of Trichoderma strains were active against all eight test cultures of plant pathogenic micromycetes. Conclusions. The studied Trichoderma strains have significant antagonistic potential both for individual strains of plant pathogenic micromycetes and for all studied plant pathogens. The involvement of a wide range of test cultures of plant pathogens, as well as significant amount (100) of Trichoderma strains allowed a more objective and systematic assessment of the antagonistic potential of fungi of this genus. Thus, our study of action of the wide range of Trichoderma strains against test cultures of different species showed that the fungi of the genus Trichoderma were effective antagonists of plant pathogenic fungi. The 38% of Trichoderma strains showed a high level of antifungal action and inhibited the growth of plant pathogens by 70% and more. Only 12% of strains showed less than 50% activity. In addition, 35% of Trichoderma strains were active against all eight tested plant pathogen test cultures. The significant amount of studied Trichoderma strains was highly active, and they can be used as a basis for further research to obtain effective biological control agents of plant pathogenic micromycetes.

An investigation of conventional microbial culture for the Naja atra bite wound, and the comparison between culture-based 16S Sanger sequencing and 16S metagenomics of the snake oropharyngeal bacterial microbiota.

Naja atra is a major venomous snake found in Taiwan. The bite of this snake causes extensive wound necrosis or necrotizing soft tissue infection. Conventional microbial culture-based techniques may fail to identify potential human pathogens and render antibiotics ineffective in the management of wound infection. Therefore, we evaluated 16S Sanger sequencing and next-generation sequencing (NGS) to identify bacterial species in the oropharynx of N. atra. Using conventional microbial culture methods and the VITEK 2 system, we isolated nine species from snakebite wounds. On the basis of the 16S Sanger sequencing of bacterial clones from agar plates, we identified 18 bacterial species in the oropharynx of N. atra, including Morganella morganii, Proteus vulgaris, and Proteus mirabilis, which were also present in the infected bite wound. Using NGS of 16S metagenomics, we uncovered more than 286 bacterial species in the oropharynx of N. atra. In addition, the bacterial species identified using 16S Sanger sequencing accounted for only 2% of those identified through NGS of 16S metagenomics. The bacterial microbiota of the oropharynx of N. atra were modeled better using NGS of 16S metagenomics compared to microbial culture-based techniques. Stenotrophomonas maltophilia, Acinetobacter baumannii, and Proteus penneri were also identified in the NGS of 16S metagenomics. Understanding the bacterial microbiota that are native to the oropharynx of N. atra, in addition to the bite wound, may have additional therapeutic implications regarding empiric antibiotic selection for managing N. atra bites.

Prevalence and antibiotic susceptibility of community acquired methicillin resistant Staphylococcus aureus from healthy students of University of Jos

Staphylococcus aureus is a virulent pathogen that is currently, not only the most common cause of nosocomial infections worldwide, but increasingly also, a community associated pathogen. This study aimed at determining the prevalence and susceptibility of methicillin resistant S. aureus (MRSA) isolated from apparently healthy university student population in Jos Nigeria. Two hundred and seventeen (217) urine samples were screened for Staphylococcus aureus. Isolates were characterized by conventional cultural and biochemical methods including rapid test kits (MicrogenID test kit). Their susceptibility profiles were determined against a panel of eleven antibiotics using the method of the Clinical and Laboratory Standard Institute (CLSI). The MRSA status were confirmed phenotypicallyusing oxacillin and cefoxitin as markers. Of the 217 samples collected, 171 (78.8%) were positive for Staphylococcal species, while 135 of these were coagulase positive and were classed as S. aureus. Seventy-three (73) of these were identified as S. aureus with the MicrogenID test kit. Isolates were resistant to at least one of the antibiotics used in the study. Seventy-one (71) of 73 S. aureus isolates were methicillin resistant. The susceptibility pattern observed in community isolates implies that any outbreak of infections caused by these strains may pose a threat to public health.&#x0D; Keywords: Staphylococcus aureus; Prevalence; Antimicrobial resistance; MRSA, Susceptibility

Isolation Chip Increases Culturable Bacterial Diversity and Reduces Cultivation Bias.

Conventional cultivation methods, including petri dish plating, are selective and biased to enrich specific microorganisms, such as big population and fast-growing bacteria. In this study, we evaluated the ability of isolation chip (ichip) to reduce cultivation bias. We used the ichip and petri dish plating methods to cultivate bacteria from soil contaminated with (contaminated soil) or without (natural soil) crude oil. Ichip improved the richness and evenness of bacterial isolates in both the natural and contaminated soil samples. Using the petri dish plating method, Pseudomonas and Lysinibacillus isolates were found to be the most abundant, with over 50% of the relative abundance in the natural and oil-polluted soil-cultured communities, respectively. In comparison, using the ichip method, the isolates with the highest relative abundances were from Bacillus and Aeromonas in natural and contaminated soil-cultured communities, which only accounted for 20% and 28% of the total isolates, respectively. Interestingly, the evenness and richness of the bacteria varied slightly between the natural and oil-polluted soil samples, indicating that ichip had the ability to reduce the cultivation bias. In addition, oil selective pressure enriched the functional bacteria isolated using the petri dish plating method. In summary, ichip allows bacteria to grow evenly, as well as allowing for substance exchange between the environment and single cells. As such, it is a very good method for increasing culturable bacterial diversity and reducing cultivation bias.

Microbiota of Chicken Breast and Thigh Fillets Stored under Different Refrigeration Temperatures Assessed by Next-Generation Sequencing.

Chicken is one of the most widely consumed meats worldwide. The exploration of the bacterial diversity of chicken meat may provide new insights into the chicken-associated microbiome that will lead to moderation of food spoilage or safety. This study was undertaken to explore the bacterial communities of chicken breast and thigh fillets stored at refrigeration (0 °C and 5 °C) and slightly abuse (10 °C) temperatures for 5 days through conventional cultural methods along with next-generation sequencing (NGS) analysis. Total viable counts (TVC), Brochothrix thermosphacta, Pseudomonas spp., and lactic acid bacteria (LAB) were enumerated, while the bacterial communities were mapped through 16S rRNA gene amplicon sequencing. Chicken breast and thigh fillets possessed a complex bacterial structure that incorporated a total of >200 Operational Taxonomic Units (OTUs) at the genus level. The core microbiota of fresh samples consisted of Acinetobacter, Brochothrix, Flavobacterium, Pseudomonas, Psychrobacter, and Vibrionaceae (family). These genera persisted until the end of storage in >80% of samples, except Psychrobacter and Flavobacterium, while Photobacterium was also identified. Hierarchical clustering showed a distinction of samples based on storage time and chicken part. Conventional plate counting with growth media commonly used in spoilage studies did not always correspond to the microbial community profiles derived from NGS analysis, especially in Pseudomonas, Acinetobacter, Photobacterium, and Vibrionaceae. Results of the present study highlight Photobacterium and Vibrionaceae, in general, as potent chicken meat spoilers and suggest the necessity to combine classical microbiological methods along with NGS technologies to characterize chicken meat spoilage microbiota.

Frequency, Distribution and Genotyping of Malassezia Species in Patients with Psoriasis vulgaris

Abstract Background: Malassezia species are reported to play a role in the etiology of Psoriasis vulgaris. Objective: The aim of this study was to determine the presence, frequency, distribution, and genotyping of skin colonization of Malassezia species in Psoriasis vulgaris and to compare with healthy individuals and to investigate its relationship with the severity of the disease. Methods: Skin samples were taken from scalp, arm, body, and leg of 34 psoriasis patients (lesional/non-lesional skin) and 30 healthy volunteers. Overall, 392 skin scraping samples were taken for the isolation of Malassezia species, which were incubated on the modified-Dixon agar. Conventional culture methods were used for Malassezia species identification. In isolates, genotyping was carried out by PCR-RFLP method. Results: In the samples from psoriatic lesions, most frequently isolated Malassezia species were M.globosa and M.furfur. Similarly, the most frequently isolated species in healthy volunteers was M. globosa; followed by M.restricta and M.sympodialis. The M.furfur isolation rate in psoriatic scalp and leg lesions of the patients was significantly higher than in healthy volunteers. There was no relationship between the severity of the disease and the isolated species. Conclusion: It was found that there was a difference between patients with psoriasis and healthy controls regarding presence and frequency of Malassezia species. Therefore, our study results support the view that Malessezia species may be associated with the etiopathogenesis of psoriasis. In addition, we surmise that the treatment applications for the regulation of skin microbiota of psoriasis patients will contribute positively to the treatment of psoriasis.

Pleural fluid investigations for pleural infections

: Over 1.5 million patients are admitted to hospital with pneumonia in the United States each year. Up to 50% of them can develop a parapneumonic effusion which is associated with higher mortality. The incidence of pleural infection continues to rise, particularly in the elderly and those with comorbidities. Parapneumonic effusions cover a spectrum of presentations from a free-flowing ‘simple parapneumonic effusion’ to a septated ‘complicated parapneumonic effusion’ (CPE) (usually from bacterial pleural invasion) and ‘empyema’ (presence of pleural pus). Pleural infection is defined as either CPE or empyema, and usually requires evacuation of the infected fluid. Laboratory investigations play an essential part of the diagnosis and management of pleural infection. A parapneumonic effusion is typically a neutrophil-rich exudate. Presence of bacteria from culture of the fluid defines pleural infection but conventional culture methods have a low yield. Surrogate pleural fluid markers are often employed to confirm a CPE, including low pleural fluid pH (<7.2) or glucose (<3.3 mmol/L) and elevated lactate dehydrogenase (LDH). Measurement of pleural fluid triglyceride and chylomicrons (for chylothorax) and cholesterol (for pseudochylothorax) may be needed to separate lipid pleural effusions from empyema. Tuberculous pleural effusions usually result from a hypersensitivity pleuritis and are lymphocyte predominant with elevated pleural fluid adenosine deaminase (ADA) and interferon gamma levels. Culture yield of mycobacteria is typically low. Caseating granulomas on pleural tissue biopsy is often considered diagnostic. Common organisms for community-acquired pleural infection include Streptococcus pneumoniae, Streptococcus anginosus group bacteria and Staphylococcus aureus. Hospital-acquired pleural infections have higher mortality and are often polymicrobial which can include S. aureus, Enterobacteriaceae and anaerobes. Antibiotics and evacuation of the infected fluid, usually by chest tube drainage, remain the mainstay of treatment. Intrapleural fibrinolytic and deoxyribonuclease therapy, or occasionally surgical drainage, may be required.

#97: Rapid, Noninvasive Detection of Kingella kingae Pediatric Vertebral Infections Using a Microbial Cell-free DNA Sequencing Test for Pathogen Identification

Abstract Background Kingella kingae is a recognized cause of bone and joint infections (BJI) in infants. The diagnosis of Kingella kingae BJI can be challenging due to its fastidious growth with conventional culturing methods even when infected tissue is obtained. Kingella kingae spinal infections are likely an underdiagnosed entity given the limitations of culture-based methods and the reluctance to biopsy spinal locations of infection (in favor of empiric treatment). This approach often necessitates MRSA coverage. A sensitive, rapid, noninvasive diagnostic approach to pediatric vertebral infections would enable targeted therapy. Detection of circulating microbial cell-free DNA (mcfDNA) in the plasma originating from areas of sequestered infection through next-generation sequencing (NGS) has shown utility in pediatric pneumonia (Farnaes et al. DMID 2019) and a wide variety of infections in the immunocompromised host (Rossoff et al. OFID 2019) and potentially offers promise in resolving the etiology of pediatric vertebral infections. Methods The Karius test is a CLIA-certified/CAP-accredited NGS plasma test that detects circulating mcfDNA in the blood. After mcfDNA is extracted and NGS performed, human sequences are removed and remaining sequences are aligned to a curated pathogen database of &amp;gt;1400 organisms. Organisms present above a statistical threshold are reported and quantified. The time to result reporting is on average 24 hours from sample receipt. Karius Test results over the prior 2 years were reviewed for detections of Kingella kingae in the context of spinal infections. Clinical chart review was performed by the treating pediatric infectious diseases physicians at each participating institution after IRB notification and approval. Results Six cases of Kingella kingae pediatric vertebral infections were identified across five institutions; clinical data were available for five cases across four institutions (see Table). Four cases were male; the average age was 15.3 months. Four of five cases had an antecedent URI. The clinical presentations were characterized by decreased mobility and relatively bland inflammatory response (lack of fever, bland inflammatory markers). The lumbar region was the most commonly affected vertebral location (80%). Blood cultures were negative in all cases; empiric anti-MRSA therapy was initiated in all cases. The time to result of Kingella kingae mcfDNA detection in the plasma was one day from sample receipt in all cases. McfDNA from co-pathogens were detected in 66.7% of cases (Haemophilus influenzae was the most common). The detection of Kingella kingae by the Karius test influenced a decision to narrow coverage in 80% of cases and a decision to forego biopsy in 60% of cases. Conclusion Plasma NGS for circulating mcfDNA offers a rapid, noninvasive means of detecting Kingella kingae pediatric vertebral infection. This culture-independent approach may enable specific diagnosis despite antibiotic pretreatment and obviate the need for an invasive procedure. Accurate identification of Kingella kingae has important implications on antibiotic stewardship enabling targeted therapy without the reliance on empiric MRSA coverage. Given the capacity to detect over 1400 organisms from a single sample NGS for mcfDNA offers a means to detect a broad variety of pathogens known to have predilection to cause pediatric spine infection.

Evaluation of Bacterial Load and Antibiotic Resistance Pattern of Staphylococcus aureus from Ready-to-Eat Raw Beef in Bahir Dar City, Ethiopia.

Background Staphylococcus aureus is one of the most important causes of food-borne intoxication and the most frequent antibiotic-resistant pathogen in the world. Regular evaluation of the current safety status of food is a proactive measure to minimize the possible danger of food-borne pathogens. Therefore, this study was conducted to assess the bacterial load and antibiotic resistance profile of S. aureus from ready-to-eat raw beef in Bahir Dar city, Ethiopia. Methodology. This cross-sectional study was conducted from October 2018 to April 2019 by collecting a total of 101 raw beef samples from butcher shops using a simple random sampling method. Isolation and microbial load determination of S. aureus use were performed by conventional culture method and an antibiotic susceptibility test was conducted by using Kirby Bauer disk diffusion method on the Mueller–Hinton agar. The data were analyzed by using STATA software version 12.0. Result Out of 101 raw beef samples, 55 (54.45%) were positive for S. aureus with a mean bacterial count of 3.40 ± 0.63 (log10 cfu/g). About 13% of butcher shops had unacceptable and potentially dangerous (above 104 cfu/g) bacterial load. High S. aureus drug resistance was observed on penicillin (92.73%) followed by cefoxitin (74.5%), tetracycline (63.63%), and clindamycin (50.9%). On the other hand, there was the highest susceptibility for ciprofloxacin (100%) followed by gentamycin (90.91%) and erythromycin (87.27%). Multidrug resistance was also found in 54 (98%) of the isolates. Conclusion In this study highly drug-resistant S. aureus was incriminated as the main meat contaminant in butcheries of Bahir Dar city. Therefore, appropriate antimicrobial use and staphylococcal control methods should be employed to prevent S. aureus intoxications in foods.